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

Transcript of Part 2: Bruton Tyrosine Kinase Signaling: The pre-B Cell Receptor and B Cell Differentiation

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.