Like B cells, T cells develop in a manner that enhances the diversity of T cell receptors while preventing self-recognition. This session provides an overview of T cell development and differentiation. It covers two important mechanisms that prevent autoimmunity – central and peripheral tolerance – and characterizes the biogenesis and function of two specialized T cells, Tregs and Th17 cells.
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,
00:19:09.14 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.
00:00:15.01 My name is Dan Littman.
00:00:16.04 I'm a professor of Molecular Immunology at the Skirball Institute,
00:00:19.10 which is part of New York University School of Medicine.
00:00:22.11 And I'm also an Investigator of the Howard Hughes Medical Institute.
00:00:26.04 What I'm going to tell you about is how the microbiota, as well as other components
00:00:33.17 of the environment, influence the immune system at barrier surfaces.
00:00:37.13 I'm going to focus mostly on the intestine, which is the area that's been best studied
00:00:43.02 by many groups around the world during in the last decade.
00:00:46.11 And I'll tell you, in my first part of the talk, about the different kinds of cells
00:00:51.11 that are involved, as well as some of the signals that are involved, particularly in the functions
00:00:56.23 of lymphocytes, in lymphocytes that are both within the innate immune system
00:01:01.16 and the adaptive immune system.
00:01:03.21 The adaptive immune system consists of B lymphocytes and T lymphocytes that differentiate
00:01:09.10 from common lymphoid progenitors.
00:01:11.09 And these are adaptive because they have rearranging genes that give rise to T cell receptors
00:01:17.20 or antibody receptors on the surface of these cells,
00:01:21.05 so that each cell has a clonally restricted type of receptor.
00:01:24.12 The innate cells... lymphoid cells also differentiate from a common lymphoid progenitor, but they
00:01:30.27 have fixed receptors, and they are basically hardwired to respond to various cues that
00:01:37.02 are presented to them, be they cytokines or be they some type of danger,
00:01:44.13 antigens that are presented to them.
00:01:45.26 So, I'll start out talking about the T cells, and in the second part of this presentation
00:01:51.00 I will tell you a little bit about some of the innate lymphoid cells.
00:01:54.25 T lymphocytes develop in the thymus from common progenitors that... that enter the thymus
00:02:02.07 as so-called double negative cells.
00:02:04.11 And the double negative cells are so called because they don't express on their cell surface
00:02:08.19 the molecules CD4 and CD8.
00:02:11.00 As they undergo development, these cells can take one of two lineages.
00:02:17.13 One lineage is to become gamma delta T cells, meaning they have receptors encoded by
00:02:22.16 the gamma and delta genes.
00:02:24.07 Or they become alpha beta T cells.
00:02:26.08 The alpha beta T cells typically express both CD4 and CD8 on the surface as
00:02:32.28 they become double positive cells.
00:02:34.17 And these cells develop, if they have appropriately rearranged receptor genes that give rise to
00:02:40.10 the protein on the surface, a heterodimer of the alpha and beta chains.
00:02:44.14 The vast majority of these cells undergo cell death, because for a cell to develop in the thymus,
00:02:49.11 it needs to have a receptor that interacts with self MHC proteins,
00:02:53.24 major histocompatibility complex proteins,
00:02:57.01 that have peptides presented to the T cell receptor.
00:03:00.10 So, since most of the cells do not have an appropriate receptor, they undergo cell death.
00:03:05.00 A few of the cells also have receptors that interact with very high affinity with self-antigen.
00:03:10.13 And these are potentially damaging cells that can lead to autoimmunity, and those are
00:03:14.23 also eliminated through a process called negative selection.
00:03:17.20 And then the few cells that make it through this gauntlet undergo positive selection,
00:03:23.04 and most of them become either CD4-positive cells or CD8-positive cells.
00:03:28.13 The CD4-positive cells are those that are selected on MHC class II molecules,
00:03:33.18 and they are typically helper cells.
00:03:35.26 And I'll talk a great deal about this throughout the rest of my presentation.
00:03:39.11 The CD8-positive cells are selected on MHC class I, and they mostly become
00:03:44.28 cytotoxic or killer T cells.
00:03:46.13 There's another type of CD4 cell that's also selected on MHC class II.
00:03:51.00 Typically, these are cells that have a higher affinity receptor.
00:03:54.20 And these cells up regulate the transcription factor Foxp3 and become regulatory T cells.
00:04:00.02 So, these are thymically derived regulatory T cells, which are essential to maintain tolerance
00:04:05.15 in the periphery and prevent autoimmune activation of the other types of cells of the immune system.
00:04:12.06 I'll tell you a little bit about another type of regulatory T cell that arises in the periphery,
00:04:18.01 the so-called induced Treg cells, in... a little bit in this presentation
00:04:24.07 and a lot more in the second part of my talk.
00:04:27.28 Once these cells develop the thymus, they are exported into the periphery.
00:04:32.16 And most of the classical alpha beta T cells, the CD4 cells and the CD8 cells
00:04:38.16 go to secondary lymphoid organs, where they are naive T cells awaiting to be activated by immune signals,
00:04:47.03 potentially from invading microorganisms, and following activation of innate immunity.
00:04:53.21 But there are also cells, such as the gamma delta T cells, as well as some subsets
00:04:58.24 of alpha beta T cells, that go directly to peripheral organs: into skin, the epithelium in the intestine,
00:05:05.09 as well as the lamina propria in the intestine, as well as the female reproductive tract, or the lung.
00:05:11.19 And these... these are cells that are very much like innate lymphoid cells, in that
00:05:18.02 they can often be activated very quickly to deliver the cytokine load that they have.
00:05:24.18 But the more conventional cells from the lymphoid organs, once they are activated by antigen
00:05:30.06 in those organs, can migrate out to the very same sites in the periphery, into these different...
00:05:36.18 different tissues.
00:05:37.18 And once these cells migrate to the different tissues, they can stay there and continue
00:05:41.28 to replenish themselves.
00:05:43.21 And these become tissue-resident memory T cells.
00:05:46.13 And they consist not only of the T lymphocytes that I just mentioned, but also of
00:05:50.26 innate lymphoid cells that I'll tell you more about, and also macrophages.
00:05:54.13 In particular, there are different populations of macrophages, some of which arise very early
00:05:59.17 during fetal development, that establish themselves into tissues and then reside in those tissues
00:06:04.19 for the life of the organism, continuing to replenish themselves.
00:06:08.02 So, these are to be distinguished from the other types of cells that are circulating
00:06:12.05 between the blood and the lymph and the secondary lymphoid organs.
00:06:16.02 And that is a distinction that one should keep in mind.
00:06:20.08 So, these are many different tissues that now harbor these tissue-resident cells.
00:06:25.08 An example is shown here from work of Daniel Mucida, in which he described T lymphocytes
00:06:31.28 that... that established themselves in the epithelium of the intestine, in this case,
00:06:37.19 the small intestine.
00:06:38.28 And you can see the tracings of these cells as they traffic through the epithelium.
00:06:44.17 They basically undergo a flossing-like movement in which they are detecting potentially harmful pathogens
00:06:51.04 as well as any kind of damage to the tissue, and then make the cytokines
00:06:56.22 and growth factors to repair the tissues, or rid the organism of the potential pathogen.
00:07:03.28 I'm going to focus mostly on the CD4-positive T cells, and most of these cells are
00:07:11.05 helper T cells, because they help the cytotoxic T cells to undergo their functions.
00:07:16.18 They also help B lymphocytes to make antibodies.
00:07:20.14 But it's... about 30 years ago, Tim Mosmann and Bob Coffman, at the DNAX Institute at that time,
00:07:26.02 first described different properties of CD4-positive T cells in that
00:07:31.16 they could make different types of cytokines.
00:07:34.02 What they found was one subset of cells made the cytokine interferon gamma.
00:07:40.10 And they and others then found that these cells are critical for killing a variety of
00:07:44.17 intracellular microbes -- bacteria, viruses, protozoa -- are controlled by these T helper 1 cells.
00:07:53.24 These cells express the transcription factor T-bet, which is required for their differentiation.
00:07:58.25 The other cell type that they identified secreted interleukin-4 and interleukin-5,
00:08:06.08 and later was shown that they also make IL-13.
00:08:08.28 And these are critical for controlling infection with helminths, or parasitic worms.
00:08:14.03 And on the other hand, these cells are also very important in allergy and in asthma,
00:08:19.25 and need to be controlled.
00:08:21.26 The Th1 cells were initially thought to be the key cells involved in autoimmune disease.
00:08:27.02 But about a decade ago, a third type of differentiated T cell was described, called the Th17 cell.
00:08:33.28 And these cells are so-called because they make the cytokines interleukin-17A and interleukin-17F.
00:08:38.23 They also make interleukin-22.
00:08:42.07 And it was found that these are actually the cells that are most often involved in autoimmune inflammation.
00:08:50.02 And these are cells that are normally needed to kill extracellular bacteria and fungi
00:08:55.26 at mucosal surfaces.
00:08:57.18 They're very important for repairing damage to mucosal tissues.
00:09:01.19 And in a number of different models for autoimmunity, they have been found to be the critical cells.
00:09:06.15 But more important, they have been found to be critical cells in autoimmunity in human.
00:09:13.13 I'm going to tell you a lot about these.
00:09:15.20 First of all, just as a way of background, the IL-17 cytokines are very important
00:09:20.26 for inducing chemoattractant cytokines, chemokines that attract neutrophils to the site of the secretion.
00:09:28.01 They're also involved in tissue remodeling.
00:09:31.17 IL-17 induces matrix metalloproteinases, as well as VEGF, which leads to angiogenesis
00:09:42.00 in a variety of tissues.
00:09:43.27 Interleukin-22, on the other hand, is more of a cytokine that leads to
00:09:49.00 proliferation of epithelial cells, and protects the barriers from damage.
00:09:55.21 There's another, fourth cell type that I want to describe here, and I mentioned it briefly already.
00:10:00.07 That is the induced regulatory T cell, which, like the one that's made in the thymus,
00:10:04.12 also expresses FOXP3.
00:10:05.12 But in the periphery, these are cells that are induced by combinations of cytokines,
00:10:12.10 particularly TGF-beta and retinoic acid, as well as interleukin-2.
00:10:16.26 And you'll note that the Th17 cells can also rely on TGF-beta for their differentiation.
00:10:24.03 And I'm going to concentrate on telling you a little bit about the requirements
00:10:28.20 for the differentiation of these cells into... in these different directions.
00:10:32.21 When a T lymphocyte is activated, it requires two signals through... in order to proliferate
00:10:40.00 and produce their cytokines.
00:10:43.06 One of the signals of course comes from the signaling pathway linked to the T cell antigen receptor,
00:10:48.06 which interacts with MHC and... either class I or class II MHC and peptide.
00:10:54.08 But the second signal is mediated through CD28, which is called a costimulatory molecule.
00:10:59.28 It interacts with ligands on antigen-presenting cells, on specialized antigen-presenting cells,
00:11:05.12 particularly dendritic cells.
00:11:06.25 And only when these two signals are integrated will the cell, now, become activated and proliferate.
00:11:12.23 But a third signal is needed for the differentiation.
00:11:15.19 And that is a signal provided by cytokines that are made, also, by these antigen-presenting cells
00:11:20.09 most of the time.
00:11:22.13 And these cytokines signal through a variety of different receptor subsets in order to
00:11:29.12 provide the cell with the... with the function that it's going to adopt.
00:11:34.20 An example of this is shown here, in which I show the critical cytokines involved in
00:11:40.17 the differentiation of Th1 cells and of Th17 cells.
00:11:45.17 And these are the cytokines interleukin-12 and interleukin-23.
00:11:49.04 IL-12 and IL-23 share a subunit, the p40 subunit shown here.
00:11:54.18 And they also share a receptor, the IL-12 receptor beta-1, you can see is present
00:12:00.05 as a receptor for both cytokines.
00:12:01.21 But then they also have unique subunits -- p35 for IL-12 and p19 for interleukin-23 --
00:12:09.27 as well as unique receptor polypeptides, which link them up to the signaling pathways downstream.
00:12:18.13 Much of the early confusion about Th1 cells was because of these shared components,
00:12:24.08 of p40 and the IL-12 receptor beta-1, and some of the functions for IL-23
00:12:30.12 were ascribed to IL-12 at the time.
00:12:33.03 But there's been a lot more clarity in the last few years, with the discovery of IL-23.
00:12:38.17 And what we now know is that these receptors both signal through the JAK-STAT pathway
00:12:45.05 of signaling... of signaling molecules.
00:12:47.23 The JAKs are cytoplasmic tyrosine kinases that are engaged by the different receptors.
00:12:53.15 And they transphosphorylate to become activated, and then phosphorylate different types of
00:12:58.19 STAT proteins, which are transcription factors, which, when they are tyrosine phosphorylated,
00:13:03.19 form dimers that then translocate to the nucleus and activate a variety of sets of genes.
00:13:09.04 In the case of IL-12, it activates the STAT4 transcription factor.
00:13:14.26 In the case of IL-23, it activates the STAT3 transcription factor.
00:13:19.14 And many of the other cytokines that I'll tell you about, such as interleukin-6,
00:13:23.28 also activate STAT3, but each of these receptors that utilizes STAT3 also has distinct signaling components
00:13:29.11 to target particular genes.
00:13:33.10 STAT4 can also, under some circumstances, be activated by IL-23.
00:13:38.20 And that is when IL-23 is engaged in pathogenic processes in vivo.
00:13:43.21 And I'll tell you a little bit about the IL-23 function in the next... in the next few slides.
00:13:50.16 The key experiment that introduced the concept of Th17 cells and showed the importance
00:13:55.10 of interleukin-23 was from the laboratory of Dan Cua at DNAX in 2003.
00:14:01.20 What they did was to use a model that's widely used for multiple sclerosis,
00:14:08.16 called experimental autoimmune encephalomyelitis.
00:14:11.11 And this is a model that I'll be talking about throughout my presentation, because it's
00:14:16.23 often used because it's fairly rapid and it's fairly robust.
00:14:20.23 In this kind of a model, a myelin protein is injected into mice along with adjuvant.
00:14:26.22 And typically, within about two weeks, the animals began to develop paralysis.
00:14:31.03 And as you can see over here, in wild type mice the paralysis develops as expected.
00:14:36.17 And in mice that are deficient for p40, which, as you recall, is absent in... in...
00:14:44.21 leads to an absence of both interleukin-12 and interleukin-23, you can see these animals are protected.
00:14:50.24 But the surprise at the time was that mice lacking just IL-12, that were deficient for p35,
00:14:56.04 not only developed disease, but they actually had even more severe disease than the wild type.
00:15:02.03 Whereas mice deficient for p19 -- that... what was then the newly discovered component
00:15:07.15 of interleukin-23 -- were completely protected.
00:15:10.20 So, that then led to the important concept of the target of p19 as being the Th17 cell.
00:15:18.11 And these cells are critical for barrier defenses against a variety of different bacteria
00:15:24.14 and fungi, fungi including Candida albicans.
00:15:28.26 But the flip side of this is that these cells can also be highly pathogenic through
00:15:33.18 the production of their variety of cytokines.
00:15:37.19 And under inflammatory conditions, they can now produce not only IL-17 and IL-22,
00:15:44.01 but they can also make interferon gamma.
00:15:46.28 And these cells have been validated to be very important in many human diseases, particularly psoriasis.
00:15:52.23 In psoriasis, antibodies against interleukin-17A are very effective in therapy.
00:15:58.01 Also, a variety of different arthritides, psoriatic arthritis and ankylosing spondylitis
00:16:03.08 in particular, can be treated by blockade of interleukin-17 and interleukin-23.
00:16:10.05 And then there are also some other... there are some other suggestions that multiple sclerosis,
00:16:17.04 as well as a variety of inflammatory bowel diseases, are Th17-mediated diseases.
00:16:23.05 For IBD in particular, it is known that polymorphisms in the Th17 signaling pathway can contribute
00:16:31.02 to the disease, again adding further argument for a role for these kinds of cells in the disease.
00:16:39.01 There's also some evidence from animal models that Th17 cells in the mother can influence
00:16:45.22 the development of the fetal brain, if they are expressed in very high levels
00:16:50.08 and can cross the placenta.
00:16:51.19 This is only an animal model that has been looked at, but I will discuss this
00:16:58.15 in the second part of my presentation.
00:17:01.20 At the center of the Th17 differentiation process is a transcription factor, ROR gamma t.
00:17:08.03 And ROR gamma t is encoded by this locus, Rorc, in which two different isoforms
00:17:13.23 can be ex... can be transcribed, depending on the promoter that's used.
00:17:18.08 And the longer form is expressed quite broadly, and it's expressed in a circadian manner.
00:17:24.26 But the shorter form of ROR gamma t is expressed exclusively in lymphoid lineage cells.
00:17:32.16 And these include the cells that develop in the thymus, the double positive thymocytes
00:17:39.18 that require ROR gamma t for their survival, as well as lymph nodes and Peyer's patches,
00:17:46.19 secondary lymphoid organs that develop in the fetus and that require the lymphoid tissue inducer cells
00:17:51.17 that are dependent on ROR gamma t.
00:17:54.27 In this slide, I show a crystal structure of ROR gamma t, of the ligand binding domain,
00:18:00.03 which regulates its function.
00:18:02.19 So, this is a nuclear receptor, very similar to estrogen receptor and glucocorticoid receptor.
00:18:08.21 And it is thought that it's regulated by ligand, but the precise ligand has yet to be defined.
00:18:14.07 What we know is that molecules in the cholesterol biosynthetic pathway are very effective
00:18:22.16 at regulating ROR gamma t function.
00:18:25.15 But we don't yet have strong genetic data to tell us which of these intermediates
00:18:32.11 are important in vivo, and in particular, in the differentiation of the different cell types,
00:18:37.07 whether there may be different ligands that are involved.
00:18:40.08 Now, in... beyond the function and development, the transcription factor, of course,
00:18:46.15 is required for Th17 cell differentiation.
00:18:50.03 It's also plays... playing a role in the differentiation of the induced regulatory T cells
00:18:58.07 found in the intestine, in particular in response to microbiota.
00:19:02.22 And I'll talk more about that in the second part of my presentation.
00:19:07.02 Also, there are gamma delta T cells that are specialized to make IL-17, and that's
00:19:12.12 also dependent on ROR gamma t.
00:19:14.11 And then there are the innate lymphoid cells, and these include the lymphoid tissue inducer cells,
00:19:18.15 both those that are involved early in the fetus, in lymphoid development,
00:19:23.25 but also those that appear... that develop postnatally and that are involved in some of
00:19:32.19 the tertiary lymphoid tissues.
00:19:34.07 I'll talk a bit more about the type III innate lymphoid cells a little bit later.
00:19:39.00 But I want to come back to the Th17 cells.
00:19:41.17 And this experiment that we did a bit more than a decade ago showed the importance of
00:19:48.08 ROR gamma t in the EAE model.
00:19:51.15 So, you can see here that animals deficient for expression of ROR gamma and ROR gamma t
00:19:57.08 are highly protected from EAE.
00:20:02.01 And you can see that there are very few IL-17 producing cells, shown by the FACS analysis,
00:20:07.14 over here.
00:20:08.24 These are cells in the central nervous system.
00:20:11.27 On the other hand, in the wild type mice, you see not only that there are IL-17-producing cells
00:20:17.03 but also cells that make both interferon gamma and interleukin-17.
00:20:21.12 And it turns out these are very important cells, because these are those cells
00:20:25.12 that are found in pathological situations, in which there is tissue destruction and autoimmunity.
00:20:33.21 I will discuss two pieces of evidence that suggest a critical role for interleukin-23
00:20:39.05 in the generation of these cells that produce both interferon gamma and interleukin-17.
00:20:46.00 One of the experiments is from an in vitro model for EAE, in which it is possible to
00:20:51.08 differentiate cells into Th17 in vitro, and if these cells are specific for a myelin protein,
00:20:59.14 then they can be injected into mice.
00:21:01.21 And within two weeks the animals get EAE.
00:21:03.28 So, people typically use transgenic mice in which the transgene for the T cell receptor
00:21:11.06 is for a T cell receptor that recognizes a myelin protein.
00:21:16.02 And typically, most people use, in vitro, interleukin-6 and TGF-beta to differentiate cells
00:21:22.13 into Th17 cells that make interleukin-17 and interleukin-22.
00:21:29.27 But it turned out that under these conditions these cells did not induce EAE.
00:21:35.04 On the other hand, when interleukin-23 was included, it was possible, now, to get EAE.
00:21:40.13 And it was even possible to do so in the absence of TGF-beta, just by including interleukin-1 beta
00:21:46.04 instead of TGF-beta.
00:21:47.15 In that case, again, EAE could be induced.
00:21:51.10 And so the interleukin-23 molecule is critical in this process.
00:21:55.18 And this is a very elegant experiment that was done by Brigitta Stockinger's laboratory in England,
00:22:01.21 in which they showed the importance of IL-23 in the generation of these Th17 cells
00:22:07.24 that make interferon gamma.
00:22:09.23 What she did was a fate mapping experiment, in which the yellow fluorescent protein is
00:22:15.13 knocked into a ubiquitous locus, but it's only expressed when a transcriptional stop signal
00:22:20.18 is excised by the action of Cre recombinase.
00:22:23.28 So, she bred these mice to animals in which the Cre recombinase was knocked into the IL-17a locus,
00:22:32.10 so only those cells that make IL-17a will have the capacity to then express YFP.
00:22:38.00 So, upon expression of Cre, the stop signal is excised.
00:22:41.27 YFP is expressed for the life of that cell, even after the cell stops making IL-17.
00:22:48.14 So, what the group then did was to gate on just those YFP-positive CD4 cells in the model of EAE,
00:22:56.13 looking now at what happens.
00:22:59.02 And of course, in the absence of interleukin-23, there is no EAE.
00:23:03.10 But they were able to now look at these YFP-positive cells and see what their phenotype was.
00:23:08.19 And you can see here in wild type mice, many of these cells, now, express interferon gamma,
00:23:14.10 or a combination of interferon gamma and interleukin-17A.
00:23:17.15 But these cells are absent in the absence of interleukin-23.
00:23:22.26 So, that really provided the critical piece of evidence that these double-producing cells
00:23:32.06 are important in the pathogenesis in this model.
00:23:35.12 But there's evidence that it's important in pathogenesis in other models as well.
00:23:39.12 So then, what is the role of TGF-beta?
00:23:42.02 Because, as I showed you from the in vitro experiment, it seemed like TGF-beta was
00:23:46.10 not necessarily needed to be able to induce EAE.
00:23:50.23 But on the other hand, if... when people looked in animals in which the TGF-beta receptor
00:23:57.22 was ablated, they saw that there was no EAE, compared to the wild type mice, here.
00:24:02.19 So, how could one explain this?
00:24:04.17 Well, a recent paper by Zhang et al has begun to shed some light on what TGF-beta is doing
00:24:11.15 during differentiation of Th17 cells.
00:24:15.18 And typically, when interleukin-6 signals by itself, through phospho-STAT3, there is
00:24:21.24 no expression of ROR gamma t and no IL-17 that is made.
00:24:26.05 But that appears to be because the... one of the targets of the TGF-beta signaling pathway, SMAD4,
00:24:32.22 recruits a co-repressor complex that includes the SKI molecule, which itself brings
00:24:39.11 in the histone... histone deacetylases.
00:24:41.24 And the histone deacetylases basically shut down chromatin at the ROR gamma t locus.
00:24:47.26 But when TGF-beta is applied, that now leads to a degradation of SKI, of SKI, and basically
00:24:57.22 the relief of the histone deacetylase function, and activation of transcription of ROR gamma t.
00:25:03.09 And now interleukin-17 and other cytokines can be produced.
00:25:09.27 We don't really know yet whether some of the other targets of the TGF-beta signaling pathway,
00:25:14.26 like phosphorylation of SMAD2 and 3 have some positive effects here, but what's clear is that
00:25:20.14 the TGF-beta relieves this negative function.
00:25:23.26 And indeed, what you can see here, in this very nice experiment...
00:25:29.04 if, in the TGF-beta receptor knockout mouse, one introduces also a mutation,
00:25:34.02 a deletion of the SMAD4 locus, now EAE can be restored
00:25:39.09 because Th17 cells can now differentiate in the absence of TGF-beta.
00:25:44.03 So it appears, then, that, in least in vivo, TGF-beta is also important in autoimmunity,
00:25:50.09 although in... even though it doesn't appear to be necessary in the in vitro models.
00:25:58.14 So, what I've just shown you suggests, then, that there are two different types of Th17 cells.
00:26:05.17 Those that can differentiate in the absence of interleukin-23, and that can make interleukin-17A and F,
00:26:12.03 and IL-22, and these we can call homeostatic or non-pathogenic Th17 cells.
00:26:18.03 They typically are induced by microbiota, and they're found at barrier surfaces.
00:26:24.00 On the other hand, in various cases of inflammation, which can be found in different types of tissues,
00:26:29.21 interleukin-23, along with IL-1-beta, but at least in some cases probably aided by TGF-beta,
00:26:35.24 leads to the differentiation of these cells that can make not only the Th17 cytokines
00:26:41.10 but also Th1-like cytokines, like interferon gamma, and that can contribute, now, to disease.
00:26:47.23 And these kinds of pathogenic Th17 cells have also called... been called Th1* cells in human.
00:26:55.26 And I'll summarize for you what we currently know about these Th17 cells, be they non-pathogenic
00:27:01.22 or pathogenic.
00:27:02.22 So, the homeostatic cells are typically induced by the commensal microbiota and protect
00:27:08.18 the mucosal barriers.
00:27:10.02 And they produce not only IL-17a and f and IL-22, but also interleukin-10,
00:27:14.24 which is an anti-inflammatory cytokine.
00:27:18.00 But the pathogenic Th17 cells are induced by selected microbial pathogens,
00:27:24.13 or what we call pathobionts, which can live in our bodies under normal circumstances
00:27:29.09 without causing disease, but then can be stimulated to cause disease in some circumstances.
00:27:35.02 They participate in the autoimmune diseases, dependent on interleukin-23.
00:27:39.10 And in addition to the Th17 cytokines, they make these other cytokines, particularly interferon gamma.
00:27:45.15 And the human equivalent was described by Federica Sallusto.
00:27:49.24 These are cells in the circulation that produce interferon gamma, but they also express target genes
00:27:54.08 for both Th1 and Th17 transcription factors, such as CXCR3 and CCR6,
00:28:02.06 which are chemokine receptors there are targets of Tbet and ROR gamma t, respectively,
00:28:08.16 the Th1 and Th17-specifying transcription factors.
00:28:14.11 So, there are many mutations that have been now described in humans that give us
00:28:20.16 some clues about the Th17 pathways.
00:28:23.04 There are individuals who have chronic cutaneous candidiasis, and oftentimes there are mutations
00:28:31.24 in interleukin-17, interleukin-17 receptor, as well as the signaling components
00:28:37.06 that have been identified.
00:28:39.02 And then there are a few patients who have been described who have disseminated mycobacterial infections
00:28:44.16 after immunization with BCG, which is used as a vaccine for tuberculosis.
00:28:50.07 And it was found that ROR gamma t mutations can account for this, in which there is
00:28:55.24 loss of both the Th1* cells and Th17 cells, even though there's no effect on Th1 cells
00:29:02.24 that can serve an anti-viral function.
00:29:05.22 And some of these mutations are depicted here in this slide.
00:29:09.25 The STAT3 mutations, both gain-of-function and loss-of-function, have been described.
00:29:15.24 And also a gain-of-function mutation in STAT1, which blocks the differentiation of Th17 cells
00:29:22.27 while promoting Th1 cell differentiation.
00:29:26.18 Those kinds of mutations can also account for some of the cases of candidiasis.
00:29:32.20 Mutations in IL-17...
00:29:34.23 IL-17F have been described, as well in the IL-17 receptor A and an adaptor molecule,
00:29:42.24 And polymorphisms in the IL-23 receptor are some of the most commonly associated polymorphisms
00:29:49.18 in inflammatory bowel disease, again providing evidence of the importance of this pathway
00:29:55.20 in autoimmune diseases.
00:29:58.13 There are many different therapeutics that are currently being brought to the market
00:30:02.09 to target this pathway.
00:30:04.06 There are therapeutics at target IL-12/IL-23, that is, p40, or just IL-23 alone, p19.
00:30:12.22 And these are very effective in psoriasis and some forms of arthritis.
00:30:16.27 Then, there are antibodies that target interleukin-17 alone, or a combination of IL-17A and IL-17F.
00:30:25.00 And again, these have been very effective for psoriasis.
00:30:28.13 And finally, there are antibodies that target IL-17 receptor A.
00:30:33.07 Now, these are a little bit more complicated, because IL-17RA is shared by the IL-17A and F cytokines,
00:30:41.02 with other cytokines, IL-17E... which is also called IL-25, which is made by... not only by type II...
00:30:49.04 which acts on type II innate lymphoid cells that I'll tell you about...
00:30:54.07 and also IL-17C acts on the IL-17RE receptor, which is found primarily on epithelial cells.
00:31:00.26 So, one needs a word of caution here, that we don't yet know that much about
00:31:06.15 the biology of some of these other cytokines and receptors.
00:31:09.25 And this particular molecule targets all of these.
00:31:12.28 ROR gamma t is also being targeted for therapeutic purposes, for the obvious reason that
00:31:18.12 it's upstream of these different cytokines, but that has not yet been tested clinically.
00:31:23.26 So, ROR gamma t, if... when it is targeted, is going to affect not only the Th17 pathway,
00:31:30.20 but also innate lymphoid cells.
00:31:32.24 And I'm going to tell you just a little bit about these innate lymphoid cells that
00:31:36.01 have been described only during the past decade.
00:31:38.18 And we believe that these may have been early evolutionary precursors of the
00:31:43.09 differentiated types of T helper cells.
00:31:45.16 So, you can see here that there are innate lymphoid cells -- ILC1, 2, and 3 --
00:31:51.19 that mirror in their transcription factors those transcription factors found on the Th1, Th2, and Th17 cells.
00:31:58.02 In addition, there are innate lymphoid cells that make both ROR gamma t and Tbet.
00:32:04.10 And these are cells that have an NK, natural killer, cell surface marker, NKp46.
00:32:10.18 And these seem to resemble very closely the pathogenic Th17 cells, the Th1* cells
00:32:18.16 that I was mentioning.
00:32:20.12 And these cells also express cytokines that are very similar to those expressed by the...
00:32:26.13 by the T helper cells.
00:32:28.15 And I won't dwell on this, but keep in mind that the T helper cells and the innate lymphoid cells
00:32:36.12 can share many different functions.
00:32:39.11 I'll show you one example here, for type II innate lymphoid cells, in which these are cells
00:32:44.18 that have a very important function in protection from parasitic worms, or helminths.
00:32:50.16 And what was found was that a very highly specialized cell in the intestinal epithelium,
00:32:55.28 in the tuft cell, responds to some product of helminths and also of protozoa by
00:33:03.13 producing interleukin-25 or IL-17E.
00:33:07.10 And that acts on a receptor on type II innate lymphoid cells.
00:33:10.25 And these cells will now make interleukin-13, which acts back on the intestinal stem cells,
00:33:16.05 leading to production of more tuft cells as well as goblet cells, secretory goblet cells,
00:33:21.12 which are very important for expulsion of the... of the parasites.
00:33:28.18 In addition, the type II innate lymphoid cells have another receptor that selectively expressed
00:33:33.19 on these cells.
00:33:34.24 It's a receptor for a neuropeptide, neuromedin-U.
00:33:38.13 And that also is important in this type of regulation.
00:33:42.02 An example of the expansion of these tuft cells is shown here, from work in Richard Locksley's lab,
00:33:48.01 in which they infected mice with a parasite, Nippostrongylus brasiliensis.
00:33:52.08 They generated mice in which the red fluorescent protein was knocked into the IL-25 locus.
00:33:58.02 And you can see, here, red fluorescence in rare tuft cells present, here, in uninfected mice,
00:34:03.22 but a great expansion of the number of cells making RFP following infection.
00:34:09.20 And this can also be shown by using a tuft cell-specific marker, showing expansion over here.
00:34:15.24 But this expansion of tuft cells is probably not only dependent on their making IL-25,
00:34:21.18 but there is also an effect on the type II innate lymphoid cells by neurons in the intestine,
00:34:26.19 by the enteric nervous system.
00:34:28.00 The enteric nervous system consists of many different cell types that are distributed
00:34:33.18 throughout different plexi in the layers of the intestine, and they can communicate locally
00:34:37.28 as well as extrinsically with the central nervous system through the vagus nerve.
00:34:44.16 You can see here, with a pan-neuronal marker, that these neurons can extend into the villi
00:34:51.23 in the small intestine.
00:34:54.07 And it was found that there's a class of neurons that respond to helminthic infection,
00:35:00.14 through detection of patterns from these... from these organisms, through the innate immune responses.
00:35:08.19 And they then produce the neuropeptide neuromedin-U.
00:35:13.21 These are cholinergic neurons that also make acetylcholine, but neuromedin-U acts on this receptor
00:35:18.04 on type II innate lymphoid cells, leading to production of interleukin-13.
00:35:23.14 And again, interleukin-13 leads to expansion of goblet cells and tuft cells.
00:35:27.25 And that leads, now, to more rapid expulsion of the parasitic worms, which are found in
00:35:35.23 both the intestine and also in the lung.
00:35:39.16 There's another example, in type III innate lymphoid cells from work that Jhimmy Talbot
00:35:44.06 in our laboratory has been doing.
00:35:46.06 And what he noted was that neurons in the small intestine, here stained in red,
00:35:54.00 are in very intimate contact with type III innate lymphoid cells found within structures called
00:36:00.09 These are sentinel posts just beneath the mucosal layer, beneath the epithelium in the intestine,
00:36:07.14 and these respond to commensal microorganisms.
00:36:11.13 And you can see that there are very close contacts made by neurons and these
00:36:16.08 type III innate lymphoid cells, which are marked here with a knockin of the green fluorescent protein
00:36:21.03 at the ROR gamma t locus.
00:36:23.07 And what we now can appreciate is that these cells are specialized for making the neuropeptide
00:36:29.24 vasoactive intestinal peptide, VIP.
00:36:33.23 And what VIP does is to act on receptors that are selectively expressed on
00:36:40.27 type III innate lymphoid cells, inhibiting their production of cytokines, particularly of interleukin-22.
00:36:47.21 So that... we think that these are neurons that respond in a way to maintain homeostasis
00:36:53.10 and prevent too much activation of this pathway, which can lead to hyperproliferation of the epithelium,
00:36:58.21 and potentially can lead to damaging consequences.
00:37:02.19 So, what I hope that I've shown you here is that the homeostasis at the mucosal barrier
00:37:10.01 involves not only a couple of lymphoid cells, but also epithelial cells,
00:37:15.13 various types of myeloid cells that make a variety of different cytokines, as well as enteric neurons,
00:37:21.17 whose relationship to each of these different cell types is only now beginning to be elucidated.
00:37:27.03 So, we are now in a very exciting period in which the various relationships between
00:37:32.26 the different cell types can begin to be explored in much greater detail.
00:37:37.25 So, I will stop there and acknowledge those in our laboratory who contributed to what
00:37:42.09 I showed you here.
00:37:43.15 Teruyuki Sano did much of the work on the Th17 cell induction in the... in the intestine.
00:37:50.02 Jhimmy Talbot worked on the... on the VIP-positive neurons in the gut.
00:37:55.24 And Ivaylo Ivanov, who is now at Columbia University, did the very important work, early on,
00:38:00.18 showing the relationship between ROR gamma t and Th17 cells.
- Diane Mathis iBioSeminar: T Cell Tolerance
- Dan Littman iBioSeminar: Th17 Cells and Innate Lymphoid Cells in Barrier Defense and Inflammatory Disease
Dan Littman is the Helen and Martin Kimmel Professor of Molecular Immunology in the Skirball Institute of Biomolecular Medicine and in the Departments of Pathology and Microbiology of the New York University School of Medicine. He is also an Investigator of the Howard Hughes Medical Institute. Littman discovered the excitement of science while he was… Continue Reading
Diane Mathis is a Professor in the Division of Immunology and the Department of Microbiology and Immunobiology at Harvard Medical School. She is also a principal member of the Harvard Stem Cell Institute and an associate member of the Broad Institute. Mathis’ lab studies the genetic, cellular and molecular mechanisms that determine immunological tolerance, and… Continue Reading