Treg Development Research Articles

Single-Cell RNA Sequencing Maps Immune Cell Heterogeneity in Mice with Allogeneic Cardiac Transplantation

Objective: Immune cells play important roles in mediating allograft rejection and tolerance after cardiac transplantation. However, immune cell heterogeneity at the single-cell level, and how immune cell states shape transplantation immunity, remain incompletely characterized. Methods: We performed single-cell RNA sequencing (scRNA-seq) on immune cells in LNs from a mouse syngeneic and allogeneic cardiac transplantation model. Nine T cell clusters were identified through unsupervised analysis. Pathway enrichment analysis was used to explore the functional differences among cell subpopulations and to characterize the metabolic heterogeneity of T cells. Results: We comprehensively determined the transcriptional landscape of immune cells, particularly T cells, and their metabolic transcriptomes in LNs during mouse cardiac transplantation. On the basis of molecular and functional properties, we also identified T cell types associated with transplantation-associated immune processes, including cytotoxic CD8+ T cells, activated conventional CD4+ T cells, and dysfunctional Tregs. We further elucidated the contribution of JunB to the induction of Th17 cell differentiation and restriction of Treg development, and identified that HIF-1a participates in T cell metabolism and function. Conclusions: We present the first systematic single-cell analysis of transcriptional variation within the T cell population, providing new insights for the development of novel therapeutic targets for allograft rejection.

GPR174 knockdown enhances blood flow recovery in hindlimb ischemia mice model by upregulating AREG expression

Regulatory T cells (Tregs) are critically involved in neovascularization, an important compensatory mechanism in peripheral artery disease. The contribution of G protein coupled receptor 174 (GPR174), which is a regulator of Treg function and development, in neovascularization remains elusive. Here, we show that genetic deletion of GPR174 in Tregs potentiated blood flow recovery in mice after hindlimb ischemia. GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs, thereby enhancing endothelial cell functions and reducing pro-inflammatory macrophage polarization and endothelial cell apoptosis. Mechanically, GPR174 regulates AREG expression by inhibiting the nuclear accumulation of early growth response protein 1 (EGR1) via Gαs/cAMP/PKA signal pathway activation. Collectively, these findings demonstrate that GPR174 negatively regulates angiogenesis and vascular remodeling in response to ischemic injury and that GPR174 may be a potential molecular target for therapeutic interventions of ischemic vascular diseases.

Construction of a T cell receptor signaling range for spontaneous development of autoimmune disease.

Thymic selection and peripheral activation of conventional T (Tconv) and regulatory T (Treg) cells depend on TCR signaling, whose anomalies are causative of autoimmunity. Here, we expressed in normal mice mutated ZAP-70 molecules with different affinities for the CD3 chains, or wild type ZAP-70 at graded expression levels under tetracycline-inducible control. Both manipulations reduced TCR signaling intensity to various extents and thereby rendered those normally deleted self-reactive thymocytes to become positively selected and form a highly autoimmune TCR repertoire. The signal reduction more profoundly affected Treg development and function because their TCR signaling was further attenuated by Foxp3 that physiologically repressed the expression of TCR-proximal signaling molecules, including ZAP-70, upon TCR stimulation. Consequently, the TCR signaling intensity reduced to a critical range generated pathogenic autoimmune Tconv cells and concurrently impaired Treg development/function, leading to spontaneous occurrence of autoimmune/inflammatory diseases, such as autoimmune arthritis and inflammatory bowel disease. These results provide a general model of how altered TCR signaling evokes autoimmune disease.

Ruxolitinib Stimulates Expansion of Regulatory T Cells in Murine Immune Bone Marrow Failure

Aplastic anemia (AA) is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow. In cases that are not constitutional or due to environmental causes, an immune-mediated etiology is inferred from the efficacy of immunosuppressive therapies and extensive work implicating cytotoxic CD8+ T cells in bone marrow destruction. To study AA experimentally, BMF is induced in mice with major histocompatibility complex-mismatched C67BL/6 (B6) ⇒ CByB6F1 lymph node (LN) cell infusion following sublethal total body irradiation (TBI). Following recent FDA approval of the JAK 1/2 inhibitor ruxolitinib (RUX) for graft-versus-host disease (GVHD), and based on its suppressive effect on T cell activation and inflammatory cytokine production, we studied RUX in our murine BMF model. Our results demonstrate that RUX attenuated BMF effectively by suppressing T cell activation and proliferation. Regulatory T cells (Tregs) are a heterogeneous subpopulation of cells that prevent autoimmunity by suppressing autoreactive T cells. Murine Tregs are identified by cell surface expression of the T-cell co-receptor CD4 and the activation marker CD25 and intracellular expression of the transcription factor FoxP3. As a master regulator of Treg development, FoxP3 associates with other transcription factors to induce the Treg functional identity. When assessing Tregs in a laboratory setting, cell fixation and permeabilization are required for antibody binding to FoxP3 due to its intranuclear location, preventing isolation of viable Tregs. To isolate intact Tregs, CD4 and CD25 double positivity alone is conventional, but some prior studies evaluating JAK inhibition used only CD4+FoxP3+ and excluded CD25. Thus, there is lack of consensus in definition of these important immunoregulatory cells in different immune contexts. Our studies revealed a RUX-mediated decrease in T-cell activation, evidenced by decreased CD25 surface expression in both CD8+ [30.5±2.71 (BMF n=7) vs 9.48±1.30 (RUX, n=10), p<0.0001] and CD4+ [33.76±2.40 (BMF n=7) vs 2.29±1.08 (RUX, n=10), p<0.0001] T cells. Concurrently, FoxP3 expression was increased [7.61±2.16 (BMF n=7) vs 16.12±0.94 (RUX, n=10), p<0.01] in CD4+ T cells, indicating a higher quantity of Tregs. We therefore postulated that, in RUX treatment, CD25 expression primarily reflects activation status rather than marking induction of Tregs. We sought to identify other surrogate surface markers for FoxP3, in order to enable Treg identification and isolation to evaluate immunoregulatory function. We investigated the surface markers GITR, CD45RB, and CD127, which have been previously described in association with Tregs, to characterize Tregs in BMF control and RUX-treated BMF mice. We observed strong co-expression of GITR and FoxP3 (Figure 1A), suggesting that CD4+GITRhi expression identified the Treg phenotype. CD4+FoxP3+ cells were consistently GITRhi but had lower CD25 expression in RUX-treated BMF samples than in BMF controls, implying that changes in CD25 expression from RUX treatment may render CD25 an unreliable marker of Tregs in this context. Tregs identified by both CD4+FoxP3+ and CD4+GITRhi showed co-expression of CD45RB and CD127. The proportions of cell populations that were CD45RB and CD127 double-negative and double-positive unexpectedly differed, according to whether BMF mice were untreated or RUX-treated, suggesting possible phenotypic changes in Tregs induced by RUX. To assess the function of Tregs in the spleen of RUX-treated BMF samples, we used flow cytometry to sort Tregs based on expression of CD4+GITRhi, and we co-cultured them with activated CFSE-labeled T cells in vitro at a 1:1 ratio for 5 days. A CFSE assay demonstrated slower CFSE dilution among T cells co-cultured with Tregs compared to those without Tregs (Figure 1B). These isolated Tregs inhibited both CD4+ and CD8+ T cell proliferation, but CD8+ T cells to a greater extent, and similarly to Tregs isolated from healthy mice. Murine Tregs can be reliably identified using CD4+GITRhi in immune contexts involving altered T-cell activation. We demonstrate phenotypic changes in the proportions of Tregs expressing surface marker combinations after RUX treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rnf20 inhibition enhances immunotherapy by improving regulatory T cell generation.

Allergic disorders are common all over the world. The pathogenesis of allergy is unclear. Therapies for allergic disorders require improvement. Endoplasmic reticulum (ER) stress is one of the factors influencing immune response. The purpose of this study is to improve the effectiveness of immunotherapy for experimental respiratory allergy by targeting the ER stress signal pathway. Committed CD4+ T cells were isolated from blood samples collected from patients with allergic rhinitis (AR) and TCR ovalbumin transgenic mice. The effects of TCR engagement and 3-methyl-4-nitrophenol (MNP) on inducing ER stress in committed CD4+ T cells were evaluated. ER stress was detected in antigen-specific CD4+ T cells (sCD4+ T cells) of AR patients. The environmental pollutant MNP increased the expression of the X-binding protein-1 (XBP1) in the committed CD4+ T cells during the TCR engagement. XBP1 mediated the effects of MNP on inhibiting regulatory T cell (Treg) generation. The effects of MNP on induction of protein 20 (Rnf20) in CD4+ T cells were mediated by XBP1. Inhibition of Rnf20 rescued the Treg development from MNP-primed sCD4+ T cells. The ablation of Rnf20 improved the immunotherapy of AR through the restoration of the Treg generation. ER stress can be detected in CD4+ T cells in TCR engagement. Exposure to MNP exacerbates ER stress in committed CD4+ T cells. Regulation of the ER stress-related Rnf20 expression can restore the generation of Treg from CD4+ T cells of subjects with allergic diseases.

LATE ONSET IPEX SYNDROME IN AN ADOLESCENT MALE

<h3>Introduction</h3> Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare disorder that presents with autoimmunity typically at an early age. We report the case of a 15-year-old male with common variable immunodeficiency (CVID) and bronchiectasis who presents with type 1 diabetes mellitus (T1DM) at age of 13. His severe presentation is concerning for an underlying monogenic immunodeficiency. This case highlights the late presentation of suspected IPEX syndrome in an adolescent male. <h3>Case Description</h3> Our patient with CVID and bronchiectasis presented with diabetes ketoacidosis at 13 years of age. He had a history of frequent sinopulmonary infections and was on immunoglobulin replacement therapy since 10 years of age for panhypogammaglobulinemia and specific antibody deficiency. Both immunologist and endocrinologist were concerned when T1DM developed and was difficult to treat. Genetic evaluation yielded hemizygous variant of uncertain significance in FOXP3[c.1036A>G(p.lle346Val)] in the forkhead domain. This variant is not present in Gnomad and deleterious by SIFT which prompted us to evaluate for IPEX. Immunophenotyping revealed reduced fraction CD4+CD25hiCD127low regulatory T cells (Tregs) with decreased forkhead box protein 3 (FOXP3) expression. More in-depth evaluations of his immune deficiency and immune dysregulation are pending. <h3>Discussion</h3> This case describes a late onset presentation of IPEX syndrome with features such as bronchiectasis, CVID, and late onset T1DM without other autoimmune manifestations. FOXP3 is essential for normal thymic Treg development. Missense mutations in nearby position Arg.347His has been described with T1DM but milder disease. The diagnostic approach of IPEX can be hindered by atypical phenotype as autoimmunity may present late.

G9a Modulates Lipid Metabolism in CD4 T Cells to Regulate Intestinal Inflammation

Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. Human naive T cells were isolated from peripheral blood and differentiated invitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The invivo role of G9a was assessed using 3 murine models. We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) invitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion invivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development invitro and invivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

Ecotoxicological studies of surface and groundwater in the adjacent territories of the Shuakhevi HPP (Adjara AR)

In the pathogenesis of autoimmune diseases, including immune thrombocytopenia (ITP), maintenance of inflammation and the loss of immune tolerance is essential. Transforming growth factor-beta (TGF-β) is a regulatory cytokine with pleiotropic function. TGF-β inhibits differentiation of T helper (Th1, Th2) cells and promotes the development of peripheral Tregs. The aim of our study was to explore the plasma levels of TGF-β and to perform its correlative analyses with peripheral blood biomarkers of systemic inflammation (NLR, PLR, PMR, SII, dNLR) in patients with ITP who did not respond to the first line treatment and had splenectomy as a second line therapy. The concentrations of TGF-β in plasma was quantified using ELISA kits by eBioscience. Statistical analyses was performed using Graph Pad Prism - Mann Whitney U test; for the correlation analyses we used spearman rank correlation by SPSS program. Our study revealed that the level of TGF-β in patients with refractory ITP is significantly diminished compared to the healthy subjects or patients undergoing splenectomy for other reasons than autoimmune or malignant hematological pathologies. There was no correlation observed between the plasma concentrations of TGF-β and the peripheral blood biomarkers of systemic inflammation.

Ecotoxicological studies of surface and groundwater in the adjacent territories of the Shuakhevi HPP (Adjara AR)

In the pathogenesis of autoimmune diseases, including immune thrombocytopenia (ITP), maintenance of inflammation and the loss of immune tolerance is essential. Transforming growth factor-beta (TGF-β) is a regulatory cytokine with pleiotropic function. TGF-β inhibits differentiation of T helper (Th1, Th2) cells and promotes the development of peripheral Tregs. The aim of our study was to explore the plasma levels of TGF-β and to perform its correlative analyses with peripheral blood biomarkers of systemic inflammation (NLR, PLR, PMR, SII, dNLR) in patients with ITP who did not respond to the first line treatment and had splenectomy as a second line therapy. The concentrations of TGF-β in plasma was quantified using ELISA kits by eBioscience. Statistical analyses was performed using Graph Pad Prism - Mann Whitney U test; for the correlation analyses we used spearman rank correlation by SPSS program. Our study revealed that the level of TGF-β in patients with refractory ITP is significantly diminished compared to the healthy subjects or patients undergoing splenectomy for other reasons than autoimmune or malignant hematological pathologies. There was no correlation observed between the plasma concentrations of TGF-β and the peripheral blood biomarkers of systemic inflammation.

IL-2 and IL-15 drive intrathymic development of distinct periphery-seeding CD4+Foxp3+ regulatory T lymphocytes.

Development of Foxp3-expressing regulatory T-lymphocytes (Treg) in the thymus is controlled by signals delivered in T-cell precursors via the TCR, co-stimulatory receptors, and cytokine receptors. In absence of IL-2, IL-15 or their receptors, fewer Treg apparently develop in the thymus. However, it was recently shown that a substantial part of thymic Treg are cells that had recirculated from the periphery back to the thymus, troubling interpretation of these results. We therefore reassessed the involvement of IL-2 and IL-15 in the development of Treg, taking into account Treg-recirculation. At the age of three weeks, when in wt and IL-15-deficient (but not in IL-2-deficient) mice substantial amounts of recirculating Treg are present in the thymus, we found similarly reduced proportions of newly developed Treg in absence of IL-2 or IL-15, and in absence of both cytokines even less Treg developed. In neonates, when practically no recirculating Treg were found in the thymus, the absence of IL-2 led to substantially more reduced Treg-development than deficiency in IL-15. IL-2 but not IL-15 modulated the CD25, GITR, OX40, and CD73-phenotypes of the thymus-egress-competent and periphery-seeding Treg-population. Interestingly, IL-2 and IL-15 also modulated the TCR-repertoire expressed by developing Treg. Upon transfer into Treg-less Foxp3sf mice, newly developed Treg from IL-2- (and to a much lesser extent IL-15-) deficient mice suppressed immunopathology less efficiently than wt Treg. Taken together, our results firmly establish important non-redundant quantitative and qualitative roles for IL-2 and, to a lesser extent, IL-15 in intrathymic Treg-development.

The Impact of Short-Chain Fatty Acids on Neonatal Regulatory T Cells.

Over the first weeks of life, the neonatal gastrointestinal tract is rapidly colonised by a diverse range of microbial species that come to form the ‘gut microbiota’. Microbial colonisation of the neonatal gut is a well-established regulator of several physiological processes that contribute to immunological protection in postnatal life, including the development of the intestinal mucosa and adaptive immunity. However, the specific microbiota-derived signals that mediate these processes have not yet been fully characterised. Accumulating evidence suggests short-chain fatty acids (SCFAs), end-products of intestinal bacterial metabolism, as one of the key mediators of immune development in early life. Critical to neonatal health is the development of regulatory T (Treg) cells that promote and maintain immunological tolerance against self and innocuous antigens. Several studies have shown that SCFAs can induce the differentiation and expansion of Tregs but also mediate pathological effects in abnormal amounts. However, the exact mechanisms through which SCFAs regulate Treg development and pathologies in early life remain poorly defined. In this review, we summarise the current knowledge surrounding SCFAs and their potential impact on the neonatal immune system with a particular focus on Tregs, and the possible mechanisms through which SCFAs achieve their immune modulatory effect.

Nature vs. nurture: FOXP3, genetics, and tissue environment shape Treg function.

The importance of regulatory T cells (Tregs) in preventing autoimmunity has been well established; however, the precise alterations in Treg function in autoimmune individuals and how underlying genetic associations impact the development and function of Tregs is still not well understood. Polygenetic susceptibly is a key driving factor in the development of autoimmunity, and many of the pathways implicated in genetic association studies point to a potential alteration or defect in regulatory T cell function. In this review transcriptomic control of Treg development and function is highlighted with a focus on how these pathways are altered during autoimmunity. In combination, observations from autoimmune mouse models and human patients now provide insights into epigenetic control of Treg function and stability. How tissue microenvironment influences Treg function, lineage stability, and functional plasticity is also explored. In conclusion, the current efficacy and future direction of Treg-based therapies for Type 1 Diabetes and other autoimmune diseases is discussed. In total, this review examines Treg function with focuses on genetic, epigenetic, and environmental mechanisms and how Treg functions are altered within the context of autoimmunity.

The long and winding road: From mouse linkage studies to a novel human therapeutic pathway in type 1 diabetes.

Autoimmunity involves a loss of immune tolerance to self-proteins due to a combination of genetic susceptibility and environmental provocation, which generates autoreactive T and B cells. Genetic susceptibility affects lymphocyte autoreactivity at the level of central tolerance (e.g., defective, or incomplete MHC-mediated negative selection of self-reactive T cells) and peripheral tolerance (e.g., failure of mechanisms to control circulating self-reactive T cells). T regulatory cell (Treg) mediated suppression is essential for controlling peripheral autoreactive T cells. Understanding the genetic control of Treg development and function and Treg interaction with T effector and other immune cells is thus a key goal of autoimmunity research. Herein, we will review immunogenetic control of tolerance in one of the classic models of autoimmunity, the non-obese diabetic (NOD) mouse model of autoimmune Type 1 diabetes (T1D). We review the long (and still evolving) elucidation of how one susceptibility gene, Cd137, (identified originally via linkage studies) affects both the immune response and its regulation in a highly complex fashion. The CD137 (present in both membrane and soluble forms) and the CD137 ligand (CD137L) both signal into a variety of immune cells (bi-directional signaling). The overall outcome of these multitudinous effects (either tolerance or autoimmunity) depends upon the balance between the regulatory signals (predominantly mediated by soluble CD137 via the CD137L pathway) and the effector signals (mediated by both membrane-bound CD137 and CD137L). This immune balance/homeostasis can be decisively affected by genetic (susceptibility vs. resistant alleles) and environmental factors (stimulation of soluble CD137 production). The discovery of the homeostatic immune effect of soluble CD137 on the CD137-CD137L system makes it a promising candidate for immunotherapy to restore tolerance in autoimmune diseases.

In Vitro Generation of Brain Regulatory T Cells by Co-culturing With Astrocytes.

Regulatory T cells (Tregs) are normally born in the thymus and activated in secondary lymphoid tissues to suppress immune responses in the lymph node and at sites of inflammation. Tregs are also resident in various tissues or accumulate in damaged tissues, which are now called tissue Tregs, and contribute to homeostasis and tissue repair by interacting with non-immune cells. We have shown that Tregs accumulate in the brain during the chronic phase in a mouse cerebral infarction model, and these Tregs acquire the characteristic properties of brain Tregs and contribute to the recovery of neurological damage by interacting with astrocytes. However, the mechanism of tissue Treg development is not fully understood. We developed a culture method that confers brain Treg characteristics in vitro. Naive Tregs from the spleen were activated and efficiently amplified by T-cell receptor (TCR) stimulation in the presence of primary astrocytes. Furthermore, adding IL-33 and serotonin could confer part of the properties of brain Tregs, such as ST2, peroxisome proliferator-activated receptor γ (PPARγ), and serotonin receptor 7 (Htr7) expression. Transcriptome analysis revealed that in vitro generated brain Treg-like Tregs (induced brain Tregs; iB-Tregs) showed similar gene expression patterns as those in in vivo brain Tregs, although they were not identical. Furthermore, in Parkinson’s disease models, in which T cells have been shown to be involved in disease progression, iB-Tregs infiltrated into the brain more readily and ameliorated pathological symptoms more effectively than splenic Tregs. These data indicate that iB-Tregs contribute to our understanding of brain Treg development and could also be therapeutic for inflammatory brain diseases.

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.

Vitamin D3 Priming of Dendritic Cells Shifts Human Neutrophil-Dependent Th17 Cell Development to Regulatory T Cells.

Vitamin D3 (VD3) is a potential adjuvant for use in tolerogenic vaccine formulations that target dendritic cells (DCs) for the treatment of chronic inflammatory disorders, e.g., autoimmune diseases. These disorders are often associated with enhanced activity of IL-17-producing T helper 17 (Th17) cells which develop in a DC-driven and neutrophil-dependent fashion. Here, we investigated the effect of VD3 on Candida albicans-specific human T-cell differentiation, since C. albicans is a model pathogen for Th17 cell development. VD3 priming of DCs restricted neutrophil-dependent Th17 cell development and neutrophil-independent Th1 cell formation from naive CD4+ T cells. In line with this, the production of Th1/Th17-polarizing cytokines IL-12 and IL-23 by DCs was reduced by VD3 priming. Development of both FoxP3+CD127lowCD25+ Tregs and IL-10-producing T cells was significantly enhanced in VD3-primed conditions, even in the presence of neutrophils. ICOS+ Tregs, major IL-10 producers, CD69+FoxP3+, and TIGIT+FoxP3+ Tregs were significantly induced by VD3 priming as well. Our data support the potential use of VD3 as an adjuvant to induce tolerance in the treatment of autoimmune disorders, including those in which neutrophils are involved in pathogenesis, since we show that Treg development is enhanced by VD3 even in the presence of neutrophils, while Th17 cell development is restricted.

The expression landscape of FOXP3 and its prognostic value in breast cancer.

BackgroundForkhead Box Protein 3 (FOXP3), as an essential marker of regulatory T cell (Treg) development, is reportedly overexpressed in invasive breast carcinoma (BRCA) and could be a potential prognostic factor for BRCA. However, the biological function of FOXP3 in BRCA is still unclear. In this study, we comprehensively explored the expression landscape of FOXP3 and its prognostic value in BRCA.MethodsFOXP3 transcriptomic expression data were mainly obtained from The Cancer Genome Atlas (TCGA). The Kaplan-Meier plotter and receiver operating characteristic (ROC) curve were used to assess the prognostic and diagnostic value of FOXP3 in BRCA. UALCAN, cBio-Portal, and MethSurv were used to evaluate the genomic variation of FOXP3. Gene set enrichment analysis (GSEA) was performed to explore the FOXP3 pathways involved in BRCA. Morover, we detected the expression of FOXP3 in 123 BRCA specimens and 5 BRCA cell lines to verify the biological value of FOXP3 in BRCA. The Kaplan-Meier method was adopted for the overall survival (OS) analysis, and a Cox proportional hazards model was used to estimate the hazard ratio (HR) for OS.ResultsFOXP3 was more highly expressed in BRCA than in normal tissues (2.808±1.020 vs. 1.409±0.656, P<0.001), and overexpressed FOXP3 was associated with a better prognosis. The ROC curve demonstrated a significant diagnostic value of FOXP3 in BRCA (area under the ROC curve, AUC: 0.877). Genomic analysis revealed that promoter hypomethylation of FOXP3 may be the underlying mechanism of FOXP3’s upregulation in BRCA. GSEA found that FOXP3 coexpressed genes were mainly involved in the Toll-like receptor pathway, JAK/STAT pathway, cell cycle, and apoptosis. Moreover, high FOXP3 expression was an independent protective factor for OS in our 123 BRCA tissues (HR: 0.367; P=0.036). In vitro, we found that FOXP3 knockdown with siRNA promoted migration and invasion in MCF-7 cells.ConclusionsThis study demonstrated that FOXP3 shows prognostic and diagnostic value for BRCA. We provided evidence that promoter hypomethylation and a high expression of FOXP3 were both related to a favorable prognosis in BRCA, which maybe associated with the Toll-like receptor pathway, JAK/STAT pathway, cell cycle, and apoptosis.

TREGking From Gut to Brain: The Control of Regulatory T Cells Along the Gut-Brain Axis.

The human gastrointestinal tract has an enormous and diverse microbial community, termed microbiota, that is necessary for the development of the immune system and tissue homeostasis. In contrast, microbial dysbiosis is associated with various inflammatory and autoimmune diseases as well as neurological disorders in humans by affecting not only the immune system in the gastrointestinal tract but also other distal organs. FOXP3+ regulatory T cells (Tregs) are a subset of CD4+ helper T cell lineages that function as a gatekeeper for immune activation and are essential for peripheral autoimmunity prevention. Tregs are crucial to the maintenance of immunological homeostasis and tolerance at barrier regions. Tregs reside in both lymphoid and non-lymphoid tissues, and tissue-resident Tregs have unique tissue-specific phenotype and distinct function. The gut microbiota has an impact on Tregs development, accumulation, and function in periphery. Tregs, in turn, modulate antigen-specific responses aimed towards gut microbes, which supports the host–microbiota symbiotic interaction in the gut. Recent studies have indicated that Tregs interact with a variety of resident cells in central nervous system (CNS) to limit the progression of neurological illnesses such as ischemic stroke, Alzheimer’s disease, and Parkinson’s disease. The gastrointestinal tract and CNS are functionally connected, and current findings provide insights that Tregs function along the gut-brain axis by interacting with immune, epithelial, and neuronal cells. The purpose of this study is to explain our current knowledge of the biological role of tissue-resident Tregs, as well as the interaction along the gut-brain axis.

Steroid nuclear receptor coactivator 2 controls immune tolerance by promoting induced Treg differentiation via up-regulating Nr4a2.

Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. While SRC1 inhibits the differentiation of regulatory T cells (Tregs) critical for establishing immune tolerance, we show here that SRC2 stimulates Treg differentiation. SRC2 is dispensable for the development of thymic Tregs, whereas naive CD4+ T cells from mice deficient of SRC2 specific in Tregs (SRC2fl/fl/Foxp3YFP-Cre) display defective Treg differentiation. Furthermore, the aged SRC2fl/fl/Foxp3YFP-Cre mice spontaneously develop autoimmune phenotypes including enlarged spleen and lung inflammation infiltrated with IFNγ-producing CD4+ T cells. SRC2fl/fl/Foxp3YFP-Cre mice also develop severer experimental autoimmune encephalomyelitis (EAE) due to reduced Tregs. Mechanically, SRC2 recruited by NFAT1 binds to the promoter and activates the expression of Nr4a2, which then stimulates Foxp3 expression to promote Treg differentiation. Members of SRC family coactivators thus play distinct roles in Treg differentiation and are potential drug targets for controlling immune tolerance.

The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

SummaryThe most frequent genetic aberration leading to infant ALL (iALL) is the chromosomal translocation t(4;11), generating the fusion oncogenes KMT2A:AFF1 and AFF1:KMT2A, respectively. KMT2A-r iALL displays a dismal prognosis through high relapse rates and relapse-associated mortality. Relapse occurs frequently despite ongoing chemotherapy and without the accumulation of secondary mutations. A rational explanation for the observed chemo-resistance and satisfactory treatment options remain to be elucidated. We found that elevated ICOSLG expression level at diagnosis was associated with inferior event free survival (EFS) in a cohort of 43 patients with t(4;-11) iALL and that a cohort of 18 patients with iALL at relapse displayed strongly increased ICOSLG expression. Furthermore, co-culturing t(4;11) ALL cells (ICOSLGhi) with primary T-cells resulted in the development of Tregs. This was impaired through treatment with a neutralizing ICOSLG antibody. These findings imply ICOSLG (1) as a relapse-predicting biomarker, and (2) as a therapeutic target involved in a potential immune evasion relapse-mechanism of infant t(4;11) ALL.