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

Transcript of Part 1: Early B Cell Development: A Look at the Defining Questions in Immunology

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

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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