Session 6: T Cells: Development and Differentiation
Transcript of Part 1: An Introduction to T Cell Tolerance
00:00:08.05 So, my name is Diane Mathis. 00:00:09.25 I'm a professor of immunology 00:00:11.21 at Harvard Medical School. 00:00:13.21 My two talks have to do with 00:00:17.00 immunological tolerance, 00:00:18.25 in particular T cell tolerance. 00:00:22.05 In the first talk, 00:00:23.29 I'll relate some general concepts 00:00:26.17 and then go on to 00:00:29.23 define and describe the major mechanisms of tolerance. 00:00:33.17 And then in the second talk, 00:00:35.17 I'll focus on one particular mechanism 00:00:39.07 of enforcing tolerance. 00:00:42.19 So, some general concepts... 00:00:46.17 the major function of the immune system 00:00:49.15 is to fight off microbial challenges. 00:00:52.10 It does this by mobilizing its two major arms: 00:00:56.12 the innate response and the adaptive response. 00:01:00.05 The innate response comes in early 00:01:02.26 and it's quite stereotypic. 00:01:04.23 It's a hardwired system 00:01:08.09 that includes cells like macrophages and neutrophils. 00:01:13.01 The adaptive system... response 00:01:18.01 comes in a little bit later 00:01:20.00 and it's a more nuanced response. 00:01:22.10 The major players in the adaptive response 00:01:26.21 are B and T lymphocytes. 00:01:29.22 Now, these cells have, displayed on their surfaces, 00:01:33.26 receptors that are specific for particular antigens. 00:01:39.19 The B cell receptor, or BCR, 00:01:42.21 for B cells, 00:01:44.20 which is also called immunoglobulin, 00:01:47.05 and the T cell receptor, or TCR, for T cells. 00:01:50.23 Now, in general, 00:01:52.29 each B or T cell 00:01:55.29 has multiple copies of a receptor 00:01:59.03 that sees one particular antigen 00:02:01.21 or a group of structurally related antigens, 00:02:06.13 so you can understand that 00:02:08.10 in order to fight off the myriad of microbial challenges, 00:02:11.27 that the repertoire must be both big and diverse. 00:02:17.02 And this happens by 00:02:19.14 random rearrangement of gene segments 00:02:24.05 encoding variable regions of the T and B cell receptors 00:02:27.27 as these cells are differentiating, 00:02:32.00 in the thymus for T cells 00:02:33.24 and in the bone marrow for B cells. 00:02:38.16 Now, since it is a random process, 00:02:41.28 just by chance, 00:02:44.11 sometimes specificities will be generated 00:02:46.27 that are able to recognize 00:02:49.08 the body's own constituents. 00:02:51.05 For example, a T cell may see insulin 00:02:55.07 or it may see the acetylcholine receptor, 00:02:59.05 or it may see myelin basic protein, 00:03:01.23 and if these T cells are let loose 00:03:05.05 they would cause autoimmune attack on the pancreas, 00:03:08.20 or myasthenia gravis, 00:03:10.24 or multiple sclerosis, 00:03:13.20 which is an autoimmune attack in the central nervous system. 00:03:17.21 Now, since this is 00:03:20.08 a very dangerous problem for the individual, 00:03:22.29 through evolution, 00:03:25.09 multiple levels of immunological tolerance 00:03:29.23 have come into play. 00:03:32.24 Now, these are generally 00:03:35.28 divided into central and peripheral mechanisms. 00:03:39.12 Central tolerance has to do with 00:03:42.16 the primary lymphoid organs 00:03:44.15 where T cells or B cells are generated, 00:03:46.08 so, for T cells, in the thymus. 00:03:48.25 So, the antigens that would be dealt with 00:03:51.17 in central tolerance 00:03:53.07 would be antigens that are expressed in the thymus 00:03:56.19 or are expressed in all cells, 00:03:59.03 or are expressed in cells which are 00:04:02.16 trafficking through the thymus, 00:04:04.00 like blood cells, for example. 00:04:06.08 Peripheral tolerance has to do with those cells, 00:04:11.05 once they've emerged from the thymus 00:04:13.15 and are now in the periphery, 00:04:15.16 and can see potential self-antigens 00:04:19.04 in the liver or in the heart or in various tissues. 00:04:25.19 Now, the major mechanisms of central tolerance are: 00:04:30.05 first, clonal deletion, 00:04:31.28 which is physical removal of that particular T cell from the repertoire; 00:04:38.12 clonal inactivation, 00:04:42.09 where the T cell is there, 00:04:44.21 but once it gets into the periphery 00:04:47.07 it's no longer able to make a response; 00:04:51.17 and then clonal diversion, 00:04:53.27 the T cell is there and it can respond, 00:04:55.21 but during its differentiation 00:04:58.21 it gave up its identity as an effector T cell, 00:05:03.04 which is the kind of cell 00:05:05.26 which actually does the damage, 00:05:08.06 and has taken on the mantle 00:05:10.23 of another kind of cell, a regulatory cell. 00:05:14.19 The mechanisms of peripheral tolerance 00:05:16.29 are somewhat similar. 00:05:19.07 So, the first three are the same 00:05:21.08 -- deletion, inactivation, and diversion -- 00:05:22.23 but onto this are added three more. 00:05:25.21 So, clonal ignorance just refers to the fact that 00:05:29.19 some T cells can escape, 00:05:31.07 but they're quite fine, 00:05:33.02 because the antigen that they see 00:05:35.00 is hidden from the lymphocyte circulation. 00:05:39.18 So, for example, it might be an antigen 00:05:42.02 which is in a cell 00:05:43.27 that's behind the blood-brain barrier 00:05:45.09 or it might be an antigen 00:05:47.06 which is in the eye, 00:05:48.18 which is thought to be "immune privileged", 00:05:52.10 or an antigen which is in the testis. 00:05:54.00 So, normally, these are not seen 00:05:55.26 unless there's some kind of damage 00:05:58.16 to these different tissues, 00:06:00.16 which releases the antigen into the circulation. 00:06:03.22 Helplessness refers to the fact that 00:06:06.07 many B cell and cytotoxic T cell responses 00:06:12.01 must have help from CD4+ helper cells 00:06:17.16 in order to respond effectively. 00:06:19.20 So, as you can imagine, 00:06:21.25 you might not need to directly 00:06:24.15 tolerize that B cell or that cytotoxic T cell, 00:06:26.13 but rather the CD4+ T cells 00:06:29.09 that are helping them. 00:06:31.21 And then, finally, suppression, 00:06:36.22 which is a major mechanism, 00:06:39.02 refers to the fact... 00:06:39.17 refers to cells which keep in check the activity 00:06:42.10 of effector T cells. 00:06:46.19 So, looking at that net of mechanisms 00:06:51.25 which I showed you, 00:06:53.00 you might be tempted to think that 00:06:55.27 this is very comprehensive 00:06:57.27 and autoimmunity should be rare. 00:07:01.10 But, breaks in tolerance do occur, 00:07:02.24 leading to autoimmunity 00:07:04.12 and, in the most extreme form, 00:07:06.22 to autoimmune disease, 00:07:08.13 and they occur quite often 00:07:12.10 -- 5-8% of the population in Western countries 00:07:16.22 have some form of an autoimmune disease -- 00:07:21.03 and they occur by different means. 00:07:23.22 So, at the last calculation, 00:07:26.04 there are more than 80 different types 00:07:28.29 of autoimmune diseases, 00:07:30.25 and probably double that 00:07:34.17 because sometimes we lump together 00:07:36.19 a particular autoimmune disease 00:07:39.00 just because they have the same manifestations, 00:07:41.10 for example, arthritis, 00:07:43.15 but the way to get there might be, 00:07:45.09 actually, quite different. 00:07:48.12 So, let's look a little bit more closely 00:07:51.19 into central tolerance. 00:07:53.22 And I'd like to focus on the first mechanism that I mentioned 00:07:59.09 -- clonal deletion. 00:08:01.02 Now, as I said, 00:08:02.19 clonal deletion is physical removal 00:08:04.09 of the self-reactive T cells from the repertoire, 00:08:07.24 and this is a quite important mechanism 00:08:12.14 because fully two-thirds of the T cells 00:08:16.08 that reach full maturity in the thymus 00:08:19.00 actually undergo clonal deletion, 00:08:22.04 because they have some self-reactivity. 00:08:24.27 And it's also the most definitive mechanism 00:08:26.18 because it ends in death, 00:08:28.04 and you can't be more definitive than that. 00:08:32.11 So, I'd like to tell you 00:08:37.22 more about this mechanism of clonal deletion, 00:08:40.22 but before I do that 00:08:43.04 I have to explain to you 00:08:44.28 how T cells see antigen 00:08:46.26 and how they differentiate in the thymus. 00:08:49.20 So, a T cell actually sees its antigen quite differently 00:08:52.25 from how a B cell sees its antigen. 00:08:56.04 B cells see antigens through their B cell receptor, 00:09:00.29 or immunoglobulins, 00:09:02.10 very directly. 00:09:03.20 They actually recognize 00:09:06.11 a piece of the three-dimensional structure 00:09:09.02 and then make a response. 00:09:11.02 For T cells, it's quite different. 00:09:13.20 They must recognize 00:09:16.09 a short peptide sequence, 00:09:18.12 a primary sequence, 00:09:20.23 presented by major histocompatibility complex molecules, 00:09:27.02 which occur at the surface of most cell types. 00:09:32.01 Now, this presentation of antigens to T cells 00:09:36.06 is a quite complicated 00:09:38.22 and very elegant mechanism, 00:09:40.04 which, if you're interested in, 00:09:43.02 you can refer to the talk by Dr. Mellman, 00:09:46.09 which goes into much more detail. 00:09:49.01 But, suffice it to say, in this context, 00:09:51.14 an antigen would be either 00:09:54.09 synthesized by a cell or taken up by a cell, 00:09:57.12 and then undergoes proteolytic degradation 00:10:01.00 to make peptides. 00:10:02.14 And according to where that peptide 00:10:04.26 was actually made, 00:10:06.06 and what enzymes were involved, 00:10:07.23 as well as the primary sequence of the peptide, 00:10:10.23 it can bind either to an MHC class II molecule 00:10:15.16 or an MHC class I molecule. 00:10:17.14 And then these molecules 00:10:21.26 shuttle the peptides to the surface. 00:10:23.26 Now, some T cell receptors 00:10:26.04 can see a peptide 00:10:29.29 in the context of an MHC class II molecule, 00:10:34.20 and it does this with the help of the CD4 co-receptor, 00:10:37.11 and these T cells turn out to be 00:10:40.10 CD4+ helper T cells. 00:10:43.07 Other T cells have receptors 00:10:45.11 that are capable of recognizing 00:10:47.16 MHC Class I molecules 00:10:49.17 and peptides within them, 00:10:51.16 and they're helped along with the co-receptor CD8, 00:10:55.26 and these cells turn into 00:10:57.29 CD8+ cytotoxic T cells. 00:11:01.26 Now, as I mentioned, 00:11:03.23 T cells undergo differentiation in the thymus. 00:11:07.05 The precursor, which gives rise to a T cell, 00:11:09.28 comes from the fetal liver 00:11:12.25 or the adult bone marrow, 00:11:14.24 and these precursors are really very ignorant. 00:11:19.27 So, they must go through a number of processes in the thymus 00:11:22.27 to educate them, 00:11:26.07 they must learn their antigen specificity 00:11:29.08 -- and they do this by rearrangement of the T cell receptor genes -- 00:11:35.00 they must have an enrichment for T cells 00:11:37.13 which are able to see the MHC molecule 00:11:39.23 that the individual is expressing, 00:11:43.02 but not see it so well 00:11:47.06 that it might cause autoimmunity. 00:11:49.15 So, these are positive selection 00:11:51.02 and negative selection events. 00:11:53.14 And then, finally, 00:11:55.24 phenotypic specialization takes place. 00:11:59.08 So, I mentioned that there are CD8+ cytotoxic T cells 00:12:02.19 and CD4+ helper T cells, 00:12:04.27 and these are what we call effector cells, 00:12:07.06 which get the job done in an immune response. 00:12:11.02 There are also CD4+ regulatory T cells, 00:12:14.01 which control the activities of these effector cells. 00:12:19.14 Now, this all takes place in the thymus 00:12:22.00 by an orchestrated series of events. 00:12:27.17 The precursors enter through the blood vessels 00:12:32.23 at the corticomedullary junction, 00:12:35.25 and then they percolate through the cortex 00:12:38.25 and the medulla, 00:12:40.20 coming into contact with various stromal cell types 00:12:43.29 that express MHC molecules 00:12:48.05 with peptide at their surface, 00:12:49.15 and also produce different cytokines 00:12:53.02 and have different other types of receptors on their surface. 00:12:56.29 And through this series of contacts, 00:13:00.24 these different processes, 00:13:03.09 which are so important for the life of the T cell, 00:13:06.24 take place. 00:13:08.15 Now, it's possible to monitor this 00:13:10.28 using flow cytometry, 00:13:12.08 using the CD4 and CD8 co-receptors 00:13:16.12 as markers of the differentiation pathway. 00:13:20.19 So, when the precursor enters from the blood, 00:13:24.19 it doesn't express either CD4 or CD8, 00:13:29.16 and during this early time period 00:13:32.08 it's basically undergoing cell division 00:13:36.13 and is starting gene rearrangement. 00:13:40.06 Now, all of this takes place inside the cortex. 00:13:45.25 Now, eventually, CD4 and CD8 are both turned on, 00:13:50.13 to have the double-positive stage, 00:13:52.23 which also takes place in the cortex. 00:13:55.18 Now, immunoglobulin... 00:13:57.23 sorry, T cell receptor gene rearrangement 00:14:00.15 is completed 00:14:03.01 during this double-positive stage, 00:14:05.12 and these cells are then 00:14:07.17 ready to be positively and negatively selected. 00:14:12.17 If they're positively selected 00:14:15.12 and deemed worthy of final maturation 00:14:19.07 they become either CD4 single-positive, 00:14:22.02 if they saw an MHC class II molecule with peptide, 00:14:24.18 or CD8+, 00:14:26.27 if they saw an MHC class I molecule with peptide. 00:14:29.02 And this final step of differentiation 00:14:33.04 happens as the cells are entering the medulla. 00:14:36.11 So, the fate of a differentiating T cell 00:14:42.28 is very much focused on these molecules 00:14:45.15 that I've portrayed here 00:14:47.11 -- the interaction between the T cell receptor and co-receptors, 00:14:52.00 and the MHC molecules. 00:14:55.03 And it has to do with 00:14:58.06 the affinity of this interaction 00:15:00.12 -- so, how strongly the T cell receptor can see this complex -- 00:15:04.21 and the avidity 00:15:07.00 -- how many T cell receptor and MHC complexes 00:15:09.14 actually become engaged on a particular set of cells? 00:15:15.26 So, with the least amount of interaction, 00:15:22.18 so, no interaction, 00:15:24.15 or with the strongest interaction, 00:15:26.13 death is the outcome. 00:15:30.00 So, with little interaction, the cell with die because of neglect 00:15:32.20 -- it doesn't get any signaling -- 00:15:34.23 within three days of becoming a double-positive cell. 00:15:39.19 Now, with very strong interaction, 00:15:43.10 clonal deletion will take place, 00:15:45.11 and that cell is removed from the repertoire. 00:15:48.24 Now, with a signal that's a little bit stronger, 00:15:50.22 that's quite weak, 00:15:52.28 but stronger than nothing, 00:15:54.28 naive T cells will 00:15:58.23 continue maturation and eventually leave the thymus. 00:16:01.21 And these will be 00:16:04.02 both CD4 and CD8-positive T cells, 00:16:06.03 depending on whether the MHC molecule 00:16:08.19 was class I or class II. 00:16:11.05 And then somewhere between 00:16:13.22 the naive T cell and clonal deletion, 00:16:16.06 there's clonal diversion, 00:16:18.02 and that signal leads to 00:16:21.04 the changing of the effector T cell phenotype 00:16:24.09 to a regulatory T cell, 00:16:26.15 as I've described before, 00:16:27.26 or perhaps a cell which gets 00:16:30.18 shunted off to the intestine, 00:16:32.02 where it's rather innocuous. 00:16:37.11 So, I'd like to show you 00:16:39.22 some examples of clonal deletion, 00:16:42.24 and the most striking ones come 00:16:45.27 from T cell receptor transgenic mice. 00:16:47.22 So, unfortunately, any particular T cell specificity 00:16:51.06 is very rare in an individual 00:16:54.03 -- it's usually only 1 in 10^4 00:16:56.25 to 1 in 10^6 T cells 00:16:59.21 are of one particular specificity. 00:17:03.23 That means that that T cell receptor sees a particular antigen. 00:17:07.09 And that's very difficult to study and follow these cells, 00:17:09.28 so the trick that immunologists play 00:17:12.15 is to take a T cell clone, 00:17:15.20 which they know the specificity for 00:17:17.25 and they know where it came from, 00:17:19.11 and they take the already rearranged T cell receptor genes 00:17:24.01 from that T cell clone, 00:17:25.13 and use those to make transgenic mice with. 00:17:28.13 And since those are already rearranged, 00:17:30.07 they shut down endogenous rearrangement, 00:17:32.14 and you end up with a transgenic mouse 00:17:35.07 where the repertoire is highly skewed 00:17:37.22 for that transgene-encoded T cell receptor. 00:17:42.19 So, in one case, umm... 00:17:45.11 investigators started with a T cell clone 00:17:48.15 that was CD8+ 00:17:53.20 and recognized an antigen which is male-specific, 00:17:56.04 and that's called the H-Y antigen 00:17:58.16 because it's encoded on the Y chromosome. 00:18:01.17 And they took those T cell receptor genes 00:18:03.14 and made TCR transgenics, 00:18:05.23 and they looked at differentiation in the thymus. 00:18:09.23 And what they saw, at steady state, 00:18:11.13 was that, yes, you get double-negatives, 00:18:14.15 as expected, 00:18:15.24 you get double-positives, 00:18:17.15 and then the single-positives are primarily CD8+, 00:18:21.08 because the clone that you started with was a CD8+ T cell. 00:18:25.07 Now, this is in females, 00:18:26.16 where the H-Y antigen does not exist. 00:18:29.27 If instead, you look in males 00:18:32.09 that are TCR transgenic, 00:18:33.23 what you find is you get double-negatives and you don't see any other T cells after that 00:18:40.02 -- they've been clonally deleted. 00:18:42.03 Now, another example 00:18:46.01 comes from a T cell clone that is CD4+ 00:18:47.23 and it sees an antigen 00:18:50.17 which is found in the blood, 00:18:52.29 and it's called the C5 antigen. 00:18:54.26 It's a complement protein. 00:18:57.16 And when people make these T cell receptor transgenics, 00:19:00.29 in a line of mice 00:19:03.19 that naturally does not have C5, 00:19:05.27 they find that there are double-negatives, 00:19:08.09 double-positives, 00:19:09.14 and primarily CD4 single-positive cells. 00:19:12.12 However, if instead, they looked on a line 00:19:16.04 where C5 does exist, 00:19:18.18 they find double-negatives and double-positives, 00:19:22.02 but not CD4 single-positives. 00:19:25.03 Now, from this one slide, 00:19:26.26 you can already learn several things 00:19:28.19 about clonal deletion. 00:19:30.13 First of all, clonal deletion 00:19:34.02 happens to both CD4+ and CD8+ T cells. 00:19:38.06 And then, secondly, it can occur in different places 00:19:41.24 at different stages 00:19:43.17 during T cell differentiation. 00:19:45.06 So, in the top case, 00:19:47.24 we had an antigen which is male-specific, 00:19:50.16 and it's a ubiquitous antigen found on all cell types. 00:19:54.03 So, as soon as T cells... as double-positives turned... 00:19:59.04 have finished the rearrangement of their T cell receptors, 00:20:01.16 they get deleted, 00:20:02.26 and there's no T cells available for continuing with maturation. 00:20:08.09 It's a different case with this C5 antigen, 00:20:10.26 which is found in the blood, 00:20:12.14 because blood circulates through the thymus, 00:20:15.10 in the medullary region 00:20:17.20 and not in the cortical region, 00:20:18.23 and so double-positives never see this antigen 00:20:21.28 and it's only when the T cells are fully mature 00:20:25.15 and move into the medulla 00:20:26.28 that clonal deletion occurs. 00:20:28.27 In fact, clonal deletion can even occur 00:20:31.28 in the periphery under certain conditions. 00:20:38.14 So, the mechanism of clonal deletion is apoptosis, 00:20:43.25 and I think this is nicely shown 00:20:47.20 by the experiment illustrated on this slide. 00:20:49.06 So, here, people are 00:20:52.06 dealing with a T cell whose receptor 00:20:56.00 sees a quite common antigen 00:20:58.07 and sees it in the medulla. 00:21:01.26 And what they find... 00:21:03.19 and the way they look for clonal deletion 00:21:05.28 is to use what's called the TUNEL assay, 00:21:08.23 which is an assay which radioactively labels 00:21:11.04 free DNA ends, 00:21:13.09 which are generated during the process of apoptosis. 00:21:18.29 And so you can see, at the top, 00:21:21.07 when the antigen is present, 00:21:23.00 that there's a lot of apoptosis occurring in the medulla... 00:21:27.21 a little bit in the cortex, but mostly in the medulla... 00:21:29.08 however, when the antigen is absent, 00:21:31.12 you don't see these apoptotic structures. 00:21:39.15 As an example of how important 00:21:43.03 clonal deletion can be, 00:21:44.16 one should recognize the fact that 00:21:48.18 there are human autoimmune diseases 00:21:50.27 which reflect deficits in clonal deletion. 00:21:56.06 So, in one example, 00:21:58.05 there's a mutation in a transcription factor 00:22:01.04 which is important as a general 00:22:07.03 effector of immunological tolerance, 00:22:11.00 and so what happens is that 00:22:13.14 these individuals get a multi-organ autoimmune disease 00:22:16.07 called autoimmune polyglandular syndrome type-1. 00:22:20.02 In the second example, 00:22:22.03 it's a quite specific deficit 00:22:24.20 in central tolerance, 00:22:26.14 having to do with expression of the insulin gene 00:22:29.21 in the thymus, 00:22:31.24 and these individuals 00:22:34.26 specifically develop type-1 diabetes. 00:22:36.29 Now, the first example 00:22:39.11 will form the basis of my second talk, 00:22:42.06 so I won't go any further on that now, 00:22:45.11 but I will give you a little bit of information, 00:22:48.11 more information about the second example. 00:22:52.29 So, people have done a number of genetic studies 00:22:55.21 and identified several genes 00:23:00.11 which predispose to the development of type-1 diabetes in humans, 00:23:04.18 or protect from the development of type-1 diabetes. 00:23:09.05 Now, the most important one of these genes 00:23:11.15 is the HLA locus, 00:23:14.11 which is the equivalent of the MHC gene, 00:23:15.29 which I introduced you to. 00:23:17.29 But the second most important 00:23:20.00 is actually the insulin gene itself. 00:23:22.03 Now, the variation in 00:23:25.15 the diabetes-predisposing 00:23:28.00 versus the diabetes-protective insulin gene 00:23:31.23 is not in the coding region of the insulin gene, 00:23:35.15 but rather in the promoter region, 00:23:38.08 where there's a class of sequences 00:23:41.15 called variable number of tandem repeats 00:23:45.14 -- just a bunch of repeated sequences -- 00:23:48.07 and depending on how many of these sequences 00:23:51.28 there are in the promoter region, 00:23:53.10 the allele is either 00:23:56.29 diabetes-promoting or diabetes-protective. 00:23:59.19 So, when there are just a few of these repeats, 00:24:01.17 it's diabetes-promoting, 00:24:03.01 and when there are more 00:24:05.22 it's diabetes-protective. 00:24:07.12 So, if we look through the population at these alleles 00:24:11.08 that have either protective or promoting VNTRs, 00:24:15.03 what we find is that those people who have the protective... 00:24:22.15 the promoting allele, the predisposing allele, 00:24:25.12 develop diabetes much more commonly 00:24:28.08 than the population in general. 00:24:32.00 And those are the examples on your left. 00:24:36.10 Now, the ones on your right 00:24:38.03 are those individuals who have 00:24:40.04 two copies of the protective allele, 00:24:42.14 and you can see that they develop diabetes 00:24:44.26 much less frequently than the general population. 00:24:49.04 And then, if you look to see 00:24:53.17 what these variations in the promoter regions 00:24:56.28 do to insulin gene expression, 00:24:58.17 what you find is that 00:25:01.06 there's not much difference in the expression of insulin 00:25:04.14 in the pancreas. 00:25:05.25 Where there is a big difference 00:25:07.11 is actually in expression of insulin 00:25:10.06 in the thymus. 00:25:11.18 And people have made the speculation 00:25:18.02 that those cases where there's higher expression, 00:25:22.00 for example, with the class III alleles, 00:25:24.27 these people have more expression of insulin, 00:25:28.26 greater clonal deletion in the thymus, 00:25:31.18 and then less development of autoimmunity. 00:25:34.11 And this has actually been modeled in mice 00:25:37.19 by making transgenic mice 00:25:40.06 which are expressing the different types of human alleles, 00:25:44.09 and the results fit very well this idea. 00:25:49.20 So, I hope I've convinced you by now that 00:25:53.22 central tolerance, and in particular clonal deletion, 00:25:55.11 is an important mechanism of immunological tolerance. 00:25:59.05 However, clonal deletion is never complete. 00:26:03.26 There are antigens which are not expressed in the thymus 00:26:07.02 or they're not expressed... 00:26:11.15 there are antigens which T cells don't see 00:26:13.28 with a high enough affinity, 00:26:15.15 or there are antigens whose concentration in the thymus 00:26:19.15 is not high enough to permit clonal deletion. 00:26:23.12 And one might actually make the statement 00:26:28.00 that it would be devastating 00:26:29.25 if clonal deletion was actually complete, 00:26:33.15 because, as you can imagine, 00:26:35.04 if you deleted all the T cells that saw any antigen 00:26:39.17 with any affinity 00:26:43.02 that there would be a very small repertoire 00:26:45.02 that would emerge into the periphery, 00:26:47.08 and not be able to fight off 00:26:49.09 the millions and millions of microbes 00:26:51.25 that the individual is going to encounter. 00:26:56.03 So, that leaves open 00:26:59.26 the space for peripheral T cell tolerance. 00:27:05.14 And I'm going to focus on one particular and important mechanism 00:27:09.23 of peripheral tolerance, 00:27:11.16 and that's suppression. 00:27:13.28 And, as I mentioned, suppression 00:27:17.28 is regulation of the behavior of self-reactive T cells 00:27:20.28 by other T cells... by other cells. 00:27:24.04 This is, again, an important mechanism, 00:27:27.01 and that can be seen very clearly 00:27:30.19 by the phenotype of either humans of mice 00:27:36.04 who are lacking a particular kind of regulatory T cell, 00:27:39.21 a suppressor T cell, 00:27:43.04 and these people get a very severe 00:27:45.13 autoinflammatory disease. 00:27:47.14 And, in fact, there are 00:27:49.14 more than just this type of suppressor cell 00:27:53.14 that keep effector T cells in check. 00:27:58.17 There are actually several types: 00:28:00.10 T cells; 00:28:02.19 some people think some B cells suppressor cells; 00:28:04.15 and also some macrophage suppressor cells. 00:28:08.15 But I'm going to focus my comments 00:28:11.06 on one very famous regulatory T cell, 00:28:14.16 the most famous T cell in immunology, actually, 00:28:18.11 and these are regulatory T cells 00:28:21.13 which express the transcription factor Foxp3, 00:28:24.11 and we call them, affectionately, 00:28:27.04 Treg cells. 00:28:28.23 So, these T cells express 00:28:31.24 the αβ T cell receptor, 00:28:33.17 the CD4 co-receptor, 00:28:34.19 and they express high levels 00:28:37.12 of the high-affinity receptor for IL-2, 00:28:42.10 and this molecule is called CD25, 00:28:45.07 and that was used for many years 00:28:47.18 as a means to distinguish them from other types of T cells. 00:28:50.21 But later, it was found that 00:28:53.16 these T cells are actually a particular lineage of T cells 00:28:56.18 and what defines that lineage 00:28:58.27 is the transcription factor Foxp3. 00:29:01.13 Now, normally circulating through the body 00:29:07.12 of just a standard person or mouse, 00:29:10.02 around 5-15% of the CD4+ T cells 00:29:14.23 are these Foxp3+ regulatory T cells. 00:29:18.19 And they're importance became very clear 00:29:21.24 when it was understood that 00:29:24.20 humans that have the IPEX disease, 00:29:27.08 and mice that have the Scurfy disease, 00:29:30.12 have a very severe autoinflammatory disease 00:29:35.01 affecting many organs, 00:29:36.13 because they are missing Foxp3 00:29:39.24 -- they have a mutation in it -- 00:29:41.14 and sub... 00:29:43.20 consequently they are missing this population of regulatory T cells. 00:29:48.12 Now, as I mentioned, 00:29:49.26 these T cells are generated... 00:29:54.17 most of them are generated in the thymus 00:29:56.21 by the process of clonal diversion. 00:30:00.12 In other words, their T cell receptors 00:30:03.03 see a self-antigen expressed in the thymus 00:30:07.13 at an intermediate affinity 00:30:10.12 between what a naive T cell sees 00:30:13.10 and allows it to undergo maturation, 00:30:16.20 and a clonally-deleted T cell 00:30:22.27 that undergoes negative selection sees. 00:30:25.21 By now, using gain-of-function and loss-of-function experiments, 00:30:29.24 it's been determined that 00:30:32.23 Tregs actually control almost all types of immune responses. 00:30:36.26 They control autoimmune diseases 00:30:39.21 like inflammatory bowel disease or type-1 diabetes. 00:30:43.07 They control inflammatory diseases like rheumatoid arthritis, 00:30:47.14 allergic diseases such as asthma. 00:30:50.07 They also keep in check graft rejection. 00:30:53.28 They promote tumor escape, 00:30:58.12 and they inhibit the response 00:31:03.13 to various types of infections. 00:31:06.18 So, from graft rejection up, 00:31:10.20 the regulatory T cells are the good guys 00:31:13.25 -- the more you have, the less disease you have. 00:31:16.24 From tumors down, 00:31:19.18 the regulatory T cells are the bad guys, 00:31:22.04 so the more Tregs you have, 00:31:23.28 the less chance the individual has 00:31:27.09 of mounting an immune response against the tumor, 00:31:30.12 and the same thing for infections. 00:31:36.00 It was found that regulatory T cells, 00:31:41.17 as I've said, 00:31:44.05 are critical for controlling all types of inflammatory responses in the body, 00:31:49.25 and actually that this happens throughout life, 00:31:52.06 and that was determined by looking at mice 00:31:57.08 where it was possible to ablate regulatory T cells 00:32:03.04 by deleting the Foxp3 gene. 00:32:05.22 And so, if this was done from birth on, 00:32:09.25 it was very clear that the mice were very sick. 00:32:13.26 They died before 25 days of age 00:32:17.05 and they had a massive expansion 00:32:24.18 of immune cell populations 00:32:26.22 in the spleen and the lymph node, 00:32:28.22 and you can see in the bottom picture that they're actually quite small 00:32:31.25 and wasting away. 00:32:33.27 And in fact, you can wait 00:32:36.03 until the mouse becomes an adult, 00:32:38.04 and so it's had Tregs during this whole time, 00:32:40.28 and then just deplete Tregs 00:32:43.23 in an adult mouse, 00:32:45.09 and within a week or so 00:32:48.18 these mice will also develop 00:32:51.13 a very severe and fatal autoinflammatory disease. 00:32:57.08 More specifically, 00:32:59.07 Tregs will control particular types of autoimmunity, 00:33:02.00 and one nice example is the 00:33:05.19 development of type-1 diabetes in a mouse model 00:33:08.08 called the NOD mouse. 00:33:10.10 Now, when Tregs are there, 00:33:13.08 these Foxp3+ regulatory T cells, 00:33:16.14 autoimmunity does not develop 00:33:20.12 or it develops very slowly. 00:33:22.17 Whereas, if you get rid of Tregs, 00:33:26.16 it will develop much, much more quickly. 00:33:30.11 You can add in Tregs 00:33:34.09 and what you find is that these animals 00:33:36.20 that got diabetes really, really quickly 00:33:40.18 would now develop it much, much later, 00:33:45.18 off-scale on the timeframe that I'm showing you. 00:33:51.15 So... and then I'd just like to finish 00:33:54.21 by talking a little bit about the different effector mechanisms 00:33:57.13 that regulatory T cells use. 00:34:00.05 There are several. 00:34:02.19 One is by the production of anti-inflammatory cytokines 00:34:06.21 such as IL-10 or IL-35. 00:34:11.04 The second is by actually killing the effector T cell. 00:34:17.19 Third, they disrupt the metabolism of the cells, 00:34:23.13 for example, 00:34:25.19 I mentioned that regulatory T cells have 00:34:28.10 high levels of the high-affinity IL-2 receptor, 00:34:30.25 so they can act as a sink 00:34:34.12 for sucking up all this IL-2, which is actually required for the health 00:34:39.09 of the effector T cells. 00:34:41.24 And then, lastly, Tregs can 00:34:44.22 affect other types of cells, 00:34:46.23 antigen-presenting cells in the region, 00:34:49.09 and stop them from 00:34:54.22 triggering an immune response, 00:34:56.28 either by killing the cell or 00:34:59.08 changing what type of cytokines these cells make. 00:35:02.07 So, this is a quite complex mechanistic scenario 00:35:11.16 and people in the field have been asking, 00:35:14.04 is it the case that every single Treg cell 00:35:17.10 is able to do these different types of inhibition 00:35:22.22 and, if so, 00:35:26.16 when does one come into play and the other replace it? 00:35:29.00 Or it is that in any kind of Treg response, 00:35:33.04 there is a heterogeneous set of cells there, 00:35:37.22 some of which are specialized in doing mechanism 1 00:35:40.23 and others in doing mechanism 4 00:35:43.06 and others in mechanism 3? 00:35:45.05 And that's something which the field is very interested in 00:35:49.29 determining at the moment. 00:35:52.03 So, I'll just finish by saying that regulatory T cells 00:35:54.11 are a very exciting field at the moment in immunology, 00:35:59.06 that there's a lot of interest in 00:36:02.17 actually using them to control autoimmune diseases 00:36:07.00 or other inflammatory diseases 00:36:09.25 by taking them out, expanding them, 00:36:13.11 and reintroducing them into individuals with various diseases. 00:36:19.01 Thank you.