Session 6: T Cells: Development and Differentiation
Transcript of Part 2: Th17 Cells and Innate Lymphoid Cells in Barrier Defense and Inflammatory Diseases
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, 00:29:41.24 ACT1. 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 00:35:59.09 cryptopatches. 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.