An Introduction to a Spiral Approach for Understanding Contemporary Immunology for the Layperson and the questioning Immunologist

Many individuals, besides professional immunologists, are interested in how the immune system works. This is partly because the immune system is related to diverse areas of medicine, and partly because it has some fascinating attributes.

I have been doing research in immunology for over fifty years. Contemporary textbooks on immunology run to several hundred, and often over a thousand, pages. I become overwhelmed, as I immerse myself in most of these texts, by the feeling that the immune system is extremely complex. My belief that the immune system can be understood in foundational terms is undermined. I then explore whether I can regain my composure by quietly re-examining the validity of some ideas I have in the past found helpful. I thereby regain my composure and am again moved by the prospect that these foundational ideas provide a framework for medical interventions. Obviously, some of the foundational ideas I am referring to are not widely held, and so not incorporated in textbooks.

Most of these foundational ideas were first published between 45 and 50 years ago. When I first became an immunologist, I met, in the late 1960s, John Howard, an older colleague and eminent researcher, who complained about the state of the field. Ten years ago, he explained, he knew almost all the immunologists worldwide and had some idea of their interests. He regretted the field was becoming too large for an individual to maintain such an intimate overview. The number of immunologists now, about 15000 I estimate, must have increased by perhaps twenty-fold since then. There are thousands of pages published a month that an individual, with broad immunological interests, “should” assimilate, if they are to know all that is pertinent to their interests. This is not possible.

One way, the only way I can conceive, of transcending the information overload, is to

concentrate on foundational issues. Whether this approach will work is a legitimate question. I would suggest an appreciation of how science has progressed, in different areas, provides grounds for believing the efficacy of this approach is worthy of exploration. The structure of DNA is simple, and was proposed to provide a molecular basis for understanding genetics. Contemporary studies on how DNA replicates, on how damage to DNA is repaired, and on the processes involved in “genetic recombination”, are complex. However, this research does not undermine the knowledge of the structure of DNA; rather this research is based on the structure. This example illustrates the power of foundational discoveries.

I have written a short book, The Foundations of Immunology and Their Pertinence to Medicine, to give my contemporary view of the immune system. I wrote this book for both laypeople and immunologists. I strove to present my view in the most accessible and convincing way. A colleague, Michael Epstein, interested in how best to conduct research and communicate science, suggested I represent my view in less than a page, and in successively longer but short and accessible forms. I have written “an elevator presentation”, less than a page, a ten and a twenty pager that can be read successively. Such a spiral process seems to me to be a novel way of transcending the information overload and in a manner that reflects the history of the subject. I offer these synopses as samples and as an introduction to my book.

An introduction to one view of contemporary immunology: an elevator presentation

Good morning,I hear you are interested in immunology and its relationship to medicine and are curious

on my take.

There are two big questions about how immune responses are regulated. The immune system responds to invaders, such as disease-causing pathogens and cancer, collectively called foreign antigens. Its potential to respond against cells and molecules of the body, to which the immune system belongs, collectively called self-antigens, is normally ablated/destroyed. How do foreign and self-antigens interact differently with the immune system to trigger immune responses or ablate this potential?” In other words: To Be, or Not To Be?

The second question is: If To Be, What To Be? This question arises because the immune system has different means of attacking foreign invaders and has a decision-making process as to which to employ. The two major means are referred to as cell-mediated and antibody-mediated immunity. So how does the immune system decide on what immunity to generate? I have ideas on answers to these two interconnected questions. My slant is pertinent to developing strategies to prevent and treat illness in the four areas of medicine related to the immune system.

Contemporary and effective vaccination works because it guarantees an increased antibody response upon infection, and is effective only against pathogens contained by antibody. However, some pathogens, such as those causing AIDS and tuberculosis, are contained only by cell-mediated immunity. We have developed strategies to ensure stable cell-mediated immunity upon infection, and so protection, and to modulate an on-going antibody response to a cell-mediated one, leading to treatment.

Individuals allergic and non-allergic to house mite dust, living in the same area, produce different “classes” of antibodies to these antigens. We therefore believe that understanding the answer to “What To Be?” will allow one to prevent and treat allergies.

Autoimmunity is an immune attack against self. It represents a failure to ablate the ability to respond to self. If we understand how antigen normally interacts differently with cells of the immune system, to ablate and to trigger immune responses, we are likely to be able to prevent and treat autoimmunity.

Cancers are contained by a sufficiently strong cell-mediated response, but not by antibody. It appears that cancers grow progressively if the cell-mediated response is insufficiently strong. Similar strategies for prevention and treatment are likely applicable against cancers as are effective against pathogens susceptible only to cell-mediated attack.

I might say, in closing, that my proposed answers to the two basic questions are different from those entertained by most immunologists, and so are my suggested strategies of prevention and treatment. I would be pleased to tell you more.

A ten pager on two foundational immunological questions pertinent to medicine

To Be or Not To Be and, if To Be, What To Be?

Medical Significance Immunity plays an intimate role in four areas of medicine: infectious diseases, allergies, autoimmune diseases, such as multiple sclerosis and autoimmune diabetes, and cancer. Immunity represents very specific attacks against targets. This specificity can be likened to that of keys for their corresponding locks.

The immune and innate systems of defense Only vertebrates have immune systems. Non-vertebrates, and vertebrates, have innate defense mechanisms. How do innate and immune

mechanisms of attack differ? Innate defense acts quickly after an insult, they are constitutive, part of the animal’s constitution. An example is acute inflammation, evident following a scratch, with reddening and swelling. In contrast, the expression of the attack mechanisms of the immune system, their effector mechanisms, is highly regulated. It takes days after an infection to generate immunity that attacks an invader, a process referred to as the induction of an immune response.

To Be or Not To Be? Immunity in healthy people is directed against targets manifestly foreign, such as pathogens, or altered self, such as cancer, collectively called foreign antigens. Significant immunity to self-antigens, i.e. to cells and molecules of the body to which the immune system belongs, is not normally generated. This preferential ability to respond against foreign antigens is called self-nonself discrimination. The underlying mechanisms sometimes fail, leading to attacks on self. Such attacks are referred to as autoimmunity, an example being multiple sclerosis.

Autoimmunity shows the immune system has the intrinsic capacity to attack self-antigens, a capacity normally ablated/destroyed. How do foreign and self-antigens normally interact differently with the immune system to induce or ablate immune responses: To Be, or Not To Be?

Different classes of immunity Some immune cells bind to cancer cells, or virally infected cells, resulting in the target cell’s destruction. These cytotoxic cells are a manifestation of cell-mediated immunity, immunity mediated by cells. Other attacks against invaders are initiated by antibody molecules. Specific antibodies bind an invader and also to the machinery that constitutes an innate effector mechanism, thus acting as connectors between the invader and this innate mechanism. One example occurs in allergies. Individuals allergic to peanuts have IgE antibody specific for peanuts. When peanut antigens locally impinge on such an individual’s body, these antigens bind to the IgE antibody and so trigger local, acute inflammation, the effector mechanism. Distinct effector mechanisms are “harnessed” by different classes of immunity, the two main classes being antibody and cell-mediated immunity. Thus vertebrates have three major forms of defense: innate defense that is constitutive, and two inducible forms, constituting the immune system, cell-mediated and antibody-mediated immunity.

If To Be, What To Be? The immune system, when it responds to an invader, does not attack with all the weapons in its arsenal. It has a decision-making mechanism. How does the immune system, once it responds, decide on what class of immunity to generate, a process referred to as immune class regulation? If To Be, What To Be?

The Pertinence of the Regulation of Immune Responses to Medicine Autoimmunity If we correctly understand how self- and foreign antigens normally interact differently with the immune system, to respectively ablate and induce immune responses, we will likely be able to understand how autoimmunity arises, to prevent it in genetically susceptible individuals, and perhaps even to treat autoimmune patients. Allergies Individuals allergic and non-allergic to particular antigens, and both exposed to these antigens because in the same environment, are immune to the antigens. Allergic individuals produce IgE antibody, able to initiate acute inflammation, whilst non-allergic individuals produce benign, non-inflammatory IgA antibody. If we understood the basis of immune class regulation, we could likely ensure the production of IgA antibody on exposure to the allergen.Infectious diseases and cancer All currently effective vaccines increase the speed and size of the antibody response that occurs upon infection. Vaccination is only effective against pathogens contained by antibody. Clinical and experimental observations show that cancers, and certain

pathogens, such as those responsible for AIDS and tuberculosis, are only constrained by cell-mediated immunity. Disease is due to too weak a cell-mediated response, either because the antigen only triggers a weak response, or because it generates an antibody response, associated with the down-regulation of the cell-mediated response. In both cases, if we immunize in a manner that guarantees a strong cell-mediated response on natural exposure, we guarantee protection. Understanding the basis of immune class regulation allows the development of such a strategy, and allows us to modulate ongoing immune response from an antibody to a cell-mediated phenotype. The Clonal Selection Theory of Antibody Formation of the 1950s Setting the scene: the two questions Antibodies are very specific and can be made against anything foreign, an attribute called universality. Specificity and universality imply an astronomical number of different antibodies can be produced. What is the origin of all these antibodies?

Ehrlich posed a second question in the early 1900s. He injected red blood cells red from one goat into another goat. The antibodies raised destroyed the donor’s red blood cells, but not those of the immunized goat, showing these red blood cells were different. This fact struck Ehrlich. He thought antibodies to the goat’s own red blood cells, if produced, would be devastating. How is this avoided? People later paid attention to Ehrlich’s concern with the discovery of autoimmunity in the early 1940s. One such disease is autoimmune hemolytic anemia: antibodies to the patient’s own red blood cells destroy them. We shall consider how autoimmunity is usually avoided.

The context provided by molecular biology The structure of DNA was discovered in 1953. This was pivotal in establishing molecular biology, that in turn provided a foundation for The Clonal Selection Theory and so for immunology. Antibodies are proteins. Molecular biology explained how the genetic material, DNA, exerted its role in heredity, in large measure by defining the proteins a cell can make. All proteins consist of one or a few polypeptide chains. A defined piece of DNA, a gene, defines, or codes for, a polypeptide chain. The prototypical unit of antibodies consists of two identical light and two identical heavy chains. Two genes thus define a prototypical antibody unit, having two identical halves, and so this prototype can bind two antigen molecules.

The problem of self-nonself discrimination Burnet proposed, in the late 1940s, that the respective presence and absence of self- and foreign antigens, early in development, ie in the history of the animal, allows the immune system to discriminate self from nonself. This conjecture was correct. The presence of a foreign antigen during development renders the animal unable, as an adult, to immunologically respond to the antigen; the immune system “now” regards it as self. This Historical Postulate became central to the formulation of The Clonal Selection Theory.

How can so many antibodies be generated? Why one cell makes only one antibody. Most new genes arise slowly through evolution, and are handed down intact from parents to their offspring. Lederberg imagined that parents provide their offspring not with antibody genes, but with assembly kits to generate such genes. I illustrate the process by a simile. Suppose there are a 100 similar but different red pieces, a 100 similar yellow pieces, and a 100 similar blue pieces. We join together one red, one yellow and one blue piece to obtain a “potential” heavy chain gene. This means 300 pieces can be combined to generate 100 x100 x 100 =106 combinations. A combination sometimes codes for a functional heavy chain, in which case the cell tries to assemble a gene that codes for a functional light chain. Once this is successful, the cell ceases

the assembly process. Many attempts fail, and the cells die. The surviving cells have assembled genes for making one antibody!

Characteristics of antibody responses accounted for by these ideas The antibody precursor cells so generated were correctly envisaged to express antibody receptors on their surface. Since the immune system can produce many antibodies, an antibody precursor cell, able to produce an antibody specific for a particular antigen, is very scarce. Immunologists envisaged that a foreign antigen could interact with, and so select, a scarce precursor cell, with surface antibody receptors to which the antigen binds, causing it to divide and so form a clone of identical cells, giving rise to many antibody-producing cells. These ideas explained two things: how very scarce cells could produce significant amounts of antibody within several days, through cellular multiplication, and why radiation of an animal compromised its ability to produce antibody. Irradiated cells were known to die when forced into cell division. Much evidence supports this Clonal Selection Theory.

Three different types of precursor cells The antibody precursor cell is generated in the marrow of the bone and is called a B cell. Two types of “T cell” are generated in the thymus. The CD8+ T cell can be activated to produce CD8+cytotoxic T cells, able to kill cancer and virally infected cells. The CD4+ T cells play a central role in immune regulation. These “helper T cells” are required to activate both B cells and CD8 T cells. These three types of cell are histologically similar, and so are called “lymphocytes”. All lymphocytes are highly specific by virtue of the specific receptors they bear on their surface.

The first foundational question: the basis of self-nonself discrimination

Two mechanisms are recognized as contributing to self-tolerance. The first, enunciated by Lederberg, and represented in Figure 1, is strongly supported by evidence. The need for a second mechanism is accepted, but this mechanism is in the eye of contemporary controversy.

Lymphocytes, when first arising, are inactivated by antigens if present, as self-antigens are envisaged to be, see Figure 1. The lymphocyte, if not “paralyzed”, develops after a few days into an inducible state. Thus inducible lymphocytes accumulate specific for foreign antigens.

Central and peripheral tolerance Evidence supports Lederberg’s proposal, with one caveat. Not all self-antigens are sufficiently present in the organs, where lymphocytes are generated, to ablate/destroy their corresponding lymphocytes. This ablation process is called central tolerance. The “missing” antigens are more prevalent outside these organs, in the periphery. One peripheral self-antigen is insulin, a target in autoimmune diabetes. Immunologists

Figure 1 The top part of the figure represents The Historical Postulate. The bottom part represents Lederberg’s model of lymphocyte development, proposed to explain how self-nonself discrimination is achieved. For explanation see text.

recognized there is a second mechanism for ablating lymphocytes specific for peripheral antigens, leading to peripheral tolerance.

Peripheral Tolerance for B and CD8+ T cells Peripheral, mature lymphocytes can interact with antigen in two distinct ways, one leading to their inactivation, the other to their activation. How do these interactions differ? Bretscher and Cohn proposed in 1970 that single lymphocytes are inactivated by antigen, whereas their activation requires antigen-mediated lymphocyte cooperation. This proposal explained diverse observations and also how peripheral tolerance may occur, in a manner consistent with the Historical Postulate, see Figure 2. The first lymphocyte generated, specific for a peripheral self-

antigen will be a loner and, according to the proposal, be inactivated by antigen. Lymphocytes specific for self-antigens are eliminated as generated, one or a few at a time. Lymphocytes specific for a foreign antigen accumulate in its absence; once the foreign antigen impinges upon the immune system, it mediates lymphocyte cooperation to generate immunity.

This one lymphocyte/multiple lymphocyte model, for the inactivation/activation of lymphocytes, is generally accepted as being pertinent in the case of B and CD8 T cells. Their activation requires helper CD4 T cells and, in the absence of this help, antigen inactivates these cells. Thus CD4 T cells are the guardians over the fate of B and CD8 T cells on their encounter with antigen. A central question of the last 30 years, with controversial answers, is this: how does antigen interact differently with CD4 T cells to activate and to inactivate them? Central to some proposals is our understanding of how innate defense systems achieve self-nonself discrimination.

Self-Nonself discrimination by innate defense Various molecular structures are commonly expressed by various microorganisms but are absent from vertebrates. Some of these “pathogen-associated molecular patterns” (also expressed by non-pathogenic microbes), are referred to as PAMPs, and are recognized by vertebrates, through their “pattern recognition receptors”, or PRRs. The PRRs thus recognize some foreign but not self-entities. PRRs are proteins and so coded for by genes, so these genes ensure self-nonself discrimination, ie self-nonself is genetically determined.

Figure 2 The one lymphocyte/multiple lymphocyte model for the antigen dependent inactivation and activation of lymphocytes. The top half represents the fateof anti-self lymphocytes, the bottom of those specific for foreign antigens. For explanation, see text

The activation and inactivation of CD4 T cells: The PAMP and Danger Models Janeway proposed in 1989 that antigen can activate CD4 T cells only if the antigen expresses a PAMP, or is administered with PAMP-expressing material. In the absence of PAMPs, antigen inactivates CD4 T cells. Janeway justified his idea by pointing out the difficulty in raising immunity to purified, vertebrate, protein antigens, unless given with PAMPs. In contrast, PAMP-expressing microbes readily induce immunity. Whether antigen activates CD4 T cells would NOT depend on whether the antigen is foreign, but on whether it contains PAMPs. Matzinger in 1994 pointed out that foreign vertebrate grafts, that are PAMP-deficient, are rejected. Grafting causes the recipient animal to express a “stress response”. Matzinger proposed that activation of CD4 T cells requires a “Danger Signal”. In the absence of Danger, antigen would inactivate the CD4 T cell. The CD4 T cell cooperation model I find these models implausible, though dominating the field for the last 25 years. Many sterile, PAMP-free, foreign, vertebrate antigens are immunogenic when delivered in a way not expected to provoke a danger signal. One example is sheep erythrocytes delivered to mice with a sharp needle. Secondly, PAMP- and Danger-signals are not antigen-specific; a requirement for such signals to activate CD4 T cells specific for foreign antigens would likely sometimes result in the activation of newly generated CD4 T cells specific for peripheral self-antigens. Lastly, PAMP/Danger Models are inconsistent with the Historical Postulate. In contrast, many observations support the proposal that the activation of CD4 T cells is facilitated by antigen-mediated CD4 T cell cooperation, and indirect evidence that antigen can inactivate single CD4 T cells. Moreover, this more detailed form of the one lymphocyte/multiple lymphocyte model for the antigen-dependent inactivation/ activation of lymphocytes, see Figure 2, explains peripheral tolerance at the level of CD4 T cells, in a manner consistent with the Historical Postulate. I favor this possibility.

The central role of CD4 T cells in immune class regulation The presence of activated CD4 T helper (Th) cells not only determines whether antigen activates B and CD8 T cells. Immunologists have shown that the activation of naïve CD4 T cells can lead to the generation of different types, or subsets of, helper T cells. These different subsets determine the class of immunity the antigen induces. For example, “Th1 cells” are exclusively generated when antigen induces an exclusive cell-mediated response, and “Th2 cells” when IgG1 and IgE antibody are exclusively produced. Activated Th1 and Th2 cells differ in the spectrum of cytokines they secrete on antigen stimulation. The cytokine molecules affect the differentiation of cells with which these Th cells come into contact. For example, one cytokine produced by Th2 cells facilitates the production by B cells of IgE antibody. Th1 cells facilitate the activation of CD8 T cells to give rise to cytotoxic T cells. The question of “What To Be?” can be translated into what subset of CD4 T cell is generated?

What different circumstances lead to the generation of Th1 and Th2 cells?The PAMP and Cytokine Milieu Hypotheses There are diverse PAMPs and corresponding PRRs on different microorganisms. A highly prevalent idea is that PAMPs are not only needed to activate CD4 T cells, but their diverse nature leads to the generation of different Th subsets. It is also apparent that the cytokine environment in which CD4 T cells are activated can affect the Th subset generated under some circumstances. This proposal leaves open the question of what determines the cytokine environment? The observations on which this cytokine proposal is based are artificial and, partly on such grounds, I question this proposal. In addition, there are more general grounds for questioning both these proposals that currently dominate thinking in the field. If one had a correct understanding of how antigen interacts differently with cells of the immune system, to result in the generation of Th1 and Th2 cells, one should be able to account

for the variables of immunization known to affect whether Th1 or Th2 cells are generated. Conversely, if one knows these variables, one might be able to make an informed guess as to how antigen interacts differently with cells of the immune system to generate Th1 and Th2 cells.

Variables of immunization affecting the differential generation of Th1 and Th2 cellsThree variables were characterized over 50 years ago, and their generality has stood the test of time. Antigens that are minimally foreign are only able to induce a cell-mediated, Th1 response. It appears that such minimally foreign antigens are only susceptible to cell-mediated attack, providing a biological rationale for this first generalization. Most cancers belong to this category. More foreign antigens, such as bacteria, can induce cell-mediated immunity or antibody, depending on both the dose of antigen and the time after immunization at which the immunity is assessed, as depicted in Figure 3. A low dose only generates a cell-mediated response. Increasing the dose results in a more rapid cell-mediated response and with time the evolution of an IgG, Th2 antibody component.

A further increase in dose results in a more rapid antibody response, and the cell-mediated response may be eclipsed. This pattern is seen with PAMP-free, foreign, vertebrate antigens, and with PAMP-expressing pathogenic bacteria and protozoa. All these observations are difficult to reconcile with the PAMP hypothesis. For example, the PAMPs do not change with time after infection, making the evolution of the response from an exclusive Th1 to a mixed Th1/Th2 mode paradoxical. Neither is the dose dependence of the nature of the immune response readily understandable.

The Threshold Hypothesis I proposed this hypothesis in 1974, partly because it accounted for the three variables of immunization, discussed above and then known to affect the cell-mediated/IgG antibody nature of the response. It also fitted in with what was then surmised about cancer/tumor cells being only susceptible to cell-mediated attack, an idea that still seems valid today. The hypothesis was formulated within the framework that the activation of CD4 T cells requires the antigen-mediated cooperation of CD4 T cells. It stated, in modern terms, that tentative and robust cooperation respectively result in the generation of Th1 and Th2 cells. The hypothesis accounted for why minimally foreign antigens only induce Th1 cells, as there will be

Figure 3 Depiction of how dose of antigen, and time after immunization, affects the nature of the immune response, after Salvin, 1958. Salvin measured delayed-type sensitivity (DTH) to assess the cell-mediated, Th1 response, and IgG antibody as reflecting a Th2 response. The generation of DTH and cytotoxic T cells occurs before IgG antibody can be detected.

relatively few CD4 T cells specific for such antigens and so, even with optimal amounts of antigen, CD4 T cell cooperation will only be tentative. There will be more CD4 T cells specific for more foreign antigens and so, in the presence of optimal antigen, cooperation will be robust and Th2 cells will be generated. Decreasing the antigen dose will decrease the cooperation and, if sufficiently decreased, cooperation will be tentative, resulting in the generation of Th1 cells. Consider what happens after antigen impacts the immune system. Antigen is known to cause the CD4 T cells to multiply and so, so long as the antigen level is sufficiently sustained, CD4 T cell cooperation will increase, explaining the evolution of the response from a Th1 towards a Th2 mode, see Figure 3. The Threshold Hypothesis thus explains how the variables of immunization affect the Th1/Th2 nature of the ensuing response. The hypothesis makes one very strong and unique prediction. Suppose we have a situation where antibody and Th2 cells are generated, indicating robust CD4 T cell cooperation. Sufficient but partial depletion of CD4 T cells, around the time of immunization, all other variables being kept constant, will result in tentative CD4 T cell cooperation, and in the generation of Th1 cells. We have successfully tested this prediction in diverse experimental systems. This finding is incomprehensible in terms of the PAMP and the Cytokine Milieu Hypotheses, as reducing the number of CD4 T cells is not expected to change the PAMPs expressed or change the cytokine milieu, but just to reduce the size of the response.

An indication of the importance of the answers to the two questions in terms of medical interventions The most studied mouse model of a human infectious disease, caused by a pathogen uniquely susceptible to cell-mediated attack, is cutaneous leishmaniasis. Infection of different strains of mice with 106 of the causative parasites has one of two outcomes: a stable Th1 response, limited growth of the parasite and resistance, or in time a Th2 response and unrestrained parasite growth and susceptibility. Infection of the latter, “susceptible” mice with 300 parasites, a “low dose”, results in a stable cell-mediated, Th1 response, and resistance. Infection of these mice, two months after this first infection, with 106 parasites, also results in a stable Th1 response and resistance. Thus infection with low numbers of parasites not only results in a stable Th1 response but results in a Th1 imprint, guaranteeing a Th-1 response upon a challenge of 106 parasites. This low dose vaccination strategy holds promise of leading to successful vaccination against pathogens uniquely susceptible to Th1 attack, such as those causing AIDS and tuberculosis. We have shown that the same strategy appears to work against transplantable tumors in animals, that serve as animal models of human cancer. Note that all these attempts at medical intervention depend upon the generalization that the Th1/Th2 nature of the response depends upon the dose of antigen administered. This generalization is accounted for by the threshold mechanism, but not by the currently popular PAMP and Cytokine Milieu Hypotheses, perhaps explaining why the low dose vaccination strategy has had little impact.

The one lymphocyte/multiple lymphocyte model for the inactivation/activation of lymphocytes also appears to be pertinent to ablating immune responses and facilitating their generation. The administration of foreign antigens that are very small indeed, and so only able to bind the receptors of one CD4 T helper cell, is known to inactivate their corresponding CD4 T cells, whereas making small antigens more foreign, by coupling the smaller antigen to a more foreign one, facilitates the activation of their corresponding CD4 T cells. These observations do not make sense in terms of the PAMPs/Danger Models. These findings may provide a platform for developing strategies to prevent and treat autoimmunity and facilitating immune responses to antigens that are inherently weak due to the paucity of helper T cells, as may occur for some antigens present, for example, on cancer cells.

A philosophical note A major question facing contemporary immunology, and more generally our attitude to

understanding the world, is how much information do we need to gain insight? I personally believe that a study of how foundational concepts arise, and are found to be valuable, demonstrates that hypothetical concepts are usually formulated, after reflection, in response to a few observations/generalizations/questions, recognized to be poignant; once formulated, we collectively find the means for testing their utility and provisional validity. To be more strict, observations can only be realized within a tentative framework, and so the information inferred inherits this tentativeness. A different mind-set is that we only achieve real insights when we have amassed a considerable body of indisputable observations. These contrasting mind-sets are clearly apparent in how people hope to achieve insight in immunology. I argue that the history of immunology provides a case study supporting the first viewpoint. No one today doubts the essential elements of The Clonal Selection Theory. The impetus for this theory was to answer some foundational questions, arising from observations and some questions they led to, as indicated above, rather than being deduced from a large number of indisputable observations. Moreover, this theory did not anticipate the enormous amounts of new information that have accumulated in the last 50 years. All immunologists, in view of overwhelming evidence, employ The Clonal Selection Theory as a framework for further understanding, and do not decry its inability to explain new observations beyond its envisaged mandate. I similarly suggest that the one lymphocyte/multiple lymphocyte model for the antigen-dependent inactivation/ activation of lymphocytes, and The Threshold Hypothesis, provide essential frameworks for further advances in the field.

A Twenty Pager: An Introduction to Immunology for the Layperson

The Foundations of Immunology and Their Pertinence to Medicine

Note: The glossary at the end indicates the page on which technical terms are indicated by being bolded and italicized and their meaning indicated by their context. It also lists the few abbreviations used.

The birth of immunology Immunology, and the study of infectious diseases, both came into being in the mid/late

1800s. Jenner had developed in the late 1700s the means to protect against smallpox, then killing one in five Englishman. Jenner harvested material from the pocks of cows suffering from cowpox, a related disease. He applied this material to a person’s scarified skin to achieve protection against small pox.

In the mid-1800s, Robert Koch and Louis Pasteur discovered that pathogens cause infectious diseases. The pathogen, isolated from a diseased animal, caused disease when injected into another animal. Such pathogens are virulent. Pasteur showed that pathogens, grown under some conditions, lost their virulence and became attenuated, i.e. no longer caused disease.

Infecting with the attenuated pathogen could protect an animal from a virulent challenge, a process Pasteur called vaccination.

Two further findings were seminal. Some pathogens cause illness/death by secreting toxins. The administration of toxoid, a modified, non-toxic form of toxin, can protect an animal from a subsequent, normally lethal challenge of the toxin These processes, leading to protection, are referred to as immunization. Some thought protection might be due to molecules in the blood. A spectacular finding demonstrated this. Blood, from an animal immune to a toxin, was allowed to clot. The cell-free serum was injected into another animal that now resisted a normally lethal challenge of toxin. Immune serum contains protective molecules, called antibodies. Anything recognized by antibodies is called an antigen. The donor of the immune serum has been actively immunized with the toxoid, and the recipient of the immune serum has been passively immunized. The immune system is a recent invention on the part of evolution

Only vertebrates express immunity, though all organisms protect themselves against invaders. How then do immune mechanisms differ from those of non-vertebrates? A description of two non-immune mechanisms will allow me to illustrate the differences.

All multicellular organisms have specialized cells able to capture and take up foreign matter/invaders. These cells normally digest the ingested material and are called phagocytes (Gr phago, to eat). Phagocytes protect against foreign invaders and constitute one of the oldest of innate defense mechanisms. Another such mechanism is evident when we get a scratch. A red line develops within seconds, and swelling within minutes. This acute inflammation brings phagocytes, and other defensive mechanisms, to the site.

The immune system’s five cardinal attributes distinguish it from innate defense The attributes of specificity and of universality Immunologists, in the early 1900s, raised antibodies to small molecules, the size of a benzene ring or two. Such small molecules are non-immunogenic, ie unable to stimulate the production of antibodies. To raise antibodies, the small molecule, the hapten, is attached to a larger, immunogenic carrier. Anti-hapten antibodies are produced by immunizing with hapten-carrier conjugates.

Immunologists raised antibodies to a hapten, whose precise structure was known. They examined whether these anti-hapten antibodies would bind to structurally similar molecules. They were astounded by how specific antibodies are, and likened this to the “specificity” of keys for their corresponding locks. Antibodies can also be raised against any foreign hapten, an attribute called universality. These two attributes mean an animal has the capacity to produce an enormous number of different antibodies, estimated to be of the order of a billion. A metaphor illustrates how remarkable specificity and universality are. Imagine a shop that has keys to all known locks as well as to all those not yet invented!

Adaptability Phagocytosis and acute inflammation are effector mechanisms. These mechanisms of innate defense are said to be constitutive, as part of the constitution of the animal. They are present on infection, or very shortly thereafter. In contrast, it takes days to produce antibody. Moreover, vaccination works because a secondary immune response, seen on a secondary infection, is more rapid and stronger than the primary response, seen on a primary infection. Thus exposure to the antigen can result in positive memory. Exposure to antigen under certain circumstances can also result in an ablation of the ability, or a diminished capacity, to respond to antigen, as we shall see. This is negative memory. Both kinds of memory reflect the adaptability of the immune system.

Adaptive processes responsible for immunological self-nonself discrimination All effector mechanisms, of both the innate defense and immune systems, normally only “fight” foreign entities, but not parts of the body to which the effector mechanism belongs. This attribute is called self-nonself discrimination. Cells of innate defense bear pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs), expressed by pathogens and benign microbes and parasites, but not by self-molecules or self-cells. Hence innate self-nonself discrimination is due to the specificity of the PRRs. PRRs are usually proteins, encoded by genes of the individual. Hence, it is said the self-nonself discrimination of innate mechanisms is genetically determined or germline encoded.

Universality means an individual can immunologically respond to their sibling’s foreign antigens, foreign due to genetic differences. However, through chance events at conception, the individual could have inherited these genes. An individual thus responds to something that could have been “self”. The lack of response to self must therefore be due to the ablation of this potential, ie due to negative memory. This ablative process can fail. One example is autoimmune, hemolytic anemia: antibodies are produced against the patient’s own red blood cells, leading to their destruction and so to anemia.

Immune Class Regulation Allergic individuals react within minutes when the allergen is injected into their skin. The injection site reddens, swelling occurs, signs of acute inflammation. Giving serum from an allergic to an unsensitized animal renders it allergic, demonstrating antibodies mediate allergies. Some other kinds of immunity are due to cells. This discovery arose from Robert Koch’s attempts to treat tuberculosis (TB).

Koch made an extract of Mycobacterium tuberculosis that causes TB. He injected this extract into the skin of patients, hoping to boost their immunity and so their recovery. Some patients took a turn for the worse, and so Koch abandoned this treatment. However, he noticed an inflammatory response at the injection site, peaking 24-48 hours after injection. This distinguished it from acute inflammation. It is referred to as delayed-type hypersensitivity (DTH). DTH can only be transferred from an immune to a naïve animal by the transfer of cells. DTH is an example of cell-mediated immunity (CMI). Immunologists thus first realized there were different kinds of immunity.

Optimal DTH and antibody responses occur under different circumstances. This is the tip of the iceberg. In people, there are seven different types antibody, the IgM, IgE, IgA and IgG classes, the latter consisting of the IgG1-4 subclasses; different classes/ subclasses of immunity are optimally generated under different conditions. These generalizations lead to two major questions: what determines which class/classes of immunity are induced under a given set of circumstances? What is the physiological and medical significance of such differential generation? The mechanisms controlling such differential regulation are collectively referred to as immune class regulation. Clinical findings provide tantalizing clues as to the physiological/pathological significance of different classes of immunity.

The five areas of medicine where immune regulation is central Autoimmunity and transplantation An understanding of self-nonself discrimination must explain how foreign antigens induce immune responses and self-antigens ablate this potential. Such an elucidation should allow one to prevent, and perhaps treat, autoimmunity in genetically susceptible individuals, and to assess the prospects for facilitating the transplantation of foreign grafts.

Allergies Non-allergic people, living in a place where allergies are prevalent, say to dust mites,

are immune. Allergic and non-allergic people respectively predominantly produce IgE and IgA antibody to the allergen. When children outgrow their allergy to milk, the response evolves from an IgE to IgA mode. Desensitization of allergic patients, by giving escalating doses of the allergen, results in a similar modulation. Immune class regulation is thus pertinent to preventing IgE responses, ie avoiding allergies, and modulating the IgE response against the allergen to an IgA mode, thus constituting treatment.

Infectious diseases Current and effective vaccination ensures a secondary antibody response upon natural infection. It is only effective against pathogens contained by antibodies. A strong cell-mediated response is required to contain some pathogens. Disease is often associated with a predominant antibody, or humoral response. In these cases, effective vaccination must cause a cell-mediated imprint upon the immune system that guarantees CMI upon infection. We later explain how this can be achieved. Treatment requires a modulation of the response from a humoral to a cell-mediated mode.

Cancer Clinical observations and animal studies lead to the conclusion that CMI, in the form of cytotoxic T lymphocytes (CTL), is protective against human cancer and animal tumors. Much immunological research, including that on immunity to tumors, requires inbred strains of animals, generated by extensive brother/sister mating. Individuals of the same strain and sex are genetically identical, allowing cells to be transferred, from one animal to another, without rejection of the donor cells. Some tumors arose in inbred strains, giving rise to transplantable tumors. Sometimes, these tumors are rejected, due to immunity against tumor-associated antigens; sometimes the tumors grow progressively, and would kill the animal unless humanely sacrificed. Rejection is most often associated with the generation of cytotoxic T lymphocytes. These cells can bind to and kill tumor cells. Vaccination against cancer would appear to require a guarantee of CMI against cancers should they arise; treatment should modulate the immune response so that sufficient cytotoxic T lymphocytes can control the cancer. The Clonal Selection Theory of antibody formationPreamble This theory was formulated in the late 1950s. The structure of DNA was proposed in 1953. The Clonal Selection Theory was developed in the wake of the newly emerging field of molecular biology, and has become the cornerstone of the discipline. Appendix I gives a synopsis of the molecular biology need to understand this text for those unfamiliar with the subject.

The theory attempted to answer two questions, both first raised in the early 1900s by Ehrlich. He immunized one goat with red blood cells from another goat. The antibodies produced destroyed the donor’s RBC, but not those of the immunized goat. Ehrlich suggested such anti-self antibodies, if produced, would be very damaging. He recognized the attribute of self-nonself discrimination. The discovery of autoimmune diseases in the 1940s, illustrated, by their absence in healthy individuals, the importance of this attribute. Burnet suggested, in 1949, that the presence of self-antigens in ontogeny, ie during the development or early history of the individual, allows the immune system to discriminate self from nonself. Medawar reported, in1953, that a fetus, exposed to foreign antigens, failed, as an adult, to mount an immune response against them, thus supporting this conjecture, that we refer to as The Historical Postulate. The stage was set for detailed proposals as to how self-nonself discrimination could be achieved.

Ehrlich also speculated on how antigen induces the formation of antibodies. He envisaged that cells bore diverse surface receptors; when antigen bound a particular receptor, the cells produced and secreted more of this receptor, hence the production of antibody. The antigen

selected the receptor/antibody to be produced.

Selective and instructive ideas Landsteiner demonstrated, in the early-mid 1900s, the universality and the specificity of antibody responses. Thus an animal can produce an astronomical number of different antibodies. Some envisaged that antigen was needed to instruct the immune system to produce antibody; others envisaged antibodies were first generated without the presence of antigen; antigen then selected the corresponding antibody. The instructive process is likened to a tailor making a suit, the antibody, to fit an individual, the antigen; the selective process to an individual, the antigen, selecting a well-fitting suit, the antibody, upon visiting a well-stocked department store. One problem unaddressed by protagonists of instructive theories was that of self-nonself discrimination. Why did the immune system not keep on making anti-self antibodies, as self-antigens are much more prevalent than foreign antigens?

A quartet creates the theory Neils Jerne detected antibodies in unimmunized rabbits. He therefore proposed, in 1955, a selective theory of antibody formation, based on three postulates. (i) A diversity of antibodies is generated in fetal life and is present in fetal blood. (ii) Those combining with self-antigens are removed, leaving only those specific for foreign antigens. (iii) When a foreign antigen later impinges upon the animal, it combines with its antibodies. These complexes are taken up by a cell, where the antibody copiously replicates. Antibodies are proteins. Jerne’s third postulate implies proteins can replicate. This was incompatible with the then emerging field of molecular biology.

Talmage proposed in 1957 a diversity of cells produce a diversity of antibodies, but one cell can only produce one chemically distinct kind: this cell contains the genes for the polypeptide chains that constitute the antibody. Each cell bore its unique antibody as a surface receptor. The interaction of antigen with this receptor, when the cell is generated in fetal development, leads to its removal, leaving cells only able to respond to foreign antigens. These cells are referred to as antibody precursor cells. Talmage imagined precursor cells specific for a given antigen to be very scarce. He suggested antigen triggers their multiplication, to more rapidly produce antibody, constituting a clone of cells. Burnet also outlined in 1957 a similar proposal, The Clonal Selection Theory. Lederberg published in 1959 the most coherent account of this theory. Burnet, Lederberg and Talmage interacted during these critical years, making the history of the ideas somewhat difficult to disentangle. We now turn to Lederberg’s additional insights.

Lederberg recognized, as autoimmunity arises in adults, that self-tolerance is not uniquely established in the fetus. Precursor cells are generated throughout life. When first generated, he surmised the cell is ablated if it interacts with antigen. If not ablated, in a day or so, it differentiates into a precursor cell that antigen can activate. Thus self-antigens, present before precursor cells are first generated, would ablate their precursor cells. Lederberg thus proposed a precise, and it turns out a prescient, explanation for self-nonself discrimination.

Note that with 1000 different light and 1000 different heavy chains, 1000,000 different

antibody molecules can be generate

d. Although the actual numbers are greater, those chosen illustrate how combinatorial mechanisms lead to a much larger number of combinations than of elements. Additional combinatorial mechanisms are exploited by nature: the gene coding for the variable part of a heavy chain is made by randomly joining one of a bank of h DNA segments, to one of a bank of d segments and to one of a bank of j segments, producing many more combinations than the number of segments. Thus, though most genes are created during evolution, Lederberg envisioned that vertebrates receive, from their parents, assembly kits for immunoglobulin genes, now known to be true. This process occurs in the cells that give rise to precursor cells. If the process leads to genes coding for a functional heavy and a functional light chain, it stops. The cell differentiates into a precursor cell. These processes give rise to a population of precursor cells that express diverse antibody receptors. Thus is universality achieved!

Confirmation of The Clonal Selection TheoryInvestigations confirmed the general processes Lederberg envisaged, concerning the

generation of immunoglobulin genes, and defined these processes in considerable detail. The picture developed is not the subject of serious challenge. It is useful to outline another general test of the theory for both its methodology and its illumination.

Irradiation of animals was long known to abrogate antibody responses. Irradiation causes the death of cells if forced into division (due to damage of the cell’s DNA molecules). Burnet had on this basis long surmised that antibody formation requires cell proliferation. Talmage incorporated this idea to explain how antibody could be produced more rapidly by the multiplication of precursor cells on their activation. Thus the transfer of precursor cells to irradiated mice is anticipated to allow them to respond to antigen. Spleen cells have this ability. Some spleen cells bear surface antibody receptors and are also required for the reconstitution of irradiated mice. This beautifully supports the idea that precursor cells bear antibody receptors. Furthermore, very scarce mouse spleen cells bind to sheep red blood cells via antibody receptors. Their removal abrogates the spleen’s ability to restore the antibody response to this antigen, when given to irradiated mice, but not the response to unrelated antigens. Thus separate cells are responsible for producing antibody to unrelated antigens. These, as well as a multitude of other observations, make the Clonal Selection Theory unassailable. It also became clear, in the 1980s, that Lederberg’s model of tolerance was valid, but incomplete.

Remarkably, Lederberg also presciently addressed the origin of the antibody genes. The prototypical antibody unit consists of two identical light and two identical heavy chains, see Figure 1. These chains contain several roughly equally sized domains, the light chain 2, heavy chains 4 or 5. The first domains of both chains are highly variable, and entwine in space, forming the two antigen-binding sites. The other domains show limited variability. These other domains of the heavy chain are collectively referred to as its constant region. There are 7 heavy chain constant regions in people. This region defines the class to which the antibody molecule belongs.

Figure 1 The prototypical unitout of which all antibodies aremade, consisting of two identical halves and containing two binding sites

Anti-self lymphocytes are inactivated by self-antigens, by the mechanism envisaged by Lederberg and described above, as they are generated in primary lymphoid organs, the organs where lymphocytes are generated. This elimination of anti-self lymphocytes is called central tolerance. Some self-antigens, such as insulin, are insufficiently present in primary lymphoid organs to eliminate their corresponding lymphocytes. Observations in the 1960s led to the idea, since substantiated, that antigen can interact with mature lymphocytes in two ways, one leading to their inactivation and the other to their activation. Presumably, peripheral self-antigens, such as insulin, normally inactivate their corresponding lymphocytes, leading to peripheral tolerance. Foreign antigens usually activate their corresponding lymphocytes.

The Simple Antigen-Binding Model of Precursor Cell ActivationMost assumed in the late 1960s that antigen can interact with a lymphocyte to either

inactivate or activate it. Most also assumed that the mere interaction of antigen with a lymphocyte’s receptors activated it. However, some findings were found to be inexplicable on this Simple Antigen-Binding Model of lymphocyte activation.

Two paradoxes illustrate the situation. Rajewsky studied the immunogenicity in rabbits of the related, 4-subunit proteins, A4, A2B2, and B4. Anti-A antibodies raised in rabbits did not react with B4, and vice versa. A strain of rabbits produced antibody to B4 but not to A4. However, immunization with A2B2 produced some antibodies specific for A4, and others for B4. This is interpretable in classical terms. “A” is the hapten and B the carrier. According to the Clonal Selection Theory, anti-A precursor cells must exist, as anti-A antibodies can be induced on challenge with A2B2. Why did A4 not activate them? A further critical point: the hapten and carrier had to be linked to generate antibody to the hapten. Thus anti-A antibody is produced upon immunization with A2B2, but not with A4 and B4. Successful cooperation requires the operational recognition of linked epitopes. We shall later see this is a crucial point.

Weigle exposed very young rabbits to an antigen B. Normal adult rabbits, but not adults pre-exposed as neonates, produce antibodies to B on challenge with B. The latter rabbits are said to be unresponsive. When Weigle immunized normal rabbits with an antigen H, he found 10% of the antibody that bound H also bound to the antigen B, reflecting similarities in structure between H and B. The antibody binding both antigens is called cross-reactive antibody. Weigle immunized adult rabbits, and adult rabbits pre-exposed neonatally to B, with H. Both types of rabbit produced cross-reactive antibody. The precursor cells, responsible for producing cross-reactive antibody, must, according to The Clonal Selection Theory, have receptors that bind to both H and to B. Why, on the Simple Antigen Binding Model, was B unable to activate these cells? This is paradoxical. The general process by which the production of anti-B antibody, in B-unresponsive rabbits, is achieved by immunizing with H, is referred to as breaking the unresponsive state. These findings are generally regarded as pertinent to understanding how autoimmunity arises. Thus, infection with group A streptococci can cause rheumatic heart disease, resulting in antibody that reacts both with heart tissue and with these bacteria. Such immune cross-reactions are believed to be a major cause of autoimmunity. We shall shortly see how autoantibodies are induced.

Cell cooperation in the antibody responseThese paradoxes were resolved with three major discoveries. Claman showed in 1966

that lymphocytes cooperate to produce antibody. Irradiated mice needed cells obtained from the bone marrow, so-called B cells, and from the thymus gland, situated just beneath the top of the rib cage, so-called T cells, to produce antibody on antigen challenge. The B cells bear antibody receptors and are the antibody precursor cells. T cells became known as helper lymphocytes. An

antibody response requires B cell/T cell cooperation.Secondly, spleen cells reconstitute the antibody response of irradiated mice. This is

because spleen contains both B and T cells. The removal of the thymus within the first day of life results in the adult mouse being immunologically deficient and deficient in spleen cells that bear the Thy antigen. The T helper cell, and most thymic cells, bear this antigen. Thus Thy+ splenic T cells are generated in the thymus. Thirdly, Mitchison found optimal secondary antibody responses require primed B cells and primed T cells. Thus primary and secondary antibody responses require B cell/T cell cooperation. The paradoxes described above were resolved. A major question remained: how does antigen inactivate lymphocytes? It was generally thought at the time that evidence was incompatible with this occurring following the mere binding of antigen to the lymphocyte’s receptors.

The Two Signal Model of Lymphocyte Activation and Peripheral Tolerance Cohn and I proposed in 1970 that the activation of all lymphocytes requires their antigen-mediated interaction with Th cells, and that antigen inactivates single lymphocytes. We proposed this model for three reasons. It accounted for Weigle’s studies on breaking the unresponsive state, regarded as paradoxical at the time. Secondly, many thought the evidence then available incompatible with the idea that antigen can inactivate a single lymphocyte. We argued that quantitative considerations could resolve these paradoxes. Moreover, other observations directly supported our proposal. Thirdly, our ideas provided an explanation for peripheral tolerance in accord with the Historical Postulate. Thus, the first lymphocyte generated in the fetus and specific for a peripheral antigen, pA, would be inactivated by pA, as would further pA-specific lymphocytes, generated one or a few at a time. Lymphocytes uniquely specific for a foreign antigen, F, would accumulate in its absence and, once F impinged upon the immune system, F could mediate the lymphocyte cooperation required to initiate an immune response. These proposals thus provide an explanation for peripheral tolerance.

We also proposed a terminology to encapsulate our concepts. The binding of antigen to the lymphocyte’s receptors generates a signal, signal 1, that, when generated alone, results in lymphocyte inactivation. Activation of a responder lymphocyte requires the generation of signal 1 and of signal 2. The generation of signal 2 requires the helper T cell to recognize antigen, thereby triggering its delivery of signal 2. We proposed signal 2 is delivered by short-range molecules and/or by cell/cell contact, thereby foreseeing a role for cytokines and costimulatory systems as signal transmitters. These proposals are in aggregate referred to as The Two Signal Model of Lymphocyte Activation.

Subsequent evidence supported, in large part, these proposals. The three main classes of naïve lymphocytes are B cells, the antibody precursor cells, CD4 T cells, the precursor helper T cells and CD8 T cells, the precursors of cytotoxic T lymphocytes. We know in 2020 that the activation of most B and CD8 T cells requires activated helper T cells; in their absence, antigen inactivates them, in accord with the Two Signal Model. The cardinal role of CD4 T helper cells is thus evident. How antigen interacts differently with CD4 T cells to inactivate and activate them has been a major question of the last 30 years.

A critical feature of the Two Signal Model is that cooperation of lymphocytes is mediated by the operational recognition of linked epitopes. If this was not so, inactivation, by a peripheral antigen, pA, of its corresponding CD4 T cells, could be interfered with by the presence of any unrelated antigen against which immune responses are occurring. This requirement is thus a way of realizing the Principle of Non-Interference. Thus people envisaged that an anti-hapten B cell would bind a hapten carrier conjugate, and the T cell, on binding the carrier, would deliver signal 2 to the B cell. This model is conveniently referred to as The Antigen-Bridge Model of the B cell/T helper cell interaction.

Progress on different frontsThe Two Signal Model is not only important in understanding peripheral tolerance, but

also immune class regulation. It is necessary, in order to describe the contemporary scene, to encapsulate the very considerable progress made in different areas of immunology in the last fifty or so years. The brevity of this outline does not reflect the significance of the advances made, but rather their substantial and so now uncontroversial nature. This context allows us to focus later on controversial and therefore pivotal issues.

The fundamentally different nature of the antigen-specific receptors of B cells and of T cells. We have seen that Landsteiner’s research led to the concept that the antibody response was universal. This was shown by the ability to generate antibody to any foreign hapten by immunizing with an immunogenic hapten-carrier conjugate. Remarkably, the carrier had to be a protein. As we shall see, T cells can only recognize proteins. T cells are universal only in the sense that they can respond to all proteins.

T cell receptors, and the related phenomena of antigen processing and presentation

Many observations came together, over several decades, to provide an insight into how the immune system monitors whether unusual events are happening inside cells. Such monitoring is critical to protection against intracellular parasites and cancer.

Another molecular system for recognizing antigen, other than antibody and T cell receptors, is centrally involved in immune responses. These are major histocompatibility complex (MHC) molecules. They bind an oligo-peptide having a length of about 10 amino acids that we will generically refer to as the deca-peptide. The deca-peptides are generated by enzymatically cutting up polypeptide chains of self or foreign proteins. As there are 20 different amino acids, 2010 ~ 1012 different potential deca-peptides exist. The genes for MHC molecules are extraordinarily polymorphic and so the MHC molecules are usually different between individuals. There are several MHC genetic loci, so we each have about 10-20 different MHC molecules. All MHC molecules have a groove to which some deca-peptides can bind. The groove has affinity for the common backbone of a deca-peptide in its extended form (see appendix 1), ie in its longest state, see Figure 2, but, if binding is to occur, the groove must accommodate the side chains of three of the amino acid residues of the deca-peptide, so-called anchor residues.

MHC molecules function in the following way. A sample of the proteins inside the cell is cut up into short peptides, a “process” called processing. They are in a compartment of the cell to which newly synthesized MHC molecules are directed. Some of the deca-peptides, so generated, bind to the groove of these MHC molecules; these loaded MHC molecules are then inserted into the cell’s external membrane. Consider two identical cells, except that one is virally infected. They both present self-peptides, bound to MHC molecules and derived from the self-proteins of the cell. The virally infected cell will also present some viral peptides bound to MHC molecules.

The T cell’s antigen specific receptors are clonally expressed and recognize peptides that are bound to MHC molecules. How this limitation on what T cells can recognize is explained by their development, as outlined, for example, in my book, The Foundations of Immunology and Their Pertinence to Medicine.

Some T cells recognize viral peptide/MHC complexes presented by virally infected cells, and can be activated to become cytotoxic for virally infected cells. In this manner, the immune system surveys the interior contents of cells to good effect.

Finally, there are two kinds of MHC molecules, and two kinds of “related” and corresponding T cells. Class I MHC molecules are present on all cells, and present deca-peptides derived from proteins synthesized within the cell, so called endogenous proteins. The CD8 T cells recognize peptide/MHC complexes where the peptide is derived from an endogenous protein.

In contrast, Class II MHC molecules are present only on cells with specialized means of taking in exogenous proteins from outside the cell. These exogenous proteins are synthesized elsewhere than the cells that take them up. Examples of cells with these specialized means are macrophages and B cells, that “take up” or endocytose the antigen that binds to their antigen receptors. These specialized cells process the endocytosed antigen and some of the resulting deca-peptides bind to newly synthesized class II MHC molecules. These peptide-loaded class II MHC molecules are then expressed on the surface of the cell, thus presenting peptides derived from an exogenous antigen. The clonally expressed receptors of CD4 T cells recognize such peptide/MHC complexes.

In summary, all cells of your body express class I MHC molecules, presenting peptides derived from endogenous antigens. CD8 T cells can recognize such peptide/MHC complexes via their clonally expressed receptors. Cells with specialized means for taking-up or endocytosing exogenous proteins express on their surface in addition class II MHC molecules, presenting peptides derived from exogenous antigens. These peptide/MHC complexes are recognized by the clonally expressed receptors of CD4 T cells.

Naïve CD8 T cells, with receptors able to bind self peptides/MHC complexes, are inactivated by central tolerance or, if the deca-peptide is derived from a peripheral self-antigen, by the two signal mechanism of peripheral tolerance already discussed: antigen inactivates CD8 T cells as they are generated, in the absence of helper CD4 T cells.

The recognition that the receptors of T cells do not bind antigen, but rather a peptide, derived from the nominal antigen, that is itself bound to an MHC molecule, had repercussions for how people envisaged that CD4 T cells help the activation of B cells. The new understanding of what the clonal receptors of T cells recognize was incompatible with the Antigen-Bridge Model of the B cell/T helper cell interaction. It became established that an anti-hapten B cell could bind a hapten-carrier conjugate, the conjugate being endocytosed and processed, see Figure 3. The B cell subsequently expressed on its surface carrier-derived peptide/class II MHC molecules. An activated T helper cell, specific for the nominal carrier, could bind these complexes and deliver signal 2 to the B cell. This description is referred to as

Figure 2 The tip of a peptide-loaded MHC molecule. The peptide, in extended form, is indicated by the black spheres.

The MHC-restricted model of the B cell/T helper cell interaction required for the activation of the B cell.

Preliminaries to a consideration of the activation of T helper cellsAs activated CD4 T helper (Th) cells are needed to activate B and CD8 T cells, the

requirements to activate precursor Th (pTh) cells bear on the earliest stages of immune responses. As the activation and inactivation of CD4 T cells requires its TcR to interact with peptide/class II MHC complexes, antigen-presenting cells (APC) are central to initiating/ablating immune responses. There are three major classes of APC: B cells, macrophages, already mentioned, and dendritic cells (DC). Immature DC (iDC) are strategically placed to capture antigen as it enters the body; they readily take up antigen, process and present it. They give rise to mature DC (mDC); mDC have lost the ability to take up antigen, but migrate to lymph nodes or spleen, secondary lymphoid organs, where antigen activates lymphocytes to induce immune responses. The mDC present antigens they took up when immature. What controls DC maturation is a subject of great and contemporary interest, as we shall examine. APC do not only process and present antigen, but they bear on their surface costimulatory (CoS) molecules. CoS molecules are recognized by counter receptors present on the surface of T cells. The regulation of the expression of CoS molecules is central to models of peripheral tolerance and immune class regulation, as we shall later discuss.

Contemporary Models of CD4 T cell activation/inactivation It became accepted in the 1980s, as outlined above, that whether antigen activates/inactivates B and CD8 T cells depends on whether activated CD4 T cells are present. In this sense, CD4 T cells were recognized as being the conductor of the immunological orchestra! So what controls whether antigen activates or inactivates CD4 T cells?

Janeway pointed out, in 1989, that vertebrate antigens are often non-immunogenic unless given with adjuvants containing microbial products. Adjuvants enhance immune responses. Janeway made the radical proposal that whether antigen activates CD4 T cells does not depend on whether the antigen is foreign, but rather whether it, or the molecules of the adjuvant given with it, express PAMPs, recognized by PRRs. He envisaged that PAMPs cause iDC to mature and express CoS molecules, and that the activation of naïve CD4 T cells requires two signals, a TcR-mediated, signal 1, and a CoS signal. Signal 1 alone results in the inactivation of the CD4 T cell. This PAMP Model is strikingly similar to the original Two Signal Model, except Janeway postulated that the second signal depends on the presence of PAMPS rather than on lymphocyte cooperation. However, foreign, vertebrate, non-PAMP bearing grafts are vigorously rejected. Matzinger therefore proposed in 1994 that what is required to activate CD4 T cells, beyond antigen, is the detection by the body of stress of one kind or another, thus leading to the Danger Model. The PAMP/Danger Models have dominated the attention of immunologists for the last 30 years. I question below the basis of the confidence placed in these models.

Figure 3 The MHC-restricted Model of the B cell/T helper cell interaction involved in theantigen-dependent activation ofB cells

I proposed, in 1999, the Two Step, Two Signal Model for CD4 T cell activation, see Figure 5. Activation of a CD4 T cell requires two steps. A CD4 T cell that only undergoes step 1 divides a few times, but its progeny die unless they undergo step 2. This model is a more detailed version of our 1970 Two Signal Model. This can be understood as a single CD4 T cell is envisaged to only undergo step 1; only if there are a minimal number of CD4 T cells available will step 2 take place.

I favor this model for three main reasons. Firstly, PAMP/Danger signals are not antigen-specific. Therefore a response against any foreign antigen would be expected to often interfere with the inactivation by a pS of its corresponding CD4 T cells. This violates the Principle of Non-Interference. These models also violate The Historical Postulate and therefore, in my view, provide an unreliable mechanism of peripheral tolerance. My 1999 model posits that the activation of a responder CD4 T cell requires its antigen-mediated cooperation with a helper CD4 T cell, a cooperation mediated by an antigen-specific B cell, and the operational recognition of linked epitopes, as is postulated to occur in step 2, see Figure 5. This mechanism both ensures non-interference and is consistent with the Historical Postulate. Secondly, diverse observations support the proposal, in different ways. For example, diverse studies show that the activation of

CD4 T cells is facilitated by CD4 T cell cooperation, mediated by a B cell. In addition, complex, vertebrate, non-PAMP bearing antigens, such as sheep RBC in mice, are immunogenic when delivered with a sharp needle and without adjuvant. There appears to be no PAMPs or Danger here. The model uniquely explains the efficacious clinical treatment of cell-mediated autoimmunity by depletion of B cells; this would undermine CD4 T cell cooperation and so CD4 T cell activation. Also, infection by group A streptococci results not only in antibody cross-

Figure 4 The PAMP/Danger Models for theAntigen-dependent activation of CD4 T cells. For explanation,see text.

Figure 5 The Two Step, TwoSignal Model for the Activationof CD4 T cells. The requirementsfor Step Two are discussed in the text, as these are critical inunderstanding how the model accounts for peripheral toleranceat the level of CD4 T cells

reactive for both heart and bacterial antigens, and also in the activation of cross-reactive CD4 T cells, readily explicable if the activation of CD4 T cells requires lymphocyte cooperation. Thirdly, the activation of a naïve CD4 T cell can result in the generation of effector cells belonging to different Th subsets. These different Th cells determine the class/subclass of immunity generated. The analysis of what controls which Th subset is generated depends upon what one envisions is required to activate a naïve CD4 T cell. I indicate below how the 1999 model of CD4 T cell activation allows a much more integrated picture than alternative models.

Feedback Regulation It is obvious that the model of CD4 T cell activation, involving the antigen-dependent cooperation of CD4 T cells, represents an escalating mechanism of such activation in the sustained presence of antigen. Such a situation would appear dangerous. Suppose some of the CD4 T cells specific for a foreign antigen also react weakly with a peripheral self-antigen. The unrestrained expansion of such a CD4 T clone by the foreign antigen would be expected with time to allow the peripheral self-antigen to activate these CD4 T cell even when the foreign antigen has been eliminated. These and other considerations make plausible the importance of feedback mechanisms of lymphocyte activation in general, and of CD4 T cell activation in particular. Diverse feedback mechanisms have been discovered. One important one relies on the expression of a molecule on activated CD4 T cells, the CTLA4 molecule, that binds very strongly to critical costimulatory molecules on APC, thereby inhibiting the generation and delivery of costimulatory signals and so further CD4 T cell activation. The importance of this molecule is demonstrated by mice who have been genetically altered so that they do not express this molecule. These mice develop rampant autoimmunity. There is currently much interest in achieving cancer immunotherapy by inhibiting these feedback mechanisms, and thus increasing the anti-cancer immune response. However, such treatments are not antigen-specific and so may have untoward side effects.

Immune Class RegulationPreamble We acknowledge the complexity of immune class regulation, in that there are several different classes/subclasses of antibody that are differentially regulated, even when neglecting cell-mediated immunity. We initially and primarily discuss how cell-mediated immunity, in the form of DTH, and IgG1 antibody in mice and people, are differentially regulated. This allows us to examine in a focused way the validity of some current ideas. This framework will also allow us to briefly consider what a broader picture of immune class regulation might look like. However, to start, we need to rely on classical studies, dating from before 1970, on how immunity, in the form of DTH and IgG antibody production, depends on various variables of immunization. The broad relevance, and the validity, of these older studies have stood the test of time.

Also, as already alluded to, the class/subclass of immunity generated is now recognized to be dependent upon what CD4 T cell subset is predominantly generated. The classical studies we initially refer to do not involve mucosal immunization, that is immunization via the intestinal, bronchiolar or urogenital tracts, but rather those involving a breaking of the skin barrier, such as immunization by the intravenous and subcutaneous routes. In relating these older studies in terms of modern concepts, we note that an exclusive DTH response correlates with the generation of Th1 cells, and of IgG1 antibody production with Th2 cells. Coffman and Mosmann first defined these subsets in the late 1980s, on the basis that Th1 cells produce interferon-g IFNγ and Th2 cells IL4. These subsets differently affect the target cells with which they intimately interact. Extensive studies have attempted to analyze what determines whether antigen preferentially generates Th1 or Th2 cells. We refer to the different ways an antigen

interacts with the cells of the immune system, to result in the generation of Th1 or Th2 cells, as the decision criterion determining the Th1/Th2 phenotype of the response.

The

The variables of immunization that affect theTh1/Th2 phenotype of the response Any viable proposal for the nature of

the decision criterion should account for the variables of immunization known to affect the Th1/Th2 phenotype of the response. We outline these variables to provide context for an analysis of possible decision criteria.

Threshold Hypothesis was formulated in the context of the assumption that the activation of a responder CD4 T cell requires its antigen-mediated interaction with helper CD4 T cells. It states that tentative and robust interactions are respectively required to generate Th1 and Th2 cells. It provides an explanation for how the variables of immunization, outlined above, affect the Th1/Th2 phenotype of the ensuing response, in the following manner. There are few CD4 T cells specific for minimally foreign antigens and so, even with optimal amounts of antigen to mediate CD4 T cell interactions, the interaction will be weak, and so Th1 cells are generated. There are more CD4 T cells specific for more foreign antigens and so, in the presence of optimal antigen concentrations, the interactions will be robust and Th2 cells generated; as the antigen is lowered, the strength of CD4 T interactions will be decreased and, if sufficiently decreased, will be tentative and only Th1 cells generated. Lastly, antigen, when it impacts the immune system, causes CD4 T cells to multiply; thus, so long as the level of antigen is sufficiently sustained, CD4 T cell interactions will increase in intensity with time, explaining how the response evolves from a Th1 towards a Th2 mode.

All these variables of immunization are quantitative; their explanation requires quantitative considerations. The Threshold Hypothesis makes a unique prediction: a sufficient reduction in number of CD4 T cells will modulate a response from a Th2 to Th1 mode, all other variables of immunization being held constant. We have successfully tested this prediction in diverse ways.

The PAMP Hypothesis is one of the two most popular, contemporary hypotheses for how the Th1/Th2 phenotype of the response is determined. It states that the PAMPs borne by the pathogen determine the Th subset generated. The hypothesis is thought plausible as PAMPs have diverse effects on cells, by, for example affecting the activation of APCs, including their expression of CoS molecules. However, the very same variables of immunization control the

Figure 6 The effects of dose of antigen, and time after immunization, on the induction of DTH and IgG antibody

Th1/Th2 phenotype of the response to PAMP-containing and PAMP-free, vertebrate antigens, see above. An explanation of these variables exclusive of PAMPs seems called for. The explanation of how these variables affect the Th1/Th2 phenotype of the response make little sense even for PAMP-containing antigens. Consider the evolution of the response from a Th1 to a Th2 mode. The PAMPs do not change during the infection, making this evolution inexplicable. The popularity of this hypothesis, and its inability to explain the variables of immunization affecting the Th1/Th2 phenotype of the response, probably contributes to why these variables are to a large extent ignored. The Cytokine Milieu Hypothesis, described below, has had, I believe, a similar influence, and for the same reasons.

The Cytokine Milieu Hypothesis is very influential in the context of Th1 and Th2 cells, and other Th subsets. It is the explanation found in all textbooks. Cytokines are short-range molecules made by diverse cells, including Th1 and Th2 cells, that influence the differentiation state of target cells with which effector cells interact. In vitro studies provided the initial support for this hypothesis. The presence of IL-12, produced by infected DC, favors the generation of Th1 cells, as does IFNγ; IL4, in contrast, favors the generation of Th2 cells. Are such in vitro observations physiologically pertinent? In vivo observations, made in the mouse model of the human parasitic disease of cutaneous leishmaniasis, provide support for such a role.

Infection of genetically “resistant” mice with 106 parasites results in a sustained Th1 response, parasite containment and resistance; similar infection of “susceptible” mice results, in time, in a predominant Th2 response, parasitemia and pathology. Administration of anti-IFNγ antibody to resistant mice, at the time of infection, results in a Th2 response and susceptibility, and of anti-IL4 antibody to susceptible mice to a Th1 response and to resistance! Thus IFNγ required to generate Th1 and IL4 to generate Th2 responses. Moreover, blocking the activity of the cytokine results in the “opposing response”. Many studies seek to find the cellular source of IL4 required to prime the generation Th2 cells, or of IL12 and/or IFNγ for Th1 cells. We showed that the IL4 required to generate Th2 cells is made by Th cells themselves! Thus “Th2” cells appear to facilitate the further generation of Th2 cells! This finding gave rise to a different idea as to the roles of cytokines.The Cytokine Implementation Hypothesis explains these observations, on the effects of cytokines on which Th subsets of Th cells are generated, in a different manner. It posits that the threshold mechanism initially defines the Th1/Th2 phenotype of the Th cells generated. Due to the different nature of the antigens of pathogens, such as bacteria, including their relative amounts, the Th1/Th2 phenotype of the Th cells generated to distinct components is anticipated to initially be quite diverse. I have suggested the cytokines produced by the predominant Th subset generated “sharpens up” this “rough and initial decision”. Four considerations support this hypothesis. Firstly, most cytokines produced by cells of one Th subset inhibit the generation of cells of other Th subsets, and/or favor their own further generation. For example, IL4 stimulates the division of Th2 but not of Th1 cells. The IFNγ made by Th1 cells inhibits the division of Th2 but not of Th1 cells. The IL10 made by Th2 cells inhibits IFNγ production by Th1 cells, thus inhibiting the inhibition of the division of Th2 cells! Secondly, the components of a “real antigen”, such as the L major parasite, are diverse. These components, as separate antigens, would generate responses of diverse Th1/Th2 phenotype. However, the anti-parasite responses are often remarkably coherent, being predominantly of a Th1 or Th2 phenotype, some days after infection. This can be explained by the physical linkage of the different components and by the “self-promoting” nature of different Th subsets. Thirdly, coherence is physiologically important. Thus, Th1 cells can protect against M tuberculosis, but this protection is undermined by Th2 cells. Thus protection not only is favored by the generation of Th1 cells but by the lack of generation of Th2 cells. Lastly, it appears that, shortly after antigen impact, the response is

less coherent than at later times. The self-promoting nature of the predominant Th cells generated explains this, and the envisaged processes can explain the coherence of immune responses to complex antigens.

The broader picture: other classes/subclasses of immunity and further Th subsetsPreamble The views I have favored above provide provisional answers as to how the Th1/Th2 phenotype of a T cell is determined. The observations on which the threshold mechanism is based involve immunization by non-mucosal routes. I now discuss more tentative ideas involving immunization by mucosal, ie intestinal, bronchiolar and urogenital routes.

Allergies, following IgE antibody production, often arise from antigen exposure via mucosal routes, bronchiolar or intestinal, and are associated with Th2 cells producing IL4. There is no doubt that route of exposure influences the class of antibody produced. This is in part due to the presence different subsets of DC at different sites of entry by antigen to the body. Thus the production of IgA antibody requires chronic mucosal exposure to antigen, associated with Th cells producing the cytokines “TGFβ” and IL-10. These cytokines respectively facilitate in humans IgA and IgG4 antibody production. IgA and IgG4 are non-inflammatory, and counter the action of antibodies belonging to other classes/subclasses, such as IgE antibody, by competing for the antigen. IgA and IgG4 antibodies are produced only after chronic exposure, and are produced against gut flora. Interestingly, chronic antigen exposure via the skin, as seen in the exposure of beekeepers to bee stings over several years, results in the spring in Th2 cells facilitating IgE production; this is quickly suppressed after the first 50 stings of the year by the generation of Th cells producing IL-10, and by a decrease of IgE and an increase of IgG4 antibody production!

Most current research, on what controls the class/subclass of immunity generated, involving Th subsets beyond Th1/Th2 subsets, accept the PAMP and/or Cytokine Milieu Hypotheses. Textbooks identify cytokines needed to generate particular Th subsets. This framework is likely incorrect in the sense imagined, and in any case leaves open the pivotal question of what determines the presence of the critical cytokines.

Immune regulation and medical interventionsAutoimmunity My1999 model for CD4 T cell activation states that CD4 T cells will be inactivated by antigen in the absence of CD4 T cell collaboration. The PAMP/Danger Models collectively predict that any antigen, if given without DAMPs or eliciting Danger, will inactivate their CD4 T cells. It should be surprising to proponents of these models that there are no reports that this latter prediction holds, except for those antigens expected to be inactivating on the 1999 model, as discussed below. Sterile sheep erythrocytes, delivered to mice with a sharp needle, are immunogenic, and do not inactivate their corresponding CD4 T cells in the absence of DAMP or Danger signals.

Consider a peptide, p, that binds to self-class II MHC molecules and whose corresponding pThp cells recognize the peptide/MHC complex. It is known that peptides resident in the grooves of MHC molecules, expressed on a cell’s surface, slowly disassociate from their MHC molecules and can be replaced by such a peptide as p, if present. The intravenous injection of p will result in the “decoration” of some MHC of many APC, many more than the number of pThp cells. This will therefore result in single pThp cells interacting with APC presenting antigen, resulting in their inactivation. Studies show this is an effective way of eliminating antigen-specific helper T cells. I discuss further approaches to ablating anti-self immune responses under Further Reading.

Allergies Desensitization is a risky process, as the allergen administered can cause acute inflammation on interacting with mast cell bound IgE. Hence desensitization consists of the progressive administration of escalating doses of the allergen, and cessation of treatment if undue inflammation occurs. A modified strategy of desensitization may be based upon the fact that antibody recognizes antigen in a very different way from T cells. Therefore, an intriguing strategy would be to employ a form of antigen to desensitize that can stimulate T cells but not interact with the preformed IgE antibody present, and so avoiding acute inflammation. This idea is more fully developed under Further Reading.

Infectious diseases and cancer Current vaccination in the west is only effective against pathogens susceptible to antibody. Strong CMI is required to contain some pathogens, such as HIV, M. tuberculosis, L major and L donavani, the latter two respectively causing cutaneous and visceral leishmaniasis. Cancers are also best contained by CMI, in the form of CTL. There are two plausible reasons for the weak CMI associated with disease. The antigens may be insufficiently immunogenic due, I suggest, to a lack of sufficient CD4 T cells; alternatively, other classes of immunity are induced and CMI is down-regulated. I suggest CMI-imprints, guaranteeing strong CMI upon infection, will ensure protection.

We successfully tried to generate a CMI-imprint and so effective vaccination. Infection of “susceptible” mice with 106 parasites results in a Th2 response and parasitemia. Infection with a lower number, 300 parasites, results in a stable Th1 response and, in time, a Th1 imprint. Thus mice, so infected, produce, on challenge with 106 parasites two months after exposure, a strong and sustained Th1 response, and parasite containment. This Low Dose Vaccination Strategy also works in a mouse model of tuberculosis and against cattle tuberculosis. We examined whether it also works in mouse models of cancer.

Studies in the 1960s showed that mice, given a lethal challenge of a tumor, resist a second challenge, given 7 days post-implantation. Indeed, at this time, the spleen and lymph nodes of the tumor bearing mouse can be shown to harbor protective cells, as assessed by the protection they provide when given to a naïve mouse, challenged with a normally lethal dose of tumor. This immunity is called concomitant immunity. Robert North in the 1980s attempted to understand why a lethal challenge is lethal, in view of its rapid stimulation of protective immunity. His extensive experiments were carried out with different tumor types. He showed that a lethal dose of tumor first generates a protective CTL response; it is subsequently down-regulated by the generation of tumor-specific CD4 T cells that suppress the generation of a sustained CTL response. We were struck by the similarity of North’s findings to those of Salvin, see the middle panel of Figure 6. North’s findings led us to formulate and successfully test the Th2-Skewing Hypothesis of Tumor Escape. Thus we found a lethal dose of tumor first generated a transient Th1 response that was down-regulated as Th2 cells arise. We demonstrated that generating a Th1 imprint led to resistance to a normally lethal challenge.

Interestingly, a generally effective means of “vaccinating” against tumors was discovered in the 1960s. Mice were injected into the skin with a lethal dose of tumor, and this tumor excised at about 7 days after implantation, a time at which North subsequently showed there was maximal expression of concomitant immunity. These mice became resistant to a normally lethal challenge. This excision priming makes sense in terms of concomitant immunity and the Th2-skewing Hypothesis of Tumor Escape. Excision of the tumor removes the bulk of the tumor when concomitant immunity is strong, so the antigen-dependent progression of the anti-tumor response, towards a Th2 mode, anticipated on the threshold mechanism, is undermined. Excision priming can thus be viewed as a form of “low dose vaccination”.

Similar principles likely allow effective treatment. We found that chronic disease, in the mouse model of cutaneous leishmaniasis, associated with a stable, mixed Th1/Th2 response, can

be treated by partial depletion of CD4 T cells. This depletion results in a modulation of the response to a Th1 phenotype and a resolution of lesions. The number of CD4 T cells thus controls the Th1/Th2 phenotype of both primary and on-going immune responses. Patients with visceral leishmaniasis, expressing a mixed Th1/Th2 response, can be treated with a short course of anti-parasite drugs, resulting in a Th1 response. Low antigen doses induce primary Th1 responses, and lowering the antigen load, by giving anti-parasite drugs, modulates an on-going response from a Th1/Th2 to a Th1 phenotype.

The envisaged threshold mechanism thus leads to strategies to guarantee, through vaccination, CMI against CMI-sensitive pathogens, and against CMI-sensitive cancers. It also allows rational approaches to treatment, resulting in a modulation of the phenotype of on-going immune responses, from a Th1/Th2 to Th1 phenotype. I discuss elsewhere the relevance of these ideas for protection against, and treatment of, tuberculosis and AIDS, see Further Reading.

EpilogueMany people, particularly immunologists, may be puzzled by, and likely even

incredulous of, the nature of this 20 pager. There is so much information, not incorporated into the framework developed, so how can it be thought plausible? I think this frequent reaction is, in large part, a result of the information overload. To understand where we are, it is helpful to understand this incredulity. Given that there are many different subsets of CD4 T cells, of DC, of natural killer (NK) cells of diverse sort, of αβ and γδ T cells, etc, etc, how can one be sure that these diverse cells are not central to any understanding of peripheral tolerance and immune class regulation? One of course cannot be, but the blanket reaction expressed is unhelpful to progress. How can one proceed in view of all this information? Some would suggest only by mining more information.

I have tried to illustrate what I consider the way forward in this 20 pager. I have attempted to find foundational concepts that allow one to bring together critical observations to explain the physiological functioning of the immune system. Ideas such as The Historical Postulate, The Principle of Non-Interference, the coherence of immune responses, and the centrality of the operational recognition of linked epitopes in lymphocyte interactions, allow one to propose models that can be tested. Such endeavors, to be successful, must be continuously reassessed. It is a long journey.

Four types of experience can provide faith in a developing and evolving framework. The first is the discovery of generalized patterns. I realized in 1973 that a bird’s eye view of the literature showed that there were three conditions under which DTH is induced (in contrast to antibody), three conditions under which T cells, inhibitory for the induction of antibody, are generated, and that these three conditions are the same. It was then natural to entertain the idea that the inhibitory T cells explained the lack of antibody production under circumstances where strong DTH is induced. Moreover, these three conditions could all be accounted for by one idea, namely the Threshold Hypothesis. The second experience is when radical changes in foundational concepts do not fundamentally undermine a framework. Thus the discovery of the MHC-restricted recognition of antigen by T cells did not undermine The Two Signal Model and the Threshold Hypothesis; the ideas could, with some new problems faced and potential solutions proposed, be reformulated to take account of this new discovery, and to make new predictions. The third experience is the successful invasion of ideas into neighboring fields. I have indicated why I believe the threshold mechanism has provided new insights into how immunity to pathogens and cancer is regulated. Lastly, developing and successfully testing medically significant strategies of prevention and treatment increases one’s faith in the underlying framework. I suggest such successes make it plausible that it is not necessary to include a full description of the role of different CD4 T cell subsets, of DC, of NK cells of

diverse kinds, to make substantial progress. I think it will be possible to accommodate their roles without dramatically changing the framework outlined.

This view is supported by history. The originators of Clonal Selection Theory were fearless and grappled with big issues. We do not cast aside their insights because the theory does not account for contemporary findings. The greatest contribution of science to culture is to reinforce the wisdom of Aristotle, who said: “It is the mark of an educated mind to entertain an idea without believing it”. And to see where it leads, I would add, and to respect the evolution of ideas, with the possibility of conserving their beauty.

Further Reading:

The Foundations of Immunology and Their Pertinence to MedicineFriesenPress, Victoria, BC, Canada, 2016

Rediscovering the Immune System as an Integrated OrganFriesenPress, Victoria BC, Canada, 2016

Glossary

This glossary provides the (page/pages) on which different words are first used and their meaning indicated in context. It also similarly includes abbreviations. The list of words/abbreviations is arranged alphabetically.

actively immunized (12)acute inflammation (12)adaptability (13)adjuvants (23)anchor (amino acid) residues (20)antibodies (12)antigen (12)Antigen-Bridge Model of the B cell/T helper cell interaction (20)antigen, nominal (22)assembly kits (17)antigen presenting cell (APC) (22)APC: antigen presenting cell (22)attenuated (12)autoimmune (13)autoimmune diseases (15)B cells (18)B cell/T cell cooperation (18)bone marrow (18)breaking the unresponsive state (18)carrier (12)CD4 T cell subsets (25)CD4 T helper cell (22)CD8 T cells (21)cell-mediated immunity (14)cell-mediated imprint (14)central tolerance (17)class I MHC molecules (21)class II MHC molecules (21)Clonal Selection Theory (16)clone (16)CMI-imprints (29)CMI: cell-mediated immunity (14)coherent (27)concomitant immunity (29)combinatorial mechanisms (17)constant region (16)CoS: costimulatory/costimulation (22)CoS signal (23)costimulatory molecules (22)costimulatory systems (19)cross-reactions, immune (18)cross-reactive antibody (18)

CTL: cytotoxic T lymphocytes (15)Cytokine Implementation Hypothesis (27)Cytokine Milieu Hypothesis (26)cytokines (19, 26)cytotoxic T lymphocytes (15)CD: dendritic cell (22)Danger Model (23)deca-peptide (20)decision criterion (25)dendritic cell (22)delayed-type hypersensitivity (14)desensitization (13)DTH: delayed-type hypersensitivity (14)effector mechanisms (13)endocytose (21)endogenous proteins (21)excision priming (29)exogenous proteins (21)genetically determined (13)hapten (13)helper lymphocytes (18)Historical Postulate (15)iDC: immature dendritic cell (22)immature dendritic cell (22) germline encoded (13)Group A streptococci (18)hapten-carrier (13)hemolytic anemia (13)humoral (13)IFN-γ (25)immune (12)immune class regulation (13)immune cross-reactions (18)immune serum (12)immunogenic carrier (13)immunization (12)immunized, actively (12)immunized, passively (12)inbred strains (15)infection, primary (13)infection, secondary (13)inflammation, acute (1)innate defense mechanisms (12)instructive process (15)interactions, robust (26)interactions, tentative (26)interferon-γ (25)interleukin-4 (25)linked epitopes, operational recognition of (18)Low Dose Vaccination Strategy (29)

lymphoid organs, primary (17)lymphoid organs, secondary (22)major histocompatibility complex (20)mature dendritic cell (22)mDC: mature dendritic cell (22)mechanisms, effector (13)memory, negative (13)memory, positive (13)MHC: major histocompatibility complex (20)MHC molecules, class I (21)MHC molecules, class II (21)MHC-restricted Model of the B cell/T helper cell interaction (22)mucosal (25)negative memory (13)nominal antigen (22)non-immunogenic (13)nonself: (15)operational recognition of linked epitopes (18)PAMP: pathogen-associated molecular patterns (13)PAMP Hypothesis (26)PAMP Model (23)passively immunized (12)pathogen-associated molecular patterns (13)pattern recognition receptors (13)peripheral self-antigens (17)peripheral tolerance (17,19)phagocytes (12)positive memory (13)precursor cells (16)present (20)primary infection (13)primary lymphoid organs (17)primary response (13)Principle of non-interference (19)processing (20)PRR: pattern recognition receptors (13)pTh: precursor Th cell (22)recognition of linked epitopes, operational (18)red blood cells (15)response, primary (13)response, secondary (13)rheumatic heart disease (18)robust interactions (26)secondary infection (13)secondary lymphoid organs (22)secondary response (13)selective process (16)selected (15)self (15)self-antigens, peripheral (17)

self-nonself discrimination (13)serum (12)Simple Antigen-Binding Model (18)specific (13)specificity (13)tentative interactions (26)Th: T helper (22)Th1 cells (25)Th1/Th2 phenotype (25)Th2 cells (25) Threshold Hypothesis (26)Th subsets (24)TB: tuberculosis (14)T cells (18)Thy-1 antigen (18)thymus gland (18)tolerance, central (17)tolerance, peripheral (19)toxins (12)toxoid (12)transplantable tumors (15)tuberculosis (15)tumor-associated antigens (15)tumors, transplantable (15)Two Signal Model of Lymphocyte Activation (19)Two Step, Two Signal Model (23)universality (13)unresponsive (18)unresponsive state, breaking the (18)vaccination (12)virulent (12)

Section I: Brief Scientific Biography

I, as many, liked the world of ideas from when I was small. I read some philosophical books in my early teens, mainly by Bertrand Russell, but came to the belief that studying physics could provide better opportunities to appreciate and come close to beauty in nature.

I studied physics whilst at Gonville and Caius, College, Cambridge, from 1961- 1964, as an undergraduate. I found the experience frustrating; there was not enough time for me to understand and appreciate the vast amount of material. I felt I could not do justice to my aspirations or the subject.

I knew I wanted to be in a field where I could try to make some significant conceptual contributions and hopefully do some related experiments. I had little confidence I could achieve this in physics. I was very interested in theory. The particular field did not seem so important to me, but rather the approach one took in an attempt to make progress. I was very fortunate. My brother, Mark, who had trained as an organic chemist, was doing his PhD under the guidance of Sydney Brenner and Francis Crick in the area of protein synthesis and the genetic code, in the Laboratory of Molecular Biology, in Cambridge, England. I was accepted to do graduate studies at the same laboratory with John Kendrew, who already had his Nobel Prize for the structure of myoglobin. My graduate studies were to be in the field of protein Xray crystallography.

To have been at The Laboratory of Molecular Biology at this time was a unique privilege and allowed one to witness history. I taught myself some genetics and took an interest in many things. Crick and Brenner were interested, among very many things, in the origin of the genes coding for antibody chains. I thought the model by Brenner and Milstein on this topic, published in Nature, implausible, and brought my considerations to Sydney Brenner, who was very open to discussion. There were several hours of discussion every Saturday over coffee in the second floor laboratory “kitchens”, on scientific or other matters, with Sydney almost always present. This was the time of the acridine mutant work using T4 phage that gave rise to the experiments on the general nature of the genetic code, of experiments on co-linearity of gene and polypeptide, of elucidation of the genetic code and of the proposal for the Wobble Hypothesis and of allostery.

We had visitors I am unlikely to forget: Jacques Monod, Seymour Benzer, Frank Stahl, Jim Gowans, and Jim Watson, for example.

One day, I was doing an experiment and wearing plastic gloves to protect myself against a nasty chemical, bromo-acetic acid. I felt a throb in a finger as I fell asleep that night. The next morning one fingertip was quite white and without feeling. It was clear that a glove had leaked. The finger-tip was in time surgically removed and skin grafted

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to the raw end of what remained of my finger. However, the first morning after the accident, I saw a sharp line begin to develop that demarked the healthy from the unhealthy tissue. I interpreted this as reflecting immunological self-nonself discrimination. I was already somewhat interested in immunology, as a result of my interaction with Sydney Brenner. Cesar Milstein had suggested I read some Burnet, but after reading a bit, I gave up as I felt much of what Burnet said was a bit wild. I started speculating about various aspects of immunology as described in the third section of this profile. I talked to Crick about several ideas. He suggested I see Avrion Mitchison, for a potential postdoc position, which I did. However, Crick suggested, as I talked to him more about my ideas, that I would find Mel Cohn of the Salk Institute a very compatible individual to do a postdoc with, given our common love of speculation. Crick was a Visiting Fellow at the Salk Institute at that time. A series of circumstances led me to realize during my PhD studies, and then find evidence in the literature that I could use to support the idea, that antigen, interacting with a single lymphocyte, inactivates it, and that activation requires the antigen-mediated cooperation of lymphocytes. I realized that these rules provided a potential explanation of immunological self-nonself discrimination, as I describe later (Section III).

Mel Cohn went to a Brooke Lodge meeting only a few (two or three?) weeks afterI arrived at the Salk Institute, California. He talked about the ideas on the induction and paralysis of immune responses, as the processes of activation and inactivation of lymphocytes were then called. We decided that we had better publish our ideas quickly. I am not sure this was the wisest decision, but we thought a formal presentation was better than leaving the point of view we had open to misconception. I wrote a covering letter explaining the situation to Nature. I recall that Nature accepted the publication one day after they received it. Never anything remotely like this has ever happened again in my life! There were some aspects of this first exposition that troubled at least me. Resolution of these aspects led us to formulate the Two Signal Model of Lymphocyte Activation, published in Science, in 1970.

To have been at the Salk Institute at this time was also a great privilege. Neurophysiologists from Harvard came every summer and gave mind bending lectures. Various visiting Fellows also came a few weeks every year, including Crick and Monod. I got to know Jacques Monod quite well, as he was always interested in new ideas in immunology. Monod and I also had some considerable, good-natured, philosophical arguments. The diversity of lectures at the Salk Institute was probably unique. Some of the most interesting and speculative lectures were on the origin of life, because one of the Fellows, Leslie Orgel, was a prominent individual in this field. Some of the most beautiful were on phage lambda. Dulbecco, whose laboratory was devoted to understanding the biology of the oncogenic viruses, polyoma and SV40, felt that lambda’s tricks might be relevant to understanding the tricks oncogenic viruses got up to. We had seminars on philosophical subjects and on animal behavior, and an attempted lecture on race and IQ. This lecture never took off the ground as it was disrupted. These were Vietnam days.

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The individuals in Mel’s laboratory for the most part had two properties in common: most (though not all) worked on immunological subjects and, of all that Iknew, none had a background in immunology, until Michael Bevan joined the laboratory,shortly before I left. The real sign of experimental sophistication in those days was if you could inject mice intravenously! I remember one individual with a background in phage genetics becoming very distraught on finding a mouse that he had killed had two spleen. He was distraught, as one cannot do genetics with dead mice (anyway, not then). He wasn’t really all that relieved when someone pointed out to him that he had been removing kidneys. It is unbelievable, in retrospect, how we lived. There was very little pressure from Mel to do experiments if you were a postdoc, and in some sense rather little guidance, unless you consider a lot of enthusiasm and more suggestions about what was important than anyone could digest. I also think Mel was fortunate in some of the young people he had. David Schubert, Sidhartha Sarkar, Alan Harris, Alan Sher, Bob Coffman, Jim Watson, Chuck Kimmel, Alan Harris, Jim Watson, Mario Caesari, and Martin Weigert were all contemporaries of mine, and I overlapped with Michael Bevan a bit at the end of my fellowship.

Besides my involvement in The Two Signal Model of Lymphocyte Activation, I did theoretical work on autoimmunity and immune class regulation. Bob Coffman was one of the individuals who kindly read and gave me comments on the manuscript describing my theory of immune class regulation. I did not publish anything from my experimental work. This was not for want of trying. I was inexperienced and Mel’s lab was in its early days. I was also a bit foolish. I developed one series of experiments testing whether a circumstance, under which lymphocytes should be inactivated according to our two signal ideas, did indeed lead to inactivation, and got the kind of result I anticipated. However, the results were not as clear-cut as I had expected. I took this to mean that my understanding of the phenomenon was inadequate, and so I thought I should not publish. Similar but even less clear work along the same lines was later published and was regarded as pretty significant. I have learnt from this to better accept that my understanding is never complete, and to therefore be both more realistic and humble.

Another aspect of our intellectual life was its freedom, unbelievable in thismodern day of urgency and competitiveness, illustrated by two endeavors. Many people became interested in neurophysiology, with the summer lectures by the gifted Harvard professors. It was hard not to be fascinated. I spent some time studying David Marr’s Theory of the Cerebellum, and gave a few talks on it, as understanding it required considerable effort. Sidhartha Sarkar and I decided to try to learn some neurophysiology together. We got a self-teaching book on neuroanatomy, and a brain we were going to dissect. The point of these tasks we had set ourselves, after a few attempts at a bit of cramming, became questionable. We decided we would only need to know neuroanatomy, once we had some interesting ideas, that would propel our learning all that stuff. We never of course did develop those interesting ideas and therefore never learnt a significant amount of neuroanatomy.

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The second endeavor had more long-term consequences. A group of us felt weshould be studying a subject that might be more relevant to the relief of human suffering than basic immunology. We decided to study together schistosomiasis, a disease that caused much suffering world-wide. Donato Cioli, Paul Knopf, Alan Sher and I were all in this group, and all took the plunge of going into immunoparasitology, except me. I decided to keep my interest in the subject, but to remain in basic immunology for the present. I hoped that my contributions in basic science might also help in some way to relieve human suffering in the longer term.

I returned to Cambridge, England, to the Babraham Institute of AnimalPhysiology, in 1972. I had established myself as a theoretical immunologist whilst in Cohn’s lab. I knew hardly any immunologists in England, so my options were limited, but I did know Cambridge well, and some immunologists there. After rather less than a year, I saw an advert for a job at The Australian National University (ANU), for a person with research interests in an area suspiciously close to mine. This was too good to be true. Moreover, the Head of the Department was a well-known immunologist, Gordon Ada, whom I had met on his way through the Salk Institute. I applied, with some anticipation. However, the job had been ear-marked for Chris Parish. Chris had actually done a lot of very interesting experiments, and these had in part stimulated my Theory of Immune Class Regulation. No wonder the job description made me think the fit with my interests was too good to be true!

Although I did not get the job advertised, Gordon Ada made other arrangements so that I could go to his department at the ANU. I was very interested in doing so, because I knew that the department really was a laboratory of professional microbiologists and immunologists, not exactly the description one would provide for Mel’s lab, and I really wanted to develop my experimental skills. Moreover, I had some clear ideas on what type of experimental approach should be explored to test some of my proposals underlying immune class regulation.

Chris had a graduate student, Ian Ramshaw, who got interested in the theory. I think his other experiments had not been going so well. We discussed different approaches as to how to test various ideas. It was a very nice journey we took together. I remember Ian coming up with a paper describing an experimental system that he thought might be useful. It was. At one stage, I decided to do a job tour that took some weeks. When I returned, I found Ian had made tremendous progress. We felt we had really accomplished something significant. The experiments seemed to fit the predictions beautifully.

The John Curtin School for Medical Research, of the Australian National University, was another special place, set up, as was The Salk Institute, with a generosity of spirit. In our Department of Microbiology, Alastair Cunningham, who kindly gave me some space in his laboratory, was exploring with Linda Pilarski whether genes,

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encoding immunoglobulin chains, hypermutate following antigen stimulation of the B cell carrying them. A few months before I arrived, Peter Doherty and Rolf Zinkernagel, working together in Bob Blanden’s lab with the type of systems Bob had established, had made the first observations demonstrating the MHC-restriction of cytotoxic lymphocytes, for which they received the Nobel prize some years later. Here too there was an unusual freedom for intellectual exploration. It is this kind of enthusiastic environment that leads to significant and bold ideas. Alastair Cunningham had been Kevin Lafferty’s first graduate student. Kevin was interested in transplantation and overcoming the transplantation barrier. This had led him and Alastair to try to understand some phenomena related to the activation of T cells. They related their work to our Two Signal Model of Lymphocyte Activation. So I came into a very interesting and very interested environment.

Australia was incredibly beautiful, and I very much liked the people. I was asked very informally if I might like to be considered for a better kind of job at the ANU. I decided that Australia was too far away from family. I often subsequently wondered whether I had been too hasty.

I had made a tour whilst at ANU looking for possibilities for my next career step. Linda Pilarski, who had done her postdoc with Alastair Cunningham, was at the University of Alberta, in Edmonton, Canada, so I visited the Department of Immunology there. The Head was Erwin Diener, who had also been in Australia at the Walter Eliza Hall Institute. I decided to go there.

I arrived at the University of Alberta in 1978, about nine years after my PhD inprotein Xray crystallography. I had done some publishable and published experimental work whilst at the ANU, but I wished to develop myself further in this respect. I was successful in this whilst at the University of Alberta. Both Ian Ramshaw and I had decided that the next critical step would be to obtain an in vitro system in which antigen could induce primary cell-mediated (DTH) and humoral responses. In this way, we could examine what circumstance favored the generation of one response over the other. No one had managed to generate primary cell-mediated responses in vitro, with the exception of those to MHC antigens, which we felt were a special case. We kept in frequent touch. We used somewhat different approaches. Ian developed his system much faster than I did. He refrained from submitting his work until I had caught up, which took quite some time. Who under today’s pressures could and would show such kindness and generosity?

I judged myself to be successful in doing significant experiments, and so felt Icould now give students guidance. I agreed to take on my first graduate student, Jane Tucker. Also, I had help in the laboratory from someone I came to greatly respect, Mohamed Dhalla. After some years, Tim Mosmann joined the department. We were

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both part of a small MRC group, that included Bhagi Singh. I also met Calliopi Havele, also an immunologist, who became my spouse.

In 1987, I moved to the Department of Microbiology and Immunology at the University of Saskatchewan. One advantage of the move was that the Department of Immunology at the University of Alberta had no microbiologists, in contrast to the Department at Saskatoon, and I really think this affects the way people think. I got a small grant shortly after arriving. This was given to new Faculty, on application, to start up a new area of investigation. I knew what to do. I had wanted to work on infection and immunity, in the mouse model of human cutaneous leishmaniasis, for some time. However, I regarded this as too risky an undertaking if using funds from my own MRC grant, obtained to study basic regulation of the immune response. Juthika Menon did her PhD developing the low dose vaccination strategy in this model.

We established, during the 1990s to early 2000s, a number of differentexperimental systems, investigating issues relevant to preventing and treating a certain class of infectious diseases. This class of diseases is defined by the ability of a strong cell-mediated response, in contrast to antibody, to contain the pathogen, and includes the leishmaniases, tuberculosis and AIDS. Cell-mediated immunity is also recognized as being uniquely effective against most cancers. The ability to develop new areas of investigation was only possible because of support in the laboratory from Goujian Wei, whose efforts, under my guidance, have opened up one new experimental system after another. This has allowed me to provide guidance to more graduate students. In addition, Juthika and I started working on human disease, mainly on tuberculosis and visceral leishmaniasis. We developed collaborative research in Ethiopia. The Saskatchewan Lung Association, formerly the Anti-Tuberculosis League, has supported our tuberculosis research in a particularly helpful way. We have studied immunity to intracellular pathogens that cause chronic disease, such as tuberculosis and the leishmaniases, in mouse models. The way these studies appear to shed light on human disease, and in some cases appear to dovetail with our human studies, is quite overwhelming. I hope I can contribute to and witness some real progress in prevention and treatment of these devastating tropical diseases. I felt and feel we understand enough about how the immune response is regulated that we are probably able to develop effective vaccination against TB and AIDS, and treat early stages of AIDS. It has been an incredible journey to go into new fields and obtain clear and striking results, consistent with one’s expectations. However, my hope is somewhat dented by my losing all research funding from national granting agencies in the field of infection and immunity despite my greatest efforts.

I have had a strong interest in tumor immunology since the late 1960s. Whenfunding for our research on immunity to infections became problematical, I tried to get money for doing studies on immunity against tumors. I had a simple hypothesis as to why the immune system often fails against cancer. We initiated these studies on tumor immunology in the early part of the 2000s. Duane Hamilton, then a graduate student,

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asked me whether he could take on these studies. Guojian Wei had made some initial and very interesting findings. Duane’s observations were very striking to both him and me and were anticipated on our hypothesis. They seem to open up new ways of guiding immunotherapy, if our mouse studies really reflect parallel situations to those that occur in human cancer. However, I have found it problematical to obtain funding for this research.

I had a heart attack in August 2006, when Calliopi Havele, my wife, and I were visiting our daughter’s family and our first grandchild in Toronto. This attack led to emergency bypass surgery. I was 63. I had three days in intensive care, where the situation I faced, and perhaps the morphine I was given, made me reflect on my past, and how I might still achieve unfulfilled aspirations that I had formed in my youth. These aspirations were to interact and communicate with my fellow human beings about how one could understand and become close to, or feel to be part of, nature and face existential questions. I had envisaged that these aspirations could be realized through doing scientific research collectively. However, I had found that doing science, the way it is practiced today, is overall a lonely process. I was not realizing my dreams of joint discovery and pleasure, except with my students. A solution seemed obvious. I had always felt that basic ideas were communicable to most of my fellow human beings. I had never tried to join scientific bandwagons. It seemed to me that I might have a fresh start of realizing my aspirations by writing a book for the non-specialist on what scientific culture meant to me. I would illustrate this by the story of my involvement in immunology. I started writing this book within a couple of days of leaving hospital.

I describe in the second section of this profile the books I have written, and amwriting now, and how I came to write them. I am 76 as I update this profile. I still have a lab with a wonderful, last PhD graduate student, Ghassan Al-Yassin. I still speculate on immunological topics. However, our experimental research over the last twelve or so years has been greatly hindered by my inability to get appropriate funding for either basic immunology, or in the related medical fields of autoimmunity, and immunity to pathogens and to tumors. I think there is a simple reason. I believe that the predominant conceptual frameworks, employed today to explore the most basic questions, are wrong and so misleading. It is not easy to get funded if one has frameworks contrary to those most widely held.

I have come to the belief that the culture supporting science has changed dramatically in the last 50 years. This change is driven in part by the information overload, and in part because most scientific careers have become lonely and competitive journeys, and so do not optimally contribute to a common realization. I believe both the influence of the information overload, and of the competitiveness that leads to individual rather than to collective journeys, are not only an impediment to progress, but undermine the spiritual inspiration that science can bring to our lives. Such influences have, to be overcome, to be diagnosed. The drive to write my third,

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non-immunological book, in progress, is to explain the reasons for my thinking these influences are not inevitable and to explore how they can be transcended.

Lastly, the change in culture supporting immunological research perhaps reflects the change in the general culture, supporting science, in a particularly exaggerated form. Immunology has been, in the last three or four decades, one of the most rapidly expanding areas of biological research. Forty years ago, it was possible for most to have a rather general knowledge of the field. Now, few immunologists know much beyond the niche they are dedicated to exploring. I deeply believe progress in both basic immunology, and its impact on medicine, would be much greater if more attention was given to formulating what are the foundational questions of the field, and in fostering attempts to answer them. As indicated above, I believe some of the major frameworks commonly employed, in exploring what are recognized as critical questions, are incorrect. A knowledge of the history of some science illustrates how a change in basic frameworks can have unimaginable and unimagined effects.

I decided, about six years ago, to visit some universities and researchinstitutions, to find colleagues with whom I could discuss the foundations of the field. I took part of my salary as a grant to support this journey. I did not give talks or accept payment for any expenses, as then I would have to give seminars and meet people on a formal basis. This would interfere with my primary aim. I wanted an opportunity to find individuals who were open to discussing foundational questions. It might seem strange to many readers that this was not possible with most researchers. However, I should point out in this context that many foundational issues were not being addressed by the immunological community, and would often involve a questioning of frameworks, commonly employed by the individuals in their own research. I had decided on a very gentle approach. I would not pursue a line of argument if I felt my colleague was at all defensive. I wanted to find individuals open to unfettered exploration of ideas.

I also went to scientific meetings with the same purpose in mind. I met two individuals through these journeys. Colin Anderson, also a Canadian, had a record of interest in basic questions and, though we had different perspectives, we found ourselves able to have meaningful discussions that influenced both our views. I met Alex Corthay, of Oslo University, at two meetings. Alex was very open to exploring ideas. The second time I met him was in Davos, Switzerland. My parents were Swiss, and I had a half ownership of a house, Casa Mispa, in the southern part of Switzerland. When I met Alex the second time, I expressed my regret I had a flight booked back to Canada; otherwise, I suggested, we could have met for a couple of days at Casa Mispa to discuss science. Alex responded by suggesting we arrange a workshop in the future. Casa Mispa is set in a truly wonderful and beautiful setting. I had always dreamt of having discussions there with colleagues. Alex proposed that Zlatko Dembic, also of Oslo University, would be a suitable and engaged participant, and agreed to approach him. I approached Colin Anderson, whom I had met on my visit to the University of Alberta. I also had been very impressed by Zoltan Nagy’s book on a History of Modern

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Immunology, and asked him whether he would like to attend, though I had never met him. Inger Oynebraten, Alex’s friend and researcher in cancer immunology, and Calliopi Havele, my wife and a researcher in transplantation immunology, were members of our first, one-week Workshop on the Foundations of Immunology, held at Casa Mispa. We have now had five successive, annual workshops. We have made a couple of reports from the second annual meeting, and now are planning our sixth. Speaking personally, I have no doubt that these meetings have contributed to the development of my ideas on immunology. The kind of interaction we have is something that, it appears to me, to be both valuable but rare in the immunological community.

August, 2019

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Section II: Books written and being written by Peter Bretscher

Rediscovering the Immune System as an Integrated Organ, FriesenPress, 2016

The Foundations of Immunology and Their Pertinence to Medicine, FriesenPress,2016

On Science, Society and the Contemporary Condition, in preparation

How I came to write some books

I had a heart attack in 2006, when 63 years old. I consequently had a bypass operation and was in Intensive Care for three days. This traumatic experience, and close brush with death, led me, whilst recovering in the Intensive Care Unit, to assess whether I had realized my youthful aspirations. I had resolved, as a youngster when twelve, to attempt to become a scientist. I was very interested in theories. I was also interested in philosophy as a teenager, and felt the scientific attitude would naturally contribute to the further development of the culture of our society.

I looked back over my life as I came to in the Intensive Care Unit: my successes,my failures, and my life’s path. I felt well about my modest contributions to science; the activity of doing science had more than fulfilled my anticipations of wonder, engagement, ecstasy, and satisfaction. I had also anticipated that doing science would, by the example, satisfy my aspirations to express how scientific attitudes can contribute to society. I recognized, whilst in the Intensive Care Unit, the illusion of this anticipation. Poignantly, I felt the scientific culture had changed dramatically over the 40 years I had been scientifically engaged, and this culture was no longer as inspirational as it had been at the beginning of my involvement. The overall scientific attitude, as represented by contemporary science, could, I felt, no longer provide guidance as to how society should face existential questions. It was also my conviction that this lack of beneficial influence would have dire consequences. This conviction led to my resolve to write a book. Recognizing I had been given another chance, I would explain my perception of how an optimal scientific culture could contribute to how society faces today’s existential questions.

I started writing within days of being discharged from hospital. As many first time authors, I tried to include all ideas and topics of interest in my first book. An editor, on

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reading a draft manuscript eight years in the making, gave me this assessment. I immediately recognized the assessment to be insightful. I feel the need to first say a bit about my life to explain how I came to write books, and why I appreciated the editor’s advice.

How I came to be interested in immunology

I studied physics as an undergraduate at Cambridge University, England. Isought, before graduating, to identify a rapidly developing science in which I might do graduate studies, to obtain a PhD, and to which I might hopefully contribute. I was lucky in being influenced to do, and being accepted into, graduate studies at the Cambridge Laboratory of Molecular Biology. My acceptance occurred in 1964, 11 years after Watson and Crick had proposed the structure of DNA. This lab was not that large at the time, though four of its senior members had recently been awarded the Nobel Prize, including Crick. Given my physics background, I was encouraged to do my graduate studies in the field of X-ray crystallography, employing X-rays to determine the three dimensional structure of proteins, under the supervision of John Kendrew. Kendrew had received the Nobel Prize for the determining the three dimensional structure of the first protein, namely “myoglobin”.

I became fascinated, during my graduate studies, by immunology, the study ofhow the immune system works. The science of immunology is pertinent to preventing infectious diseases, by effective vaccination, besides being also pertinent to the prevention and curing of several other kinds of disease. My fascination took the form of trying to answer some of the most basic questions of the field. Crick had a notice board outside his office. He had pinned there an invitation, to those who wished to discuss science, to enter his office. I availed myself of this invitation a number of times to discuss ideas concerning various immunological issues. So began my life as an immunologist.

During the last 50 years, my students and I have been engaged both in making theories and in experimental studies in the field of immunology. The way I approach my research is influenced by the philosophical ideas I had developed as a teenager and undergraduate. Thus, in the first draft of my book, I gave an outline of our immunological research. This outline, I thought, could provide a case study for showing how the culture of science has changed over the last four decades, and provide a forum for a discussion of the effects of this change. I would try to explain how this change of culture undermined the effectiveness of the scientific approach, as now practiced.

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My first book: Rediscovering the Immune System as an Integrated Organ

However, the constructive criticism of an editor, as already described, made me realize this was an ineffective way to proceed. I therefore decided to first write a book on how

broad considerations, in the context of experimental studies, could give rise to a coherent view as to how the immune system functions. It took me a couple of years, after receiving the editor’s advice, to write this first book. I might add two significant features about the book and its realization. Firstly, I wanted to stress the value of certain foundational ideas as to how the immune system functions in a coherent fashion. We probably all know that scientists stress the importance of observation and of experiments to the success of their research, and I was

very mindful in this book to explain not only foundational ideas, but the

substantial observations supporting them, as well as the observations makingalternative ideas seem less plausible. I anticipated that the book, for an outsider to the field of immunology, would be a pretty heavy read.

I initially submitted the draft to various University Presses, but had no takers. I therefore had to go with a non-university publisher, or find a firm that would allow me toself-publish. I had been in email contact with an immunologist, Zoltan Nagy, who hadrecently written a book, entitled “A History of Modern Immunology: the Pathway toward Understanding”. Zoltan Nagy’s book was produced by a commercial publisher. The cost

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of his book was about five times the cost of what my book would cost if self-published. A main motivation in writing the book was to foster discussion, a process probably not optimally fostered by an expensive book. I therefore decided to self-publish with a Canadian firm, FriesenPress, in order to make the book broadly accessible. I outline below a couple of responses to this book.

My second book: The Foundations of Immunology and Their Pertinence to Medicine

I was quite surprised when, having seen my first book to press, I found myself ina virtual trance, committed to writing a second book. This book would stress the ideas I regarded as important to the field. I would employ observations to illustrate some ideas or to argue against the plausibility of others, but I would not attempt to observationally justify the foundational ideas I proposed. I was comfortable doing this, as my first book had provided such a justification. This justification gave me the freedom to primarily appeal to the reader’s analytical abilities and imagination. Moreover, I could explain in the text how foundational ideas were pertinent to the prevention and treatment of many medical conditions. I found writing this text to be exhilarating. It was completed in three months. I felt I had achieved accessibility, brevity and clarity in my exposition.

The release of energy, to write the second book, initially took me by surprise. Ihad not even envisioned writing a second book on immunology. My energy arose from my intuition that the information overload of contemporary life has undermined imagination and faith in careful analysis, to the detriment of gaining real insights. My attempt to appeal to the reader’s intuition and analytical ability, to overcome the weight of this overload, presented me with an inviting challenge.

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I would like to explain a technical consideration concerning the production of my second book. Modern immunological texts are often between 500 to 1000 pages long, with fairly small type, and do not appear to me to optimally address the most foundational issues of the field. They actually often employ frameworks that I consider implausible, which makes the picture developed, of how the immune system functions, complicated. I find these modern texts to be overwhelming. I wanted the physical nature of my second book to reflect the idea that keeping to the basics allows the functioning of the immune system to be seen as simple and coherent. I told my Editor that I wanted the book to be printed in as large a type as possible. This would allow the reader, as they encountered the text, to feel they were making progress, as they turned the pages!

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She suggested a fairly large print, and told me such a book would be about 180 pages. She also told me that, if the print were larger, the book would only be stocked in the children’s section!

Comments on the books

I wrote these books with the hope they would substantially contribute to the fieldand be of interest to some of the general public. I made enquires concerning whether the books might be reviewed in various journals, but to no avail. This quite frankly surprised and somewhat shocked me. I submitted the first book to the New England Journal of Medicine, perhaps the premier medical journal. They were much politer than most. They explained to me that they no longer reviewed books. They had done surveys and found most of their readers were relatively uninterested in book reviews, and so had decided, as editorial policy, not to do reviews. Another journal gave me a similar reply. Most journals did not respond to my enquiry.

Reviews/Comments on Rediscovering the Immune System as an Integrated Organ

One colleague, to whom I sent a very late draft of the book, was both enthusiastic and generous. Ted Steele wrote: “Those who have followed Peter Bretscher’s work these past 50 years will recognize this book for what is it – a rare achievement, a scientific masterpiece. It is a must read for all those Immunologists and Clinicians who want to find effective immunological cures for the many debilitating health issues that confront us. Peter Bretscher has produced a lucid and logical expression of the rules governing how the adaptive immune system responds to all foreign antigens, whether bacterial, viral or modifications of self, which emerge in cancer or autoimmune conditions. This book is a testimonial to Louis Pasteur’s dictum that there is no applied science, just the application of basic science.”

Dr Alex Corthay, Head, Tumor Immunology Unit, University of Oslo, wrote a review for the Scandinavian Journal of Immunology.

I quote part of the review below:

The immune system may seem incredibly complex. Researchers in immunologyare amassing enormous amounts of detailed information without gaining proportional insights. Why might this be? So asks Peter Bretscher near the start of his book Rediscovering the Immune System as an Integrated Organ. He argues that contemporary immunology fails to provide understanding at the level of the system

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because it is dominated by molecular and cellular considerations. He reminds us of a famous quotation: Not everything that counts can be counted and not everything that can be counted counts, before stating the ambitious aim of his book: to make plausible an integrated and readily accessible view of how the immune system functions. By Peter Bretscher. FriesenPress, 2016. 288 pp. ISBN: 978‐1‐4602‐7406‐4.

Peter Bretscher is a well‐known immunologist who has been doing research for almost fifty years. His most famous contribution to the field is the two‐signal model of lymphocyte activation, which he published in 1970, together with Melvin Cohn. This model, which provided an explanation for how self/non‐self discrimination is realized, constitutes a pillar of modern immunology thinking. - - - - - Although very ambitious in its content, with both theoretical and applied immunology, this book is very didactic and well written. Results from key experiments are illustrated by simple figures which allow the reader to comprehend the original observations. Similarly, major theories are illustrated in a clear fashion. An extensive reference list invites to further reading. Each chapter is summarized by a brilliantly written synopsis. It is a must read for anyone interested in immunology, a classic book already.

Reviews/Comments on Foundations of Immunology and their Pertinence to Medicine

I sent a copy of a draft of this book to Dr Zoltan Nagy, immunologist and authorof A History of Modern Immunology: A Pathway toward Understanding”, even though I had not personally met him. Zoltan kindly offered to write a comment on the book for the back cover: “Foundations, despite its small size, is a monumental edifice of immunological thinking which could not have been written by anyone else but Peter Bretscher.”

Rachel Jagareski reviewed the book for Foreward in February, 2017. She gave the book five stars (*****), and headed her review with:

From an innovative theorist and an elegant writer, this book is a valuable contribution to literature on medicine and disease

Excerpts from the review:

In The Foundations of Immunology and Their Pertinence to Medicine, research scientist Peter Bretscher summarizes historical discoveries and foundational concepts of immunology to encourage potential medical breakthroughs among non-specialists, clinicians, and “broadly educated” thinkers. The book presents overspecialization among scientists and medical professionals as a trend that limits the creative and

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synergistic study of human illness. Bretscher sees many possibilities for treating and curing a variety of human ailments, from seasonal allergies to leishmaniasis to cancer, through an understanding of the ways immune system components variously attack pathogens and cells within the body to cause autoimmune diseases. Most of the book forwards a historical overview of immunology, from its beginnings in the eighteenth century with Edward Jenner’s observations of smallpox immunity among cowpox survivors through to the very latest research into the molecular structure and function of cells within the immune system. The chapters gradually build up the layers of information needed to understand how lymphocytes and other cells are triggered into action to fight off infection—and conversely, why they don’t in other circumstances.

Bretscher analyzes mountains of immunology literature and points out areaswhere further research could clarify previous findings or explain contradictory results. His is a thorough review that tosses out enough exciting ideas to occupy graduate students around the globe.- - - - - - The final chapters are the most exciting and reveal specific avenues for potential immunological treatment for a number of diseases. The author’s analysis suggests that immune responses might be very specifically regulated to release certain antibodies, or to unleash one of the range of cell-mediated responses in specific situations, holding promise for many medical conditions. He envisions immunological scenarios allowing for the creation of vaccines against HIV and tuberculosis; targeted medical treatments for arresting multiple sclerosis and other autoimmune diseases; screening for susceptibility to certain kinds of cancers and inoculation to trigger immune protection; and protocols for protecting against the rejection of transplanted organs and tissue. Bretscher is an innovative theorist and an elegant writer, and this book is a valuable contribution to literature on medicine and disease. It provides ideas for further research into harnessing the body’s highly individual immune response to vanquish ailments that have plagued humankind for centuries.

Dr Alex Corthay, Head, Tumor Immunology Unit, University of Oslo, also wrote a review of Foundations for the Scandinavian Journal of Immunology. I quote part of the review below:

We are in the era cursed with overspecialization. Charles Darwin's foundationalOrigin of Species, published in 1859, was accessible to the broadly educated individual. Starting with this statement, Peter Bretscher, in his new book, aims at explaining the foundational concepts of immunology to a broad public. His standpoint is that foundational ideas usually can be simply expressed. This book describes first how foundational concepts of immunology have evolved over the last two and a half centuries. The importance of self–non‐self‐discrimination by the immune system is acknowledged, and models for immunological tolerance are compared. A main focus concerns the understanding of immune class regulation. According to Bretscher's Threshold Hypothesis, a low antigen dose or low degree of foreignness results in Th1

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immunity whereas a high antigen dose or high degree of foreignness induces Th2 responses. A novel vaccination strategy is proposed with an ultra‐low number of live, attenuated organisms. It is described in detail how this approach may potentially be used to prevent or treat HIV infection, tuberculosis and cancer. The book is very well written in a clear, simple and stimulating style, which makes it accessible not only to immunologists but also non‐specialists, teachers, medical students and clinicians. It will convince many that the immune system is not too complex to be understood. By Peter Bretscher. FriesenPress, 2016. 200 pp. ISBN: 978‐1‐4602‐9656‐1.

Peter Bretscher is a renowned immunologist who has been doing bothconceptual and experimental immunology for almost fifty years. He is famous for thetwo‐signal model of lymphocyte activation, which he published in 1970 together with Melvin Cohn. This model provided an explanation for how self/non‐self‐discrimination is realized by the immune system. Bretscher is an outstanding communicator whose ambition in his new book is to explain both basic immunological concepts and their relevance to medicine to a broad public. It is in many ways a shorter, simpler version of his previous and very successful book Rediscovering the Immune System as an Integrated Organ, which was published at the beginning of 2016. ………………………The book ends with a very stimulating 33 pages‐long chapter dedicated to novel strategies for medical interventions. Bretscher makes a number of concrete suggestions for how conceptual immunology may be used clinically in domains as different as infectious diseases, allergy, autoimmunity, transplantation and cancer. The potential of immunological imprinting is first described. The Concept is based upon mouse experiments and human observations suggesting the immune response to a given antigen can be locked into a Th1 or Th2 mode.……... …………………………………………… …It is described in detail how this approach may potentially be used to prevent or treat HIV infection, tuberculosis and cancer.

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Section III: A Personal Perspective on the History of Two Signal Models of Lymphocyte Activation

This influential model, first published in Science in 1970, evolved, as put forwardby Cohn and myself and others, as the years passed. Cohn also gave accounts of the history of how this model originally arose. I found these accounts in some respects to be misleading. I also felt that most of the modifications of the model, proposed by Cohn after I left his lab, to be implausible, and so undermining the appeal of the original model. I therefore decided, in the early 2010s, to write an article reassessing and reaffirming the 1970 formulation of the two signal model, in the context of then contemporary evidence and ideas. Cohn responded to this article, explaining why he disagreed with several of my proposals, and also making again what I considered inaccurate statements on its history. As a result of this, I decided to post my personal perspective of the history of the 1970 model in 2014 on my web profile. This was 44 years after the model was first published, and decades after Cohn’s first accounts of its history that I felt to be inaccurate. This personal perspective constitutes the main body of the third section of the profile. I have not brought this section up to date, as I wanted it to stand as originally written. However, I think it appropriate to add some new facts here. Cohn died in October, 2018, at the age of 96. Robert Coffman, Stephen Hedrick and Alan Sher wrote a two page In Memoriam of Cohn, for the March/April Newsletter of the American Association of Immunologists. They described there how I came to Cohn’s lab in 1968, as a postdoctoral fellow, with the basic ideas of the two signal modelalready formulated. This accords with my memory.

August 2019

The account below was written from November 2014 to April 2015

There are two related reasons for wishing to have an accurate history of how significant advances in a scientific field are made. The most obvious and prosaic is to give credit where credit is due. The second reason is more important and interesting. A knowledge of how important advances comes about is germane to understanding how progress occurs. Such understanding can be inspirational.

I think it useful to clarify at the outset what different people mean by “two signal models” in the context of lymphocyte activation, as my reading of the literature leads me to recognize there are two different and sometimes potentially confusing uses. Dresser and Mitchison, in their 1968 article in Advances in Immunology1, clearly state the idea that competent lymphocytes can interact with antigen in two ways, one way leading to their “paralysis”, or inactivation, and the other leading to lymphocyte activation. In this case, there are two separate kinds of signal a lymphocyte can receive, each with different consequences. I shall refer to this use as “a model for the two signals involved in the paralysis and activation of lymphocytes”. Sometimes people refer to this idea

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simply as a two-signal model, but it is clearly not a “two signal model of lymphocyte activation.” The term “two signal model” as now used is most often taken to mean a two- signal model of lymphocyte activation. It refers to the view that the activation of a lymphocyte requires antigen to interact with the receptor of a lymphocyte, generating signal 1, that, if generated alone for a sustained time, leads to the inactivation of the lymphocyte; the activation of the lymphocyte requires the generation of this signal 1, and a second signal, in addition. This second signal is referred to as signal 2. A major impetus for the genesis of this two signal model of lymphocyte activation was to provide an understanding, in 1969/1970, of how self-nonself discrimination might be achieved2, as outlined below. This was a time when it was not recognized, as also outlined below, that there are central and peripheral mechanisms of tolerance.

The account I give here was stimulated in part by other accounts of how the two signal model of lymphocyte activation came about. It seems to me that these accounts are inaccurate, and therefore mitigate against a correct appropriation of credit. More importantly, these accounts obscure the considerations underlying the initial proposals, and so undermine an understanding of the imaginative, rational and observational considerations involved. A real appreciation of these considerations might stimulate other individuals, of a susceptible temperament, to be empowered to be brave, a quality required to be creative. This concern for accuracy may seem strange to some. For me this concern is very important, as my intellectual braveness, such as it is, was fostered by appreciating the braveness of others by following their intellectual journeys. This fostering led me to believe in the subtlety of argument. I felt I was a part of a community and culture that recognized the value of subtle thoughts. I hope a correct account of the origin of the two-signal model can help others to be brave, and thus beneficially contribute to this culture. I would like to add, in parentheses and so not to be misunderstood, that the best kind of braveness is probably unselfconscious; it arises from intense engagement with a problem and an unconscious faith, nurtured through experience, that subtle considerations can yield enormous dividends.

Cohn wrote an invited first article of reminiscences for the 1994 Annual Review of Immunology, entitled the Wisdom of Hindsight3. I read that part describing the origin of our Two Signal Model of lymphocyte activation shortly after Cohn’s article was published. Cohn’s account was much different from my recollections. I decided not to read the rest of the article. I had often found, after leaving the Salk Institute in 1973, Cohn’s papers difficult to read. I found them both difficult to understand and, when I thought I understood what Cohn meant, I often found the proposals put forward to benon-compelling or implausible. I therefore did not make the effort to give them greatattention. I have thus not been much engaged in getting to know and critically assessing Cohn’s ideas after I left the Salk Institute in 1973. This might be surprising to some. There was also a more emotional reason. I found it difficult to read Cohn’s papers when I felt his proposals often muddied what we had proposed together in our 1970 Science article2. I also felt, for most of my career, that the best response, to my perception that our vision was being distorted in an unfortunate way, was to do the best science I could,

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and not become distracted by taking on a personal agenda to put matters right. I found support for this course with my happiness at how we had expressed our vision in the Science article3, our most cited, joint publication. When I have read it decades after it was published, I have no misgivings.

I had a year of administrative leave, starting in June 2013, from my normal duties at the University of Saskatchewan. In my normal university life I had been busy teaching, doing research and some administration. However, this administrative leave encouraged me to reconsider my priorities, particularly in view of my age of 70, and my frustration, over decades, that ideas, that I considered may be valuable to the field, were barely considered by the immunological community, particularly those ideas on immune class regulation. I also envisaged these ideas could have a considerable impact on medicine and the relief of human suffering. I therefore decided to devote considerable energy in this year to explore ways to get my views on two subjects more broadly considered. In the end, I found a way to get two reviews, describing my current thinking, accepted by the Scandinavian Journal of Immunology, as “Discussion Forums”, one on the activation/inactivation of CD4 T cells4, the other on Immune Class Regulation5. I did this on receiving the same suggestion from two individuals I substantially met for the first time during this year, Colin Anderson, of the University of Alberta, Canada, and Alexandre Corthay, of the University of Oslo, Norway. Both suggested I submit these articles through the Associate Editor, Zlatko Dembic, also of the University of Oslo. There is clearly a tradition, through the forums of this journal, to open up discussion of fundamental questions.

My first Discussion Forum paper, on the Activation/ Inactivation of CD4 T cells4, drew a long response from Cohn6. I was asked to review it, as he had been asked to, and did, review my corresponding submission. I have therefore, in the last more than twelve months, become more familiar with both the details of some of Cohn’s scientific proposals and also with his description of how the 1970 Two Signal Model of lymphocyte activation3,6 came about. In view of the statements on the history of the two signal model for lymphocyte activation that Cohn made in his 2014 response6to my Discussion Forum article4, and in his 1994 article in Annual Reviews of Immunology3, I decided to embark on this project, to outline those statements of Cohn, on the history of the two signal model of lymphocyte activation, with which I disagree, and contrast them with my recollections of the pertinent events, with supporting evidence. I have not read other accounts of the history that Cohn has written since 1970. I consider that the two accounts I have before me is a sufficient basis for me to comment on.

Cohn, in his “The Wisdom of Hindsight” article3, has a section entitled “The Origin of a ‘Two Signal Model’ of the Self-Nonself Discrimination”. This section starts off with:

“Peter Bretscher joined my laboratory in 1967; he was, by way of background, an X-ray crystallographer who had a passionate curiosity about immunology as well as a clear,

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crisp way of analyzing complex problems. This made the difficult question of the self- nonself discrimination an ideal one to answer; we were aware that it had to be analyzed correctly if a next step was to be made in immunology.

“We had two prior formulations, those of Lederberg and Forsdyke………..Forsdyke gave us the barest hints as to what a competing theory would entail…... Forsdyke proposed a situation where an interaction leading to a doublet [antigen binding to two distinguishable antibody sites of identical specificity in close proximity] results in inactivation, whereas one leading to a singlet leads to activation, in any given cell. ….A two signal mechanism distinguishing self from nonself by each antigen-responsive cell was required, and Bretscher and I set out to develop just such a theory.” I take Cohn to mean here a model for the two signals involved in the paralysis and activation of lymphocytes, not a two signal model of lymphocyte activation.

I would now like to give an account of my recollections concerning the origin of the two signal model of lymphocyte activation. I apologize for going into considerable detail; such an exposition seems important for others to both understand the basis, and assess the legitimacy, of what I say.

My involvement

I became interested in immunology when a graduate student, under the supervision of John Kendrew, doing research in the field of protein X-ray crystallography, at the Cambridge Laboratory of Molecular Biology. I started my graduate studies in 1964. From the very beginning, I was much more interested in what was going on in the Division of Molecular Genetics than in the analysis of protein structure by X-ray diffraction. The Division of Molecular Genetics was headed jointly by Brenner and Crick. A group of research students and postdoctoral fellows, typically about 5 to 7 individuals, primarily from this division, met semi-regularly on Saturday mornings for a few hours to chat, with Sydney Brenner and over Nescafe, about diverse scientific topics. I joined my brother Mark in attending these get-togethers when I first became a graduate student. Mark soon left for a postdoctoral fellowship with Paul Berg, at Stanford, and I continued attending these meetings.

It was in 1966 that Sydney talked about a paper written by himself and Cesar Milstein on a model for “The origin of antibody variation”, that had just been accepted by Nature. I thought about this model over the next week and came to believe it implausible, and the next Saturday I tried to explain my reasons to Sydney. Sydney was marvelous. He considered my reasons and thought them interesting. He took my proposal that there were likely to be invariant amino acid residues in the variable region of immunoglobulin chains to Cesar, a possibility unanticipated on and in weak conflict with their model; Cesar in time confirmed the validity of this proposal. The prevalent existence of such

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invariant residues made the Brenner/Milstein model less plausible. This experience spurned my interest in immunology.

In time, I got into the habit of very occasionally making use of the invitation, placed on a notice-board outside Crick’s and Brenner’s joint office, to discuss science with Crick. I probably knocked on Crick’s door on at least ten occasions.

As I developed various ideas about immunological subjects and about my PhD research, I took the opportunity to discuss these with Francis. One of my earliest immunological ideas now appears obvious. However, at the time, Francis appeared to think it new and was enthused. This is perhaps not so surprising, as at the time some reasonable people were unsure about the plausibility of the Clonal Selection Theory. My ideas buttressed the plausibility of some of the critical features of the Clonal Selection Theory.

I thought that when antigen interacted with antibody receptors on an antibody precursor cell, there must be “an interaction sensing site” that “recognizes” this interaction via changes in the structure of the antibody. I considered the situation that would occur as the genes coding for immunoglobulin chains are generated, in evolutionary time or somatically. I considered that this “interaction sensing unit” must be complementary to an invariant part of the antibody molecule; if it were complementary to a variable part, there would have to be two “complementary” mutations, one in the antibody genes, the other in the gene encoding the interacting sensing unit, an unlikely event. Assume, as seemed plausible, that the “interaction sensing unit” was indeed complementary to an invariant part of the antibody molecule. In this case, if the cell had two chemically different antibody receptors, the cell could not distinguish which of the two interacted with antigen, and so could not, for example, uniquely only activate the transcription of genes coding for the antibody receptor that had interacted with antigen. Thus I was led to the proposition one cell “must” be committed to the synthesis of antibody of just one specificity. This consideration was obviously another way of making plausible some essential aspects of the Clonal Selection Theory. These considerations formed part of the two papers Cohn and I subsequently published in Nature in 1968 and in Science in1970.

Crick encouraged me, once he understood what I was driving at. Francis could initially not understand what I was saying, given my nervous disposition. He recommended I put my ideas in written form on one side of a page. I did this and gave him my effort. The argument was dense, and as rigorous as I could make it. He read it and in time understood what my considerations were. I of course remember the details of this interaction vividly, as it was so important to me. Francis gently chastised me. I did not need to take his recommendation of one page so literally, and so make the argument so dense. Neither did I have to prove my proposal, just to make it plausible. My interactions with Francis determined to a considerable extent my scientific fate.

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On getting interested in immunology, I asked Cesar Milstein for advice on an introduction to the subject. He suggested Burnet, and lent me his copy (presumably) of The Clonal Selection Theory of Acquired Immunity. I read some it, but I was quite naïve. I remember I found it in parts implausible, and so did not read it in toto. I clearly remember though that I took notice of one remark made. Burnet pointed out that an antigen, in order to be immunogenic, i.e. able to induce the formation of antibody, had to be a macromolecule. This caught my attention, as small molecules could interact with antibody. The inference was, perhaps, that the simple interaction of antigen with antigen receptors was insufficient to activate an antibody precursor cell. I must have gleaned from Burnet the importance of the attribute of self-nonself discrimination. I probably was also introduced to the idea that the immune system relies on the early presence of self antigens in development, and their continuous presence thereafter, in ablating the ability to launch anti-self immune responses. This idea seemed so obvious to me that I now cannot remember its source. I also searched and read other articles, particularly after I had an accident.

This accident must have also occurred sometime later but still in 1966, I think. I was using a chemical that reacted predominantly with the free amino group of lysine residues of proteins. This chemical was somewhat dangerous, as it readily permeated skin. I wore gloves in using it. The gloves leaked. The tip of my right index finger started throbbing one night as I fell asleep, on a day I had used the chemical. The next morning the tip was white and quite dead. In the long run, the tip was surgically removed and skin, obtained from my arm, was grafted onto the raw end. However, before this, during the next few days after the mishap, a sharp line developed between what I imagined was healthy me and the proteins of my finger tip, altered by reacting with this chemical and so becoming “foreign”. This personal experience stimulated my interest in how the immune system might manage to respond to foreign but not to self-antigens.

My limited reading, or a conversation with Cesar Milstein, I now do not know which, led me to believe that antigen could interact with antibody precursor cells in two ways, one leading to the antibody precursor cell’s inactivation, the other to its activation, i.e. multiplication and the differentiation of its progeny into antibody secreting cells. There is one circumstance I recall that is surely significant. I had already decided to try to become an immunologist if I managed to get my PhD. In talking about this with Francis Crick, he suggested I might do postdoctoral studies with Avrion Mitchison, at that time at Mill Hill, London. I therefore visited Av, as he was known to all, sometime in 1966 or perhaps early in 1967. Av explained his lab was very full but that, if I really wanted to come, he could probably squeeze me in. I remember very clearly from this visit a big picture Av had high up on a wall. It represented a conclusion drawn from some of his recent studies. Av had harvested spleen cells from mice primed with the hapten carrier conjugate, h-C, and could show that such primed cells, when exposed to h-C in vitro for some time, could mount a secondary response to the hapten when subsequently given to irradiated mice without more antigen. Av found the presence of a monovalent form of h, when given in sufficient amounts with h-C during the in vitro stimulation of the primed

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cells, could block the production of anti-hapten antibody when the primed cells were given to irradiated mice. This “again” implied that the interaction of a small molecule, the hapten, with an antibody receptor on the memory antibody precursor cell, was insufficient to “activate” the antibody precursor cell; actually, the hapten could block the precursor cell’s activation by h-C. I recall that I was at this stage already very interested in what was required to activate antibody precursor cells, but I had not yet formulated any clear ideas that were appealing under sustained examination. Av was in the future (at the 1967 CSH symposium) to articulate his view that the generation of a secondary antibody response required antigen to mediate cell-collaboration between hapten- specific antibody precursor cells and carrier-specific cells. I believe that Av had not yet come to this conclusion on my visit, most likely, or was reticent in openly expressing his views. I remember asking him what more was required to activate the antibody precursor cell than its interaction with the hapten/antigen?

I had some ideas, most probably in1967, and certainly after my meeting with Av, about how antigen might interact differently with antibody precursor cells to result in their activation and in their inactivation. I realized that the possibility that single precursor cells are inactivated on interacting with antigen, but that their activation required theantigen-mediated interaction between specific lymphocytes, had three appealingcharacteristics. Firstly, it was the only proposal I knew of that attempted to account for how antigen interacted differently with the same antibody precursor cell to result in either its inactivation or its activation. Secondly, it “explained” why antigens had to be macromolecular to be immunogenic. Thirdly, it accounted for how self non-self discrimination could be achieved, as outlined elsewhere2,4,7. Briefly, anti-self lymphocytes were envisaged to be inactivated when they were first generated, one, or a few, at a time, due to the early presence of self antigens in ontogeny and their continuous presence thereafter. Lymphocytes only able to recognize foreign antigens would accumulate during ontogeny in the absence of the foreign antigen and, once the foreign antigen impacted upon the immune system, this antigen could mediate the lymphocyte cooperation needed to initiate an immune response against the foreign antigen. The ideas concerning the activation and inactivation of lymphocytes occurred to me before I had come across any observations on “carrier effects”. However, I searched the literature for observations bearing on this proposal for one lymphocyte/multiple lymphocytes for the antigen-dependent inactivation/activation of lymphocytes. I did not know what journals to look at, so I initially randomly leafed through various periodicals on the shelves, looking for immunological articles. I found a paper in the Journal of Experimental Medicine that seemed to strongly support the proposal I envisaged. This was a paper by Benacerraf’s group on guinea pig non- responders to poly-L-lysine (PLL). These animals could make a response to PLL when they were immunized with PLL coupled to an immunogenic carrier, namely bovine serum albumin (BSA). There was no interpretation of the observations in the paper by the authors along the lines I envisaged. This was the first paper, among several, that seemed to support the ideas I had come up with.

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Two review articles were very important to the further development of my ideas as a graduate student in Cambridge. Firstly, the article in the 1968 Advances in Immunology by David Dresser and Av Mitchison gave a very broad overview of observations and ideas pertinent to generating antibody responses and generating unresponsive states. Particularly important to me were the references to articles and the description of studies by Bill Weigle from 1961 onwards, as I have described elsewhere 4. Many observations of Weigle fit in with the proposal I was entertaining, and had been in the literature for several years. There were no suggestions I could find anywhere, along the lines I envisaged for how these observations might be interpreted, for example in the Dresser and Mitchison article. This appreciation of the explanatory power of the ideas certainly increased my perception of their plausibility and potential significance.

I also came across an article by Gell and Kelus on Anti-antibodies in Advances in Immunology, 1967 (Adv Immunol 6:461-78.). I was very interested to know under what circumstances the interaction of antibody with antigen resulted in conformational changes in the antibody molecule, as I envisaged such changes had the potential for signaling to the precursor cell that its antibody receptor had interacted with antigen. I found the description, of antibodies that could recognize sites on other antibody molecules that were complexed with antigen, but these sites were not present on unbound antibody, very intriguing from this perspective. These anti-antibodies provided a means of detecting changes in antibody conformation that occurs when antibodies formed complexes with antigen.

As my reading of the immunological literature advanced, I kept Francis informed of how my ideas progressed. He changed his advice on where I might do my postdoctoral studies. Francis was now annually visiting the Salk Institute as a Non-Resident Fellow and, given his appreciation of my great interest in theory, suggested I might do a postdoc with Mel Cohn at the Salk Institute. Naturally, I accepted Francis’ advice.

Mel Cohn also naturally accepted me on Francis’ recommendation. Cohn had a major experimental, NIH-sponsored, program for producing myeloma tumors in BALB/c mice. He already had a substantial collection. I was encouraged to apply for a Damon Runyon Postdoctoral Fellowship whilst a graduate student in Cambridge. I had to outline a plausible research proposal. I suggested that the cause of the myeloma cancer might be because the cells produced a myeloma protein that had an abnormal structure, reflecting a structure representative of antibody complexed with antigen. I envisaged that if such abnormal “myeloma antibodies” were generated in an antibody precursor cell they might give rise to precursor cells that behaved as continually activated, and so constitute a cancer of this cell. The prediction was that myeloma proteins, not complexed with antigen, would react with antibodies that normally only recognize antibody sites present on antigen/antibody complexes. Although this idea was not experimentally explored, as I came to feel it implausible once I had been in Mel’s lab for several months, it was part of my successful application for a postdoctoral fellowship, and this idea was reflected in my first publication with Cohn7.

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Before leaving Cambridge for southern California for my postdoc, I submitted a letter to Nature on a non-immunological topic. This letter was received by Nature on July 8, 1968. I surmise I arrived in Cohn’s lab in late July, 1968, at the very earliest.

My impression, on arriving at the Salk Institute, was that Cohn was mainly pre-occupied with the problem of the origin of the diversity of antibody genes. He was less aware of the literature on the requirements to generate antibody responses and generate unresponsive states than I was. I had not come across, as a graduate student, Lederberg’s classic Science paper of 19598. I became aware for the first time of the proceedings of the 1967 Cold Spring Harbor Meeting. The two major “new” discoveries for me, from reading the proceedings of this meeting, was that others, namely Mitchison, Rajewsky and Jerne, had come to the view that the generation of an antibody response most probably required the antigen-mediated interaction between lymphocytes. Secondly, Rajewsky’s contribution described observations, novel to me, concerning his work with porcine lactic dehydrogenase (LDH) isozymes as antigens in rabbits. Rajewsky’s observations were of particular interest to me, as they were interpreted as showing that non-immunogenic molecules were non-tolerogenic, a proposition inconsistent with the ideas I had developed in Cambridge. I thought this conclusion misleading, for reasons we outlined in our 1968 Nature7 and 1970 Science2papers, and that I have also recently discussed4. I might add that, in some real sense, Rajewsky’s observations and their interpretation were not radically new, as somewhat similar observations had been made earlier, as recorded in the Dresser and Mitchison article, and been similarly interpreted.1 I have recently given a detailed account of these observations and the considerations they led to.4

I was very motivated, on arriving in Cohn’s lab, to quietly and thoroughly review the literature to see how my ideas might stand up to current findings and other people’s ideas, and to explore how they might be further developed before “going public”. This was not to be.

Cohn told me shortly after my arrival that he had been invited to a small meeting of about 40 individuals, on Immunological Tolerance, to be shortly held at Brook Lodge, Michigan. Moreover, Mel was asked to summarize the meeting. The invited participants to this meeting included many of the leading investigators, including Av Mitchison, Klaus Rajewsky, Bill Weigle and Benacerraf. The meeting was held from September 18 to September 20, 1968, surely less than two months after I had arrived in Mel’s lab. It was natural for Cohn, in his summary of the meeting, to try to account for the major findings, outlined during the meeting, in terms of the ideas we were developing.

Cohn, on returning to the Salk Institute, gave me the original transcript of the meeting. I still have this transcript. On reading this transcript at the time, two things seemed clear. (All this is from memory; I have not gone back to check). Cohn’s description of some critical observations from the literature was challenged, and he was not that clear in his

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response to these challenges. In particular, I believe, he was unclear about the nature of Weigle’s observations that showed that challenges with an antigen that crossreacts with the tolerogen could break the unresponsive state. This was a most critical finding that supported our proposals. This concerned me as it meant our model was not as clearly enunciated as it should have been. The major contention was caused by our view that non-immunogenic molecules could cause unresponsiveness. Most thought there were carrier effects not only in generating an antibody response, but in generating unresponsive states, or in causing the inactivation of the antibody precursor cell, a proposition to which I shall return. Cohn refers to this analysis on our part in a way that seems historically incorrect and to reflect an unawareness of the paradoxes of the late 1960s. He says (top of page 35, Annual Reviews of Immunology3 volume 12: “The second choice [of a requirement for recognition of a carrier for paralysis] was initially rejected on experimental grounds (i.e. recognition of the carrier is required for induction, not unresponsiveness).” This statement misses, as far as I am concerned, the critical point evident to me at the time. Those few who had come to grips with carrier effects, namely Mitchison and Rajewsky, agreed that there was lymphocyte cooperation in the induction of antibody, but argued that there were also carrier effects in generating unresponsiveness. It seems to me Cohn misses the essential clarification “our” proposals brought about in recounting these events. It required careful, subtle and quantitative analysis to realize why the observations reported by, for example, Rajewsky, did not mean that there were carrier effects in the inactivation of antibody responses, as I have carefully recently outlined.4

Both Cohn and I were uneasy about how our ideas had been presented at Brook Lodge. Moreover, the proceedings of the meeting were to be published as a book. We decided in the circumstances to write an account of our ideas, as then formulated, as a paper to be submitted to Nature. Nature received our submission on October 17, 1968, less than a month after the Brook Lodge Meeting. It was published in the November 2, 1968, issue.

I received a personal letter from Av Mitchison, shortly after our Nature paper was published. He said “I think you deeply cloud the issue by using the carrier effect to account for the immunity/tolerance decision. I don’t at all see why one cannot have all sorts of lovely carrier effects and also something else, e.g. non-specific stimulation from macrophages.” Klaus Rajewsky had come to the Salk Institute straight from the Brook Lodge meeting to talk science. As clear from our Nature and Science papers, we disagreed with a conclusion Klaus had come to in his studies in rabbits with porcine lactic dehydrogenases as antigens. His observations were a particularly clear example of those employed by immunologists to support the proposition that non-immunogenic molecules were also non-tolerogenic. We argued that such observations were likelymis-interpreted, as quantitative considerations2,4,7 could be employed to suggest theobservations were not against the ideas I had formulated as a graduate student and, moreover, other observations supported these ideas. Furthermore, it seemed most plausible that most self-antigens were tolerogenic but not immunogenic.

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The description above gives my best recollection of the events leading up to the publication of our Nature paper.

Our paper was submitted to Nature on October 17, 1968. I had been in Cohn’s laboratory for less than four months, perhaps only two, and did not arrive in 1967, as Cohn states3. Our Nature paper pretty well reflected the ideas I brought with me, except there were references to the ideas of Mitchison, Rajewsky and Jerne on cell cooperation in the generation of antibody responses, and to Rajewsky’s LDH observations, referred to above. We (meaning I) thanked Crick for discussions related to this paper.

Cohn says, in the quote from his 1994 Annual Reviews article, that I came to his lab in 1967, but I came in late July, 1968, at the earliest. He talks about the influence of Donald Forsdyke’s paper, published in the Lancet in early 1968, as though we were aware of it. This is a rarely quoted paper that I first came across when reading Cohn’s 1994 Annual Review article. I have to say I was really surprised when I first read what Cohn stated. I should say that Forsdyke’s paper does not refer to any of the observations on carrier effects, observed in generating antibody responses, observations which supported the ideas I had formulated as a graduate student and which we discussed at great length in our Nature and Science articles. Neither do Cohn’s accounts of how the two signal model of lymphocyte activation came to be formulated prominently refer to these observations on carrier effects. Cohn’s account is at odds with the fact that I came to his lab with the idea that one lymphocyte/multiple lymphocytes are required for the antigen-dependent inactivation and activation of lymphocytes, and with the considerations that this proposal was supported by a number of observations in the literature.

I have never met Donald Forsdyke. However, circumstances arose where Donald Forsdyke and I communicated by email early in the 2000s. When circumstances led to our email interaction, I decided I had to let him know my perception of the impact of his 1968 Lancet paper on the origin of our ideas, as Cohn inferred3 we were inspired by his ideas, but we had not quoted Forsdyke’s paper in either our 1968 Nature6 or 1970 Science paper2. The account Cohn gave of Forsdyke’s influence has been taken up by some philosophers/historians of science10,11, which is most unfortunate, as it obscures the grounds for the original proposal. Moreover, if influenced by Forsdyke’s proposal, we surely should have and would have acknowledged his paper. Thus not only is Cohn’s account incorrect, but his account makes it appear that Cohn and I were amiss in not giving credit where credit was due. I wrote the following email to Donald Forsythe in 2002.

02:27 PM 2002-11-14 -0600, you wrote:

Dear Donald,

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Thank for your response. I am pretty interested in how ideas develop as youseem to be. The Coombs response [to Donald Forsdyke’s ideas] is not so surprising. Why are scientists not enamored of proposals for clear answers to basic questions, independently of whether they are "correct"? My youthful dream was that discussing science would be like playing really well in a quartet, where the music is divine, and one's own contribution so fused as to be unknown!

I hope it is alright to be straightforward. I had the idea of the one hit/two hit whilst a graduate student in Cambridge, and talked to Crick about it. Mel went to the Brook Lodge symposium about two weeks (?) after I got there. He did not seem at that time to be so involved in the activation/inactivation questions, but he summarized the model in his summation of the meeting. This report of the summation went through much development after the meeting. (I must have the original transcript somewhere). In fact, Mel Cohn did not know many of the experiments on which I had based the model. I thanked Crick for discussions in this first paper. I was quite surprised to read Mel's account of how the two signal model developed in Ann Rev Immunol., because it does not fit in with my memory of how things occurred in major ways. I first learned of your Lancet paper in 1994. This is of course not to deny its insight, just to say I am very surprised about Mel's account.

I will make an effort to visit you if I get close to Kingston. I think we might have a really good time. Peter

The 1968 Nature paper was written fast and under the considerable pressure I have described. I was very soon consciously uneasy with its content. I consider its strengths to be its articulation that antibody precursor cells would be inactivated upon interacting with antigen alone, and that their activation required a second site be recognized by antigen-specific “carrier antibody”. However, its weakness was the envisaged mechanism by which the activation of the antibody precursor cell was “made to be dependent” on the recognition of a second site on the antigen by the carrier antibody. We suggested that the carrier antibody held the antigen in a way that, when the antibody receptor of the antibody precursor cell bound to two antigen molecules, the receptor took on a “distorted” conformation, recognized by an “interaction recognition unit” of the cell. This proposal reflected ideas in my postdoctoral research proposal. The weakness of this model was that it predicted that self-antigens, with repeating epitopes that could distort the antibody receptors on interacting with them, would induce autoantibodies.

Cohn and I very soon realized, as soon as we relaxed (!), the indicated weakness of the model described in our Nature paper. We realized in time that there was really only one secure way of ensuring a requirement that the activation of an antibody precursor cell

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needed the recognition of a second site on the antigen by carrier antibody. Such a recognition would be required if the activation of the antibody precursor cell was obligatorily dependent on events following the recognition of a second site on the antigen by carrier antibody. We envisaged these events could involve the delivery of short-range soluble molecules to the antibody precursor cell, and/or membrane/ membrane interactions between the antibody precursor cell and a cell to which the carrier antibody was attached. These suggestions were highly novel and appeared at first to be “extravagant”; however, they were supported by the subsequent discovery of interleukins and costimulaory systems.

Jacques Monod was a non-resident Fellow of the Salk Institute. Jacques was very interested in immunology and he and Mel knew each other very well. Mel had done a postdoc with Jacques, working on the lac operon. I very much enjoyed Jacques’ scientific company, and I think he enjoyed mine. As a non-resident Fellow, Jacques visited the Salk Institute for a few weeks every year. In our discussions in 1969, Jacques suggested to us the importance, in getting our message over, that we develop a way of expressing the essence of our views independently of the particular details of how they might be realized. We recognized the wisdom of this suggestion. We therefore started to say that the interaction of antigen with the antigen-specific receptor of the antibody precursor cell resulted in the generation of signal 1, and the recognition of a second site on the antigen by carrier antibody resulted in the generation of signal 2. We of course envisaged that the sustained generation of signal 1 alone resulted in inactivation of the antibody precursor cell. Its activation required the generation of signals 1 and signal 2, constituting a two signal model/ mechanism of precursor cell activation. Signal 2 would be mediated by what came to be recognized as interleukins and costimulatory systems. I might add here that Forsdyke suggests10 his model was one of the first two signal models. It was one of the first two signal models, one signal leading to lymphocyte activation and the other signal leading to lymphocyte inactivation, but it was not a model that proposed two distinct signals were required for the activation of lymphocytes.

By 1970, when we wrote our Science article, it was clear the antibody precursor cell was a B cell and that the helper cell a T cell. The Science article was written with the care one likes to bestow upon what one hopes is a significant paper. We thanked Monod for important discussions, and extended our considerations to both B and T cells. This article constitutes a careful justification of the original two signal mechanism/model of lymphocyte activation, written without pressure.

Cohn states6 in 2014: “When, in 1968, I presented the “Two Signal” model, as Goran Moller had dubbed it, to a meeting that was meticulously recorded and published, all bedlam broke out.” I am again surprised. The “Two Signal” model (of lymphocyte activation) was not formulated in 1968. Mel used a two signal formulation in his first presentation at Brook Lodge, in the sense of two different signals, one for the paralysis and one for the activation of lymphocytes, but this formulation was part of current

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concepts, as evident from the Dresser/Mitchison review1. This two signal model does not imply the requirement of two distinct signals for lymphocyte activation. The formulation of the two signal model of lymphocyte activation was a subsequent, important and heuristic development that did not, to my knowledge, involve Goran Moller.

I make one final point for clarity. It became apparent from various observations, made after 1970, that there were at least two different processes contributing to self-tolerance. There was central tolerance, occurring in primary lymphoid organs, by self antigens present at sufficient levels in these organs to cause efficient clonal ablation of their corresponding lymphocytes. Other self antigens are more prevalent in the periphery. Lack of reactivity against such “peripheral” self antigens is achieved by mechanisms of “peripheral” tolerance. The two signal model of lymphocyte activation attempts to address how peripheral tolerance is achieved.

References

1. DW Dresser and NA Mitchison, 1968. The Mechanism of immunological paralysis.Adv Immunol 8:129

2. PA Bretscher and M Cohn, 1970. A Theory of Self-Nonself Discrimination. Science169: 1042

3. M Cohn, 1994. The wisdom of hindsight. Ann Rev Immunol 12: 14. PA Bretscher, 2014. The activation and inactivation of mature CD4 T cells: a case

for peripheral self-nonself discrimination. Scand J Immunol 79: 3485. PA Bretscher, 2014. On the Mechanism Determining the Th1/Th2 Phenotype of an

Immune Response, and its Pertinence to Strategies for the Prevention, and Treatment, of Certain Infectious. Scand J Immunol 79: 361

6. M Cohn, 2015. Thoughts engendered by Bretscher’s Two Step, Two Signal Modelfor a peripheral self-nonself discrimination and the origin of primer effector T- helpers. Scand J Immunol, 81: 87

7. PA Bretscher and M Cohn, 1968. A minimal model for the mechanism of antibodyinduction and paralysis by antigen. Nature 220: 444

8. J Lederberg, 1959. Genes and Antibodies. Science 129: 16499. K Rajewsky and E Rottlander, 1967. Tolerance specificity and the immune

response to lactic dehydrogenase isoenzymes. Cold Spring Harbor Symp Quant Biol 32: 547

10. DR Forsdyke, 2012. Immunology (1955-1975): The Natural Selection Theory, theTwo Signal Hypothesis and Positive Repertoire Selection. Journal of the History of Biology 45: 139

11. SH Podosky and AI Tauber, 1997. The Generation of Diversity. Clonal SelectionTheory and the Rise of Modern Immunology, Cambridge, MA. Harvard University Press

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Section IV: Selected Publications

This section contains a list and description of some of my publications, with the aim of providing a forum for discussion of some current, important issues in immunology (email: peter.bretscher@usask.ca). I include narrative comments to indicate the historical context of the observations made or of the ideas discussed, as well as comments directed at indicating features pertinent to current debate. My aim is to provide a description that is understandable by the interested, non-specialist.

Papers are listed under different headings, indicating their primary area of relevance

Area A: Lymphocyte activation/inactivation and immunological self- nonself discrimination

A1. Bretscher, P.A. and M. Cohn. 1968. Minimal Model for the Mechanism of Antibody Induction and Paralysis by Antigen. Nature. 220:444-8.

Synopsis: It was proposed that lymphocyte activation by an antigen A requires the antigen-mediated cooperation between the resting lymphocyte to be activated and otherA-specific lymphocytes, and that the interaction of the resting lymphocyte with A aloneresults in its inactivation (paralysis in then current terms). It was argued that these proposals incorporate a mechanism by which the immune system can respond to foreign antigens and normally remain unresponsive or “tolerant” towards self-antigens, see A2 below under Explanation of Self-Nonself Discrimination.

A2. Bretscher, P.A. and M. Cohn. 1970. A Theory of Self-Nonself Discrimination. Science. 169:1042-49.

Synopsis: Develops the grounds for the two signal model of lymphocyte activation. This model represents a more correct formulation, according to my mind, of the potentially valid ideas of the 1968 paper (A1 above). It is proposed that an interaction of antigen with a resting lymphocyte, through its antigen-specific receptors, leads to the generation of signal 1, which, when generated alone, leads in time to the inactivation of the lymphocyte, rendering it refractory to activation.

Activation was envisaged to involve the obligatory generation of this signal 1, and a second signal, signal 2, delivered directly or indirectly following the recognition of a second site on the antigen by the cooperating or “helper” lymphocyte, see the Figure 1. (Note that terminology was then different from now. It was envisaged that thymus derived cells helped via the production of a particular class of antibody called “carrier” antibody.) Thymus derived T cells were identified as the cells that cooperate with B cells and allow the B cells to divide and produce humoral antibody. It was explicitly predicted that the antigen-dependent activation of helper T cells (e.g. resting CD4 T cells in

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current terms) also requires similar antigen-mediated cooperation between these specific T cells and other specific “helper” T cells (see A5 and A6 below for tests of this prediction).

Figure 1 The original Two Signal Model of lymphocyte activation

Figure 2 Explanation of self-nonself discrimination according to the two signal lymphocyte model of lymphocyte activation. For explanation, see text.

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Explanation of self-nonself discrimination

The originators of the Clonal Selection Theory (Burnet, Jerne, Lederberg and Talmage) were preoccupied not only with how the genes encoding antibody chains were generated, but how the induction of anti-self reactivity could be prevented, see, for example, the classic and incomparably insightful paper by J. Lederberg, entitled Genes and Antibodies, Science, 129:1649 (1959). These individuals envisaged that the ability of the immune system to tolerate self- and respond to foreign antigens must rely on the fact that (most) self-antigens, in contrast to foreign antigens, appear early in development and are continuously present thereafter. This central idea is neglected or rejected by much contemporary analysis (as discussed under The HistoricalPostulate in A9 below). The two signal model accounted for peripheral self-tolerancethus, see Figure 2 above: lymphocytes specific for a continuously present self-antigen, S, are inactivated as they are generated, one or a few at a time. Lymphocytes specific for a foreign antigen, F, will accumulate in the absence of F. Once F impinges upon the immune system, F can mediate the interaction between the accumulated anti-F lymphocytes that is required to generate an anti-F immune response.

Current context: It was not appreciated, at the time this 1970 paper was published, that there are (at least) two levels of tolerance, namely central and peripheral tolerance. We now know that self-antigens, present in the primary lymphoid organs where lymphocytes are generated, i.e. the bone marrow for B cells and the thymus for T cells, can interact with their specific lymphocytes and inactivate them. This is called central tolerance. We also know that T cells are generated in the thymus specific for “peripheral”, organ specific antigens, not present in the thymus at levels to cause reliable the reliable inactivation of these lymphocytes. These T cells emigrate to the periphery and secondary lymphoid organs, where they are kept in check by mechanisms collectively referred to as “peripheral tolerance”. For example, a break- down in peripheral tolerance can lead to an immune response against the insulin- producing β-islet cells of the pancreas, resulting in diabetes. It seemed that, once the existence of both central and of peripheral tolerance was recognized, that the two signal model provided an explanation of peripheral tolerance.

A3. Bretscher, P.A. 1972. The Control of Humoral and Associative Antibody Synthesis. In G. Moller (ed.), Lymphocyte Activation by Mitogens, Transplantation Reviews, 11:217-267.

Synopsis: A general analysis of possible models of lymphocyte activation/inactivation in the context of achieving peripheral self-nonself discrimination is outlined. This conceptual analysis, together with references to pertinent observations, leads to the two signal model of lymphocyte activation. The antigen-recognition molecule made by T helper cells is here referred to not as the T cell receptor (TcR), as in current usage, or as “carrier antibody” (as in A2), but as “associative antibody”. The paper also includes a

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simple quantitative formulation of the two signal model. This formulation considers how the generation of signal 1 and signal 2 might depend on a variety of variables, such as antigen dose and the number/density of specific helper T cells. This quantitative formulation was central to the development of A Theory of Immune Class Regulation, see B1 below.

A4. Bretscher, P.A. 1975. The Two Signal Model for B Cell Induction. In G. Moller (ed.), Concepts of B Lymphocyte Activation, Transplant. Reviews, 23:37-48.This volume represents the then current views of different investigators as to therequirements for B cell activation. I attempt to critically examine the quality of the evidence, then available, for the two signal model, as it applies to B cells.

A5. Tucker, M.J. and P.A. Bretscher. 1982. The T Cells Cooperating in the Induction of Delayed-type Hypersensitivity act via the Linked Recognition of Antigenic Determinants. Journal of Experimental Medicine. 155:1037-49.

Synopsis: Tests of a critical prediction of the two signal model of lymphocyte activation as it pertains to T cells. This paper shows that the generation of (CD4+) T cells, able to mediate DTH and specific for an antigen A, could be helped by (CD4+) T cells specific for a non-crossreacting antigen B in the presence of the conjugate A-B, but not when A and B are present but unconjugated. This form of recognition between interacting lymphocytes is referred to as the recognition of linked epitopes.

A6. Bretscher, P.A. 1986. The Primary Induction of Helper T Cells Involves the Linked Recognition of Antigenic Determinants. J of Immunology. 137:2726-2733.

Synopsis: This paper demonstrates the existence of a cascade of T cell/T cell interactions in the generation of helper T cells. Each step in the cascade involves theantigen-mediated interaction of T cells with other T cells. Each of these interactionsoccurs via the recognition of linked epitopes by the interacting T cells. See synopsis of A5 above for an operational definition of recognition of linked epitopes, and A8 below for a possible mechanism by which such an operational recognition could be realized.

A7. Bretscher. P.A. 1992. The Two-signal Model of Lymphocyte Activation Twenty-one Years Later. Immunology Today 13:74-76.

Synopsis: Much evidence supports the two signal model, in that CD4 T helper cells are required to facilitate the antigen-dependent activation of most B cells and at least some CD8 T cells. In the absence of such CD4 T helper cells, antigen can inactivate the B cells and CD8 T cells, via the generation of signal 1 alone. These findings are consistent with the envisaged role of CD4 T helper cells as the guardian over the activation/ inactivation of other lymphocytes. The requirements for the activation/inactivation of CD4 T helper cells thus seems ever more central.

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Evidence is reviewed showing that the activation of CD4 T cells most probably requires two signals. Signal 1 is generated when the T cell receptor (TcR) binds a peptide/MHC class II molecule complex on the antigen presenting cell (APC). The CD4 T cell is operationally inactivated when this signal is generated alone. Evidence supports the suggestion that CD4 T cell activation requires the T cell to receive a second signal, generated when a receptor on its surface binds to a “costimulatory molecule” on the APC. Both the costimulatory molecule and the T cell receptor recognizing it are envisaged in one model to be constitutively present on mature APC and on CD4 T cells respectively. According to some other models, the costimulatory molecule on the APC is inducible under some circumstances (discussed under A8 and A9). The model in Figure 3, in which the costimulatory molecule is constitutively expressed, is therefore conveniently referred to as the Constitutive Model.

Figure 3 The Constitutive Model of CD4 T cell activation

I found this constitutive model problematical as a description of the activation/ inactivation of CD4 T cells. The description conforms very nearly to what was envisaged in the “original” Two Signal Model, see A2 above and Figure 2. However, although two signals are envisaged as being required for CD4 T cell activation in both the constitutive model and the “original” Two Signal Model of CD4 T cell activation, the nature of the second signal is critically different in these two models. In the original two signal model, signal 2 was generated and delivered following the recognition of a second site on the antigen by an antigen-specific lymphocyte. In the constitutive model, the second signal is delivered by the APC. The APC, being non-specific, will deliver signal 2 to CD4 T cells specific for self or foreign peptides alike. APC such as dendritic cells cannot distinguish between self and foreign peptides by themselves. It would appear such APC cannot deliver signal 2 in a manner that ensures the inactivation of CD4 T cells specific for peripheral self and the activation of CD4 T cells specific for foreign peptides. Also, this model ignores the evidence for T cell cooperation in the activation of T helper cells (e.g. see A5 & A6 above). This paper expresses the anguish of this predicament.

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A8. P.A. Bretscher. 1999. A two step, two signal model for the primary activation of precursor helper T cells. Proc. Natl. Acad. Sci, 96:185-190.

Figure 4 The Two Step, Two Signal Model for the activation of precursor T helper (pTh) cells

Context and synopsis: It was generally accepted, at the time this model was proposed, that the “original” two signal model of lymphocyte activation (see A2 above) applied to most B cells and at least some CD8 T cells (see A7 above). Thus CD4 T helper cells were required for antigen to activate most B cells and at least some CD8 T cells, and antigen could inactivate these B and CD8 T cells in the absence of “helper T cells”. Thus CD4 T cells controlled, in large measure, the fate of other lymphocytes.

This two step, two signal model, see Figure 4, was an attempt to account for the pertinent, extant observations, and to resolve 5 problems.

(i) Addressing the problem associated with the constitutive model Observations support the idea that mature macrophages and/or dendritic cells constitutively express (B7) costimulatory molecules, and that antigen presented by these antigen presenting cells (APC) can induce immune responses. These observations led to the “constitutive model” (see A7). However, this constitutive model cannot account for peripheral CD4 T cell self-nonself discrimination (see A7 above). Suggested resolution: This “constitutive

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model” describes the first step in the activation of naïve CD4 T cells, or precursor T helper cells (pTh) cells, thus accounting for most of the observations supporting the constitutive model. The first step is envisaged to result in the proliferation of the pTh cell, but these progeny are postulated to die or become anergic unless they successfully complete the second step. The second step is obligatory if these progeny are to give rise to effector Th (eTh) cells. The second step incorporates a mechanism of self- nonself discrimination, see (ii) below.

(ii) How to achieve peripheral self-nonself discrimination for CD4 T cells? The “original” two signal model (A2) proposed that (peripheral CD4) T cell tolerance could be achieved if inactivation occurred when antigen acts on a single CD4 T cell, and if the activation of a CD4 T cell required collaboration with other CD4 T cells. These “rules” incorporate a mechanism of self-nonself discrimination, as explained in A2 above. These “rules” are embodied in step two, see Figure 4, of the Two Step, Two Signal Model. Activation to yield functional effector Th (eTh) only occurs if the second step is completed, requiring the presence of other CD4 T cells (eTh) cells specific for the antigen.

(iii) The scarcity problem A problem recognized when the original two signal model was formulated arose from the fact that antigen-specific lymphocytes are expected to be very scarce in an “unprimed” animal, and yet the activation of pTh cells was envisaged to require the antigen-mediated interaction between such scarce Th cells. The first step results in an expansion of pTh cells, making these cells less scarce; this may favor their interaction with other scarce cells, thereby contributing to solving the scarcity problem.

(iv) The priming problem Biology contains many “priming” problems, the major one being the origin of life. We know life can beget life, but how did the first life arise? We need ribosomes to make ribosomes, but how did the first ribosome arise? We need, according to the Two Step, Two Signal Model, activated, i.e. effector Th (eTh) cells, to activate pTh cells to yield more eTh cells. How does the first eTh cell arise? We call this the priming problem. In this context, we first note that there is evidence for a requirement for eTh cells to initiate immune responses, as reviewed in this paper. Secondly, various ways of “solving” the priming problem are discussed in the full text referred to in A3 above.

(v) How can the recognition of linked epitopes between interacting CD4 T cells be achieved? Observations demonstrate that the antigen-mediated interaction between a pTh cell and the eTh, required to activate the pTh cell, occurs through a mechanism involving the recognition of linked epitopes (see A5 and A6). This was a critical prediction of the “original” two signal model. We first consider why linked recognition is so critical for understanding CD4 T cell peripheral tolerance. Then we discuss potential mechanisms of linked recognition.

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Consider the situation where two pTh cells are newly generated, one specific for a peripheral self-antigen, S, and the other specific for a foreign antigen F, and where both F and S are present. According to the model proposed, there must be eTh cells specific for F in order for F to be able to activate the anti-F pTh cell. This would also result in the activation of the anti-S pTh unless the interaction between the anti-F pTh cell and the anti-F eTh cell required the recognition of linked epitopes. Recognition of linked epitopes is required to ensure that the process of activating pTh cells specific for foreign antigens does not inevitably interfere with the inactivation of pTh cells specific for peripheral self antigens. We refer to this desirable situation as the Principle of Non-Interference. Versions of two signal models that violate this principle seem to have thevery same characteristic that makes third signal models (see A3) so unappealing: initiation of an immune to foreign antigens provides circumstances under which immune responses to peripheral self antigens would inevitably occur. Moreover, we appear to be able to rest in peace, as observation demonstrates a requirement for linked recognition!

To imagine a mechanism responsible for linked recognition has seemed problematical in the context of modern knowledge: CD4 T cells recognize a peptide, derived from the nominal antigen, bound to an MHC class II molecule. Consider a protein antigen Q that is processed into peptides q1,q2, q3,…..qn, recognized by CD4 T cells in the context of the host’s class II MHC molecules. How can we imagine a mechanism by which an anti- q3 pTh cell can only be helped by an anti-q2 eTh cell, when Q must be broken down into q peptides before either Th cell can recognize its peptide? It seems the linkage, deemed so important, must be destroyed before the Th cells can interact.

I have been able to come up with only one plausible solution to resolve this issue. It consists of the proposition that the antigen presenting cell in the second step has to be an antigen-specific B cell. Consider the interaction between two CD4 T cells, one specific for the nominal antigen Q and the other for R, where Q and R do not crossreact. This CD4 T cell interaction would be mediated only by the Q-R conjugate. The antibody receptors of a B cell, specific for Q or for R, will be able bind the conjugate Q-R, and present peptides derived from processing Q and R. Thus a specific B cell could mediate the interaction between two CD4 T cells, one specific for Q and the other for R. Such an interaction would not take place in the absence of the Q-R conjugate and in the presence of separate Q and R molecules. The proposal that an antigen-specific B cell acts as the APC thus explains why the interaction between the CD4 T cells requires the recognition of linked antigenic epitopes. We suppose the requirement for a B cell can be assured by its expression, when activated, of a particular constellation of costimulatory molecules (and/or cytokines) required to activate the step one primed pTh cells through the second step. We have tested various predictions of this model, see A10.

A9. Bretscher, P.A., 2000. Current models for peripheral tolerance and the "classical historical postulate", Seminars in Immunology, M Cohn and R Langman, Editors, 12:221-229, 273-276, 325-329

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This volume consists of initial statements by the invited participants, which were then made available to the other participants for their comments, and so on for a total of three rounds.

Synopsis The originators of Clonal Selection Theory envisaged that the ability of the immune system to tolerate self and respond to foreign antigens must rely on the fact that (most) self antigens, in contrast to foreign antigens, appear early in development and are continuously present thereafter. Consider, from this perspective, what must happen when two CD4 T cells, newly generated in the thymus, and one being specific for a peripheral self antigen, S, and the other for a foreign antigen F, emigrate to the periphery and encounter an environment containing both F and S. We would hope that S and F can inactivate and activate their respective pTh cells. Moreover, these different consequences should reflect the fact that S, in contrast to F, has been continuously present during the past history of the individual whose immune system we are considering. We refer to this idea, put forward by the originators of Clonal Selection Theory, as the “classical historical postulate”.

The two step, two signal model of pTh activation provides an explanation of peripheral, CD4 T cell tolerance consistent with the classical historical postulate, see A2. Janeway proposes that the initiation of an immune response requires the activation of innate defense mechanisms through their recognition of pathogen-associated molecular patterns (PAMPs) of microbial pathogens (infectious entities), and Matzinger envisages that initiation requires the generation of a “danger signal”. According to these “third signal” models, the most widely entertained models of the day, the third signal activates the APC to generate an effective costimulatory signal; antigen is envisaged to inactivate pTh cells in the absence of costimulation, i.e. in the absence of signal 3.

These third signal models postulate that only the circumstances at a particular time determine whether antigen activates or inactivates pTh cells at this time, i.e. the critical circumstance is whether a third signal is generated at this time, and the past history of the individual with respect to exposure to the antigen in question is not central or even relevant. These third signal models thus violate the classical historical postulate. I personally believe that activation of innate defense mechanisms, the occurrence of inflammation, or events arising from circumstances covered by the rather flexible concept of danger, can significantly affect immune responses. It is implausible to me, however, that third signals play the critical and universal role ascribed to them in third signal models, as these signals are not antigen-specific. I also discuss in this contribution observations difficult to reconcile with these third signal models.

A10 Testing predictions of the Two Step, Two Signal Model (A8)

A10a. Power CA, Grand CL, Ismail N, Peters NC, Yurkowski DP, et al. (1999) Avalid ELISPOT assay for enumeration of ex vivo, antigen-specific, IFN-gamma producing T cells. J Immunol Methods 227: 99–107.

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A10b Peters NC, Hamilton DH, Bretscher PA (2005) Analysis of cytokine-producing Th cells from hen egg lysozyme-immunized mice reveals large numbers specific for ‘‘cryptic’’ peptides and different repertoires among different Th populations. Eur J Immunol 35: 56–65.

A10c Peters NC, Kroeger DR, Mickelwright S, Bretscher PA (2009) CD4 T cellcooperation is required for the in vivo activation of CD4 T cells. Int Immunol 21: 1213–1224.

A10d Kroeger DR, Rudulier CD, Peters NC, Bretscher PA (2012) Directdemonstration of CD4 T cell cooperation in the primary in vivo generation of CD4 effector T cells. Int Immunol. 2012/04/25 ed.

A10e David R. Kroeger, Christopher D. Rudulier, Peter A. Bretscher (2013)Antigen Presenting B Cells Facilitate CD4 T Cell Cooperation Resulting in Enhanced Generation of Effector and Memory CD4 T Cells, PLoS ONE 8: e77346

A10f Peter A Bretscher The Activation and Inactivation of Mature CD4 T cells: ACase for Peripheral Self–Nonself Discrimination Scand J Immunol 79: 348

A10g Ghassan A. Al-Yassin and Peter A. Bretscher (2018) Does T Cell ActivationRequire a Quorum of Lymphocytes? J Immunol 201: 2855

A10h Peter A Bretscher (2019) The history of the two‐signal model of lymphocyteactivation: A personal perspective Scan J Immunol 89:e12762

Synopsis. We describe the above 8 papers together, as they are all pertinent to testing predictions and discussing the plausibility of the Two Step, Two Signal Model of CD4 T cell activation (A9)

We set out to test whether the in vivo activation of CD4 T cells requires CD4 Tcell collaboration on immunization with antigen but without microbial adjuvant. Our previous studies on CD4 T cell cooperation had all been conducted in vitro. We judged we would need a very sensitive assay to detect activated CD4 T cells, and decided to employ the ELISPOT assay, developed by others to detect single, antigen-specific, cytokine-producing cells. We established the assay in the lab, but found that the number of “antigen-dependent spots detected”, corresponding to antigen-specific cytokine- producing cells, dropped much more rapidly than the dilution of spleen cells plated. We

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found that, if we supplemented the sensitized spleen cells with unprimed spleen cells, so the total number of cells plated was a constant, including the APC involved in the antigen-dependent stimulation of CD4 T cells, the sensitivity of the assay increased and the number of spots developed was now linear with the number of sensitized cells plated. This paper thus describes how the standard ELISPOT assay can be modified to validly enumerate antigen-specific cytokine producing cells (A10a).

We wanted to define the peptide specificity of all the activated CD4 T cells generated, upon in vivo immunization with a protein antigen, and we chose hen egg lysozyme (HEL) as the antigen. We thought that such a definition might allow us to examine whether T cells specific for one peptide helped in the activation of T cells recognizing another peptide, on immunization with HEL. We employed the ELISPOT assay to define the peptide specificity, in both CBA and BALB/c mice, of the CD4 T cells generated upon immunizing with HEL. We validated our analysis by showing that the sum of the number of CD4 T cells generated to non-overlapping HEL peptides was equal to the number of CD4 T cells generated against the whole protein, in a spleen cell population from a mouse immunized to HEL (A10b). Important for our further studies was the fact that, in BALB/c mice, half the CD4 T cells, generated upon immunization with HEL, were specific for the “major peptide”, and the rest for “minor peptides” we identified.

HEL is a small protein, and not very immunogenic. We envisioned that CD4 Tcells specific for HEL were sparse. We hypothesized that, if we ablated the CD4 T cells specific for the “major peptide” before immunizing with HEL, not only would the generation of the CD4 T cells specific for the major peptide be ablated, but the CD4 T cell response to minor peptides would be considerably diminished; we envisaged that the activation of CD4 T cells specific for these minor peptides were most probably facilitated, in a mouse with a normal complement of CD4 T cells specific the major peptide, by major peptide-specific CD4 T cells. We tested this prediction. We showed that when we ablated the CD4 T cells specific for the major peptide, the CD4 T cell responses to both the major and minor peptides were dramatically diminished following a challenge with HEL (A10c).

Our next study demonstrated that CD4 T cells specific for a peptide of ovalbumin (OVA) could facilitate the activation of effector CD4 T cells specific for the major HEL peptide (A10d). We further examined the nature of the antigen-presenting cell (APC) mediating this cooperation. In order to approach the nature of the APC, we gave the peptide antigens in the form of APC loaded in vitro with these peptides. We successfully tested two main predictions. Firstly, a mixture of different APC types could only facilitate the cooperation between the CD4 T cells if the APC were loaded in vitro with both peptides; a mixture of two populations of APC, each loaded with one of the two peptides, did not facilitate the cooperation. This study tests the idea that the two CD4 T cells interact with each other via an APC that presents both peptides. We also tested

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the ability of dendritic cells (DC) and B cells, loaded with the two peptides, to mediate the cooperation between the two CD4 T cell populations. We found that loaded B cells, but not loaded DC, could mediate the cooperation (A10e). This study confirms a strong prediction of The Two Sep, Two Signal Model (A8).

The Two Signal Model of Lymphocyte Activation is widely accepted by the immunological community in the context of the activation of B cells and CD8 T cells, but not in the context of CD4 T cells. The most widely entertained, contemporary models for the activation of CD4 T cells are the Danger and PAMP (pathogen-associated molecular pattern) models. Moreover, since the publication of our two signal model in Science, in 1970 (A2), several variations of the original model have arisen. I felt and feel most of these variations undermine the coherence of the model as originally proposed. I wrote an article in 2014, 44 years after the model was first published, to re-examine and then support the essential ideas of the 1970 two signal model, both in the context of CD4 T cells, and of then contemporary information and ideas (A10f). It also seems that contemporary studies are almost exclusively undertaken at the molecular and/or cellular level, ignoring past and current studies and ideas that are best described as at the level of the system. I shall shortly explain what I mean by “at the level of the system.” My PhD graduate student, Ghassan Al-Yassin, and I wrote a paper in support of the view that older studies, many at the level of the system, conflict with the Danger and PAMP models. I will illustrate our view with one example. William Weigle published several papers in the early 1960s, employing rabbits unresponsive to bovine serum albumin (BSA), following the administration of massive doses of this antigen on the first few days of life. The rabbits, as young adults, would not produce antibody on challenge with BSA, but they produced antibody to human serum albumin (HSA) on immunization with this antigen. Some of the antibody so raised to HSA bound to BSA. These are some of the classical experiments showing that immunization, with antigens cross-reacting with the antigen the animal is unresponsive to, can (sometimes) break the unresponsive state. There was, at the time, no cellular or molecular interpretation by Weigle, or anyone else, as to how this might occur. These studies were in this sense carried out at the level of the system. Such studies would not be considered important today by most immunologists, as they do not lead, as described, to a picture of what is happening in terms of cells and molecules. However, we argued that this and related studies really supported the proposition that the activation of helper T cells requires lymphocyte cooperation. We describe many older studies, many at the level of the system, that are difficult to reconcile with the Danger and PAMP Models, but support the idea the activation of T helper cells requires T cell collaboration (A10g).

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Area B: Immune class regulation

B1a. Bretscher, P.A. 1974. On the Control Between Cell-mediated, IgM and IgG Immunity. Cellular Immunology. 13:171-195

B1b. Bretscher, P.A. 1981. The Significance and Mechanisms of the Cellular Regulation of the Immune Response. Theoretical Immunology Symposium of the 64th Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, California, April 16, 1980, Federation Proceedings, 40:1473-8.

Synopsis These two papers provided a conceptual framework that attempts to describe how antigen interacts with lymphocytes to determine the class of immunity induced. At the time, the expression of delayed-type hypersensitivity (DTH) was used to indicate the generation of a cell-mediated response, and IgG antibody production to indicate humoral immunity. Much experimental work of my students and myself over decades has been directed at testing the theory I proposed here, and using it in attempts to achieve practical aims. I describe the theory in a manner to highlight predictions highly characteristic of it. I refer for convenience to the theory as a Theory of Immune Class Regulation. (It should be noted that the MHC-restricted recognition of antigen by T cells had not been discovered in 1974.) This theory is easily recast now, taking into account this feature of T cell recognition, by employing The Two Step, Two Signal Model, see B6 below, as a context for discussing ideas on immune class regulation. However, the original description and that given here is made within the context of the assumption that T cells recognize antigen in a similar way as antibody. It is convenient to consider three different types of consideration that were incorporated into the Theory of Immune Class Regulation.

(i) The mechanism of exclusion between cell-mediated and humoral immunity

It had been known for decades that different circumstances of immunization give rise to different kinds of immunity, i.e. either to predominant cell-mediated or humoral immunity, or to a mixed response. Then, in the mid 60s, a number of reports showed that the generation of humoral immunity precluded the subsequent generation of cell- mediated immunity in the form of DTH. Asherson and Stone christened this phenomenon with the name “immune deviation”. We now refer to this as “humoral immune deviation” to indicate the nature of the deviation. Chris Parish and colleagues described the related phenomenon of “cell-mediated immune deviation” in the early 70s. They repeatedly immunized rats with a protein antigen, in a manner that lead to cell- mediated immunity alone. Such sensitized rats, when given a challenge of the protein that generated a brisk antibody response in naïve rats, produced little antibody but the rats expressed DTH, i.e. expressed cell-mediated immunity to the protein. This represents a state of cell-mediated immune deviation. The phenomena of cell-mediated

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and humoral immune deviation show that the immune response to a particular antigen can become locked into a particular mode, either humoral or cell-mediated. It was proposed in the Theory of Immune Class Regulation, partly on the basis of observation, partly on conceptual grounds, that the operation of distinct “suppressor” T cells were responsible for cell-mediated and humoral immune deviation. It was envisaged that chronic immunization, giving rise to a predominant cell mediated response, also gave rise to the generation of antigen-specific T cells that suppress or inhibit the corresponding humoral response. We referred to these inhibitory T cells as TsAb cells. It was also envisaged that the generation of a predominant antibody response gave rise to antigen-specific T cells that suppress or inhibit the corresponding DTH response. We refer to such cells as TsDTH cells see Figure 5.

Figure 5 How TsAb and TsDTH are envisaged to account for the tendency for exclusivity between DTH and antibody responses

(ii) The Pathophysiological Significance of Immune Class Regulation It is perhaps initially surprising that the immune system incorporates decision making processes controlling which, of the diverse means it has of fighting foreign invaders, to employ. One might imagine that, once the “decision” has been made to fight a foreign invader, it would be advantageous to employ all means available. Observation shows this not to be so. Why?

A series of observations are suggestive of a possible answer. Antibody is ineffective against antigens that have few, or a low density (if a cell), of foreign sites recognized by the antibody. One example is the IgG antibody-dependent complement-mediated lysis of foreign red blood cells. A few IgG molecules, bound close together on the cell surface of the red cell, forming an IgG complex, are required to initiate the complement cascade. This initiation leads to the local generation of a hydrophobic “donut” that inserts itself into the red cell membrane, leading to the lysis of the red cell. Antibody can

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similarly attack bacteria. The requirement for an IgG complex to activate complement means that several 100,000 IgG molecules have to bind to one red cell in order for there to be a 50% chance of an effective IgG complex to form. Thus antibody is ineffective against cells with few foreign sites. Antibody is usually ineffective, for example, against cancer cells. However, cancer cells can be lysed by the cytotoxic T lymphocytes (CTL), an expression of cell-mediated immunity. I suggested that antibody is not normally induced to soluble antigens/cells with few foreign sites. This suggestion was made on the basis of observations by others, and because, in addition, it made sense in that it appeared antibody is likely to be ineffective in damaging such foreign invaders. Moreover, the presence of antibody might block the effectiveness of the cell-mediated response. This consideration provides a rationalization for why antibody production is often inhibited during the course of a cell-mediated response. However, antigens with many foreign sites can be attacked effectively by antibody-dependent mechanisms and can induce antibody. It is explained in this paper why the inhibition of cell-mediated immunity, during the course of a strong antibody response, can be rationalized as a means of minimizing the damaging effects of any autoreactivity induced.

Two potential insights follow from these considerations. Sometimes ineffective antibody is generated against tumors, or against pathogen-infected cells bearing pathogens that cause chronic disease (e.g. HIV-1 leading to AIDS, Mycobacterium tuberculosis leading to tuberculosis), resulting in stable or progressive disease. It was suggested that vaccination/ treatment might be achieved by ensuring a predominant cell mediated response. Approaches to achieve such ends are discussed in section C. It was also anticipated that humoral, antibody-mediated autoreactivity might sometimes be benign, whereas cell-mediated autoreactivity to the same antigens would more likely cause damage and hence autoimmune disease. This also seems to be sometimes the case.

(iii) The decision criterion controlling the cell-mediated/humoral nature of the immune response: the necessity for quantitative considerations

What determines whether antigen preferentially generates a cell-mediated (with associated TsAb cells) or an antibody, humoral response (with associated TsDTH cells)? Firstly, it was argued that TsAb and TsDTH cells probably inhibit each other’s generation so that, once one class of cell becomes a bit dominating, it inhibits its adversary and so tends to become more dominant, see Figure 5. It was also argued that if antigens with few foreign sites (or a low density of such sites for cellular antigens) could only be contained by cell-mediated immunity, the decision criterion should ensure that such antigens normally only generate a cell-mediated response. It was encouraging that Pearson and Raffel had previously made the generalization, based upon observations, that antigens with few foreign sites could induce DTH but not antibody. In addition, although an antigen with many foreign sites could induce antibody formation, the dose of antigen administered was known to be critical. The administration of low doses of such antigens gave rise to exclusive cell-mediated immunity, and of higher doses to antibody production, following an initial, exclusive cell-mediated phase.

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I proposed that the immune system uses the number of antigen-specific CD4 T cells that are present to gauge an antigen’s degree of foreignness. This proposal assumes that there are no or relatively few CD4 T cells specific for self-antigens. This is an assumption with which most would now concur. I also assumed that the activation of (CD4) precursor T helper (pTh) cells requires other CD4 Th cells, according to the two signal model or more contemporary formulations (see A2, A3, A5-A8 above). The proposal that weak antigen-mediated CD4 T cell/ CD4 T cell interactions would lead to the generation of relatively few signal 2s, and to the activation of the pTh cell to expresscell-mediated immunity (or give rise to Th1 cells in modern terminology) and thatstronger interactions would lead to the generation of more signal 2s, and the generation of antibody (and of Th2 cells in modern terminology), seemed attractive. In this case, the threshold number of (helper T cell dependent) signal 2s required to generate cell- mediated (Th1 cells) and antibody (Th2 cells) responses would be different. This hypothesis is currently referred to as the Threshold Hypothesis for the nature of the decision criterion controlling the Th1/Th2 nature of the immune response. I argued in favor of the Threshold Hypothesis on the following grounds: (a) It made physiological sense (see (ii) immediately above). (b) It accounted for the fact that antigens with few foreign sites could only induce cell-mediated, Th1 responses. (c) It explained why

Figure 6 For explanation, see accompanying text

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administration of antigens with many foreign sites, for which there were more CD4 T cells, could generate antibody responses and Th2 cells. (d) It explained why administration of low doses of such antigens with many foreign sites only induce cell- mediated, Th1 responses, as strong antigen-mediated interaction of the CD4 T cells cannot take place when antigen is very limiting; under these circumstances the signal 2s are not optimally generated or delivered. (e) It was known that antigen activated CD4 helper T cells. Thus antigen-mediated CD4 T cell/CD4 T cell interactions should improve with time, explaining how the immune response can evolve into an antibody, Th2 mode after passing through an exclusive cell-mediated, Th1 phase, see NOTE in Figure 6.

Finally, it was predicted that all cell interactions between antigen specific lymphocytes and involved in immune class regulation were mediated via the recognition of linked epitopes (see A5 for definition). This mechanism ensures the specificity of regulation of the class of immunity is specific and has important consequences, see B5 below.

B2a. Ramshaw, I.A., P.A. Bretscher and C.R. Parish. 1976. Regulation of the Immune Response I. Suppression of Delayed-type Hypersensitivity by T cells for Mice Expressing Humoral Immunity. European J Immunology. 6:674-79.

B2b. Ramshaw, I.A., P.A. Bretscher and C.R. Parish. 1977. Regulation of the Immune Response II. Repressor T Cells in Cyclophosphamide-induced Tolerant Mice. European Journal of Immunology. 7:180-185;

B2c. Ramshaw, I.A., I.F.C. McKenzie, P.A. Bretscher and C.R. Parish. 1977. Discrimination of Suppressor T Cells of Humoral and Cell-mediated Immunity by Anti-Ly and Anti-Ia Sera. Cellular Immunology.

Synopsis These three papers tested the predictions that cell-mediated and humoral immune deviation were mediated by TsAb and TsDTH cells respectively (see B1 for a definition of these cells) and established some of the characteristics of these cells.

Mice were induced to produce a strong antibody response to an antigen A.These mice could no longer be induced to express cell-mediated immunity in the form of DTH to a challenge of A that generated a DTH response in naïve mice. They were therefore in a state of humoral immune deviation. We showed, by transfer studies, that such mice contained in their spleen Ly1+Ly2- (CD4+ in current terms) T cells able to inhibit the generation of DTH in an antigen-specific manner. In addition, we showed that these TsDTH cells acted by linked recognition. Thus lymphocytes from mice made humorally immune to foreign haemocyanin (Hm) could inhibit the DTH response to horse red blood cells (HRBC), in the presence of Hm chemically attached to HRBC(Hm-HRBC), but not when Hm was present but not coupled to HRBC (i.e. in thepresence of both HRBC and Hm coupled to mouse red blood cells (Hm-MRBC))(B2a).

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Mice were also induced to express strong cell-mediated immunity to an antigen C. These mice could no longer produce antibody following an antigen challenge that resulted in antibody production in naïve mice. They were therefore locked into a state ofcell-mediated immunity, called a state of cell-mediated immune deviation (see B1). Weshowed, by transfer studies, that such mice contained in their spleen Ly1-Ly2+ (CD8+ in current terms) T cells able to inhibit the antibody response in an antigen-specific manner. In other words, TsAb cells do exist and have a CD4-CD8+ phenotype (B2b).

These experiments defined two classes of regulatory T cell, TsAb and TsDTH cells, that differ in three characteristics. They are generated under different circumstances, they bear different surface markers (B2c), and they inhibit different types of immune response, see Figure 7.

Figure 7 Summary of the experimental findings of B2a, B2b and B2c

We also examined the surface markers on cells mediating DTH. These were Ly1+Ly2- T cells, i.e. CD4 T cells in modern terms. We had therefore defined two classes of CD4 T cell, one that mediates DTH, and another, generated under conditions that result in antibody production, that does not express DTH but acts to inhibit the induction of DTH (B2c). It is natural to associate these two classes of CD4 T cell with the two types of CD4 T cell clones, Th1 and Th2, characterized considerably later by Mosmann and Coffman, on the basis of the lymphokine spectrum they express and the biological activities they mediate.

In the contemporary context, it seems to be almost universally held that Th1 cells inhibit the generation of Th2 cells and vice versa. I feel the experimental basis for the inhibition of Th2 responses by Th1 cells not to be impeccable. This inference comes from fairly artificial systems. Indeed, some of the evidence that led to a Theory of Immune Class Regulation was that the three conditions under which exclusive DTH is induced are just those giving rise to the generation of CD8 TsAb cells (see B1).

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However, these experiments by others were done in diverse systems. No one, as far as I know, had looked at a situation where both DTH was induced and the animal was unresponsive for the induction of antibody, and had tried to define the nature of the TsAb cells responsible, until we did so. We found the TsAb to be CD8 T cells. I feel this question of whether CD4 or CD8 T cells, or both cell types, inhibit the generation of Th2 cells, is still unresolved (see B4).

B3a Bretscher, P.A. 1979. In-vitro Induction of delayed-type hypersensitivity. European Journal of Immunology. 9:311-16

B3b Bretscher, P.A. 1983. In Vitro Analysis of the Cellular Interactions Between Unprimed Lymphocytes Responsible for Determining the Class of Response an Antigen Induces: Specific T Cells Switch a Cell-mediated Response to a Humoral One. Journal of Immunology. 131:1103-7

B3c. Bretscher, P.A. 1983. Regulation of the Immune Response Induced by Antigen. I. Specific T Cells Swith the In-vivo Response from a Cell-mediated to a Humoral Mode. Cellular Immunology. 81:345-56.

Synopsis We wished to develop in vitro and in vivo systems in which we could generate both primary cell-mediated and antibody responses. Such systems should allow one to alter various experimental parameters to determine how such parameters affect the class of immunity generated. Such systems should allow one to test various hypotheses as to the nature of the decision criterion controlling the cell-mediated/humoral (or Th1/Th2) nature of the immune response.

We first found conditions in vitro under which we could generate primary DTH responses, involving a medium density of unprimed spleen cells. We then found that, holding the amount of antigen per culture well constant, that low densities of unprimed spleen cells did not support the generation, during six days of culture, of cells mediating DTH or of cells producing antibody. Similar cultures but consisting of a medium density of unprimed spleen cells supported the generation of DTH-mediating T cells but few cells producing antibody. Similar cultures but consisting of a high density of unprimed spleen cells supported substantial antibody production but not substantial generation of DTH mediating cells. In other experiments, lethally irradiated mice were given a constant amount of antigen and reconstituted with different numbers of unprimed spleen cells. Mice given a medium number of spleen cells supported the generation of DTH- mediating cells, and little antibody production. Mice reconstituted with greater numbers of unprimed spleen cells supported strong IgG antibody responses and the generation of DTH-mediating cells was poor. The in vitro and in vivo findings were remarkably parallel, giving us confidence in the validity of the in vitro system. Higher densities or numbers of unprimed spleen cells were required to generate antibody than DTH responses. Further observations showed that it was the greater number of T cells in

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higher densities/numbers of unprimed spleen cells, rather than the greater number of APC, that was required to obtain antibody production and a decreased generation ofDTH-mediating cells. We shall return to this system when discussing a more recentstudy employing more sensitive techniques (B6 below), and discuss its relevance to critical testing of the Threshold Hypothesis (see B1 above).

B4a S.A. LeCercq and P.A. Bretscher. 1987. T Cells Expressing Delayed-typeHyper-sensitivity can be Derived from a Humorally Immune Lymphocyte Population. Eur. J. Immunol. 17, 949-954.

B4b Tuttosi, S. and P.A. Bretscher. 1992. Antigen-specific CD8+ T Cells Switch the Immune Response Induced by Antigen from an IgG to a Cell-mediated Mode. J. Immunol. 148:397-403.

Synosis In vivo experiments show that immune responses can become locked into humoral or cell-mediated modes (see B1). These “locks” are mediated, at least in part, by TsAb and TsDTH cells (see B2). If we understand these locks, we may be able to pick them. In addition, effector cells of a given type are likely to be in a dynamic state during the course of an on-going immune response, dying and being regenerated. If we can find conditions inimical to their regeneration, we may be able to get rid of them. The potential clinical importance of such modulation is evident in cancer and in some infectious diseases, as discussed later, see C7-C9. We explored in these papers the possibility of modulating responses from an antibody (now known to be a mixed Th1/Th2 response) to a Th1 mode.

We cultured humorally immune spleen cells under various conditions and, after a few days, assessed the production of antibody and the presence of DTH mediating cells. The in vivo immunized spleen cells were producing much antibody and expressing very little DTH at initiation of culture. Little immunological reactivity was sustained by these cells if the spleen cells were cultured without antigen. Under all conditions realizable in vitro, antibody production decreased and DTH reactivity appeared after several days of culture in the presence of antigen (B4a). Restimulation under certain conditions resulted in the virtually complete absence of antibody production and the expression of massive DTH. We showed that this cell population contains CD8 T cells that can switch in vivo immune responses from an antibody to a cell-mediated mode, i.e. CD8 TsAb cells(B4b). We conclude that mixed Th1/Th2 responses can be readily modulated into apredominant Th1 mode. These findings influenced our therapeutic strategy in the murine model of cutaneous leishmaniasis (see C7) and have implications for treatment of AIDS, as discussed in my two immunology-books.

B5. N. Ismail and P.A. Bretscher, 1999. The Th1/Th2 nature of concurrent immune responses to unrelated antigens can be independent. J. Immunol. 163:4842-4850.

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Synosis It is recognized that the Th1/Th2 nature of the immune response generated against many pathogens is critical to whether containment of the pathogen is achieved. It also seems plausible that concurrent, subclinical infections by different pathogens often occur under natural conditions. Given these likelihoods, it would seem important that the Th1/Th2 nature of concurrent immune responses are independently determined under most circumstances. For example, it would seem physiologically detrimental if a Th2 response to a subclinical infection of a helminth deviated an immune responseto Mycobacterium tuberculosis towards the Th2 pole, thereby resulting in tuberculosis.We test here the “Independence Hypothesis”. This hypothesis postulates that theTh1/Th2 nature of simultaneous responses to non-cross-reacting antigens, even when generated in the same secondary lymphoid organ, can be independently determined. We show that a low dose of xenogeneic red blood cells (XRBC) given by itself i.v. generates an exclusive Th1 splenic response; the administration of a high dose of a non-cross-reacting XRBC by itself generates a predominant Th2 splenic response. Mice immunized with both antigens simultaneously mount splenic responses indistinguishable from that generated in singly immunized mice. This provides support for the Independence Hypothesis.

B6 Testing predictions of the Threshold Hypothesis

B6a. N. Ismail and Peter Bretscher, 2001, More antigen-dependent CD+ Tcell/CD4+ T cell Interactions are required for the Primary Generation of Th2 than of Th1 cells, European Journal of Immunology, 31:1765-1771.

B6b Ismail N, Basten A, Briscoe H, Bretscher PA. Increasing the foreignness of an antigen, by coupling a second and foreign antigen to it, increases the T helper type 2 component of the immune response to the first antigen. Immunology. (2005) 115:34–41.

Synopsis The experiments reported represent stringent tests of the Threshold Hypothesis (B1).

We employed lethally irradiated mice reconstituted with various populations of unprimed spleen cells and challenged them with different doses of antigen (see B3c). We then measured at day 6 after irradiation, reconstitution and immunization, the generation of antibody producing cells, of antigen-specific Th1, IFN-γ and of antigen-specific Th2, IL-4 producing CD4 T cells, employing an ELISPOT assay for enumerating single, antigen- specific cytokine producing cells (see A10a).

We initially employed a substantial dose of antigen that generates a strong IgG antibody response and substantial Th2 cells in intact mice. Irradiated mice reconstituted with a medium number of unprimed spleen cells generated little antibody and mounted a predominant Th1 response upon immunization, whereas mice reconstituted with a large

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number of unprimed spleen cells produced much IgG antibody and substantial numbers of Th2 cells were generated. Clearly a higher number of unprimed spleen cells are required to generate Th2 cells and antibody producing cells than are required to generate Th1 cells. Further experiments showed that it is the greater number of CD4 T cells in a large than in a medium number of spleen cells that are required to obtain IgG antibody production and the generation of Th2 cells. There was no detectable role for CD8 T cells or for antigen presenting cells. Moreover, the number of CD4 T cells and the antigen dose were interdependent variables in determining whether Th2 cells were generated. For example, a slight decrease in antigen dose, that by itself would result in a loss of the generation of Th2 cells, could be compensated for by increasing the number of CD4 T cells, so that Th2 cells were still generated. These observations are expected if more antigen-mediated CD4 T cell/CD4 T cell interactions are required for the generation of Th2 than for the generation of Th1 cells. These observations are highly characteristic predictions of the Threshold Hypothesis, and appear to support its plausibility (B6a).

We also decided to test a critical prediction of the Threshold Hypothesis in intact mice. Basten and Goodnow had made different mice transgenic for the antigen hen egg lysozyme (HEL). In collaboration with Tony Basten, we employed one of their transgenic mice known to be tolerant to HEL at the T cell level. We immunized normal and transgenic with a low dose of foreign red blood cells (FRBC), chosen to generate a Th1 response. We coupled HEL to the FRBC and immunized normal and transgenic mice with a low dose of the conjugated-FRBC. HEL transgenic mice generated a Th1 response to the FRBC, upon such immunization, but the response of normal mice had a greater Th2 component. The responses of normal and transgenic mice were identical on challenge with a low dose of FRBC to which ovalbumin had been conjugated, both generating a response with an increased Th2 component (B6b). It thus appears that the Th2 component of the response to the FRBC can be increased in normal mice by immunizing with the FRBC-HEL conjugate, making the antigen more foreign, but this increase is not seen in the response generated in HEL transgenic mice, tolerant to HEL at the T cell level.

B7 Rudulier CD, McKinstry KK, Al-Yassin GA, Kroeger DR, Bretscher PA. The Number of Responding CD4 T Cells and the Dose of Antigen Conjointly Determine the Th1/Th2 Phenotype by Modulating B7/CD28 Interactions (2014). The Journal of Immunology. 192: 5140-50

This paper describes critical predictions of the Threshold Hypothesis in both invivo and in vitro systems. We show that TcR transgenic CD4 T cells specific for an antigen R will modulate the in vivo response to the antigen Q, in the presence of the Q- R conjugate, but not in the presence of Q and R as separate molecules. We showed that the same Q-specific CD4 T cells could facilitate the generation of R-specific Th1 cells with a small, limiting dose of the conjugate Q-R. The very same Q-specific CD4 T cells could modulate a Th1 response towards a Th2 mode when the dose of the Q-R conjugate was chosen to generate a Th1 response in the absence of the Q-specific

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CD4 T cells.The in vitro experiments were carried out with TcR transgenic CD4 T cells in a

system based on the study with polyclonal CD4 T cells (B3b). We found, in the presence of a standard number of APC, that different numbers of transgenic CD4 T cells generated different effectors, lower numbers of CD4 T cells giving rise to Th1 cells and higher numbers of CD4 T cells to predominant Th2 responses. The Th1/Th2 phenotype of the response also depended in the expected way on antigen dose. We made the hypothesis that the immune system “counted” the CD4 T cells present by a mechanism involving the up-regulation of CoS molecules as CD4 T cells interacted with them. Our experiments support the idea that a number of potential costimulatory molecules and their counter receptors are not involved, and that the B7/CD28 costimulatory signals are involved.

B8 Reviews on how the Th1/Th2 phenotype of the response is determined

B8a Peter Bretscher (2014) On the mechanism determining the TH1/TH2 phenotype of an immune response, and its pertinence to strategies for the prevention, and treatment, of certain infectious diseases. Scand J Immunol.2014;79(6):361-76.

B8b Peter Bretscher (2019) On analyzing how the Th1/Th2 phenotype of an immune response is determined: classical observations must not be ignored, Frontiers in Immunology

I was motivated to write these two papers in view of the fact that virtually all analysis, on the mechanism by which the Th1/Th2 is determined and carried out by others during the last three decades, were in the context of two frameworks that I find implausible: The Cytokine Milieu Hypothesis and the pathogen-associated molecular pattern (PAMP) Hypothesis. These two hypotheses are inconsistent with many quantitative observations on the variables of immunization that affect the Th1/Th2 phenotype of the response, as I argue in these papers. These quantitative variables are those that are accounted for by the Threshold Hypothesis (B1a and B1b). Moreover, these two frameworks are employed in trying to develop strategies of prevention and treatment of many clinical situations, related to infectious disease, allergies, autoimmunity and cancer. I illustrate in this paper how the Threshold Hypothesis can be employed to develop such strategies. Such strategies are also described under Area C below.

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Area C Vaccination/modulation of the immune response against intracellular pathogens causing chronic disease: studies in humans and in model systems

Introductory Background

Vaccination against polio and small pox is extremely effective. Immunologists believe they know why. The immune response that ensues following natural infection is larger and more rapid in vaccinated than in naïve individuals. This increased speed and intensity of the immune response provides the vaccinated individual with the edge in the race between making an immune response that contains the infection and the unrestrained multiplication of the pathogen that leads to disease. The effectiveness of vaccination against small pox and polio contrasts with our inability to vaccinate effectively against the bacterium, Mycobacterium tuberculosis, that can cause tuberculosis, or the virus, HIV-1, that can cause AIDS. Medical observations provide clues as to why this might be so. Some individuals that are infected by these pathogens do not become ill. An examination of their immune status shows that they often have a predominant cell-mediated, Th1 response, often with the production of little antibody. In contrast, the immune response of patients often has a substantial Th2 component. Is this fortuitous or significant? The only way of telling is to find a way of vaccinating individuals in a manner that guarantees a strong and predominant Th1 response upon natural infection, and ascertaining whether indeed this results in protection. Such experiments have been successfully carried out in mice. These experiments suggest it is not just the size but the type of response that is critical in determining whether an immune response is effective.

The murine model of human cutaneous leishmaniasis has become the most developed animal model for examining the regulation of immunity against intracellular pathogens that can cause chronic disease. The hope is that we shall uncover general principles applicable in diverse systems.

Leishmania parasites multiply inside macrophages. Infection of some strains of mice with a million parasites results in an initial increase in parasite burden, the generation of a stable and predominant Th1 response, and containment of the parasite. The parasite does not spread to sites distal from the initial site of infection. Such mice are called “resistant”. Mice of other strains, similarly infected, produce in time a predominant Th2 response against the parasite. This response is associated with increasing parasite burden and the spread of the parasite to sites distal from the site of initial infection and, in the long run, death. Such mice are called “susceptible”. Is the association between a Th1 response and resistance, and between a Th2 response and susceptibility, merely accidental or causal? Various manipulations at the time of infection of susceptible mice result in a change in the long term nature of the immune response, from a Th2 to a Th1 mode, and these manipulations also allow the mice to resist the infection. Manipulations

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to resistant mice at the time of infection, that change the response from a Th1 to Th2 mode, result in susceptibility. These observations provide compelling evidence that the correlations between a Th1 response and resistance, and between a Th2 response and susceptibility, are not accidental but meaningful.

C1 Bretscher, P.A., G. Wei, J.N. Menon and H. Bielefeldt-Ohman. 1992. Establishment of Stable Cell Mediated Immunity Makes "Susceptible" Mice Resistant to Leishmania major, Science, 13:342.

Menon, J.N. and P.A. Bretscher. 1996. Characterization of the immunological memory state generated in mice susceptible to Leishmania major following exposure to low doses of L. major and resulting in resistance to a normally pathogenic challenge. Eur. J. Immunol. 26:243-49.

Synopsis BALB/c mice are susceptible to infection with a million leishmania parasites. Such an infection generates a small and transient DTH response, and parasites grow in number and spread as parasite-specific Th2 are generated with an ever decreasing presence of parasite-specific Th1 cells. In contrast, infection of the same mice with about a thousand parasites results in a prolonged and sustained Th1, DTH response, little antibody production, and containment of the parasite. Lesions, if they form at all, are small, transient and the mice heal. We examined whether we could establish cell- mediated immune deviation by this low dose infection, in a manner reminiscent to that developed by Parish employing a protein antigen in rats. Infection of mice with a million parasites, that had been infected with a low number of parasites about two months previously, reliably resulted in remarkable resistance. Pre-exposure to a low dose infection resulted in a modulation of the response, following infection with a million parasites, from a Th2 to a Th1 mode. Lesion formation was transient. Low dose infection thus constitutes effective vaccination. These studies show that “susceptibility” is not an absolute trait, but one conditional on parasite dose. Also, “susceptibility” is also not absolute in the sense that it is modifyable, in this case by pre-exposure to a low dose infection. Conditionality and modifyability are essential attributes of susceptibility if vaccination is to be feasible. We refer to the process of vaccinating in a manner that guarantees a Th1 response to subsequent infections as Th1 imprinting.

C2 Bretscher, P.A. 1992. A Strategy to Improve the Efficacy of Vaccination against Tuberculosis and Leprosy, Immunology Today, 9:342-3454

Synosis This paper reviews BCG vaccination trials against tuberculosis, and proposes,in view of our findings in the mouse model of cutaneous leishmaniasis (see C1 above), that lower doses of BCG may be more efficacious in preventing tuberculosis and leprosy than the standard dose of BCG currently employed. Our studies in murine leishmaniasis had shown that infection with a low number of parasites could generate a Th1 response and a Th1 imprint upon the immune system, guaranteeing a protective,

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Th1 response upon infection with a high of parasites that was lethal in normal mice. A recognized and major problem in developing a standard vaccination protocol that is universally effective is due to the genetic diversity of the vaccinated population. This diversity means that the immune response to a standard vaccination protocol will generally be different in different people. A strategy to overcome this problem is suggested. It is proposed that vaccination should be carried out with a very low dose of BCG that does not generate a Th2 response in any member of the population. It will then either generate a Th1 response and a Th1 imprint, or be below the threshold required to do so. In the latter case, the BCG bacteria, being alive, will grow unimpeded, until they reach the threshold level needed to induce a Th1 response. They should then induce such a response and a Th1 imprint. This strategy is supported by the study described in C4 below.

C3 Salk, J., Bretscher, P.A., Salk, P., Clerici, M., Shearer, G.M. 1993. A Strategy for Prophylactic Vaccination against HIV. Science 260:1270-127; Salk, J., Bretscher, P.A., Salk, P., Clerici, M., Shearer, G.M. 1993. Protection from HIV infection or AIDS? Science 262:1075-76, under "Technical Comments".

Synopsis Discusses the possibility of developing an effective AIDS vaccine by Th1 imprinting and describes evidence supporting the plausibility of this approach.

C4 Menon, J.N. and P.A. Bretscher, 1998. Parasite dose determines the Th1/Th2 nature of the response to Leishmania major independently of infection route, strain of host or of parasite. Eur. J. Immunology 28:4020-4028.

Synopsis We examined the dose dependence of the Th1Th2 phenotype of the immune response under a variety of conditions. We examined different parasite strains, three different strains of mice and two different routes of infection (subcutaneous and intravenous). We found in all cases studied that we could define a “transition number” of parasites, tn. Infection with a number of parasites below tn resulted in a stable Th1 response; with a number above tn in a Th2 response and progressive disease. The value of tn could vary dramatically between hosts. We found a case where the value of nt for the same parasite strain, same site of infection, but different hosts, differed by about a million fold. These studies strongly support the generality of the dependence of the Th1/Th nature of the response upon antigen dose or parasite number. This is potentially very important as it might provide the means of achieving universally effective vaccination by a standard vaccination strategy in a genetically diverse population (see C2 above).

C5 Power, C., G. Wei and P.A. Bretscher. 1998. Mycobacterial Dose Defines the Th1/Th2 Nature of the Immune Response Independently of Whether Immunization is by the Intravenous, Subcutaneous or Intradermal Route. Infection and Immunity, 66:5743

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Synopsis Shows that the Th1/Th2 nature of the response in BALB/c mice to mycobacteria depends upon the mycobacterial dose independently of whether infection is by the intradermal, subcutaneous or intravenous route. Lower doses induce predominant Th1 responses, and higher doses mixed Th1/Th2 responses.

C6 Jude E. Uzzona, Guojian Wei, Dean Yurkowski, and Peter Bretscher, 2001. Immune elimination of Leishmania major in mice: implications for immune memory, vaccination and reactivation disease, J. Immunol. 167:6967-6974

Synopsis Provides evidence that BALB/c mice, infected with a low number of parasites, can generate a predominant Th1 response and can sometimes eliminate the parasite, previously thought never to occur. We also describe a reliable system in which elimination can be conclusively demonstrated and the consequences of such elimination examined. Elimination results both in loss of resistance and the disappearance of parasite-specific lymphocytes, as assessed by limiting dilution analysis. These observations show, in a very natural situation, the dramatic role of antigen in maintaining immunological memory. This has obvious implications for vaccination strategies, and for the possibility of controlling reactivation disease. (Reactivation disease occurs when a chronic, subclinical infection, held in check by the immune system, erupts due to some immunosuppressive events. Reactivation tuberculosis, that can occur in AIDS patients, could be prevented if ways were found by which the immune system could be conditioned to eliminate M. tuberculosis).

C7 Jude E. Uzonna, and P.A. Bretscher, 2001, Anti-IL4 antibody therapy causes regression of chronic lesions caused by medium-dose Leishmaniamajor infection, European Journal of Immunology, 31:3175-3184 (2001)

Synopsis BALB/c mice are susceptible to an infection with a million parasites. Several manoevres, carried out at the time of this infection, result in resistance. These include treatment with anti-IL-4 antibody, with anti-CD4 antibody and administering IL-12. These manoevres are ineffective once these infections become established. The conventional wisdom is that such simple manoevres do not provide models for how human disease might be treated.

Previous work shows that mixed Th1/Th2 responses may be readily modulated to a predominant Th1 mode (see B4). In addition, the mouse model of infecting BALB/c mice with a million leishmania parasites may not realistically reflect much human disease. The immune response in this particular mouse model becomes fairly rapidly of a very dominant Th2 phenotype, whereas responses may well be less polarised in much human disease. We therefore employed infection of BALB/c mice with about 10,000 leishmania parasites, which we knew (C1 above) often resulted in large, stable lesions, associated with a mixed Th1/Th2 response. We refer to such a diseased state as a state of borderline leishmaniasis. We felt these immune states and lesions might better

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correspond to much human disease. We found borderline leishmaniasis readily treatable with simple manoevres, such as administration of neutralising anti-IL-4 antibody or of anti-CD4 antibody. Such treatment resulted in a complete resolution of very large lesions, and also a modulation of the immune response to a predominant Th1 mode. Recovered mice were completely resistant to a challenge of a million parasites that is progressive in normal mice. This acquisition of resistance seems analogous to what happens in patients with visceral leishmaniasis who are treated with anti-parasite drugs (see C8).

C8 A. Hailu, J.N. Menon, N. Berhe, L. Gedamu, T. Hassard, P.A. Karger, J. Olobo, A. Senthilsen, and P. A. Bretscher, 2001, Distinct immunity in visceral leishmaniasis patients from that in healthy contacts and drug-cured people: implications for the mechanism underlying drug cure, J. Infectious Diseases, 184:112-115Synopsis We examined the levels of anti-leishmania parasite antibody belonging todifferent IgG subclasses in (a) normal individuals (b) patients, (c) healthy contacts and (d) drug-cured individuals. Dramatic differences are readily seen between the antibody levels of patients and healthy contacts. Patients had higher levels of IgG1, IgG3 and IgG4 antibodies, but similar levels of IgG2 antibody. Most interestingly, the immune state of drug cured individuals and healthy contacts were indistinguishable. This explains the known resistance of most individuals, that have undergone anti-parasite drug therapy, to re-infection. Our observations demonstrate that human immune responses can be modulated. We argue that modulation of the response from a mixed Th1/Th2 to predominant Th1 mode probably occurs due to a lowering of the antigen load following drug treatment and parasite death.

C9. P.A. Bretscher, N. Ismail, J.N. Menon, C.A. Power,. J. Uzonna and G. Wei, 2001. Vaccination against and treatment of tuberculosis, the leishmaniases and AIDS: perspectives from basic immunology and immunity to chronic intracellular infections. Cellular and Molecular Life Sciences, 58: 1879-1896

Synopsis We outline our view on how concepts derived from basic studies might have a beneficial impact upon strategies of vaccination. This is in the context of intracellular pathogens that can cause chronic disease. We try to justify the concept of “coherence”, as follows. Suppose we represent the proteins of a parasite as Q, giving rise to q1,q2,q3,….qn peptides recognised by CD4 T cells in the context of host class II MHC molecules. The idea of coherence reflects the fact that the Th1/Th2 nature of the response to q1, q2. ….qn are not independently determined but are interdependent. The response to all the peptides tends to be predominantly Th1, or predominantly Th2, or predominantly mixed under a given set of circumstances. We discuss regulatory mechanisms that account for coherence. It is argued that we can ensure effective protection by ensuring that certain global features of the immune response are appropriate, such as its strength and its Th1/Th2 nature, without the need for defining protective antigens and the detailed specificity of protective T cells. We discuss in

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general terms some of our then unpublished findings in tuberculosis and our unpublished findings in leprosy. We outline our favored strategies in preventing and treating tuberculosis, AIDS and the leishmaniases.

C10a P.A. Bretscher , J.N. Menon, C. Power, J. Uzonna, and G. Wei, 2001, A case for neonatal, low dose BCG vaccination, Scandinavian Journal of Infectious Diseases, 33:253-257

C10b TG Kiros, CA Power, G Wei, and P Bretscher, 2010, Immunization of newborn and adult mice with low numbers of BCG leads to Th1 respnses, Th1 imprints and enhanced protection on BCG challenge. Immunotherapy, 2: 25-35

Synopsis C10a gives what was then the most recent and complete presentation of the case for a neonatal, low dose BCG vaccination trial against tuberculosis. We tested this strategy (C10b). At the time of these studies, a leading investigator, in discussing immunization of mice with BCG, called immunization with a million colony forming units (cfu) of BCG as immunization with a low number of BCG. We show in this paper that immunization of very young mice with 33 cfu of BCG generates very strong Th1 responses and the best Th1 imprints! Buddle and colleagues have experimentally found a way to protect cattle against experimental tuberculosis. Part of the secret of their success was that they reduced the vaccination dose by about 106 fold!

C11 A hypothesis for the existence of two types of tuberculosis, reflecting two distinct types of immune failure to control the pathogen, based upon prevalence of mycobacterium‐specific IgG subclasses, 2018, J Menon, VH Hoeppner, A Judd, CA Power, PA Bretscher, Scand J Immunol e12665

Tuberculosis (TB) is among the greatest killers of all infectious diseases worldwide. Although many think Th1 responses are protective, much of the pathology associated with TB is caused by lung granulomas, also caused in large part by a Th1 response to bacterial antigens. These facts illustrate a major quandary of TB research: what is different about the Th1 response in healthy infected individuals and in patients? We need to know what immunological parameters discriminate between responses in patients and in healthy infected individuals if we are to rationally design vaccines against TB. In addition, these correlates are important when considering immunotherapy of this infectious disease.

We approached this question by measuring the relative prevalence of different subclasses of IgG antibodies, among those specific for mycobacterial antigens, in patients and healthy infected individuals. We calculated the ratio of IgG1 to IgG2 antibodies. We found that this ratio varied about a thousand fold among healthy infected (0.001 to 1) and about hundred, thousand fold among patients (0.001 to 100). It was evident that patients with IgG1 to IgG2 ratios between about 1 to 100 had a greater Th2 component to their anti-mycobacterial-specific immune response than patients and

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healthy infected individuals with lower ratios (0.001 to 1). This led us to suppose that the immune response of these patients had a down-regulated Th1 response, and so could not contain the pathogen. We refer these patients as having type 2 tuberculosis. Why might there be other patients with qualitatively similar responses to those of healthy infected individuals?

We were very impressed by our findings that the transition number for theresponse to Leishmania major varied over almost a million-fold range (C4). We argue that if there is anything like a similar variation in humans, in their response to Mycobacterium tuberculosis, the nature of the response will be widely different upon exposure to an infectious dose of M tuberculosis. We argue in this paper that people with a very high transition number will only make a response to M tuberculosis once the bacterial burden has become considerable. Such individuals, having a greater bacterial burden, would also need a much stronger Th1 response to win the war against the pathogen. We thus propose that patients, with IgG1 to IgG2 ratios between 0.001 to 1, have a similar response to healthy infected individuals, but the type I TB patients have too small a Th1 response to contain the infection. We discuss how this idea allows several paradoxes concerning immunity to M tuberculosis to be resolved. There are implications of this hypothesis for both vaccination and treatment.

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Area D: Tumor Immunology

D1 The Th2 Skewing Hypothesis of tumor escape: implications for vaccination against and immunotherapy of cancer

D1a Hamilton, D. H. and P. A. Bretscher (2008). "Different immune correlates associated with tumor progression and regression: implications for prevention and treatment of cancer." Cancer Immunol Immunother 57(8): 1125-36.

D1b Hamilton, D. H. and P. A. Bretscher (2008). The commonality in the regulation of the immune response to most tumors: the prevalence of immune class regulatio as a tumor escape mechanism. Cancer Therapy 6:745-754

D1c Duane Hamilton, Nahed Ismail, David Kroeger, Christopher Rudulier, Peter Bretscher(2009). Macroimmunology and immunotherapy of cancer. Immunotherapy 1:367-383

Synopsis Recent reviews of the relationship between cancers and the immune system provide a somewhat dismal prospect for vaccination against and immunotherapy of cancer. This is because many and diverse mechanisms of tumor escape from the surveillance of the immune system have been described. How can we provide protection against all these diverse escape mechanisms?

We felt and feel differently about the prospects for vaccination and immunotherapy. There are clearly diverse mechanisms of escape, but we strongly argue that in mouse systems cell-mediated, Th1, cytotoxic T lymphocyte immunity is protective, and tumor progression correlates very frequently with the generation of a Th2 component of the anti-tumor response. We tested this Th2-skewing hypothesis in two different and well studied murine tumor systems and found it held (D1a). We discuss the significance of this finding for understanding the immunological basis of various empirical therapies and for developing effective vaccnation and immunotherapy. We discuss the possibility that a similar situation holds in human cancer (D1b and D1c).

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Area E: Miscellaneous

E1 Bretscher, P.A. 1987. Signs and Meaning in the Study of the Immune and Higher Nervous Systems, in "The Semiotics of Cellular Communication in the Immune System". E.E. Sercarz, F. Celada, N.A. Mitchison & T. Tada, Eds., NATO ASI Series, Springer-Verlag, Publ. pp 281-304.

E2 Bretscher, P.A. 1988. Decision Criteria and Coherence in the Regulation of the Immune Response, in Theoretical Immunology, A.L. Perelson, Editor, Part I, 295- 307, Addison-Wesley Publishing Co.

Synopsis A discussion of how simple observations imply that there must be sophisticated interactions between antigen-specific lymphocytes. lymphocytes do not behave as independent entities, but as well regulated members of a society with rules. I argue that the behavior of lymphocyte populations must reflect the existence of more interactions between lymphocytes that usually envisaged.

E3 Bretscher, P.A. and C. Havele. 1992. Cyclosporine A can Switch the Immune Response Induced by Antigen from a Humoral to a Cell-mediated Mode. European Journal of Immunology. 22:349-355.

Synopsis We show that the very same dose of cyclosporin A (CsA) can, under different circumstances, have very different and what at first appears to be contradictory effects. A given concentration of CsA can inhibit the generation of cell-mediated immunity in the form of delayed type hypersensitivity (DTH). The very same concentration of CsA can inhibit an antibody response and increase the induction of DTH. These diverse effects of the same concentration of CsA on the generation of cell-mediated immunity can be understood if CsA inhibits the generation of eTh, and if the generation of antibody responses are more helper T cell dependent than is the generation of cell-mediated immunity in the form of DTH.

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