Lecture 8 Vertebrate immunity Lymphocyte receptor diversity Humans have about 30,000 genes, so theres clearly not one gene for each of the tens of millions of different receptors on our T-cells Instead we have a combination of three things: 1.Receptors (at least B-cell ones) are composed of two protein chains, each different Lymphocyte receptor diversity Each chain is built of multiple segments that are combined by specially controlled recombination (somatic recombination) Heavy chains have three regions that affect recognition (receptor binding), variable (V), diversity (D), and joining (J) Light chains have only V and J regions In humans there are about 100 different V genes, 12 D genes, and 4 J genes Lymphocyte receptor diversity Each progenitor of a B-cell clone undergoes somatic recombination that brings together a V-D-J combination for the heavy chain There are 100X12X4 = 4,800 V-D-J combinations Similar recombination events lead to the light chain How many possible light chain combinations are there? And heavy plus light chain combinations? Lymphocyte receptor diversity 4,800 V-D-J combinations for the heavy chain 400 V-J combination for the light chain = 1,920,000 different B-cell receptors (aka immunoglobulins, aka antibodies) Plus there are random DNA bases added between segments, so the possible diversity is pretty much infinite There are lymphocytes with around 100 million specificities floating around inside each of us Lymphocyte receptor diversity Finally, the six areas of the genes that code for the parts of the receptor that do the recognizing can undergo further small changes due to mutations within individual lymphocytes The V-D-J shuffle will be different for each lymphocyte, and is then locked in for that lymphocyte The glass slipper doesnt changemuch. But it changes a bit through somatic hypermutation (Haldanes idea) Somatic recombination gives a combinatorial pool of diversity which is then fine tuned Lymphocyte receptor diversity Upon infection, one of the clones generated by VDJ recombination might fit a pathogen epitope like Cinderellas slipper This stimulates amplification of that clone The new generation of clones increase their mutation rate at recognition site This creates slight variation in the clone population, and variants with tighter binding are stimulated to divide more rapidly = affinity maturation Remind you of anything? Lymphocyte receptor diversity Clonal selection The process that underlies lymphocyte specificity and differentiation is akin to natural selection only those lymphocytes that encounter an antigen to which their receptor binds will be activated to proliferate and differentiate into effector cells This selective mechanisms was first proposed in the 1950s by the Australian biologist Frank MacFarlane Burnet at a time when nothing was known about lymphocyte receptors, or even that lymphocytes were important Clonal selection It wasnt until the 1960s that James Gowans removed lymphocytes from rats and noticed that their adaptive immunity disappeared Peter Medawar removed the last conceptual problem in the 1950s by showing how the problem of immune responses to self is solved How? (Burnet and Peter Medawar were co-recipients of the 1960 Nobel Prize in Physiology or Medicine for demonstrating acquired immune tolerance. This research provided the experimental basis for inducing immune tolerance, the platform for developing methods of transplanting solid organs.) Clonal selection Exposure to foreign tissues during embryonic development of mice caused them to become tolerent of those tissues later (I.e. no immune response) Led to the idea that developing lymphocytes that are potentially self-reactive are removed before they can mature = clonal deletion these sorts of experiments are why we call MHC MHC (major histocompatibility complex) Figure 1-15 Figure 1-14 part 1 of 2 Figure 1-14 part 2 of 2 Clonal selection The proliferation of lymphocytes after clonal selection leads to immunological memory After a lymphocyte is activated, it takes 4-5 days of proliferation before clonal expansion is complete Thats why adaptive responses occur only after a delay of several days After this primary response, some antigen-specific cells persist and lead to a more rapid and effective secondary response, and lasting immunity = immunological memory Clonal selection, adaptive immunity, and diversity generation The proliferation of lymphocytes after clonal selection leads to immunological memory (and vaccines) Types of lymphocytes B-cells produce immunoglobulins, molecules produced by adaptive immunity to dispose of particular threats Antibody = immunoglobulin = free-floating B-cell receptor. B-cells main job is to produce humoral immunity, to neutralize pathogens floating anywhere outside of cells (extracellular) Figure 3-11 T-cell receptors (TCRs) have a very similar structure to BCRs/immunoglobulin molecules Formed by the same sort of somatic recombination, but no affinity maturation Figure 1-27 Host cells continually break up intracellular proteins into little peptides around 10 aa long The host MHC molecules bind these little peptides within the cell The cell then transports the MHC/peptide combination to the cell surface for presentation to roving T-cells The two main classes of MHC molecules present antigen from cytosol (MHC class I) and vesicles (MHC class II) Figure 8-10 TCRs recognize (bind) the MHC/antigen complex presented by infected cells TCRs also recognize antigen presented by cells (e.g. B-cells) that have ingested antigen Figure 3-23 MHC class I molecule presenting an epitope Figure 3-24 Peptides eluted from two different MHC molecules One MHC can bind multiple epitopes, often with similar anchoring residues Figure 1-28 MHC class I molecules present antigen derived from proteins in the cytosol Basically, MHC I is all about viruses, obligate intracellular parasites As the virus synthesized proteins in the cytosol, they are transported to the cell surface Contrast this with early (wrong) theories that postulated that T-cells had some kind of finger with which they could probe the interior of a cell (supported with electron microscopy) CD8-bearing T-cells (aka cytotoxic T lymphocytes, CTLs, killer T-cells) then kill the infected cell Figure 1-28 part 1 of 2 Figure 1-28 part 2 of 2 Figure 1-30 Figure 1-28 MHC class II molecules present antigen derived from proteins originating in intracellular vesicles Some bacteria (like M. tuberculosis) infect cells and reside in intracellular vesicles Peptides derived from them they are transported to the cell surface by MHC II molecules CD4-bearing T-cells, specifically T H 1 cells and T H 2 cells (T H 2 are more commonly called helper T-cells ) then facilitate the disposal of the pathogen Figure 1-29 part 1 of 2 Figure 1-31 part 1 of 2 T H 1 cells tell an infected macrophage to go ahead and fuse lysozomes with the vesicles This leads to the breakdown of the pathogens Figure 1-29 part 2 of 2 Figure 1-31 part 2 of 2 Figure 3-19 Every nucleated cell in the body expresses MHC (class I always present: why?) Pattern of MHC class, expression level varies among cell types Erythrocytes do not express MHC. This may be one reason for success of Plasmodium spp. Why is the lack of MHC class I on the surface of RBCs not a problem with respect to viral infection? The architecture of immunity The white blood cells of the immune system derive from precursors in the bone marrow. The main cell types are macrophages, T- lymphocytes of various subclasses, and B- lymphocytes. Other cells types are involved in innate immunity and allergic responses (eosinophils, neutrophils, basophils The architecture of immunity Distribution of lymphoid tissues in the body: Central lymphoid tissues. Bone marrow: houses stem cells Thymus gland: where T-lymphocytes differentiate from stem cells. Figure 1-7 The peripheral lymphoid organs are specialized to trap antigen and allow the initiation of adaptive immune responses The architecture of immunity Peripheral lymphoid tissues. 1.Lymph glands: T-cells and B-cells migrate and occupy lymph glands. Macrophages and dendritic cells are present to trap antigens entering the glands (inducer cells) 2.Spleen: another filter of the blood and lymph 3.GALTs: gut associated lymphoid tissues are aggregates of cells found in various organs. Especially associated with mucosal membranes of respiratory and gastrointestinal tract (e.g. tonsils) 4.Blood and lymph: where B- and T-lymphocytes circulate, passing continuously in and out of the system and through lymphoid organs via lymphatic circulation Figure 1-8 part 1 of 2 Lymph nodes are highly organized structures that are the sites of convergence of an extensive system of vessels that collect extracellular fluid (lymph) from tissues and return it to the blood The architecture of immunity Lymphatic circulation. Allows access of lymphocytes to body tissues and organs to respond to any invading foreign antigen Allow recruitment of lymphocytes to an inflammatory site and thus enables the initiation of the immune response Allows replenishment of lymphoid organs damaged by trauma or infection Figure 1-11 Figure 1-3 part 1 of 4 Figure 1-3 part 2 of 4 Figure 1-3 part 3 of 4 Figure 1-3 part 4 of 4 Evolution of the immune system In its present state, the immune system of mammals is a coherent system of interacting cells and molecules, but this state has been pieced together by tinkering Its components have been co-opted from different physiological functions and shaped by evolutionary pressures to fit relatively seamlessly together The distribution of the components of the immune system in different species can shed light on its overall evolutionary history Evolution of the immune system The most ancient immune defenses lie within the innate immune system Drosophila spp. Have well developed innate immune system The first defense molecules in evolutionary terms were probably antimicrobial peptides, produced by plants and animals Evolution of the immune system For a long time the evolution of adaptive immunity was obscure, because it seemed to emerge suddenly as a complete biological system at roughly the same time as the vertebrates The picture is now becoming clearer. Evolution of the immune system Adaptive immunity appeared abruptly in the cartilagenous fishes Its been known for 50 years that all jawed fish can mount an adaptive immune response Jawed fish have B-cells, T-cells, MHC, Lymphoid organs, immunological memory Evolution of the immune system hagfish and lampreys lack most signs of an adaptive immune system; no organized lymphoid tissue, no immunological memory, no T-cells, etc. Evolution of the immune system Both prokaryotic and eukaryotic genomes contain mobile DNA elements known generally as transposable elements Transposable elements can move themselves, or copies of themselves to different positions on chromosomes: selfish DNA TEs contain two essential elements: a sequence encoding transposase, a DNA recombinase that is able to cut DNA and excise the element; and terminal repeat sequences that are recognized by the transposase and are required for excision and insertion Evolution of the immune system The recombination-activating genes that catalyze somatic recombination are called RAG-1 and RAG-2, and they make RAG-1 and RAG-2 proteins They are essential for receptor gene rearrangements Mice lacking either gene cannot form lymphocyte receptors and hence dont have adaptive immunity RAG-1 and RAG-2 are unusual genes in vertebrates in that they dont have introns They look an awful lot like transposons Evolution of the immune system The evolution of adaptive immunity seems to have been made possible by the invasion of a putative immunoglobulin-like gene by a transposable genetic element This conferred on the ancestral immunoglobulin gene the ability to undergo somatic gene rearrangement, and thus to create genetic diversity Evolution of the immune system When a mobile DNA element excises itself from a piece of DNA, alterations in the original sequence are introduced into the host DNA when cut ends are rejoined This is the origin of antigen receptors in the adaptive immune system! Evolution of the immune system By luck, a normally selfish DNA element, a sort of genomic pathogen, gave vertebrates the machinery to cut and join independent loci This paved the way for the full-blown somatic gene rearrangement seen in the immunoglobulin and T-cell receptor genes today Landmark papers: Is V(D)J recombination irreducibly complex? We can look high or we can look low, in books or in journals, but the result is the same. The scientific literature has no answers to the question of the origin of the immune system. -Michael Behe Evolution of the immune system Why did adaptive immunity arise in vertebrates? Brainstorming contest Recapitulation The immune system defends the host against infection Innate immunity serves as a first line of defense but lacks the ability to provide the specific protective immunity that prevents re-infection Adaptive immunity is based on the clonal selection of lymphocytes bearing highly diverse antigen- specific receptors Adaptive immunity is an evolutionary novelty relative to innate immunity, and likely arose after a transposon invaded a proto-immunoglobulin-like gene Recapitulation In the adaptive immune response, antigen-specific lymphocytes proliferate and differentiate into effector cells that eliminate pathogens Host defense requires different recognition systems and a wide variety of effector mechanisms to seek and destroy the wide variety of pathogens in their various habitats within and on the body Recapitulation Not only can the adaptive immune response eliminate a pathogen, but can deliver immunological memory This depends on a pool of differentiated memory lymphocytes generated through clonal selection Memory cells engender a more rapid and effective response upon re-infection