Session 5: B Cells: Development, Selection, and Function
Transcript of Part 3: Immunology: The Basics of Antibody Diversity
00:00:07;23 My name is Hidde Ploegh. 00:00:08;23 I'm an investigator at Boston Children's Hospital in the program of Cellular and Molecular Medicine. 00:00:15;20 I will present two talks today. 00:00:18;17 The first one provides you with a more general introduction to certain aspects of the immune system. 00:00:25;03 And in the second half of the talk, I'll speak about some evolutionary anomalies in the immune system 00:00:30;24 that we've been able to leverage into a new class of tools that I think will be 00:00:35;20 of more general interest. 00:00:36;27 So, let me begin by giving you an introduction of host defense. 00:00:43;23 We generally consider host defenses composed of three layers. 00:00:48;18 Mechanical and chemical defenses, depicted in this diagram, as line 1, 00:00:53;04 probably hold at bay 98% or more of viruses and pathogenic bacteria. 00:00:59;18 But because these organisms come equipped with special tricks to cir... circumvent these barriers, 00:01:05;11 we have a backup system. 00:01:08;08 This is the combination of innate and adaptive immunity. 00:01:11;23 Layer 2, innate immunity in this cartoon, should be considered the rapid-deployment forces 00:01:18;02 of the immune system. 00:01:19;27 They can distinguish between pathogenic entities such as bacteria and our own tissues, 00:01:26;11 but do so with a limited degree of specificity. 00:01:29;14 The nice thing is that they respond very quickly. 00:01:32;13 And so, should defenses of the mechanical and chemical nature fail, usually innate immunity 00:01:38;11 deals with the ensuing problem. 00:01:41;02 But given the sophistication of pathogens and the tricks they've evolved, some of these 00:01:46;21 require stronger measures. 00:01:49;12 And for that reason we have adaptive immunity kick in. 00:01:52;18 This is a time-consuming process, but it allows us to distinguish, truly with pinpoint precision, 00:01:59;01 between pathogenic microorganisms and our own tissues. 00:02:02;15 So, what I'll do in the next segment is to describe one particular aspect of 00:02:09;04 the adaptive immune system, because this will become relevant when we discuss, 00:02:12;24 in the second part of my presentation, 00:02:15;01 some of the unusual properties of antibodies made by other vertebrate species. 00:02:21;08 This is an amplification of the cartoon that I've just shown you, and it provides 00:02:25;27 a little bit more specificity. 00:02:28;13 You have the pathogens coming in. 00:02:31;09 They come equipped, as I've said, with enzymes that would allow one to 00:02:35;22 break down these mechanical defenses. 00:02:38;04 They can inactivate some of these chemical defenses. 00:02:40;22 And so when layer 1 fails, innate immunity kicks in. 00:02:44;17 And here we have a combination of cells -- such as macrophages and dendritic cells -- 00:02:49;18 as well as molecules -- proteins of the complement cascade and hormone-like substances referred to as cytokines -- 00:02:55;15 that collaborate to provide protection. 00:03:00;09 In turn, the output of the innate immune system synergizes with adaptive immunity. 00:03:06;16 And this layer of defense really becomes important when innate immunity fails. 00:03:10;21 So, the products elaborated in the course of an innate defense prime the pump, 00:03:16;04 so to speak, and facilitate the ensuing adaptive response. 00:03:20;08 This comprises types of lymphocytes that I'll discuss in a moment. 00:03:24;08 But it's really the synergy between innate and adaptive immunity that makes a key contribution 00:03:29;19 to host defense. 00:03:32;20 If we look at the kinetics with which these processes unfold, it recapitulates some of 00:03:37;20 the items that I've already spoken to you about. 00:03:40;25 Innate immunity consists of molecules such as type-1 interferons, natural killer cells... 00:03:47;08 and these kick in literally within hours to days of exposure to the pathogen. 00:03:54;00 If we look at what happens to the virus titer -- if we deal with, say, an influenza virus infection -- 00:03:59;12 we see that innate immunity can rapidly reduce the number of circulating virus particles, 00:04:04;27 albeit not to zero. 00:04:06;23 And it is at this point that adaptive immunity must kick in. 00:04:10;09 We have virus specific CTLs; the abbreviation stands for cytotoxic T lymphocytes. 00:04:17;00 And we have antibody titers that rise as the infection is being resolved. 00:04:23;09 In a first exposure, the rise in antibody titers is relatively modest. 00:04:30;09 And in a phenomenon referred to as immunological memory or recall response, the secondary exposure 00:04:36;13 rapidly leads to massive induction of both antibody titers, we have memory killer T cells 00:04:44;00 that kick in, and it's the combined action, again, of these antibodies and T cells 00:04:50;21 that manages to control the infection. 00:04:54;27 If we think of where these processes occur in the human body, we must consider the circulatory system, 00:05:01;07 which includes arteries and veins. 00:05:03;25 It's the high arterial pressure that allows some fluids to leave the bloodstream, 00:05:09;02 which must be returned to the circulation in the form of lymph. 00:05:13;00 This lymphatic fluid is filtered through specialized structures called lymph nodes. 00:05:18;02 And it's really in these lymph nodes that the immune responses of the adaptive type 00:05:21;24 take place. 00:05:23;06 We should consider the circulatory system as a means of trafficking. 00:05:28;03 It's the vehicle via which lymphocytes, from their site of origin, arrive at their final destination. 00:05:34;22 And so, by monitoring what happens in the bloodstream, we can only get a transient snapshot 00:05:39;24 of what a real immune... immune response looks like. 00:05:42;14 So importantly, all of the important events that start an adaptive immune response 00:05:49;11 take place in specialized lymphoid structures called lymph nodes. 00:05:56;22 On the right, you see the organization of the lymphatic structures in a human. 00:06:02;14 The little ball-like structures are the lymph nodes, through which lymph fluid is filtered. 00:06:07;07 And it's really in these specialized structures that adaptive immunity is initiated. 00:06:14;10 One important cell type that we will revisit later on in this presentation are 00:06:20;19 so-called dendritic cells, thus named because they have spines that very much resemble what one finds 00:06:25;13 on neurons. 00:06:27;06 And these dendritic cells are positioned throughout the body. 00:06:31;06 They are really the first point of encounter of a foreign invader with the immune system. 00:06:36;18 And it's the ability of dendritic cells to assess the presence of an invader, 00:06:42;04 to then process that information, and present it to the appropriate cell types within the immune system 00:06:46;20 that is responsible for proper orchestration of these immune responses. 00:06:52;28 If we ask, what cell types contribute to adaptive immunity? 00:06:57;02 They are really the lymphocytes that I'll speak about most. 00:07:01;08 If we consider the origin of lymphocytes, they all derive from a stem cell that arises 00:07:05;24 in the bone marrow. 00:07:06;24 These so-called hematopoietic stem cells give rise to all bloodborne cells, 00:07:11;20 including platelets, red blood cells, and so forth, as shown on the left branch of this slide. 00:07:17;02 But importantly, for the remainder of the discussion, will consider mostly the lymphocytes. 00:07:21;26 They originate from a common lymphoid precursor and, through a series of carefully orchestrated 00:07:27;09 differentiation steps, they give rise to both B lymphocytes and T lymphocytes, thus named 00:07:32;20 because of their bone marrow and thymic origin, respectively. 00:07:37;04 The output of B lymphocytes are so-called antibodies or immunoglobulins, 00:07:40;23 a diagram of which is shown in the top. 00:07:42;24 And I'll return in some detail to the structural features of this class of molecule. 00:07:48;17 But let me point out that these antibody molecules, or immunoglobulins, exert a number of functions 00:07:53;27 that can contribute to protection. 00:07:57;02 First of all, they enhance phagocytosis. 00:08:00;15 This is the process by which the dendritic cells that I've just mentioned can 00:08:03;27 acquire particulate matter and process it to cells of the immune system. 00:08:09;04 Antibodies can also assist the function of elements of the innate immune system. 00:08:13;17 On the bottom left, I've shown natural killer cells. 00:08:16;13 They can bind immunoglobulins through receptors specific for them. 00:08:20;14 And once their union has occurred, they can assist in the killing of targets to which 00:08:24;28 the antibody is bound. 00:08:27;25 On the top right, you see yet another mechanism by which antibodies can confer protection. 00:08:33;07 And this is complement-mediated cytotoxicity. 00:08:36;24 In addition to immunoglobulins that circulate in the bloodstream, there's a class of proteins 00:08:41;25 called the complement proteins that, when properly activated, can directly exert 00:08:46;19 a cytolytic effect, either on bacteria or, as shown in this particular example, on tumor cells. 00:08:54;22 And then finally -- and this is one of the earliest discoveries as far as immunoglobulin 00:09:00;08 function is concerned -- immunoglobulins or antibodies can neutralize bacterial toxins. 00:09:06;28 They can bind to virus particles. 00:09:08;24 And by covering the surface of these structures, render them pretty much innocuous. 00:09:13;17 So, these are the many functions of immunoglobulins. 00:09:16;27 And the one that I've left out so far is the one on the top left. 00:09:21;05 We also have practical applications of immunoglobulins. 00:09:24;15 And spectacular recent examples include the immunotherapy of cancer. 00:09:28;21 And, as I'll show in the second half of my talk, we can make derivatives of these antibody fragments 00:09:34;04 and use them for purposes such as imaging of immune responses, non-invasively. 00:09:40;12 So, what about the structure of immunoglobulins? 00:09:43;02 As this cartoon illustrates, they are proteins abundantly present in serum. 00:09:48;11 They're glycoproteins composed of two identical heavy chains, in dark blue, 00:09:52;27 and two identical light chains, in light blue. 00:09:55;26 The heavy chains are glycosylated, and the light chains and heavy chains are held together 00:09:59;24 by disulfide bonds. 00:10:02;00 Biochemists would like to shrink the immunoglobulin molecules into units that retain the capacity 00:10:06;26 to bind antigen. 00:10:08;08 And for this purpose, proteolytic digestion has been used. 00:10:12;03 On the bottom left, you see the products that result from digestion with the protease papain. 00:10:16;13 It results in the release of fragments that are so-called Fab fragments. 00:10:21;24 They are monovalent and retain the capacity to bind antigen. 00:10:25;28 If you wish to retain the capacity of bivalent binding, 00:10:29;26 an intrinsic property of the immunoglobulin molecule, pepsin digestion may be used. 00:10:34;11 And this allows the two antigen-binding fragments to remain linked through disulfide bonds, 00:10:39;27 as indicated on the bottom right. 00:10:42;25 If we look at the diversity of immunoglobulins as they occur in the typical mammalian species, 00:10:48;24 there is massive diversity in structure and function. 00:10:52;00 I won't have the time to discuss all of these diverse functions, but I do want to highlight 00:10:56;09 a few of the salient structural differences. 00:10:59;08 We have here this massive pentameric structure of a class called immunoglobulin M or IgM. 00:11:06;03 We have a version of immunoglobulins that's found in secretions such as tear fluid, 00:11:10;10 held together by an unusual protein called the J chain. 00:11:13;28 We have the IgE molecule, implicated in allergic reactions. 00:11:19;05 And what most of you are probably familiar with are the immunoglobulins of the IgG classes, 00:11:24;11 of which several subclasses exist. 00:11:27;24 Now, when we look at the ability of an antibody molecule to bind a foreign substance, 00:11:34;08 also called an antigen, we realize that the immunoglobulin contacts the antigen 00:11:39;02 at the very tip of this Y-shaped structure. 00:11:42;15 And because structural biologists have been able to solve the three-dimensional structure 00:11:46;11 of antibody fragments in complex with antigen, we know at atomic resolution exactly how these 00:11:52;17 acts of binding occur. 00:11:54;02 So, in this box here, you see at higher magnification the typical mode of interaction of an immunoglobulin 00:12:01;07 with its antigen. 00:12:03;00 You'll realize that the immunoglobulin, composed of two identical heavy chains 00:12:07;06 and two identical light chains, 00:12:09;00 uses elements of both to achieve this specific recognition. 00:12:12;22 So, in light blue, the variable region of the light chain; 00:12:16;08 in dark blue, the variable region of the heavy chain. 00:12:18;25 And it is through the tips of these very subunits that interactions occur with the antigen. 00:12:25;24 These include hydrophobic interactions, salt bridges, van der Waals interactions... 00:12:30;23 a perfectly complementary surface is created to confer specificity. 00:12:35;22 And we know that antibodies can achieve a degree of specificity that allows them 00:12:39;17 to distinguish between molecules that differ in as little as one proton. 00:12:44;04 The presence or absence of a hydrogen atom can make all the difference 00:12:47;20 -- whether or not an antibody recognizes its target or not. 00:12:52;12 So, if we consider the ability of the immune system to mount an immune response against 00:12:57;27 pretty... any... pretty much any foreign substance we throw at it, we must ask the question, 00:13:03;06 how does the immune system achieve this remarkable result? 00:13:07;09 So, first of all, biochemists, without recourse to any molecular genetic tools, 00:13:16;28 accumulated large numbers of sequences of immunoglobulin proteins. 00:13:21;04 And this allowed them to relate the primary structure 00:13:24;09 -- that is to say, the amino acid sequence of the immunoglobulin variable regions -- 00:13:29;16 to their antigen-binding properties. 00:13:30;24 And by aligning multiple sequences of either the heavy chin or light chain variable regions, 00:13:36;13 several salient features emerged. 00:13:39;03 The so-called hypervariable regions, indicated in red, are precisely those regions in the molecule 00:13:45;23 that contact the antigen. 00:13:47;27 And if one compares a large number of different sequences, that is also where 00:13:52;04 the majority of sequence diversity is concentrated. 00:13:56;02 This is not to say that other residues cannot vary, as is clear from the gray bars, 00:14:00;23 which indicate the variability index -- the extent to which different variable regions might 00:14:05;06 differ from one another -- but the bulk of the variation occurs in these three hypervariable regions, 00:14:11;19 also called complementarity-determining regions because that is exactly where 00:14:16;28 the binding of the antigen occurs. 00:14:19;11 Now, if one were to consider a million different antigens against which we would like to 00:14:25;08 raise an antibody, and you calculate the amount of genetic information required to encode 00:14:30;19 that information in the germline of an organism, you quickly reach the conclusion that 00:14:35;17 you run out of sequence space. 00:14:37;17 There is simply not enough DNA to encode, at the DNA level, the structure of a million 00:14:44;15 distinct antibody fragments. 00:14:46;21 And this is a question that has puzzled immunologists for decades until, in the '70s, 00:14:51;23 the molecular mechanisms by which diversity is generated became to be understood. 00:14:58;02 It turns out that immunoglobulin genes are, like many eukaryotic genes, genes in pieces. 00:15:05;18 But there's an additional element of surprise, here. 00:15:08;24 In fact, when we create a functional immunoglobulin gene, it's not just about introns and exons 00:15:14;18 that require splicing to create a functional messenger RNA. 00:15:18;22 The very cells that produce these immunoglobulins reshuffle their genetic information. 00:15:23;12 This is called somatic gene rearrangement, and it accounts for much of 00:15:27;20 the diversity of the immunoglobulins as proteins. 00:15:31;17 On the top of this diagram, you'll see our current understanding of how the light chain locus operates. 00:15:38;08 In mice and humans, there are two types of light chains called kappa and lambda, 00:15:42;12 and I'll confine myself to a quick description of what happens for the kappa light chain. 00:15:47;14 We have a battery of variable region sequences, separated by intervening DNA, followed by 00:15:53;22 so-called joining segments, and, at some distance downstream of it, the remainder of 00:15:59;16 the kappa light chain, the so-called constant region. 00:16:03;01 In the course of B cell development, somatic gene rearrangements occur, and this allows 00:16:07;28 juxtaposition of a randomly chosen V gene element with a randomly chose chosen J segment. 00:16:14;15 And it's not until this rearrangement process is complete that we arrive at 00:16:17;28 a functional light chain. 00:16:20;15 You'll notice that I've indicated the presence of an enhancer. 00:16:23;23 The promoters that drive expression of a functionally rearranged heavy chain do not come 00:16:29;00 within controlling distance of these enhancers unless and until somatic gene rearrangement has occurred. 00:16:34;11 So, the rearrangement process achieves two things. 00:16:37;10 First, it creates a functional unit that can be transcribed and translated into what 00:16:42;21 we know to be a light chain. 00:16:44;19 And second, its expression, its transcription, is controlled by an enhancer, 00:16:49;14 the function of which requires the rearrangement process. 00:16:52;24 For the immunoglobulin heavy chain locus, the situation is somewhat more complex. 00:16:58;04 In addition to this battery of these V segments and J elements, we have interposed 00:17:03;22 a battery of so-called diversity elements. 00:17:06;15 And in this case, the rearrangement process makes use of V, D, and J rearrangement 00:17:11;27 to arrive at a functional heavy chain variable region. 00:17:16;24 There is, again, an enhancer, the reach of which does not extend into those V genes 00:17:23;09 that have yet to rearrange. 00:17:25;04 And it's only upon completement... completion of the rearrangement process that the VDJ combination 00:17:30;18 is placed within controlling distance of this enhancer to enable expression of 00:17:35;27 a functional heavy chain. 00:17:39;11 This process is perhaps best compared to the one-armed bandit. 00:17:43;04 Think of V, D, and J elements as three independently spinning wheels on a slot machine. 00:17:49;17 The B cell, in the course of development, pulls the handle, and some random combination 00:17:54;08 of these Vs, Ds, and Js emerges. 00:17:57;24 This is not the whole story. 00:18:00;15 In this particular diagram, I've recapitulated what I've just told you -- for the heavy chain locus, 00:18:06;24 a battery of these Vs, Ds, and Js. 00:18:09;17 And in the course of B cell development, these rearrangements to which I referred occur 00:18:14;12 in highly ordered fashion. 00:18:16;14 First we have the D-to-J rearrangement. 00:18:19;04 And what I've indicated here by this little segment of rainbow-colored material in between 00:18:23;19 is a phenomenon called junctional imprecision. 00:18:27;11 When a D and a J element are juxtaposed, the act of recombination itself produces 00:18:33;05 some imprecision at the joint, adding and subtracting nucleotides in an unpredictable fashion. 00:18:40;04 And as you might imagine, if you disrupt the reading frame, you have what is called 00:18:44;08 a non-productive rearrangement. 00:18:46;15 If you add multiple nucleotides, you can affect the primary structure of the final product. 00:18:52;26 And so this imprecision in the course of V, D, and J rearrangement contributes to 00:18:59;02 diversity of the final product. 00:19:01;19 Not only do we see this junctional imprecision when Ds and Js rearrange, it also applies 00:19:06;23 when Vs are brought in to hook up with the newly generated DJ combination. 00:19:13;07 And if that weren't enough, there is an enzyme called terminal deoxynucleotidyl transferase 00:19:18;15 or TdT. 00:19:20;09 And this enzyme, in template-independent fashion, adds random nucleotides whenever Ds and Js, 00:19:26;21 or Vs and Ds, are joined together. 00:19:29;22 This massively expands the diversity of the final product. 00:19:33;16 And so if we consider the problem of antibody diversity, it is the combination of 00:19:38;07 a random choice of Vs, Ds, and Js, but that information is strictly germline encoded. 00:19:42;21 But the very act of somatic recombination introduces an element of imprecision 00:19:48;04 whenever joining occurs. 00:19:49;21 And this allows massive expansion of diversity of the immunoglobulin variable regions. 00:19:55;00 So, this slide summarizes much of what I've told you already. 00:19:59;10 In this case, for the light chain, I've indicated the positions of variability. 00:20:05;02 On the bottom, you see these hypervariable regions to which I made reference. 00:20:09;09 And the constant region, as the name suggests, is invariant in sequence and doesn't 00:20:14;00 make contact with antigen. 00:20:15;25 It serves to mediate interactions between the various building blocks of the immunoglobulin 00:20:21;01 molecule itself. 00:20:22;11 These ovals are referred to as immunoglobulin domains, and they all share a conserved sequence. 00:20:30;16 If we consider the different manifestations of immunoglobulins as they occur on the 00:20:36;11 surface of a B cell, we realize that there's an important cell biological distinction to be made. 00:20:42;02 B cells make both membrane-bound immunoglobulin, and that very same immunoglobulin can be secreted 00:20:47;13 as well. 00:20:48;20 This is a process that's controlled by alternative polyadenylation. 00:20:52;24 Depending on which poly-A addition site is used, the B cell either produces 00:20:57;25 the secreted version or the membrane-bound version of that one-and-the-same immunoglobulin. 00:21:04;03 This foreshadows the important role of the B cell receptor in perceiving antigen and 00:21:08;10 allowing B cells to expand, but also to allow that very same B cell to release immunoglobulins 00:21:13;08 into the bloodstream, where they can exert their effect, for example, by neutralizing 00:21:18;04 a virus. 00:21:20;05 The B cell receptor also plays a key role in orchestrating the processes that I've just summarized. 00:21:25;15 So, in the absence of a functional heavy chain rearrangement, B cells fail to complete development. 00:21:30;28 The discrete developmental stages are characterized by the presence of so-called surrogate light chains, 00:21:35;24 in this diagram depicted as VpreB and lambda-5. 00:21:40;00 And only when those subunits all come together and form a properly assembled pre-B cell receptor 00:21:46;28 does the B cell enable rearrangement of the missing piece, which is the light chain. 00:21:51;17 So, this pre-B cell receptor, depicted on the left, is a necessary condition for B cells 00:21:57;19 to engage light chain rearrangement. 00:21:59;27 And it's only when all these processes are executed perfectly that we arrive at 00:22:05;04 a fully assembled B cell receptor at the surface of a B lymphocyte. 00:22:10;04 You'll notice these little red and yellow stubs. 00:22:12;26 These are coreceptors, referred to as Ig-alpha and Ig-beta. 00:22:17;01 And they're absolutely crucial, because the B cell receptor itself 00:22:20;06 -- the immunoglobulin subunits -- 00:22:22;22 lack the cytoplasmic tails required for signal transduction. 00:22:26;10 It's the non-covalent association with these accessory subunits -- Ig-alpha and Ig-beta -- 00:22:32;01 that allow so-called immunoreceptor tyrosine-based activation motif, or ITAMs, 00:22:38;18 cytoplasmically disposed, to recruit the requisite kinases that initiate internalization, 00:22:43;19 proliferation of B cells that properly engage the antigen, and so forth. 00:22:47;19 So, to summarize, this would be the structure of a B cell receptor as you would find it 00:22:52;19 on the typical resting B lymphocyte. 00:22:54;23 A membrane-bound version of the IgM molecule in non-covalent association with these 00:23:00;23 accessory subunits, Ig-alpha and Ig-beta. 00:23:03;04 And it's through these accessory subunits that B cell receptors fulfill most of their functions. 00:23:09;22 There's an added layer of complexity. 00:23:11;18 And we'll have to use that when we discuss, in the second part, the unusual attributes 00:23:16;14 of certain antibody molecules made by camelid species, and this is a phenomenon 00:23:20;27 referred to as class switch recombination. 00:23:23;05 Recall that at the outset I referred to the different classes of immunoglobulins -- 00:23:28;03 the hugely complex pentameric IgM all the way down to the more simple IgG molecules. 00:23:34;19 It turns out that a given VDJ combination can be put in juxtaposition with the information 00:23:41;05 that provides the IgM molecule, the so-called new chains. 00:23:45;19 And by a process called class switch recombination, that rearranged VDJ cassette can be placed 00:23:51;06 upstream of whatever constant region you might require to execute the necessary functions. 00:23:57;25 This class switch recombination requires the involvement of the other major class of lymphocytes, 00:24:02;16 this... the T lymphocytes or T helper cells. 00:24:06;00 And there are accessory molecules such as the cytokine, IL-4, and enzymatic functions, 00:24:11;11 activation-induced deaminase expressed in the B lymphocyte, that are an absolute prerequisite 00:24:15;20 to execute the class switch recombination. 00:24:18;20 So at the end of the day, you might end up with an IgG-producing B lymphocyte which takes 00:24:24;11 this VDJ cassette and places it in juxtaposition, in my example, with the gamma-2 constant region. 00:24:32;22 In yet another example, you might take that very same VDJ combination and instead 00:24:37;10 hook it up to the alpha constant region, so that you may suit... that so that you may produce 00:24:42;11 this secreted version of the IgA molecule. 00:24:46;00 Now, how... how is all of this arranged? 00:24:50;17 It turns out that we have a detailed molecular understanding of how this somatic rearrangement process, 00:24:56;01 as well as the class switch recombination, occurs. 00:25:00;02 And unlike the enzymes involved in putting together V, D, and J elements, class switch recombination 00:25:06;00 requires the activity of activation-induced deaminase, expressed in B cells only when 00:25:11;28 properly contacted by T helper cells. 00:25:15;20 In a looping-out reaction, the rearranged VDJ combination is put in juxtaposition 00:25:22;09 with whatever constant region the B cell demands at that point in time. 00:25:26;11 And by physical excision of the intervening DNA, we may now connect the functionally rearranged 00:25:32;17 VDJ combination to whatever constant region we require. 00:25:37;00 Now, importantly, I refer to the role of helper T cells to execute this reaction. 00:25:46;11 To understand a little bit more about how these T cells operate, let me give you 00:25:51;06 the following information. 00:25:54;09 The professional antigen-presenting cells -- think of the dendritic cells which I showed at the very outset -- 00:26:00;04 may acquire antigen, a foreign substance, by a process called phagocytosis. 00:26:05;14 Once the phagocytosed antigen has been internalized and delivered to the appropriate endocytic compartments, 00:26:11;17 these antigens are attacked by proteolytic enzymes and converted 00:26:16;11 into short peptide fragments that will be displayed on the surface of the so-called antigen-presenting cell. 00:26:23;09 There's a special class of molecules involved in this process. 00:26:26;14 These are the products encoded by the major histocompatibility complex, 00:26:30;23 to which I'll return as well. 00:26:32;20 And it's really the combination of these unique peptide- MHC combinations that will be recognized 00:26:38;03 by T lymphocytes by means of antigen-specific receptors. 00:26:44;01 The B cell is a specialized case. 00:26:46;21 It too can bind to antigen by virtue of the fact that expresses, at its surface, 00:26:53;06 the B cell receptor for antigen. 00:26:55;12 The B cell receptor for antigen is really the high-affinity capture device that 00:26:59;28 allows the B lymphocyte to probe what's in the external environment and bind only those protein antigens, 00:27:06;01 or other foreign substances, for which it is specific. 00:27:09;26 It does so by virtue of what we call an epitope. 00:27:13;25 This is a structural feature of the antigen itself that can be seen by the B cell receptor. 00:27:19;06 Now, B cells can internalize the B cell receptor when complexed with antigen. 00:27:24;00 And by the same mechanism that I've just described, proteolytic activity will chop up the foreign protein 00:27:29;28 into short synthetic fragments, which are bound by these MHC products and presented 00:27:35;18 on the surface of the B lymphocyte. 00:27:38;04 It is the T cell that now recognizes, by means of its antigen-specific receptor, the unique 00:27:44;19 combination of peptides derived from the original antigen, presented by products of the MHC. 00:27:50;26 And the key concept to understand here is that the features of structure that 00:27:55;25 allowed the B cell to recognize antigen in the first place may well be distinct from the fragments 00:28:00;22 generated from that antigen and presented via MHC molecules to T lymphocytes. 00:28:06;13 This phenomenon is called linked recognition, and it ensures that only those B cells that 00:28:11;11 have acquired antigen and present peptides derived from it to appropriately specific T cells 00:28:16;22 that an antibody response can ensue. 00:28:20;01 So, to integrate all of this, and without going through the details... on the far left, 00:28:25;19 you'll see dendritic cells acquiring antigen and presenting it to T helper cells. 00:28:30;13 In the right half, you'll see B cells acquiring antigen and presenting peptides to T cells 00:28:35;04 of appropriate specificity. 00:28:36;17 And when all is said and done, we have a productive interaction between the T helper cell, 00:28:41;27 which is antigen specific, and the B cell, that is antigen specific. 00:28:46;25 And so this is how we can orchestrate an immune response. 00:28:50;22 I mentioned the fact that there are two major classes of lymphocytes: the B lymphocytes, 00:28:55;19 which we just discussed, and T lymphocytes, which as we saw provide necessary help 00:29:01;17 and also generate so-called killer T cells, or cytotoxic T cells. 00:29:06;19 They have antigen receptors very much like the B cell receptors we discussed. 00:29:11;09 And they make use of very similar rearrangement processes, in fact employing the exact same 00:29:16;20 enzymatic machinery. 00:29:18;02 So, the T cell receptor, like its immunoglobulin counterpart, is composed of two subunits: 00:29:22;23 alpha and beta subunits. 00:29:25;04 And they, like their immunoglobulin counterparts, make use of V-to-J and V-to-DJ rearrangements, 00:29:32;08 as diagrammed in this cartoon. 00:29:33;28 Each element is flanked by the appropriate recognition signal sequences, 00:29:38;01 features of structure that are shared with the immunoglobulin variable regions 00:29:42;06 of the heavy and the light chain. 00:29:44;18 Now, T cells, as I've said, recognize antigen not in solution but bound to the products 00:29:50;28 of the major histocompatibility complex. 00:29:53;24 As diagrammed in this cartoon, you see a T cell receptor with its two subunits engaging 00:29:59;26 a class-I MHC product, thus named because it spans the lipid... lipid bilayer only once. 00:30:06;14 And these MHC products present these short snippets of foreign protein to antigen-specific receptors 00:30:12;15 on T cells. 00:30:14;03 In the second part, I'll have a few words to say about these so-called co-stimulatory 00:30:18;16 or checkpoints. 00:30:20;02 These are molecules that can fine-tune immune responses, and either enhance or inhibit 00:30:24;20 immune recognition by T lymphocytes. 00:30:27;00 Now, the MHC products are unique in structure because, notwithstanding the fact that 00:30:33;25 they are of unique and fixed sequence, they can nonetheless bind a vast diversity of peptides 00:30:41;12 by virtue of the fact that the architecture of the peptide binding pocket is designed 00:30:45;15 such that many peptides of different sequence can fit into one-and-the same peptide binding pocket. 00:30:52;14 The overall global structure of a class-I MHC product is composed of a heavy chain 00:30:58;18 in non-covalent association with its light chain, beta-2 microglobulin. 00:31:03;00 And it's this assembly that creates the peptide binding pocket -- this is the top view of 00:31:07;04 the very same molecule shown here -- into which peptides bind for presentation 00:31:12;20 to these antigen-specific receptors. 00:31:17;12 The way in which this system functions is that T cells are test-driven on MHC products 00:31:23;00 that present peptides from our own self proteins, which you ideally would like to ignore. 00:31:28;13 And it's not until a stressful situation such as cancer or infection occurs that 00:31:33;08 new peptides derived either from pathogen-specific proteins or tumor-specific antigens 00:31:39;18 make their appearance. 00:31:40;18 So, the immune system is taught to ignore peptides of our own proteins. 00:31:47;03 And what remains at the end of the day is a repertoire of T lymphocytes uniquely capable 00:31:51;18 of recognizing peptide-MHC complexes that differ from our own self-MHC products. 00:32:00;13 If you think of an infectious situation, in the absence of any immune recognition, 00:32:08;20 unopposed infection might result in the organism’s death. 00:32:11;23 We have lytic infections. 00:32:13;07 We have massive virus production. 00:32:15;19 And it is for this reason that we have components of the adaptive immune system, to fight specifically 00:32:21;01 these kinds of events. 00:32:22;14 I've mentioned the fact that antibodies can neutralize virus particles in the circulation. 00:32:27;05 That is one means of protection. 00:32:29;14 I've indicated the existence of so-called killer T cells, the CD8-bearing T lymphocytes. 00:32:36;02 CD8 is a glycoprotein marker uniquely confined to these killers. 00:32:41;06 And by means of their antigen-specific receptors, they recognize class-I MHC products that present, 00:32:46;18 for example, viral peptides as in this example. 00:32:50;20 But because many pathogens have replication times vastly shorter than the host, 00:32:57;02 they can acquire mutations that allow them to elude immune attack. 00:33:00;17 And that's depicted by the transition of this somewhat innocuous pink virus to the nasty red. 00:33:07;09 Many of these viruses do so by, for example, altering expression of class-I MHC products, 00:33:12;26 and that also happens to be one of the mechanisms by which cancerous cells can evade detection 00:33:18;02 by T lymphocytes. 00:33:19;23 If you eradicate expression of class-I MHC products, you're essentially invisible 00:33:25;22 to the cytotoxic T lymphocyte, and that gives you the upper hand in terms of virus production 00:33:31;11 or, in the case of a cancerous cell, replication. 00:33:34;11 Now, we know a great deal about the molecular details by which the class-I proteins acquire 00:33:40;00 their peptide cargo. 00:33:41;27 From a cell biological perspective, this is a very unusual and interesting series of reactions. 00:33:47;01 And it focuses on the function of the ubiquitin pathway. 00:33:51;06 Proteins in the cytoplasm are modified by ubiquitin in an enzymatic cascade that involves 00:33:56;06 these three classes of enzymes: E1s, E2s, and E3s. 00:34:00;15 And having modified our protein with multiple ubiquitin molecules, now these proteins 00:34:05;20 are poised for recognition by the proteasome, which in a highly processive fashion 00:34:10;16 destroys these proteins and produces peptides capable of being recognized by T lymphocytes. 00:34:15;23 The problem, however, is the fact that the entire machinery for the generation of peptides 00:34:21;03 is located in the cytoplasm, whereas the molecule charged with antigen presentation 00:34:26;14 lives in extracellular space. 00:34:28;18 So somehow we must deliver peptides to extracellular space. 00:34:32;18 And this is the function of a dedicated transporter referred to as the transporter associated 00:34:37;23 with antigen presentation, or the TAP protein, indicated by this array of helical segments here. 00:34:47;03 Once peptides are translocated into the endoplasmic reticulum, they become part of 00:34:52;01 a nascent class-I MHC product, which itself requires the action of a panoply of chaperones to ensure its 00:35:00;04 proper folding. 00:35:01;04 But when all is said and done, we make this peptide-MHC complex, which is then free 00:35:05;09 to travel to the cell surface. 00:35:07;01 And as I've suggested in the preceding slide, viruses are masters of deception. 00:35:12;07 They've evolved numerous countermeasures with which to frustrate this process of antigen presentation. 00:35:18;09 And here's just an example taken from herpes viruses, one class of pathogens that 00:35:23;09 once you acquire them stay with you for the rest of your life. 00:35:26;26 We have proteins that in... such as pp65 that involve... that interfere with ubiquitylation 00:35:34;25 of possible targets. 00:35:37;24 The virus that is the causative agent of mononucleosis, Epstein-Barr virus, produces a protein 00:35:43;22 that renders viral products insensitive to proteolytic digestion by the proteasome. 00:35:49;03 We have other herpes virus-encoded proteins that impede peptide translocation into 00:35:53;08 the endoplasmic reticulum, detain class-I molecules at the site of synthesis, 00:35:58;23 or even reverse the process of membrane insertion and target those very same MHC products 00:36:03;19 for proteasomal degradation. 00:36:06;01 The process is more complex than this. 00:36:09;04 We have meanwhile figured out some of the details. 00:36:11;14 This is the mating dance between the viral protein US2 and the class-I molecule it destroys. 00:36:17;26 And in a process referred to as retrotranslocation, a newly assembled class-I heavy chain is 00:36:23;16 sent back to the cytoplasm for proteasomal degradation. 00:36:27;01 This is just one example of the many tricks viruses can use to frustrate adaptive immunity. 00:36:33;02 And such interference may apply to other surface proteins, cytokines released from the cell, 00:36:39;10 aspects of innate immunity. 00:36:40;11 I need to emphasize the fact that the constant interplay between the immune system, 00:36:45;27 which exerts a selective pressure, and pathogens, which have the capacity to rapidly evolve, 00:36:51;22 results in this per... perpetual chess game between host and pathogen. 00:36:57;13 Much of this work enables cell biological explorations that would be difficult to achieve otherwise. 00:37:03;17 And to put some molecular detail on this particular cartoon, this would be our current understanding 00:37:09;04 of how this complicated machine operates. 00:37:11;25 We have this centrally positioned class-I MHC product and a host of other cofactors 00:37:17;12 that together ensure that this class-I protein in a virus-infected cell can be extracted 00:37:22;24 from the endoplasmic reticulum and ultimately targeted for proteasomal degradation. 00:37:27;27 So, after this whirlwind tour of the immune system, let me return to where we started. 00:37:33;19 We have a multi-layered immune defense system, of which the mechanical and innate immune defenses 00:37:39;13 are probably the most important on a daily basis. 00:37:42;20 But once these systems fail, adaptive immunity kicks in. 00:37:46;26 And the remarkable precision with... with which the adaptive immune system can recognize antigens 00:37:51;23 has allowed the explorations which I've tried to summarize in the preceding 00:37:56;17 thirty minutes or so. 00:37:58;02 Key features: ability to distinguish between structures that differ by very little -- as few as an atom, perhaps; 00:38:06;06 the ability to respond rapidly; 00:38:09;25 and the ability to adjust the specificity of the ensuing response to whatever the needs of the day may be. 00:38:17;14 In the second part of my talk, I will highlight one specific element of this adaptive immune system. 00:38:23;22 And we'll see how this can be leveraged into tools that might be useful, 00:38:28;24 both for basic cell biology as well as for biomedicine.