 So, thank the organizers for the invitation and giving me the chance to participate and learn from all of you here. I am an immunologist and I'm going to give you a little bit of background as to where we come from and that needs to be switched. But I should be connected. I was before. Just one. Shall I close? Yeah. Okay. Thank you. So, I'm going to talk about the relationship between the microbiota and the immune system and give you some examples of homeostatic interactions and what we think may occur in states of disease. And David Rellman showed the entire painting earlier, but years ago when I was in the Prado, I found that Hieronymus Bosch painting and found this little fragment of it where this person is analyzing a model animal system. And David, if you look carefully, I think you'll find that somewhere in the middle of the painting. And so we've been studying for a long time the experimental mouse to try to give us insights into human physiology and been focusing in particular on different types of T lymphocytes in the immune system and more recently at mucosal surfaces. So this is just a very general review of some of the more important immune cell types at the mucosal border in the gut. We know that there are signals that are transmitted through different types of dendritic cells that can influence the balance of potentially pro-inflammatory and anti-inflammatory cells. The anti-inflammatory cells are regulatory T cells that express FoxB3. The pro-inflammatory cells can be TH1 or TH17 cells. I have the TH17 cells listed here which are particularly abundant in the laminopropia. And there are also innate lymphoid cells that make very similar types of cytokines as the different types of T cells. And all of these function together normally to protect the barrier and in large part through production of antimicrobial peptides that can regulate the levels of the commensal microbiota as well as potentially any potential pathogens. So I'm going to just very briefly tell you about how we think different modes of interaction of microbiota with the host immune system can occur. So we believe there are certain bacteria such as the segmented filamentous bacteria that I showed you in the Mof at the outset that can homeostatically induce cells of the immune system presumably for beneficial purpose both through the microbiota as well as hopefully to the host. And I'll show you an example of this through T-helper 17 cells. But there are other examples very likely in the case of regulatory T cells. And maybe Sarkis-Masmanian will tell us a little bit about Bifragilis and its role in regulatory functions. But then we also think that there is a process of homeostatic inhibition of the activation of the immune system in which microbiota can signal to prevent the activation of effective responses at inductive sites. And in the absence of this kind of inhibitory signal that presumably puts a stop signal to certain cells of the immune system such as dendritic cells, these would now become released taking up components of the microbiota and leading to an inflammatory induction of immune responses. And maybe something like this could be going on in some of the IBDs. So we have certain examples and I'll just cite a couple from our own laboratory of how the immune system is compartmentalized from the microbiota. So one of this avoidance of microbial-listed immune responses in which a particular type of myeloid cell that expresses the chemokine receptor CXVCR1 seems to be particularly important in preventing and avoiding activation of immune responses. So these cells seem to be inhibited by my88 regulated microbial signals from migrating to inductive sites such as the mesenteric lymph nodes. So that is a kind of a functional compartmentalization. Then there's another kind of compartmentalization that's enforced by regulatory T-cells, recruitment of T-regs to the laminopropia. And then another mechanism is this selective micro-specific immune response that I mentioned. We know from work of Kenya Honda that there can be regulatory T-cell expansion in response to certain clostridia. And then Chi-She's group in St. Louis, Lathropetal paper a couple of years ago, showed that there can be micro-specific regulatory T-cells, although their specificity was not demonstrated. And I'm going to tell you about segmentofilamentous bacteria-mediated induction of antigen-specific T-helper 17 cells. So just an example of this compartmentalization by negative signals, Gretchen Diel in our laboratory found very surprisingly that when mice are treated with antibiotics and then given bacteria like a non-invasive salmonella, or in this case just E. coli K12, and then looked at E. coli titers in this case, as well as an IGA antibody response in the feces, surprisingly after antibiotic treatment there was now E. coli in the mesentery nodes, as well as the induction of a E. coli specific IGA response. And in brief what we think happens is that the microbiota give this kind of a negative signal through MiD88, presumably through tall-like receptors, keeping these CX3CR1 positive mononuclear facocytes in the attached to the epithelium in the laminopropria. And in the absence of this signal, there's a CCR7 mediated migration of these cells. These cells basically become untethered, they pick up bacterial content, and they transport actually full bacteria that we can now culture from the mesentery nodes. They transport these through the afferent lymphatics where they can induce both a TSA response and an antibody IGA response, and we'd like to propose that a dysregulation of this kind of a process in a dysbiotic gut may result in inflammation through this kind of a mechanism. This is just one example, and San Juan Kim has been doing work on regulatory T-cell recruitment to the laminopropria, and he found that a orphan G-protein cover receptor, GPR15, that's expressed preferentially on regulatory T-cells is involved in the migration of these cells to the large intestine laminopropria where we think that these promote homeostasis. So I'll now tell you a bit about selective micro-specific immune responses. So a few years ago, Ivo Ivanov in our laboratory found that animals from Jackson Lab versus the Teconic Farms providers of mice differed in the level of TH17 cells, and he found that that was because of the absence versus the presence of segmented filamentous bacterium showed here attaching to the ilium. Now this was present only in the Teconic Black Six mice here, and not in the Jackson mice, and just to remind you, these are gram-positive anaerobes that are spore-forming. They have yet to be cultured, and they most closely resemble clostridia, but they're still very, very different from typical clostridia, and they have a very reduced genome size. And we collaborated with Kenya Honda and Yoshio Masaki's group in Japan because they were able to provide us with mono-associated feces from mice colonized just with SFB. So when these were provided to germ-free mice within a week to 10 days, we could now see the induction of T-cells, CD4 T-cells that make both interleukin-22 and interleukin-17. So you can see this induction occurring over here. So that proved really that SFB itself is the inducer of TH17 cells. So how does this now influence TH17-mediated processes? We know that TH17 cells are involved in a number of autoimmune diseases and other inflammatory processes, and can this also be beneficial? And this is just showing a couple of examples. So in colonization of animals with citrobacter, and citrobacter will grow primarily in the colon, you can see that animals that have SFB are relatively protected from the growth of citrobacter in the colon. On the other hand, as shown here with Diane Mathis and Christophe Benoît in a spontaneous arthritis model that they have developed, you can see that germ-free mice basically do not get arthritis. And animals that have now been given SFB within days begin to develop the arthritis. And this is a TH17-mediated process. It's absolutely dependent on T-helper-17 cells. And Sarkis has shown in a different model in the EAE model something very similar that SFB greatly exacerbates the disease process in EAE. So that indicates then that TH17 cells that are induced locally in the gut can contribute somehow at a systemic level to autoimmune disease, and potentially can also contribute at a systemic level to protective processes, because SFB is present in the small intestine and may induce a protective response also for the colon. But that is something that still needs to be studied. So how do SFB elicit a TH17 cells then provide specific protection or exert pathogenic functions? So there are several questions we can ask within this larger question here. And one is, what is the antigen specificity of the SFB-elicited TH17 cells? And we can think of several possibilities. One is that these cells are nonspecific so that any T-cell in an SFB-conditioned microenvironment could become a T-helper-17 cell. Alternatively, these could be cells that are particularly reactive to self-antigen, but the SFB-dependent microenvironment may sensitize, may actually lower the threshold for their activation as auto-reactive TH17 cells. Or alternatively, this microenvironment may curb regulatory T-cell mediated tolerance. And the third possibility is that these T-cells are specific for SFB or other commensal antigens. And I'll tell you about some of the experiments we did to look at this. Another question which we have not addressed is, do SFB-induced T-helper-17 cells circulate to lymphoid tissues that are draining the specific organs involved in autoimmunity? And if so, how do these circulate out of the gut, out of the inductive site, to go to, say, tissues that drain the CNS, synovium for rheumatoid arthritis or the skin and, for example, psoriasis? Okay, so in order to do this, we decided to look at the repertoire of the TH17 cells in the gut. This is work that Benny Yang in our laboratory did. And we're very fortunate to get mice made by Mohamed Uka and Vijay Kutru, in which the green fluorescent protein was knocked into the R23 receptor locus. So the wonderful thing about this tool is that all TH17 cells express GFP now. And so we can look at GFP positive TH17 cells and at GFP negative non-TH17 cells and look at various T-cell receptor subunits on the surface, in this case, V-beta-14. And I show V-beta-14 in particular because you see here that there is a prevalence of V-beta-14 positive cells among TH17 cells compared to non-TH17 cells. So looking at many animals here, you can see that it's roughly a 3-to-1 preference for TH17 in this particular type of T-cell, whereas V-beta-6 cells you can see are at a 1-to-1 ratio. Initially, we thought this might be due to some kind of a superantigen, but upon much more work, we figured that this indeed has something to do with a predisposition of this subset of T-cell receptors for antigens on SFB, as I will show you. So the way that we learned this, and this is work done by Benny with help from Miriam Tarkczynski, was to take a reporter, T-cell hybridoma, that reports on activation through the T-cell receptor by expressing GFP driven by NFAT. So when these cells have an introduced T-cell receptor along with co-receptor CD4, if they can be activated as a control with anti-T-cell receptor antibody, or with antigen-presenting cells and colonic contents, we can look for the presence or absence of GFP induction. And so we introduced then into these cells various pairs of alpha-beta T-cell receptors that are cloned from individual T-cells that were either GFP-plus or minus in the colon, either TH-17-plus or non-TH-17 cells. And something very surprising to me, at least, happened when we looked at this. First of all, the non-TH-17 cell T-cell receptors did not respond at all, or responded very poorly here to colonic content. But the TH-17 cell receptors, you can see almost all of them, respond in some very, very strongly, only in this case to SFB mono-associated fecal content. And this then allowed us to look in more detail here at activation of T-cells. There were either GFP-positive TH-17 or non-TH-17. And you can see, essentially, all the V-beta-14 positive cells in the gut, if they are TH-17, now respond to SFB-containing microbiota, whereas the non-TH-17 cells do not respond. But this is not restricted to V-beta-14. You see V-beta-8 cells, also a large proportion of these cells respond. Also, V-beta-6 cells are shown here. So this is just showing non-V-beta-14 cells responding as well. But only the TH-17 cells are responding. So this is really telling us there's something about SFB that elicits a TH-17 response and not other types of T-cells. So to really try to narrow this down a bit more, we then did shotgun cloning of the SFB genome into E. coli and looked to see whether there were colonies that could stimulate these T-cell-reported hybridomas in the presence of syngenic splenocytes. And what you see here, for example, is a negative well where these CD3 cells that are not activated. This is a positive well here in which GFP is being turned on. And that allowed us to show that among this 11 or so T-cell receptor hybridomas, they fell about half and half into two different categories. Again, these are just V-beta-14 hybridomas, but they recognize two different proteins. One, a very large protein that we predict to be extracellular, that is expressed at a fairly high level within SFB in the gut of the mouse. And one, it's a smaller protein, also thought to be extracellular, that's expressed at a much lower level. But yet, there are a lot of T-cells specific for this. So that then gave us some tools that we could make, one of which was to make a T-cell receptor transgenic mouse with one of these receptors that was specific for this more prevalent protein. And this is the 3340 protein here. So what we could do is basically inject these T-cell receptor transgenic cells into a mouse that either is given SFB or is not. And then we can look at the lamina-appropriate T-cells or mesenteric nodes at various times after transfer. And one of the things that we found right away is that when we do this kind of a transfer, we see expansion of these SFB specific T-cells only in SFB colonized animals. So you can see these are donor derived cells, these are host derived cells. And we see this expansion in the lamina-appropriate of the small intestine only in the SFB plus mice and not in the SFB negative mice. And if we now look at, in this case, we looked with just very small numbers of cells that are injected that are now expanding there. And we can look at also different times. What we see is that, essentially, all the donor derived cells become ROR gamma-T positive cells, which is a mark transcription factor that marks the TH17 cells. Whereas in the host derived cells, typically about 20% to 30% of the cells will be TH17 cells, ROR gamma-T positive cells. So the reactivity to SFB makes these cells become TH17 cells. This is looking at multiple animals. And you can see always this kind of a distribution which the T-cell receptor transgenic derived cells all become T-helper 17 cells. And we developed another tool in collaboration with Mark Jenkins, which was to make a tetramer because we mapped the peptides that are being recognized within the, for example, the 3340 protein that are being recognized with MHCIA of B by the T-cell receptors. And these tetramers now allow us to stain unmanipulated animals for the presence of T-cells specific for this antigen. And you see now when we look in the lamina-appropriate at ROR gamma-T positive cells and negative cells down here and tetramer positive cells, you see essentially all the tetramer positive cells are in the ROR gamma-T positive TH17 compartment. Whereas we see here in the negative ROR gamma-T negative compartment, typically we do see some of, of course, some TH17 cells that have other specificities, obviously. So when we do this again in multiple animals, we see again the 20 to 30% distribution of ROR gamma-T positive cells in the tetramer negative cells and more than 90% in the tetramer positive cells. So, surprisingly then, when we ask what is the antigen specificity of SFB-elicited TH17 cells, we find that they are, these are SFB specific by and large and maybe in other cases there could be specificities for other commensals. That does not rule out that these other mechanisms may not be involved. And in fact, if we look in colon, I don't show you the data here, we find that there are very few TH17 cells in colon in animals that have this tetramer positive kind of specificity. So there may be different mechanisms, perhaps some kind of a, there could be environmental related induction of TH17 cells as well. So that really remains to be figured out. So, let me move on here quickly because I want to make sure I have time for some relevant questions here. So what we think then is that there may be a specific niches where particular types of antigen presenting cells will bring microbial antigens to the inductive site, in this case, say a mesentery lymph node, where there will be induction, for example, of Tregs if we start with Clostridia here. And if SFB are stimulating maybe a different type of niche where a different antigen presenting cell would be present that would now induce, make the types of cytokines that would induce TH17 cells. By using the TCR transgenic mice, we find that this induction occurs first in the mesentery lymph node around day one to three. And what then would happen is presumably these cells would redistribute by turning on homing receptors and after day four we see them scattered through the laminopropria and with Tregs, induction of GPR-15 at least would lead to these cells going to the large intestine in large number where we find them in the largest numbers. And then what happens in autoimmunity? Well, one possibility is that these cells are redirected apparently to either to joints, for example, for arthritis or to lymphoid tissues that are draining these sites. And for some reason, these would either through cross reactivity or through near neighbor kind of functions would induce disease at these sites. So of course then we wanted to look at TH17 cells in human disease and I won't be labored at this point but we know that many autoimmune diseases in human and asthma, steroid resistant asthma are associated with TH17 cells. So Jose Scherr who is a rheumatology fellow at NYU along with Steve Abramson who is now a chair of medicine but he was the head of rheumatology joined us in looking at commensal 16S in rheumatoid arthritis patients and we collaborated with Carlos Ubeda and Eric Pamer initially to do the 16S analysis and we're very fortunate that we had access to new onset RA patients at Bellevue Hospital but we also had chronic RA patients and psoriatic arthritis along with healthy control. And what came out immediately that was really striking is that only in these new onset RA patients and NORA patients there was a very high abundance of a prevetella in the feces of these patients whereas in the healthys and the chronic RA and psoriatic RA we saw only 15 to 20% of these with high levels of prevetella. And if we looked then more closely by metagenomic sequencing we saw something that was very similar to what was observed in the HMP analysis and Michael Fischbach provided me this analysis here but Curtis who is following me and his talk he may have one slide that shows this as well. There really is a bimodal distribution of prevetella copri in the healthy population. Typically around 15% of people have it but 85% of it do not. In our case we see about 70% of the NORA patients who have this and it is the most closely related to p-copri based on the metagenomic analysis. And what we also find is that there are some sequences that we find associated with the patient sample prevetella and others primarily with a healthy sample prevetella bringing up the possibility that actually there may be some virulence associated with this particular species in the patients. Finally we don't have any proof of causality here but the way we've been looking is by introducing prevetella in this case a reference strain p-copri into mice that have been first treated with antibiotics. We confirm colonization and then we treat these mice with disulfate sodium which is basically an irritant and we can see that there is a more severe weight loss and more severe colonic inflammation as well that Randy Longman in the lab found if the animals have received prevetella. We see something very similar also with collagen induced arthritis. So then to close here what we think then is that there may be different mechanisms of achieving homeostasis, achieving a balance between T-regs and effector T-cells by having responses to different types of bacteria in different niches within the gut but in addition there is a way of restraining bacterial content from reaching the inductive sites. So a few questions then that we can ask right away. Are there microbiota specific TH-17 cells expanded in RA and do they contribute to pathogenesis? We don't have an answer to this but also another what I think is an interesting question is could commensals specialize for homeostatic activation of adaptive immune cells be used for protective or towerogenic vaccination as an example to control early HIV induced depletion of TH-17 cells that occurs in the intestinal lamina propria. So these are just some of the questions that we're interested in but we think that there are many outstanding questions here in the field. So in terms of what might be the microbiota influence on the immune system and I'll just read quickly through these as I'm going a minute or two over here. Is there a subset of microbes that influence differentiation of discrete components of the immune system? So I showed you an example for SFB. SFB may exist in human and I'd like to have a discussion about that later but is SFB an outlier or are there many such bacteria like this? And this would involve induction of T-cells subsets, B-cells in immunoglobulins of course in eight cells that are both lymphoid and myeloid and the specific microbial metabolites such as secondary by-lacids influence immune cells and we think that that is going to be the case. What sets apart these microorganisms that influence immune responses such as the bacteria that induce TH-17 cells? Are there regional differences in inductive events say TH-17 cells in the small intestine versus the large intestine? And what's the role of the microbiota in the TB cell functional repertoire? I showed you one example here but are there circulating BNT cells specific for antigens encoded by the microbiome? Are there associated effector functions? What's the proportion of such cells in our circulation? Could such cells account for the repertoire of T-cells and potentially B-cells with effector memory phenotypes? Okay, so these are experienced cells presumably that are anticipatory for potential pathogens and Mark Davis recently showed that there are anti-HIV memory T-cells in people who are naive to HIV. Could these be induced by microbiota and could there be evolutionary pressures for these kinds of microbiota interactions with the host? And could individual commensals be exploited then to induce specific antimicrobial protective immune responses such as commensal HIV vaccines, I mentioned? And some other question is what is the relationship of microbiota composition to host genetics at steady state? And we heard a little bit about this from Ruth Lay but in particular is there a link of MHC haplotypes or immune system gene polymorphisms to the composition of the microbiota and do dysbiosis and the abundance of particular microbes contribute to diverse autoimmune diseases and other inflammatory conditions? I gave you an example of Prevotella and the association with Nuance at RA but as David Relman told us this morning, there can, we don't know if there's a causal effect, if it's initiating or propagating. In our case, we think it's very likely it would be initiating if it is causal because we don't see it in the chronic RA patients. Is it necessary and or sufficient? And can micro-specific T cells and antibodies be detected at steady state and in dysbiosis associated disease? And are these present systemically? I think these are all questions that we are now in a position to have the tools to begin to answer. I've mentioned many of the people who've been involved in this work already. I want to particularly point out Nicola Segata and Curtis Huttenhauer have helped us enormously on the Prevotella work most recently, and these folks here have helped us a great deal with the SFB. So thank you very much. Sorry for going over. Yeah, thank you so much, Dr. Litman. And since he already asked himself the questions and put on the slides, I think we're gonna move on to the next speaker and at the end, when we have the open floor discussion, maybe you can ask other questions. So our next speaker is Dr. Curtis Huttenhauer from Harvard School of Public Health and he's gonna present functional analysis of human microbiome, metagenomes, metatranscriptomes, and multiomics.