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Masterarbeit Universität Innsbruck 2024

Low-grade inflammation is closely linked to obesity and obesity-related comorbidities; therefore, immune cells have become an important topic in obesity research. Here, we performed a deep phenotypic characterization of circulating T cells in people with obesity, using flow cytometry. Forty-one individuals with obesity (OB) and clinical criteria for bariatric surgery were enrolled in this study. We identified and quantified 44 different circulating T cell subsets and assessed their activation status and the expression of immune-checkpoint molecules, immediately before (T1) and 7-18 months after (T2) the bariatric surgery. Twelve age- and sex-matched healthy individuals (nOB) were also recruited. The OB participants showed higher leukocyte counts and a higher percentage of neutrophils. The percentage of circulating Th1 cells were negatively correlated to HbA1c and insulin levels. OB Th1 cells displayed a higher activation status and lower PD-1 expression. The percentage of Th17 and Th1/17 cells were increased in OB, whereas the CD4+ Tregs' percentage was decreased. Interestingly, a higher proportion of OB CD4+ Tregs were polarized toward Th1- and Th1/17-like cells and expressed higher levels of CCR5. Bariatric surgery induced the recovery of CD4+ Treg cell levels and the expansion and activation of Tfh and B cells. Our results show alterations in the distribution and phenotype of circulating T cells from OB people, including activation markers and immune-checkpoint proteins, demonstrating that different metabolic profiles are associated to distinct immune profiles, and both are modulated by bariatric surgery.

Inflammatory bowel disease (IBD) is an umbrella term for the chronic immune-mediated idiopathic inflammation of the gastrointestinal tract, manifesting as Crohn's disease (CD) or ulcerative colitis (UC). IBD is characterized by exacerbated innate and adaptive immunity in the gut in association with microbiota dysbiosis and the disruption of the intestinal barrier, resulting in increased bacterial exposure. In response to signals from microorganisms and damaged tissue, innate immune cells produce inflammatory cytokines and factors that stimulate T and B cells of the adaptive immune system, and a prominent characteristic of IBD patients is the accumulation of inflammatory T-cells and their proinflammatory-associated cytokines in intestinal tissue. Upon antigen recognition and activation, CD4 T-cells differentiate towards a range of distinct phenotypes: T helper(h)1, Th2, Th9, Th17, Th22, T follicular helper (Tfh), and several types of T-regulatory cells (Treg). T-cells are generated according to and adapt to microenvironmental conditions and participate in a complex network of interactions among other immune cells that modulate the further progression of IBD. This review examines the role of the CD4 T-cells most relevant to IBD, highlighting how these cells adapt to the environment and interact with other cell populations to promote or inhibit the development of IBD., Instituto de Salud Carlos III, European Regional Development Fund, Depto. de Inmunología, Oftalmología y ORL, Fac. de Medicina, TRUE, pub

Interleukin-1 (IL-1) is a pleotropic cytokine with complex biology. Because of its potent pro-inflammatory properties, several mechanisms have evolved to regulate its systemic and local effects. At the tissue level, a non-signaling decoy receptor, Interleukin-1 receptor 2 (IL-1R2) plays a major role in attenuating IL-1 mediated signaling. IL-1R2 is highly expressed on regulatory T cells (Tregs) that infiltrate both mouse and human tumors. Tregs respond to cytokines in their local environment to modulate their activity. IL-1 signaling is thought to regulate Treg effector function in the context of Type 1 and Type 17 inflammation and has primarily been characterized in autoimmune diseases. However, the function of IL-1 in tumor Treg biology is relatively unknown. Here, we show that a subset of highly activated Tregs induce IL-1R2 expression upon migration into tumors. In contrast to myeloid cells, induction of IL-1R2 on Tregs was mediated by T cell receptor (TCR) engagement and not IL-1-mediated signaling. Signaling through the canonical IL-1 receptor (IL-1R1) activated Tregs to express a unique gene expression signature and IL-1R2 potently attenuated this activation module in tumors. Accordingly, deletion of IL-1R2 on Tregs resulted in a more suppressive tumor microenvironment and increased tumor growth. In translational studies, we developed a novel IL-1R2 depleting antibody which was highly selective for tumor infiltrating Tregs. Tumor Treg depletion using this approach potently inhibited tumor growth without generating systemic autoimmunity. Taken together, we define the biology of IL-1R2 expression on tumor Tregs and provide the foundation for a novel therapeutic strategy to augment anti-tumor immunity.

BackgroundProgressive age-associated change in frequencies and functional capacities of immune cells is known as immunosenescence. Despite data linking chronic environmental, physiological, and psychosocial stressors with accelerated aging, how stress contributes to immunosenesence is not well characterized.ObjectiveTo help delineate the contribution of cumulative physiological stress on immunosensence we assessed relationships between a composite measurement of cumulative physiological stress, reflecting the functioning of the hypothalamic-pituitary-adrenal axis, sympathetic nervous system, cardiovascular system, and metabolic processes, and lymphocyte changes typically affiliated with aging in a cohort of healthy volunteers ranging from 18 to 66 y.ResultsPhysiological stress load positively correlated with subject age in the study cohort and was significantly higher in adults 50-66 y compared to adults 18-33 y and 34-49 y. Using physiological stress load, we identified a significant age-dependent association between stress load and frequencies of circulating regulatory T lymphocytes (Tregs). Frequencies were higher in younger participants, but only in participants exhibiting low physiological stress load. As stress load increased, frequencies of Tregs decreased in young participants but were unchanged with increasing stress load in middle and older age individuals. Follow-up analysis of stress load components indicated lower circulating DHEA-S and higher urinary norepinephrine as the primary contributors to the effects of total stress load on Tregs. In addition, we identified age-independent inverse associations between stress load and frequencies of naïve Tregs and naïve CD4 T cells and positive associations between stress load and frequencies of memory Tregs and memory CD4 T cells. These associations were primarily driven by stress load components waist circumference, systolic and diastolic blood pressure, CRP, and HbA1c. In summary, our study results suggest th

BACKGROUND/AIM: Regulatory T cells (Treg) play a crucial role in maintaining immune tolerance and preventing autoimmune diseases. Recent data also indicate that type 1 regulatory T (Tr1) and regulatory B (Breg) cells play an inhibitory (i.e., protective) role in autoimmune diseases. Conventional synthetic disease-modifying antirheumatic drugs (csDMARD) are a first-line therapy for rheumatoid arthritis (RA), and our aim was to predict clinical responses of this treatment using immunophenotyping. MATERIALS AND METHODS: We first detected the presence of immune cells in fresh blood from 16 healthy controls (HC) and 26 patients with RA (14 drug-naive and 12 csDMARD-experienced). Then, we recorded immunophenotypic changes in 14 drug-naive RA (naive RA) patients prior to csDMARD treatment (i.e., day 0) and after receiving treatment for 6 months. The observed changes were also compared with other clinical indicators, including the presence of anti-citrullinated peptide antibodies (anti-CCP), rheumatoid factor (RF) levels, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. RESULTS: Naive RA patients had significantly lower Tregs than HC and csDMARD-experienced patients (both p<0.0001) and the number of Tregs correlated with the diagnosis of RA and therapeutic efficacy of csDMARD treatment. Furthermore, lower baseline levels of Treg, memory Treg, Tr1, and higher PD-1+ Marginal B, Breg cells were significantly associated with decreased development of the 28-joint Disease Activity Score (DAS28) (all p<0.05), revealing better medical response. Multiple regression and principal component analysis identified Treg, Tr1, and Breg as potential predictors of csDMARD responses (Area under curve: 0.9; Accuracy: 92.86%). Furthermore, elevated Treg, Tr1, and Breg cells were associated with decreased DAS28, ESR, and CRP (all p<0.05); changes in Treg and Breg cell expression were also more pronounced among double negative anti-CCP and RF in RA patients wi

Introduction: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which is highly expressed in mucosal tissues - by epithelial cells and immune cells such as Th17 CD4+ and T reg- ulatory cells (Treg). Besides its function of clearing environmental pollutants from the body, it was also revealed that AhR has immunoregulatory effects, thus becoming a potential therapeutic target for mod- ulating the immune response. For that purpose we tested a novel synthetic AhR modulator under the code name C43. Methods: CYP1A1 (downstream effector of AhR) activation was tested by the EROD assay. Sort-purified CD4+ cells from mesenteric lymph nodes (MLN) were treated with C43 for 24 h. Zebrafish embryos were used to test the toxicity of C43. Male C57BL/6 mice orally received C43 (10 mg/kg) for 5 consecutive days, after which MLN were harvested. Phenotype and function of the cells were analyzed by flow cytometry. Results: C43 showed mild AhR agonistic activity. After treating the sort-purified CD4+ cells with C43, there was a shift in the Th17/Treg ratio in favour of the latter. C43 showed no signs of toxicity when tested on zebrafish embryos. MLN cells from mice that received C43 revealed a shift in the Th1/Treg ratio in favour of Tregs, with a documented rise of the portion of Tregs that expressed CYP1A1 in comparison with the control group of mice. Conclusion: C43 can modulate the immune response through the intestine by promoting the im- munosuppressive Treg population.

New-generation mRNA and adenovirus vectored vaccines against SARS-CoV-2 spike protein are endowed with immunogenic, inflammatory and immunomodulatory properties. Recently, BioNTech developed a noninflammatory tolerogenic mRNA vaccine (MOGm1 psi) that induces in mice robust expansion of antigen-specific regulatory T (Treg) cells. The Pfizer/BioNTech BNT162b2 mRNA vaccine against SARS-CoV-2 is identical to MOGm1 psi except for the lipid carrier, which differs for containing lipid nanoparticles rather than lipoplex. Here we report that vaccination with BNT162b2 led to an increase in the frequency and absolute count of CD4(pos)CD25(high)CD127(low) putative Treg cells; in sharp contrast, vaccination with the adenovirus-vectored ChAdOx1 nCoV-19 vaccine led to a significant decrease of CD4(pos)CD25(high) cells. This pilot study is very preliminary, suffers from important limitations and, frustratingly, very hardly can be refined in Italy because of the >90% vaccination coverage. Thus, the provocative perspective that BNT162b2 and MOGm1 psi may share the capacity to promote expansion of Treg cells deserves confirmatory studies in other settings.

Autoimmune diseases affect many people worldwide. Current treatment modalities focus on the reduction of disease symptoms using anti-inflammatory drugs which can lead to side effects due to systemic immune suppression. Restoration of immune tolerance by down-regulating auto-reactive cells in an antigen-specific manner is currently the “holy grail” for the treatment of autoimmune diseases. A promising strategy is the use of nanoparticles that can deliver antigens to antigen-presenting cells which in turn can enhance antigen-specific regulatory T cells. In this review, we highlight some promising cell targets (e.g. liver sinusoidal endothelial cells and splenic marginal zone macrophages) for exploiting natural immune tolerance processes, and several strategies by which antigen-carrying nanoparticles can target these cells. We also discuss how nanoparticles carrying immunomodulators may be able to activate tolerance in other antigen-presenting cell types. Finally, we discuss some important aspects that must be taken into account when translating data from animal studies to patients.

Abstract Kidney transplantation is the preferred treatment for individuals with kidney failure offering improved quality and quantity of life. Despite significant advancements in short term graft survival, longer term survival rates have not improved greatly mediated in large by chronic antibody mediated rejection. Strategies to reduce the donor kidney antigenic load may translate to improved transplant survival. CD39 on the vascular endothelium and on circulating cells, in particular regulatory T cells (Treg), is upregulated in response to hypoxic stimuli and plays a critical role in regulating the immune response removing proinflammatory ATP and generating anti-inflammatory adenosine. Herein, the role of CD39 in reducing ischaemia–reperfusion injury (IRI) and on Treg within the context of kidney transplantation is reviewed.

Regulatory T cells (Tregs) are vital for regulating immune homeostasis and for preventing immunopathology. The immunosuppressive functions of Tregs have raised hope for their potential use in clinical applications. This thesis investigates features of Treg that may be relevant for their use in clinical applications and includes the first-in-man study of Treg infusion in clinical islet transplantation. In paper I we explored the immunological profile of haemodialysis patients and young healthy individuals; haemodialysis patients are a prospective target for adoptive Treg therapy following kidney transplantation. Flowcytometric gating strategies were analyzed to optimize the isolation of Tregs. We found that both groups presented a similar Treg profile, and sorting for CD25 in combination with CD127low was preferable in terms of Treg yield and purity. In paper II we compared the effects of mTOR inhibitors Azithromycin (AZM) and Rapamycin (RAP) on in vitro Tregs cultures, as compounds that improve the quality of Treg cultures are sought. While RAP can improve the purity of Treg expansions by suppressing the proliferation of non-Treg cells, the effects of AZM on Treg expansions had not been previously studied. We found that RAP induced a FoxP3+Helios + phenotype and increased suppressive function, but may also inhibit Treg expansion. In comparison, AZM promoted a FoxP3+ phenotype, but to a lesser extent than RAP and the AZM treated Tregs are possibly less suppressive. In Paper III we performed the first-in-man study of autologous Treg infusion in clinical allogenic pancreatic islet transplantation. Patients underwent leaukapheresis from which polyclonal Tregs were purified by magnetic-activated cell sorting (MACS) and cryopreserved until transplantation. The Tregs were thawed and co-infused with pancreatic islets in the portal vein. No negative effects were seen related to the Treg infusion, regardless of cell dose. This indicates the procedure is safe and feasible. Fut

The role of CD4 T regulatory cells is well established in peripheral tolerance and the pathogenesis of the murine model and human autoimmune diseases. CD4 T regulatory cells (CD4 Tregs) have been investigated in common variable immunodeficiency (CVID). Recently, additional members have been added to the club of regulatory lymphocytes. These include CD8 T regulatory (CD8 Tregs), B regulatory (Bregs), and T follicular helper regulatory (TFR) cells. There are accumulating data to suggest their roles in both human and experimental models of autoimmune disease. Their phenotypic characterization and mechanisms of immunoregulation are evolving. Patients with CVID may present or are associated with an increased frequency of autoimmunity and autoimmune diseases. In this review, we have primarily focused on the characteristics of CD4 Tregs and new players of the regulatory club and their changes in patients with CVID in relation to autoimmunity and emphasized the complexity of interplay among various regulatory lymphocytes. We suggest future careful investigations of phenotypic and functional regulatory lymphocytes in a large cohort of phenotypic and genotypically defined CVID patients to define their role in the pathogenesis of CVID and autoimmunity associated with CVID.

Expression of PD-1 ligands PD-L1 and PD-L2 on the surface of tumour and immune cells has led to the widespread success of checkpoint blockade immunotherapy, yet despite decades of research, knowledge of the underlying mechanisms tumour cells implement to avoid recognition by the immune system is still evolving. Research from our laboratory has validated that human Mo-DCs can increase surface expression of PD-L1 and PD-L2 in the presence of inflammatory stimuli. PD-L1 on APCs has been implicated in the conversion of conventional T cells into Tregs, however the role that PD-L2 may play in this system has not been explored. Furthermore, the mechanism by which tumours can elicit expression of PD-1 ligands on the surface of APCs, and the impact that this may have on infiltrating T cell phenotype and function is incompletely characterised. In this study, human Mo-DCs were generated and assessed for their ability to simultaneously upregulate PD-L1 and PD-L2 in response to stimulation with proinflammatory cytokines. It was discovered that IL-1 could elicit upregulation of both PD-1 ligands more effectively than TNF, and IFN-gamma could induce low levels of PD-L1 but was unable to modulate PD-L2 expression. Other members of the IL-1 superfamily did not have the same ability as IL-1, and it appeared that the cellular response was limited to Mo-DCs as lymphocytes and macrophages did not respond similarly. While attempting to reproduce these results in a more biologically relevant system, it was discovered that A375 melanoma cells were able to lose their ability to modulate PD-L1 and PD-L2 expression, however modification of the culture conditions to mimic features of the tumour microenvironment partially restored this function. Further analysis of the supernatants of tumour cell-lines resulted in the identification of an inhibitory factor which antagonised the IL-1beta-mediated PD-L1 and PD-L2 upregulation by Mo-DCs, and the efficacy of this factor could be modulated by cultur

The IL-17A inhibitor secukinumab is efficacious for the treatment of psoriasis. To better understand its mechanism of action, we investigated its impact on psoriatic lesions from 15 patients with moderate-to-severe plaque psoriasis undergoing secukinumab treatment. We characterized the longitudinal transcriptomic changes of whole lesional skin tissue as well as cutaneous CD4+ and CD8+ T effector cells and CD4+ T regulatory cells across 12 weeks of treatment. Secukinumab was clinically effective and reduced disease-associated overexpression of IL17A , IL17F, IL23A, IL23R, and IFNG in whole tissue as soon as 2 weeks after initiation of treatment. IL17A overexpression in T-cell subsets, primarily CD8+ T cells, was also reduced. Although secukinumab treatment resolved 89‒97% of psoriasis-associated expression differences in bulk tissue and T-cell subsets by week 12 of treatment, we observed expression differences involved in IFN signaling and metallothionein synthesis that remained unresolved at this time point as well as potential treatment-associated expression differences involved in IL-15 signaling. These changes were accompanied by shifts in broader immune cell composition on the basis of deconvolution of RNA-sequencing data. In conclusion, our study reveals several phenotypic and cellular changes within the lesion that underlie clinical improvement from secukinumab.

Tregs are able of suppressing tumor-specific effector cells, such as lymphocytes CD8+, CD4+ and Natural Killer cells. Different drugs, especially different schedules of administration, like metronomic chemotherapy (mCHT), seem to be able to increase anticancer immunity, by acting on downregulation of Tregs. Most of the data available regarding the immunomodulating effect of mCHT have been obtained with Cyclophosphamide (CTX). Aim of the present study was to explore the effects of mVRL and mCAPE administration, alone or in combination, on T cells. Observation of 13 metastatic breast cancer patients lasted controlling for 56 days, where Treg frequencies and function, spontaneous anti-tumor T-cell responses were monitored, as well as the clinical outcome. No depletion in Treg absolute numbers, or percentage of T lymphocytes, was observed. Only in 5 patients, a modest and transient depletion of Tregs was observed during the first 14 days of treatment. To better describe the effect on Tregs, we subsequently looked at the variations in Memory, Naïve and Activated Treg subpopulations: we observed a trend in reduction for memory Treg (Treg MEM) and an increase for Treg Naïve (Treg NAIVE) and Treg Activated (Treg ACT) components. We finally analyzed the average trend of Treg in the Treg depleted patients and non-depleted ones, without fiding any significant differences. The trend of the Treg MEM appeared different, showing a reduction during the first 14 days, followed by an increase at the levels before treatment at Day 56 in the group of depleted patients and a progressive substantial reduction in the group of non-depleted patients along the entire course of treatment. Opposed to the data known, treatment with mVRL w/o mCAPE did not show any effect on Tregs.

Regulatory CD4 + T cells (Treg) are essential for maintaining peripheral tolerance, preventing autoimmune diseases like dermatomyositis, and play a role in the anti-tumor response and prevention of graft versus host disease (graft versus host disease - GVHD) after allogeneic bone marrow transplantation. Using two markers associated with ATP metabolism, CD26 and CD39, and the CD45RA maturation marker, we separated the Treg into 5 distinct subpopulations, each representing a different stage of maturation. Micro-environmental factors dictate this maturation as well as the functions of these different sub-populations. Dermatomyositis is an autoimmune disease primarily affecting the skin and muscle. During this pathology, we observed a phenotypic modification of Treg with a decrease in CD25 expression and an accumulation of CD39+ populations. The study of immune polarization revealed the disappearance of circulating MAIT (mucosal-associated invariant T cells) cells, the existence of a similar lymphocyte profile between the classic form and the amyopathic form and a skin "type I interferon” polarization during anti-MDA5 associated with the presence of interferon kappa. Finally, we were interested in patients presenting with relapsing MDS or AML after allogeneic transplantation treated with azacytidine (AZA), a hypomethylating agent. We were able to show that the Treg phenotype was modified compared to ungrafted individuals, but that it did not differ between relapse and non-relapse. Treatment with AZA appears to have a different dose-dependent effect on Tregs, both in vivo and in vitro, resulting in particular in the acquisition of FOXP3 and CD39., Les cellules T CD4+ régulatrices (Treg) sont essentielles au maintien de la tolérance périphérique, la prévention des maladies auto-immunes comme la DM et jouent un rôle dans la réponse anti-tumorale et la prévention de la maladie du greffon contre l’hôte (graft versus host disease – GVHD), après allogreffe de moelle. Grâce à deux marqueurs associés au métabolisme de l’ATP, le CD26 et le CD39, et au marqueur de maturation CD45RA, nous avons séparé les Treg en 5 sous-populations distinctes, chacune représentant un stade de maturation différent. Les facteurs micro-environnementaux dictent cette maturation ainsi que les fonctions de ces différentes sous-populations. La DM est une maladie auto-immune affectant principalement la peau et le muscle. Au cours de cette pathologie, nous avons observé une modification phénotypique des Treg avec une diminution d’expression du CD25 et une accumulation de populations CD39+. L’étude de la polarisation immunitaire a révélé la disparition des cellules MAIT (mucosal-associated invariant T cells) circulantes, l’existence d’un profil lymphocytaire similaire entre la forme classique et la forme amyopathique et une polarisation cutanée « interféron de type I » importante au cours de la forme avec anticorps anti-MDA5 en lien avec la présence d’interféron kappa. Enfin, nous nous sommes intéressés à des patients présentant un SMD ou une LAM en rechute après allogreffe traités par azacytidine (AZA), un agent hypométhylant. Nous avons pu montrer que le phénotype des Treg était modifié par rapport à des individus non greffés mais qu’il ne différait pas entre rechuteurs et non rechuteurs. Le traitement par AZA semble exercer un effet sur les Treg différent en fonction de la dose, à la fois in vivo et in vitro, aboutissant notamment à l’acquisition du FOXP3 et du CD39., (MED - Sciences médicales) -- UCL, 2021

Peripheral nerve injury-induced neuropathic pain is a chronic and debilitating condition characterized by mechanical hypersensitivity. We previously identified microglial activation via release of colony-stimulating factor 1 (CSF1) from injured sensory neurons as a mechanism contributing to nerve injury-induced pain. Here, we show that intrathecal administration of CSF1, even in the absence of injury, is sufficient to induce pain behavior, but only in male mice. Transcriptional profiling and morphologic analyses after intrathecal CSF1 showed robust immune activation in male but not female microglia. CSF1 also induced marked expansion of lymphocytes within the spinal cord meninges, with preferential expansion of regulatory T-cells (Tregs) in female mice. Consistent with the hypothesis that Tregs actively suppress microglial activation in females, Treg deficient (Foxp3DTR) female mice showed increased CSF1-induced microglial activation and pain hypersensitivity equivalent to males. We conclude that sexual dimorphism in the contribution of microglia to pain results from Treg-mediated suppression of microglial activation and pain hypersensitivity in female mice.

The search of prognostic factors is a priority in diffuse large B-cell lymphoma (DLBCL) due to its aggressiveness. We have recently found that the level of circulating MDSCs is a good marker of survival in a translational study based on a trial (EudraCT Number: 2014-001620-29), using lenalidomide combined with R-GDP (rituximab plus gemcitabine, cisplatin, and dexamethasone). Since Vitamin D is a known immunomodulator, we have studied blood levels of these cell populations comparing patients with deficit of vitamin D levels (<15 ng/mL with those with normal levels >15 ng/mL. Mann–Whitney U test was used to compare cells distributions between groups, Wilcoxon test to compare cells distribution at different times and Spearman test to measure the association between cell populations. Patients with vitamin D deficit maintained the increased level of immune suppressor cells, whereas we observed a depletion of all immune suppressor cells in patients with normal vitamin D levels. In conclusion, we have confirmed the importance of vitamin D in the response to treatment in R/R DLBCL, suggesting that vitamin D deficit may be involved in the immune deficit of these patients, and thus, vitamin D supplementation in these patients may help to obtain a better response, warranting further investigation.

Corneal transplantation (CT) is the most frequent type of solid organ transplant (SOT) performed worldwide. Unfortunately, immunological rejection is the primary cause of graft failure for CT and therefore advances in immune regulation to induce tolerance remains an unmet medical need. Recently, our work and others in pre-clinical studies found that cyclophosphamide (Cy) administered after ("post-transplant," PTCy) hematopoietic stem cell transplantation (HSCT), i.e., liquid transplants is effective for graft vs. host disease prophylaxis and enhances overall survival. Importantly, within the past 10 years, PTCy has been widely adopted for clinical HSCT and the results at many centers have been extremely encouraging. The present studies found that Cy can be effectively employed to prolong the survival of SOT, specifically mouse corneal allografts. The results demonstrated that the timing of PTCy administration is critical for these CT and distinct from the kinetics employed following allogeneic HSCT. PTCy was observed to interfere with neovascularization, a process critically associated with immune rejection of corneal tissue that ensues following the loss of ocular "immune privilege." PTCy has the potential to delete or directly suppress allo-reactive T cells and treatment here was shown to diminish T cell rejection responses. These PTCy doses were observed to spare significant levels of CD4+ FoxP3+ (Tregs) which were found to be functional and could readily receive stimulating signals leading to their in vivo expansion via TNFRSF25 and CD25 agonists. In total, we posit future studies can take advantage of Cy based platforms to generate combinatorial strategies for long-term tolerance induction.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may produce a systemic disease, the coronavirus disease-19 (COVID-19), with high morbidity and mortality. Even though we do not fully understand the interaction of innate and adaptive immunity in the control and complications of the viral infection, it is well recognized that SARS-CoV-2 induces an immunodepression that impairs the elimination of the virus and favors its rapid dissemination in the organism. Even less is known about the possible participation of inhibitory cells of the innate immune system, such as the myeloid-derived suppressor cells (MDSCs), or the adaptive immune system, such as the T regulatory cells (Tregs). That is why we aimed to study blood levels of MDSCs, as well as lymphocyte subpopulations, including Tregs, and activated (OX-40+) and inhibited (PD-1) T lymphocytes in patients with mild COVID-19 in comparison with data obtained from control donors. We have found that 20 hospitalized patients with COVID-19 and no health history of immunosuppression had a significant increase in the number of peripheral monocytic MDSCs (M-MDSC), but a decrease in Tregs, as well as an increase in the number of inhibited or exhausted T cells, whereas the number of activated T cells was significantly decreased compared with that from 20 healthy controls. Moreover, there was a significant negative correlation (r = 0.496) between the number of M-MDSC and the number of activated T cells. Therefore, M-MDSC rather than Tregs may contribute to the immunosuppression observed in patients with COVID-19.

Data observed a decrease of Treg percentages as well as the IL-10 production in response to the combination of TLR7lig-Pp3 plus TLR4lig-Pp3 compared to each TLRnlig-Pp3 in allergic patients

Data indicated significantly higher percentages of Treg cells and IL-10 production in allergic patient compared to tolerant controls when stimulated with both TLRnlig-Pp3. Moreover, these effects were higher compared to Pp3 peptide alone, TLRnlig as well as TLRnlig plus Pp3 only in allergic patients except with TLR4lig-Pp3 for the IL-10 production. Regarding the TLR4 activation, they can induce a low production of IL-10.

T cells form immunological synapses with professional antigen-presenting cells (APCs) resulting in T cell activation and the acquisition of peptide antigen-MHC (pMHC) complexes from the plasma membrane of the APC. They thus become APCs themselves. We investigate the functional outcome of T-T cell antigen presentation by CD4 T cells and find that the antigen-presenting T cells (Tpres) predominantly differentiate into regulatory T cells (Treg), whereas T cells that have been stimulated by Tpres cells predominantly differentiate into Th17 pro-inflammatory cells. Using mice deficient in pMHC uptake by T cells, we show that T-T antigen presentation is important for the development of experimental autoimmune encephalitis and Th17 cell differentiation in vivo. By varying the professional APC:T cell ratio, we can modulate Treg versus Th17 differentiation in vitro and in vivo, suggesting that T-T antigen presentation underlies proinflammatory responses in conditions of antigen scarcity.

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth., SCOPUS: ar.j, info:eu-repo/semantics/published

The cells of the immune system, particularly the T lymphocytes, have two main features that distinguish them from the cells of other tissues. They proliferate after activation and have the ability to move in tissues and organs. These characteristics compel them to develop metabolic plasticity in order to fulfil their immune function. This review focuses on the different known mechanisms that allow T cells to adapt their metabolism to the real-life circumstances they operate in, whether it is to exit quiescence, to differentiate into effector cells, or to participate in immune memory formation. Some of the metabolic adaptations to environmental variations that T cells are likely to undergo in their immune monitoring function are also discussed., SCOPUS: re.j, info:eu-repo/semantics/published

Selective IgA deficiency (SIgAD), characterized by a serum IgA level below 0.07 mg/ml, while displaying normal serum levels of IgM and IgG antibodies, is the most frequently occurring primary immunodeficiency that reveals itself after the first four years after birth. These individuals with SIgAD are for the majority healthy and even when they are identified they are usually not investigated further or followed up. However, recent studies show that newborns and young infants already display clinical manifestations of this condition due to aberrancies in their immune defense. Interestingly, there is a huge heterogeneity in the clinical symptoms of the affected individuals. More than 50% of the affected individuals do not have clinical symptoms, while the individuals that do show clinical symptoms can suffer from mild to severe infections, allergies and autoimmune diseases. However, the reason for this heterogeneity in the manifestation of clinical symptoms of the individuals with SIgAD is unknown. Therefore, this review focusses on the characteristics of innate immune system driving T-cell independent IgA production and providing a mechanism underlying the development of SIgAD. Thereby, we focus on some important genes, including TNFRSF13B (encoding TACI), associated with SIgAD and the involvement of epigenetics, which will cover the methylation degree of TNFRSF13B, and environmental factors, including the gut microbiota, in the development of SIgAD. Currently, no specific treatment for SIgAD exists and novel therapeutic strategies could be developed based on the discussed information.

Worldwide, the prevalence of allergies in young children, but also vitamin D deficiency during pregnancy and in newborns is rising. Vitamin D modulates the development and activity of the immune system and a low vitamin D status during pregnancy and in early life might be associated with an increased risk to develop an allergy during early childhood. This review studies the effects of vitamin D during gestation and early life, on allergy susceptibility in infants. The bioactive form of vitamin D, 1,25(OH)2D, inhibits maturation and results in immature dendritic cells that cause a decreased differentiation of naive T cells into effector T cells. Nevertheless, the development of regulatory T cells and the production of interleukin-10 was increased. Consequently, a more tolerogenic immune response developed against antigens. Secondly, binding of 1,25(OH)2D to epithelial cells induces the expression of tight junction proteins resulting in enhanced epithelial barrier function. Thirdly, 1,25(OH)2D increased the expression of anti-microbial peptides by epithelial cells that also promoted the defense mechanism against pathogens, by preventing an invasive penetration of pathogens. Immune intervention by vitamin D supplementation can mitigate the disease burden from asthma and allergy. In conclusion, our review indicates that a sufficient vitamin D status during gestation and early life can lower the susceptibility to develop an allergy in infants although there remains a need for more causal evidence.

Los pacientes trasplantados renales reciben tratamiento inmunosupresor para evitar el rechazo del órgano; se sabe que el riesgo de padecer cáncer es de 2 a 6 veces mayor que en la población general, siendo el cáncer de piel no melanoma el que requiere de una consideración especial. El objetivo principal de este trabajo es saber si existe relación entre los niveles de células TREG con la posterior aparición de carcinoma de células escamosas (CCE), para poder llevar a cabo medidas de prevención y diagnóstico precoz del mismo si fuesen necesarias., Grado en Medicina

The intestinal gut microbiota is essential for maintaining host health. Concerns have been raised about the possible connection between antibiotic use, causing microbiota disturbances, and the increase in allergic and autoimmune diseases observed during the last decades. To elucidate the putative connection between antibiotic use and immune regulation, we have assessed the effects of the antibiotic amoxicillin on immune regulation, protein uptake, and bacterial community structure in a Brown Norway rat model. Daily intra-gastric administration of amoxicillin resulted in an immediate and dramatic shift in fecal microbiota, characterized by a reduction of within sample (α) diversity, reduced variation between animals (β diversity), increased relative abundance of Bacteroidetes and Gammaproteobacteria, with concurrent reduction of Firmicutes, compared to a water control group. In the small intestine, amoxicillin also affected microbiota composition significantly, but in a different way than observed in feces. The small intestine of control animals was vastly dominated by Lactobacillus, but this genus was much less abundant in the amoxicillin group. Instead, multiple different genera expanded after amoxicillin administration, with high variation between individual animals, thus the small intestinal α and β diversity were higher in the amoxicillin group compared to controls. After 1 week of daily amoxicillin administration, total fecal IgA level, relative abundance of small intestinal regulatory T cells and goblet cell numbers were higher in the amoxicillin group compared to controls. Several bacterial genera, including Escherichia/Shigella, Klebsiella (Gammaproteobacteria), and Bifidobacterium, for which the relative abundance was higher in the small intestine in the amoxicillin group than in controls, were positively correlated with the fraction of small intestinal regulatory T cells. Despite of epidemiologic studies showing an association between early life antibio

Regulatory T cells (Treg) are a suppressive subset of helper T cells that controls immune responses using a variety of mechanisms. Despite initially being thought of as a homogenous population, Treg cells were recently found to adapt their function to their environment and acquire specialized phenotypes depending on their tissue of residence. The molecular mechanisms underlying this functional and tissular adaptation remain to be fully elucidated.In the course of this work, we studied the role of transcription factor c-Maf in the differentiation and the function of Treg cells. For this, we used a murine model invalidated for c-Maf specifically in Treg cells. Transcription factor c-Maf is preferentially expressed by intestinal Treg cells, particularly among the RORγt+ Treg subset, which controls immune responses directed towards the gut microbiota. We have shown that the differentiation of RORγt+ Treg cells results from the integration of multiple environmental signals, highlighting the plasticity of Treg specialization. Furthermore, we have shown that c-Maf is required for the differentiation of RORγt+ Treg cells as well as the expression of the anti-inflammatory cytokine IL-10 by intestinal Treg cells, thus endowing Treg cells with the ability to control homeostatic Th17 responses in the intestine. Mice deficient for c-Maf in Treg cells exhibit an exacerbated Th17 response and spontaneously develop colitis. However, c-Maf-deficient mice develop fewer polyps in a model of colitis-associated colon cancer. This suggests that c-Maf expression in Treg cells plays a beneficial role on intestinal homeostasis at steady state, but is detrimental in a tumoral context.Our results allow a better understanding of the mechanisms involved in the specialization of intestinal Treg cells. The knowledge of these mechanisms is crucial for the identification of new therapeutic targets in the context of inflammatory bowel disease and colon cancer., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Regulatory T (Treg) cells are a subset of adaptive immune cells known for their immunosuppressive functions. Emerging evidence demonstrates that Treg exist throughout the body in tissue-specific sub-populations with specialized functions. RNA-seq analysis performed on mice during systemic autoimmune inflammation revealed high expression levels of Nod-like receptor protein 3 (NLRP3) in gut-resident Treg cells. NLRP3 is part of well-characterized inflammasome commonly associated with innate immune cells, but is beginning to be appreciated in adaptive immune cells as well. To determine the role of NLRP3 in gut-resident Treg-mediated immunosuppression, we generated bone marrow chimeras (BMC) and found that NLRP3-deficiency in Treg cells does not contribute to Treg development or homeostasis, but does result in increased Teff production of IL-17A in the colon. Moreover, in an adoptive T cell transfer model of colitis, co-transfer of NLRP3KO Treg cells failed to rescue Rag-/- recipient mice from weight loss and led to increased Teff production of IFN- and IL-17A in the colon. Moreover, to gain further insights into the role of NLRP3 in Treg cells, in addition to the aforementioned studies with NLRP3KO Treg cells, we also generated a mouse model in which NLRP3 is constitutively-activated in a Treg-specific manner (Foxp3creNLRP3iCA). After immunologically challenging these mice via Citrobacter rodentium, we have shown that colonic Foxp3creNLRP3iCATreg cells exhibit enhanced control over Teff production of IFN- which could potentially impact bacterial clearance. Collectively, by taking both loss-of-function and gain-of-function approaches, our data reveals a pivotal role of NLRP3 in gut-associated Treg cells in maintaining intestinal homeostasis.

Regulatory T cells (Treg) play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg development and function are dependent on the transcription factor Foxp3. Although a large body of work has been devoted to dissecting the molecular mechanisms regulating Foxp3 expression, a systematic, genome-wide approach has not been elucidated. In this study, we performed genome-wide CRISPR loss-of-function screens to identify Foxp3 regulators in mouse primary Treg cells. The screen results not only confirmed a number of known Foxp3 regulators but also revealed many novel factors that control Foxp3 expression. Gene ontology analysis showed Foxp3 regulators were highly enriched in genes encoding subunits of the SWI/SNF nucleosome remodeling and SAGA chromatin modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc)BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Gene ablation or chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg function. Brd9 ablation compromised Treg function in inflammatory disease and tumor immunity. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes, indicating that Brd9 actively participates in the Foxp3-dependent transcriptional program. In summary, this work provides an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg function.

Regulatory T cells (Tregs) maintain immune homeostasis and prevent autoimmunity. Serine stimulates glutathione (GSH) synthesis and feeds into the one-carbon metabolic network (1CMet) essential for effector T cell (Teff) responses. However, serine’s functions, linkage to GSH, and role in stress responses in Tregs are unknown. Here, we show, using mice with Treg-specific ablation of the catalytic subunit of glutamate cysteine ligase ( Gclc), that GSH loss in Tregs alters serine import and synthesis and that the integrity of this feedback loop is critical for Treg suppressive capacity. Although Gclc ablation does not impair Treg differentiation, mutant mice exhibit severe autoimmunity and enhanced anti-tumor responses. Gclc-deficient Tregs show increased serine metabolism, mTOR activation, and proliferation but downregulated FoxP3. Limitation of cellular serine in vitro and in vivo restores FoxP3 expression and suppressive capacity of Gclc-deficient Tregs. Our work reveals an unexpected role for GSH in restricting serine availability to preserve Treg functionality.

Background There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear. Methods In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFN gamma for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumour by flowcytometry. Results We discovered that IFN gamma increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumour microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes. Conclusions Here we clarify for the first time an additional mechanism of anti-tumour effect-as exerted by anti-PD-1 antibody decreasing Treg- we anticipate that our findings will lead to the development of new methods for cancer treatment., Article, BMC CANCER. 20(1):25 (2020)

Epoxyeicosatrienoic acids (EET) and related epoxy fatty acids (EpFA) are endogenous anti-inflammatory compounds, which are converted by the soluble epoxide hydrolase (sEH) to dihydroxylethersatrienoic acids (DHETs) with lessened biological effects. Inhibition of sEH is used as a strategy to increase EET levels leading to lower inflammation. Rheumatoid arthritis is a chronic autoimmune disease that leads to destruction of joint tissues. This pathogenesis involves a complex interplay between the immune system, and environmental factors. Here, we investigate the effects of inhibiting sEH with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) on a collagen-induced arthritis model. The treatment with TPPU ameliorates hyperalgesia, edema, and decreases the expression of important pro-inflammatory cytokines of Th1 and Th17 profiles, while increasing Treg cells. Considering the challenges to control RA, this study provides robust data supporting that inhibition of the sEH is a promising target to treat arthritis.

This work supports the massive presence of potential immunomodulatory peptides in the human gut metaproteome. These peptides were identified through the MAHMI database as potentially anti-inflammatory, and sixteen of them synthesized for characterize their mechanism of action. From them, peptide HM14 was encrypted in an extracellular protein produced by Bifidobacterium longum, a common member of the human microbiota, and displayed the highest anti-inflammatory capability. Molecular mechanism of action of HM14 pointed to a specific interaction between this immunomodulatory peptide and antigen presenting cells, which resulted in a higher formation of iTreg cells. Moreover, HM14 was effective in decreasing pro-inflammatory parameters in PBMCs isolated from a cohort of Crohn’s patients. Finally, non-targeted metabolomics confirmed the ability of HM14 to modulate the metabolic activity of PBMCs to fulfil its energy and biosynthetic requirements. Overall, our combined in silico/multiomics approach supports the human gut metaproteome as a source for immunomodulatory peptides.

Background: Seasonal influenza virus infection is a significant cause of morbimortality in the elderly. However, there is poor vaccine efficacy in this population due to immunosenescence. We aimed to explore several homeostatic parameters in the elderly that could impact influenza vaccine responsiveness. Methods: Subjects (> 60 years old) who were vaccinated against influenza virus were included, and the vaccine response was measured by a haemagglutination inhibition (HAI) test. At baseline, peripheral CD4 and CD8 T-cells were phenotypically characterized. Thymic function and the levels of different inflammation-related biomarkers, including Lipopolysaccharide Binding Protein (LBP) and anti-cytomegalovirus (CMV) IgG antibodies, were also measured. Results: Influenza vaccine non-responders showed a tendency of higher frequency of regulatory T-cells (Tregs) before vaccination than responders (1.49 [1.08–1.85] vs. 1.12 [0.94–1.63], respectively, p = 0.061), as well as higher expression of the proliferation marker Ki67 in Tregs and different CD4 and CD8 T-cell maturational subsets. The levels of inflammation-related biomarkers correlated with the frequencies of different proliferating T-cell subsets and with thymic function (e.g., thymic function with D-dimers, r = − 0.442, p = 0.001). Conclusions: Age-related homeostatic dysregulation involving the proliferation of CD4 and CD8 T-cell subsets, including Tregs, was related to a limited responsiveness to influenza vaccination and a higher inflammatory status in a cohort of elderly people

The average age of onset of multiple myeloma (MM) is in the 7th decade coinciding with the development of immunosenescence. As a consequence, lymphocyte recovery after lymphopenia-inducing anti-MM therapies (most notably autologous stem cell transplant, ASCT) is reliant on homeostatic proliferation of peripheral T cells rather than replenishment by new thymic emigrants. It has been shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced T cell receptor repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which retain IFNg production but lose proliferative potential. A reduction in CD4:8 ratio is characteristic of MM disease progression, due to high IL-15 levels in lymphopenic states preferentially driving expansion of CD8+ memory T cells. The first section of this thesis describes changes that occur in the T cell population in MM from diagnosis and throughout treatment, and compares with age-matched healthy donors (HD) and RRMM. ASCT and lymphopenia-inducing therapies in MM have a largely irrecoverable effect on the CD4+ T cell population, with a permanent reduction in CD4:8 ratio and loss of naïve T cells. Furthermore, there is a substantial and sustained increase in the proportion of PD-1 expressing T cells post-ASCT. Regulatory T cells increase with treatment in NDMM and RRMM patients. Utilising bisulfite sequencing, it is demonstrated that these cells are peripherally- rather than thymically-derived, which has implications for the stability of the regulatory T cell population. In the second part of this thesis, RNA-Seq is utilised to investigate differences in CD4+ T cells from NDMM, RRMM and HD. These results demonstrated upregulation of mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Using qPCR of key rate-limi