RNF213 promotes Treg cell differentiation by facilitating K63-linked ubiquitination and nuclear translocation of FOXO1.

Autoreactive CD4+ T helper cells are critical players that orchestrate the immune response both in multiple sclerosis (MS) and in other neuroinflammatory autoimmune diseases. Ubiquitination is a posttranslational protein modification involved in regulating a variety of cellular processes, including CD4+ T cell differentiation and function. However, only a limited number of E3 ubiquitin ligases have been characterized in terms of their biological functions, particularly in CD4+ T cell differentiation and function. In this study, we found that the RING finger protein 213 (RNF213) specifically promoted regulatory T (Treg) cell differentiation in CD4+ T cells and attenuated autoimmune disease development in an FOXO1-dependent manner. Mechanistically, RNF213 interacts with Forkhead Box Protein O1 (FOXO1) and promotes nuclear translocation of FOXO1 by K63-linked ubiquitination. Notably, RNF213 expression in CD4+ T cells was induced by IFN-ß and exerts a crucial role in the therapeutic efficacy of IFN-ß for MS. Together, our study findings collectively emphasize the pivotal role of RNF213 in modulating adaptive immune responses. RNF213 holds potential as a promising therapeutic target for addressing disorders associated with Treg cells.

FOXP3 snatches transcription factors depending on the context.

FOXP3 hijacks DNA-binding proteins to regulate gene expression. In this issue of JEM, He et al. (https://doi.org/10.1084/jem.20232068) propose a dynamic model in which FOXP3 associates with DNA-binding proteins to regulate Treg cell function in response to environmental cues.

Interleukin-17 directly stimulates tumor infiltrating Tregs to prevent cancer development.

Background: Interleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance, but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses. Methods: We generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice (Il17ra ΔTreg mice). Subsequently, we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer (Cdx2-Cre +/Apc F/+), to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors, and compared to those of human tumor infiltrating Treg cells. Results: IL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA, specifically in Tregs, resulted in increased Th17 cells, and exacerbated tumor development. Mechanistically, tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation, maturation, and suppression function, and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming, we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing, and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function. Conclusion: IL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC.

Immune interactions and regulation with CD39+ extracellular vesicles from platelet concentrates.

Introduction: CD39 plays an important role in the immunoregulation and inhibition of effector cells. It is expressed on immune cells, including Tregs, and on extracellular vesicles (EVs) budding from the plasma membrane. Platelet transfusion may induce alloimmunization against HLA-I antigens, leading to refractoriness to platelet transfusion with severe consequences for patients. Tregs may play a key role in determining whether alloimmunization occurs in patients with hematologic disorders. We hypothesized that CD39+ EVs might play an immunoregulatory role, particularly in the context of platelet transfusions in patients with hematologic disorders. Such alloimmunization leads to the production of alloantibodies and is sensitive to the regulatory action of CD39. Methods: We characterized CD39+ EVs in platelet concentrates by flow cytometry. The absolute numbers and cellular origins of CD39+ EVs were evaluated. We also performed functional tests to evaluate interactions with immune cells and their functions. Results: We found that CD39+ EVs from platelet concentrates had an inhibitory phenotype that could be transferred to the immune cells with which they interacted: CD4+ and CD8+ T lymphocytes (TLs), dendritic cells, monocytes, and B lymphocytes (BLs). Moreover, the concentration of CD39+ EVs in platelet concentrates varied and was very high in 10% of concentrates. The number of these EVs present was determinant for EV-cell interactions. Finally, functional interactions were observed with BLs, CD4+ TLs and CD39+ EVs for immunoglobulin production and lymphoproliferation, with potential implications for the immunological management of patients.

Complex Role of Regulatory T Cells (Tregs) in the Tumor Microenvironment: Their Molecular Mechanisms and Bidirectional Effects on Cancer Progression.

Regulatory T cells (Tregs) possess unique immunosuppressive activity among CD4-positive T cells. Tregs are ubiquitously present in mammals and function to calm excessive immune responses, thereby suppressing allergies or autoimmune diseases. On the other hand, due to their immunosuppressive function, Tregs are thought to promote cancer progression. The tumor microenvironment (TME) is a multicellular system composed of many cell types, including tumor cells, infiltrating immune cells, and cancer-associated fibroblasts (CAFs). Within this environment, Tregs are recruited by chemokines and metabolic factors and impede effective anti-tumor responses. However, in some cases, their presence can also improve patient's survival rates. Their functional consequences may vary across tumor types, locations, and stages. An in-depth understanding of the precise roles and mechanisms of actions of Treg is crucial for developing effective treatments, emphasizing the need for further investigation and validation. This review aims to provide a comprehensive overview of the complex and multifaceted roles of Tregs within the TME, elucidating cellular communications, signaling pathways, and their impacts on tumor progression and highlighting their potential anti-tumor mechanisms through interactions with functional molecules.

Effect of Short Peptides of Pregnancy-Specific ß1-Glycoprotein on Differentiation of Human Regulatory T Cells In Vitro and on Their Cytokine Profile.

Pregnancy-specific ß1-glycoprotein (PSG), one of the most important proteins of pregnancy, has a pronounced immunosuppressive effect. Short peptides of PSG, the so-called SLiMs (short linear motifs), are promising molecules for mild immunosuppression. We studied in vitro effect of short PSG peptides (YACS, YQCE, YVCS, and YECE) on differentiation and cytokine profile of human T-regulatory lymphocytes (Treg). T helpers isolated from the peripheral blood and polarized into the Treg phenotype with a T-cell activator (anti-CD2/3/28) and the cytokines IL-2 and transforming grown factor ß (TGFß) were used. PSG peptides were shown to have no direct modulatory effect on Treg differentiation in a culture of CD4+ cells polarized to the Treg phenotype. At the same time, PSG peptides had no effect on the viability and number of CD4+ cells in the in vitro culture. PSG peptides also had no effect on the levels of TNFα, IL-8, IL-2, macrophage inflammatory protein 1ß, IL-17, IL-10, IL-6, granulocyte-macrophage CSF, monocyte chemoattractant protein 1, IL-13, IL-5, IL-7, IL-12(p70), IL-1ß, granulocyte CSF, IL-4, but decreased IFNγ levels. The observed ability of the YQCE peptide to reduce the production of this proinflammatory Th1 cytokine by T helper cells can be interpreted as a positive effect. Our findings can be used for further development of safe peptide drugs based on SLiMs sequences.

Extracellular vesicles from seminal plasma interact with T cells in vitro and drive their differentiation into regulatory T-cells.

Seminal plasma induces immune tolerance towards paternal allogenic antigens within the female reproductive tract and during foetal development. Recent evidence suggests a role for extracellular vesicles in seminal plasma (spEVs). We isolated spEVs from seminal plasma that was donated by vasectomized men, thereby excluding any contributions from the testis or epididymis. Previous analysis demonstrated that such isolated spEVs originate mainly from the prostate. Here we observed that when isolated fluorescently labelled spEVs were mixed with peripheral blood mononuclear cells, they were endocytosed predominantly by monocytes, and to a lesser extent also by T-cells. In a mixed lymphocyte reaction, T-cell proliferation was inhibited by spEVs. A direct effect of spEVs on T-cells was demonstrated when isolated T cells were activated by anti-CD3/CD28 coated beads. Again, spEVs interfered with T cell proliferation, as well as with the expression of CD25 and the release of IFN-γ, TNF, and IL-2. Moreover, spEVs stimulated the expression of Foxp3 and IL-10 by CD4+CD25+CD127- T cells, indicating differentiation into regulatory T-cells (Tregs). Prior treatment of spEVs with proteinase K revoked their effects on T-cells, indicating a requirement for surface-exposed spEV proteins. The adenosine A2A receptor-specific antagonist CPI-444 also reduced effects of spEVs on T-cells, consistent with the notion that the development of Tregs and their immune suppressive functions are under the influence of adenosine-A2A receptor signalling. We found that adenosine is highly enriched in spEVs and propose that spEVs are targeted to and endocytosed by T-cells, after which they may release their adenosine content into the lumen of endosomes, thus allowing endosome-localized A2A receptor signalling in spEVs targeted T-cells. Collectively, these data support the idea that spEVs can prime T cells directly for differentiation into Tregs.

Mesenteric lymph nodes: a critical site for the up-regulatory effect of hUC-MSCs on Treg cells by producing TGF-ß1 in colitis treatment.

BACKGROUND: Mesenchymal stem cells (MSCs) demonstrate a wide range of therapeutic capabilities in the treatment of inflammatory bowel disease (IBD). The intraperitoneal injection of MSCs has exhibited superior therapeutic efficacy on IBD than intravenous injection. Nevertheless, the precise in vivo distribution of MSCs and their biological consequences following intraperitoneal injection remain inadequately understood. Additional studies are required to explore the correlation between MSCs distribution and their biological effects. METHODS: First, the distribution of human umbilical cord MSCs (hUC-MSCs) and the numbers of Treg and Th17 cells in mesenteric lymph nodes (MLNs) were analyzed after intraperitoneal injection of hUC-MSCs. Subsequently, the investigation focused on the levels of transforming growth factor beta1 (TGF-ß1), a key cytokine to the biology of both Treg and Th17 cells, in tissues of mice with colitis, particularly in MLNs. The study also delved into the impact of hUC-MSCs therapy on Treg cell counts in MLNs, as well as the consequence of TGFB1 knockdown hUC-MSCs on the differentiation of Treg cells and the treatment of IBD. RESULTS: The therapeutic effectiveness of intraperitoneally administered hUC-MSCs in the treatment of colitis was found to be significant, which was closely related to their quick migration to MLNs and secretion of TGF-ß1. The abundance of hUC-MSCs in MLNs of colitis mice is much higher than that in other organs even the inflamed sites of colon. Intraperitoneal injection of hUC-MSCs led to a significant increase in the number of Treg cells and a decrease in Th17 cells especially in MLNs. Furthermore, the concentration of TGF-ß1, the key cytokine for Treg differentiation, were also found to be significantly elevated in MLNs after hUC-MSCs treatment. Knockdown of TGFB1 in hUC-MSCs resulted in a noticeable reduction of Treg cells in MLNs and the eventually failure of hUC-MSCs therapy in colitis. CONCLUSIONS: MLNs may be a critical site for the regulatory effect of hUC-MSCs on Treg/Th17 cells and the therapeutic effect on colitis. TGF-ß1 derived from hUC-MSCs promotes local Treg differentiation in MLNs. This study will provide new ideas for the development of MSC-based therapeutic strategies in IBD patients.

[Overexpression of ubiquitin-conjugating enzyme 2T induces radiotherapy resistance in hepatocellular carcinoma by enriching regulatory T cells in the tumor microenvironment].

OBJECTIVE: To investigate the effect of overexpression of ubiquitin-conjugating enzyme 2T (UBE2T) on radiosensitivity of hepatocellular carcinoma (HCC). METHODS: Hepa1-6 cells were transfected with a UBE2T-overexpressing or a control lentiviral vector, and the changes in their radiotherapy sensitivity and concentrations of glucose and lactate in the supernatant were assessed using colony-forming assay and colorimetric assay. The transfected cells were inoculated subcutaneously in nude mice or C57BL/6 mice, and tumor growth following irradiation were recorded. The xenografts were collected for analyzing infiltration of CD4+ T cells and regulatory T cells (Tregs) using flow cytometry and detecting expressions of HK1 and LDHA using Western blotting. The correlations of UBE2T expression with immune cell infiltration, glycolysis and Tregs in HCC were analyzed using CIBERSORT algorithm and TCGA database, and the results were verified in a co-culture system of Hepa1-6 cells and Tregs. RESULTS: UBE2T overexpression caused radiotherapy resistance in both cultured Hepa1-6 cells and xenografts in the tumor-bearing mouse models (especially in C57BL/6 mice). CIBERSORT analysis suggested that a high expression of UBE2T was associated with increased percentages of dendritic cells, T follicular helper cells, M2 macrophages, monocytes, lymphocytes and Tregs in HCC. The UBE2T-overexpressing xenografts showed an increased percentage of Tregs and enhanced expressions of HK1 and LDHA, and irradiation increased infiltration of CD4+ T cells and Tregs in the tumor microenvironment. Hepa1-6 cells overexpressing UBE2T showed a decreased glucose concentration and an increased lactate concentration. GSEA analysis suggested that a high UBE2T expression was positively correlated with increased glycolysis and Tregs infiltration in HCC. In the cell co-culture system, UBE2T overexpression significantly enhanced lactate production, proliferation and immunosuppressive functions of Tregs. CONCLUSION: A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.

miPEP31 alleviates Ang II-induced hypertension in mice by occupying Cebpα binding sites in the pri-miR-31 promoter.

BACKGROUND: Previous studies have shown that peptides encoded by noncoding RNAs (ncRNAs) can be used as peptide drugs to alleviate diseases. We found that microRNA-31 (miR-31) is involved in the regulation of hypertension and that the peptide miPEP31, which is encoded by the primary transcript of miR-31 (pri-miR-31), can inhibit miR-31 expression. However, the role and mechanism of miPEP31 in hypertension have not been elucidated. METHODS: miPEP31 expression was determined by western blot analysis. miPEP31-deficient mice (miPEP31-/-) were used, and synthetic miPEP31 was injected into Ang II-induced hypertensive mice. Blood pressure was monitored through the tail-cuff method. Histological staining was used to evaluate renal damage. Regulatory T (Treg) cells were assessed by flow cytometry. Differentially expressed genes were analysed through RNA sequencing. The transcription factors were predicted by JASPAR. Luciferase reporter and electrophoretic mobility shift assays (EMSAs) were used to determine the effect of pri-miR-31 on the promoter activity of miPEP31. Images were taken to track the entry of miPEP31 into the cell. RESULTS: miPEP31 is endogenously expressed in target organs and cells related to hypertension. miPEP31 deficiency exacerbated but exogenous miPEP31 administration mitigated the Ang II-induced systolic blood pressure (SBP) elevation, renal impairment and Treg cell decreases in the kidney. Moreover, miPEP31 deletion increased the expression of genes related to Ang II-induced renal fibrosis. miPEP31 inhibited the transcription of miR-31 and promoted Treg differentiation by occupying the Cebpα binding site. The minimal functional domain of miPEP31 was identified and shown to regulate miR-31. CONCLUSION: miPEP31 was identified as a potential therapeutic peptide for treating hypertension by promoting Treg cell differentiation in vivo. Mechanistically, we found that miPEP31 acted as a transcriptional repressor to specifically inhibit miR-31 transcription by competitively occupying the Cebpα binding site in the pri-miR-31 promoter. Our study highlights the significant therapeutic effect of miPEP31 on hypertension and provides novel insight into the role and mechanism of miPEPs.

[Terminalia chebula water extract reduces joint inflammation by regulating cellular immunity in spleen cells of collagen-induced arthritis (CIA) rats through up-regulation of PD-1/PD-L1].

Objective To investigate the effect of Terminalia chebula water extract (TCWE) on the cellular immunity and PD-1/PD-L1 pathway in rats with collagen-induced arthritis (CIA). Methods SD rats were randomly divided into four groups: a control group, a CIA group, a TCWE group and a methotrexate (MTX) group, with 15 rats in each group. Except for the control group, SD rats in other groups were subcutaneously injected with type II collagen to establish the model of collagen-induced arthritis (CIA). The rats in the TCWE group were treated with 20 mg/(kg.d) TCWE and the rats in the MTX group were treated with 1.67 mg/(kg.d) MTX. After 14 days of treatment, the cartilage morphology was examined using hematoxylin-eosin (HE) staining, and splenic T lymphocyte apoptosis and Treg/Th17 cell ratio were detected by flow cytometry. The mRNA expressions of retinoid-related orphan nuclear receptor γt (RORγt), forkhead box P3 (FOXP3), PD-1 and PD-L1 in spleen were detected by reverse transcription PCR. The expression and localization of RORγt and FOXP3 were detected by immunohistochemical staining. The protein expressions of PD-1 and PD-L1 in splenic lymphocytes were detected by Western blot, and the levels of serum interleukin 17 (IL-17) and transforming growth factor ß (TGF-ß) in rats were detected by ELISA. Results Compared with CIA group, the pathological changes of cartilage and synovium were significantly alleviated in the TCWE group and the MTX group. Both the apoptosis rate of T lymphocytes in spleen and the ratio of Treg/Th17 cells increased. The expression of RORγt decreased, while the expressions of FOXP3, PD-1 and PD-L1 increased in spleen lymphocytes. The level of serum IL-17 decreased, while the level of serum TGF-ß increased. Conclusion TCWE treatment may activate PD-1/PD-L1 pathway in spleen cells to regulate cellular immunity, thus reducing cartilage injury in CIA rats.

Mesenchymal stem cells-derived extracellular vesicles ameliorate lupus nephritis by regulating T and B cell responses.

BACKGROUND: Human umbilical cord mesenchymal stem cells-derived extracellular vesicles (hUCMSC-EVs) have potent immunomodulatory properties similar to parent cells. This study investigated the therapeutic effects and immunomodulatory mechanisms of hUCMSC-EVs in an experimental lupus nephritis model. METHODS: The hUCMSC-EVs were isolated by using differential ultracentrifugation. In vivo, the therapeutic effects of hUCMSC-EVs in lupus-prone MRL/lpr mice were investigated, and the mechanisms of treatment were explored according to the abnormal T and B cell responses among both the spleen and kidney. RESULTS: MRL/lpr mice treated with hUCMSC-EVs reduced proteinuria extent, serum creatinine and renal pathological damage; decreased splenic index and serum anti-dsDNA IgG level; and improved survival rate. hUCMSC-EVs lowered the percentage of T helper (Th)1 cells, double-negative T (DNT) cells, and plasma cells among splenocytes; inhibited the infiltration of Th17 cells but promoted regulatory T (Treg) cells in the kidney, followed by a reduction in pro-inflammatory cytokine levels(IFN-γ, IL-2, IL-6, IL-21, and IL-17 A). In addition, hUCMSC-EVs inhibited the activation of STAT3 and down-regulated IL-17 A protein levels in the kidney. CONCLUSION: The results of this study demonstrated that hUCMSC-EVs had therapeutic effects on experimental lupus nephritis (LN) by regulating Th1/Th17/Treg imbalance and inhibiting DNT and plasma cells. Additionally, hUCMSC-EVs inhibited Th17 cell differentiation in kidney by regulating the IL-6/STAT3/IL-17 signal pathway, which might be an important mechanism for alleviating renal injury. Taken together, we demonstrated that hUCMSC-EVs regulating T and B cell immune responses might represent a novel mechanism of hUCMSCs in treating LN, thus providing a new strategy for treating LN.

STING trafficking activates MAPK-CREB signaling to trigger regulatory T cell differentiation.

The Type-I interferon (IFN-I) response is the major outcome of stimulator of interferon genes (STING) activation in innate cells. STING is more abundantly expressed in adaptive T cells; nevertheless, its intrinsic function in T cells remains unclear. Intriguingly, we previously demonstrated that STING activation in T cells activates widespread IFN-independent activities, which stands in contrast to the well-known STING-mediated IFN response. Here, we have identified that STING activation induces regulatory T cells (Tregs) differentiation independently of IRF3 and IFN. Specifically, the translocation of STING from the endoplasmic reticulum to the Golgi activates mitogen-activated protein kinase (MAPK) activity, which subsequently triggers transcription factor cAMP response element-binding protein (CREB) activation. The activation of the STING-MAPK-CREB signaling pathway induces the expression of many cytokine genes, including interleukin-2 (IL-2) and transforming growth factor-beta 2 (TGF-ß2), to promote the Treg differentiation. Genetic knockdown of MAPK p38 or pharmacological inhibition of MAPK p38 or CREB markedly inhibits STING-mediated Treg differentiation. Administration of the STING agonist also promotes Treg differentiation in mice. In the Trex1-/- autoimmune disease mouse model, we demonstrate that intrinsic STING activation in CD4+ T cells can drive Treg differentiation, potentially counterbalancing the autoimmunity associated with Trex1 deficiency. Thus, STING-MAPK-CREB represents an IFN-independent signaling axis of STING that may have profound effects on T cell effector function and adaptive immunity.

Dynamic Foxp3-chromatin interaction controls tunable Treg cell function.

Nuclear factor Foxp3 determines regulatory T (Treg) cell fate and function via mechanisms that remain unclear. Here, we investigate the nature of Foxp3-mediated gene regulation in suppressing autoimmunity and antitumor immune response. Contrasting with previous models, we find that Foxp3-chromatin binding is regulated by Treg activation states, tumor microenvironment, and antigen and cytokine stimulations. Proteomics studies uncover dynamic proteins within Foxp3 proximity upon TCR or IL-2 receptor signaling in vitro, reflecting intricate interactions among Foxp3, signal transducers, and chromatin. Pharmacological inhibition and genetic knockdown experiments indicate that NFAT and AP-1 protein Batf are required for enhanced Foxp3-chromatin binding in activated Treg cells and tumor-infiltrating Treg cells to modulate target gene expression. Furthermore, mutations at the Foxp3 DNA-binding domain destabilize the Foxp3-chromatin association. These representative settings delineate context-dependent Foxp3-chromatin interaction, suggesting that Foxp3 associates with chromatin by hijacking DNA-binding proteins resulting from Treg activation or differentiation, which is stabilized by direct Foxp3-DNA binding, to dynamically regulate Treg cell function according to immunological contexts.

The Expression of Forkhead Box P3 T Regulatory Lymphocytes as a Prognostic Factor in Malignant Melanomas.

Since transcription factor Forkhead Box P3 (FoxP3) was identified as a specific regulatory T cell (Treg) marker, researchers have scrutinized its value as a potential novel therapeutic target or a prognostic factor in various types of cancer with inconsistent results. The present analysis was performed to assess the influence of Treg FoxP3 expression on the prognosis of primary melanoma and to evaluate the correlations with various clinicopathological prognostic factors. We analyzed all eligible patients with stage pT3 primary malignant melanomas treated in a tertiary cancer center. Immunohistochemical staining for Treg FoxP3 expression was performed on retrospectively identified paraffin blocks and subsequently correlated with the outcomes of the patients. A total of 81% of the patients presented a positive Treg FoxP3 expression, being correlated with a higher risk of lymph node metastasis, tumor relapse, and death. Moreover, positive expression was statistically associated with a shorter OS. The tumor relapse rate was estimated at 36.7%. A positive expression of Treg FoxP3 and lymph node metastasis were associated with a higher risk of death based on multivariate analysis. Treg FoxP3 expression may be used as an independent prognostic factor in patients with malignant melanoma to evaluate tumor progression and survival.

Defining Human Regulatory T Cells beyond FOXP3: The Need to Combine Phenotype with Function.

Regulatory T cells (Tregs) are essential to maintain immune homeostasis by promoting self-tolerance. Reduced Treg numbers or functionality can lead to a loss of tolerance, increasing the risk of developing autoimmune diseases. An overwhelming variety of human Tregs has been described, based on either specific phenotype, tissue compartment, or pathological condition, yet the bulk of the literature only addresses CD25-positive and CD127-negative cells, coined by naturally occurring Tregs (nTregs), most of which express the transcription factor Forkhead box protein 3 (FOXP3). While the discovery of FOXP3 was seminal to understanding the origin and biology of nTregs, there is evidence in humans that not all T cells expressing FOXP3 are regulatory, and that not all Tregs express FOXP3. Namely, the activation of human T cells induces the transient expression of FOXP3, irrespective of whether they are regulatory or inflammatory effectors, while some induced T cells that may be broadly defined as Tregs (e.g., Tr1 cells) typically lack demethylation and do not express FOXP3. Furthermore, it is unknown whether and how many nTregs exist without FOXP3 expression. Several other candidate regulatory molecules, such as GITR, Lag-3, GARP, GPA33, Helios, and Neuropilin, have been identified but subsequently discarded as Treg-specific markers. Multiparametric analyses have uncovered a plethora of Treg phenotypes, and neither single markers nor combinations thereof can define all and only Tregs. To date, only the functional capacity to inhibit immune responses defines a Treg and distinguishes Tregs from inflammatory T cells (Teffs) in humans. This review revisits current knowledge of the Treg universe with respect to their heterogeneity in phenotype and function. We propose that it is unavoidable to characterize human Tregs by their phenotype in combination with their function, since phenotype alone does not unambiguously define Tregs. There is an unmet need to align the expression of specific markers or combinations thereof with a particular suppressive function to coin functional Treg entities and categorize Treg diversity.

CTLA-4-expressing ILC3s restrain interleukin-23-mediated inflammation.

Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1-4. However, the mechanisms that shape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5-7, contributes to chronic inflammation.

9-cis-retinoic acid signaling in Sertoli cells regulates their immunomodulatory function to control lymphocyte physiology and Treg differentiation.

BACKGROUND: Testis is an immune privileged organ, which prevents the immune response against sperm antigens and inflammation. Testicular cells responsible for immune tolerance are mainly Sertoli cells, which form the blood-testis barrier and produce immunosuppressive factors. Sertoli cells prevent inflammation in the testis and maintain immune tolerance by inhibiting proliferation and inducing lymphocyte apoptosis. It has been shown that 9-cis-retinoic acid (9cRA) blocks ex vivo apoptosis of peripheral blood lymphocytes and promotes the differentiation of Treg cells in the gut. However, the role of retinoid signaling in regulating the immune privilege of the testes remains unknown. OBJECTIVE: The aim of this study was to determine whether 9cRA, acting via the retinoic acid receptors (RAR) and the retinoic X receptors (RXR), controls the immunomodulatory functions of Sertoli cells by influencing the secretion of anti-inflammatory/pro-inflammatory factors, lymphocyte physiology and Treg cell differentiation. METHODS: Experiments were performed using in vitro model of co-cultures of murine Sertoli cells and T lymphocytes. Agonists and antagonists of retinoic acid receptors were used to inhibit/stimulate retinoid signaling in Sertoli cells. RESULTS: Our results have demonstrated that 9cRA inhibits the expression of immunosuppressive genes and enhances the expression of pro-inflammatory factors in Sertoli cells and lymphocytes, increases lymphocyte viability and decreases apoptosis rate. Moreover, we have found that 9cRA blocks lymphocyte apoptosis acting through both RAR and RXR and inhibiting FasL/Fas/Caspase 8 and Bax/Bcl-2/Caspase 9 pathways. Finally, we have shown that 9cRA signaling in Sertoli cells inhibits Treg differentiation. CONCLUSION: Collectively, our results indicate that retinoid signaling negatively regulates immunologically privileged functions of Sertoli cells, crucial for ensuring male fertility. 9cRA inhibits lymphocyte apoptosis, which can be related to the development of autoimmunity, inflammation, and, in consequence, infertility.

Matrine Ameliorates DSS-Induced Colitis by Suppressing Inflammation, Modulating Oxidative Stress and Remodeling the Gut Microbiota.

Matrine (MT) possesses anti-inflammatory, anti-allergic and antioxidative properties. However, the impact and underlying mechanisms of matrine on colitis are unclear. The purpose of this research was to examine the protective impact and regulatory mechanism of matrine on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. MT alleviated DSS-induced UC by inhibiting weight loss, relieving colon shortening and reducing the disease activity index (DAI). Moreover, DSS-induced intestinal injury and the number of goblet cells were reversed by MT, as were alterations in the expression of zonula occludens-1 (ZO-1) and occludin in colon. Simultaneously, matrine not only effectively restored DSS-induced oxidative stress in colonic tissues but also reduced the production of inflammatory cytokines. Furthermore, MT could treat colitis mice by regulating the regulatory T cell (Treg)/T helper 17 (Th17) cell imbalance. We observed further evidence that MT alleviated the decrease in intestinal flora diversity, reduced the proportion of Firmicutes and Bacteroidetes, decreased the proportion of Proteobacteria and increased the relative abundance of Lactobacillus and Akkermansia in colitis mice. In conclusion, these results suggest that MT may mitigate DSS-induced colitis by enhancing the colon barrier integrity, reducing the Treg/Th17 cell imbalance, inhibiting intestinal inflammation, modulating oxidative stress and regulating the gut microbiota. These findings provide strong evidence for the development and application of MT as a dietary treatment for UC.

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.