Suppressive Capacity Of Tregs Research Articles

Proteobacteria impair anti-tumor immunity in the omentum by consuming arginine

Gut microbiota influence anti-tumor immunity, often by producing immune-modulating metabolites. However, microbes consume a variety of metabolites that may also impact host immune responses. We show that tumors grow unchecked in the omenta of microbe-replete mice due to immunosuppressive Tregs. By contrast, omental tumors in germ-free, neomycin-treated mice or mice colonized with altered Schaedler's flora (ASF) are spontaneously eliminated by CD8+ Tcells. These mice lack Proteobacteria capable of arginine catabolism, causing increases in serum arginine that activate the mammalian target of the rapamycin (mTOR) pathway in Tregs to reduce their suppressive capacity. Transfer of the Proteobacteria, Escherichia coli (E.coli), but not a mutant unable to catabolize arginine, to ASF mice reduces arginine levels, restores Treg suppression, and prevents tumor clearance. Supplementary arginine similarly decreases Treg suppressive capacity, increases CD8+ Tcell effectiveness, and reduces tumor burden. Thus, microbial consumption of arginine alters anti-tumor immunity, offering potential therapeutic strategies for tumors in visceral adipose tissue.

Multiply restimulated human cord blood-derived Tregs maintain stabilized phenotype and suppressive function and predict their therapeutic effects on autoimmune diabetes

BackgroundRegulatory T cells (Tregs) are involved in the maintenance of immune homeostasis and immune regulation. Clinical trials on the adoptive transfer of Tregs have been ongoing for > 10 years. However, many unresolved issues remain in the production of readymade Treg products and selection of patients. Hence, this study aimed to develop a method to expand off-the-shelf Tregs derived from umbilical cord blood (UCB-Tregs) in vitro without changing their phenotype and inhibitory function. In addition, the study intended to design an approach to precisely select patients who are more likely to benefit from the adoptive Treg transfer therapy.MethodsUCB-Tregs were isolated and cultured in a medium containing human recombinant IL-2 and rapamycin and then multiply restimulated with human T-activator CD3/CD28 dynabeads. The phenotype and suppressive capacity of Tregs were assessed on days 18 and 42. The relationship between the suppressive function of UCB-Tregs in vitro and clinical indicators was analyzed, and the ability of the in vitro suppressive capacity to predict the in vivo therapeutic effects was evaluated.ResultsUCB-Tregs expanded 123-fold and 5,981-fold at 18 and 42 days, respectively. The suppressive function of UCB-Tregs on the proliferation of immune cells at 42 days was not significantly different compared with that of UCB-Tregs obtained at 18 days. The suppression rate of UCB-Tregs to PBMCs was negatively correlated with the course of diabetes. Moreover, the high-suppression group exhibited a better treatment response than the low-suppression group during the 12-month follow-up period.ConclusionsMultiply restimulated UCB-Tregs expanded at a large scale without any alterations in their classical phenotypic features and inhibitory functions. The suppressive function of Tregs in vitro was negatively correlated with the disease duration. The present study revealed the possibility of predicting the in vivo therapeutic effects via the in vitro inhibition assay. Thus, these findings provided a method to obtain off-the-shelf Treg products and facilitated the selection of patients who are likely to respond to the treatment, thereby moving toward the goal of precision treatment.

Improving regulatory T cell production through mechanosensing.

Induced Tregs (iTregs) have great promise in adoptive immunotherapy for treatment of autoimmune diseases. This report investigates the impacts of substrate stiffness on human Treg induction, providing a powerful yet simple approach to improving production of these cells. Conventional CD4+ human T cells were activated on materials of different elastic modulus and cultured under suppressive conditions. Enhanced Treg induction was observed on softer materials as early as 3 days following activation and persisted for multiple weeks. Substrate stiffness also affected epigenetic modification of Treg specific genes and Treg suppressive capacity. Tregs induced on substrates of an optimal stiffness balance quantity and suppressive quality.

Altered Regulatory T cell-mediated Natural Killer Cells Suppression may lead to Generalized Vitiligo

Generalized vitiligo(GV) is characterized by white patches due to autoimmune loss of melanocytes. Regulatory T cells(Tregs) maintain immune homeostasis, while NK cells eliminate pathogens and tumors. Increased NK cell frequency and reduced Treg frequency and suppressive capacity are observed in vitiligo patients. However, studies assessing Treg-mediated suppression of NK cell functions in GV are lacking. Therefore, our study aimed to assess in vitro Treg-mediated suppression of NK cells function over K562 and SK-Mel-28 cells in 31 GV patients and 30 controls using the BrdU-cell proliferation assay. We found decreased Treg-mediated suppression of NK cell function in GV patients(p=0.0289). Moreover, increased NK cell-mediated K562 and SK-Mel-28 cells’ suppression was observed in GV patients(p=0.0207,p=0.0419). Disease activity-based analysis, suggested reduced Treg-mediated suppression of NK cell function and increased NK cell function in active vitiligo patients(p=0.03,p=0.0436). Interestingly, age-based analysis suggested decreased Treg-mediated suppression of NK cell function in 1-20 and 21-40 years age groups compared to 41-60 years age group of GV patients(p=0.005,p=0.0380). Overall, our study, for the first time, suggests that decreased Treg-mediated suppression of NK cells may lead to increased destruction of melanocytes in GV, and this knowledge may help in developing effective therapeutics based on Tregs and NK cells for GV.

Deciphering the immune landscape of head and neck squamous cell carcinoma: A single-cell transcriptomic analysis of regulatory T cell responses to PD-1 blockade therapy

Immunotherapy is changing the Head and Neck Squamous Cell Carcinoma (HNSCC) landscape and improving outcomes for patients with recurrent or metastatic HNSCC. A deeper understanding of the tumor microenvironment (TME) is required in light of the limitations of patients’ responses to immunotherapy. Here, we aimed to examine how Nivolumab affects infiltrating Tregs in the HNSCC TME. We used single-cell RNA sequencing data from eight tissues isolated from four HNSCC donors before and after Nivolumab treatment. Interestingly, the study found that Treg counts and suppressive activity increased following Nivolumab therapy. We also discovered that changes in the CD44-SSP1 axis, NKG2C/D-HLA-E axis, and KRAS signaling may have contributed to the increase in Treg numbers. Furthermore, our study suggests that decreasing the activity of the KRAS and Notch signaling pathways, and increasing FOXP3, CTLA-4, LAG-3, and GZMA expression, may be mechanisms that enhance the killing and suppressive capacity of Tregs. Additionally, the result of pseudo-temporal analysis of the HNSCC TME indicated that after Nivolumab therapy, the expression of certain inhibitory immune checkpoints including TIGIT, ENTPD1, and CD276 and LY9, were decreased in Tregs, while LAG-3 showed an increased expression level. The study also found that Tregs had a dense communication network with cluster two, and that certain ligand-receptor pairs, including SPP1/CD44, HLA-E/KLRC2, HLA-E/KLRK1, ANXA1/FPR3, and CXCL9/FCGR2A, had notable changes after the therapy. These changes in gene expression and cell interactions may have implications for the role of Tregs in the TME and in response to Nivolumab therapy.

Harmine and Kaempferol treatment enhances NFATC1 and FOXP3 mediated regulatory T-cells’ suppressive capacity in generalized vitiligo

BackgroundGeneralized vitiligo (GV) is an autoimmune disease characterized by the progressive loss of melanocytes. ObjectivesCurrent study was undertaken to assess in-vitro therapeutic potential of Harmine and Kaempferol for GV. MethodsCalcium, calcineurin, NFATC1 levels, cell proliferation were assessed by various kits and ORAI1, PEIZO1, Calcineurin, GSK3B, DYRK1A transcripts and IFN-γ,IL-10,TGF-β protein levels were assessed by qPCR and ELISA in blood and skin biopsy samples from Tregs of 52 patients and 50 controls. ResultsHarmine and Kaempferol treatment enhances Treg suppressive capacity, NFATs and FOXP3 expression in blood and skin Tregs of GV patients (p < 0.05). Furthermore, Harmine and Kaempferol treatment in Tregs increased calcineurin and NFATC1 activity and decreased DYRK1A transcripts in blood and skin Tregs of GV patients(p < 0.05). In-silico analysis revealed that Harmine and Kaempferol might boost Treg suppressive capacity by increasing calcineurin dephosphorylation activity leading to increase NFATs activation and also increase nuclear retention of NFATs by inhibiting DYRK1a phosphorylation activity. Moreover, calcineurin and NFATC1 activity in Tregs were positively correlated with Treg suppressive capacity, NFATC1 and FOXP3 expression (p < 0.05), whereas, DYRK1A transcripts were negatively correlated with Treg suppressive capacity, NFATC1 and FOXP3 expression (p < 0.05). These compounds significantly increased melanocytes’ survival and proliferation in Treg:CD4+/CD8+:SK-Mel-28 cell line co-culture system from GV patients (p < 0.0001). ConclusionsFor the first time the study suggests that Harmine and Kaempferol treated Tregs could control the CD8+ and CD4+T-cells’ proliferation and IFN-γ production, leading to melanocytes’ survival and proliferation. These compounds may serve as novel Treg-based therapeutics for GV; however, in vivo studies are warranted to assess the safety and efficacy of these compounds.

Regulatory T cells in autoimmune primary adrenal insufficiency.

Primary adrenal insufficiency (PAI) is most often caused by an autoimmune destruction of the adrenal cortex resulting in failure to produce cortisol and aldosterone. The aetiology is thought to be a combination of genetic and environmental risk factors, leading to breakdown of immunological tolerance. Regulatory T cells (Tregs) are deficient in many autoimmune disorders, but it is not known whether they contribute to development of PAI. We aimed to investigate the frequency and function of naive and expanded Tregs in patients with PAI and polyendocrine syndromes compared to age- and gender-matched healthy controls. Flow cytometry was used to assess the frequency and characterize functional markers of blood Tregs in PAI (N = 15). Expanded Treg suppressive abilities were assessed with a flow cytometry based suppression assay (N = 20), while bulk RNA-sequencing was used to examine transcriptomic differences (N = 16) and oxygen consumption rate was measured by a Seahorse cell metabolic assay (N = 11). Our results showed that Treg frequency and suppressive capacity were similar between patients and controls. An increased expression of killer-cell leptin-like receptors and mitochondrial genes was revealed in PAI patients, but their expanded Tregs did not display signs of mitochondrial dysfunction. Our findings do not support a clear role for Tregs in the contribution of PAI development.

A target-based discovery from a parasitic helminth as a novel therapeutic approach for autoimmune diseases

Regulatory T cells (Tregs) can alleviate the development of autoimmune and inflammatory diseases, thereby proposing their role as a new therapeutic strategy. Parasitic helminths have co-evolved with hosts to generate immunological privilege and immune tolerance through inducing Tregs. Thus, constructing a "Tregs-induction"-based discovery pipeline from parasitic helminth is a promising strategy to control autoimmune and inflammatory diseases. The gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) were used to isolate immunomodulatory components from the egg extracts of Schistosoma japonicum. The extracted peptides were evaluated for their effects on Tregs suppressive functions using flow cytometry, ELISA and T cell suppression assay. Finally, we carried out colitis and psoriasis models to evaluate the function of Tregs induced by helminth-derived peptide invivo. Here, based on target-driven discovery strategy, we successfully identified a small 3kDa peptide (SjDX5-53) from egg extracts of schistosome, which promoted both human and murine Tregs production. SjDX5-53 presented immunosuppressive function by arresting dendritic cells (DCs) at an immature state and augmenting the proportion and suppressive capacity of Tregs. In mouse models, SjDX5-53 protected mice against autoimmune-related colitis and psoriasis through inducing Tregs and inhibiting inflammatory T-helper (Th) 1 and Th17 responses. SjDX5-53 exhibited the promising therapeutic effects in alleviating the phenotype of immune-related colitis and psoriasis. This study displayed a screening and validation pipeline of the inducer of Tregs from helminth eggs, highlighting the discovery of new biologics inspired by co-evolution of hosts and their parasites. This study was supported by the Natural Science Foundation of China (82272368) and Natural Science Foundation of Jiangsu Province (BK20211586).

Chronic hypoxia disrupts memory T regulatory cell phenotype which contributes to the pathogenesis of pulmonary hypertension

Pulmonary hypertension (PH) caused by chronic hypoxia (CH) is a multifaceted disease most often caused by sleep apnea, prolonged high-altitude exposure, or chronic obstructive pulmonary disease (COPD), with minimal treatment options, leading to right heart failure and death. One major contributor in the pathogenesis of PH that requires more characterization is the accompanying immune response.Our previous studies have demonstrated the importance of inflammation and self-immunity in CH-induced PH. We demonstrated pulmonary-perivascular localization of pro-inflammatory Th17 cells as well as an increase in total Th17 cells in the lung, while finding no overall changes in the number of anti-inflammatory T regulatory (Treg) cells present in the lung. Imbalance between Th17 and Treg cells has been shown to drive multiple inflammatory and metabolic diseases. One area of interest related to this balance has been the recent discovery and attempted characterization of exTregs, or cells that, at one point in time, were Tregs but lost their phenotype transitioning to pro-inflammatory Th17 cells, due to decreases in the expression of the transcription factor FoxP3. Therefore, we hypothesized that the increase in Th17 cells seen in CH is due to a decrease in Tregs and Treg suppressive capacity as well as a switching of Tregs to Th17 cells. Using a mouse model that allows the study of lineage stability and genetic mapping of Tregs, together with single-cell RNA sequencing (scRNA SEQ), we initially established that CH resulted in a gene expression profile consistent with exTregs or Tregs with less suppressive activity, such as downregulation of Il2ra gene and upregulation of Tnfrsf4 (adjusted p-value 0.03). To further establish a loss in Tregs as well as an increase in exTregs following CH exposure, we performed spectral flow cytometry of splenocytes on a Cytek Aurora to look for changes in protein expression and cell prevalence. Finally, we used a similar lineage mapping mouse model to look at Th17 cells. Following these experiments, we found supporting evidence of our scRNA SEQ data, finding a decrease in Tregs in both male and female mice following CH exposure, as well as an increase in exTregs in both groups exposed to CH. We also further supported this data finding Th17 cells tracked through our model to be increased following CH. These results support the importance of immune cell balance and inflammation in the pathogenesis of CH-induced PH. R56 HL153065-01 NIH, AHA 18TPA34170037, AHA967925 &amp; T32 HL07736 NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute Downregulation but Not Genetic Ablation of Murine MCU Impairs Suppressive Capacity of Regulatory CD4 T Cells.

By virtue of mitochondrial control of energy production, reactive oxygen species (ROS) generation, and maintenance of Ca2+ homeostasis, mitochondria play an essential role in modulating T cell function. The mitochondrial Ca2+ uniporter (MCU) is the pore-forming unit in the main protein complex mediating mitochondrial Ca2+ uptake. Recently, MCU has been shown to modulate Ca2+ signals at subcellular organellar interfaces, thus fine-tuning NFAT translocation and T cell activation. The mechanisms underlying this modulation and whether MCU has additional T cell subpopulation-specific effects remain elusive. However, mice with germline or tissue-specific ablation of Mcu did not show impaired T cell responses in vitro or in vivo, indicating that 'chronic' loss of MCU can be functionally compensated in lymphocytes. The current work aimed to specifically investigate whether and how MCU influences the suppressive potential of regulatory CD4 T cells (Treg). We show that, in contrast to genetic ablation, acute siRNA-mediated downregulation of Mcu in murine Tregs results in a significant reduction both in mitochondrial Ca2+ uptake and in the suppressive capacity of Tregs, while the ratios of Treg subpopulations and the expression of hallmark transcription factors were not affected. These findings suggest that permanent genetic inactivation of MCU may result in compensatory adaptive mechanisms, masking the effects on the suppressive capacity of Tregs.

Abstract 5712: Anti-TIGIT antibody tiragolumab leverages myeloid cells and regulatory T cells to improve PD-L1 checkpoint blockade

Abstract Background: TIGIT is a co-inhibitory receptor and immune checkpoint associated with T cell and natural killer (NK) cell dysfunction in cancer. Tiragolumab is an anti-TIGIT antibody with an active IgG1/kappa Fc. In a randomized double-blind phase 2 clinical trial in non-small cell lung cancer (NSCLC), tiragolumab + atezolizumab (anti-PD-L1) combination treatment demonstrated significant improvement relative to atezolizumab alone. However, the mechanisms underlying the efficacy of this combination are not well understood. Methods: In CITYSCAPE (phase 2, NCT03563716), chemotherapy-naïve patients with locally advanced/metastatic NSCLC received either placebo + 1200 mg atezolizumab or 600 mg tiragolumab + 1200 mg atezolizumab q3w IV. We collected tumor pretreatment samples and serum samples (baseline and on-treatment) from patients enrolled in the trial, which were subject to bulk RNA-seq and proteomics, respectively. In the mouse tumor model, BALB/c mice were implanted with syngeneic CT26 tumors. After tumor establishment, mice were randomized by tumor volume and then treated with control IgG2a, anti-PD-L1, anti-TIGIT IgG2a-LALAPG (Fc-inactive) ± anti-PD-L1, anti-TIGIT IgG2b ± anti-PD-L1, or anti-TIGIT IgG2a ± anti-PD-L1. Three days after treatment, CD45+ immune cells were collected from the peripheral blood and tumor from selected treatment groups and underwent single cell RNA sequencing or flow cytometry profiling. Tumor growth was also monitored to determine efficacy. Results: Here, we show that tiragolumab functions as both a conventional checkpoint inhibitor and, via Fc gamma receptor engagement, as a modulator of immunosuppressive myeloid cells and T regulatory (Treg) cells. Gene expression analysis of patient tumor samples revealed high levels of these cell subsets, were instead associated with treatment benefit in the tiragolumab + atezolizumab arm but not atezolizumab arm. Analysis of patient serum proteins suggested an association of myeloid cell activation with clinical benefit mediated by the combination therapy. In mouse tumor models, treatment with anti-PD-L1 + anti-TIGIT IgG2a (but not anti-TIGIT IgG2b or Fc-silent anti-TIGIT) led to effective tumor control in mice, suggesting a pivotal role for activating Fc receptors. Phenotypic profiling by single cell RNAseq and flow cytometry revealed that Fc-active anti-TIGIT remodels the tumor microenvironment, most prominently by inducing antigen presentation machinery in myeloid cells, counteracting of anti-PDL1-enforced CD8+ T cell exhaustion, and reducing Treg suppressive capacity. Conclusions: These findings reveal that FcR engagement is one of several distinct mechanisms by which tiragolumab unleashes antitumor immune responses, and inform further clinical development of anti-TIGIT therapies. Citation Format: Namrata S. Patil, Shyam Srivats, Yoonha Choi, Xiangnan Guan, Barzin Nabet, Lisa McGinnis, Eugene Chiang, Alexis Dunkle, Bill O’Gorman, Patrick S. Chang, Ruozhen Hu, John Silva, Joy Han, Amelia Au-Yeung, Chikara Takahashi, Nandini Molden, Pallavi Daggumati, Wendy Connolly, Melissa Johnson, Delvys Rodriguez Abreu, Byoung Chul Cho, Antoine Italiano, Ignacio Gil-Bazo, Enriqueta Felip, Ira Mellman, Raymond Meng, Sanjeev Mariathasan, Robert Johnston, David S. Shames. Anti-TIGIT antibody tiragolumab leverages myeloid cells and regulatory T cells to improve PD-L1 checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5712.

Decreased GZMB, NRP1, ITPR1, and SERPINB9 Transcripts Lead to Reduced Regulatory T Cells Suppressive Capacity in Generalized Vitiligo Patients

Generalized vitiligo (GV) is an autoimmune skin disease characterized by bilateral white patches over the entire body. Regulatory T cells (Tregs) maintain peripheral tolerance; however, they are found to be reduced in numbers and function in vitiligo patients. The exact mechanism for reduced Treg suppressive capacity is unknown. Therefore, we aimed to assess transcript levels of Tregs-associated immunosuppressive genes (GZMB, NRP1, PDCD1, FASLG, and TNFRS18), regulatory molecules of Tregs suppressive function (SERPINB9, ITPR1, and UBASH3A), and Treg-associated transcription factors (GATA2, GATA3, RUNX1, STAT3, and STAT5) in 52 GV patients and 48 controls by real-time PCR (qPCR). We found significantly reduced GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV Tregs compared to controls (p = 0.03, p = 0.023, p = 0.0045, and p < 0.0001, respectively). There were 0.44-, 0.45-, 0.32-, and 0.54-fold decrease in GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV Tregs. Additionally, disease activity and severity-based analyses revealed significantly decreased GZMB (p = 0.019 and 0.034), SERPINB9 (p = 0.031 and p = 0.035), and ITPR1 (p = 0.0003 and p = 0.034) transcripts in active vitiligo and severe GV patients' Tregs. Interestingly, we found a positive correlation for ITPR1 with GZMB (r = 0.45, p = 0.0009) and SERPINB9 (r = 0.52, p = 0.001) transcripts in GV Tregs. Moreover, we found positive correlation for percentage Treg mediated suppression of CD4+ and CD8+T cells with ITPR1 (r = 0.54; r = 0.49), GZMB (r = 0.61; r = 0.58), NRP1 (r = 0.55; r = 0.52), and SERPINB9 (r = 0.56; r = 0.48) in GV Tregs. Further, calcium treatment of Tregs resulted into significantly increased ITPR1, SERPINB9, and GZMB transcripts in GV Tregs (p = 0.023, p = 0.0345, p = 0.02). Overall, our results for the first time revealed the crucial role of GZMB, NRP1, SERPINB9, and ITPR1 transcripts in decreased Treg suppressive capacity leading to GV pathogenesis, progression, and severity. In addition, our study highlighted that ITPR1 might be linked with decreased GZMB and NRP1 expression in GV Tregs. Moreover, our study for the first time suggest that increased SERPINB9 transcripts may lead to endogenous granzyme B-mediated Tregs apoptosis, and calcium treatment of Tregs may improve the Treg suppressive capacity. These findings may further aid in development of Treg-based therapeutics for GV.

Calcium controlled NFATc1 activation enhances suppressive capacity of regulatory T cells isolated from generalized vitiligo patients.

NFATs and FOXP3 are linked with impaired regulatory T-cells (Tregs) in generalized vitiligo (GV). To elucidate calcium mediated NFATc1 signalling pathway and its effect on Treg suppressive capacity in GV. Calcium levels, calcineurin, NFATc1 and GSK-3β activity and cell proliferation were assessed in 52 GV patients and 50 controls by calcium assay kit, calcineurin phosphatase assay kit, TransAM NFATc1 kit, GSK-3β ELISA and BrdU cell proliferation assay. Transcripts (CNB, CAM, GSK3B, DYRK1A and calcium channel genes) and protein (IFN-γ, IL-10 and TGF-β) expressions were assessed by qPCR and ELISA, respectively. Reduced plasma and intracellular Tregs calcium levels and ORAI1 transcripts suggested altered calcium homeostasis in GV Tregs (p=0.00387, p=0.0048, p < 0.0001), which led to decreased calcineurin and NFATc1 activity in GV Tregs (p=0.0299, p < 0.0001). CNB and CAM transcripts were reduced in GV Tregs (p < 0.0001, p=0.0004). GSK-3β activity, GSK3B and DYRK1A transcripts significantly increased in GV Tregs (p=0.0134, p < 0.0001 and p < 0.0001). Plasma (p=0.0225, p=0.032) and intracellular Treg (p=0.0035, p=0.005) calcium levels, calcineurin (p=0.001) and NFATc1 (p=0.001, p < 0.0001) activity and ORAI1 (p=0.0093, p < 0.0001), CAM and CNB (p=0.0214) transcripts significantly decreased in active vitiligo (AV) and severe GV (sGV) Tregs. Calcium treatment significantly increased intracellular calcium and ORAI1 transcripts in GV Tregs (p=0.0042, p=0.0035). Moreover, calcium treatment enhanced calcineurin and NFATc1 activity in GV Tregs (p=0.0128, p < 0.0001). Remarkably, calcium treatment increased Treg mediated suppression of CD4+ and CD8+ T-cells (p=0.015, p=0.006) in GV and increased Tregs associated cytokines: IL-10 (p=0.0323, p=0.009), TGF-β (p=0.0321, p=0.01) and decreased IFN-γ production (p=0.001, p=0.016) by CD4+ and CD8+ T-cells. Intracellular calcium levels positively correlated with calcineurin (r=0.83; p < 0.0001) and NFATc1 (r=0.61; p < 0.0001) activity, suggesting the enhanced Treg immunosuppressive capacity after calcium treatment. Our study for the first time suggests that reduced plasma calcium and ORAI1 transcripts are linked to calcium uptake defects in Tregs, which leads to reduced calcineurin and NFATc1 activation, thereby contributing to decreased Tregs immunosuppressive capacity in GV. Elevated GSK-3β activity and GSKB and DYRK1A transcripts are involved in reduced NFATc1 activity in GV Tregs. Overall, the study suggests that calcium-NFATc1-signalling pathway is likely to be involved in defective Tregs function and can be implicated for development of effective Treg mediated therapeutics for GV.

Tregs in Autoimmunity: Insights Into Intrinsic Brake Mechanism Driving Pathogenesis and Immune Homeostasis.

CD4+CD25highFoxp3+ regulatory T-cells (Tregs) are functionally characterized for their ability to suppress the activation of multiple immune cell types and are indispensable for maintaining immune homeostasis and tolerance. Disruption of this intrinsic brake system assessed by loss of suppressive capacity, cell numbers, and Foxp3 expression, leads to uncontrolled immune responses and tissue damage. The conversion of Tregs to a pathogenic pro-inflammatory phenotype is widely observed in immune mediated diseases. However, the molecular mechanisms that underpin the control of Treg stability and suppressive capacity are incompletely understood. This review summarizes the concepts of Treg cell stability and Treg cell plasticity highlighting underlying mechanisms including translational and epigenetic regulators that may enable translation to new therapeutic strategies. Our enhanced understanding of molecular mechanism controlling Tregs will have important implications into immune homeostasis and therapeutic potential for the treatment of immune-mediated diseases.

MicroRNA-92a promotes CNS autoimmunity by modulating the regulatory and inflammatory T cell balance.

A disequilibrium between immunosuppressive Tregs and inflammatory IL-17–producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, the molecular mechanisms underlying the Treg and Th17 imbalance in CNS autoimmunity remain largely unclear. Identifying the factors that drive this imbalance is of high clinical interest. Here, we report a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. miR-92a was elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuated EAE. Mechanistically, miR-92a mediated EAE susceptibility in a T cell–intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses, by directly repressing the transcription factor Foxo1. Although miR-92a did not directly alter Th1 differentiation, it appeared to indirectly promote Th1 cells by inhibiting Treg responses. Correspondingly, miR-92a inhibitor therapy ameliorated EAE by concomitantly boosting Treg responses and dampening inflammatory T cell responses. Analogous to our findings in mice, miR-92a was elevated in CD4+ T cells from patients with MS, and miR-92a silencing in patients’ T cells promoted Treg development but limited Th17 differentiation. Together, our results demonstrate that miR-92a drives CNS autoimmunity by sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS.

MicroRNA control of inflammatory and regulatory T cells in CNS autoimmunity

Abstract A disequilibrium between immunosuppressive regulatory T cells (Tregs) and inflammatory interleukin (IL)-17-producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, molecular mechanisms underlying Treg and Th17 imbalance in the central nervous system (CNS) autoimmunity remain largely unclear and thus identifying factors which drive this imbalance is of high clinical interest. Recently, we found a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. MiR-92a is elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuates EAE. Mechanistically, miR-92a mediates EAE susceptibility in a T cell-intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses by directly repressing the transcription factor, Foxo1. Correspondingly, miR-92a inhibitor therapy ameliorates EAE by modulating the balance between Tregs and Th17 cells. Analogous to mice, miR-92a is elevated in MS patient CD4+ T cells, and miR-92a silencing in patient T cells promotes Treg development while limiting Th17 differentiation. Together, our results identify a previously unknown function by which miR-92a drives CNS autoimmunity via sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS. Supported by NIH R01 R01AI127853 NMSS RG-1507-05164

The Amphiregulin/EGFR axis protects from lupus nephritis via downregulation of pathogenic CD4+ T helper cell responses

Systemic lupus erythematosus (SLE) is a common autoimmune disorder with a complex and poorly understood immuno-pathogenesis. Lupus nephritis (LN) is a frequent and difficult to treat complication, which causes high morbidity and mortality. The multifunctional cytokine amphiregulin (AREG) has been implicated in SLE pathogenesis, but its function in LN currently remains unknown. We thus studied the model of pristane-induced LN and found increasing renal and systemic AREG expression during the course of disease. Importantly, renal injury was significantly aggravated in the absence of AREG, revealing a net anti-inflammatory role. Analyses of immune responses showed dual effects. On the one hand, AREG enhanced activation of pro-inflammatory myeloid cells, which however did not play a major role for the course of LN. More importantly, on the other hand, AREG strongly suppressed pathogenic cytokine production by T helper effector cells. This effect was more general in nature and could be reproduced in response to antigen immunization. Since AREG has been postulated to downregulate T cell responses via enhancing Treg suppressive capacity, we followed up on this aspect. Interestingly, however, in vitro studies revealed potential direct and Treg independent effects of AREG on T helper effector cells. In favor of this notion, we found significantly enhanced T cell responses and consecutive aggravation of LN, only if epidermal growth factor receptor (EGFR) signaling was abrogated in total T cells, but not if the EGFR was absent on Tregs alone. Finally, we also found enhanced AREG expression in plasma and renal biopsies of patients with LN, supporting the relevance of our findings for human disease. In summary, our data identify AREG as an anti-inflammatory mediator of LN via broad downregulation of pathogenic T cell immunity. These findings further highlight the AREG/EGFR axis as a potential therapeutic target.

Cure of syngeneic carcinomas with targeted IL-12 through obligate reprogramming of lymphoid and myeloid immunity.

Therapeutic IL-12 has demonstrated the ability to reduce local immune suppression in preclinical models, but clinical development has been limited by severe inflammation-related adverse events with systemic administration. Here, we show that potent immunologic tumor control of established syngeneic carcinomas can be achieved by i.t. administration of a tumor-targeted IL-12 antibody fusion protein (NHS–rmIL-12) using sufficiently low doses to avoid systemic toxicity. Single-cell transcriptomic analysis and ex vivo functional assays of NHS–rmIL-12–treated tumors revealed reinvigoration and enhanced proliferation of exhausted CD8+ T lymphocytes, induction of Th1 immunity, and a decrease in Treg number and suppressive capacity. Similarly, myeloid cells transitioned toward inflammatory phenotypes and displayed reduced suppressive capacity. Cell type–specific IL-12 receptor–KO BM chimera studies revealed that therapeutic modulation of both lymphoid and myeloid cells is required for maximum treatment effect and tumor cure. Study of single-cell data sets from human head and neck carcinomas revealed IL-12 receptor expression patterns similar to those observed in murine tumors. These results describing the diverse mechanisms underlying tumor-directed IL-12–induced antitumor immunity provide the preclinical rationale for the clinical study of i.t. NHS–IL-12.

Study on the preparation and function of regulatory T cells from human peripheral blood.

BackgroundRegulatory T cells (Tregs) are an important cell subgroup of CD4+ T cells. Treg cells are critically involved in inducing immune tolerance, maintaining immune environment homeostasis, and preventing the occurrence of autoimmune diseases. Under normal conditions, the number of Tregs in the body is very small. This research was designed to establish an effective method to expand human peripheral blood Tregs in vitro and to analyze phenotype, purity, and function of Treg cells post-expansion.MethodsPeripheral blood was obtained from healthy donors. CD4+CD25+CD127dim/− Treg cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic-activated cell sorting (MACS), and an optimized culture system was used for amplification. The in vitro amplification ability of Treg cells was evaluated to determine the expression and purity of Treg cell-specific surface markers in different culture cycles. The suppressive function of Treg was determined by in vitro lymphocyte proliferation assay.ResultsTreg cells could be successfully isolated by magnetic activated cell sorting (MACS). After 21 days of in vitro culture, the mean expansion fold was 2,009±452.2 in ≤60 years, and there was a significant difference between the younger group and the older than 60 years group (1,238±330.0). Flow cytometry analysis revealed that the percentages of CD4+CD25+ cells and FOXP3+ cells were (93.25±3.05)% and (94.19±4.21)% on day 14, and (92.86±4.36)% and (91.55±5.62)% on day 21, respectively. In addition, the proportions of CD8+ T, CD19+ B, CD3−CD56+ natural killer cell (NK), and CD3+ CD56+ natural killer T cell (NKT) were extremely low. Lymphocyte proliferation assay demonstrated that Tregs could inhibit the proliferation of CD8+ T cells more effectively than that of CD4+ T cells. Furthermore, the suppressive capacity of Tregs was correlated with Treg-to-PBMCs ratios.ConclusionsWe successfully established a technical protocol for manufacturing a large quantity of Tregs with high efficiency in vitro. The expanded Tregs have a steady FOXP3 expression and exhibited a potent immune suppression, which might have great significance in adoptive Treg therapy for treating graft-versus-host disease and autoimmune diseases.

BT-11 (omilancor), a first-in-class, topical therapeutic for psoriasis, ameliorates disease severity and inflammation through activation of LANCL2 pathway

Abstract BT-11 (omilancor) is a small molecule therapeutic that activates the LANCL2 pathway in immune and epithelial cells, resulting in regulatory effects at the intersection of immunity and metabolism. LANCL2 modulates the plasticity between Th17 and regulatory T cells (Tregs), favoring the stability and suppressive capacity of Tregs. Given the implication of the LANCL2 pathway in autoimmune disease and the demonstrated clinical efficacy and safety of BT-11 in a Phase 2 clinical trial of UC, we evaluated the therapeutic efficacy of BT-11 in psoriasis. In an imiquimod-induced mouse model of psoriasis, pharmacological activation of LANCL2 through topical BT-11 treatment ameliorates disease severity, resulting in over 60% reduction of PASI score. Indeed, BT-11 treatment decreased acanthosis and immune cell infiltration, with no signs of parakeratosis. The loss of LANCL2 in mice resulted in increased hyperkeratosis and parakeratosis. Local LANCL2 activation by BT-11 resulted in decreased TNF, IL17, IL6 and IL21-producing cells in the spleen. Additionally, LANCL2 activation regulated keratinocyte metabolism and function. Keratinocytes from BT-11-treated mice presented lower metabolic activation, reported as decreased ATP production, mitochondrial respiration and glucose metabolism. In translational studies with human PBMCs, BT-11 modulated the metabolic profile of CD4+ T cells at late state glycolysis, favoring oxidative pathways, and decreased expression of proinflammatory cytokines (IFNγ, TNFα), while upregulating IL-10. These results merit further exploration into the implication of LANCL2 signaling in human psoriasis and leverages the development of BT-11, as a first-in-class, topical therapeutic for psoriasis.