Session 5: B Cells: Development, Selection, and Function
Transcript of Part 1: Early B Cell Development: A Look at the Defining Questions in Immunology
00:00:07;06 I'm gonna be talking today about some of the defining questions in immunology. 00:00:11;15 And I'm gonna take you back to the 19th century. 00:00:17;07 So, the Franco-Prussian war ended in 1872 with the siege of Paris. 00:00:25;13 And this rivalry between the French and the Germans carried on into the field of science 00:00:31;28 for the next few decades. 00:00:33;20 Two of the most important investigators of the time were Robert Koch in Berlin and 00:00:41;00 Louis Pasteur in Paris. 00:00:42;25 And their laboratories had helped establish the germ theory of disease, showing us 00:00:47;24 for the first time that disease was actually caused by pathogens, by microbes, and not by some 00:00:53;28 unknown imbalance of bodily humors. 00:00:57;19 The pace of discovery was truly remarkable. 00:01:00;24 To just cite one example, in 1884, Loeffler in Berlin described the Bacillus that causes diphtheria. 00:01:09;15 In 1887, Roux and Yersin in Paris showed that these bacteria secreted a toxin 00:01:15;21 into culture supernatants, and this was a lethal toxin. 00:01:19;06 In 1890, Brieger and Frankel, in Koch's laboratory in Berlin, showed that they could 00:01:25;16 inject the stocks in... into small animals and showed some form of immunity. 00:01:31;00 Later that same year, also in Koch's laboratory, Shibasaburo Kitasato and Emil von Behring 00:01:38;24 chemically inactivated the diphtheria toxin, injected it into large animals in increasing doses, 00:01:44;10 and showed the presence of a principle in the serum of these animals which they called 00:01:50;15 an antitoxin, which could neutralize the diphtheria toxin. 00:01:55;22 Within a year of the... of this discovery, children who were dying -- gasping for breath 00:02:01;11 from diphtheria -- were being miraculously saved by the injection of this antitoxin. 00:02:09;19 Serum therapy caught on all across the world. 00:02:12;02 This was a remarkable new form of therapy, which was the first scientific approach 00:02:18;10 to actually treating a disease. 00:02:19;28 A young man called Paul Ehrlich returned to the laboratory from Egypt, came back to Koch's laboratory. 00:02:27;08 He saw all this excitement about serum therapy and antitoxins. 00:02:31;28 And he realized, while his colleagues had made a remarkable discovery, a life-saving discovery, 00:02:36;22 what they had missed was an underlying principle. 00:02:41;28 And the underlying principle was that when a foreign substance is injected into a vertebrate 00:02:49;11 it creates complementary molecules. 00:02:53;27 Ehrlich would name these molecules that were injected into animals as antigens. 00:03:00;17 And the complementary molecules, which were then produced by the recipient animal, 00:03:05;24 he called antibodies or antikorpers. 00:03:09;17 Ehrlich was a remarkable scientist. 00:03:12;03 He would make many discoveries in his lifetime. 00:03:14;07 As a medical student, he described the existence of mast cells. 00:03:18;25 He had devised stains, and he looked and found a new cell type which he then described, 00:03:23;27 which is an important cell in immunity. 00:03:26;06 He would go on to describe antigens and antibodies. 00:03:29;12 He would also describe a phenomenon that he called horror autotoxicus, or autoimmunity. 00:03:35;04 He predicted that there might be immune phenomena where we attacked ourselves. 00:03:39;25 He's the first person to think about the immune surveillance of cancer. 00:03:44;12 He was also the first person to start the field of chemotherapy. 00:03:48;22 The entire field of cancer chemotherapy and antibody therapy owes a debt to Ehrlich, 00:03:54;15 who came up with the first chemotherapeutic. 00:03:57;22 But his most remarkable contribution was to think about a model for how the immune system 00:04:04;24 worked. 00:04:06;03 And this he called the side chain hypothesis. 00:04:09;15 And in a sense, you have to think about a time 60 years before the fluid mosaic model 00:04:16;01 of the membrane is known, more than 65 years before we have the first polypeptide 00:04:22;12 hormone receptor described, and Ehrlich comes out with the model where he imagines 00:04:26;26 an immune cell has antibodies on the surface as receptors. 00:04:31;25 And then he visualizes antigens coming, triggering the immune cell -- so, triggering a receptor -- 00:04:37;04 and then the cell induces more of the same antibody. 00:04:40;21 So, this remarkable hypothesis, which is described in this slide, is to suggest, as Ehrlich did, 00:04:49;02 that we already have all these different antibodies on the cell surface and that an antigen 00:04:54;28 comes by, interacts with one of these an... anti... antibodies, and triggers the release of 00:05:00;28 the same antibody into the blood. 00:05:03;13 Now, at this time, when he came out with this model, Ehrlich had also done experiments 00:05:09;15 to show that he could modify a chemical at a single atom and he could create a new antibody. 00:05:17;17 Karl Landsteiner, who was one of Ehrlich's greatest opponents, intellectually, had done 00:05:22;10 similar experiments. 00:05:24;04 He had taken chemicals, also modified them at a single atom, and he had made new antibodies. 00:05:29;12 So, there were antibodies which are highly specific and could recognize 00:05:33;26 a single atom difference between two molecules. 00:05:36;27 It was clear that there was an immense number of antibodies. 00:05:42;04 The number of antibodies within us was probably infinite. 00:05:46;26 How could you possibly conceive of the fact that we already have all these antibodies 00:05:52;01 within us? 00:05:53;03 That was what Ehrlich was suggesting. 00:05:55;00 So to reframe this, the clear picture that emerged at that time was, how can we 00:06:02;04 create complementary structures? 00:06:04;03 Are these pre-existing? 00:06:06;10 Or have they to be induced? 00:06:09;01 To give you an analogy, imagine you have a hundred people showing up for a job at UCSF, 00:06:14;11 or they are going to start at UCSF today, or maybe at Google or wherever you want. 00:06:19;25 Each one of these people goes to an office and gets an ID card made. 00:06:24;24 And the ID card requires that he has a photograph taken, and then it gets laminated, 00:06:30;01 and then you have an ID. 00:06:32;02 Now, this is a model where you show up, you get your photograph taken 00:06:36;26 -- so, this is an induced model -- 00:06:39;15 and then you get your ID. 00:06:41;27 Imagine on the other hand, if you showed up at Google and they already had your picture 00:06:46;22 on file. 00:06:47;22 In fact, they had your great-grandchildren's pictures on file, and your grandfather's pictures 00:06:52;06 on file. 00:06:53;06 They had pictures on file for everybody who is around in the world today, or who will 00:06:58;05 ever be around in the universe. 00:06:59;19 And that was the model Ehrlich was proposing, that we already have all the antibodies 00:07:04;26 within us before an antigen shows up. 00:07:07;22 And this seemed inconceivable, so most thinkers in the field moved away from Ehrlich, 00:07:14;18 and the first people to actually formulate this, they were Haurowitz and Breinl, who came up 00:07:20;07 with the model for a direct template. 00:07:23;11 Linus Pauling, the great chemist, took this further and essentially suggested that antibodies 00:07:28;10 came from one of these pink primordial proteins within the cell. 00:07:32;28 An antigen enters the cell. 00:07:34;28 The antigen then reacts with the antibody. 00:07:37;05 And the... the antibody, the preformed antibody, folds around the antigen, forms a new shape, 00:07:43;02 and then is secreted. 00:07:44;21 And this was the model that existed 'til 1957, because people couldn't accept that you could 00:07:50;26 already have pre-existing repertoires which went into 10^12 different possibilities, 00:07:58;18 and you have these many different antibodies within you. 00:08:03;05 In the 1950s, Jerne would first suggest that maybe there's a model where we already 00:08:08;12 have all the antibodies secreted from us and provided some data for that. 00:08:13;11 And then David Talmage would come up with another model, which is now called 00:08:18;13 the clonal selection hypothesis. 00:08:19;20 And this was further refined by... by Frank MacFarlane Burnet. 00:08:24;04 And the model essentially states that we already have a range of immune cells, 00:08:30;10 each with a different receptor. 00:08:33;07 An antigen comes by, identifies one of those cells, the cell gets triggered 00:08:38;10 -- so, this is now a clone of lymphocytes -- the clone expands -- so, we have clonal expansion -- 00:08:45;04 and then after clonal expansion we have these cells secrete antibodies, which are then going to 00:08:50;08 be the effector molecules of the immune system. 00:08:53;01 So, this was a model, which is now the accepted model of the immune system. 00:08:57;11 We... we already have pre-existing immune cells, each with a different receptor. 00:09:04;14 And then antigen comes and triggers one of them. 00:09:06;18 And this holds true both for T cells and for B cells. 00:09:11;09 When the clonal selection hypothesis was put forward, we didn't know what the immune cells 00:09:18;03 for adaptive immunity were. 00:09:20;20 At this time in 1957, we thought these were just macrophages, or some macrophage-like cell. 00:09:28;02 We would soon learn that there were different cells that mediated these functions for the immune system. 00:09:35;14 So by the 1960s... so, antibodies were discovered in 1890... but by the 1960s, we have 00:09:43;17 the first ideas of the structure of antibodies. 00:09:46;23 And this is from the work of Gerald Edelman and Rodney Porter. 00:09:50;17 So, Edelman would show that antibodies are made up of two chains. 00:09:53;24 So, we have these two pink chains, which are the heavy chains, and these two orange chains 00:09:57;26 are the light chains. 00:10:00;06 And these were associated and linked to each other by disulfide bridges. 00:10:05;10 Porter would actually show that the antibody had different domains, and this is shown 00:10:11;06 on the next slide. 00:10:12;06 So, this is a much more modern slide. 00:10:13;24 And you can... you'll note that in the antibody there are heavy chains and light chains, 00:10:19;04 but they can be cleaved by certain proteases to give you fragments called Fab fragments. 00:10:24;24 So, Fab fragments are basically the part of the antibody that binds antigen. 00:10:30;10 And Fc fragments, which are really the tail of the antibody. 00:10:36;05 Crystal structures would then tell us the structure of an antibody domain. 00:10:41;13 And this will all come in the '70s. 00:10:43;08 So, if you looked at an antibody domain... so, here we have a schematic view of a domain, 00:10:50;01 in which you can notice that there is basically a ribbon. 00:10:53;05 So, it's a beta sheet, which is folded over to form a beta barrel. 00:10:59;12 And sticking out on top are some loops, labeled here a CDR1, CDR2, and CDR3. 00:11:06;08 Okay? 00:11:07;08 So basically, you have a ribbon, the ribbon is joined together in a fold, and then 00:11:13;04 each strand of the ribbon is linked the next by a loop. 00:11:16;28 And when you looked at the sequence of antibodies -- so, if you looked at this part of the slide -- 00:11:21;10 you'll notice that there are three regions. 00:11:23;16 We've compared 100 antibodies... there are three regions of the antibody sequence 00:11:29;02 where the sequence varies a lot. 00:11:30;22 And these are called CDR1, 2, and 3. 00:11:34;00 And these correspond to those three loops that stick out from the top of the Ig domain. 00:11:39;24 So, immunoglobulin domains can be variable domains like this, with the CDRs, 00:11:44;19 or they can be constant domains, which make up the rest of the antibody molecule. 00:11:49;12 So, if you think about it differently, the CDRs are like fingers. 00:11:54;19 You have three fingers for the variable domain of the light chain, three fingers for the 00:12:00;20 variable domain of the heavy chain. 00:12:02;25 And the fingers can come close together to bind a small molecule, or they can 00:12:06;20 splay out widely to form a surface that can accommodate a protein. 00:12:10;27 So, on the right, you can see there is actually, over here, an antigen bound to an antibody. 00:12:16;03 It's a large protein antigen. 00:12:17;21 So, you can assume that CDR1, 2, and 3 -- the fingers -- have splayed out to form 00:12:23;00 a big surface to accommodate the antigen. 00:12:27;17 This is another view of the Fab part of an antibody, shown here in yellow and blue. 00:12:33;11 And all the red residues that you see on the Fab correspond to those six fingers, 00:12:39;20 CDR1, 2, and 3 from the heavy chain and CDR1, 2, and 3 from the light chain. 00:12:45;01 One the other side is hen egg lysozyme, in green. 00:12:49;03 And there too, the red residues are the residues on hen egg lysozyme that interact with 00:12:54;21 the red residues on the antibody, on the Fab of the antibody. 00:12:59;12 The glutamine that's shown in purple is actually going to burrow deep into the groove in 00:13:03;22 the middle of the red residues in the antibody. 00:13:09;02 Antibodies have many functions. 00:13:10;19 And most of... most of you are aware of them. 00:13:13;20 They can neutralize viruses and toxins. 00:13:16;22 So, neutralization means that the antibody binds to the virus or to the toxin and 00:13:23;05 prevents it from entering our cells or binding to a receptor. 00:13:27;12 Antibodies can medi... mediate opsonization. 00:13:30;17 So, opsonization really means that a pathogen might be coated with an antibody, and then 00:13:36;27 a receptor on the phagocyte -- which is for the Fc part of the antibody, so, the tail of the antibody -- 00:13:43;17 recognizes the antibody and ingests the pathogen. 00:13:47;15 We call that opsonization. 00:13:49;24 There's another phenomenon called ADCC. 00:13:52;13 Now, in ADCC, you have a virally infected cell. 00:13:56;26 An NK cell recognizes an antibody that's coating the virally infected cell. 00:14:03;28 And this triggers a receptor on the NK cell, which then causes the killing of the 00:14:08;23 virally infected cell. 00:14:10;11 And so that's another function of antibodies, where the antibody coats the target cell, 00:14:15;01 and then an NK cell kills it. 00:14:17;04 And then finally, one of the other functions of antibodies is mediated through complement. 00:14:22;00 So, the complement proteins can lyse the microbes -- so, this is over here -- 00:14:27;03 or fragments of complement can serve as opsins and help internalize pathogens. 00:14:32;26 And you can also have complement fragments which drive inflammation. 00:14:38;07 To summarize this part of the talk, antibodies protect you. 00:14:45;02 But antibodies kill. 00:14:47;24 Autoantibodies can make you very ill. 00:14:52;22 Antibodies coat pathogens; they coat the infected cell. 00:14:55;20 With NK cells and phagocytes, they give those microbes hell. 00:15:01;24 When antibodies fix complement, the shit really hits the fan. 00:15:05;27 You'll be blown to bits, little microbe. 00:15:09;05 Run away, if you can. 00:15:11;27 You take a set of beta strands, you get a beta-pleated sheet. 00:15:16;12 Fold the pleated sheet in two, then the barrel is complete. 00:15:20;04 Clip it with a disulfide bond and you have an Ig fold. 00:15:26;22 Found even in archaebacteria, this domain is old. 00:15:32;25 Loops make connections between the beta strands. 00:15:37;16 If you want to make an Fab, please use both your hands. 00:15:43;01 The loops are like fingers. 00:15:44;22 They stick out at the top. 00:15:48;14 Complementarity-determining regions, the cream of the crop. 00:15:54;16 Three fingers from the heavy, three fingers from the light, come together to create 00:16:01;25 an antigen-binding site. 00:16:04;07 Bring the fingers close together, you get a cleft with a purpose. 00:16:08;12 Splay the fingers widely, you get a protein-binding surface. 00:16:15;13 Antibodies protect you, but, baby, antibodies kill. 00:16:21;22 Autoantibodies can make you very ill. 00:16:25;22 Oh, you Y-shaped globular proteins, with a name that Ehrlich gave, 00:16:32;02 if the good doctor could hear this doggerel, he'd be turning in his grave. 00:16:38;21 The next question I'm going to turn to was long known as the GOD Question, and GOD refers to 00:16:46;11 the generation of diversity. 00:16:49;14 It was impossible, until the late 1960s, to imagine how this question would ever be answered. 00:16:56;09 The question essentially was this. 00:16:58;00 We know we have, now, about 20,000 genes. 00:17:01;14 But we can make maybe 10^14 different antibodies. 00:17:06;00 If you believe that one gene gives rise to one polypeptide, how is this ever going to 00:17:12;18 be conceived of being possible? 00:17:15;08 How can you use a limited number of genes to give you proteins that can recognize 00:17:21;20 10^14 different things? 00:17:23;14 So, this question boggled everybody's imagination. 00:17:26;28 It became one of the central questions of biology, because no one could understand 00:17:31;23 how this could actually happen. 00:17:33;08 And this question remained so mysterious that it was assumed -- I think this was assumed by the early 1970s -- 00:17:41;13 that it would be one of those things that would never be solved. 00:17:45;07 We would never know the answer. 00:17:46;13 So, it was a religious question, okay? 00:17:49;23 Now, we did have some idea about antibody structure. 00:17:52;26 I told you that Porter and Edelman had out with the structure of two heavy chains and 00:17:57;24 two light chains, and the existence of light chains was understood. 00:18:01;25 There were plasmacytomas. 00:18:03;08 So, Henry Kunkel's lab at Rockefeller had described a lot of plasmacytomas. 00:18:07;12 These are tumors that make a single antibody. 00:18:10;11 So, there were... there was the ability to have a pure antibody, a pure light chain, 00:18:15;20 to try to sequence it. 00:18:17;17 But by the early '60s, people still couldn't sequence an entire light chain protein. 00:18:22;19 I mean, insulin had been sequenced, but this was a difficult task. 00:18:26;23 And many groups were trying to sequence light chains. 00:18:30;02 Now, Edelman... his mentor was Henry Kunkel, and Kunkel and Edelman didn't quite get along. 00:18:36;26 And there was a great meeting that was going to be held in California in Warner Springs 00:18:40;10 -- I mean, I... to me it's like Woodstock when I think about Warner Springs -- 00:18:45;10 and everybody who was anything in biology... from Seymour Benzer, to Chris Anfinsen, 00:18:49;23 everybody was invited 00:18:51;03 to come and discuss and describe to others what they could think about how we 00:18:56;11 created diversity in the immune system. 00:18:58;26 So, Mel Cohn was the organizer of this meeting, and he got a call from Rodney Porter, 00:19:03;28 who was in Oxford. 00:19:05;25 And Rodney Porter made this call saying, there is this postdoc at Rockefeller, 00:19:11;07 you don't know who he is, but I've heard about his existence from Henry Kunkel. 00:19:16;14 And this postdoc actually was with a... worked with a friend of Kunkel's called Lionel Craig. 00:19:20;11 And he said, you should call him. 00:19:22;12 His name is Norbert Hilschmann, and he'll have something interesting to tell you. 00:19:26;14 Call him to your meeting in Warner Springs. 00:19:28;20 So, at Warner Springs, many people went and presented their knowledge, what they knew 00:19:35;06 about antibody light chains. 00:19:36;27 They had peptide maps. 00:19:38;06 They couldn't quite figure out what the maps told them. 00:19:41;10 They didn't have the sequence of a light chain. 00:19:43;26 The heavy chains were too difficult. 00:19:45;19 They were too big. 00:19:46;24 And then there was this talk from Norbert Hilschmann, this unknown person, never seen before, 00:19:52;17 never heard of before. 00:19:54;20 He shows up, and he gives his talk. 00:19:58;06 And on his first slide -- and these days... those days you had real slides -- he showed 00:20:02;20 the complete sequence of two antibody light chains. 00:20:06;19 Okay? 00:20:07;19 So, we have two antibody light chains. 00:20:10;27 And the remarkable thing about the sequence was that the light chains were 00:20:15;16 identical in sequence for most of the molecules, but the top third, the top... the N-terminal parts 00:20:21;15 were different. 00:20:23;16 This was a remarkable finding. 00:20:25;28 Everyone was excited. 00:20:27;03 For the first time, there was some sense about how antibodies differed from each other. 00:20:34;00 People stopped Hilschmann. 00:20:35;12 They asked him to go back to his earlier slides. 00:20:38;22 But Hilschmann did not. 00:20:41;20 He moved rapidly through the rest of the slides, he was not collegial, and he left the meeting. 00:20:47;21 And that's probably the reason why he was not the third recipient of the Nobel Prize, 00:20:53;15 along with Porter and Edelman. 00:20:55;06 Because he made a remarkable discovery. 00:20:58;05 But Hilschmann disappeared from public view. 00:21:00;24 He was somewhat concerned that his data would be taken up... taken on by others and so on. 00:21:06;09 But he didn't interact. 00:21:08;11 But someone in the audience listened carefully to this talk. 00:21:11;15 His name was Bill Dreyer, who's a polymath. 00:21:14;23 He's no longer around, but he was at Caltech as a professor, and an assistant professor 00:21:19;05 at the time. 00:21:20;09 And he looked at the data and said. 00:21:22;03 I understand how diversity is created. 00:21:26;26 So, he went back to his lab. 00:21:29;19 Most people didn't understand what he was trying to explain, but someone in his lab 00:21:33;06 understood him. 00:21:34;15 And they sat down and wrote a paper together. 00:21:36;25 And that's called the Dreyer and Bennett hypothesis, published in 1965. 00:21:42;07 And if I can give you an analogy to explain what the hypothesis says, imagine that you 00:21:47;22 have a... a lady with a strange wardrobe. 00:21:51;28 She has just one black skirt, but maybe she has a thousand different tops. 00:21:58;10 So, by mixing and matching a thousand tops with one black skirt she has a thousand outfits. 00:22:05;05 Now, imagine that she has twenty different, very different-looking belts. 00:22:09;16 Again, by mixing and matching these, she could get you twenty thousand different outfits. 00:22:14;20 And this is essentially what Dreyer and Bennett postulated, that genes might come in pieces, 00:22:21;09 and then you have these cassettes, and you can join together different variable cassettes 00:22:26;10 with one constant cassette and create different antibodies in different cells. 00:22:31;11 Okay? 00:22:32;11 You can prove this. 00:22:33;13 It was impossible to prove this at the time. 00:22:35;25 The recombinant DNA revolution hadn't occurred. 00:22:39;21 Finally, when recombinant DNA couldn't be performed in California or in... or in the 00:22:46;20 city of Cambridge, Massachusetts, Susumu Tonegawa went off and worked at 00:22:52;10 the Basel Institute of Immunology, and he did the crucial experiments 00:22:56;26 to show that immunoglobulin genes come in pieces. 00:23:00;08 This is just a Southern blot showing that DNA is that of a different size, so the DNA 00:23:07;19 for the antibody genes is of a different size in all cells other than B cells. 00:23:12;01 So, in a liver cell it's different from B cells. 00:23:14;19 And he did a slightly different experiment, but I'm using this Southern blot to show you this, 00:23:18;22 to show you that DNA had been cut and joined, and something had been done to it 00:23:24;15 in a B cell. 00:23:25;15 And this was Tonegawa's big discovery. 00:23:27;23 Okay? 00:23:28;23 So, when you look at immunoglobulin genes, we now know that these genes come in pieces. 00:23:34;00 We have a whole range of V segments, and then we have D segments and J segments. 00:23:39;20 We have these different settings for the heavy chain. 00:23:41;27 For the light chains, also, we have V segments and J segments. 00:23:45;09 And these can be joined together in different cells. 00:23:47;26 So... and I'll illustrate this in the next few slides. 00:23:50;15 So, this is the big picture view. 00:23:52;21 You have a bunch of V segments, D segments, and J segments upstream of a constant region segment. 00:24:00;01 And then these... we're gonna join together one V, one D, and one J to create an antibody gene. 00:24:07;02 And then at the junctions between the V's and D's, and D's and J's, we can 00:24:11;01 add or remove bases and create some junctional diversity. 00:24:14;10 And then when this DNA is transcribed, you're going to get a messenger RNA which actually 00:24:18;21 has this rearranged DNA encoding the variable part of the antibody. 00:24:24;00 And this is what's going to give you an antibody gene. 00:24:26;20 Okay? 00:24:27;20 So again, this is another view of light chains, where we have only V segments and J segments. 00:24:34;04 So, now the V's are going to be joined to the J's. 00:24:36;01 And seen below, you have V-kappy-29 joined to J-kappa-3. 00:24:40;21 And that creates a V-J exon. 00:24:44;07 And that's going to be upstream of the constant exons and give you a kappa light chain, 00:24:48;27 for instance. 00:24:50;03 Okay? 00:24:51;08 This is a view that... 00:24:52;18 Tonegawa looked carefully at the sequences of the antibody genes. 00:24:57;12 And what he was trying to ask was, how do you know where to cut? 00:25:00;22 How does the cell know where it should cut a V segment and a J segment so they can 00:25:05;10 be joined together? 00:25:06;18 And what he showed was that adjoining the constant... adjoining the coding region 00:25:11;22 -- so, if you look at the V in... in orange -- you'll notice that there is a 7-base pair sequence. 00:25:17;28 It's called a heptamer, CACAGTG. 00:25:20;20 Then, that's followed by a spacer, in this case it's about 12 base pairs. 00:25:25;14 And then it's followed by a nonamer, which is nine base pairs. 00:25:29;00 But the heptamer is always constant. 00:25:32;01 It's always CACAGTG. 00:25:34;08 And this is going to be joined to another gene segment downstream. 00:25:38;28 So, that's the one you see in green, down there. 00:25:41;21 So, the green segment has also next to it a CACAGTG, which is also the heptamer. 00:25:49;03 A spacer... this time, the spacer has 23 base pairs. 00:25:52;13 And then you have a 9-base pair region which is AT-rich. 00:25:56;00 So, this sequence, of a heptamer, a spacer, and a nonamer, was called a 00:26:02;04 recombination signal sequence. 00:26:04;13 And the spacer corresponds to either 12 base pairs -- that's one turn of the DNA helix -- 00:26:10;02 or 23 base pairs -- it's two turns of the DNA helix. 00:26:14;16 And the rule was you always joined an RSS containing a 12-base pair spacer to 00:26:21;22 another coding segment which has an RSS which contains a 23-base pair spacer. 00:26:26;28 Never 12 to 12; never 23 to 23. 00:26:30;26 This assures that V joins to J, and V does... or D joins to J, and V doesn't join to J, 00:26:37;04 depending on the locus you're looking at. 00:26:40;16 Okay? 00:26:41;16 So, going back to this... so, the first stage of VDJ recombination... so, now, 00:26:45;21 in David Baltimore's lab, David Schatz and Marjorie Oettinger discovered RAG-1 and RAG-2. 00:26:51;25 And they showed that these two proteins actually helped the chromosome ribbon... 00:26:57;03 so, the first step of VDJ recombination, which requires RAG-1 and RAG-2, is it allows the chromosome 00:27:02;10 to ribbon between the two segments that are going to be joined. 00:27:05;07 So, you form a big loop. 00:27:08;00 And then you have synapsis. 00:27:09;13 Just bringing these two guys close to each other, though they were far away on the chromosome 00:27:13;19 to begin with. 00:27:15;17 Once this happens, RAG-1 then is gonna make a nick. 00:27:18;16 So, RAG-1 makes a nick, and the 3' hydroxyl then attacks the other strand and forms a hairpin, 00:27:26;05 so that the coding sequence now ends in a hairpin, whereas the signal sequence 00:27:31;04 with the CACAGTG is released as a clean double-strand break. 00:27:35;04 Okay? 00:27:36;04 So, the first step was synapsis, then there was cutting -- so we have synapsis, then we 00:27:42;00 have cutting... sorry, I have to go back to this slide. 00:27:44;25 The seconds step is cleavage. 00:27:46;24 The next step is opening up... you made hairpins, so you have a hairpin and you need to 00:27:52;11 open up the two hairpins, one from the coding region for the V, the other from the coding region for the J, 00:27:57;12 and join them together. 00:27:58;24 So, that's called hairpin opening and end-processing. 00:28:01;04 And the final step is repair, or ligation, where you join these pieces together. 00:28:06;15 Okay? 00:28:07;15 So, just to explain what happens in junctional diversity, you have two hairpins. 00:28:11;22 An enzyme called artemis cuts the hairpin maybe eccentrically. 00:28:16;00 So now you have a flap created, where you have DNA from the bottom strand going to 00:28:20;24 the top strand after the flap is created. 00:28:23;05 Polymerase fills in, so now you've filled in the gap. 00:28:25;25 So, those added nucleotides are called P nucleotides. 00:28:29;02 So, they were created in a templated manner. 00:28:31;26 And then at the blunt ends, we have another enzyme called TdT, terminal deoxynucleotidyl transferase, 00:28:38;05 which can add additional bases. 00:28:40;04 We call them N nucleotides. 00:28:41;15 So now, the two happens, instead of just joining them together, you actually have created 00:28:46;26 more diversity at the junction. 00:28:48;05 So, even if you join the same V and the same J in two different cells, the junctions 00:28:53;04 are going to be different. 00:28:54;22 Okay? 00:28:55;22 So, I'm going to summarize this part of the lecture. 00:29:00;15 CACAGTG, the generation of diversity. 00:29:06;14 A one-turn J kissed a two-turn V. They were brought into proximity by that lovely couple, 00:29:14;18 RAG-1/RAG-2. 00:29:16;01 RAG-1 says, I'm gonna cut you. 00:29:18;20 A pair of genes the RAGs do pick. 00:29:22;23 At each heptamer, they make a nick. 00:29:26;14 The 3' hydroxyl then must bend, to make a hairpin at the coding end. 00:29:32;21 Artemis cuts the pretty hairpin. 00:29:35;07 TdT puts N regions in. 00:29:38;17 It's time to shut the DNA door, bring in XRCC and ligase-IV. 00:29:45;14 CACAGTG, the generation of diversity. 00:29:51;01 Now you know your G-O-D. 00:29:54;19 Next time, all of immunology. 00:29:57;20 So, if you go back and think about B cell development in the context of understanding 00:30:03;21 VDJ recombination, we can understand that we have an early stage called a pro-B cell. 00:30:09;06 So, at the pro-B stage, you start to rearrange the antibody genes. 00:30:13;18 And you start with the heavy chain. 00:30:16;13 By the large pre-B stage, you have completed the rearrangement of the antibody heavy chain gene. 00:30:22;28 And you're gonna form a structure called the pre-BCR. 00:30:25;00 I'll come back to that. 00:30:27;16 Then the cell is going to go on, eventually, to become an immature B cell with IgM on the surface. 00:30:33;10 So, it has heavy chains and light chains. 00:30:34;27 This cell is going to emigrate from the bone marrow to the spleen, and then become 00:30:40;09 a general garden-variety follicular B cell. 00:30:45;04 So, one important checkpoint during B cell development is called the pre-BCR checkpoint. 00:30:51;07 So, when you go through VDJ recombination, you reach this point where you become 00:30:56;22 a large pre-B cell. 00:30:57;22 The large pre-B cell is a cell that has correctly rearranged the antibody heavy chain gene. 00:31:03;07 You know, when you add these bases to the junctions, you can go out of frame, so only 00:31:07;25 roughly half the cells are going to do this right. 00:31:10;01 So, the cells that have done it right on one chromosome are going to make a heavy chain protein, 00:31:14;09 they're going to make something called a pre-B receptor, and these cells are 00:31:19;15 going to survive and expand, and become a huge population of selected pre-B cells. 00:31:26;15 Each one of them will then... will then go on to rearrange a different light chain, 00:31:30;28 so that you now have B cells which have heavy chain and light chain. 00:31:34;09 And the pre-BCR checkpoint is very important in the context of B cell development and disease. 00:31:40;08 So, the pre-BCR... so, when the heavy chain is made, it associates with something called 00:31:46;03 surrogate light chains, which will be described in a subsequent lecture in some detail. 00:31:50;07 And it associates to form a receptor. 00:31:53;02 And this receptor signals constitutively. 00:31:55;03 The moment it's made, it's not looking for a ligand. 00:31:57;25 It's saying, you've done it right. 00:32:00;03 You have the right reading frame. 00:32:01;16 You deserve to live. 00:32:02;17 So, the signals cause the expansion and survival of the cell. 00:32:06;20 It also mediates a phenomenon called allelic exclusion, which I'll describe later in 00:32:11;10 a subsequent lecture. 00:32:14;13 So finally, the last question I'm going to talk about very briefly is about self-nonself recognition. 00:32:19;16 I'm going to give you a narrow view of this. 00:32:20;21 So, you create this diverse repertoire of B cells and T cells. 00:32:25;13 Each sees a different antigen. 00:32:27;08 But sometimes these are going to be self-reactive. 00:32:29;09 In fact, about 75% of the time they are self-reactive. 00:32:33;01 So, how do you get rid of the self-reactive guys? 00:32:35;25 I'm blood group A. If I make a blood group a B cell, it has the potential to kill me. 00:32:40;28 I need to do something about it. 00:32:42;21 So, one of the mechanisms that this happens during development is that at this immature B cell stage 00:32:48;11 you actually have this phenomenon of tolerance, or central tolerance. 00:32:54;08 And central tolerance in B cells is mainly mediated by a process called receptor editing. 00:33:00;13 And what I'm showing you here is that... imagine that this is a self-reactive B cell over there, 00:33:05;17 which has this orange light chain. 00:33:08;07 It sees a self-antigen -- let's say that's a red blood cell with that group A on it. 00:33:12;20 It triggers the cell and the cell then changes its light chain. 00:33:17;22 It no longer expresses the orange light chain. 00:33:20;13 It rearranges a new light chain, so now it has the yellow light chain. 00:33:24;08 And this combination of the heavy chain and the yellow light chain may be no longer self-reactive. 00:33:29;00 This process is called receptor editing. 00:33:31;03 It's a politically correct approach to tolerance. 00:33:34;00 You just don't bump off the self-reactive cell; you allow to reform itself. 00:33:38;24 Okay? 00:33:39;24 So, here you see in receptor editing, you'll notice that we have, let's say, V-kappa-29 00:33:44;04 has rearranged to V... 00:33:45;21 J-kappa-3. 00:33:46;24 But if I... if this cell were to edit, it might use V-kappa-25 to go and rearrange 00:33:52;02 to J-kappa-2, something down... or, sorry... 00:33:55;15 J-kappa-4 or J-kappa-5, something downstream of J-kappa-3. 00:33:58;20 And this would delete the bad light chain and bring in a new light. 00:34:02;08 This could also happen on the other kappa light chain chromosome, or it could happen 00:34:06;11 on a lambda light chain chromosome. 00:34:08;04 So, if a cell is self-reactive, it has a few opportunities to reform itself, 00:34:13;08 make a new light chain, and become no longer self-reactive. 00:34:17;11 So, this is one mechanism of central tolerance, which is in... in B cells, this is a major mechanism. 00:34:24;07 It's called receptor editing. 00:34:25;28 The other mechanism is deletion. 00:34:27;03 So, if you look at a big picture view of what happens during lymphocyte development 00:34:31;11 for B and T cells, we have assembly of receptors through VDJ recombination; then cells go through 00:34:38;16 an immature stage, when they are like teenagers, where they have to be tested; 00:34:42;20 and the ones which are self-reactive are going to be eliminated or edited. 00:34:47;23 And that's tolerance. 00:34:49;13 And then the cells are allowed to mature and become naive cells, go to the lymph nodes, 00:34:54;20 and be ready to do battle with pathogens. 00:34:56;13 So, in this lecture, we talked about the three central questions that have shaped immunology. 00:35:04;00 We first discussed the phenomenon of having pre-existing or induced antibodies. 00:35:10;25 How does a vertebrate actually make complementary shapes? 00:35:15;11 And we described this phenomenon as being explained best by the clonal selection hypothesis, 00:35:22;22 which is now an accepted fact in immunology. 00:35:26;13 We then asked the question... we have these different immune cells, each with 00:35:30;15 a different receptor... how do you create this incredible diversity? 00:35:34;22 And we answered by explaining that this has now been explained by VDJ recombination. 00:35:41;10 And finally, we asked, if you have this incredible diversity, which can see almost every shape 00:35:46;02 known to man -- and in fact, any shape that might be created in the next century as well, 00:35:51;05 we have receptors to recognize them -- how do you get rid of cells which are self-reactive? 00:35:56;07 How do you mediate this phenomenon of self-nonself recognition? 00:36:00;26 And we explained that in B cells the major way this was done in central tolerance 00:36:06;17 was through receptor editing. 00:36:07;18 There is another mechanism called deletion, which occurs in B cells but is much more important in T cells. 00:36:14;18 What we didn't talk about, because we didn't cover this, was the phenomenon of 00:36:19;24 peripheral tolerance, which happens after you've made your B and T cells. 00:36:24;00 And T regulatory cells, or regulatory T cells, are described as the cells that mediate a 00:36:30;19 peripheral lot of peripheral tolerance, which basically squelch self-reactive B and T cells in the periphery. 00:36:39;06 In the following two lectures, I'm gonna talk about some research results that go back to 00:36:45;13 the discovery, a few decades ago, of the pre-B receptor and of BTK signaling, 00:36:52;06 in the next lecture, which will explain some of the concepts of the early stages of B cell development. 00:36:59;06 And then, in the final lecture, I will talk about how we have used this kind of knowledge 00:37:04;10 about VDJ recomb... 00:37:06;06 VDJ recombination and signaling and other things to try to understand human disease.