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.