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.