I, uh, I spent a lot of time wondering actually how I was gonna introduce Alex today. Um, but I decided to keep it professional. And so it is my great honor to introduce our very own Doctor Alex Cuenca as today's grand round speaker. Alex pursued his pre-medical studies at the New College of Florida, where he studied Biology, followed by Medical School at the University of Florida, where he obtained his MD and PhD in Immunology and microbiology. He pursued training in general surgery at the University of Florida, Shan's Teaching Hospital, where he discovered his love of pediatric, hepatobiliary and transplant surgery. Alex completed his training in pediatric General Surgery here with us and transplant surgery subsequently at MGH. Before joining the faculty here, where he's maintained a busy clinical schedule, as well as overseeing a growing lab uh in basic science, and we are very fortunate to hear about some of those insights and discoveries that he and his team have made over the past several years. So, without further ado, my friend and our colleague, Alex Cua. Uh, thank you, Doctor Lee, for that gracious introduction. Um, and before I get started, I just wanna wish the fellows good luck on their mocks today. You guys are gonna kill it. It's gonna be great. Um, so I've hopefully, uh, I can go through some of, uh, the concepts that I've been looking at over the past several years and, and some of my interests with allograft tolerance, um. I unfortunately have no disclosures at this time, but we'll work on that, um, but I thought I'd go over some definitions that I'll be saying, uh, throughout the, the purpose of the talk and, and, and what the differences between some of the more commonly. Used themes and or uh uh sort of suggestions which are what it you know I I'll I'll use the concept of operational tolerance which is different than what we think of as just straight tolerance operational tolerance essentially is what we observe sometimes clinically with our patients after we've been able to withdraw immunosuppression. And they're able to have what we consider a um normal allograft function and histologically if you look at them theoretically no signs of rejection or chronic rejection and so we consider that to be an operational tolerance as opposed to just straight tolerance um and then chimerism which is uh you know a term that we use a lot in uh in with bone marrow transplants and and looking at. Um, what percentage of leukocytes are of the donor and which percentage of leukocytes are of the recipient, and we use it all the time, uh, to sort of discuss that and that that concept will come also, uh, throughout the, the talk. So I thought I'd start with sort of an interesting sort of case scenario. Um, so this is a patient that we had here that uh Doctor Kim knows very well. It's a 2.5 year old that developed uh Langerhan Langerhan cell histotocytosis and received a significant amount of my ablative chemotherapy. Um, and progressed to develop liver failure. Uh, the patient underwent a split liver transplant in 2009, um, and then afterwards, uh, within the month ensuing months, developed GVHD of the skin and GI tract about a month after transplant, was hospitalized for many months with slow resolution. And interestingly, uh, several months afterwards, uh, the peripheral, uh, sort of look at the at the at the chimerism because there's some thought that maybe increased donor chimerism would lead to a GVHD as opposed to not, but it was only about 3%. And then in subsequent uh looks at this with the most with the last uh uh sort of uh assay to look at this was in 2010, um, showed that the that the patient actually had 100% donor chimerism and interestingly, uh, aside from a little bit of oral steroids afterwards, uh, although it was on the normal immunosuppression at the beginning of the transplant, uh, for the majority of his post-transplant course has been maintained off of. Immunosuppression meds. He's had some intermittent flares of like vitiligo, which we think of as obviously like a skin type of GVHD but has been mainly treated and out outpatient and has done really quite well. So, um, the the concepts that I'll be exploring or that I've tried to explore at least in the lab kind of surround that that process and trying to bring together different areas of the immune system and how they may educate the peripheral immune system to potentially provide tolerance and so I'll first start out with a. Around and where we are maybe some clinical advances in immune tolerance, uh, and refresh our, our, our minds on some of these trials, uh, how the immune system trains and educates just as a, as a more background and then how I put it all together and, and I've sort of looked at things with regards to, uh, my basic science endeavors, um, uh, sorry, and then as well as, uh, future directions and things that I'm looking at and things concepts that I'm, I'm gonna explore. So, um, there have been a dramatic amount of improvement in in allograft survival in patient survival with transplant over the last 50 to 7 years, uh, and liver and kidney allograft at 5 survival at 5 years is greater than 80% at this point, and that's really due to a number of different advances including advances in, uh, static cold storage and storage techniques for livers and now even using machine perfusion and normothermic perfusion. Advances in things like microvascular techniques and other techniques to put vessels together and as well as immunosuppression. Although with regards to this, we've sort of been stagnant since about the 90s and some of these survival and allograft survival curves reflect that. So the national outcomes are actually quite good um as I've said, but can we do better? And I think the answer to that is absolutely. So, um, as I said, we're, we're, we've gotten really good at trying to get patients through the acute phase of the transplant, getting them through, uh, the, the rough physiologic insult that transplant brings, but it's really the maintenance period that that we're really focused on. How do we promote this longer term success, uh, if we look at uh some of the data from the OPTN SGTR, which is our large database looking at, uh, liver and all the different organ systems and, and what their outcomes are. We can see that over time really for the you know, whether it's 10 years although there was a blip here we did poorly, so this is essentially a graph failure um over time really it's, it's stayed pretty stagnant. There's not really been much movement and I think a lot of that is related to uh likely uh uh poor or no advances with regards to immune suppression and, uh, maintenance therapy. And so with regards to immune suppression, we know that it's necessary but also brings toxic and or untoward morbidity. There's a high side effect profile with high dose corticosteroids that can lead to any number of things, even with our most innocuous induction agent of basaliximab. You do still get like a mild surge response, although none of this is that significant or clinically relevant. Uh, with regards to what we see, so, uh, but the maintenance therapy that happens afterwards, uh, either with corticosteroid weans, calcineurin inhibitors, which is where the majority of our, um, uh, morbidity comes from, which leads to things like renal vasoconstriction and AKI, CKD, diabetes, PTLD, uh, and neurotoxicities. Uh, the, uh, anti-metabolites, uh, uh, like, uh, mycophenolate, which leads to GI distress, it's teratogenic, uh, hypertension, electrolyte disturbances, or even the MTORI inhibitors which we think as as being more benign of a of a maintenance therapy, uh, but has a lot of whole host of side effects including poor wound healing, uh, ulcers, myelosuppression, hepatic artery thrombosis, strangely enough, and hyperlipidemia. And with regards to pediatric immunosuppression in particular, there are a host of challenges including differences in metabolism and bioavailability depending on hepatic enzyme activity. There's differences when you have to account for growth and they're changing meds based off of BSA and have to keep on top of that. We're sometimes dependent on caregivers to administer the meds appropriately and sometimes they don't. The meds may not be palatable and so patients may need an enteral tube. For administration and of course just like with adults, there's a lifetime increased cancer risk across the board. So maybe it's just that we're giving too much. So can we possibly take it away or decrease it? And some investigators have looked at this, and probably the most well known study to do this was performed by a multi in a multi-center trial led by Sandy Feng. Uh, where they looked at 20, uh, living parental living donor recipients, uh, and with a variety of liver disease except for autoimmune disease, uh, which is a common theme throughout a lot of these liver, uh, sort of tolerance trials and, and, and makes sense with given the pathologies. Uh, the transplant had to occur within 44 years or more prior to enrollment, um, and biopsies were performed to make sure that there was no histologic evidence of chronic rejection or acute rejection. Of the 20 patients that were enrolled, 12 patients were able to be weaned completely from IS, uh, and that was again from an original study population of 129, so it's quite small, the number of patients that were actually, uh, originally enrolled. Um, and DSA or donor specific antibody, which we think is a marker of the development of, uh, sort of, uh, uh, allo immunity in these patients, was detectable in 4 of the 12 quote unquote operationally tolerant patients and 7 of the 8 of the non-tolerant patients by the end of the study. A follow-up study to this, uh, 5 years later looked at those 12 operationally tolerant patients and interestingly found no progressive inflammation or fibrosis, um, and, but these, uh, still same sort of, uh, DSAs or donor specific antibodies, uh, will have unclear significance obviously since there was, uh, very, there was very little inflammation or fibrosis. So this is not widely generalizable. Clearly there's a very small percentage. There's a uh estimate of about 10 to 20% of patients that undergo transplants, and liver transplants in particular, um, that are able to potentially be withdrawn, and I think 20% is quite high of a, of a number, but some studies have suggested it. Um, and it's, it may not be, uh, the, the way that to go in, in a lot of circumstances. So in terms of trying to understand where we're at with things, I thought we would just review some of the things that happen following solid organ transplant with regards to the physiologic insults that occur, uh, just to sort of see what are the limitations or what are potentially providing these obstacles for us. So after the donor organ procurement, we know that. And and uh implantation, we know that there's a significant amount of ischemia reperfusion injury, the release of danger associated molecular patterns or damps, which are basically proteins inside of cells that lice after the ischemia reperfusion uh insult, um, and these could consist of a whole host of things that essentially are important for initiating an inflammatory response. This inflammatory response recruits a whole host of immunovector cells such as macrophages and neutrophils. Uh, which then are engaged by all of these different types of receptors that are that the ligands are released for, um, and you get a significant amount of inflammation post which then these antigen presenting these cells that now become antigen presenting cells go towards the recipient lymphoid tissues and then subsequently produce allo reactive effector T cells. And we think that maybe this process happens over the course of uh the first week uh in uh in in particular. So the obstacles we really need to overcome are things like ischemia reperfusion, uh, trying to understand and uncouple self versus non-self, and how do we trick the immune system into recognizing the into recognizing the allograft as self. Uh, and this is usually done through the recognition of these, uh, aloe MAC, uh, uh, molecules or human leukocyte antigens such as HLAs, HLAA, BC, which are class one antigens, or DRDQ, etc. which are the class two antigens, recognition of those allo antigens that are being presented in the context of these MHC uh MHC molecules, as well as trying to control adaptive and innate immune responses. So it's a lot to go, it's a lot to, uh. To control when you're trying to uh uh sort of harness or understand what's happening uh with uh tolerogenic responses and trying to get tolegenic responses in our patients. So, uh, following transplant just to try to understand where uh where these uh responses and or how we may sort of understand or target these different areas to produce a suppressive response, we have to look back at at at how things happen through development, uh, which would be uh through the process of of education and or uh how the body learns about self versus non-self which first happens in hematopoietic organs like. The bone marrow, uh, which then produce lymphoid progenitors, which are the progenitors of B cells and T cells, specifically T cells in this case, which then migrate to the thymus, uh, and then from the thymus go to the periphery when they've uh when we've sort of deleted all of the uh self-responsive cells. So to start really in the tolerance reduction of the thymus is where all of the T cells sort of get educated initially. Um, there's obviously a robust response and or exposure earlier in life, and then that subsequently fades as the thymus involutes, but, uh, the first process is as the common lymphoid progenitor migrates to the thymus, the first check is, is via positive selection to make sure that the cells that are being produced can recognize these HLA or MHC complexes first, and once that happens, it then goes through a process of negative selection. Where if the cells can recognize MHC but don't do it too strongly, then they're allowed to sort of survive. If they do it intermediate, they become a group of cells known as regulatory T cells which we'll talk about a whole lot in the rest of this talk. And if they get recognized and if it's if the binding is quite strong, then again they get deleted uh and because those are thought to be allo reactive or self-reactive T cells. In the bone marrow, I think that the process is slightly more complex. There's a whole host of cells that are what we consider to be toleogenic. So as these cells sort of migrate back and are being educated in bone marrow, we think of things that like torogenic dendritic cells. Or myeloid derived suppressor cells or even the T-regs that were created in the thymus that have then migrated back to the bone marrow to then sort of surveil the peripheral blood to try to delete some of these alloy reactive T cells or self T cells and control tolerance from that aspect. And then in the periphery or in the secondary lymphoid organs, uh, these Tregs are really sort of the workhorse or the the engine of, of that. Uh, these regulatory T cells either create an allergic response in those T cells that are reactive towards self, they suppress it, uh, or they delete it, and really this is, this is the prime mechanism by which, uh, we would. prime mechanism by which we think at least is the end product or the end point of where we would, uh, try to target, uh, to to try to achieve operational tolerance. And then coincidentally this is also the target unfortunately, or one of the targets unfortunately that cancers sometimes use to evade detection, but hopefully I can convince you by the end of this that we can potentially have long lasting surveillance of of of cancer antigens and non-self while maintaining self unresponsiveness. So what happens when we use these Tregs in clinical studies? Well, a whole, a whole group of investigators have done that. Um, this, and I'm I'll go through some trials in both liver and kidney, but basically uh some a group out of Japan. Uh, looked at, uh, using T. regs in a small number of haplo-identical versus non-matched patients. So in a small study of about 10 patients that underwent a hap, sorry, not haplo identical, fully matched, uh, living donor living transplant, uh, living donor liver transplants, uh, they showed that there was, uh, that subsequently underwent an immunosuppressive wean over 18 months and followed for about 2.5 to 22 to 2.5 years. Uh, of 10 patients, 4 patients remained operationally tolerant at the end of the group. Initially, the, the study was planned for 40 patients, but they ended up closing early because many of the patients developed, uh, acute celery rejection episodes, and so they thought maybe this is not the best process to, to, to, to go through. Uh, they have other studies ongoing that they talked about in this, in this, uh, uh, in this manuscript, uh, and one of which is looking at hap identical and what the outcome is, and they have many more patients than that. Uh, it'll be interesting to sort of see if they actually get a similar type of response, which I would imagine they would since it's they're less matched than the full, uh, you know, sort of sibling matched, uh, uh, allografts that they initially transplanted, but, uh, more to come on that. And then this was an interesting strategy actually that was born out of some of the literature in inflammatory bowel disease. Some groups have looked at administering low dose IL-2, which is a stimulant of TRGs, to try to expand T-RGs peripherally in IBD patients, actually have shown a pretty good effect in IBD. However, when they use the same sort of strategy in transplant. You can see that uh now again not that many patients, uh, but had very little to no effect over the course of a month after a low dose IL-2 so nobody, uh, so no, none of the patients in this group ended up with any uh uh sort of tolerance or reduction in in their immunosuppression. In kidney, um, a couple of groups have looked at this, and I just selectively chose this one because, uh, I, uh, Doctor Markman is one of my mentors, but, uh, he'd also looked at the expansion of T. regs from renal transplant patients. So at the time of, uh, of, uh, of listing. Uh, they would harvest patient PBMCs, um, and then subsequently expand them ex vivo in in the culture to generate aloe, uh, energized T. regs, uh, and then subsequently. Reinfuse them back at different intervals to provide tolerance again, small numbers and not quite operationally tolerant, but 3 patients were maintained on tack monotherapy versus triple therapy like we would usually think of. Uh, in patients after, uh, 6 years, uh, suggesting that even with the, the T. regs, uh, that they've infused that you still need some amount of immunosuppression and it's not it's still not quite there in that in this group. The next sort of concepts on the horizon that people are thinking about using and borrowing again from the cancer literature like a lot of the transplant studies do is looking at instead of CAR T cells, which are chimeric antigen receptor generated T cells against tumors and now generating CAR Tregs for use in. Transplant and studies are ongoing and we'll sort of see, but the concept is obviously that you generate these receptor antigens towards specific organ antigens that then when they're presented in the context by antigen presenting cells in the recipient will produce buckets of immunosuppressive cytokines and thenfore affect peripheral tolerance. So as I said, you know, many investigators have sort of looked at this from the concept of going from straight from the solid organ straight to the end product, which is the regulatory T cell, bypassing the bone marrow, and maybe that's the missing piece in terms of her long lasting tolerance. Some groups have actually also looked at this and have done simply bone marrow infusions and with withdrawal of immunosuppression of adult liver transplants. This is probably the largest study that that that has gone through this and originally published in AJT in 2005, and I tried to look to see if there were other follow-up studies. But uh there to this or follow up uh cohorts to this, but there were not, um, but anyways, they initially enrolled 104 patients uh after allograft transplant, um, they harvested at the same time of deceased donor, uh, bone marrow from the vertebral bodies of the donor. Owners themselves and then subsequently infused them back in again they had to have the the recipients had to have no history of autoimmune disease um and there were 45 that received this bone marrow and 59 controls and they said after about 5 years there was essentially no difference between the two unfortunately. Um, and then in a similar fashion looking at these another set of cohorts with that that received a bone marrow transplant after total lymphocyte radiation and antithymocyte globulin again sibling only transplants of this they had a little bit better success with 17 of 22 patients able to be completely withdrawn. Uh, so, so some suggestion that if you deplete these memory T cells that are against self and then maybe repopulate with bone marrow, that's probably the best way to do it, uh, although there, there is the morbidity associated with obviously total lymphocyte irradiation. Um, in this, in this process, but theoretically this might be the best way to go about it because you can essentially re-educate and repopulate the peripheral, uh, T cell compartment and re-educate them to alo alo antigens while the, the graft is in place. This is the trials that have gone through the different with the summary of some of the ones that we've just talked about that have gone through looking at these different aspects of giving bone marrow transplants with the allografts in kidney transplant and probably the one that's done that had the most patients was in Stanford again they were looking at trying to provide persistent mixed uh chimerism. At MGH we always when when I was there we always talked about providing a transient chimerism meaning uh you would see uh the the the population or a population of donor uh derived cells initially but then it would subsequently go away uh versus a full donor uh chimerism which is what they see in uh in Northwestern. You know, the concern with these, these chimerisms, as I've said before, is the potential for graft versus host disease. And so, which is why, uh, my colleagues at MGH have been sort of leaning very heavily on the mixed chimerism concept and the desire for mixed chimerism. Because theoretically you could get the education of the uh peripheral T cells and or adaptive immune responses without and have that happen initially and then have it go away and have them have uh long lasting results without there being this this risk or worry of GVHD, um, and I will say that updates from this, uh, the, the numbers of patients with graft loss is a little bit higher than 6 at this point, uh, but, uh. Uh, there's still this, this, uh, desire to try to pursue this and to look and see what they can do to make this better, uh, on the outcome using, uh, some pre-clinical models. So again, why don't we just do this for everyone? Well, uh, you know, obviously total body irradiation, total lymphocyte irradiation is not, uh, the least morbid thing that we can do. Um, there's a lot of, of risks that go into that. Um, and a lot of these studies that did, uh, that moved from completely my ablative regimens to total lymphocyte radiation, they're still targeting the thymus and above the diaphragm lymph nodes as well as the spleen. Uh, all of which should theoretically deplete memory T cells and then allow for repopulation reconstitution and it does seem to give the most durable effect but obviously is, is, is not that easy and then with all of the, uh, a lot of the other studies, uh. They, they've used sibling matched transplants for a full, uh, full match, and obviously this is not a this is not a possibility for every transplant and again the generalizability of this that we to, to most of the transplant that we that we have that we do or perform is quite poor. Um, and then this is just going through again the mix for this Keimerism strategy, which one of these is the best, and again I, I think the, the, the jury is still unclear and out on that. Um, so, and then the other thing I would say is that even in the setting where they're able to withdraw the, the, uh, immunosuppression completely and they've looked at these sort of patients long term, uh, even beyond the five years that I initially said, so this is the, this is just harkening back to the original immunosuppression withdrawal studies done by uh uh Sandy Fang. What they found was actually even after in the long term, even though there wasn't anything after 5 years, after 10 years or so, there was still a good number of patients with some amount of chronic rejection, suggesting that even in operational tolerance we're not quite tolerant and we're still having organ and organ damage. So what about using other regulatory cell populations that have been described in other pre-clinical studies? A lot of groups have focused on a whole host of them. Um, Bregs was another one that uh Jim Markman had looked at significantly. I'll talk about innate lymphoid cells and myeloid derived suppressor cells, but a group uh in uh in Pittsburgh is very excited about regulatory DCs and have published a recent study as of this year of essentially. Where they followed 13 patients underwent a living donor liver transplant that were subsequently where the donors underwent a leukapheresis and they matured these Dregs or DC regs in vitro and the patients were. Compared to are being compared to standard immunosuppression protocols and then subsequently weaned after 12 months, and the subsequent data hopefully will be interesting and may yield some important insights into how we can provide operational tolerance using innate immune cells. So just to go back a little bit to the drawing board again, we're still kind of, I think one of the challenges that we're we're still having is trying to understand what when we do a solid organ transplant. Even though we have regulatory T cells, how do we bridge this or how do we make this better, and what is the involvement of the bone marrow in this and how do we involve the bone marrow, because it seems to be that obviously if you can delete, uh, all of your peripheral allo reactive T cells or allo reactive adaptive immune cells, then maybe you can produce or allow your regulatory T cells to function long-term and provide allograft tolerance. One of the things that I became interested in in the lab was these innate lymphoid cells, uh, a group 2 in particular. These cells are found in all our resident, uh, sort of innate immune cells that are found in all sorts of tissues and most and most importantly in all of the organs that we transplant. These cells are thought to provide local immunoregulatory function. Um, but, uh, are still being sort of, uh, uh, understood with regards to what their actual role is. Again, just like a lot of our transplant literature, they looked at this first in the tumor setting or in the cancer setting and have shown that these cells can communicate with myeloid derived suppressor cells as well as T-regulatory cells to sort of support this sort of oncogenic potential in in. During tumor growth, and it does so through a variety of paracrine factors, but we don't know what that effect may be in solid organ transplant. I said that I was looking mainly at group 2s, but there's actually 3 groups in total. I'm going to exclude these two groups because these two groups are mainly relegated to the intestine and really focus on this group 2, which have really been thought to play a significant or important role in wound healing and immunoregulation. So some people have sort of have looked at them with regards to what their actual function is, and initially we thought that they couldn't migrate hematogenously after stress or physiologic insult, but we now know that not to be true. As I said, they're found in all of the organs that we transplant in specific heart, liver, kidney. And some secondary lymphoid organs, and they facilitate tissue repair and pretty much are poised to be potentially ideal for providing a sort of local immuno regulation following transplant. And in one study they showed that it facilitated some graft tolerance in an islet model, although there were some questions with regards to how they did the study, but overall showed that there was some paracrine effect of having those innate lymphoid cells in place and how that promoted tolerance. So we ask the questions, are they factors? Are they facilitating rejection and facilitating this this response, uh, or are they trying to suppress rejection? What is their role in the in in the process of the organ, and how do they affect this normal interaction between the T cells? So we know we have two T cells, CD4s and CD8s, that interact with antigen presenting cells. How do they affect that, uh, in, in the transplanted organ? To look at this, uh, we developed a really nice model using a heterotopic heart transplant whereby we transplant the heart into the abdomen of a mouse and then can follow it and look at different immune interactions. And so for the first case, uh, for the first set of experiments we looked at simply what happens when you do a heart transplant, uh, into a mouse that has these innate lymphoid cells versus one that does not, and then look at, uh, the response 10 days later. Uh, and interestingly what we found is on these cells that subsequently migrated into the transplanted heart, there was this strong upregulation of MHC class 2 which as you. You may recall is the molecule that's responsible for presenting antigen to the affector T cells or to T cells that are alloy reactive T cells that are coming to destroy the graft. What's interesting is that even though they strongly up-regulated MAC class 2, they didn't up-regulate B71 or B72, and again recalling back to immunology, you need to have signal one with signal 2 to have a productive immune response. And so what they're getting is signal one but no signal 2, which we think of as leading towards an allergic or tolerogenic response. So then the next question is, what happens when we add T cells to this system. And look at graph survival, and I apologize for the busyness of the slide, but I'll walk you through it. Essentially we have survival on the y axis in days post, uh, uh, essentially post transplant. In the blue lines here we see the setting where there are ILCs or innate lymphoid cells. In the red line here we see where the setting where there are no innate lymphoid cells. Suggesting that when innate lymphoid cells are present, you're able to provide actually long lasting graft survival versus when they're not and there's rejection of the organ. And then as a as a bolster to this because one thought may be, well, you're not deleting NK cells, maybe the NK cells are playing a role and just as a primer, NK cells are thought to recognize non-self essentially cells and delete them. So it's important, it would have been important for us to figure out whether or not NK cells were playing a role, and you can see that when we delete NK cells we get the same response essentially. So it's not an NK mediated effect, it's actually an ILC or innate lymphoid cell, a group 2 innate lymphoid cell mediated effect. And then this is just immunofluorescence sort of suggesting again in the presence where you have ILCs you can see that there are some ILCs here above it it's a little faint in the periphery, and then a few CD4 T cells versus when you don't have ILCs there's again there's no red here but a ton of green where you have these T cells, so suggesting that maybe locally these cells are actually controlling the influx of all reactive T cells. And then what was interesting is that when you use a cytokine to expand these cells within the animal, you're able to actually improve that initial observation of survival, so therapeutically you can get them to expand and provide better allograft tolerance. So what does this mean? Uh, so essentially we think, or at least the, the one of the concepts in the lab, or projects in the lab is that ILC2s are very important immuno immunomodulatory roles, uh, that, uh, directly inhibit allo reactive T cells, uh, in activity in the graft, uh, and I haven't shown you this data, but they do it not only in the direct fashion like we sort of suggest. but also indirectly with cytokines um uh through paracrine paracrine means, but, uh, if you, if you know those, those models that I was that I was just showing you the RAG models, the RAG 2 models, and the, uh, the, the problem with those, uh, conceptually is that those models essentially are very, um, uh, are somewhat artificial because they don't have adaptive immune cells and so I had to add those adaptive immune cells back to look at this. Um, and so when we look at this setting, uh, when we look at these cells in the normal setting, it's very difficult because you can't actually just delete those cells, which is why we had to use the very artificial system to look at it. But we're we're now developing models to be able to just specifically delete those cells in the models and look at them in an immunocompetent setting as opposed to the immunocompromised setting. Um, and, uh, the next phase of the, of the research is going to try to understand like that original picture that I showed you, do ILCs have cross talk with other of these other immunoregulatory cell populations to provide uh allograft tolerance or what we would see in our model as tolerance. So we think that the the ILCs at least are playing a role locally at the level of the transplanted organ. What about at the level of the bone marrow? Is there maybe a cell that could be providing this signal and helping and sort of coordinating responses with the Tregs and the ILCs? For this we turn to the myeloid derived suppressor cell. These cells essentially are a natural part of the the immune stress response. It's essentially what we consider to be the left shift peripherally, as you might with either steroids or infection, you get a dramatic increase in. Innate immune cells into your bloodstream and what that does in the bone marrow is it frees up niches in space that allow for the expansion of progenitor cells that then turn into these quote unquote myeloid derived suppressor cells. These myeloid derived suppressor cells can produce a whole host of immuno. suppressive factors that can then control innate adaptive and innate immune responses and have been identified in both mice and humans, although the majority of the work is in mice but are definitely seen in humans and have been worked through. The the issue is essentially that these cells are progenitors and so even though they they expand quite robustly. Over time they then ultimately mature into mature into innate immune cells and so it's hard to officially track them, but for our purposes you can definitely identify them when they expand and look at what their effect is in our mind. A lot of people have looked at it in preclinical models of transplant, specifically with cardiac allografts, skin grafts, and it basically just shown increased survival when looking at these cells, and increased survival with looking at either direct or indirect mechanisms of immune suppression. And similarly, although not directly as mechanistic as the pre-clinical models have shown an association between the presence of these cells in patients that have had long-term allograft survival versus the absence of these cells in those patients that have complicated, more rejection and poor allograft survival. So at the time that I that I came, uh, we, this was one of the the the innate immune cell populations that I was interested in, and at the same time, uh, another group in University of Maryland was also looking at this and even though we use different methods to get the end product of of providing improved Lgraft survival. Survival, there was something that was quite interesting that happened, but essentially we identified a way of trying to condition the transplant recipient so that we could prevent rejection of aloe in an aloe islet model. We showed that we were able to expand them with this adjuvant conditioning regimen. Um, and that interestingly, in addition to myeloid derived suppressor cells, we were able to get an expansion of Tregs, um, and that these T-regs would be able to be induced also in vitro as well as in vivo. If you blocked MDSCs, you removed this protective effect, suggesting that this, this adjuvant conditioning was important for preventing the expansion of these cells, and if you adoptively transferred these cells into mice that were not adjuvant condition, you could also get protection. And what we saw was that even though we had some protection here or delay and rejection, again this is more immunosuppression as opposed to tolerance, we still had a drop off that happened sometime about 3 weeks after we did this, and this was almost exactly the same as what the University of Maryland group found. Um, and the thought is, why is this happening, um, and so the, the concept that I think is, is developing in, uh, in with regards to innate immunity and trying to understand responses that happen post or at least trying to link this together with T regulatory T cells. Is that there is some interaction between myeloid derived suppressor cells and T-RGs that's been demonstrated again in tumor models. There's the show that if you affect myeloid derived suppressor cells, you can expand Tregs. You can get them to sort of provide some amount of tolerrogenic activity. And so is it possible that MDSCs may link the ILCs back to the bone marrow again going back to this original figure where there's crosstalk between different paracrine and soluble factors to stop allo reactive T cells, and I think that's something that's sort of. An investigation, but what's interesting is that if you do expand these, these cells early, which is what we, what, what happens with any transplant or with any physiologic stress, um, you do get this expansion of T. regs. Now how we harness those T-regs or get them to be antigen specific to provide long term tolerance is something that that I'm looking at currently. So where are we at today? So, uh, somebody wise, when I was telling who found out, uh, what I was, uh, talking about today, said you guys have spent like 50 years on this, and I've only had 2 patients off meds, and I know it sounds depressing, but we are literally 50 years closer. So I, I do think that they were on the horizon within the next 1010 years or so, uh, but it's, uh again, hopefully what I've tried to explain to you or show you today is that there are a lot of players and a lot of sort of interactions that we're still trying to understand and every time that we. Think about this. We have to also look back at what we're doing to our patients. And so here's all of the, all of the most commonly used immunosuppressive medicines that we use and how they affect all those immuno regulatory cells. You can see sometimes it's positive, sometimes it's negative, sometimes they suppress these cells, sometimes, and so with all of these different mechanisms or schemes that we're using. We were probably affecting the immune system in untoward ways and then luckily are able to get patients off of meds in some cases but only in a very, very small number of those cases. So some of the future directions that I'll be looking at are how do we get more ILCs into organs, uh, into the organs, and so one of the things that I didn't show you is that there's been a lot of talk about again with this migration and do these cells actually migrate into the organ after any sort of physiologic stress and hopefully I've shown you that they do. The problem is that the number is limited, which is probably also goes back to the reason why I have to use such an artificial system to look at them. Uh, to see a huge effect, uh, a lot of, uh, investigators have looked at what controls that and have thought that, you know, maybe it's related to, uh, you know, a soluble factor that we, that a lot of us know is interferon gamma, and if we block interferon gamma, can we now get now these ILCs to come into the cells, uh, sorry, come into the allografts more to provide uh local uh immuno regulatory functions. Um, and then of course the more complicated question is how do all these cells communicate and, uh, one of the, you know, trying to tease this apart, uh, one of the major projects in the lab is trying to look at how T. regs and ILCs, uh, communicate and, and what, what, uh. Uh, receptors for that are important. Again, looking back at the literature and trying to see what has been looked at and what has been found in other types of models of autoimmunity or even cancer to see how how we can improve that communication and crosstalk and and and transplant. And so even though we think of the bone marrow is still kind of a black box, there have been many studies that are still coming out, and this one most recently in Nature that I've looked at specific. stem cells or hematopoietic stem cells within the bone marrow and through chemokines, which are basically essentially proteins that allow for the trafficking and movement of of white blood cells into different organs, as well as nitric oxide may play a significant role in providing immune tolerance and getting cells to where they need to go and to provide that local immunoregulatory role. And different things that are being uncovered about how innate immune responses actually function. We now know that innate immune responses have actually two types of memory, quote unquote memory. One type is thought to be this trained immune responses, which is what we think potentially may happen in things like biliary tresia, where you have a prolonged sort of inflammation which then over time causes innate immune responses to have a lower threshold of activation when stimulated. Compared to those that are completely compared to those innate immune cells that are completely naive, and even as I said, innate immune memory, and a lot of this has been worked out again by a lot of elegant work out of Pittsburgh with the Fati Laus group where they looked at how certain cell surface markers can recognize non. Self MHC and can do so and have a lower threshold of activation and rejection response, suggesting a kind of memory and so one of the thought processes here is I always like to think of of all of these things having a a counter regulatory role as well as a regulatory role if you think about a recipient monocyte having. These cell surface markers where Pier A, which activates this pro-inflammatory cascade or pro-rejection response, uh, these recipient monocytes also should express what's known as Pier B, which is suppresses an inflammatory response, and with certain stimuli you'll get like the transplant, you get recognition of non-self. And the creation of trained immunity and or innate immunity, innate immunity and memory that has up regulation of all of these pure A molecules and a reduced activation of pro-inflammatory response. But can you have a coordinated immunoregulatory response where you have monocytes that are actually tolerogenic as opposed to just immunogenic, and can we use that therapeutically? So can we train tolerance and transplant and can we condition transplant recipients to donor antigens to facilitate this operational tolerance, uh, that is that work is in progress. Um, so with that I just like would like to finish by, uh, thanking, uh, my lab in particular, uh, Wei Kong and Maddie, uh, MacArthur who have been instrumental in a lot of the recent, uh, findings, uh, looking at ILCs and, uh, the epigenetic studies looking at memory and innate immunity in, in, uh, in the lab, as well as, uh, my, my, uh, uh, mentors, uh, uh, Doctor Markman, who's not at MGH anymore, is at UPenn, and, uh, Jon Madsen. Uh, and, uh, my local mentor here, uh, Yvonne Zanonni, uh, and as well as my, uh, uh, funding agencies in the department for their continued support, and with that I'm happy to take questions. Well, thanks, Alex. I, I, um. I'm almost wondering if I went to medical school today if, if I would enjoy immunology as opposed to the terrifying experience I had where it seemed like, uh, imaginary magic to me, um, not quite 50 years ago, but long enough ago, um, and thanks for a little, little bit of education. I actually have some questions to ask offsite because I think I understood a little bit. I wanna open it up to, to, uh, to questions, um, from, uh, people smarter than myself. Yeah, Alexa, thanks for that talk, uh, and thanks for bringing me, bringing me back, uh, down memory lane. Uh, I worked in this area for quite a while, uh, eventually gave it up. And, uh, but, uh, I appreciate you continuing this, uh, pursuit of tolerance. I wanna zoom out and ask you a high, high-level question. Uh, now as an observer, uh, more of a, a sort of educated observer and reading more, uh, lay press kind of, um, things about tolerance and transplant. What do you think uh is more likely to occur in, in our lifetimes? Uh, xenotrans successful xenotransplantation on a widespread basis or allograft tolerance, uh, in, uh, on a widespread basis? So when you mean successful xenograft transplant, do you mean that the xenograft will last long term? I mean, or just as I mean a xeno xenotransplant as a standard of care. Yeah, well, I think xenotransplant in particular is, is already gaining traction, but as a bridge right now, um, in, in certain settings, I think that we're still sort of trying to understand, I mean, similar to, uh, the miogenicity that we see with allographs, with xenographs, and I think that if you're able to provide tolerance with. Allographs, the next step would be looking at potentially xenographs and what what you could do to get that to last long term. And if you can theoretically have a xenograph that would function that way, then yeah, that would probably solve a lot of the organ shortage. But I still think we're, we're a ways away from that because there's a significant amount of of xenoimmunity that occurs, uh, following the transplant and right now it's really meant as a bridge, uh, more so than, um. You know, as a long term solution, but I think both using both would, uh, as a long term solution for, you know, a, a patient who needs an organ is important. So you're voting for tolerance, uh, allograft tolerance. Probably. Yes. Do you know what's going on at Mass General on the Xeno front? Uh, probably as much as you do because I haven't talked to them about these trials. I know they've, uh, they've done at least 2. I can't remem I, I can't, I haven't talked to them about uh 2 kidneys. Uh, I haven't talked to them about the other potential, uh. Recept donor matches that they may have done. Great, thanks. Hi. Um, so thank you for your talk. Uh, it seems like from, from what you're describing, a lot of what is old is new again, right, in, in the sort of bone marrow transplant with kidney or liver, which was sort of how Starzel did a lot of this when he was attempting for tolerance, and at the IPTA, uh, meetings, it seemed like a lot of the Uh, thought was sort of combined liver with other stuff. Again, because the liver has a, a special place in, in sort of graft tolerance or helping to keep other grafts surviving. I guess from your perspective, In looking at the literature, are some of these data when they're looking at, you know, there's a lot of Sandy Fang with the liver trials and stuff, can that be recapitulated to other organs, you know, organs where they are more prone to rejection? Like we're studying liver a lot, but the liver already has sort of a Privileged privileged place for whatever sort of portal flow or whatever innate immune system in the liver. And what we're doing to achieve tolerance in liver, can that be applicable to other organs that have been testier in terms of their long-term outcomes for tolerance. That's a very interesting question because one of the things that I, you know, that I had thought about. Uh, uh, when I started was, what makes, aside from the fact that, you know, in fetal life the liver was essentially the bone marrow, uh, what makes these organs more prone to extramedullary hematopoiesis, and can these organs by themselves like the liver, it's not just, you know, this sort of nebulous concept that we think of, oh, we put a liver in and it can act like a sponge in a kidney for a kidney transplant, right? Um, is there actually a mechanism to induce tolerance, not just like as a sponge, but as a as like tolerogenic. Location for and yes I, I, I, I don't know that anybody has done that or has looked at, you know, I, I think people are still looking at why extramedullaryatopsis happens and like how it happens, uh, but I think that that's definitely a, a potential possibility that you could maybe get away with something like that without having such strong, you know, like this like complete lymph, uh, bone marrow myablative lympho ablative, uh, concept. Um, Yeah. I think It's just a quick question, Alex, uh, from someone who's not in the field at all. Uh, I always had a gut feeling that Keimerism might be a good thing, but what you've shown is that it might be a good thing or a bad thing. Is it, is it, is it time for us to just forget about that, uh, concept as a whole and think that we need to break this down into its rudiments and not saying you're a chimera or not. Um, Break it down into two so that so so you go into the each element like a different like aspect, um, I do think that there are more immunogenic aspects and things that we need to control more so than others and maybe we can, I, I, I think you might be suggesting that maybe there are components of that, uh, sort of like the HLAs that are more important than others, uh, maybe some, um. Uh, sort of polymorphisms in different types of there's like this HLAG now that a lot of people have that are that that people are looking at as being a more tolergenic phenotype in humans. Is it possible that we could do that? That's probably getting there, yes. Um, I do think that, that there is, there is this element every time I, I, so one of the, the, the people at, at, at MGH has looked at this a lot, uh, is Tatsuo Kawai, who was the one who published the initial, uh, MGH trials on bone marrow and kidney transplant, and I've asked him a bunch about this, you know, what's the mechanism behind mixed chimer or transient chimerism, right, because it almost, there's a little bit of it that doesn't make any sense unless you're completely like repopulating the peripheral immune system. Um, and I don't think, I don't think we understand it completely. Um, there's been some talk about how you get selective apoptosis of different cell populations, but I have no idea how that would happen, and I one more probing, I don't think they do either. Um, so I think it, you know, I think we're still, as you're suggesting, at the sort of the forefront of or at the cusp of, of trying, of almost getting there, but then, you know, having to do a lot more work to try to understand the intricacies of it. Thank you. Alex, thanks so much. Um, you led off with an example of your patient who achieved 100% chimerism, um, after an episode of GVHD, um, and yet it strikes me that, uh, there's been a relatively Underwhelming success with bone marrow transplants and things like that, so it seemed, it would seem that chimerism may be an imperfect marker for eventual. Uh, organ tolerance, um, is there a better marker, or should we be searching for that, or is it all based, or is tolerance a, a clinical, uh, definition at this point? Well, for sure in our patients it's definitely, it's just the definition, right? Uh, because like in some of the immunosuppressive withdrawal trials, none of the bone marrow, uh, the things that, that we looked at or that, that, that patient achieved. Uh, are being looked at necessarily, it's just simply can you come off of, uh, immunosuppression and is there any sort of enzymatic activity or biochemical disturbance when you come off of immunosuppression. So I think right now it's just, um, a clinical definition of tolerance which is this operational tolerance. Uh, I don't think Heimerism is, is, as you say, as you're suggesting, a, a marker of tolerance, uh, because you have different aspects and different types of Keimerism that can lead to the same thing, but I just don't think we understand what, what's in between. Well, I think we can see that true chimerism is quite rare, and I would say that is true, um, but you've proven that there are, uh, still surgeons who are equally, uh, versed in the language of basic science as they are in clinical care and very complex surgical care. Um, yes, maybe, um, you and people like you've been working for 50 years, but, um. People have been working longer than that on nuclear fusion, and it's always a decade away, but sometimes that inflection point happens, um, and, um. Doctor Folkman isn't here to see the benefits, uh, all the clinical benefits, um, but what he was saying was correct and his progeny have made enormous impact. And so, um, having surgeons having a role in this kind of investigation with the understanding of the clinical impact is, uh, well demonstrated here and, um, um, keep, keep plugging that along with your colleagues. Um, I wanna thank you for, um, educating us and for your persistence, um, in such an incredibly challenging field. Um, thank you very much and thank you.
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