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The tumour microenvironment (TME) is a complex system composed of cancer cells, stromal cells and immune cells. Regulatory T cells (Tregs) in the TME impede immune surveillance of tumours and suppress antitumor immune responses. Transcription factor forkhead box protein 3 (FOXP3) is the main marker of Tregs, which dominates the function of Tregs. FOXP3 was originally thought to be a Tregs‐specific expression molecule, and recent studies have found that FOXP3 is expressed in a variety of tumours with inconsistent functional roles. This review summarizes the recent progress of infiltrating Treg‐FOXP3 and tumour‐FOXP3 in TME, discusses the communication mechanism between FOXP3+ cells and effector T cells in TME, the relationship between FOXP3 and clinical prognosis, and the potential of FOXP3‐targeted therapy. [ABSTRACT FROM AUTHOR]

We recently reported that lactoferrin (LF) induces Foxp3+ Treg differentiation through binding to TGFβ receptor III (TβRIII), and this activity was further enhanced by TGFβ1. Generally, a low T‐cell receptor (TCR) signal strength is favourable for Foxp3+ Treg differentiation. In the present study, we explored the effect of lactoferrin chimera (LFch, containing lactoferricin [aa 17–30] and lactoferrampin [aa 265–284]), along with TGFβ1 on Foxp3+ Treg differentiation. LFch alone did not induce Foxp3 expression, yet LFch dramatically enhanced TGFβ1‐induced Foxp3 expression. LFch had little effect on the phosphorylation of Smad3, a canonical transcriptional factor of TGFβ1. Instead, LFch attenuated the phosphorylation of S6 (a target of mTOR), IκB and PI3K. These activities of LFch were completely abrogated by pretreatment of LFch with soluble TGFβ1 receptor III (sTβRIII). Consistent with this, the activity of LFch on TGFβ1‐induced Foxp3 expression was also abrogated by treatment with sTβRIII. Finally, the TGFβ1/LFch‐induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch robustly enhances TGFβ1‐induced Foxp3+ Treg differentiation by diminishing TCR/CD28 signal intensity. [ABSTRACT FROM AUTHOR]

Epigenetics connects genetic and environmental factors: it includes DNA methylation, histone post‐translational modifications and the regulation of chromatin accessibility by non‐coding RNAs, all of which control constitutive or inducible gene transcription. This plays a key role in harnessing the transcriptional programs of both innate and adaptive immune cells due to its plasticity and environmental‐driven nature, piloting myeloid and lymphoid cell fate decisions with no change in their genomic sequence. In particular, epigenetic marks at the site of lineage‐specific transcription factors and maintenance of cell type‐specific epigenetic modifications, referred to as 'epigenetic memory', dictate cell differentiation, cytokine production and functional capacity following repeated antigenic exposure in memory T cells. Moreover, metabolic and epigenetic reprogramming occurring during a primary innate immune response leads to enhanced responses to secondary challenges, a phenomenon known as 'trained immunity'. Here, we discuss how stable and dynamic epigenetic states control immune cell identity and plasticity in physiological and pathological conditions. Dissecting the regulatory circuits of cell fate determination and maintenance is of paramount importance for understanding the delicate balance between immune cell activation and tolerance, in healthy conditions and in autoimmune diseases. [ABSTRACT FROM AUTHOR]

NFATs and FOXP3 are linked with impaired regulatory T‐cells (Tregs) in generalized vitiligo (GV). To elucidate calcium mediated NFATc1 signalling pathway and its effect on Treg suppressive capacity in GV. Calcium levels, calcineurin, NFATc1 and GSK‐3β activity and cell proliferation were assessed in 52 GV patients and 50 controls by calcium assay kit, calcineurin phosphatase assay kit, TransAM NFATc1 kit, GSK‐3β ELISA and BrdU cell proliferation assay. Transcripts (CNB, CAM, GSK3B, DYRK1A and calcium channel genes) and protein (IFN‐γ, IL‐10 and TGF‐β) expressions were assessed by qPCR and ELISA, respectively. Reduced plasma and intracellular Tregs calcium levels and ORAI1 transcripts suggested altered calcium homeostasis in GV Tregs (p = 0.00387, p = 0.0048, p < 0.0001), which led to decreased calcineurin and NFATc1 activity in GV Tregs (p = 0.0299, p < 0.0001). CNB and CAM transcripts were reduced in GV Tregs (p < 0.0001, p = 0.0004). GSK‐3β activity, GSK3B and DYRK1A transcripts significantly increased in GV Tregs (p = 0.0134, p < 0.0001 and p < 0.0001). Plasma (p = 0.0225, p = 0.032) and intracellular Treg (p = 0.0035, p = 0.005) calcium levels, calcineurin (p = 0.001) and NFATc1 (p = 0.001, p < 0.0001) activity and ORAI1 (p = 0.0093, p < 0.0001), CAM and CNB (p = 0.0214) transcripts significantly decreased in active vitiligo (AV) and severe GV (sGV) Tregs. Calcium treatment significantly increased intracellular calcium and ORAI1 transcripts in GV Tregs (p = 0.0042, p = 0.0035). Moreover, calcium treatment enhanced calcineurin and NFATc1 activity in GV Tregs (p = 0.0128, p < 0.0001). Remarkably, calcium treatment increased Treg mediated suppression of CD4+ and CD8+ T‐cells (p = 0.015, p = 0.006) in GV and increased Tregs associated cytokines: IL‐10 (p = 0.0323, p = 0.009), TGF‐β (p = 0.0321, p = 0.01) and decreased IFN‐γ production (p = 0.001, p = 0.016) by CD4+ and CD8+ T‐cells. Intracellular calcium levels positively correlated with calcineurin (r = 0.83; p < 0.0001) and NFATc1 (r = 0.61; p < 0.0001) activity, suggesting the enhanced Treg immunosuppressive capacity after calcium treatment. Our study for the first time suggests that reduced plasma calcium and ORAI1 transcripts are linked to calcium uptake defects in Tregs, which leads to reduced calcineurin and NFATc1 activation, thereby contributing to decreased Tregs immunosuppressive capacity in GV. Elevated GSK‐3β activity and GSKB and DYRK1A transcripts are involved in reduced NFATc1 activity in GV Tregs. Overall, the study suggests that calcium‐NFATc1‐signalling pathway is likely to be involved in defective Tregs function and can be implicated for development of effective Treg mediated therapeutics for GV. [ABSTRACT FROM AUTHOR]

Regulatory T (Treg) cells are essential for control of inflammatory processes by suppressing effector T‐cell functions. The actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we employed pharmacological and genetic approaches to examine whether PGE2 had a direct action on T cells to modulate de novo differentiation of Treg cells. We found that TGF‐β‐induced Foxp3 expression and iTreg cell differentiation in vitro is markedly inhibited by PGE2, which was mediated by the receptors EP2 and EP4. Mechanistically, PGE2‐EP2/EP4 signalling interrupts TGF‐β signalling during iTreg differentiation. Moreover, EP4 deficiency in T cells impaired iTreg cell differentiation in vivo. Thus, our results demonstrate that PGE2 negatively regulates iTreg cell differentiation through a direct action on T cells, highlighting the potential for selectively targeting the PGE2‐EP2/EP4 pathway to control T cell‐mediated inflammation. [ABSTRACT FROM AUTHOR]

CD4+ regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti‐inflammatory function. High levels of expression of chemokine (C‐C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti‐CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8−/− mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour‐infiltrating Treg cells which were abolished in Ccr8−/− mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour‐infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8+ Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy. [ABSTRACT FROM AUTHOR]

Summary: The fetal and neonatal immune systems are uniquely poised to generate tolerance to self, maternal and environmental antigens encountered in the womb and shortly after birth. However, the tolerogenic nature of fetal and neonatal immunity can be detrimental in the context of pathogens, leading to overwhelming bacterial infections or chronic viral infections. A variety of mechanisms contribute to fetal and neonatal tolerance, including a propensity to generate Foxp3+ regulatory T cells (Treg cells). However, the mechanism(s) of fetal Foxp3+ T‐cell differentiation, the specific antigen‐presenting cells required and factors that inhibit Treg generation after the neonatal period are poorly understood. Here, we demonstrate that a subset of CD14+ monocytes expressing the scavenger molecule, CD36, can generate CD4+ and CD8+ T cells that coexpress Foxp3 and T‐bet from both umbilical cord blood. These Foxp3+ T‐bet+ T cells potently suppress T‐cell proliferation and ameliorate xenogeneic graft‐versus‐host disease. CD14+ CD36+ monocytes provide known Treg‐inducing signals: membrane‐bound transforming growth factor‐beta and retinoic acid. Unexpectedly, adult peripheral blood monocytes are also capable of inducing Foxp3+ T cells from both cord blood and adult peripheral naïve T cells. The induction of Foxp3+ T cells in umbilical cord blood by monocytes was inhibited by the lymphoid fraction of adult peripheral blood cells. These studies highlight a novel immunoregulatory role of monocytes and suggest that antigen presentation by CD36hi monocytes may contribute to the peripheral development of Foxp3+ T‐bet+ T cells with regulatory functions in both neonates and adults. [ABSTRACT FROM AUTHOR]

Summary: Activated T‐cells make both interleukin‐2 (IL2) and its high‐affinity receptor component CD25. Regulatory CD4 T‐cells (Treg cells) do not make IL2, and the IL2‐CD25 circuit is considered a paracrine circuit crucial in their generation and maintenance. Yet, all T‐cells are capable of making IL2 at some stage during differentiation, making a cell‐intrinsic autocrine circuit additionally possible. When we re‐visited experiments with mixed bone marrow chimeras using a wide range of ratios of wild‐type (WT) and IL2−/− genotype progenitors, we found that, as expected, thymic Treg cells were almost equivalent between WT and IL2−/− genotypes at ratios with WT prominence. However, at WT‐limiting ratios, the IL2−/− genotype showed lower thymic Treg frequencies, indicating a role for cell‐intrinsic autocrine IL2 in thymic Treg generation under IL2‐limiting conditions. Further, peripheral IL2−/− naive CD4 T‐cells showed poor conversion to inducible Tregs (pTregs) both in vivo and in vitro, again indicating a significant role for cell‐intrinsic autocrine IL2 in their generation. Peripherally, the IL2−/− genotype was less prominent at all WT:IL2−/− ratios among both thymic Tregs (tTregs) and pTregs, adoptively transferred IL2−/− Tregs showed poorer survival than WT Tregs did, and RNA‐seq analysis of WT and IL2−/− Tregs showed interesting differences in the T‐cell receptor and transforming growth factor‐beta‐bone morphogenetic protein‐JNK pathways between them, suggesting a non‐titrating role for cell‐intrinsic autocrine IL2 in Treg programming. These data indicate that cell‐intrinsic autocrine IL2 plays significant roles in Treg generation and maintenance. [ABSTRACT FROM AUTHOR]

Summary: The transcription factor Foxp3 controls the differentiation and function of regulatory T‐cells (Treg). Studies in the past decades identified numerous Foxp3‐interacting protein partners. However, it is still not clear how Foxp3 produces the Treg‐type transcriptomic landscape through cooperating with its partners. Here I show the current understanding of how Foxp3 transcription factor complexes regulate the differentiation, maintenance and functional maturation of Treg. Importantly, T‐cell receptor (TCR) signalling plays central roles in Treg differentiation and Foxp3‐mediated gene regulation. Differentiating Treg will have recognized their cognate antigens and received TCR signals before initiating Foxp3 transcription, which is triggered by TCR‐induced transcription factors including NFAT, AP‐1 and NF‐κB. Once expressed, Foxp3 seizes TCR signal‐induced transcriptional and epigenetic mechanisms through interacting with AML1/Runx1 and NFAT. Thus, Foxp3 modifies gene expression dynamics of TCR‐induced genes, which constitute cardinal mechanisms for Treg‐mediated immune suppression. Next, I discuss the following key topics, proposing new mechanistic models for Foxp3‐mediated gene regulation: (i) how Foxp3 transcription is induced and maintained by the Foxp3‐inducing enhanceosome and the Foxp3 autoregulatory transcription factor complex; (ii) molecular mechanisms for effector Treg differentiation (i.e. Treg maturation); (iii) how Foxp3 activates or represses its target genes through recruiting coactivators and corepressors; (iv) the 'decision‐making' Foxp3‐containing transcription factor complex for Th17 and Treg differentiation; and (v) the roles of post‐translational modification in Foxp3 regulation. Thus, this article provides cutting‐edge understanding of molecular biology of Foxp3 and Treg, integrating findings by biochemical and genomic studies. [ABSTRACT FROM AUTHOR]

Summary: A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild‐type littermates at different time‐points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time‐points after Treg cell depletion in DEREG mice from samples of early time‐points before Treg cell depletion in these mice and from gut microbiota samples of wild‐type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies. [ABSTRACT FROM AUTHOR]

Summary: Regulatory T (Treg) cell‐specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3YFP‐Cre strain of mice being one of the most widely used. In an attempt to generate Treg cells that lacked expression of the insulin receptor (Insr), we crossed Foxp3YFP‐Cre mice with Insrfl/fl mice. Using a conventional two‐band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios. We therefore developed a quantitative PCR‐based genotyping method to investigate possible ectopic recombination outside the Treg lineage. With this method we found that ~50% of the F1‐generation mice showed evidence of ectopic recombination and that ~10% of the F2‐generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion. Use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg cell‐specific Insr deletion. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3YFP‐Cre allele, particularly when studying genes that are systemically expressed. [ABSTRACT FROM AUTHOR]

Summary: Although regulatory T‐cells (Tregs) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of Tregs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4+ CD25+T‐cells (FoxP3+ cells). Dengue virus (DENV) viral loads were measured by quantitative real‐time polymerase chain reaction (PCR) and DENV‐specific T‐cell responses were measured by ex‐vivo interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV‐NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T‐cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte‐antigen 4 (CTLA‐4) and Fas. While the frequency of FoxP3+ cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3+ cells did not correlate with either ex‐vivo IFN‐γ DENV‐NS3‐, NS5‐ or NS1‐specific T‐cell responses. FoxP3+ cells of patients with acute dengue were predominantly CD45RA+ FoxP3low, followed by CD45RA‐FoxP3low, with only a small proportion of FoxP3+ cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T‐cells, with only CCR4 only being expressed at high levels in the effector Treg population. Therefore, although FoxP3+ cells are expanded in acute dengue, they predominantly consist of naive Tregs, with poor suppressive capacity. [ABSTRACT FROM AUTHOR]

Immune homeostasis requires the tight, tissue-specific control of the different CD4+ Foxp3+ regulatory T (Treg) cell populations. The cadherin-binding inhibitory receptor killer cell lectin-like receptor G1 (KLRG1) is expressed by a subpopulation of Treg cells with GATA3+ effector phenotype. Although such Treg cells are important for the immune balance, especially in the gut, the role of KLRG1 in Treg cells has not been assessed. Using KLRG1 knockout mice, we found that KLRG1 deficiency does not affect Treg cell frequencies in spleen, mesenteric lymph nodes or intestine, or frequencies of GATA3+ Treg cells in the gut. KLRG1-deficient Treg cells were also protective in a T-cell transfer model of colitis. Hence, KLRG1 is not essential for the development or activity of the general Treg cell population. We then checked the effects of KLRG1 on Treg cell activation. In line with KLRG1's reported inhibitory activity, in vitro KLRG1 cross-linking dampened the Treg cell T-cell receptor response. Consistently, lack of KLRG1 on Treg cells conferred on them a competitive advantage in the gut, but not in lymphoid organs. Hence, although absence of KLRG1 is not enough to increase intestinal Treg cells in KLRG1 knockout mice, KLRG1 ligation reduces T-cell receptor signals and the competitive fitness of individual Treg cells in the intestine. [ABSTRACT FROM AUTHOR]

Regulatory T (Treg) cells are a suppressive CD4+ T-cell subset. We generated induced Treg ( iTreg) cells and explored their therapeutic potential in a murine model of rapidly progressive glomerulonephritis. Polyclonal naive CD4+ T cells were cultured in vitro with interleukin-2 ( IL-2), transforming growth factor- β1, all- trans-retinoic acid and monoclonal antibodies against interferon- γ and IL-4, generating Foxp3+ iTreg cells. To enhance their suppressive phenotype, iTreg cultures were modified with the addition of a monoclonal antibody against IL-12p40 or by using ROR γt-/- CD4+ T cells. Induced Treg cells were transferred into models of delayed-type hypersensitivity and experimental glomerulonephritis. The iTreg cells exhibited comparable surface receptor expression and in vitro suppressive ability to natural Treg cells, but did not regulate antigen-specific delayed-type hypersensitivity or systemic inflammatory immune responses, losing Foxp3 expression in vivo. In glomerulonephritis, transferred iTreg cells did not prevent renal injury or modulate systemic T helper type 1 immune responses. Induced Treg cells cultured with anti- IL-12p40 had an enhanced suppressive phenotype in vitro and regulated dermal delayed-type hypersensitivity in vivo, but were not protective against renal injury, losing Foxp3 expression, especially in the transferred cells recruited to the kidney. Use of ROR γt-/- CD4+ T cells or iTreg cells generated from sensitized CD4+ Foxp3- cells did not regulate renal or systemic inflammatory responses in vivo. In conclusion, iTreg cells suppress T-cell proliferation in vitro, but do not regulate experimental glomerulonephritis, being unstable in this inflammatory milieu in vivo. [ABSTRACT FROM AUTHOR]

Vitamin D deficiency is associated with increased incidence and severity of various immune-mediated diseases. Active vitamin D (1α,25-dihydroxyvitamin D3; 1,25(OH)2D3) up-regulates CD4+ T-cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti-inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)2D3 on expression of the downstream ecto-5′-nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor- β (TGF- β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10−8 to 10−7 m, 1,25(OH)2D3 significantly increased expression of CD73 on peripheral human CD4+ T cells. Although 1,25(OH)2D3 did not affect the mRNA expression of latent TGF- β1, 1,25(OH)2D3 did up-regulate expression of TGF- β-associated molecules [latency-associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin-1, thrombospondin-1 and αv integrin] which is likely to have contributed to the observed enhancement in TGF- β bioactivity. CD73 was highly co-expressed with LAP and GARP following 1,25(OH)2D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4+ Foxp3- T cells, rather than CD4+ Foxp3+ T cells. Notably, neutralization of TGF- β significantly impaired 1,25(OH)2D3-mediated induction of CD73. Collectively, we show that 1,25(OH)2D3 enhances expression of CD73 on CD4+ Foxp3- T cells in a process that is at least partially TGF- β-dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune-mediated disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antigen-specific immunotherapy aims to selectively restore tolerance to innocuous antigens in cases of autoimmune or allergic disease, without the need for general immune suppression. Although the principle of antigen-specific immunotherapy was discovered more than a century ago, its clinical application to date is limited, particularly in the control of autoimmunity. This has resulted mainly from a lack of in-depth understanding of the underlying mechanism. More recently, the differentiation of extra-thymically induced T regulatory ( Treg) cell subsets has been shown to be instrumental in peripheral tolerance induction. Two main types of inducible Treg cells, interleukin-10-secreting or Foxp3+, have now been described, each with distinct characteristics and methods of therapeutic induction. It is crucial, therefore, to identify the suitability of either subset in the control of specific immune disorders. This review explores their natural function, the known mechanisms of therapeutic differentiation of either subset as well as their in vivo functionality and discusses new developments that may aid their use in antigen-specific immunotherapy, with a focus on autoimmune disease. [ABSTRACT FROM AUTHOR]

Foxp3+ regulatory T (Treg) cells are essential to maintain immune homeostasis, yet controversy exists about the stability of this cell population. Bcl6-deficient (Bcl6−/−) mice develop severe and spontaneous T helper type 2 (Th2) inflammation and Bcl6-deficient Treg cells are ineffective at controlling Th2 responses. We used a lineage tracing approach to analyse the fate of Treg cells in these mice. In the periphery of Bcl6−/− mice, increased numbers of Foxp3-negative 'exTreg' cells were found, particularly in the CD25+ population. ExTreg cells from Bcl6−/− mice expressed increased interleukin-17 ( IL-17) and extremely elevated levels of Th2 cytokines compared with wild-type exTreg cells. Although Treg cells normally express only low levels of cytokines, Treg cells from Bcl6−/− mice secreted higher levels of IL-4, IL-5, IL-13 and IL-17 than wild-type conventional T cells. Next, Treg-specific conditional Bcl6-deficient (Bcl6Foxp3−/−) mice were analysed. Bcl6Foxp3−/− mice do not develop inflammatory disease, indicating a requirement for non-Treg cells for inflammation in Bcl6−/− mice, and have normal numbers of exTreg cells. We induced Th2-type allergic airway inflammation in Bcl6Foxp3−/− mice, and found that while exTreg cytokine expression was normal, Bcl6-deficient Treg cells expressed higher levels of the Th2-specific regulator Gata3 than Bcl6+ Treg cells. Bcl6Foxp3−/− mice had increased numbers of Th2 cells after induction of airway inflammation and increased T cells in the bronchoalveolar lavage fluid. These data show both Treg-intrinsic and Treg-extrinsic roles for Bcl6 in controlling Treg cell stability and Th2 inflammation, and support the idea that Bcl6 expression in Treg cells is critical for controlling Th2 responses. [ABSTRACT FROM AUTHOR]

Regulatory T (Treg) cells play an important role in the maintenance of immune self-tolerance and homeostasis. We previously reported that neonatal CD4+ T cells have an intrinsic 'default' mechanism to become Treg (neoTreg) cells in response to T-cell receptor ( TCR) stimulation. However, the underlying mechanisms are unclear and the effects of neoTreg cells on regulating immune responses remain unknown. Due to their involvement in Foxp3 regulation, we examined the role of DNA methyltransferase 1 ( DNMT1) and DNMT3b during the induction of neoTreg cells in the Foxp3gfp mice. The function of neoTreg cells was assessed in an acute allograft rejection model established in RAG2−/− mice with allograft cardiac transplantation and transferred with syngeneic CD4+ effector T cells. Following ex vivo TCR stimulation, the DNMT activity was increased threefold in adult CD4+ T cells, but not significantly increased in neonatal cells. However, adoptively transferred neoTreg cells significantly prolonged cardiac allograft survival (mean survival time 47 days, P < 0·001) and maintained Foxp3 expression similar to natural Treg cells. The neoTreg cells were hypomethylated at the conserved non-coding DNA sequence 2 locus of Foxp3 compared with adult Treg cells. The DNMT antagonist 5-aza-2′-deoxycytidine (5-Aza) induced increased Foxp3 expression in mature CD4+ T cells. 5-Aza-inducible Treg cells combined with continuous 5-Aza treatment prolonged graft survival. These results indicate that the 'default' pathway of neoTreg cell differentiation is associated with reduced DNMT1 and DNMT3b response to TCR stimulus. The neoTreg cells may be a strategy to alleviate acute allograft rejection. [ABSTRACT FROM AUTHOR]

Regulatory T (Treg) cells act to suppress activation of the immune system and thereby maintain immunological homeostasis and tolerance to self-antigens. The frequency and suppressing activity of Treg cells in general are high in different malignancies. We wanted to identify the role and regulation of CD4+ CD25+ FoxP3+ Treg cells in B-cell acute lymphoblastic leukaemia (B- ALL). We have included patients at diagnosis ( n = 54), patients in clinical remission ( n = 32) and normal healthy individuals ( n = 35). These diagnosed patients demonstrated a lower number of CD4+ CD25+ cells co-expressing a higher level of FoxP3, interleukin-10, transforming growth factor- β and CD152/ CTLA-4 than the normal population. Treg cells from patients showed a higher suppressive capability on CD4+ CD25 − responder T (Tresp) cells than normal. The frequency and immunosuppressive potential of CD4+ CD25+ FoxP3+ Treg cells became high with the progression of malignancy in B- ALL. Relative distribution of Tresp and Treg cells was only ~5 : 1 in B- ALL but ~35 : 1 in normal healthy individuals, further confirming the elevated immunosuppression in patients. A co-culture study at these definite ex vivo ratios, indicated that Treg cells from B- ALL patients exhibited higher immunosuppression than Treg cells from normal healthy individuals. After chemotherapy using the MCP841 protocol, the frequency of CD4+ CD25+ cells was gradually enhanced with the reduction of FoxP3, interleukin-10 positivity corresponded with disease presentation, indicating reduced immunosuppression. Taken together, our study indicated that the CD4+ CD25+ FoxP3+ Treg cells played an important role in immunosuppression, resulting in a positive disease-correlation in these patients. To the best of our knowledge, this is the first detailed report on the frequency, regulation and functionality of Treg cells in B- ALL. [ABSTRACT FROM AUTHOR]

Loss of ζ-associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T-cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N-ethyl-N-nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR-induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3+ regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70A243V variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70rps allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null. [ABSTRACT FROM AUTHOR]

The mammalian target of rapamycin (m TOR) pathway is an important integrator of nutrient-sensing signals in all mammalian cells, and acts to coordinate the cell proliferation with the availability of nutrients such as glucose, amino acids and energy (oxygen and ATP). A large part of the immune response depends on the proliferation and clonal expansion of antigen-specific T cells, which depends on m TOR activation, and the pharmacological inhibition of this pathway by rapamycin is therefore potently immunosuppressive. It is only recently, however, that we have started to understand the more subtle details of how the m TOR pathway is involved in controlling the differentiation of effector versus memory CD8+ T cells and the decision to generate different CD4+ helper T-cell subsets. In particular, this review will focus on how nutrient sensing via m TOR controls the expression of the master transcription factor for regulatory T cells in order to maintain the balance between tolerance and inflammation. [ABSTRACT FROM AUTHOR]

Regulatory T ( Treg) cells are important in the regulation of immune response, but the exact regulation of Treg-cell function in vivo is still not well known. In the present study, we investigated the functional activity of CD4+ CD25+ Treg cells as well as the frequency and number of CD4+ CD25+ Fox P3+ Treg cells in the spleens of experimentally infected mice with a tissue-migrating parasite, sparganum (plerocercoid of Spirometra mansoni) for 3 weeks. The results demonstrated fluctuations in the Treg-cell function during the parasite infection, being up-regulated at day 3, down-regulated until day 14, and thereafter up-regulated again at day 21. We also investigated the cytokine-producing capability of the splenocytes to study the pattern of immune response of the mice to the parasite. The results showed decreased capabilities of interleukin-2 ( IL-2), interferon-γ ( IFN-γ) and IL-17α production, whereas IL-4-producing and IL-10-producing capabilities were increased along with the parasitic infection. Meanwhile, IL-6-producing capability was increased to reach a peak at week 2, and thereafter was decreased to the baseline level. As a regulatory mechanism, we found that Treg-cell function was attenuated in the presence of the crude extracts of sparganum, but was enhanced in the presence of the excretory-secretory products, suggesting that sparganum products were involved in the triggering and regulation of immune response in the acute and chronic phases, respectively. Results show that Treg cells are central in the immune homeostasis in vivo that is maintained by host-parasite interactions during the parasitic infection. [ABSTRACT FROM AUTHOR]

The aim of this study is to investigate the clinical significance of the ratio between interleukin-17 (IL-17) secreting cell and FOXP3-positive regulatory T cell (FOXP3+ Treg) infiltration in renal allograft tissues with acute T-cell-mediated rejection (ATCMR). Fifty-six patients with biopsy-proven ATCMR were included. Infiltration of FOXP3+ Treg and IL-17-secreting cells was evaluated with immunostaining for FOXP3 or IL-17 on the biopsy specimens, and the patients were divided into the FOXP3 high group (Log FOXP3/IL-17 > 0·45) or the IL-17 high group (Log FOXP3/IL-17 < 0·45). We compared the allograft function, severity of tissue injury, and clinical outcome between the two groups. In the IL-17 high group, allograft function was significantly decreased compared with the FOXP3 high group ( P < 0·05). The severity of interstitial and tubular injury in the IL-17 high group was higher than the FOXP3 high group ( P < 0·05). The proportions of steroid-resistant rejection, incomplete recovery and recurrent ATCMR were higher in the IL-17 high group than in the FOXP3 high group (all indicators, P < 0·05). The IL-17 high group showed lower 1-year (54% versus 90%, P < 0·05) and 5-year (38% versus 85%, P < 0·05) allograft survival rates compared with the FOXP3 high group. Multivariate analysis revealed that the FOXP3/IL-17 ratio was a significant predictor for allograft outcome. The FOXP3/IL-17 ratio is a useful indicator for representing the severity of tissue injury, allograft dysfunction and for predicting the clinical outcome of ATCMR. [ABSTRACT FROM AUTHOR]

In this study we investigated the impact of several factors on the expansion of natural regulatory T (nTreg) cells by tumours, including antigen specificity, transforming growth factor-β (TGF-β) signalling and the antigen-presenting cell subsets responsible for expansion. We found that antigen non-specific expansion of nTreg cells is tumour cell line-dependent. Although both antigen-specific and non-specific pathways can contribute to expansion, the migration of activated nTreg cells to tumour tissues is strictly antigen-dependent. Intact TGF-β signalling on nTreg cells is also essential for tumour-induced expansion. Finally, for stimulation of resting antigen-specific CD4 T cells, CD11c cells purified from tumour-draining lymph nodes were more potent than CD11b cells, suggesting that dendritic cells are the key antigen-presenting cell subset involved in cross-presentation of tumour antigens. This study not only provides an in vivo system in which cross-talk between nTreg cells and tumours can be explored but also reveals novel aspects of tumour immune evasion. [ABSTRACT FROM AUTHOR]

Statins are widely used drugs for the treatment of hypercholesterolaemia. A number of recent studies have suggested that statins also have pleiotropic effects on immune responses and statins have proven to be effective in the treatment of autoimmune diseases in animal models. Foxp3+ T regulatory cells are a unique subset of CD4+ T cells that mediate immunosuppression. Foxp3+ T cells develop in the thymus, but can also be induced in peripheral sites in the presence of transforming growth factor-β (TGF-β). We demonstrate here that simvastatin blockade of the mevalonate pathway can mediate induction of mouse Foxp3+ T cells and that simvastatin can synergize with low levels of TGF-β to induce Foxp3+ T cells. The effects of simvastatin are secondary to a blockade of protein geranylgeranylation, are mediated at late time-points after T-cell activation, and are associated with demethylation of the Foxp3 promoter. One major effect of simvastatin was inhibition of the induction of Smad6 and Smad7, inhibitory Smads that inhibit TGF-β signalling. Our results suggest that one mechanism responsible for the immunosuppressive effects of statins is the ability to promote the generation of Foxp3+ T regulatory cells. [ABSTRACT FROM AUTHOR]

T-cell receptor (TCR)-derived peptides are recognized by the immune system and are capable of modulating autoimmune responses. Using the myelin basic protein (MBP) TCR 1501 transgenic mouse model, we demonstrated that TCR CDR3 peptides from the transgenic TCR can provide a protective effect when therapy is initiated before the induction of experimental autoimmune encephalomyelitis (EAE). More importantly, TCR CDR3 peptide therapy can ameliorate the disease when administered after EAE onset. Concurrent with the therapeutic effects, we observed reduced T-cell proliferation and reduced interleukin-2 (IL-2) levels in response to stimulation with MBP-85-99 peptide in splenocyte cultures from mice receiving TCR CDR3 peptides compared with that of control mice. Moreover, we found that Foxp3+ CD4 T cells from mice protected with TCR CDR3 peptide are preferentially expanded in the presence of IL-2. This is supportive of a proposed mechanism where Foxp3+ T-regulatory cells induced by therapy with MBP-85-99 TCR CDR3 peptides limit expansion and the encephalitogenic activity of MBP-85-99-specific T cells by regulating the levels of secreted IL-2. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system and a defect in the regulatory T-cell subset seems to be involved in the pathogenesis of the disease. Foxp3 is a transcription factor that is selectively expressed in CD4+ CD25+ regulatory T cells and is required for their development and function. T-bet is a key transcription factor for the development of T helper 1 (Th1) cells. We found that both the percentage of circulating CD4+ CD25+ Foxp3+ cells and Foxp3 expression were lower in relapsing-remitting (RR) MS patients during relapses than during remission. Otherwise, the percentage of CD4+ T-bet+ T cells and T-bet expression in CD4+ T cells were higher in relapsing than in remitting RRMS patients. CD4+ CD25+ T cells both from relapsing and from remitting RRMS patients showed significantly less capacity than corresponding cells from healthy subjects to suppress autologous CD4+ CD25− T-cell proliferation, despite a similar Foxp3 expression level. CD4+ CD25+ T cells from healthy subjects and patients in remission clearly reduced T-bet mean fluorescence intensity (MFI) in CD4+ CD25− T cells up to a ratio of 1:10, whereas CD4+ CD25+ T cells from patients in relapse were able to reduce T-bet expression only at a high ratio. Our data indicate that the increased number of regulatory T (T-reg) cells and the increased Foxp3 expression in circulating CD4+ CD25+ T cells may contribute to the maintenance of tolerance in the remission phase of MS. Moreover, the inhibitory capacity of CD4+ CD25+ T cells seems to be impaired in relapsing patients under inflammatory conditions, as shown by the high levels of T-bet expression in CD4+ T cells. [ABSTRACT FROM AUTHOR]

The role of naturally occurring regulatory T cells (Treg), known to be phenotypically heterogeneous, in controlling the expression of systemic lupus erythematosus (SLE) is incompletely defined. Therefore, different subpopulations of CD4+ FoxP3+ Tregs in patients with active or inactive SLE were investigated and compared with those of healthy subjects and patients with ankylosing spondylitis (AS). Characterization of different subsets of circulating CD4+ FoxP3+ Tregs was examined using flow cytometry. CD4+ CD25high T cells were sorted and examined for suppressive activity in vitro. The results showed first that a significant decrease in the frequency of CD4+ CD25high FoxP3+ T cells was present in patients with active SLE ( n = 58), compared with healthy controls ( n = 36) and AS patients ( n = 23). In contrast, the frequencies of CD25low FoxP3+ and CD25− FoxP3+ CD4+ T cells were significantly increased in patients with active SLE by comparison with the control subjects. The elevation of these two putative Treg subpopulations was associated with lower plasma levels of complement C3 and C4 in patients with SLE. In addition, the ratios of the three subsets of CD4+ FoxP3+ Tregs versus effector T cells (CD4+ CD25+ FoxP3−) were inversely correlated with the titer of anti-double-stranded DNA IgG in patients with inactive, but not active, SLE. These results suggest that the pathogenesis of SLE may be associated with a defect in the homeostatic control of different Treg subsets. [ABSTRACT FROM AUTHOR]

Crohn’s disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Its pathogenesis is not completely understood, though the prevailing model is that the intestinal flora drives a strong intestinal T helper 1 (Th1)/Th17 type immune response and inflammation in the genetically susceptible host. This leads to overly aggressive T-cell responses to normal bacteria causing tissue damage. Intestinal homeostasis and maintenance of tolerance to harmless antigens in the intestine has been shown to be maintained by CD4+ CD25+ T regulatory cells (Treg) in animal models of inflammatory bowel diseases. Here we investigated whether Infliximab, a chimeric monoclonal antibody directed against tumour necrosis factor (TNF)-α shown to be highly effective in the treatment of CD, has any effect on mucosal CD4+ CD25+ (FOXP3+) Tregs. Colonic mucosal biopsies from children with active Crohn’s disease treated in vivo with Infliximab and controls were analysed to determine FOXP3 expression by immunofluorescence and reverse transcription–polymerase chain reaction. We observed that FOXP3+ T cells were significantly reduced in mucosa of CD patients with active disease compared with controls and restored to normal following Infliximab treatment. The frequency of FOXP3+ cells and mRNA expression was significantly increased in CD mucosa from patients treated in vivo with Infliximab compared with CD patients treated with conventional therapies. In conclusion, we show that Infliximab treatment does not solely neutralize soluble TNF-α, but also affects activation and possibly expansion of mucosal regulatory T cells. We suggest that anti TNF-α immunotherapy can also restore mucosal homeostasis in Crohn’s disease. [ABSTRACT FROM AUTHOR]

The identification of CD4+ CD25+ Foxp3+ regulatory T (Treg) cells as natural regulators of immunity in the periphery and tissues has stimulated tremendous interest in developing therapeutic strategies for autoimmune diseases. In this review, the site of origin, antigen specificity, homing markers and cytokine profiles of Treg cells were evaluated in autoimmune colitis and type 1 diabetes, two examples in which Treg cells were effective as therapy. These studies were compared with studies of Treg cells in experimental autoimmune encephalomyelitis and multiple sclerosis, where successful therapy has not yet been achieved. Antigen-specific Treg cells appear to have more potent activity than polyclonal Treg cells and therefore hold more promise as therapeutic agents. However, Treg cells specific for the pathogenic T effector cells themselves have largely been overlooked and deserve consideration in future studies. [ABSTRACT FROM AUTHOR]

A growing body of evidence has shown that professional antigen-presenting cells (APC) treated with transforming growth factor-β (TGF-β) can induce a systemic antigen (Ag)-specific tolerance, similar to anterior chamber-associated immune deviation (ACAID). However, the exact mechanism for immune tolerance induced by TGF-β-treated APC has not been elucidated. In this study, we showed that intravenous injection of ovalbumin (OVA)-pulsed APC treated with TGF-β2 induced a peripheral tolerance as evidenced by an impaired delayed-type hypersensitivity (DTH) response. CD4+ T cells from mice receiving an intravenous injection of TGF-β2-treated APC pulsed with OVA could adoptively transfer a specific tolerance to naïve mice. An increased frequency of FoxP3-expressing CD4+ CD25+ T cells was observed in mice with tolerance. CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice produced a large amount of TGF-β1 and exhibited an in vitro antigen-specific suppressive activity. CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice were able to inhibit the antigen-specific DTH response significantly when adoptively transferred to naïve mice. These results indicate that FoxP3-expressing CD4+ CD25+ T cells might be actively involved in the development of tolerance induced by TGF-β2-treated APC. [ABSTRACT FROM AUTHOR]

CD4+ CD25high regulatory T cells (Tregs) of patients with relapsing-remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single-cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4+ CD25high FOXP3+ T cells and lower FOXP3 protein expression per cell in RR-MS patients than in SP-MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)-β-treated RR-MS patients showed restored numbers of FOXP3+ Tregs. Furthermore, a higher percentage of CD4+ CD25high FOXP3+ Tregs in RR-MS patients, as compared with controls and SP-MS patients, expressed CD103 and CD49d, adhesion molecules involved in T-cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27+ CD25high CD4+ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR-MS patients. Taken together, these data show aberrant FOXP3 expression at the single-cell level correlated with Treg dysfunction in RR-MS patients. Our results also suggest that Tregs accumulate in the CSF of RR-MS patients, in an attempt to down-regulate local inflammation in the central nervous system. [ABSTRACT FROM AUTHOR]

Circulating CD4+ CD25+ regulatory T cells (Tregs) have been demonstrated to maintain immunotolerance and suppress the antigen-specific or antigen-non-specific T-cell responses, but their role in chronic hepatitis B (CHB) infection in humans has not been well characterized. In this study, we analysed the frequency and phenotypic characteristics of CD4+ CD25+ Tregs in patients of different hepatitis B virus (HBV) infection status, and investigated the effect of Tregs on antiviral immune responses in CHB patients, and the mechanism of this effect. A total of 137 subjects, including 79 CHB patients, 26 asymptomatic HBV carriers (ASCs), 12 acute hepatitis B (AHB) patients and 20 healthy controls, were enrolled in the study. We found that the frequency of CD4+ CD25high Tregs in AHB patients was comparable to that in healthy controls, while it was significantly increased in CHB patients. CD4+ CD25+ Tregs produced interleukin (IL)-10 but little or no interferon (IFN)-γ under anti-CD3 stimulation. In CHB patients, the frequency of CD4+ CD25high Tregs positively correlated with serum viral load, and the Tregs were capable of suppressing the proliferation and IFN-γ production of autologous peripheral blood mononuclear cells (PBMC) mediated by HBV antigen stimulation in vitro. However, combined administration of anti-programmed death-1 (PD-1) and anti-cytotoxic lymphocyte antigen-4 (CTLA-4) monoclonal antibody slightly enhanced the cellular proliferation and significantly increased the IFN-γ production of PBMC cocultured with Tregs at a ratio of 2 : 1. Thus, the frequency of circulating CD4+ CD25+ Tregs is increased in patients with CHB, and this may play an important role in viral persistence by modulating virus-specific immune responses. [ABSTRACT FROM AUTHOR]

Tolerance to self and harmless antigens is one of the central features of the immune system, and it is obtained through a combination of multiple mechanisms. Discriminating between pathogens and non-pathogenic antigens is especially important in the intestine, which constitutes the main contact surface between the body and the outside environment. Recently, the role of Foxp3+ regulatory T cells (Treg) in the establishment and maintenance of tolerance has been the focus of numerous studies. In this review, we briefly discuss the historical background leading to the identification of Foxp3+ Treg and give an overview of their role in controlling systemic and mucosal immune responses. [ABSTRACT FROM AUTHOR]

The role of regulatory T cells (Treg) in maintaining tolerance to self has been intensively scrutinized, particularly since the discovery of Foxp3 as a Treg-specific transcription factor. The BDC2·5NOD transgenic mouse is an excellent model of immunoregulation because it has a very low incidence of diabetes despite a highly autoreactive T-cell repertoire. It has previously been shown that reactivity against islets decreases with age in BDC2·5NOD mice. Here we show that there is a markedly higher frequency of Foxp3+ Treg in the CD4+ subset of 16–20-week-old mice compared with 4- or 8-week-old mice. This phenomenon can be observed in the spleen, thymus, pancreatic draining lymph nodes and the pancreas itself. We show that this early age-related increase in the frequency of Foxp3+ cells does not occur in wild-type NOD, BALB/c or C57BL/6 mice. Further, we show that, in contrast to some reports on Treg in wild-type NOD mice, the suppressive function of BDC2·5NOD Treg from 16- to 20-week-old mice is intact and comparable to that from 4- to 8-week-old mice both in vitro and in vivo. Our data offer insights into the long-term protection of BDC2·5NOD mice from diabetes and an explanation for the age-related decrease in anti-islet responses seen in BDC2·5NOD mice. [ABSTRACT FROM AUTHOR]

The role of regulatory T cells (Tregs) in maintaining self tolerance has been intensively researched and there is a growing consensus that a decline in Treg function is an important step towards the development of autoimmune diseases, including diabetes. Although we show here that CD25+ cells delay diabetes onset in non-obese diabetic (NOD) mice, we found, in contrast to previous reports, neither an age-related decline nor a decline following onset of diabetes in the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. Furthermore, we demonstrate that CD4+ CD25+ cells from both the spleen and pancreatic draining lymph nodes of diabetic and non-diabetic NOD mice are able to suppress the proliferation of CD4+ CD25– cells to a similar extent in vitro. We also found that pretreatment of NOD mice with anti-CD25 antibody allowed T cells with a known reactivity to islet antigen to proliferate more in the pancreatic draining lymph nodes of NOD mice, regardless of age. In addition, we demonstrated that onset of diabetes in NOD. scid mice is faster when recipients are co-administered splenocytes from diabetic NOD donors and anti-CD25. Finally, we found that although diabetic CD4+ CD25+ T cells are not as suppressive in cotransfers with effectors into NOD. scid recipients, this may not indicate a decline in Treg function in diabetic mice because over 10% of CD4+ CD25+ T cells are non-Foxp3 and the phenotype of the CD25– contaminating population significantly differs in non-diabetic and diabetic mice. This work questions whether onset of diabetes in NOD mice is associated with a decline in Treg function. [ABSTRACT FROM AUTHOR]