MicroRNAs regulate T-cell production of interleukin-9 and identify hypoxia-inducible factor-2a as an important regulator of T helper 9 and regularoty T-cell differentiation

MicroRNAs (miRNAs) regulate many aspects of helper T cell (Th) development and function. Here we found that they are required for the suppression of interleukin‐9 (IL‐9) expression in Th9 cells and other Th subsets. Two highly related miRNAs (miR‐15b and miR‐16) that we previously found to play an important role in regulatory T (Treg) cell differentiation were capable of suppressing IL‐9 expression when they were over‐expressed in Th9 cells. We used these miRNAs as tools to identify novel regulators of IL‐9 expression and found that they could regulate the expression of Epas1, which encodes hypoxia‐inducible factor (HIF)‐2α. HIF proteins regulate metabolic pathway usage that is important in determining appropriate Th differentiation. The related protein, HIF‐1α enhances Th17 differentiation and inhibits Treg cell differentiation. Here we found that HIF‐2α was required for IL‐9 expression in Th9 cells, but its expression was not sufficient in other Th subsets. Furthermore, HIF‐2α suppressed Treg cell differentiation like HIF‐1α, demonstrating both similar and distinct roles of the HIF proteins in Th differentiation and adding a further dimension to their function. Ironically, even though miR‐15b and miR‐16 suppressed HIF‐2α expression in Treg cells, inhibiting their function in Treg cells did not lead to an increase in IL‐9 expression. Therefore, the physiologically relevant miRNAs that regulate IL‐9 expression in Treg cells and other subsets remain unknown. Nevertheless, the analysis of miR‐15b and miR‐16 function led to the discovery of the importance of HIF‐2α so this work demonstrated the utility of studying miRNA function to identify novel regulatory pathways in helper T‐cell development

β7 Integrin Inhibition Can Increase Intestinal Inflammation by Impairing Homing of CD25hiFoxP3+ Regulatory T Cells.

Background & aimsIntegrin α4β7 mediates lymphocyte trafficking to the gut and gut-associated lymphoid tissues, a process critical for recruitment of effector lymphocytes from the circulation to the gut mucosa in inflammatory bowel disease (IBD) and murine models of intestinal inflammation. Antibody blockade of β7 integrins generally is efficacious in IBD; however, some patients fail to respond, and a few patients can experience exacerbations. This study examined the effects of loss of β7 integrin function in murine models of IBD.MethodsIn a mouse IBD model caused by lack of interleukin 10, a cytokine important in CD25hiFoxP3+ regulatory T cell (Treg) function, genetic deletion of β7 integrin or antibody blockade of α4β7-mucosal addressin cell adhesion molecule-1 interaction paradoxically exacerbated colitis.ResultsLoss of β7 impaired the capacity of Tregs homing to the gut and therefore suppress intestinal inflammation in an adoptive T-cell transfer model; however, the intrinsic suppressive function of β7-deficient Tregs remained intact, indicating that the β7 deficiency selectively impacts gut homing. Deletion of β7 integrin did not worsen colitis in an acute dextran sodium sulfate model in which Treg number and function were normal.ConclusionsIn Integrin subunit beta (Itgb)7-/-Il10-/- mice, loss of β7-dependent Treg homing to gut-associated lymphoid tissues combined with loss of intrinsic Treg function exacerbated intestinal inflammation. These results suggest that IBD patients with reduced CD25hiFoxP3+ Treg numbers or function or lack of interleukin 10 could be at risk for failure of α4β7 blocking therapy

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy

Notch and NF-kB: Coach and Players of Regulatory T-Cell Resposnse in Cancer

The Notch signaling pathway plays multiple roles in driving T-cell fate decisions, proliferation, and aberrant growth. NF-kB is a cell-context key player interconnected with Notch signaling either in physiological or in pathological conditions. This review focuses on how themultilayered crosstalk between different Notches and NF-kB subunits may converge on Foxp3 gene regulation and orchestrate CD4+ regulatory T (Treg) cell function, particularly in a tumor microenvironment. Notably, Treg cells may play a pivotal role in the inhibition of antitumor immune responses, possibly promoting tumor growth. A future challenge is represented by further dissection of both Notch and NF-kB pathways and consequences of their intersection in tumor-associated Treg biology. This may shed light on themolecularmechanisms regulating Treg cell expansion andmigration to peripheral lymphoid organs thought to facilitate tumor development and still to be explored. In so doing, new opportunities for combined and/or more selective therapeutic Q25 approaches to improve anticancer immunity may be found

Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory\ud T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-c chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.\ud Methodology/Principal Findings: CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21)\ud using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/ mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p,0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg6RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/ mTOR pathway.\ud Conclusions/Significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation

Metabolic regulation of regulatory T cell development and function

It is now well established that the effector T cell (Teff) response is regulated by a series of metabolic switches. Quiescent T cells predominantly require ATP-generating processes, whereas proliferating Teff require high metabolic flux through growth-promoting pathways, such as glycolysis. Pathways that control metabolism and immune cell function are intimately linked, and changes in cell metabolism at both the cell and system levels have been shown to enhance or suppress specific T cell effector functions. Furthermore, functionally distinct T cell subsets have been shown to require distinct energetic and biosynthetic pathways to support their specific functional needs. In particular, naturally occurring regulatory T cells (Treg) are characterized by a unique metabolic signature distinct to that of conventional Teff cells. We here briefly review the signaling pathways that control Treg metabolism and how this metabolic phenotype integrates their differentiation and function. Ultimately, these metabolic features may provide new opportunities for the therapeutic modulation of unwanted immune responses

Circulating gluten-specific FOXP3 + CD39 + regulatory T cells have impaired suppressive function in patients with celiac disease

Background Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. Objective We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3)+ Treg cells. Methods Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4+ T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4+ T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134). Results Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. Conclusion This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten-specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten-specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis

Regulatory T cells in rheumatoid arthritis : contributions from different functional subsets

Rheumatoid arthritis (RA) is a complex and multifactorial disease characterized by chronic joint inflammation and tissue destruction, which can affect all ethnic groups with a prevalence of 0.5-1%. FOXP3+ regulatory T cells (Treg cells) are crucial for the maintenance of self-tolerance and loss of function or reduced frequencies have been implicated in chronic inflammatory and autoimmune diseases. In patients with inflammatory arthritis including RA, Treg cells are significantly enriched at the site of inflammation compared with levels in the circulation, and are further functional in suppressing autologous effector T cells from both peripheral blood and joint origin. Given the accumulation of functional Treg cells in the rheumatic joint, an unresolved question is why local inflammation processes persist in a chronic way. In this thesis, we investigated the presence, frequency and functionality of different Treg-cell subsets in patients with inflammatory arthritis, and further studied the impact of commonly used treatment regimes on the suppressive capacity of Treg cells. We could show that synovial FOXP3+ Treg cells were increased in frequency compared with peripheral blood, displayed a high degree of FOXP3 demethylation and a low capacity of secreting pro-inflammatory cytokines upon stimulation. Moreover, the activation status of effector T cells and locally produced pro-inflammatory cytokines reduced regulatory Treg cell function in vitro and presumably in the rheumatic joint. Furthermore, expression of CD39, an ecto-nucleotidase, which together with CD73 generates anti-inflammatory adenosine, was significantly increased on synovial FOXP3+ Treg cells. Such FOXP3+CD39+ Treg cells did not produce pro-inflammatory cytokines and were good suppressors of several effector T-cell functions including secretion of IFN-γ and TNF, but did not limit IL-17A, a cytokine implicated in RA pathogenesis. Additional investigations of FOXP3+ Treg cells in the context of Helios, a suggested marker of thymus-derived Treg cells, revealed that synovial Helios+FOXP3+ T cells were abundant in the joint, displayed a more classical Treg-cell phenotype with regard to expression of surface markers and cytokine secretion capacity compared with Helios-FOXP3+ T cells. Finally, biologicals commonly used for the treatment of RA were shown to have profound effects on Treg-cell function, however by different mechanisms. Blocking of IL-6 and TNF by tocilizumab or adalimumab increased suppressive capacity of synovial Treg cells. Abatacept, in contrast, had no beneficial effect on Treg-cell function, but due to its mutual effect on effector and regulatory T cells, the inflammatory pressure in the joint could still be alleviated. In summary, our data suggest that joint-derived Treg cells in general are not impaired in their function and rather the inflammatory pressure needs to be reduced to allow for optimal Treg-cell functionality. Further, this work emphasizes the importance of dissecting synovial Treg-cell subsets to gain a better understanding on how Treg cells could be targeted for the treatment of chronic arthritis. ::doi::10.1002/eji.201041004. ::pmid::21607944 ::isi::00029326430001

Siah2 control of T-regulatory cells limits anti-tumor immunity.

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2-/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2-/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2-/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy