A Bacterial Artificial Chromosome Reporter System for Expression of the Human FOXP3 Gene in Mouse Regulatory T-Cells

The transcription factor FOXP3 plays key roles in the development and function of regulatory T cells (Treg) capable of preventing and correcting immunopathology. There has been much interest in exploiting Treg as adoptive cell therapy in man, but issues of lack of nominal antigen-specificity and stability of FoxP3 expression in the face of pro-inflammatory cytokines have been a concern. In order to enable fundamental studies of human FOXP3 (hFOXP3) gene regulation and to provide preclinical tools to guide the selection of drugs that might modulate hFOXP3 expression for therapeutic purposes, we generated hFOXP3/AmCyan bacterial artificial chromosome (BAC) transgenic mice and transfectants, wherein hFOXP3 expression was read out as AmCyan expression. Using the transgenic mice, one can now investigate hFOXP3 gene expression under defined experimental conditions used for mouse Foxp3 (mFoxp3) studies. Here, we demonstrate that hFOXP3 gene expression in BAC transgenic mice is solely restricted to CD4+ T-cells, as for mFoxp3 gene expression, showing that hFOXP3 expression in Treg cells depends on fundamentally similar processes to mFoxp3 expression in these cells. Similarly, hFOXP3 expression could be observed in mouse T-cells through TCR stimulation in the presence of TGF-β. These data suggest that, at least in part, cell type-specific human and mouse foxp3 gene expression is regulated by common regulatory regions which for the human, are located within the 110-kb human FOXP3 BAC DNA. To investigate hFOXP3 gene expression further and to screen potential therapeutics in modulating hFOXP3 gene expression in vitro, we also generated hFOXP3/AmCyan expression reporter cell lines. Using the reporter cells and transcription factor inhibitors, we showed that, just as for mFoxp3 expression, inhibitors of NF-κB, AP1, STAT5, Smad3, and NFAT also block hFOXP3 expression. hFOXP3 induction in the reporter cells was also TGF-β dependent, and substantially enhanced by an mTOR inhibitor, Torin1. In both the reporter transgenic mice and cell lines, histone H4 molecules in the hFOXP3 promoter and enhancers located in human CNS1 and CNS2 regions were highly acetylated in natural Treg and TCR/TGF-β-induced Treg, indicating hFOXP3 gene expression is regulated by mechanisms similar to those previously identified for the mFoxp3 gene

Blockade of MCP-1/CCR4 signaling-induced recruitment of activated regulatory cells evokes an antitumor immune response in head and neck squamous cell carcinoma

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PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep−/− iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.Bio-organic Synthesi

Expression of CD39 on FoxP3+ T regulatory cells correlates with progression of HBV infection

BACKGROUND: Although it is known that regulatory T cells (Tregs) can suppress the function of effector T cells, and may contribute to impaired immune response, the precise role of Tregs during the course of hepatitis B virus (HBV) infection remains to be elucidated. A newly identified subset of the CD4(+)Foxp3(+ )Tregs, the CD39(+ )Tregs, has been associated with viral infections and autoimmune diseases. Therefore, we hypothesized that this discrete Treg subset may contribute to the chronic infection of HBV. RESULTS: Initial characterization studies of healthy peripheral CD39(+)FoxP3(+)CD4(+ )T cells revealed that the majority were CD45RA(- )Treg cells. Subsequent analysis of HBV-infected patients (38 asymptomatic HBV carriers (AsCs), 37 chronic active hepatitis B (CAH), 29 HBV-associated acute-on-chronic liver failure (ACLF)) and healthy individuals (25 controls) was conducted to assess association with HBV copy number and the liver injury marker alanine aminotransferase (ALT). A higher percentage of CD39(+ )Tregs was detected within the population of FoxP3(+)CD4(+ )T cells in peripheral blood of AsCs patients. Moreover, the percentage of CD39(+ )Tregs was significantly less in CAH and ACLF patients. The increased proportions of circulating CD39(+ )Tregs were positively correlated with serum viral load, but inversely correlated with serum ALT level. CONCLUSION: These findings not only suggest that CD39(+ )Treg cells may be involved in HBV disease progression but also identify CD39(+ )Tregs as a dynamic immune regulatory cell population that may represent a new target of immunomodulatory therapeutic interventions

Monoclonal antibodies against GARP/TGF-β1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo

peer reviewedRegulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor–b1 (TGF-b1). On the Treg cell surface, TGF-b1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-b1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137–139 within GARP/TGF-b1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-b1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy

T-cell number and subtype influence the disease course of primary chronic lymphocytic leukaemia xenografts in alymphoid mice.

Chronic lymphocytic leukaemia (CLL) cells require microenvironmental support for their proliferation. This can be recapitulated in highly immunocompromised hosts in the presence of T cells and other supporting cells. Current primary CLL xenograft models suffer from limited duration of tumour cell engraftment coupled with gradual T-cell outgrowth. Thus, a greater understanding of the interaction between CLL and T cells could improve their utility. In this study, using two distinct mouse xenograft models, we investigated whether xenografts recapitulate CLL biology, including natural environmental interactions with B-cell receptors and T cells, and whether manipulation of autologous T cells can expand the duration of CLL engraftment. We observed that primary CLL xenografts recapitulated both the tumour phenotype and T-cell repertoire observed in patients and that engraftment was significantly shorter for progressive tumours. A reduction in the number of patient T cells that were injected into the mice to 2-5% of the initial number or specific depletion of CD8+ cells extended the limited xenograft duration of progressive cases to that characteristic of indolent disease. We conclude that manipulation of T cells can enhance current CLL xenograft models and thus expand their utility for investigation of tumour biology and pre-clinical drug assessment

Role of activated Protein C in modulating cellular metabolism and epigenetic control of T-Cell

The current findings indicate that activated protein C (aPC) can induce FOXP3 generation and regulatory T cells (Tregs) through epigenetic modulation and metabolic reprogramming. The current findings show that preincubation of T cells with aPC increased the frequency of Treg markers, CD4+FOXP3+ T cell frequency and suppressive function of T-cells, suggesting an increase of Tregs. The emergence of FOXP3 expression and Treg-like characteristics is linked to alterations in the epigenetic profile of T cells, characterized by a decline in overall DNA methylation, a decrease in the repressive histone marks H3K27me3 , and a reduced methylation of the FOXP3 promoter region. In addition, the induction of Tregs by aPC is accompanied by changes in mitochondrial metabolism. T lymphocytes that were preincubated with aPC exhibit a decline in mitochondrial respiration, a decrease in mitochondrial membrane potential, and a transition towards metabolic quiescence. The metabolic alterations are associated with a reduction of crucial metabolites, specifically α-ketoglutarate (αKG) and glutamine, that are known to regulate T cell function and epigenetic regulation. Reversal of the aPC-induced Treg-like phenotype and associated altered mitochondrial metabolism can be achieved through the supplementation of exogenous αKG or glutamine, supporting a functional interaction of these reduced metabolites and altered T-cell function. The current results show that chronically elevated levels of aPC in transgenic APChigh mice lead to a higher incidence of CD4+FOXP3+ Tregs in the spleen without alterations in thymic Tregs (tTregs), thereby indicating that aPC facilitates the development of Tregs in peripheral lymphoid organs but does not influence primary T-cell development. T cells derived from APChigh mice display a decrease in mitochondrial metabolism, which is consistent with the findings observed in vitro. Supplementation of αKG in cultured T cells derived from APChigh mice restores mitochondrial function and decreases the frequency of CD4+FOXP3+ Tregs. These findings support a model in which aPC alters T-cell metabolism, possibly by reducing expression of glucose (GLUT1) and glutamine (ASCT2 and SNAT1) transporters and thus the availability of metabolic substrates in T-cells. The reduced availability of these substates alters epigentic gene-expression and favors Treg development. In summary, the findings indicate that activated protein C (aPC) promotes the induction of regulatory T cells (Tregs) via metabolic modulation and altered epigenetic gene-expression. The results provide new insights into the long-lasting effects of aPC and indicate the possibility of aPC as a therapeutic target for regulating immune responses and fostering immune tolerance in diverse pathologies. Additional research is necessary to investigate the potential therapeutic applications

Immune checkpoints expression in chronic lung allograft rejection

Chronic lung allograft dysfunction (CLAD) is the main cause of poor survival and low quality of life of lung transplanted patients. Several studies have addressed the role of dendritic cells, macrophages, T cells, donor specific as well as anti-HLA antibodies, and interleukins in CLAD, but the expression and function of immune checkpoint molecules has not yet been analyzed, especially in the two CLAD subtypes: BOS (bronchiolitis obliterans syndrome) and RAS (restrictive allograft syndrome). To shed light on this topic, we conducted an observational study on eight consecutive grafts explanted from patients who received lung re-transplantation for CLAD. The expression of a panel of immune molecules (PD1/CD279, PDL1/CD274, CTLA4/CD152, CD4, CD8, hFoxp3, TIGIT, TOX, B-Cell-Specific Activator Protein) was analyzed by immunohistochemistry in these grafts and in six control lungs. Results showed that RAS compared to BOS grafts were characterized by 1) the inversion of the CD4/CD8 ratio; 2) a higher percentage of T lymphocytes expressing the PD-1, PD-L1, and CTLA4 checkpoint molecules; and 3) a significant reduction of exhausted PD-1-expressing T lymphocytes (PD-1pos/TOXpos) and of exhausted Treg (PD-1pos/FOXP3pos) T lymphocytes. Results herein, although being based on a limited number of cases, suggest a role for checkpoint molecules in the development of graft rejection and offer a possible immunological explanation for the worst prognosis of RAS. Our data, which will need to be validated in ampler cohorts of patients, raise the possibility that the evaluation of immune checkpoints during follow-up offers a prognostic advantage in monitoring the onset of rejection, and suggest that the use of compounds that modulate the function of checkpoint molecules could be evaluated in the management of chronic rejection in LTx patients