SECTION 8

INNATE IMMUNITY TO VIRUSES AND ANTIGEN RECOGNITION

CONTACT INFORMATION

Anthony DeFranco, PhD (Email)

READING

Basic Immunology: Functions and Disorders of the Immune System. Abbas, Abul K., and An-drew H. Lichtman. -- Chapter 2 and Chapter 4 (pp. 67-76)

OBJECTIVES

• Describe the role of RIG-I-like receptors (RLRs) in the induction of type 1 interferon by virus-infected cells

• Describe in general terms how type 1 interferons provide anti-viral innate immunity.

• Describe how natural killer cells can recognize virus-infected cells and kill them.

• Understand the basic functional features of antibody molecules, including their domain structure, the location of variable and constant domains, the location of hypervariable regions within the variable domains, the different types of heavy and light chains, and the identity of those parts that confer antigen binding and effector functions (Note: effector functions will be covered in detail in a subsequent lecture).

• Explain the difference between “affinity” and “avidity”. Describe how these related concepts apply to the different classes of antibodies and how antibodies change during an immune response in this regard (the latter issue will be covered in more detail in one of Dr. Cyster’s lectures).

• Enumerate the main ways in which antibodies are used in medicine.

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• Explain the difference between polyclonal antibodies and monoclonal antibodies, and describe the main advantages of monoclonal antibodies.

• Appreciate that constant parts of antibody molecules can be incorporated into protein-based therapeutic agents and describe the advantage conferred by doing so.

• Describe how antibodies are used in laboratory testing (RIA, hemagglutination, ELISA, and flow cytometry. NOTE: antibody tests are the subject of the Immunology laboratory on 9/3).

• Describe how the B cell antigen receptors differ from secreted antibody molecules.

• Describe the T cell antigen receptor (TCR) subunit structure. Explain the role of the CD3 component of the TCR. Compare and contrast the structure of the TCR α/β chains to the structure of antibodies.

KEY WORDS:

• INTERFERON

• NATURAL KILLER (NK) CELL

• IMMUNOGLOBULIN/ANTIBODY

• T CELL RECEPTOR FOR ANTIGEN (TCR)• AVIDITY

• AFFINITY

• ANTIBODY STRUCTURE

• MONOCLONAL ANTIBODY

• ELISA

MAIN IDEAS:

Virus infection is typically recognized by intracellular sensors in the cytoplasm of cells that recognize the nu-cleic acids of the replicating virus, such as double-stranded (ds) RNA and induce synthesis of type 1 inter-ferons. The two sensors of viral RNA are RIG-I and Mda5, which collectively are called RIG-I-like recep-tors (RLRs). They have different fine specificity for vi-ral RNA molecules. There are also sensors for DNA vi-rus infection that induce production of type 1 inter-feron, but their molecular identities are in the process of being characterized at this time. Type 1 interferons

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are secreted from the infected cell and act on neighbor-ing cells to limit the subsequent replication of virus. This mechanism represents a second distinct type of cellular innate response to infection, in addition to the induction of inflammation.

A key mechanism for fighting most virus infections is the killing of infected cells that are producing virus. This role is provided early after infection by the natural killer (NK) cell, a lymphocyte that is part of innate im-munity. Later in infection, cytotoxic T cells perform this function; NK and cytotoxic T cells use the same ba-sic molecular mechanism for killing infected cells.

B cells and T cells use very similar molecules for recog-nizing antigens, but the nature of the antigen recog-nized is very different (free antigen in native [unproc-essed] form vs. peptide-MHC complexes [generated by processing of protein antigens]).

A membrane-bound form of the antibody molecule is expressed on B lymphocytes and serves as a receptor for antigen, allowing the immune system to selectively activate those B cells that make antibodies able to bind to parts of an infecting pathogen.

The structures of antibodies are well tailored to their functions. Antibodies have an antigen-binding region, which exists in a great many variations, and a “con-stant” region which is responsible for many of the effec-tor functions of the antibody and which exists in 5 dif-ferent types, corresponding to the five isotypes (classes) of antibodies: IgM, IgD, IgG, IgA, and IgE. In addition, IgG has four subtypes called IgG1, IgG2, IgG3, and IgG4, each encoded separately in the ge-nome. They differ somewhat in effector function, with IgG1 and IgG3 being especially good at promoting phagocytosis and killing of microbes and being more in-flammatory in their action.

Antibodies are useful as therapeutics. “Polyclonal” anti-bodies pooled from many donors (intravenous immune globulin “IVIG”) are used to treat certain immunodefi-ciencies and also have efficacy for some autoimmune diseases (Note: the mechanism of action in autoim-mune diseases is not well established). Therapies based on a single antibody (“monoclonal antibodies”), are useful for treating an increasing number of condi-tions (especially cancers and inflammatory diseases) . Monoclonal antibodies are also frequently useful for di-agnostic and research purposes given their reproduci-

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bility and unlimited availability, in contrast to poly-clonal antibodies.

There is a specific transmembrane Fc receptor, called FcRn, that is used by the body for maintaining IgG in the bloodstream and for transmitting IgG from mother to fetus. The latter function is critical for immune pro-tection in newborn infants. In addition, some therapeu-tic molecules are designed to be able to bind to FcRn in order to increase their half-life in the blood and make them work better.

OVERVIEW AND ADDITIONAL COMMENTS BEYOND THE TEXTBOOK

Innate Immunity to Viruses

Induction of interferon by dsRNA in the cyto-plasm: Mammalian cells have two related molecules (called RIG-I and Mda5; collectively these are referred to as “Rig-I-like receptors” or RLRs) that recognize vi-ral RNA and/or replication intermediates (double-stranded RNA molecules) in the cytoplasm. These molecules signal to induce production of interferon-α and interferon-β, which signal to neighboring cells via

the interferon-α receptor (the receptor for both interferon-α and interferon-β). Note that there is also an intracellular sensor for viral DNA genomes in the cell, which also induces interferon production by a similar pathway.

Anti-viral effects of type 1 interferons (interferon-α and -β): Interferon-induced genes that participate in limiting virus replication are many, and include the dsRNA-dependent protein kinase (PKR), which blocks cellular protein synthesis in in-fected cells, oligo-A synthetase, which in infected cells makes oligo-A, which activates an RNase to degrade mRNAs, again inhibiting protein synthesis, and the Mx proteins, which are less well understood, but are thought to inhibit virus assembly and/or virus tran-scription (synthesis of mRNAs).

ANTIGEN RECOGNITION

In general, this material is covered well in the textbook and you should start there. The following is designed to cover aspects of the lecture that go beyond the text-book material.

Antibody structure: An editorial comment: it is hard to overstate how important it is for you to mas-

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ter the basics of antibody structure. The understand-ing of immunology and the practical use of it build on these basic concepts in many ways. For example, many of the effector functions of antibodies make use of rec-ognition of the constant parts of antibody molecules by cell surface receptors of macrophages, neutrophils, mast cells, NK cells, etc. and these receptors are called “Fc receptors”, which makes sense if you recall which part of the antibody molecule is the Fc part. Novel protein-based therapeutics are starting to come into use that employ Fc parts of antibodies in order to give the therapeutic protein a longer half-life in the blood. The differences between IgM and IgG are critical for un-derstanding the consequences of blood group incom-patibilities, etc. etc.

Antibodies in medicine: Antibodies have been im-portant for medical practice for many years. The suc-cess of vaccination relies almost entirely on the produc-tion of protective antibodies. The understanding of the ABO blood groups (which we will describe in further de-tail later in I-3) and their detection using antibodies was essential to making blood transfusions practical. Antibodies are used to aid in diagnosis in many ways. We shall examine some examples of this in an upcom-ing laboratory session. Antibodies can be transferred

from one individual (or even from some animal spe-cies) to another person, which is referred to as passive immunity, since the person’s own immune system did not make the protective immune response. Anti-body from an immune individual (or animal) is used for protection against some infectious diseases, for treatment against tetanus exposure of an unvaccinated individual, and for treatment of poison snake bites. Pooled antibodies from many individuals (intravenous immune globulin “IVIG”) are used to treat immunodefi-ciencies in which antibody production is defective, and increasingly have been found in be useful in some other disease states (autoimmune diseases in particu-lar). Increasingly, monoclonal antibodies are being used both as diagnostic reagents and as therapies.

Polyclonal antibodies vs. monoclonal antibod-ies: The antibodies produced in a normal immune re-sponse result from the activation of many B cells, each of which may recognize different parts of the antigen (different epitopes), and each of which may have a dif-ferent affinity for antigen. Such antibodies are called polyclonal, because they result from the combined ac-tions of multiple B cells each activated to multiply and produce a clone of progeny that go on to secrete anti-body. In contrast, monoclonal antibodies are the result

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of a single immortalized B cell, so all the antibodies have the same amino acid sequence, recognize the same epitope and have the same affinity. Monoclonal antibodies can be produced in essentially unlimited quantities. Whereas polyclonal antibodies are by their nature biological products which vary from batch to batch, monoclonal antibodies are chemically defined and always the same. Monoclonal antibodies are used frequently in diagnostic procedures, including ELISA and flow cytometry, which we will describe in detail in an upcoming lab session. In recent years, they are be-coming increasingly successful as therapeutic agents as well.

Humanized monoclonal antibodies. An impor-tant limitation of the first generation of monoclonal an-tibodies, dating back to the 1980s, is that they were made in rodents and injection of most such mono-clonal antibodies into people led to an immune re-sponse. Often patients made antibodies against the parts of the rodent antibodies that were different in amino acid sequence from human antibodies. Once that happened, the therapeutic antibody was rapidly cleared from the body and lost efficacy. In addition, the formation of large amounts of immune complexes can be deleterious in several ways. To solve this prob-

lem, there are several approaches to making mono-clonal antibodies less immunogenic and reducing the fraction of patients that make enough antibody against them to be a problem. In addition, there is a standard-ized nomenclature that attaches a suffix to the name of the therapeutic to indicate which approach was used, so that the physician can readily know which therapeu-tic is of which type. The approaches range from gene splicing methods of swapping out the constant parts of the monoclonal antibody for their human equivalents (chimeric monoclonal antibody, -ximab), to addition-ally replacing the “framework regions” of the V regions of the heavy and light chains (“humanized”, -zumab) to starting with a fully human monoclonal antibody (-mumab). A fully mouse monoclonal antibody thera-peutic has the suffix –omab, which can be fine for a single use product.

More recent monoclonal antibody therapeutics are mostly of the humanized or fully human types, whereas the chimeric antibodies tend to have come into medi-cine earlier, as they were easier to create. While the more human the therapeutic, the less immunogenic it will be on average, this is not a perfect correlation, as other factors influence the immunogenicity, including how unusual the V regions are and details of the formu-

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lation, such as how carefully aggregated monoclonal an-tibody is excluded from the product, since aggregated proteins are more immunogenic than soluble unaggre-gated forms. Currently, regulatory agencies require that the degree of immunogenicity be tested and re-ported in the product literature for monoclonal anti-body therapeutics, but this is insufficiently standard-ized at this point to be particularly useful. Until this changes, there should be skepticism about claims of relative degree of immunogenicity of one product ver-sus another, unless the comparison is based on very similar methodology.

Immunogenicity is also a potential complication of other types of therapeutic proteins. For example, older versions of type 1 interferons, which are used to treat certain cancers and hepatitis C virus infections, are con-siderably more immunogenic than some newer formu-lations, probably due to changes in the formulation it-self. As a clinician, if you have a patient who has been getting some efficacy from a protein therapeutic but the therapeutic stops working, one possible explana-tion is that the patient made antibodies against the therapeutic.

Anti-TNF therapeutics: When we get to the Rheu-matology lectures of I-3, we shall hear about the suc-cessful use of anti-TNF therapeutics to control certain inflammatory diseases. There are currently two types of TNF-blocking protein therapeutics on the market, and they represent two strategies that are used more widely in other therapeutics against other targets. In-fliximab (Remicade) and Adalimumab (Humira) are monoclonal antibodies that are specific for the pro-inflammatory cytokine TNF (and two additional mono-clonal anti-TNF therapies have been approved). Etanercept (Enbrel), in contrast is a chimeric protein in which the extracellular domain of a TNF receptor (TNFR2) is used as the part that binds to and inhibits TNF. By itself, this receptor fragment turned out to have a very short half-life in the blood, making its use for prolonged periods of time impractical. By adding to the end of this molecule the Fc portion of a human IgG molecule, the long half-life of IgG in the blood was conferred upon the chimeric protein. This long half-life is due to the actions of an Fc receptor (called FcRn) expressed by endothelial cells and some other cell types. FcRn decreases loss of IgG from the blood, possi-bly by decreasing its catabolism. FcRn is also responsi-ble for transmission of IgG between mother and fetus.

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T cell antigen receptor: The basic structures of the TCR and the BCR are similar, although note that only B cells secrete their antigen-binding molecule. This is be-cause whereas antibody is a soluble recognition ele-ment that acts all over the body by coupling to effector mechanisms of innate immunity (complement activa-tion, phagocytosis, etc.), T cells generally act locally on cells in a lymph node or at the site of infection. Hence recognition needs to occur where the T cell is and be coupled to activation of the T cell to secrete effector molecules such as cytokines or, in the case of cytotoxic T cells, molecules involved in killing the antigen-expressing cell.

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