I'm Dr. Mabel Ryder, and it's a privilege to chair this session. Thank you, Dr. Franco, and the Programming and Steering Committee for organizing this. I dabbled in immune cells in my life in the lab, and so I'm still tied to this field, both now clinically. And it's a privilege to share the chair with Dr. Yaron Tomer. He and I have circled each other's paths. He was my mentor when I was a fellow in Cincinnati, so it's good to see you, Yaron. Thank you, Mabel. I'm Yaron Tomer. I'm the chair of the Department of Medicine at Albert Einstein College of Medicine in New York, and it's a pleasure to see you all here. And we are in for a treat today, a terrific session with a great, also catchy name, When the Immune System Gets It Wrong, and It Does. So without further ado, I'll have Mabel start the session. Yes, so our first speaker is Dr. Melissa Lek, she's an MD-PhD and assistant professor of medicine in the Division of Endocrinology, Diabetes, and Metabolism at UCLA David Geffen School of Medicine. She has clinical and research interest in immunology and endocrinology, and as it relates to changes in cancer, cancer treatment, and autoimmunity. She has a particular interest in immune-related adverse effects of immune checkpoints, and she and I are members on the NCCN panel for managing IRAEs of immune checkpoints. And Melissa's gonna, her title of her talk today is Keeping the Immune System in Check in the Thyroid. Welcome, Dr. Lekner. Thank you so much for the opportunity to be here. I'm excited to see so many people interested in immunology. I'm gonna talk about mechanisms of immune tolerance and when they fall apart in the thyroid. I have no disclosures. So the aims of our study are to talk about when there's immune dysregulation in the thyroid leading to autoimmune disease in a couple different circumstances, and then how we can think about that to address the care of our patients. I'll start by just laying out the idea that the immune system is this balance of activation and tolerance. We need immune activation, antigen recognition, recruitment of immune cells in order to clear pathogens. Alternatively, we also have situations in physiology where we need tolerance so that we don't have autoimmune disease. We have pregnancy where there's a foreign host. And then we have pathologic tolerance, such as in the case of tumor immune suppression. So with that background, we're gonna launch into immune checkpoint inhibitor thyroiditis, which is now a relatively well-recognized new form of thyroiditis, and think about what the mechanisms of it might be compared to more traditional forms of spontaneous thyroid disease. As a context, cancer immunotherapy, as many of you know, is a form of cancer treatment where we leverage the body's own immune system to attack cancer cells. There's many different forms. I'm gonna focus on immune checkpoint inhibitors. These are a class of monoclonal antibodies, as well as some drugs in the pipeline, that specifically block checkpoint or endogenous regulatory mechanisms, largely on T-cells, and increase immune activation on T-cells by removing the breaks. There's cytotoxic T-lymphocyte antigen, which is commonly expressed on activated T-cells following co-stimulation. Antigen-presenting cells, like dendritic cells, have the ligand for it, and it turns to sort of attenuate immune responses. In addition, there's programmed death protein, or PD-1, and its ligands, L1 and L2. These also serve to down-regulate and attenuate, promote tolerance in T-cells, and the ligands can be on endogenous tissues that are maybe immune-privileged, as well as tumor cells. So the currently approved inhibitors target these, as well as a recently approved LAG-3 inhibitor, another checkpoint that we won't discuss today. And since the initial approval of ipilimumab, the first CTLA-4 inhibitor in 2011, there's been just a whole host of inhibitors. We now have eight approved agents in this class. And I'll highlight for you that immune checkpoint inhibitors are now first-line therapy for a number of solid malignancies shown here, and many, many clinical trials in the works. So I wanna emphasize to you that, as endocrinologists, we're gonna be seeing many patients treated with immune checkpoint inhibitors and there was a nice study done in about 2019 that showed that almost 50% of US cancer patients are eligible for immune checkpoint inhibitors. The rise of this has been largely due to some durable and prolonged immune responses that have been very exciting and previously untreatable or largely metastatic disease. However, with the increasing use of these, we also now know that immune activation has consequences, as many of you in thyroid autoimmunity probably could have told us to expect. And specifically, when we start to activate the immune system and reverse tolerance, we now recognize immune-related adverse events as a consequence. These are autoimmune toxicities that resemble autoimmune disease occurring in many different tissues of the body, including the endocrine system, but also lungs, kidneys, liver, gut. And approximately 60% of patients treated with combination immune checkpoint inhibitors develop toxicities. These toxicities impact the benefits and use of immune checkpoint inhibitors. They can lead to hospitalizations, treatment-related mortality, they can cause interruption of cancer treatment, and in the case of many endocrine organs, permanent organ dysfunction. Specific to the thyroid, about 10 to 25% of patients, depending upon the regimen, develop thyroid autoimmune toxicities. So I hope that you find them relevant. And one of the areas of research, and by many others, including our research group, is to try to understand what the mechanisms of immune-related toxicities are so that we can reduce toxicity of cancer treatment, but importantly, not impair the anti-cancer efficacy. Because remember, we're treating often stage four or very sick cancer patients, and this may be the last treatment option. So with that context, there's been some great work done to try to understand what causes toxicities. We focus on the thyroid because it's my favorite organ, but I think a lot of this is relevant and may be applicable to other endocrine organs, as well as other tissues in the body affected by toxicities. So we know that one hypothesis is that maybe we're just releasing autoreactive T-cells that are already in the body and already in the gland. This comes from some nice data that's shown that patients with pre-existing thyroid autoantibodies, specifically TPO and thyroglobulin, have a very increased risk. There's been a nice paper by Neuron colleagues that showed almost 100% penetrance of thyroid autoimmunity toxicities while on checkpoint inhibitors if you had pre-existing thyroid peroxidase. So we can say perhaps subclinical autoimmunity. In addition, Dr. Kotwal et al. showed using thyroid biopsy of patients with checkpoint inhibitory thyroiditis that there was an accumulation of PD-1 positive T-cells in the thyroid gland, suggesting perhaps a mechanism where PD-1 inhibitors directly act on these already autoreactive T-cells and then unleash an immune response in the gland, leading to the inflammation that we see. In addition, there's some wonderful other basic science work that's been done. This was a study by Sierra Alvarez and colleagues that looked at in patients with multinodular gordri versus Hashimoto's thyroiditis that indeed thyroid tissue itself may use PD-L1 as a mechanism to sort of protect or confront autoimmune disease. This is an immunofluorescence diagram. We're shown in red. You can see that thyroid follicular cells are upregulating PD-L1 in the face of a challenge from CD45 positive immune cells. Indeed, 80% of patients with Hashimoto's had this and you could see then where a PD-L1 blockade could reverse the sort of detente that exists between the immune system and the thyroid. The other thing we think about is we know a lot about spontaneous Hashimoto's thyroiditis. This has been well studied by many in the field. And so is it possible that this is just an extension of Hashimoto's thyroiditis? So we know that interleukin-17, a pro-inflammatory cytokine that's driven under the transcription factor ROR-gamma-T that comes from T cells such as DH helper cells, CD4, also CD8, gamma deltas, and now we understand N8 lymphoid cells. Is this something that can contribute? In Hashimoto's thyroiditis, there's been a number of studies done. I'll highlight a couple here. That in patients with Hashimoto's as well as Graves, there's increased circulating lymphocytes that expressed IL-17. And indeed, this group looked in the thyroid and showed that the thyroid infiltrating lymphocytes also strongly expressed this inflammatory cytokine. Subsequent studies in patients confirmed this and showed supporting data that interleukin-17 and the cytokine IL-23, which drives Th17 differentiation, were also increased in the patient's serum with Hashimoto's and then some really elegant work done by Hori and colleagues using a mouse model where genetic deletion of interleukin-17 protected the mice against thyroiditis. So hopefully we've made the case for you that interleukin-17 is one pathway that is known to be important in Hashimoto's. And so we can ask the question, well, what about checkpoint inhibitor thyroiditis? Can't it just be the same? And so we used a model that was piloted by Trevor Angel and Anupam Kotwal, where you did thyroid biopsy of patients with thyroid autoimmune disease. We subsequently used it for flow cytometry as well as single-cell sequencing, which allows in-depth profiling of the immune system. And we found some, see if the pointer works. There we go. So confirming what our colleagues have shown previously, we indeed see shown on the left in thyroid autoimmune toxicity compared to patients who have Hashimoto's. We have primarily an enrichment of CD3 T-cells, very similar to Hashimoto's, as well as B-cell shown in the middle and monocyte shown on the right, suggesting that this is a T-cell predominant but diverse immune context in the thyroid infiltrate of these patients. And then we looked at single-cell sequencing. For those of you who may not have used this technique before, it takes single cells and provides the entire gene transcriptome of what each cell is producing, and you can map out cells. This was a data set where we took thyroid binatal aspiration biopsies from five patients with acute checkpoint inhibitor thyroiditis, as well as five patients with Hashimoto's thyroiditis, and then several healthy controls that had normal thyroid function, negative thyroid autoantibodies and normal ultrasound. And we did single-cell sequencing on the immune cells in the thyroid gland. This is what's called a U-map, and what it does is it spatially lays out for you the relationships between the cells based upon their transcriptional profile. You can see that there is quite a diversity of immune cells, a large context of B-cells, as well as monocytes macrophages. And then we highlight here for you, again, primarily a T-cell cluster, suggesting, again, similarities between Hashimoto's. So now we get to the good stuff. So we can do pathway analysis to try to understand with all this genetic data what is going on. And so one way you can do that is with pathway analysis. We query the KEGG pathways to look at what the gene transcriptomes were showing us in patients with checkpoint inhibitor thyroiditis compared to normals. And indeed, we do see pathways like the Th17 signaling, very similar to what we'd expect in Hashimoto's. In addition, in order to better study mechanisms, we developed a mouse model of immune checkpoint inhibitor toxicities. The absence of sort of robust clinical models and preclinicals has really been a challenge to this field. And so we actually used a mouse with a genetic predisposition based upon its HLA to autoimmunity, the Nod mouse. I think often thought about as a diabetes model, but great for autoimmunity, including the thyroid. We gave the mice tumors and then gave them cycles of checkpoint inhibitor, just like one would for a patient. And then about four to eight weeks later, we looked at the mice to see what tissue autoimmunity they had. And we saw thyroid autoimmunity as well in the checkpoint inhibitor mice, but rare to absent in the isotype-treated mice. And then just as a side note, but coming out in our paper shortly, we also get a model of multi-organ autoimmunity. So much like patients with checkpoint inhibitors, we see gut, lung, kidney, and other autoimmunity. And then again, similar to patients, we have increasing rates of autoimmunity across the tissues with combination checkpoint inhibitors compared to monogenal lens. So we think it's a pretty robust model of autoimmune toxicities. And then using this model, we queried the type three immune response genes related to IL-17 to see if there was a lineup. And indeed, in checkpoint inhibitor-treated mice, the gene expression in the thyroid does show increased type three immune response genes, suggesting an IL-17 pathway. And then when we look specifically in the thyroid cells, thyroid-infiltrating immune cells, we see increased ROR gamma TT cells, which again, RORT is the transcription factor canonically driving IL-17. So I think moving towards a story of potentially an IL-17 pathogenesis, at least in part. And indeed, when we look at the cytokine production, here I'm showing you the thyroid-infiltrating cells making IL-17 in checkpoint inhibitor-treated versus isotype-treated mouse across two different tumor models, a B16 melanoma and then also a MC38 colon. And what we found is that there is indeed T cells increased and interestingly, in a tumor-free system that we have, perhaps representing a patient who's getting this in the adjuvant setting, it's primarily an innate gamma delta T cell making 17 that we see. In contrast, in a tumor-bearing host, we see more activation of the adaptive immune system and so more of the TH17. So I think that's just kind of an interesting thing we can do more with going forward. So what do we do with this to actually help patients? So we then treated our mice with a neutralizer antibody for IL-17, which is important for us because this is actually a clinically available therapy used to treat psoriasis. And we assessed not only the changes in thyroid autoimmune infiltrate, but we also looked at tumor growth because remember, at the end of the day, we're hoping to treat cancer patients. And so as a summary, we did see that there was a decrease in thyroid autoimmune infiltrate. I'm showing you a decrease in T cells here. We saw it across other populations as well. And I've done it across some other tissues as well. And then importantly, we preserved the anti-tumor efficacy. So shown in black on this tumor growth curve, you see that untreated or mock-treated mice have tumors that grow. In purple, you see a nice response coming down over time with immune checkpoint inhibitor therapy, but that with the addition in blue of anti-IL-17, we don't lose that efficacy. I'm showing you the data here for our colon tumor. We saw similar data in our melanoma model. And also with CNF-alpha, which is another place we can block the IL-17 access. So I think some exciting stuff in possible therapeutic avenues. So I think there's some evidence that much like Hashimoto's, IL-17 has a potential role in the pathogenesis of immune checkpoint inhibitors. But what about different stuff? So as many of you know, clinically there's quite a few differences between immune checkpoint inhibitor toxicities. They tend to show a more even male-to-female ratio compared to Hashimoto's, which is approximately eight to one female to male. In addition, many fewer patients have thyroid autoantibodies that we can detect at diagnosis. It's much more rapid, suggesting perhaps some different mechanisms and kinetics. And then finally, we really still don't know much about the mechanisms, but we're starting to learn. So because of the prominence of T cells that we saw on our flow cytometry in our single cell, we really focused in again and did a subset analysis of the T cells to try to get a more detailed look. And what I hope you see here, and again, this UMAP of just the T cells, is really the incredible diversity of autoimmune populations, including sort of our Th17. You've got some memory cells, CD8 cytotoxic T cells, and then also some T follicular and T peripheral helper cells that have been described in other autoimmune diseases like rheumatoid arthritis. I won't have time to talk about those today, but to be continued. I wanna focus a little bit on the CD8 T cells. So when we did an analysis of which subsets were preferentially expanded in the checkpoint inhibitor treated thyroids compared to Hashimoto's or normal controls, one group that we were able to infer was increased was the CD8 population marked by interfering gamma and CXCR6, suggesting that it was more of a type one immunity phenotype. And so shown here is another UMAP. It's a feature plot. Purple means more expression. And I've circled for you that cluster three of CD8 T cells of interest. And you can see that there is strong expression of interfering gamma as well as perfrin, which is a cytotoxic molecule. We also saw a lot of granzyme B and fast ligand suggesting that this might be a type one immune response as well as sort of a cytotoxic population. And then very interesting to us, we were able to identify two critical chemokine receptors, CXCR3 and CXCR6, which have been described in other autoimmune diseases as mechanisms by which T cells may go into an inflamed tissue. And again, we're thinking therapeutically here. So we're trying to find ways we can break the chain. So when we look at predicted ligand receptor interactions, which can be done with single cell datasets to think about how cells might be interacting, one thing that popped out is we saw a very strong signal between the monocyte or myeloid population and these chemokine receptors, specifically to the CD8, possibly pathogenic T cell population of interest. So we see signaling through CXCL16, an important and unique ligand for CXCR6 from monocytes, as well as communication through the chemokine CXCL9 to really bring in a bunch of different T cell populations. Similarly, we see reciprocal talk that interfering gamma might be talking to those same myeloid cells, perhaps stimulating this chemokine response, as well as CSF1 colony stimulating factor, a strong monocytic promoting factor. We did a little in vitro work here. I'm showing you macrophages in vitro that we stimulated with interferon gamma and looked at their gene expression. And interestingly, we see that CXCL9 and 10 ligands for this chemokine receptor, as well as 16, are strongly upregulated following interferon gamma. In green, you see the LPS just as a control. So that's great. I told you there's T cells in the thyroid. I told you there's myeloid cells in the thyroid. I think we beg the question then, but does it matter? Are they doing anything pathogenic? Because we're all about mechanisms. And so one way we can ask this question with single cell data is to look at the T cell receptor clonality. So bear with me. CD8 T cells we know have T cell receptors and they undergo rearrangements and each T cell has its own unique T cell receptor. You would imagine that if there's a response to antigen, that T cell expands and undergoes clonal expansion. You see on the right, I've shown you a UMAP of T cells that have no clonal expansion because it's normal healthy tissue. And you'll also see that there's a scale. And so when we look at the T cell clonal expansion overlaid on immune checkpoint inhibitors, or Hashimoto's, you see here in the bottom, let's see if my pointer works, whoops, I'll just circle it for you, there we go. So I've circled for you there that there are yellow dots representing clonally expanded T cells, meaning response to antigen and expansion, localizing to that CD8 population with a type one response and sort of a cytotoxic phenotype, suggesting that they may be responding to antigen. By comparison, if we look at Hashimoto's, we see clonal expansion at low levels across, but largely in the CD4 compartment. And so this suggests that maybe this is a population of CD8 T cells that we can further query to see if they are indeed responding to thyroid antigens or something in the thyroid microenvironment and possibly pathogenic. So I'll propose to you this model. We also see these same cells in our mouse model and have more work to do, of course. But so far, I think we've identified that there seems to be a role for IL-17 primarily through CD4 T cells as well as gamma delta T cells, depending upon the presence or absence of a tumor. And we have clinically feasible inhibitors to use and in our mouse models and hopefully to be studied soon in patients, we can block these pathways and reduce toxicities without impairing the antitumor responses. In addition, we've recently identified that there may be a interaction of myeloid cells that serve to recruit T cells. And that's another place where we can intervene with blockade of chemokines or chemokine receptors to reduce tissue autoimmunity. And then finally, we've identified a population of CD8 T cells that have this type one immunity, cytotoxic phenotype that appear to be responding to antigens in the thyroid microenvironment. The next question, of course, is what are they responding to and can we show that they're responding to thyroid tissue? And then finally, we've identified that there may be a potential mechanism by which we can address thyroid autoimmunity. So in summary, there appears to be an immune checkpoint inhibitor thyroiditis, both shared and distinct mechanisms between spontaneous Hashimoto's and checkpoint inhibitor associated thyroid autoimmunity, including these two groups here. I'm sure there are more yet to be found. And also that we should be thinking about how we might be using immune modulating therapies such as inflammatory bowel disease and psoriasis to maybe start addressing autoimmune thyroid disease like an autoimmune disease. And I think this is maybe a first step. And then of course, I don't do any of this by myself. I have a wonderful group of researchers and colleagues who contribute to this, as well as funding from a number of organizations. Thank you so much for your time. Thank you. All right, we open the floor for questions. We ask that you please come up to the microphone. For our live streaming audience, please speak into the microphone. Dr. Haugen, welcome. Hi, Brian, Haugen, Colorado. That was wonderful, Melissa, thank you. Really elegant work. You know, the thing that with treating patients as well and then getting some of these side effects, I saw in your animal models, you definitely did CTLA-4, anti-PD-1, and then the combination. I've always been a bit fascinated with the anti-PD-L1 blockade as well, because I think there's a lot of overlap, but there may be some distinct things as well. And have you had a chance to look at that in your model? That's a great question. So I think something to keep in mind with these biologic therapies is that the specific affinity of the monoclonal antibody used as well as the FC receptor, as you know, really changes the efficacy. We don't use PD-L1 yet in our model, but a lot of our collaborators do. And what they find is that PD-L1 is expressed in a lot of really important tissues, like the thyroid, and especially the liver, where there's a lot of toxicity. So I think you're right, we probably need to also test PD-L1 inhibitors. And the good thing about the PD-L1 inhibitors is that a lot of them have actually shared efficacy in mice and humans, which makes it even more relevant to patients that you can know for sure that what you're studying is gonna be translatable. That's a really good point. Thank you, this was a great talk. This is Dr. Sater. In your mouse model, when you gave a neutralizing antibody for interleukin-17, you used it after you started the immune therapy. Did you experiment, how would giving this before starting immune therapy affect the outcome? And can interleukin-17 neutralizing antibodies be used as prophylaxis in high-risk patients who have other autoimmune diseases or maybe positive TPO or thyroglobulin antibodies? Thank you so much for that question. You're absolutely right. So we initially did studies using it as a late start. I think our concern was that we needed to get things going before we blocked it, but we've done both. There doesn't seem to be a difference in the tumor-free animals. However, in the animals that are already tumor-bearing and have greater immune activation, we find that prophylactic use of it is more effective, I think because there is already probably a Th17. And importantly, we were afraid that if we used it prophylactically, we would impair the antitumor responses because there is a mixed role in terms of what the role of IL-17 is. But thus far, I think it can be used prophylactically, and I think especially for those high-risk patients that were previously excluded from immunotherapy trials because of it, and I think we're all clinically now seeing those patients. I know there's a study that the NCI is doing to look at that, and I think that would be a great population for prophylactic IL-17 or other. I think it's gonna be better than using prednisone, that it's gonna suppress everything over the world. Anything has to be better than prednisone. Thank you so much. Thank you. Josh Cahaley, MATCH Germany. Blocking interleukin-17a has been actually used to treat autoimmune disease, autoimmune thyroid eye disease, and we're doing this since years. Have you tried, actually, to block interleukin-17a in your mouse model to see if you can actually prevent these changes? So when we treat the mice to get checkpoint inhibitor with IL-17, we look at the thyroid and we prevent thyroid autoimmunity. We have yet to see thyroid eye disease in our checkpoint inhibitor-treated mice, so we have looked for uveitis because that's a common toxicity seen in cancer patients. We do have a mouse model of thyroiditis, Hashimoto's. It's not the typical iodine model. It's one where there's a difference in thymic regulation. It's an air gene-dominant negative that was actually based out of populations in Italy as its basis, and we are starting to look at IL-17 blockade there because I think you're right. I think we have to start somewhere, but I'm so glad to hear that you guys are doing it in patients. That's wonderful news. Thank you. Hi, Melissa. I'm Emily Gallagher from MedSignEye. I know you've published, as have other people, that actually if you develop the autoimmune thyroid disease you might live longer and have better outcomes from the cancer perspective, so is this kind of a theoretical question, but do you think it's wise, then, to try and prevent the autoimmune side effects if patients are doing better? Are you a plant? Yeah, so no, I think that's really the crux of it. So I think there's been some work, obviously. We know that T cells are involved. You can't knock out T cells in a checkpoint inhibitor-treated patient, right? They're gonna die of their cancer. So I think that is the crux. So the focus of our research has really been on that fine line. What can we do that's toxic and autoimmune but isn't integral for the checkpoint inhibitor tumor? So I think that's why, as painful as it was to get it, we had to have a model with the tumor because otherwise what are we doing? That was really the key part. So we think with IL-17 you have a viable target. With TNF-alpha, also known to be very anti-tumor or pro-tumor, I think that one we see working. And that one we see working in gut toxicities and patients do quite well. For the CXCR6 CD8 T cells and interferon gamma, right, we can't knock out interferon gamma. So there we're actually looking at a corollary cytokine, IL-21, which probably drives it, which might be a viable way. Hi, Dr. Davies. Hi, Terry Davies in New York. It was a very nice presentation. But I don't want to leave here confused, right? That's the idea, right? No, we don't want you to be confused, Terry. We should leave less confused than when we arrived. But it's immunology, Dr. Davies. Patients have thyroid antibodies and patients don't have thyroid antibodies. Are you suggesting they're both the same? I think we haven't yet figured out what the antibodies are for. So one of the things is we clearly know that there must be antibodies. We see B cells, we see B cell signaling, we see plasma cells and thyroid infiltrates. I think they may not have TPO and thyroglobulin antibodies in all cases. I think the question for us is, are they making different antibodies? So we're using, I think so. Let me rephrase this and say before they get treatment. Okay. If they have antibodies, that's one group of patients. And if they don't have antibodies, that's another group of patients. Are you suggesting that the etiology of the thyroiditis in both these groups is the same? I think there will be similar and distinct mechanisms from what we can see so far. Right. So the genetics doesn't matter. So actually they've done studies that shows the genetics does matter. So Zoe Quant did a nice study looking at polygenic risk score. So it seems like it probably does. Okay. Thank you. I think I've sufficiently confused everyone at this point and I am done. Thank you. That was great, Melissa. I think this is really fascinating and it's bridging both worlds. Thank you. Thank you. Okay. We're going to our next speaker and I have the pleasure to invite Dr. Romana Netea-Mayer. Professor Netea-Mayer is a professor of endocrine tumors and the chair of the Multidisciplinary Endocrine Tumor Board at the Radboud Tumor, practiced it so many times and I still can't say it, Nijmegen in the Netherlands. So without further ado, welcome Dr. Netea-Mayer. Thank you very much for the kind introduction. And I'm very happy to be here and thank you very much for the invitation to give this talk here. So what a kickoff it has been with the previous talk. So for the ones of you who are not immunologists, I will get you back to the basics. So let's start with homeostasis. So we all probably know that homeostasis is a dynamic state of equilibrium in which is actually fundamental for the functioning of every organism. And it is a very tightly regulated process in which different systems interplay with each other through different signaling molecules. So these are the immune system, endocrine system and nervous system. So today I will focus on the bidirectional relationship between the immune system and the endocrine system specifically on the regulation of the immune functions by thyroid hormones in maintaining the homeostasis, the immune homeostasis. So there are several indications that there is this bidirectional relationship between the immune system and the HPT axis. First of all, we know that severe illness such as an autoimmunity can be associated with non-thyroid illness syndrome. The other way around, when there is an abnormal amount of thyroid hormones such as in hypothyroidism or hyperthyroidism, this has been associated also with impaired immune responses and an increased risk to infections. So physiologically, the thyroid hormone production is regulated by the hypothalamus and pituitary gland through the well-known feedback mechanism. However, in serious and severe infectious diseases, for instance, there is activation of immune responses, high amount of pro-inflammatory cytokines which can affect also thyroid hormone concentrations, particularly the T3 hormone. And this happens through changes in the set point of the hypothalamus, for instance, but also in reducing the amount of bioavailable T3 at tissue level. So basically, we distinguish two types of immunity, the innate immunity, which is rapid effective, not specific, indiscriminate, and lacks immunological memory. And it is effectuated by macrophages, dendritic cells, neutrophils, basophils, eosinophils, mast cells, and NK cells. And secondly, we have the adaptive immunity, which needs 10 to 14 days to occur. It is a specific activation against particular microorganisms. It enhances the effectivity of the immune response and builds immunological memory. And the cells that are responsible for the adaptive immune responses are the lymphocytes, the B and the CT cells. And basically, when a pathogen is invading the organism or when danger molecules are released by tissue damage, for instance, innate immune cells, the monocytes and macrophages, they recognize these strange molecules and they become activated. So they produce large amounts of pro-inflammatory cytokines and these result in release of acute phase proteins in the liver, but also at the level of hypothalamus. They cause, in fact, the systemic symptoms of infection, such as fever, somnolence, anorexia, pain. Another part of the activation of an innate inflammation is played by the activation of the endothelium and this leads to increased permeability of the capillaries, which improve migration of immune cells to the place where the inflammation takes place. And moreover, there is also activation of complement. So when these mechanisms fail to remove the pathogen, the adaptive immunity is activated. And this happens because macrophages or other antigen-presenting cells are incorporating the pathogens and they are processing the antigen and they migrate to lymph nodes where they activate the T-cells and the adaptive immune responses are onset. And the infection is finally cleared by specific antibodies, T-cell-dependent macrophage activation and cytotoxic T-cells. So when we, in our experimental models, we use as readouts for the immune functions, particularly in experiments that we do in humans, circulating mediators, concentrations, platelet functions, cell numbers, immune cell numbers, I mean, production capacity of cytokines, immunoglobulins, and phagocytic capacity. So to go back to the question whether the thyroid hormones or the thyroid, the TSH impact on immune system, I would say there are some arguments for this. First of all, hypothyroidism and hyperthyroidism have been associated with disrupted immune responses. Also, there are in vivo and in vitro studies that indicate the expression of proteins which are involved in thyroid hormone actions in immune cells. And there are some mechanistic studies also suggesting functional changes in immune cells through genomic and non-genomic signaling by thyroid hormones. So the knowledge on the modulation of immune functions by thyroid hormones and TSH has been recently summarized in a number of reviews, among which this by Gerard et al. And these results are schematically summarized in this slide. So as you can see, both thyroid hormones and the TSH affect the function of numerous immune cell types, both innate immune cells but also adaptive immune cells. So for instance, the thyroid hormones can affect, for instance, the proliferation and the survival of lymphocytes. However, the effect is dependent on the model where this has been studied on the duration of the exposure. And similarly, the TSH has been shown to be able to differentiate the monocytes in macrophages and thus influence the bactericidal function of these cells. And together with T3 and T4, they are able to functionally reprogram or polarize the macrophages. Most of the experiments that have been published have been done in in vitro studies, and there are just a few in vivo animal studies. And most of the knowledge that we have on the thyroid hormones on immune cells reflect, in fact, their effect on the innate immune cells. And these effects are summarized in this slide. So as you can see, the T4 and T3 have been associated with an increase in respiratory burst activity and ROS production in neutrophil and macrophages, and thus affecting the bacterial killing of these cells. On the other hand, the T3 in physiological concentration has been shown to activate NK cell activity and interfere in gamma response. However, in hyperthyroidism, also reduced NK cell activity has been shown. And also in macrophages, both increase and decrease of inflammatory responses have been described. So apparently opposed effects. So how can we explain these? So first of all, it is important to realize that the effects of thyroid hormones on immune responses depends on numerous factors, such as circulating thyroid hormone concentration, expression of thyroid hormone transporters, and the affinity to thyroid hormone, intracellular thyroid hormone metabolism, and thyroid hormone to thyroid hormone receptor. Also, these effects can be cell and tissue specific, and they may differ strongly in healthy versus diseased states. As an example, this is the effect of thyroid hormones on macrophages. It is a very simplified slide, but just to give you an idea, in physiological situation, when there is sufficient T3 available, this activates through via the TSH, thyroid hormone receptor. So genomic activation, actually, and induced anti-inflammatory effects. However, in hypothyroidism, or when there is insufficient T3 available or less intracellular T3 available, T4 is responsible for the non-genomic activation because T4 has better affinity to integrins and through this mechanism, it increases the activity of PI3 kinase, AKD and MAP kinase and induces pro-inflammatory effects and increased cytokine production and ROS production. So what about the effects in humans, in healthy individuals? So a few years ago, we have undertaken this study, this comprehensive study in which we have looked at the interplay between thyroid function and immune function in healthy volunteers and we did this together with people from Broad Institute and from Groningen University and we did that within the Human Functional Genomics Project. Now what is this Human Functional Genomics Study? This is a large comprehensive study that has been undertaken at my institution and it has a general aim to determine, to describe the factors, endogenous and exogenous factors that are involved and are responsible for the variation in immune responses and another aim was to see how this correlates with susceptibility to different diseases and the idea is that the variation in immune responses is very large when you compare it between individuals. However, in one individuals, it is quite, the responses are quite stable so the question is whether discrete phenotypes could be actually identified which can in fact be associated to differentiated, differential susceptibility to diseases. So in this, within this project, we have looked at the role of immune, of endocrine systems specifically for today, for the talk of today, I will focus on thyroid hormones and the DSH on immune responses. So for this, in this study, we included three, two large cohorts of healthy volunteers. One was the discovery cohort of 500 volunteers and the other one was a validation cohort, a smaller one of 300 volunteers. And so in these patients, these were actually 65, 56% of these were female, half of them were using contraceptives, 13% were smokers and so in these volunteers, we took blood and we measured all kind of inflammatory mediators and also the cytokine production, respectively six cytokines production capacity after stimulation with 19 different microbial and non-microbial stimuli. And so as you can see here below, I mean there was no, not a good distribution with respect to age and BMI because most of the volunteers that were included were students. So we had mostly young patient, young volunteers and so the older individuals were underrepresented and also we had very few volunteers that have had obesity or were overweight. But these factors were taken into account as was also the seasonality which is also known to influence the immune responses. So now looking at the results, so these were the first results that I want to show you, quite disappointing actually. No association of DSH and 3D4 with inflammatory mediators and platelet function in healthy volunteers except for positive correlations, very slight positive correlation with leptin and between leptin and DSH. Also there was no effect of DSH and 3D4 on the innate immune cell population. So we visualized this population using a cell network model in which the size of the dots and the color indicates the magnitude of the association and the magnitude of the significance. Also there was actually no effect on the cytokine production of these healthy individuals. So you can see here, I mean I realized that the letters are very, very small but you can very clearly see in the red bulk that those are the effects of hormones on all those cytokines which were measured after stimulation with different stimuli and compared those with the first column which indicates for instance the role of genetic factors in cytokine production. I mean you can see immediately that the effect of hormones was negligible. So the first conclusion was that D4 and DSH have a minimal impact on innate immune responses in healthy volunteers because we found no effect on circulating mediators of inflammation on the innate immune cell populations and on the cytokine production capacity. So what about the adaptive immunity? So here the picture was completely different. So on the left side you can see the HIT map with the cell population that were associated in fact with the level of DSH and the D4 in our study and on the right side you can see this cell map model that I've shown you before. So lower in red you can see again the innate immune populations that I've magnified in the previous slide and on the right side, so in violet you can see the B cells, cell population and in green you can see the T cell population. What's immediately obvious is that the DSH concentration was strongly associated with T cells and this did not influence the differentiation of the T cells in different T cell subtypes but it was the lymphopoiesis which was in fact associated with the DSH level. With respect to D4 you see exactly, a completely different picture because we see here that D4 impacted specifically the B cell populations and also in this case it did not influence the differentiation of the B cells in different subtypes but the lymphopoiesis of the B cells. So these were very interesting results and we wanted to see whether we can validate those in a separate cohort. So we had this separate cohort of 300 healthy volunteers with similar demographic characteristics and these were students who participated in a BCG vaccination trial and at baseline they had exactly the same investigations that we performed also in the 500 BCG, 500 FG cohort. So we could use it as validations and as you can see here we could validate at least the DSH association with the T cell counts. So in order to assess whether there is a causal interference between the cell counts and the DSH and 3D4 we performed also a Mendelian randomization and using two different approaches we found that there was a significant association between the DSH level and CD8 cytotoxic cells and there was also a negative association of these T cells with a 3D4 level. So this implies or suggests a causal relationship. So in order to, because I showed you that the genetic factors are very, very strong contributors to the immune responses, we wanted to see whether the genes that were, or the gene variants that were associated with high DSH or T4 levels in our cohort were also involved in immune functions and you can see in this Manhattan plot the four genes that were, gene variants that were the strongest associated with the DSH and 3D4 level in our cohort. And if you look at the function of these genes and their EQTL, so the genes that are also influenced by these variants, you can see that the first two were in fact also involved in immune responses, the prostaglandin E receptor and TNF alpha receptor. So from this we, this data, we concluded that there are arguments for an influence of DSH and T4 on the immune phenotypes. Again, in order to understand more the biological relevance of these findings, we performed pathway analysis and in doing that we found that among the top 10 enriched pathways which were associated with 3D4 levels, there were two which were also involved in immune responses in CD4 for the B cell activation and activation of TNF alpha receptor. Which also of course supported our findings and also indicated that there might be a bidirectional association between the T4 and immune cell phenotypes. So our conclusion, second conclusion was that there was a strong interaction between DSH and T4 and adaptive immune responses because the DSH associated mainly with T cell numbers, T4 with B cell numbers and genetics informs about the direction of the association. And these data with seemingly independent effects of DSH and T4 may suggest new biology related to these hormones and their effect on immune function. So there are a number of data, animal data from previous studies that support these findings. So in different models, animal models that lack TSH or thyroid hormone, it has been shown that they also have defective lymphobiosis, they also have small thymus sizes and small spleen sizes. And more interestingly, only T4 administration corrected the B cell development completely in these animal models in the studies where an intervention has taken place. And also, anecdotally and also maybe still interesting to show you is this study, this is a study that was performed in Bajau population in Indonesia. It is a population, these are nomads that have evolved big spleens for diving. These people are able to dive for at least, for up to 13 minutes in order to fish underwater. And they really need this huge, they have developed this huge, large spleens. And interestingly, the single nucleotides which were associated with the top spleen size were also associated with higher 3T4 and lower TSH level in our cohorts. So this was the physiology, but what about the pathology model during the COVID-19 time? We have noticed that patients with COVID-19 infections also very often have lymphopenia. And we also know that when patients are critically ill, they also have low T3 levels. So we wanted to see whether there is an association between these two. And we had also access to a cohort of patients with bacterial sepsis from the ICU. And we selected in these two cohorts the patients who had the lowest number of lymphocytes and the highest number of lymphocytes during the acute phase of their infection. And when we looked at the thyroid hormones and the TSH levels in these patients, we found indeed that the patients who had lower lymphocytes numbers also had lower TSH, lower T4, lower 3T4, and T3. And this was the case in actually both of the two cohorts. However, they also had higher levels of inflammatory mediators, such as interleukin-6 and CRP and ferritin, which indicates that these patients actually were more severely ill than the other ones. But the question is, what about a normalization of lymphopenia? So does it go in parallel with normalization of thyroid function in these patients? In other words, can we have indications of causality? So unfortunately, this was not the case. So we can conclude from these that lymphopenia and thyroid dysfunction are highly prevalent in patients with severe infections, but the causal relationship could not be established in this context. And I think that this illustrates again how important is the model in which we look at these parameters. So to conclude my talk, I would like to state that there is growing evidence supporting a bidirectional interaction between the immune system and the thyroid hormones. The DSH and thyroid hormones are important regulators of the lymphoid compartment and immune homeostasis. But an in-depth understanding of this interaction is still lacking, and studies in robust in vivo and human models are needed in order to address the clinical implications and pathological conditions and to improve therapeutic approaches. And by this, I also would like to mention my collaborators from my university and also from Groningen and from Broad Institute. And I would like to also mention the Our European Thyroid Journal on behalf of our head editor. As an associate editor, I am very happy to and proud to mention this journal and to invite you to send your valuable work to our journal. So thank you very much for your attention. Thank you. Thank you, Dr. Netea Mayer. This is an incredible talk, very, very innovative, lots of food for thought. I have only 15 questions, but I'll start with the first one, just a technical one. The levels in these volunteers, I assume, were all in the normal range. All the volunteers had normal range. We excluded the ones who had TSH and T4 outside the normal range. And we excluded also patients, people who used medication that interfered with either thyroid function or the immune function, so corticoids or... So basically, you're seeing effects of thyroid hormones within the normal range. Yes, yes. My second question is on the genetics. So genetic factors that predispose to autoimmunity, like HLA, CTLA-4, PTPN-22, did not come in your GWAS? No, they did not come in our GWAS, but I must say that this was probably underpowered to assess these associations. I would recommend, since if you only do selective candidate genes, then you will have power. Yes. If you're doing GWAS, but if you took only five or six common autoimmunity genes, so I would be very curious to... Okay, we have questions from the audience. Thank you so much for this wonderful talk. While you were presenting your data, I got this question that we know that thyroid hormone contributes to the regulation of the beta-2 receptor that conducts the catecholamine signal into the cells. And we know as well that one of the beta-2 receptor is present on adaptive immune cells and on T-cells and B-cells. And I was wondering if the thyroid hormone could increase the catecholamine signal into immune cells that is known to affect the differentiation, polarization, metabolism, and action. It's a very good question. We have never looked at it in depth, but it's a very interesting and intriguing question. I don't know. I don't have an answer for you. Thank you. Thank you so much. Dr. Kahali. Romana, good job. Very good job. Thank you. Thank you. I would like to start with your trial comparing sepsis and COVID. My understanding is that the viral infection and in viral infections, you always see lymphopenia. And actually you may also have low T3 and low T4 syndrome. So this is what you were actually showing in contrast to sepsis. Exactly. Yeah. Yeah, exactly. So I mean, we were very, well, actually we were intrigued by the fact that these patients with COVID had actually more often lymphopenia than the bacterial sepsis patients. And I must say that this correlated absolutely with the disease severity also, but as a whole cohort, they were less sick. At least we had less people on the ICU, for instance, than in the bacterial sepsis cohort, which were all in fact patients from the ICU. And in the bacterial sepsis series, we found, I think there were more than 80% of those who had abnormal thyroid function test, whereas in the COVID cohort, there were only approximately 35% that had disturbed thyroid hormone. But still we could find in those two extremes, in fact, so in the patients who had very, very deep lymphopenia, they also had these T3, T4, but they did not improve together. So we could not, I think this was really both of them. So both lymphopenia and the low T3 level was an expression, in fact, of their severe disease. Thank you. A very short second question, if I may, regarding what Yaron was saying. I mean, you had two collectives, 500 and 300. And you showed us a few polymorphisms. Were these polymorphisms, let's say, known in patients without immune thyroid disease? Did they look, did you look that these were already published or these are new polymorphisms? So I did not find them in other studies, but again, so the study was under power. So most probably we could not, well, we have to look further, but we could not find a direct association with thyroid diseases. Okay, thank you. I have one more question. So, there's been a controversy for many years whether antiviral drugs actually help get Graves' disease into remission. Now, you're showing that T4 activates or at least increases the number of B cells, so I'm wondering what's your thought. Maybe it's not their effect on the immune system, but through lowering the T4, you're actually increasing the chance of remission. I think there are also some publications that have suggested that the effect might be, in fact, the lowering of the T4, which could be possible. I think what's very difficult in these kind of models is that the whole regulation of the immunity is completely different when you look at the pathogenic condition than in healthy volunteers, but it could be the fact, yeah. Okay, thank you very much again. All right, thank you. So, we're gonna continue with our international theme and welcome Dr. Graciela Gramacchi. She is coming from Argentina, just arrived, as I understand, and might be a little jet-lagged. She's the director of the Biomedical Research Institute at Catholic University of Argentina and the National Research Council of Argentina. She's also the director of the Neuromodulation and Molecular Oncology Lab at Biomed. Her research interests focus on understanding the neuroendocrine regulation of the immune system and its impact on cancer pathology, searching for molecular targets for diagnostic and therapeutic tools. So, welcome. Her talk today is titled Thyroid Hormones, T-Up Immune Function, Shaping the Antitumor Response. Welcome. Thank you, all of you, and thank you for the nice introduction. And I want to special thanks the organizers of this amazing congress to invite me here and to show you some of our results. Well, as everybody knows, stress triggers the growth of tumors, so many cancer patients use mindfulness or meditation interventions to promote emotional and physical well-being by reducing their psychological stress. And this, and very well introduced by Dr. Netea Mayer, it's rational in the fine balance that exists in the body between the neuroendocrine and the immune system that is necessary to keep health and overall homeostasis. And the most studied interaction between the neuroendocrine and the immune system is that of HPA axis, in which stress, sustained stress, triggers the release of stress hormones, leading to a diminish in the function and number of immune cells. And I want you to go to the tumor microenvironment, because I will show how this neuroendocrine immune network affects tumor microenvironment, to show that being this adrenaline and cortisol, this catecholamines, these stress hormones, solvable factors of tumor microenvironment, they are able not only to diminish the anti-tumor immune response, but also to exert a direct action on cancerous cells and blood vessels, leading to tumor growth and angiogenesis. So what my lab was working in several years ago, was to try to answer whether thyroid hormones are players in these neuroendocrine immune networks, affecting cancer growth and dissemination. The first question we want to answer was if thyroid hormones mediate regulation of immunity. And several experimental evidence point that thyroid hormones do exert a direct action on immune cells, as thyroid hormone receptors and their signaling pathways are present in cells of the immune system. We look for these receptors in T lymphocytes, as they are major components of the adaptive immune cells, as it was said in this session, activating different effector functions, like cytotoxic T cells that are positive for CD8 marker, that direct kill infected or tumor host cells, or T helper cells, being CD4 positive, that produce cytokines and activate other immune cells, but also as T cells can regulate immune responses, like T regulatory immunosuppressor cells can do. And in these cells, we do not only evaluate the presence of the nuclear classical thyroid hormone receptors, that while binding T3 activates gene transcription, but also of the membrane receptor, described by the group of Dr. Davis, to be near the RGD binding domain of the integrin alpha B beta 3, that while binding T3 or T4 with different affinities, depending on the cell type, they are able to activate intrasignals, that's leading to the activation of transcription factors and of gene transcription. When murine T lymphocytes purified from lymph nodes, and in human lymphocytes purified from peripheral blood, we found that these cells express both tier alpha and tier beta nuclear receptors. However, the levels of alpha B beta 3 integrin were very low or undetectable in resting murine T lymphocytes, as well as in T lymphocytes purified from human peripheral blood. Nevertheless, thyroid hormones, despite not inducing T cell proliferation per se, they were able to increase in a dose-dependent manner. Oh, I have to point there. The proliferation of T cells activated by mitogens. The other experimental evidence is that frequently, thyroid hormones fluctuations, physiological or pathological fluctuations of thyroid hormones can lead to alterations in immunity. In general, hyperthyroidism, several scientific work shows that hyperthyroidism leads to immunosuppression, while hyperthyroidism leads to increased immune responses. Please don't take this on advice to become hyperthyroid, to fight for infections. But I will show you, for example, the upregulation of thyroid hormones. High levels of thyroid hormones can upregulate, for example, the function of dendritic cells as they can lead to the maturation of dendritic cells. I will show you our results on T lymphocytes by using experimental models of animals with different thyroid status. L-thyroid mice, hyperthyroid treated with T4, or hypothyroid treated with PTU, the antithyroidation PTU. And in these animals, we first evaluate what happens with T cell proliferation when stimulated by antigen-specific or polyclonal activation. Here, I'm showing you the results of hyperthyroid lymphocytes, lymphocytes obtained from hyperthyroid mice, that have a higher proliferation against an allergenic stimulus, an antigen-specific, in mixed lymphocyte cultures. And we can see that hyperthyroidism leads to an increase in the proliferative response, while hypothyroidism decreases with respect to control. Similar findings were observed when we use a polyclonal activation by T cell-selective mitogens, or by anti-CD3, anti-CD28 antibodies, triggering the T cell receptor complex. In these cases, oh, sorry, hyperthyroidism leads to an increase, proliferative response, hypothyroidism decreases, and in some cases, we also use a reversion of PTU treatment by T3 administrations, and we found that we can revert the lower response to polyclonal activation. This was accompanied also by the release of key cytokines, like interferon gamma and EL2, interferon gamma and important cytokines in antitumor immunity, and hyperthyroid mice display a higher release of these hormones, of these cytokines, while lower release was found in hypothyroid conditions. And so we want to know whether this was associated to the intracellular signals that are commonly activated upon polyclonal activation in T cells, and I will look for, I'm showing here, perkinase that is activated, is phosphorylated upon T cell activation, and I'm showing here the results for hypothyroid conditions, and we found that PTU was able to impair ERG phosphorylation, and that this was reverted by the T3 treatment. So perhaps the differences we see in hyperthyroid and hypothyroid lymphocytes activation would be related to intracellular signals normally triggered during their activation. The other question we tried to answer was if thyroid status was able to alter the balance between lymphocytes and lymphocytes of populations. We found no difference between B and T cell subtypes, and also we do not find any difference between CD4 and CD8 frequencies in euthyroid, hypothyroid, PTU treated, and PTU treated plus T3 lymphocytes. However, when we look for the frequency of CD4, CD25, FOXP3, teregulatory cells, we found that hypothyroid conditions led to an increased interregulatory frequency, and this was accompanied by an increased interregulatory activity, as Tregs purified by cell sorting from the spleens of hypothyroid mice induced a higher suppression on both CD4 and CD8 responder cells. We want to know whether this hypothyroid microenvironment was able to alter the differentiation of naivety cells to teregulatory cells, and so we purified ten naivety cells from the spleens of euthyroid and hypothyroid mice and differentiate them ex vivo with TGF-beta and interleukin-2, two cytokines that lead to teregulatory differentiation, and we found that hypothyroid conditions were able to increase the induction of teregulatory cells. To further demonstrate the role of tyrodormins in these effects, we then evaluate T-naive, euthyroid T-naive differentiation in the presence of tyrodormins, and found that both T3 and T4 in physiological concentrations, as both hormones added together as occurs in circulations, were able to impair the differentiation of teregulatory cells. So, T-lymphocyte physiology is centered by the fluctuation of tyrodaxis in vivo. Hypothyroidism upregulates, while hypothyroidism downregulates the responses of T cells to antigen or polyclonal activation, and hypothyroid-driven immunosuppression is related to the high frequency and activity of teregulatory cells. These results highlight the role of tyrodormins in upregulated T-lymphocyte physiology and provide a rational basis for evaluating the thyroid status and the restoration of euthyroidism in immunocompromised patients. Taking this into consideration and thinking that tyrodormins are hormonal factors of tumor microenvironment, it's logic to think that they would affect the immune component of tumor microenvironment. But also, what we want to know was if tyrodormins are able to direct affect cancer growth and spreading as seen with stress hormones. To the same, we work with T-lymphoma cells. We look what happens in T-normal, so then we're going to look what happens in T-tumor cells, and we look for the direct tyrodormin's actions on murine and human T-cell lymphoma cells. I'm showing here results in human TCLs as they are a very aggressive lymphoproliferative disorder that can originate from immature and mature T-lymphocytes and they are very heterogeneous, so we have to use nine different cell lines to cover the different subtypes of T-cell lymphoma in humans, and we found that T-lymphoma cells only show TR-alpha, but not TR-beta receptors as normal cells do, and also they overexpress both integrin-alpha, b-beta three genes as well as the integrin dimer, so overexpression of tyrodormin receptors may suggest an enhanced response to tyrodormin's effects of malignant T-cells, and these hormones have functional consequences on T-cell lymphoma cells as both T3 and T4, or both hormones added together, I'm showing here results of both hormones added together as in circulation in physiological concentrations as in circulation, are able to increase the proliferation different to what happened in normal cells where tyrodormins per se do not have any action, and here they do increase the proliferation of T-cell lymphoma cells. And moreover, these effects were also observed when using tyrodormins coupled to agarose and so being cell impermeable, only able to trigger the integrin membrane receptor. To further demonstrate that the integrin was the receptor involved in these effects of tyrodormin actions, we used three-dimensional cultures and the selective integrin alpha-B beta-3 blocker, silangetide, and see that the increment in tyrodormin's proliferation was impaired by silangetide. We also performed interfering RNA experiments in T-cell cells, downregulating the integrin alpha-B and beta-3 and found similar results. And moreover, when we studied the transcriptional programs mediated by tyrodormins in T-cell cells, via the integrin alpha-B beta-3 receptors using agarose coupled tyrodormins and performing RNA sequencing analysis, we found that several proliferation and survival genes were upregulated, but also some angiogenesis-related genes were upregulated, like BGF. We not only found that increased release of BGF from T-cell cells, but also we found, to give this a clinical significance, a positive correlation between integrin alpha-B and beta genes and BGF-A and BGF-B in RNA microarrays from TCL primary samples of peripheral T-cell lymphoma patients. We also studied tyrodormin effects on breast cancer cell line, on a triple negative breast cancer cell line, as we will use this cell line in vivo studies, but several authors showed that tyrodormins can directly stimulate the proliferation of breast cancer cells. We looked for the action of tyrodormins on a 41 triple negative breast cancer, murine triple negative breast cancer cell line, and we found that only supra-physiologic concentrations of tyrodormins were able to increase the proliferation of these breast cancer cells. So tyrodormins can directly affect tumor growth and angiogenesis. The question was then, which was their effect on the immune component of tumor microenvironment? And to this end, we have to go in vivo. And we found that several case control and population-based studies show that both hyperthyroidism and hypothyroidism influences cancer development and progression. However, results are controversial and seem to depend on the sample population and on the tumor type. In none of these studies, the effect that tyrodormins can exert on anti-tumor immunity was studied. So we go back to our experimental models of mice with different thyroid status and inoculate them with syngeneic T-cell lymphoma cells, L4 cells in C57 black mice, or 41 cells, the ones that we have checked in vitro, in BALC-C mice. And in these tumor-bearing mice, we evaluate tumor growth, metastatic spreading, metastatic dismenation, and also the anti-tumor immune response. What we found was that hyperthyroid tumor-bearing mouse, both for T-cell lymphomas or breast cancer tumors, showed largest tumors with the largest volume and the largest weight. In T-cell lymphoma cells, we have to end experiments at 15 days because this is a very aggressive model. And for ethical reasons, we have to end the experiments here. And we did not found, at this time point, differences between the apothyroid and authyroid group. However, at the 35 post-injection of breast cancer cells, we also found decreased volume of breast cancer tumors in hypothyroid conditions. Then we want to look for tumor dissemination. In the tumor lymphoma, in the TCL lymphoma, the TCL tumor model, we were not able to find spontaneous metastasis at day 14, 15, but we performed the experimental metastasis test. It means to inoculate tumor cells intravenously and then evaluate how they extravasate and colonize distinct organs. And we found that these cells are able to colonize liver and kidney, and despite having the highest tumors, hypothyroid, a fewer number of hypothyroid mice display liver metastasis. And also, hypothyroid mice display a greater number of renal metastasis, thus showing that hypothyroidism favors TCL cells dissemination. When we look what happened in breast cancer tumors, also we found spontaneous metastasis in lung at 35 days post-injection, and found that hypothyroid mice displays a greater number of lung metastasis. In the experimental metastasis test, we also found that hypothyroid mice display lower number of metastasis as well. So, which could account for these differences between tumor growth and this controversial resulting tumor growth and tumor dissemination? We look to see what happened with tumor, with anti-tumor immunity. First, we evaluate what happened with the lymphocyte infiltrates to tumors. I'm showing here results for breast cancer, but similar results account for TCL model. And we found that hypothyroid mice display a greater infiltration of lymphocytes in the tumors, being maximum at 35 days post-injection of breast cancer cells in the breast cancer model, when we found the lowest tumors in hypothyroid conditions. And this was accompanied by an increment in the CD8-activated lymphocytes and a greater production of the anti-tumor cytokine interferon gamma. So, this increased cytotoxic environment of hypothyroid tumors would explain why they are smaller than the other two groups. And in hypothyroid conditions, probably the low number of infiltrating lymphocytes than hypothyroid conditions, together with the direct effects that we found of thyroid hormones, supraphysiological concentrations of thyroid hormones on tumor cells would explain why these tumors are greater, are largest. But surprisingly, in the breast cancer model, we also found that condition media from tumor explants display high amounts of CCL2 chemokine, or MCP1, that is a chemokine related to cancer invasiveness and metastasis, homing, for example, stromal components, mesenchymal stem cells that were shown to be immunosuppressive in breast cancer tumors. And this can give us a clue why these animals have a greater dissemination in the hypothyroid conditions. Then we want to look, then we go to look what happens with the spleen content as it reflects systemic changes in circulating lymphocytes. And we here study the lymphoid compartment, myeloid-derived suppressor cell from the myeloid compartment, and in breast cancer tumors, we also evaluate mesenchymal stem cells homing to tumors. I will show this picture to summarize our main findings. We found that hypothyroid mice display greater amount and activity of natural killer cells, splenic natural killer cells, and also of activated T-cytotoxic cells. And the importance of these cytotoxic cells in tumor dissemination was revealed as downregulation of CD8 cells in hypothyroid mice bearing solid T-cell lymphomas tumor was able to induce an increment of kidney metastasis respect to mice untreated with the anti-CD8 antibody. This was also accompanied by a decrease in myeloid-derived suppressor cells. So this systemic, the systemic study of immune cells that are similar to those found in tumor microbiome would explain why the lower dissemination in hypothyroid conditions. On the contrary, in hypothyroid mice, we found decreased number and activity of T-cytotoxic cells with an increment in T-regulatory and myeloid-derived suppressor cells that would explain why the greater dissemination in these conditions. And also when studying the homing of mesenchymal stem cells to tumors in animals with different thyroid status and to the same, we inoculate pre-stained mesenchymal stem cells at day 28 of tumor development and then at day 35 when we found the differences in the volumes, we look ex vivo for their homing to tumors. We found that hypothyroid tumors having lower CCL2 content also displays a lower homing of mesenchymal stem cells that can, for example, differentiate to tumor-associated fibroblasts leading to immunosuppression. So thyroid hormones through non-canonical actions are able to increase tumor growth and angiogenesis but they are also able to activate antitumor immunity, systemic antitumor immunity to control tumor spreading. As you can remember, I said that normal T-cell express low levels of the integrin alpha-B-beta-3, probably these actions on T-cells involved in antitumor immunity would be related to the actions of thyroid hormones on nuclear receptors. So the use of a specific inhibitor of the integrin alpha-B-beta-3 would lead to a lower increase in tumor growth without affecting tumor autoimmunity. And in fact, is what we've seen here in this model of PDX, T-cell cells from human patients with two different T-cell informal subtypes inoculated in immunodeficient mice, treated or not with silangetide, the integrin inhibitor, showed that tumors were smaller in the presence of silangetide. So hormones exert a real yin-yang in cancer. Being important regulators of cancer development and progression, directly regulating tumor cell biology but also regulating the antitumor immune response. And these results provide a therapeutic potential target for cancer treatment, the membrane receptor for thyroid hormones, integrin alpha-B-beta-3, and highlight the importance of monitoring thyroid axis in patients with cancer. These are my collaborators, the lab collaborators, external collaborators, and thank you very much for your attention. Thank you. Okay, the floor is open. We have a question coming. Thank you so much for this interesting talk that made me a little bit unable to translate it clinically. So if I want to ask you your opinion since you've done all these experiments. So we have two conditions, right? We have the thyroid cancer patient that we use supraphysiologic T4 to suppress the TSH. Does this theoretically increase metastasis? And in patients with, for example, on checkpoint inhibitors or immunotherapy, what is the optimal thyroid status? So is it good to give T4? So do we have a TSH level that is permissive? Is it individualized? What do you say about that? Thank you so much for your answer. Well, we are releasing, sooner it's in press, a manuscript showing, for example, that many drugs in chemotherapy induce thyroid hormones, adverse effects, as a side effect, they induce hypothyroidism. And so we gave them thyroid hormones to restore thyroid conditions that are very important for keeping metabolism and to keep the anti-tumor immune response so we cannot get out of those hormones that will probably exert a direct action inducing the proliferation and inducing angiogenesis in tumor cells. So what we can do is to use something that blockade the integrin receptors, or like what Dr. Herbert's proposed, is to get hypothyroxinemic condition, only having normal T3 values to have a good anti-tumor immune response and having not so important direct actions on tumor cells. Most tumor cells, it's not the case of lymphomas, but in breast cancer, T4 is more critical for direct actions of thyroid hormones on cancer cells. So in these conditions, if you lower T4 values but keep a good level of T3, it would be the best because you will have no effect, the adverse direct action, but you will not have the problem with anti-tumor immunity, especially if anti-tumor immunity is related to nuclear thyroid hormone receptors actions. So you will have T3 that is the most important. Thank you so much. This is really very, very important for us to use clinically. And the next question is how long, what's the duration of hyper or hypothyroidism that is needed to exert these actions? Because based on that, we would know how often we need to intervene and maybe guide oncology colleagues to check early to prevent prolonged states, or maybe what is the timeframe? Well, we need 30 days for hypothyroidism to develop. And in these 30 days, we just see the effects in immunity. And we just see, we always insert, we inoculate the tumors once the animals have the hypothyroid condition. So when the hypothyroid condition is already established, you will have this yin-yang action of thyroid hormones. Yes, it's the direct action of, in 24 hours we have six hours in the transcriptional actions for the integrin, several RNA sequencing, six hours are already okay for having the increment in angiogenic and pro-survival and pro-proliferative actions of thyroid hormones on those genes. No, thank you for your question. Yeah, I'll steal your spot for a second. I want to tie the first talk with your talk. So there is data to suggest, I think actually Dr. Ryder published, that if you get severe thyrotoxicosis with immune checkpoints, the outcomes of the cancer are better. Most people assume that that shows that there is better response immunologically to them, but the question is, these patients probably had much more severe thyrotoxicosis, and do you think that has to do with better responses to immune checkpoints? Well, something similar happens with direct target with thyroxine inhibitors. For example, sunitinib is one of the thyroxine kinase inhibitors that produce hypothyroidism, and if immune checkpoints induce hypothyroidism, it was shown that in those cases, the effects on cancer were lower. That you have the problem of immunity. Well, with immune checkpoints, you will have that solved. Thank you. The whole talk is very interesting, and there's a divide there between the whole animal experiments and then the cellular level experiments, and I'm always wondering, in the whole animal experiments, what's the role of TSH? How do you control for a change in TSH influencing systemic value? Yes, I do not show these results, but your question is, I have no time to add them to my talk, but I have submitted the results for T-Rex cells, and in this, we have also studied what happened in transgenic mice overexpressing TRH gene. In these mice, TRH, as well as TSH, upregulate the immune response, and in hypothyroid conditions, you have high levels of TSH and low levels of thyroid hormones, and transgenic mice have upregulated cytokine production, upregulated T cell proliferation. The contrary occurs in hypothyroid conditions, and also, hypothyroid conditions are reverted by T3 administration, so it's very probable that there are circulating levels of thyroid hormones, the one that, in this non-autoimmune condition, because I don't want autoimmunity, because this, per se, will affect immune function. In these non-autoimmune models, like happens in congenital hypothyroidism, we found that the actions are exerted by circulating levels of thyroid hormones. But you haven't tried adding back TSH to your hyperthyroid. TSH, as well as T3 and T4, do not exert a direct action on T lymphocytes, but when you culture in vitro with those hormones, you found an upregulation of polyclonal activation of T cells. Very good, thank you very much for your response. Thank you very much for a very nice presentation. So I have two observations. First of all, I would like to mention that we have some data to link your findings of high T regulatory cells in relation with hypothyroidism. In a human model, so a few years ago, we have investigated together with colleagues from India, a cohort of 850 people who are living in households, actually, of patients with TB. And we have looked whether, so which factors influence or are related to developing active TB in these households. And it was very striking that the ones that developed TB, active TB, indeed had also higher T regulatory cells in circulation, but also lower 3T4 level within the normal range. So again, linking your findings to my findings, it was very interesting to see those. And the second one is that I think in light of your presentation, it's very interesting to look critically at our studies also in the effect of immunity in thyroid cancer, because our thyroid cancer patients are, by definition, patients who have no T3 and a lot of T4, because we give them substitution with T4 alone, the large majority of the patients. And the animal models in which we study thyroid cancer, at least the immune-competent models that Dr. Ryder also used, that is a hypothyroid model. As far as I know. Thank you very much for this question, because, well, we are not at a low, at a, how do you say, the final step of cancer, because at that time, we have differences in thyroid hormone levels, but at the time points I have shown here, we checked the thyroid hormone status of all of our animals, and we look for T3, T4, because T3, T4 is not very good, we do not found very good, very, with a lot of intra-error determination in our measurements in the laboratory, so we look for T3, T4, TSH in circulation, and in that, we also study the encephalic production of TRH, and they were, hypothyroid have high levels of TRH, high levels of TSH, and low levels of T3 and T4, the contrary to hyperthyroid conditions, and all the time, till the end of experiment. But it's right, in the last, when the severe disease, you have also a decline in T3 levels, yes. And this will especially affect, I think, thyroid and anti-tumor immunity. All right, well, congratulations to all three speakers, this was really a fascinating and really helpful talk. Thank you to all the audience members.

immune checkpoint inhibitors

immune checkpoint inhibitor thyroiditis

IL-17

mouse model

thyroid autoimmunity

immune modulating therapies

autoimmune thyroid disease

hormones

T-cell populations

thyroid hormones

cancer pathology

tumor growth

euthyroidism