Session 5: B Cells: Development, Selection, and Function

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

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.
00:02:40;13 Okay?
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.
00:03:02;15 Okay?
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.
00:04:21;14 Okay?
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.
00:04:48;02 Okay?
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.
00:05:24;00 Okay?
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.
00:06:38;15 Okay?
00:06:39;15 So, he just took the whole rearranged heavy chain gene and he made a transgenic mouse.
00:06:44;26 Okay?
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.
00:07:58;09 Okay?
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.
00:08:43;23 Okay?
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.
00:11:33;04 Okay?
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.
00:11:47;22 Okay?
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.
00:12:55;19 Okay?
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.
00:14:29;12 Okay?
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.
00:14:40;09 Okay?
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.
00:15:41;15 Okay?
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.
00:16:22;24 Okay?
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.
00:17:26;22 Okay?
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.
00:19:21;08 Okay?
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.