The body needs to generate a broad diversity of antibodies in order to recognize a vast number of pathogens, while preventing recognition of self. This process is secured by safety checkpoints introduced during B cell differentiation. This session showcases the generation of antibody diversity (via V, D, J recombination), and the mechanisms that ensure the proper function of B cells (pre-BCR signaling, and tolerance). In addition, generation of B cell diversity is compared with the generation of diversity in T cell receptors during T cell development.
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
00:04:06;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.
00:11:07;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.
00:20:07;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.
00:22:32;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.
00:23:28;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.
00:24:27;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.
00:24:51;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.
00:26:41;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.
00:27:36;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.
00:28:07;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.
00:28:55;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,
00:29:16;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.
00:33:39;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...
00:33:46;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.
00:00:07;06 In this lecture, I'm gonna talk a little bit about our work from a few decades ago,
00:00:12;03 initially in David Baltimore's lab and then in my own lab, looking at the discovery of the pre-B receptor
00:00:18;20 and at BTK signaling, and how this influenced B cell differentiation.
00:00:24;04 So, the accepted view today about B cell development is that we have two different B cells subsets.
00:00:32;02 So, from a fetal liver stem cell, you can get B-1 cells.
00:00:36;13 And so, B-1 cells are self-renewing B cells which have a generic function in dealing with
00:00:43;25 a certain set of pathogens.
00:00:46;19 B-2 B cells are the garden-variety B cell.
00:00:50;23 They are derived from a bone marrow-derived stem cell, an adult stem cell.
00:00:56;13 And then they go through various stages of differentiation, and we eventually get
00:01:01;00 two subsets: follicular B cells and marginal zone B cells.
00:01:05;24 And marginal zone B cells are also self-renewing, but then the garden-variety B cell we normally talk about
00:01:11;14 is a follicular B cell. Okay?
00:01:14;24 In 1983, our understanding of B cell development consisted of the following stages.
00:01:21;09 We knew there were cells which are committing to the B lineage, so those were called pro-B cells.
00:01:27;13 They hadn't yet rearranged their antibody genes.
00:01:30;15 Then we had pre-B cells, which had completely rearranged their antibody genes and which
00:01:36;14 contained intracellular mu, IgM heavy chains... so intracellular mu.
00:01:44;06 And then we had a stage of development called the immature B cell stage, which had IgM
00:01:49;03 on the surface, just heavy chain and light chain.
00:01:51;24 Then we had mature B cells, which have both IgD and IgM on the surface.
00:01:57;18 And then, once these cells were activated, we know a lot more in between right now,
00:02:02;02 which I'm not getting into.
00:02:03;11 Once these cells were activated, we knew we would... eventually we would get plasma cells,
00:02:08;01 which are factories for the secretion of antibodies.
00:02:11;18 This was our view.
00:02:12;18 Now, one of the questions that had come up early in people's thinking about the immune systems was,
00:02:17;12 though we have two chromosomes, maternal and paternal, and we could make
00:02:22;12 two antibodies in every cell, two antibody heavy chains and so on, somehow on each cell
00:02:28;06 we only express one antibody or one antigen receptor.
00:02:33;10 And this is important because, if we express two receptors,
00:02:36;21 where would clonal specificity be?
00:02:38;17 We wouldn't have the clonal selection theory.
00:02:41;13 You need to have a single receptor on a single cell.
00:02:44;11 So, the phenomenon, unknown at the time as to how this happened, by which we made
00:02:50;24 sure that in... in immune cells we expressed only the paternal or only the maternal copy of
00:02:58;00 the antibody heavy and light chain genes, was called allelic exclusion.
00:03:03;15 And allelic exclusion is central to having specificity in the immune system.
00:03:09;08 And when I came out of the postdoc, I wanted to work on allelic exclusion because I thought,
00:03:13;07 if this goes wrong then you'll get autoimmunity, and I was interested in the phenomenon.
00:03:18;08 And allelic exclusion... the experiments that had been done earlier on, and this is
00:03:23;02 one example of such an experiment, was to take two mice which have different polymorphic forms
00:03:29;12 of the antibody heavy chain gene.
00:03:31;24 So, in this case, we have IgHa and IgHb.
00:03:36;16 When you cross these mice, you now have an F1 mouse which is IgHa and b.
00:03:42;07 It has both the a allele and the b allele.
00:03:45;25 But when you look at individual B cells, each B cell expresses either the a allele or the b allele,
00:03:52;01 never both.
00:03:53;00 So, this proved that there was truly a phenomenon called allelic exclusion, and you could
00:03:58;22 describe it in these terms.
00:03:59;24 But what was the mechanism?
00:04:01;06 How did this happen?
00:04:02;12 How did we actually achieve this?
00:04:04;26 So, in order to understand this, in the Baltimore lab, Rudi Grosschedl did an experiment
00:04:12;23 where he made transgenic mice.
00:04:14;19 That is to say, he made a mouse which contained a rearranged antibody heavy chain gene.
00:04:22;14 So this is... just to remind you that the heavy chain locus contains, you know,
00:04:27;01 V, D, and J segments, but if it's rearranged you have one V joined to one D joined to one J,
00:04:33;11 upstream of the constant regions.
00:04:34;20 So, he took a rearranged gene, after the gene had been, you know, put together in
00:04:40;24 developing B cells.
00:04:42;03 And he put this gene into the fertilized egg of a mouse.
00:04:49;02 So basically, just to remind you again why this phenomenon is important, is we do have
00:04:54;13 a phenomenon called junctional diversity as well.
00:04:56;22 Okay, we have... when you... and we discussed this in the previous lecture...
00:05:00;21 when you join two pieces of DNA, you can create diversity at the junctions, and we describe
00:05:07;10 how you create diversity at the junctions, adding P and N nucleotides, okay?
00:05:12;19 And we also wanted to understand, how do you select cells which have done the right rearrangements?
00:05:20;11 And we wondered if this could be linked to allelic exclusion.
00:05:25;00 So, if you didn't add a multiple of three bases at a junction, then that cell is
00:05:31;02 not going to be able to make an antibody heavy chain gene that means anything.
00:05:34;03 So, if I added 11 bases or 17 bases, then I'm not going to get an antibody protein
00:05:40;16 that's correct.
00:05:41;16 If I added 12 or 15 bases, it's fine.
00:05:43;16 Now, how do you make out the difference?
00:05:44;28 How do you know which cells are good and which cells are going to survive?
00:05:48;09 And these were all the questions that were in our minds.
00:05:52;16 So now, when you look at the antibody heavy chain gene, and this is an example of
00:05:57;05 a rearranged heavy chain gene at the bottom.
00:05:59;01 So, if you look over here.
00:06:00;28 So, we've put VDJ in together.
00:06:02;26 And then this is going to be transcribed to give you two messenger RNAs:
00:06:08;04 a longer one, which can give you the membrane form of the heavy chain,
00:06:11;13 and a shorter one that can give you the secreted form of the heavy chain.
00:06:14;19 So, the antibody can function both as a receptor and as a secreted molecule.
00:06:19;06 So, by alternative splicing, you can get two different forms of the heavy chain RNA.
00:06:24;15 And then this will give you two different proteins, and this is just shown in this slide,
00:06:27;27 that you can have a secreted antibody -- in this case, I'm showing you IgG --
00:06:31;25 or you could have a membrane IgG, which has a transmembrane region that goes across the membrane
00:06:37;00 with a little cytoplasmic tail.
00:06:39;15 So, he just took the whole rearranged heavy chain gene and he made a transgenic mouse.
00:06:45;26 So, this transgenic mouse... so, here you have a heavy chain gene, so just
00:06:51;02 a whole heavy chain gene, which has both the membrane and secreted form capable of being made,
00:06:55;20 injected into the male pronucleus of a fertilized egg.
00:06:59;12 This is put into a pseudopregnant female.
00:07:01;17 Then the female has pups.
00:07:04;13 And then the pups... the founder is the pup which actually carried the transgene.
00:07:08;19 Not everyone would be lucky... not every cell would actually carry the transgene.
00:07:12;20 And then the... the founder was then bred.
00:07:15;16 And then we look at all the progeny of this founder mouse, the one which carried the transgene in it,
00:07:19;27 and you discovered that, now, endogenous heavy chain genes are not rearranged.
00:07:25;03 So, by putting in a rearranged heavy chain gene into an animal such that it would
00:07:31;03 be expressed in every B cell in that animal, now the endogenous maternal and paternal chromosomes
00:07:38;01 for the heavy chain gene are not rearranged.
00:07:39;27 So, this suggested that this may be a mechanism of allelic exclusion, and that there was
00:07:45;20 a feedback, that somehow heavy chain... rearranged heavy chain proteins could send
00:07:52;09 some feedback signal to allow the prevention of heavy chain gene rearrangement.
00:07:59;15 So, another experiment was done -- this was from Phil Leder's lab by Michel Nussenzweig --
00:08:04;11 where they put in only the membrane form of the heavy chain gene.
00:08:09;17 After these first experiments had been done.
00:08:11;28 When you just put the membrane form of the heavy chain gene, again you got allelic exclusion.
00:08:16;16 If you put the secreted form, the one that doesn't function as a receptor, the one that's secreted,
00:08:21;13 it did not give you allelic exclusion.
00:08:23;24 So, somehow, the membrane form of the heavy chain gene made some protein,
00:08:29;09 which signals somehow, which prevented rearrangement of the immunoglobulin genes.
00:08:34;14 So, we talked about VDJ recombination in the previous lecture.
00:08:37;18 So basically, that entire phenomenon at the immunoglobulin heavy chain locus was
00:08:42;07 somehow blocked.
00:08:45;10 So, if the membrane form of the heavy chain signals to mediate allelic exclusion,
00:08:51;28 the question asked is, what does it bind to?
00:08:54;02 How does it signal?
00:08:55;02 What is it doing?
00:08:56;21 And one of the experiments I did -- and I'm not gonna go into all the details here
00:09:01;25 -- was to show that in pre-B cells -- so, if you look at these lanes over here, which are labeled
00:09:07;01 pre-B cells -- we found that there was a protein associated with the heavy chain
00:09:11;11 -- it's labeled omega at the bottom --
00:09:14;05 and this protein is not the kappa light chain.
00:09:16;22 So, this is a pre-B cell.
00:09:18;01 It doesn't...
00:09:19;01 hasn't yet rearranged its kappa and lambda light chain genes.
00:09:22;00 But the pre-B cells did have some other protein that was associated with the heavy chain.
00:09:27;13 It ran small.
00:09:29;04 It labeled poorly.
00:09:30;04 It's small, so it doesn't pick up as much methionine.
00:09:32;04 These were radioactively labeled cells.
00:09:34;19 And then, when you look on the other side, we have an experiment where I took
00:09:38;09 a cell line which contains D-mu.
00:09:40;25 D-mu is a truncated form of the mu heavy chain.
00:09:44;14 It did not contain that protein.
00:09:46;08 I looked at another cell line in which... it's called an L cell -- it's a fibroblast --
00:09:50;20 which I transfected with the membrane form of mu.
00:09:54;00 So, it has the mu heavy chain, but it does not have any light chain.
00:09:58;13 So, only the pre-B cells had this other protein, which we'd called omega.
00:10:04;25 This was in our fanciful thinking.
00:10:06;16 We said, it's the last light chain.
00:10:08;14 We'll call it omega, okay?
00:10:11;06 So, not... not everybody in the lab believed this meant anything.
00:10:14;27 There was this funny, funky band.
00:10:16;11 No one had seen it before.
00:10:17;25 What does it mean?
00:10:19;00 So the way we convinced people that this meant something was by doing a two-dimensional gel.
00:10:23;28 So, what we did here is... remember, antibody is linked to its light chain...
00:10:28;04 the heavy chain is linked to the light chain by disulfide bridges.
00:10:31;19 So, we ran these 2-D gels.
00:10:34;01 So, if you look at the pre-B cell, over here... so, in the pre-B cell, the first dimension
00:10:39;20 we ran non-reducing, so that's... so, we ran the sample without adding a reducing agent.
00:10:46;01 We then... after the sample had run out, we ran it in the next dimension with two... beta-mercaptoethanol.
00:10:53;12 So, it will break disulfide bridges.
00:10:56;06 And you can see there's a diagonal in the middle.
00:10:58;21 And there are some proteins that have fallen off the diagonal.
00:11:01;18 And that shows that it's linked to something else.
00:11:03;14 And the diag... off diagonal we see the mu-2/omega-2 dimers of that size.
00:11:08;27 Then we see just mu-2 with one omega.
00:11:11;02 Then we see mu-2 alone.
00:11:13;04 And then we just see mu with one omega, and so on.
00:11:15;10 Okay, so we saw all the properties of having an antibody, though we were looking at
00:11:20;28 a pre-B cell, but we were seeing a disulfide-linked tetrameric structure with two heavy chains
00:11:27;24 and two new types of light chains, which we then called surrogate light chains.
00:11:34;04 If you do this experiment in a B cell -- this was of course the traditional way of looking at things --
00:11:38;06 you found heavy chain with kappa, so it would form mu-2/kappa-2 dimers.
00:11:41;26 Tetramers, basically, or you just had mu/kappa dimers or mu-2 alone.
00:11:48;22 So, this established quite clearly that the heavy chain protein was physically in
00:11:57;28 disulfide linkage with the light chain-like protein in pre-B cells.
00:12:03;10 And these pre-B cells had not gone through any VDJ recombination for the light chain gene.
00:12:08;26 The light chain genes, kappa and gamma, were completely germline.
00:12:11;26 But they contained this other protein, which behaved like a light chain, which we called
00:12:16;02 a surrogate light chain.
00:12:18;00 Okay, so this is to show that this protein was also on the surface of these cells.
00:12:23;00 We did surface iodination.
00:12:24;24 Probably not many people do this anymore, but we basically took cells,
00:12:28;08 radioactively labeled them with iodine on the outside, and we showed, again in pre-B cells,
00:12:33;00 we could find these mu-2/omega tetramers running at the right size.
00:12:37;26 And in a normal B cell you would see mu light chain tetramers.
00:12:41;02 Okay, so we showed that, yes, the heavy chain associates with the surrogate light chain,
00:12:46;24 and it goes to the cell surface -- it's the membrane form -- so this is likely
00:12:51;00 a new type of receptor that's found in pre-B cells.
00:12:56;19 We went one step further, and we found a second protein.
00:13:01;00 And I'd actually seen this earlier on, but we'd not put it into the first paper.
00:13:05;04 We found a second protein which we called iota, for the smallest protein,
00:13:10;06 which was also in the complex, but this was not disulfide-linked to the mu chain.
00:13:15;17 Okay, so we've... we're seeing two surrogate light chains, omega and iota,
00:13:21;18 associated with the heavy chain.
00:13:24;08 So, this was the presumed structure.
00:13:27;07 And this is based on some knowledge, now, that I draw it this way.
00:13:30;26 We have two heavy chains.
00:13:32;03 We had two surrogate chains which were disulfide linked.
00:13:35;15 I haven't shown you the disulfide linkages.
00:13:37;11 That was the omega chains.
00:13:39;15 And then there were the two iota light chains.
00:13:41;17 This is what we know of the structure now.
00:13:43;10 Now, Fritz Melchers' lab, a year before we'd done our work, had published some papers showing
00:13:50;06 that there were some immunoglobulin light chain-like genes that were found in
00:13:55;05 pre-B cells.
00:13:56;17 And he found the genes but didn't look to see whether they made a protein, at that time,
00:14:00;22 that associated with mu or anything else.
00:14:02;17 So, we assumed that maybe what we were finding associated with the heavy chain was actually
00:14:07;11 the product of those genes.
00:14:09;04 So, we sequenced... we did... we did radiolabel sequencing of both omega and iota, and showed
00:14:15;24 that they were identical to the genes that he had called lambda-5 and V-PreB.
00:14:21;27 And we graciously agreed to go with his names, so we now call those proteins
00:14:26;23 lambda-5 and V-PreB.
00:14:30;12 So, we have surrogate light genes associated with a heavy chain, and the surrogate light chains
00:14:34;21 are lambda-5, which is covalently associated, and V-PreB.
00:14:41;09 And in some reviews and papers, we...
00:14:44;12 I remember coming out with hypotheses as to how these worked.
00:14:48;00 And I liked one name for these hypotheses, and we called it the ligand-independent activation of receptor
00:14:54;05 or the Liar hypothesis.
00:14:56;28 And this essentially said that this is a receptor that is not sensing the environment.
00:15:04;00 It can form a complex.
00:15:05;10 It might form a complex on the cell surface, it might form a complex of intracellular,
00:15:10;13 but it's going to, when it's assembled, it is on... in the on mode, and it's going to signal.
00:15:16;00 All it's doing is sensing whether it's the right reading frame.
00:15:18;17 It's not trying to see whether there's some new thing in the environment.
00:15:21;26 And so this model, which we put forward a long time ago, is now the accepted model
00:15:28;06 for both the pre-B receptor and the pre-T receptor, that these signal constitutively.
00:15:32;26 The moment you assemble them, they are in the on mode, they signal, and they tell the cell
00:15:37;19 to move on in differentiation.
00:15:42;14 So, we showed the veracity of this model in one study, where we looked for
00:15:47;21 activated, tyrosine-phosphorylated proteins.
00:15:50;05 So, if we look in a B cell, we see these tyrosine-phosphorylated proteins only when the B cell is activated.
00:15:56;24 So, in the last lane of panel A, we can see we have all these activated proteins
00:16:01;26 which bind to SH2 domains of other signaling proteins.
00:16:05;26 But we see them only after the B cell is activated.
00:16:08;20 However, in the pre-B cell, we don't have to activate anything.
00:16:12;14 Okay, so shown in panel B, without activation, the pre-B cell has these proteins, these tyrosine-phosphorylated proteins,
00:16:19;12 which you can capture from the cell.
00:16:24;10 The pre-BCR... this is the model of the pre-BCR which is in the textbooks now.
00:16:28;20 And this is basically... it's the heavy chain, the surrogate light chains, and then the
00:16:33;15 two signaling proteins, Ig-alpha and Ig-beta, which are also signaling proteins for the
00:16:37;28 B cell receptor.
00:16:38;28 So, this is now the accepted pre-B receptor, and it does... it signals constitutively to
00:16:44;16 keep cells alive, if they have actually made it.
00:16:47;14 So, they're in the right reading frame; they deserve to live.
00:16:50;01 It allows the expansion of these cells.
00:16:51;18 So, the biggest expansion of the B lineage comes from the pre-B cell receptor.
00:16:56;22 It also mediates allelic exclusion.
00:16:58;18 So, it sends signals to shut off rearrangement at the other allele, mediating allelic exclusion.
00:17:05;17 Signals also induce the rearrangement of the light chain at the next stage,
00:17:08;27 and shut off expression of the surrogate light chains.
00:17:11;25 So, this cell will transition from being a pre-B cell with a pre-B cell receptor
00:17:17;26 into a cell that has no surrogate light chains and no light chain, which will rearrange
00:17:22;25 the light chain, and then it will become an immature B cell.
00:17:27;22 So, there's a disease called X-linked agammaglobulinemia.
00:17:31;14 It's the first human immunodeficiency described.
00:17:33;28 It was described by Colonel Ogden Bruton in 1952.
00:17:38;04 And these were boys who had no antibodies.
00:17:41;04 And it was later discovered they had no antibodies because they had no B cells.
00:17:44;23 In 1952, we didn't know about B cells, but we knew they had no antibodies.
00:17:48;16 But then we later discovered that these boys don't have antibodies.
00:17:53;04 They get a lot of pyogenic infections -- infections with pus-forming bacteria.
00:17:58;12 And they don't have B cells in the blood.
00:18:00;25 Okay, the gene for this disease -- it's an X-linked gene -- was worked to by one group.
00:18:07;16 So, the group in Sweden and Britain.
00:18:09;25 So, this is Edvard Smith.
00:18:11;08 They worked to this gene and identified it as being a tyrosine kinase.
00:18:15;28 So, it was called Bruton's tyrosine kinase.
00:18:18;19 Owen Witte at UCLA, using a different rule... he wasn't looking for the...
00:18:24;05 the gene in X-linked agammaglobulinemia...
00:18:27;13 he found a new tyrosine kinase, which also turned out to be the same kinase, BTK.
00:18:31;15 So, he put two and two together and said that, maybe the reason why these kids get this disease
00:18:38;05 is that their pre-B receptors need to signal through BTK.
00:18:42;19 And in the absence of BTK, the pre-B receptor doesn't signal, the cells don't survive
00:18:48;06 this checkpoint, and they end up with no B cells.
00:18:51;15 So to address this, we started to look at BTK in pre-B cells and in B cells.
00:18:57;20 So, if you look at the panel called anti-pY -- that's for anti-phosphotyrosine --
00:19:03;25 and if you look at the band that's labeled BTK... so, you have to look at the panel that
00:19:08;15 is on the left, and you look at the middle lane.
00:19:11;23 That is a B cell that hasn't been activated.
00:19:14;25 U stands for unactivated.
00:19:16;06 There is no tyrosine-phosphorylated BTK in that lane.
00:19:22;08 However, the pre-B cell, without activation, already has tyrosine-phosphorylated BTK.
00:19:28;23 If I activate the B cell and I go to the third lane in the left panel, you'll notice
00:19:33;00 there's a phosphorylated band.
00:19:34;13 So, I have to activate a B cell to get BTK activated.
00:19:37;22 But in the pre-B cell, BTK is constitutively activated, which is in keeping with our
00:19:44;08 previous thinking about the pre-B receptor.
00:19:46;11 On the right lane, we're just showing you that all three lanes had BTK in them, okay?
00:19:52;11 Here's another experiment.
00:19:53;11 Now, in this experiment, you're looking at B cells.
00:19:56;01 So, at this time, BTK had not been connected to B cells, right?
00:19:59;02 So, this is why we did this experiment.
00:20:00;18 When we activate the B cells... we're looking at stimulation and zero is no stimulation,
00:20:06;07 and then we're looking at one minute, three minutes, five minutes, and ten minutes
00:20:10;16 after we trigger the B-cell receptor.
00:20:12;20 And you can notice that by the time it's about five minutes -- three to five minutes --
00:20:16;19 you see active, phosphorylated BTK showing up.
00:20:20;27 And then it will also phosphorylate enolase, which is a substrate for any kinase.
00:20:25;00 So, it's showing you that there's increased kinase activity.
00:20:27;16 So, we are bringing down the BTK molecule, showing that it's tyrosine- phosphorylated...
00:20:33;11 phosphorylated, and showing that it can also phosphorylate a target.
00:20:36;08 So, we are showing the activation of BTK after BCR ligation in B cells,
00:20:41;23 but constitutive activation in pre-B cells.
00:20:45;04 So, this made the connection between the human disease, where the pre-B receptor wasn't known
00:20:51;20 to be defective, but we are saying now the pre-B receptor is defective,
00:20:56;07 because the pre-B receptor signals through BTK and these boys are born with a defective BTK.
00:21:01;15 So, this was the broad pathway of pre-B cell activation we could think about at the time.
00:21:09;00 The pre-BCR was going to signal constitutively.
00:21:11;03 It could happen from the cell surface, or we thought, even from an intracellular membrane.
00:21:15;26 It activates downstream kinases like Src-family kinases or Syk.
00:21:20;05 And then it activates BTK.
00:21:23;12 And when BTK is activated, then the cell gets the signals to mediate allelic exclusion,
00:21:28;20 to survive, to proliferate, to differentiate further.
00:21:32;25 So, now we go back to the checkpoints during B cell development.
00:21:37;24 You have pro-B cells, which start to rearrange the antibody genes.
00:21:42;14 When they come through the late pro-B stage to the pre-B stage, they make the pre-BCR.
00:21:48;16 The cells that make the pre-BCR... so, it's not gonna be every cell...
00:21:52;00 roughly 50% of the cells... you get three chances at each chromosome, ends up as being roughly 50%...
00:21:57;12 about half of them will survive.
00:21:58;28 They will make the pre-BCR.
00:22:00;23 They will expand.
00:22:02;09 Then they'll go into the small pre-B state, where they rearrange the light chain gene.
00:22:06;20 Then they'll go on to become immature B cells, where they'll be tested for receptor editing,
00:22:11;20 in case they're self-reactive.
00:22:13;15 And then they'll go on into the periphery, to the spleen and to the lymph nodes,
00:22:17;12 and become mature B cells.
00:22:18;26 So, most of this lecture has revolved around the pre-BCR checkpoint.
00:22:24;12 And the pre-BCR checkpoint is designed to actually gauge proper reading frame,
00:22:31;01 to see whether the antibody heavy chain gene was made in frame.
00:22:35;07 So, the cells that make the pre-B receptor are the cells that are going to survive, proliferate,
00:22:42;24 expand into small pre-B cells, which will then rearrange light chain genes.
00:22:48;13 This checkpoint is intimately connected to signaling through BTK.
00:22:53;11 So, the pre-B receptor activates BTK.
00:22:57;06 And BTK is the tyrosine kinase encoded by a gene on the X chromosome, which is
00:23:03;01 mutated in boys with Bruton's disease, or X-linked agammaglobulinemia.
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.
- Shiv Pillai iBioSeminar: B Cell Development, Fundamental Questions in Immunology
- Hidde Ploegh iBioSeminar: The Importance of Antibody Diversity
Dr. Shiv Pillai is a professor of medicine and health sciences and technology at Harvard Medical School, and a Ragon Institute investigator. Pillai completed his medical studies at Christian Medical College Vellore, India (1976), and subsequently obtained his doctorate in biochemistry at Calcutta University, India. He continued his postdoctoral training at David Baltimore’s lab at… Continue Reading
Dr. Hidde Ploegh is an immunologist at Boston Children’s Hospital. His love for immunology began when he was an undergraduate at the University of Groningen in the Netherlands. As a student, he wrote a letter to renowned immunologist Jon van Rood but never heard back. However, as an undergraduate researcher, he had an opportunity to… Continue Reading