Suppressive Activity Of Treg Cells Research Articles

Semaphorin 4A Stabilizes Human Regulatory T Cell Phenotype via Plexin B1.

We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T (Treg) cell numbers in vivo; however, the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T cell lines (HuT78 and HuT102), human PBMCs, and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T effector-like phenotype (CD4+CD25lowFoxp3-), whereas HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. HuT102 cells expressed Sema4A counter receptor Plexin B1, whereas HuT78 cells were Sema4A+. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both NRP-1 and -2. However, NRP-1 and Sema4A were detected on CD3negativeCD4intermediate human monocytes. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMCs and CD4+ T cells, which were NRP-1negative but PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before recombinant Sema4A addition significantly decreased Treg cell numbers as compared with cultures with recombinant Sema4A alone. Sema4A was as effective as TGF-β in inducible Treg cell induction from CD4+CD25depleted cells but did not enhance Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases. ImmunoHorizons, 2019, 3: 71-87.

Tumor Necrosis Factor and Regulatory T Cells.

CD4+CD25+FoxP3+ regulatory T (Treg) cells play major roles in the maintenance of immune homeostasis. In this review, we comprehensively describe the relationship between tumor necrosis factor (TNF) and Treg cells, focusing on the effects of TNF on Treg cells and on TNF-producing Treg cells. Contradictory results have been reported for the effect of TNF on the suppressive activity of Treg cells. In patients with rheumatoid arthritis, TNF has been shown to reduce the suppressive activity of Treg cells. Meanwhile, however, TNF has also been reported to maintain the suppressive activity of Treg cells via a TNFR2-mediated mechanism. In addition, Treg cells have been found to acquire the ability to produce TNF under inflammatory conditions, such as acute viral hepatitis. These TNF-producing Treg cells exhibit T helper 17-like features and hold significance in various human diseases.

α‐galactosylceramide generates lung regulatory T cells through the activated natural killer T cells in mice

Our previous study showed that intraperitoneal injection of α‐galactosylceramide (α‐GalCer) has the ability to activate lung iNKT cells, but α‐GalCer‐activated iNKT cells do not result in airway inflammation in wild‐type (WT) mice. Many studies showed that iNKT cells had the capacity to induce Treg cells, which gave rise to peripheral tolerance. Therefore, we examined the influence of intraperitoneal administration of α‐GalCer on the expansion and suppressive activity of lung Treg cells using iNKT cell‐knockout mice and co‐culture experiments in vitro. We also compared airway inflammation and airway hyperresponsiveness (AHR) after α‐GalCer administration in specific anti‐CD25 mAb‐treated mice. Our data showed that intraperitoneal injection of α‐GalCer could promote the expansion of lung Treg cells in WT mice, but not in iNKT cell‐knockout mice. However, α‐GalCer administration could not boost suppressive activity of Treg cells in WT mice and iNKT cell‐knockout mice. Interestingly, functional inactivation of Treg cells could induce airway inflammation and AHR in WT mice treated with α‐GalCer. Furthermore, α‐GalCer administration could enhance iNKT cells to secrete IL‐2, and neutralization of IL‐2 reduced the expansion of Treg cells in vivo and in vitro. Thus, intraperitoneal administration of α‐GalCer can induce the generation of lung Treg cells in mice through the release of IL‐2 by the activated iNKT cells.

Abstract 713: Upregulation of immune checkpoint molecules on Treg cells in tumor microenvironment reinforces immune exhaustion in cancer patients

Abstract Purpose: Regulatory T (Treg) cells perform the immune suppressive function in cancer, but their suppressive mechanism in tumor microenvironment (TME) has not been clearly elucidated. We aim to identify the phenotype and functional mechanism of Treg cells in TME from cancer patients. Experimental Design: We collected 72 malignant effusion (ME) and peripheral blood (PB) specimens from stage IV cancer patients and 10 tumor tissue (TM) and PB from lung cancer patients who underwent surgery. Lymphocytes from ME, TM, and PB were analyzed for subtype of Tconv and Treg cells, and their expressions of immune checkpoint (IC) molecules including programmed death (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), and T cell immunoglobulin containing molecule-3 (TIM-3) using by flow cytometry. To examine the functional role of PD-1 expressed Treg cells, TC-1 lung cancer mouse model was used. Result: Upregulation of PD-1 on Treg cells in TM and ME compared to PB was even more distinguishable than that on CD4+ and CD8+ Tconv cells. To clarify the characteristics of tumor infiltrating Treg cells, we comprehensively examined the characteristics of Treg cells from tumor, peri-tumor, and PB in lung cancer patients by analyzing the expression of IC molecules including PD-1, TIM-3, CTLA-4, and TIGIT, of which PD-1 is the highest expressed on tumor infiltrating Treg cells. To investigate the mechanism by which Treg cells mediate immune suppression, we compared the suppressive activity of Treg cells expressing high and low levels of PD-1 by co-culturing each population with naïve CD8+ T cells with or without αCD3/CD28 stimulation. This was more potently inhibited in co-cultures with PD-1high tumor infiltrating Treg cells than in those with PD-1low Treg cells. PD-1 blocked tumor infiltrating Treg cells demonstrated a significantly decreased suppressive function in the proliferation of CD8+ cells and their IFN-γ production. These results implicate that PD-1 expressed tumor infiltrating Treg confers the suppressive function of proliferation in CD8+ T cells through PD-1: PD-L1 interaction. Conclusion: Our study evidently demonstrated that upregulated PD-1 on tumor infiltrating Treg cells and their PD-1: PD-L1 interaction could be potential cause of T cell suppression, which might be helpful to completely understand their suppressive mechanism in cancer patients. Citation Format: Hye Ryun Kim, Hyo Jin Park, Jimin Son, Hyo Sup Shim, Byoung Chul Cho, Sun Young Rha, Sang-Jun Ha. Upregulation of immune checkpoint molecules on Treg cells in tumor microenvironment reinforces immune exhaustion in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 713.

End‐stage renal disease, dialysis, kidney transplantation and their impact on CD4+ T‐cell differentiation

Premature aging of both CD4+ regulatory T (Treg) and CD4+ responder-T (Tresp) cells in patients with end-stage renal disease (ESRD) is expected to affect the success of later kidney transplantation. Both T-cell populations are released from the thymus as inducible T-cell co-stimulator-positive (ICOS+ ) and ICOS- recent thymic emigrant (RTE) Treg/Tresp cells, which differ primarily in their proliferative capacities. In this study, we analysed the effect of ESRD and subsequent renal replacement therapies on the differentiation of ICOS+ and ICOS- RTE Treg/Tresp cells into ICOS+ CD31- or ICOS- CD31- memory Treg/Tresp cells and examined whether diverging pathways affected the suppressive activity of ICOS+ and ICOS- Treg cells in co-culture with autologous Tresp cells. Compared with healthy controls, we found an increased differentiation of ICOS+ RTE Treg/Tresp cells and ICOS- RTE Treg cells through CD31+ memory Treg/Tresp cells into CD31- memory Treg/Tresp cells in ESRD and dialysis patients. In contrast, ICOS- RTE Tresp cells showed an increased differentiation via ICOS- mature naive (MN) Tresp cells into CD31- memory Tresp cells. Thereby, the ratio of ICOS+ Treg/ICOS+ Tresp cells was not changed, whereas that of ICOS- Treg/ICOS- Tresp cells was significantly increased. This differentiation preserved the suppressive activity of both Treg populations in ESRD and partly in dialysis patients. After transplantation, the increased differentiation of ICOS+ and ICOS- RTE Tresp cells proceeded, whereas that of ICOS+ RTE Treg cells ceased and that of ICOS- RTE Treg cells switched to an increased differentiation via ICOS- MN Treg cells. Consequently, the ratios of ICOS+ Treg/ICOS+ Tresp cells and of ICOS- Treg/ICOS- Tresp cells decreased significantly, reducing the suppressive activity of Treg cells markedly. Our data reveal that an increased tolerance-inducing differentiation of ICOS+ and ICOS- Treg cells preserves the functional activity of Treg cells in ESRD patients, but this cannot be maintained during long-term renal replacement therapy.

Forkhead box protein 3 demethylation is associated with tolerance induction in peanut-induced intestinal allergy

Regulatory T (Treg) cells play an essential role in the maintenance of immune homeostasis in allergic diseases. We sought to define the mechanisms underlying induction of tolerance to peanut protein and prevention of the development of peanut allergy. High or low doses of peanut extract were administered to pups every day for 2weeks before peanut sensitization and challenge. After challenge, symptoms, Treg cell numbers, and forkhead box protein 3 (Foxp3), TH2 and TH17 cytokine, and Tgfβ expression in mesenteric lymph node (MLN) CD4+ T cells and jejunum were monitored. Treg cell suppressive activity and Foxp3 methylation in MLN CD4+ T cells were assayed. Feeding high but not low doses of peanut before sensitization induced tolerance, as demonstrated by prevention of diarrhea and peanut-specific IgE responses, increases in the percentage of CD4+CD25+FoxP3+ cells in MLNs, and Foxp3 mRNA and protein expression in CD4+ cells from MLNs or jejunum. Feeding high doses of peanut before sensitization decreased percentages of CD3+CD4+IL-13+ and CD3+CD4+IL-17+ cells in MLNs and decreased Il13 and Il17a and increased Tgfβ mRNA expression in the jejunum; numbers of CD103+ dendritic cells in MLNs were significantly increased. Treg cell suppression was shown to be antigen specific. Foxp3 methylation was increased in peanut extract-sensitized and challenged mice, whereas in tolerized mice levels were significantly reduced. Feeding high doses of peanut to pups induced tolerance to peanut protein. Foxp3 demethylation was associated with tolerance induction, indicating that Treg cells play an important role in the regulation of peanut sensitivity and maintenance of immune homeostasis.

What's Hot, What's New From the 2016 American Transplant Congress.

From June 11-15, 2016 the American Transplant Congress, the joint meeting of the American Society of Transplantation and the American Society of Transplant Surgeons, held its annual meeting in Boston, MA. The meeting, attended by 5200 registrants, included pre-meeting conferences, focused topic sessions, and hundreds of high-quality presentations from the transplant field. This meeting report highlights key findings from specific basic science, translational, and clinical research presentations deemed to have notable impact in thematic areas. In particular, there were a number of transformative studies indicating important advances in the understanding of alloimmunity, chronic rejection, tolerance, and organ-specific outcomes. Many of these results are discussed in the context of the published literature to showcase rapid advances in the transplant field.

Peroxisome proliferator-activated receptor alpha deficiency impairs regulatory T cell functions: Possible application in the inhibition of melanoma tumor growth in mice.

Regulatory T (Treg) cells are important to induce and maintain immunological self-tolerance. Although the progress accomplished in understanding the functional mechanism of Treg cells, intracellular molecules that control the mechanisms of their suppressive capacity are still on investigation. The present study showed that peroxisome proliferator-activated receptor-alpha deficiency impaired the suppressive activity of Treg cells on CD4+CD25- and CD8+ T cell proliferation. In Treg cells, PPARα gene deletion also induced a decrease of migratory abilities, and downregulated the expression of chemokine receptors (CCR-4, CCR-8 and CXCR-4) and p27KIP1 mRNA. Treg cells from PPARα-/- mice also lost their anergic property. Since low Treg activity, as observed in PPARα-/- mice, is known to be associated with the inhibition of tumor growth, we inoculated these mice with B16 melanoma cells and assessed tumor proliferation. In PPARα-/- mice, cancer growth was significantly curtailed, and it was correlated with high expression of granzyme B and perforin mRNA in tumor bed. Degranulation of cytolytic molecules by CD8+ T cells, assessed by a perforin-release marker CD107a expression, was higher in PPARα-/- mice than that in wild-type mice. Tumor-infiltrating lymphocytes (TIL) in melanoma tumors in PPARα-/- mice exhibited high pro-inflammatory Th1 phenotype. Consistently, adoptive transfer into lymphopenic RAG2-/- mice of total PPARα-/-splenic T cells inhibited more the growth rate of B16 tumor than the wild type splenic T cells. Our findings suggest that PPARα deficiency, by diminishing Treg cell functions and upregulating pro-inflammatory T cell phenotype, exerts an invivo anti-cancer properties.

MicroRNA-17 Modulates Regulatory T Cell Function by Targeting Co-regulators of the Foxp3 Transcription Factor

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, andits expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 invitro and invivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.

Alemtuzumab long-term immunologic effect: Treg suppressor function increases up to 24 months.

Objective:To analyze changes in T-helper (Th) subsets, T-regulatory (Treg) cell percentages and function, and mRNA levels of immunologically relevant molecules during a 24-month follow-up after alemtuzumab treatment in patients with relapsing-remitting multiple sclerosis (RRMS).Methods:Multicenter follow-up of 29 alemtuzumab-treated patients with RRMS in the Comparison of Alemtuzumab and Rebif Efficacy in Multiple Sclerosis (CARE-MS) I and CARE-MS II trials. Peripheral blood (PB) samples were obtained at months 0, 6, 12, 18, and 24. We evaluated (1) mRNA levels of 26 immunologic molecules (cytokines, chemokines, chemokine receptors, and transcriptional factors); (2) Th1, Th17, and Treg cell percentages; and (3) myelin basic protein (MBP)–specific Treg suppressor activity.Results:We observed 12 relapses in 9 patients. mRNA levels of the anti-inflammatory cytokines interleukin (IL)–10, IL-27, and transforming growth factor–β persistently increased whereas those of proinflammatory molecules related to the Th1 or Th17 subsets persistently decreased after alemtuzumab administration throughout the follow-up period. PB CD4+ cell percentage remained significantly lower than baseline while that of Th1 and Th17 cells did not significantly change. A significant increase in Treg cell percentage was observed at month 24 and was accompanied by an increase in Treg cell suppressive activity against MBP-specific Th1 and Th17 cells.Conclusions:The long-lasting therapeutic benefit of alemtuzumab in RRMS may involve a shift in the cytokine balance towards inhibition of inflammation associated with a reconstitution of the PB CD4+ T-cell subsets that includes expansion of Treg cells with increased suppressive function.

Dietary Vitamin D Increases Percentages and Function of Regulatory T Cells in the Skin-Draining Lymph Nodes and Suppresses Dermal Inflammation.

Skin inflammatory responses in individuals with allergic dermatitis may be suppressed by dietary vitamin D through induction and upregulation of the suppressive activity of regulatory T (TReg) cells. Vitamin D may also promote TReg cell tropism to dermal sites. In the current study, we examined the capacity of dietary vitamin D3 to modulate skin inflammation and the numbers and activity of TReg cells in skin and other sites including lungs, spleen, and blood. In female BALB/c mice, dietary vitamin D3 suppressed the effector phase of a biphasic ear swelling response induced by dinitrofluorobenzene in comparison vitamin D3-deficient female BALB/c mice. Vitamin D3 increased the percentage of TReg (CD3+CD4+CD25+Foxp3+) cells in the skin-draining lymph nodes (SDLN). The suppressive activity of TReg cells in the SDLN, mesenteric lymph nodes, spleen, and blood was upregulated by vitamin D3. However, there was no difference in the expression of the naturally occurring TReg cell marker, neuropilin, nor the expression of CCR4 or CCR10 (skin-tropic chemokine receptors) on TReg cells in skin, SDLN, lungs, and airway-draining lymph nodes. These data suggest that dietary vitamin D3 increased the percentages and suppressive activity of TReg cells in the SDLN, which are poised to suppress dermal inflammation.

Abstract B051: Control of regulatory T (Treg) cell function by protein kinase C-eta (PKCη): A novel target for cancer immunotherapy

Abstract Modulating regulatory T (Treg) cell functions to specifically promote anti-tumor immune responses is a desirable strategy for cancer immunotherapy. Blockade of checkpoint molecules, CTLA-4 and PD-1, has proven clinical efficacy in advanced metastatic melanoma and other malignancies. We recently found that protein kinase C-eta (PKCη), a member of the PKC enzyme family, physically associated with CTLA-4, an inhibitory receptor highly expressed on Treg cells, which is important for Treg cell-mediated suppression. PKCη is recruited to the Treg cell immunological synapse, and mediates contact-dependent suppression by Treg cells. PKCη-deficient (Prkch-/-) Treg cells are defective in the suppression of anti-tumor immunity against B16 melanoma but retain the ability to control the development of autoimmune colitis. Here, we extended our findings regarding the importance of the CTLA-4-PKCη complex in contact-dependent Treg cell suppressive activity to an additional tumor model, i.e., the slow growing prostate adenocarcinoma TRAMP-C1, indicating that the novel CTLA-4-PKCη signaling pathway may be relevant to a broad spectrum of solid cancers. We further found that in the tumor microenvironment, the loss of PKCη in Treg cells substantially promoted the infiltration of CD4+ and CD8+ effector T cells without affecting the Treg cell numbers. As a result, there was a significant increase in the intratumoral CD8+ to Treg cell ratio, a parameter considered to be predictive of a better clinical outcome. Thus, our data reveal that the novel CTLA-4-PKCη signaling pathway, which we identified, is critical for suppressing anti-tumor immunity and, hence, implicate this pathway as an attractive cancer immunotherapy target for a broad spectrum of clinically debilitating malignancies. Citation Format: Christophe Pedros, Kok-Fai Kong, Amnon Altman. Control of regulatory T (Treg) cell function by protein kinase C-eta (PKCη): A novel target for cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B051.

Association of periostin expression with eosinophilic inflammation in nasal polyps

by GSK-3b leads to the instability of b-catenin, reducing translocation of b-catenin into the nucleus, which can be associated with tumorigenesis. In this study we established the critical link between GSK-3b and Foxp3 by means of tissue examination and in vitro analysis. Previously, Graham et al reported that GSK-3b inhibition is able to potentiate the suppressive activity of Treg cells, but whether other forms of posttranslational modifications, such as phosphorylation, can affect Foxp3 stability and Treg cell function has been largely unknown. In a recent study on rheumatoid arthritis, Nie et al investigated the relationship between Treg cells with pathogenic T cells at the site of chronic inflammation. They identified Ser 418 as a key phosphorylation site of Foxp3 that was responsible for the impaired Treg cell function in response to TNF-a. In this study we found a novel phosphorylation motif (Ser 270/274) of Foxp3, which can be recognized by proinflammatory GSK-3b. Interestingly, when we examined the p-Foxp3 protein using specific antibody against Ser 270 in NP tissues, we found p-Foxp3 (Ser 270) and showed a positive association of GSK-3b and p-Foxp3 (Ser 270) protein levels. Therefore these findings propose a molecular mechanism regulating inhibitory function of Treg cells, which might expand our knowledge on the pathogenesis of CRSwNP. Xingmei Wu, MD* Sihua Wang, PhD* Miaomiao Han, PhD* Bin Song, PhD Ping Ye, MD Shanshan Ma, MD Jun Li, MD Fenghong Chen, MD Geng Xu, PhD Qingqing Ding, PhD Jiahong Xia, PhD Huabin Li, PhD From the Allergy Center, Otorhinolaryngology Hospital, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; the Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; the Allergy Division, Department of Otolaryngology, Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; the Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai, China; the Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; and the Department of Pathology, University of Missouri, Columbia, Mo. E-mail: qingqing_ding@ hotmail.com. Or: jiahong.xia@hust.edu.cn. Or: allergyli@163.com. *These authors contributed equally to this work. Supported by grants from the National Natural Science Foundations of China (nos. 81172512, 81271054, 81271056, and 81470673) and the Shanghai Committee of Science and Technology (no. 14DZ2260300). Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

Critical link between glycogen synthase kinase 3β and forkhead box P3 in patients with chronic rhinosinusitis with nasal polyps

Critical link between glycogen synthase kinase 3β and forkhead box P3 in patients with chronic rhinosinusitis with nasal polyps

Inflammation-associated genes: risks and benefits to Foxp3+ regulatory T-cell function.

Foxp3+ regulatory T (Treg) cells prevent the development of autoimmunity and immunopathology, as well as maintaining homeostasis and tolerance to commensal microorganisms. The suppressive activity of Treg cells is their defining characteristic, generating great interest in their therapeutic potential. However, suppressive and effector functions are not entirely exclusive. Considerable evidence points to the ability of supposedly anti-inflammatory Foxp3-expressing Treg cells to also express transcription factors that have been characterized as cardinal drivers of T effector cell function. We will consider the mounting evidence that Treg cells can function in non-suppressive capacities and review the impetus for this functional change, its relevance to developing immune and autoimmune responses and its significance to the development of Treg-based therapies.

T-Regulatory Cells as Part of Strategy of Immune Evasion by Pathogens.

Under physiological conditions, regulatory processes can suppress the immune response after elimination of a pathogen and restore homeostasis through the destruction and suppression of obsolete effector cells of the immune system. The main players in this process are T-regulatory cells (Tregs) and immature dendritic cells, which suppress the immune response by their own products and/or by inducing synthesis of immunosuppressive interleukins IL-10, IL-35, and transforming growth factor (TGF-β) by other cells. This mechanism is also used by widespread "successful" pathogens that are capable of chronically persisting in the human body - herpes virus, hepatitis viruses, human immunodeficiency virus, Mycobacterium tuberculosis, Helicobacter pylori, and others. During coevolution of microbial pathogens and the host immune system, the pathogens developed sophisticated strategies for evading the host defense, so-called immune evasion. In particular, molecular structures of pathogens during the interaction with dendritic cells via activating and inhibitory receptors can change intracellular signal transduction, resulting in block of maturation of dendritic cells. Immature dendritic cells become tolerogenic and cause differentiation of Tregs from the conventional T-cell CD4+. Microbial molecules can also react directly with Tregs through innate immune receptors. Costimulation of Toll-like receptor 5 (TLR5) by flagellin increases the expression of the transcription factor Foxp3, which increases the suppressive activity of Treg cells. From all evasion mechanisms, the induction of immunosuppression by Treg through IL-10, IL-35, and TGF-β appears most effective. This results in the suppression of inflammation and of adaptive immune responses against pathogens, optimizing the conditions for the survival of bacteria and viruses.

Th1-Like ICOS+ Foxp3+ Treg Cells Preferentially Express CXCR3 and Home to β-Islets during Pre-Diabetes in BDC2.5 NOD Mice.

Type 1 diabetes (T1D) occurs through a breakdown of self-tolerance resulting in the autoimmune destruction of the insulin producing β-islets of the pancreas. A numerical and functional waning of CD4+Foxp3+ regulatory T (Treg) cells, prompted by a pancreatic IL-2 deficiency, accompanies Th1 autoimmunity and T1D progression in non-obese diabetic (NOD) mice. Recently, we identified a dominant subset of intra-islet Treg cells that expresses the ICOS costimulatory receptor and promotes self-tolerance delaying the onset of T1D. ICOS co-stimulation potently enhances IL-2 induced survival and proliferation, and suppressive activity of Treg cells in situ. Here, we propose an ICOS-dependent mechanism of Treg cell homing to the β-islets during pre-diabetes in the NOD model via upregulation of the CXCR3 chemokine receptor. The islet-specific ICOS+ Treg cell subset preferentially expresses CXCR3 in the pancreatic lymph nodes (pLN) in response to Teff cell-mediated pancreatic inflammation, an expression correlating with the onset and magnitude of IFN-γ production by Teff cells in pancreatic sites. We also reveal that intra-pancreatic APC populations and insulin-producing β, but not α nor δ, islet cells secrete the CXCR3 chemokines, CXCL9, 10 and 11, and selectively promote ICOS+CXCR3+ Treg cell chemotaxis in vitro. Strikingly, islet-derived Treg cells also produce these chemokines suggesting an auto-regulation of homing by this subset. Unlike ICOS- cells, ICOS+ Treg cells adopt a Th1-like Treg phenotype while maintaining their suppressive capacity, characterized by expression of T-bet and CXCR3 and production of IFN-γ in the draining pLNs. Finally, in vivo neutralization of IFN-γ blocked Treg cell CXCR3 upregulation evincing its role in regulating expression of this chemokine receptor by Treg cells. Thus, CXCR3-mediated trafficking of Treg cells could represent a mechanism of homeostatic immunoregulation during diabetogeneesis.

Lineage-Plasticity and Inflammatory Change of Human FoxP3+ Regulatory T Cells in Acute Viral Infection: Implication in Immune-Mediated Tissue Injury

FoxP3+ CD4+CD25hi regulatory T (Treg) cells play a major role in maintaining the immune homeostasis by preventing the activation of self-reactive T cells as well as in controlling a series of immune responses in viral infections. Recent studies suggest that lineage-commitment of CD4+T cells, including Treg cells, is not a fixed fate, rather a status with a wide range of plasticity. Functional changes and lineage-plasticity of Treg cells during acute viral infection, especially of human, have not been reported so far. Herein, we investigated whether Treg cells show the functional plasticity and whether such changes can affect the regulation of immunopathology in a human acute viral infection. As a model of human acute viral infection, we used a cohort of patients with acute hepatitis A (AHA), since tissue (liver) injury in AHA is mediated exclusively by activated T cells, not by direct cytopathic effect of virus.To assess the plasticity of Treg cell lineage, first, we examined the production of a variety of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining and multicolor flow cytometry. We found that a significant proportion of FoxP3+ CD4+CD25hi Treg cells produced TNF-α following TCR stimulation in patients with AHA, but not in heathy subjects. Analyses at multiple time points during the course of infection showed that TNF-α production from Treg cells decreased in convalescent phase. Likewise, we observed that liver-infiltrating Treg cells also produced TNF-α after TCR stimulation. Moreover, highly-purified CD4+CD25hiCD127lo/-Treg cells could also produce TNF-α following TCR stimulation, indicating that Treg cells of AHA patients can produce TNF-α in direct response to TCR stimulation.Next, to exclude the possibility that TNF-α might be secreted from transiently FoxP3-expressing activated non-Treg CD4+ T cells, we examined the expression level of CD127 on TNF-α-secreting FoxP3+ CD4+ T cells. TNF-α+ Treg cells expressed CD127 in the level similar to conventional TNF-α- counterpart, and CD127 expression levels of both Treg populations were much lower than FoxP3- CD4+ T cells. Furthermore, DNA methylation analysis of Treg cell-specific demethylated region (TSDR) after sorting TNF-α+ Treg cells revealed completely demethylated pattern in highly conserved CpG island of FOXP3 gene. These findings support that TNF-α is produced from bona fide Treg cells, not from FoxP3-expressing activated non-Treg CD4+T cells.In analysis of immunophenotypes, TNF-α+ Treg cells were enriched in CD45RA-FoxP3lo population, implying their reduced in vivo suppressive activity. Along with the lower level of FoxP3, TNF-α+ Treg cells showed lower level of CD39 expression, a surrogate marker of Treg cell suppressive activity, compared to TNF-α- Treg cells. Furthermore, TNF-α+Treg cells showed a robust evidence of lineage-plasticity toward Th17 lineage, expressing a key transcription factor RORγt. Consistently, they expressed CCR6 and co-produced IL-17A following TCR stimulation, which are the hallmark of Th17 effector function.To analyze the clinical implication of attenuate suppressive function and plasticity shown by TNF-α+ Treg cells, we examined correlation between production of proinflammatory cytokines from Treg cells and severity of liver damage in AHA. As a result, proportion of TNF-α-producing Treg cells closely and linearly correlated with severity of liver damage, suggesting the critical role of TNF-α+ Treg cells in the immunopathogenesis of AHA. However, Treg cell suppression assay in the absence or presence of anti-TNF-α antibody showed that Treg cell suppressive function was not affected by TNF-α blockade. This indicates that attenuated function of TNF-α+Treg cells is not attributed simply to production of a kind of inflammatory cytokine, rather to more complicated reprogramming mechanism.Taken together, these data provide a clear evidence of attenuated suppressive activity and Th17-toward lineage plasticity of FoxP3+ Treg cells, represented by TNF-α production, in a human acute viral infection. Also, we suggest one possible mechanism that lineage plasticity and inflammatory changes of Treg cells could be implicated in the immunopathogenesis of human diseases. DisclosuresNo relevant conflicts of interest to declare.

CD4+ Foxp3+ regulatory T cells in autoimmune orchitis: phenotypic and functional characterization.

The phenotype and function of regulatory T (Treg) cells in rats with experimental autoimmune orchitis (EAO) was evaluated. Distribution of Treg cells in draining lymph nodes from the testis (TLN) and from the site of immunization (ILN) was analysed by immunohistochemistry. The number, phenotype and proliferative response (5-bromo-2'-deoxyuridine incorporation) of Treg cells were evaluated by flow cytometry and Treg cell suppressive activity by in vitro experiments. TGF-β expression was evaluated by immunofluorescence. Absolute numbers of Treg cells and BrdU+ Treg cells were increased in LN from experimental compared to normal and control rats. These cells displayed a CD45RC(-), CD62L(-), Helios(+) phenotype. CD4(+) CD25(bright) T cells from TLN of experimental rats were able to suppress T cell-proliferation more efficiently than those derived from normal and control rats. Cells isolated from TLN and ILN expressed TGF-β. Our results suggest that Treg cells with a memory/activated phenotype proliferate extensively in the inflamed testis and LN of rats with EAO exhibiting an enhanced suppressive capacity. TGF-β may be involved in their suppressive mechanism.

Increased production of interleukin-17 over interleukin-10 by treg cells implicates inducible costimulator molecule in experimental spondyloarthritis.

HLA-B27/human β2 -microglobulin (hβ2 m)-transgenic (B27-transgenic) rats develop an inflammatory disorder resembling spondyloarthritis, with accumulation of proinflammatory Th17 cells. Because Treg cells and Th17 cells have opposing effects in inflammatory disorders, we sought to determine whether biased expansion of Th17 cells could result from altered Treg cell frequency and/or function in B27-transgenic rats. We characterized the phenotype and function of Treg cells from B27-transgenic rats in comparison with those from control rats, by examining their expression of cell surface markers, suppressive activity, cytokine production, and differentiation pattern. In B27-transgenic rats, the preferential accumulation of CD4+ Teff cells over Treg cells was not associated with a defect in Treg cell differentiation or suppressive activity. The expression of Treg cell markers was similar between B27-transgenic and control rats, with the exception of the inducible costimulator (ICOS) molecule, which was overexpressed in B27-transgenic rats. High levels of ICOS are considered to be a hallmark of Treg cells with heightened suppressive activity and interleukin-10 (IL-10) expression. Paradoxically, the production of IL-10 by Treg cells was reduced in B27-transgenic rats, whereas the production of IL-17 was enhanced. Moreover, the addition of anti-ICOS monoclonal antibodies during Treg cell differentiation in the presence of dendritic cells from B27-transgenic rats reversed this cytokine profile, restoring the balance between IL-10 and IL-17 in Treg cells from B27-transgenic rats. We observed dysregulated production of IL-10 and IL-17 by Treg cells from B27-transgenic rats, which may contribute to disease development. Moreover, our data highlight a key role for ICOS signaling in the generation of imbalanced production of IL-10 and IL-17 by Treg cells in this experimental model of spondyloarthritis.