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The characteristics and functions of CD4 + CD25 + regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4 + CD25 + Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4 + CD25 + Tregs on the maturation and function of monocyte-derived DCs were examined. The results showed that CD4 + CD25 + render the DCs inefficient as antigen-presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL-10 and TGF-β. There were an increased IL-10 and TGF-β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4 + T cells, CD4 + CD25 + may modulate the immune response through DCs in CHB patients., (© 2018 John Wiley & Sons Ltd.)

Soluble fibrinogen-like protein 2, sfgl2, is the new effector of CD4(+)CD25(+)FOXP3(+) regulatory T cell (Treg) and exerts immunosuppressive activity. We design this study to investigate the possible role of sfgl2 in atherosclerosis. A total of 58 acute coronary syndrome (ACS) patients, together with 22 stable angina (SA) patients and 31 normal coronary artery (NCA) people were enrolled in our study. Serum level of sfgl2 and plasma level of Treg were respectively measured. In line with the change of Treg, serum level of sfgl2 in ACS (8.70 ng/mL) was significantly decreased (P = 0.003), compared with that in SA (11.86 ng/mL) and NCA (17.55 ng/mL). Both sfgl2 and Treg level were obviously decreased in ACS; Sfgl2 may play a protective role in atherosclerosis., (© 2016 by the Society for Experimental Biology and Medicine.)

Background: We and others have previously reported that cell membrane-bound TGFβ (mTGFβ) on activated T regulatory (Treg) cells mediates suppressor function. Current findings suggest that a novel protein known as Glycoprotein A Repetitions Predominant (GARP) anchors mTGFβ to the Treg cell surface and facilitates suppressor activity. Recently, we have described that GARP+TGFβ+ Treg cells expand during the course of FIV infection. Because Treg cells are anergic and generally exhibit poor proliferative ability, we asked how Treg homeostasis is maintained during the course of feline immunodeficiency virus (FIV) infection., Results: Here, we report that Treg cells from FIV+ cats express GARP and mTGFβ and convert T helper (Th) cells into phenotypic and functional Treg cells. Th to Treg conversion was abrogated by anti-TGFβ or anti-GARP treatment of Treg cells or by anti-TGFβRII treatment of Th cells, suggesting that Treg cell recruitment from the Th pool is mediated by TGFβ/TGFβRII signaling and that cell-surface GARP plays a major role in this process., Conclusions: These findings suggest Th to Treg conversion may initiate a cascade of events that contributes to the maintenance of virus reservoirs, progressive Th cell immunosuppression, and the development of immunodeficiency, all of which are central to the pathogenesis of AIDS lentivirus infections.

Various proteins are expressed during different stages of schistosome development that are essential for cercarial penetration of vertebrate skin and evasion of host immune response. CD4(+)CD25(+) regulatory T cells are important in modulating immune responses towards helminth infections. Schistosoma japonicum protein Sj16 present in the secretions of schistosomula has been shown to have anti-inflammatory effects; however, it is uncertain whether Sj16 can induce CD4(+)CD25(+) regulatory T cells to participate in the regulation of early infection. In this study, we demonstrate a relationship between recombinant Sj16 (rSj16) and the induction of CD4(+)CD25(+) Foxp3(+) regulatory T cells. An increase in CD4(+)CD25(+) T cells was observed both in splenic cells from mice injected with rSj16 and the cells pretreated with rSj16, respectively. The induced CD4(+)CD25(+) T cells suppressed CD4(+)CD25(-) T-cell proliferation; furthermore, IFN-γ and IL-10 released from rSj16-stimulated cells contribute to this suppression. Additionally, rSj16-treated bone marrow dendritic cells (BMDCs) demonstrate an immature phenotype and play a role in the conversion of CD4(+)CD25(-) T cells into suppressive CD4(+)CD25(+) regulatory T cells. Our study identified a new CD4(+)CD25(+) T-cell population that induced by rSj16 and suggests that an IFN-γ-biased microenvironment during early infection of schistosome may favour the establishment of infection., (© 2012 Blackwell Publishing Ltd.)

The increase of CD4+CD25+ regulatory T cells in patients with ovarian carcinoma has been verified. Here we investigated the effects of supernatant derived from ovarian carcinoma cell SKOV3 on peripheral regulatory T cells. Supernatant from SKOV3 was collected and fractionated into three different molecular weight fractions (MWFs). The proliferation of the CD4+CD25+ regulatory T cells cultured in complete RPMI 1640 medium with the different stimulators was detected. The phenotype (GITR and CTLA-4) of natural and expanded CD4+CD25+ T cells was detected by flow cytometry. Foxp3 mRNA expression of low MWF-expanded CD4+CD25+ T cells was detected by RT-PCR. Those expanded CD4+CD25+ regulatory T cells showed enhanced capacity to suppress CD4+CD25- T proliferation and increased expression of GITR and CTLA-4. In brief, low molecular weight fraction of supernatant secreted by SKOV3 could expand peripheral CD4+CD25+ regulatory T cells and enhance their suppressive function.

Chronic hepatitis B (CHB) virus hepatitis B virus (HBV) infection is the key cause of hepatocellular carcinoma (HCC) in Asians. Recent studies have shown that levels of CD4(+)CD25(+) regulatory T cells (Tregs) were increased and were linked to an impaired immune response in patients with CHB. Evaluating whether Tregs are involved in the progression of CHB to HCC will provide insight into the immunopathogenesis of HCC. In the present study, we showed that circulating and liver-residing Tregs increased in CHB (n = 15) and HCC (n = 49) patients, particularly in the peripheral blood of HCC patients with HBV infection (n = 29). The increased Tregs in CHB patients suppressed the specific immune response induced by not only HBV antigen, but also by HCC tumour antigen. When peripheral blood mononuclear cells (PBMC) were co-cultured with human hepatoma cell lines that are stably transfected with HBV (HepG2.2.15), CD4(+)CD25(+) Treg populations increased and upregulated the expression of forkhead box P3 transcriptional regulator (FoxP3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid-induced tumour necrosis factor (TNF) receptor family gene (GITR). In contrast, PBMCs co-cultured with HepG2 cells (the parental cell line of HepG2.2.15) did not. CD4(+)CD25(+) Tregs isolated from PBMCs that were co-cultured with HepG2.2.15 cells also had a greater suppressive ability with respect to the tumour antigen-specific immune response induced by NY-ESO-1 or MAGE-A3 compared with CD4(+)CD25(+) Tregs isolated from PBMCs co-cultured with HepG2 cells. The results offer evidence that the expansion of CD4(+)CD25(+) Tregs and the enhancement of the suppressor function of CD4(+)CD25(+) Tregs induced by HBV infection-related factors could suppress the anti-tumour immune response to HCC tumour antigen and inhibit tumour immuno-surveillance against HCC, which may be involved in the immunopathogenesis from CHB to HCC.

Although signaling through certain TLRs is known to modulate the function of T lymphocytes, the effect of TLR7 stimulation on CD4(+)CD25(+) T(reg) cell activity has not yet been elucidated. In this study, we show that mouse CD4(+)CD25(+) T(reg) cells express TLR7 mRNA and protein. We therefore used the TLR7 agonists imiquimod, gardiquimod, and single-stranded poly(U) to show that TLR7 stimulation enhanced the ability of murine T(reg) cells to suppress anti-CD3/anti-CD28 mAb-coated bead-stimulated proliferation of syngeneic CD4(+)CD25(-) T(resp) cells. In contrast, imiquimod failed to enhance the suppressor function of T(reg) cells from mice deficient in the MyD88 adaptor protein involved in TLR7 and other TLR signal transduction. Imiquimod increased murine T(reg) cell-mediated suppression of T(resp) cell proliferation induced by anti-TCRbeta mAb in the presence of syngeneic BMDCs, and T(reg) cells from gardiquimod-treated mice exhibited enhanced in vitro suppressor function. Moreover, levels of T(resp) cell-secreted IL-2 and IFN-gamma were reduced further in the presence of T(reg) cells plus imiquimod in comparison with T(reg) cells alone. In addition, imiquimod treatment increased CD25 expression by T(reg) cells and caused exogenous IL-2 to enhance T(reg) cell suppressor function. Furthermore, combined treatment with imiquimod and IL-2 increased Foxp3 expression by T(reg) cells. Collectively, these findings suggest that TLR7 signaling enhanced the suppressor function of T(reg) cells by sensitizing T(reg) cells to IL-2-induced activation. We speculate that TLR7-stimulated enhancement of T(reg) cell suppressor function may modulate host T cell responses against ssRNA viruses.

Background & Aims: Orally induced tolerance to environmental allergens prevents deleterious, systemic, delayed-type hypersensitivity responses via immune suppression mechanisms believed to include either anergy/deletion of specific effector T cells or active suppression by CD4(+)CD25(+) regulatory T cells (Tregs). The aim of this study was to investigate whether and how antigen (Ag) penetration through the gut orchestrates these 2 distinct mechanisms., Methods: Using a model of allergic contact dermatitis (ACD) mediated by hapten-specific cytolytic CD8(+) T cells and a T-cell transfer model of contact hypersensitivity in CD3epsilon-deficient mice, we studied the outcome of Ag gavage on CD8(+) effectors and Tregs., Results: Full protection from ACD by gavage with the relevant allergen required 2 coordinated events taking place first in gut-associated lymphoid tissues and then systemically. Allergen gavage induced deletion by plasmacytoid dendritic cells of a large fraction of Ag-specific CD8(+) T cells in liver and mesenteric lymph nodes and also triggered the suppressive function of Treg of secondary lymphoid organs. Residual Ag-specific CD8(+) T cells conditioned during this mucosal step are fully susceptible to suppression by activated Treg, which completely prevented their differentiation into ACD effectors, upon re-exposure to the allergen via the skin., Conclusions: Thus, oral tolerance initiated in gut-associated lymphoid tissues by the plasmacytoid dendritic cells-mediated deletion of Ag-specific T cells is completed systemically by CD4(+)CD25(+) T cells. Biotherapies able to increase the susceptibility of effector T cells to the suppressive function of Treg may be valuable for the treatment of autoimmune and inflammatory diseases.

Variety of positive as well as negative regulatory signals are provided by antigen presenting cell in particular by dendritic cells. In this research, we studied the capacity of dendritic cells to expand antigen-specific T regulatory cells.We also investigated the role of TGF-beta in induction inhibitory functions of dendritic cells in mixed leukocyte reactions.Dendritic cells were generated from blood CD14(+) monocytes with granulocyte-Monocyte colony stimulating factor and interleukin-4 with or without TGF-beta (TGF-beta-GM-DC or GM-DC). CD4(+) T cell were isolated to assess lymphocyte proliferation by lymphocyte transformation test assay and the ratio of CD4(+)FOXp3(+) CD25(+) T cells were determined by fluorescene-activated cell sorter. T cell proliferation responses in GM-DC showed a significance antigen-specific proliferative response comparing with TGFbeta-GM -DC. T Cell proliferation was inhibited in co-culture system containing DC-treated TGF-beta. It can be suggested that the expsansion of T regulatory by TGF-beta-GM-DC provides a means for antigen specific control of unwanted immune reactions.

Staphylococcal enterotoxin B (SEB) activates T cells via non-canonical signalling through the T cell receptor and is an established model for T cell unresponsiveness in vivo. In this study, we sought to characterize the suppressive qualities of SEB-exposed CD4(+) T cells and correlate this with genetic signatures of anergy and suppression. SEB-exposed CD25(+) and CD25(-)Vbeta8(+)CD4(+) T cells expressed forkhead box P3 (FoxP3) at levels comparable to naive CD25(+) T regulatory cells and were enriched after exposure in vivo. Gene related to anergy in lymphocytes (GRAIL), an anergy-related E3 ubiquitin ligase, was up-regulated in the SEB-exposed CD25(+) and CD25(-)FoxP3(+)Vbeta8(+)CD4(+) T cells and FoxP3(-)CD25(-)Vbeta8(+)CD4(+) T cells, suggesting that GRAIL may be important for dominant and recessive tolerance. The SEB-exposed FoxP3(+)GRAIL(+) T cells were highly suppressive and non-proliferative independent of CD25 expression level and via a glucocorticoid-induced tumour necrosis factor R-related protein-independent mechanism, whereas naive T regulatory cells were non-suppressive and partially proliferative with SEB activation in vitro. Lastly, adoptive transfer of conventional T cells revealed that induction of FoxP3(+) regulatory cells is not operational in this model system. These data provide a novel paradigm for chronic non-canonical T cell receptor engagement leading to highly suppressive FoxP3(+)GRAIL(+)CD4(+) T cells.

Purpose of Review: Although a plethora of data indicate the importance of regulatory T cells (Tregs) in experimental and clinical transplantation, are we any closer to seeing these cells as therapeutic tools in the clinic? This review discusses the functional and practical aspects of using CD4+CD25hiFoxp3+ Tregs as cellular therapeutic products in clinical transplantation, focusing on the requirements in terms of phenotype, antigen specificity and preparation of Tregs., Recent Findings: Following the emergence of new phenotypic markers of Tregs as well as improved isolation methods, a few milestone clinical trials employing the adoptive transfer of Tregs are now underway. Although mounting data suggest that alloantigen-specific Tregs may provide higher therapeutic benefits in solid organ transplantation compared with polyclonal Tregs, it seems that the specificty of Treg selected for use will need to be tailored to each clinical transplantation setting. In addition, recent findings imply that immunosuppressive regimes will also need to be reevaluated in order to complement this therapeutic strategy., Summary: Although many key questions about Tregs remain, we are undoubtedly entering an exciting era of Treg research in clinical transplantation. As renewed efforts focus on translational medical research, it seems as though, whether ready or not, Tregs are finally crossing from bench to bedside.

A growing body of evidence has shown that professional antigen-presenting cells (APC) treated with transforming growth factor-beta (TGF-beta) 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-beta-treated APC has not been elucidated. In this study, we showed that intravenous injection of ovalbumin (OVA)-pulsed APC treated with TGF-beta(2) 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-beta(2)-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-beta(2)-treated APC-injected mice produced a large amount of TGF-beta(1) and exhibited an in vitro antigen-specific suppressive activity. CD4(+) CD25(+) T cells from TGF-beta(2)-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-beta(2)-treated APC.

CD4+CD25+ T regulatory cells (Tregs) can actively suppress immune responses and thus have substantial therapeutical potential. Clinical application is, however, frustrated by their scarcity, anergic status, and lack of defined specificity. We found that a single injection of a small number of expanded but not fresh HY-specific Tregs protected syngeneic male skin grafts from rejection by immune-competent recipients. The expanded Tregs were predominantly located in the grafts and graft-draining lymph nodes. In vitro expanded Tregs displayed a phenotype of CD25highCD4lowFoxp3+CTLA4+, and also up-regulated IL10 and TGFbeta while down-regulating IFN-gamma, GM-CSF, IL5, and TNF-alpha production. Furthermore, expanded Tregs appeared to express a reduced level of Foxp3, which could be prevented by adding TGFbeta to the culture, and they also tended to lose Foxp3 following the repeated stimulation. Finally, a proportion of expanded HY-specific Tregs secreted IL2 in response to their cognate peptide, and this finding could be confirmed using Tregs from Foxp3GFP reporter mice. We not only demonstrated that expanded Tregs are superior to fresh Tregs in suppressing T cell responses against alloantigens, but also revealed some novel immunobiological properties of expended Tregs which are very instructive for modifying current Treg expansion procedures.

CD4+CD25+ human regulatory T cells (Treg cells), which express the transcription factor FoxP3, suppress T cell activation. In this study, we sought to define cellular and molecular mechanisms of human Treg cell differentiation. A subset of human naive CD4+ T cells that are CD25+ express high levels of FoxP3. We show that upon activation through the TCR, these FoxP3-expressing naive T cells (termed TNreg cells) greatly expand in vitro. Expanded TNreg cells acquire a full Treg phenotype with potent suppressive activity and display low IL-2 production upon TCR stimulation. TNreg cells in which FoxP3 expression was reduced through RNA interference lost their suppressive activity, but retained their low IL-2 secretion in response to TCR stimulation. Furthermore, in support of the notion that TNreg cells represent a separate lineage of naive cells, we found that they were more susceptible to HIV infection as compared with naive CD4+ T cells. Based on these findings, we propose that TNreg cells are precursors for human Treg cells and that these cells require a high level of FoxP3 expression to maintain their suppressive function. Accordingly, modulation of TNreg cell numbers during infections such as HIV may disrupt human Treg cell development, and contribute to chronic immune activation.

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are thought to suppress the natural and vaccine-induced immune response against tumor-associated antigens (TAA). Here, we show that Treg accumulate in tumors and tumor-draining lymph nodes of aging transgenic adenocarcinoma of the mouse prostate (TRAMP) male mice, which spontaneously develop prostate cancer. TAA overexpression and disease progression associate also with induction of TAA-specific tolerance. TAA-specific T cells were found in the lymphoid organs of tumor-bearing mice. However, they had lost the ability to release IFN-gamma and kill relevant targets. Neither in vivo depletion of Treg by PC61 monoclonal antibody followed by repeated vaccinations with antigen-pulsed dendritic cells nor the combined treatment with 1-methyl-L-tryptophan inhibitor of the enzyme indoleamine 2,3-dyoxigenase, PC61 antibody, and dendritic cell vaccination restored the TAA-specific immune response. Treg did not seem to control the early phases of tolerance induction, as well. Indeed, depletion of Treg, starting at week 6, the age at which TRAMP mice are not yet tolerant, and prolonged up to week 12, did not avoid tolerance induction. A similar accumulation of Treg was found in the lymph nodes draining the site of dendritic cell vaccination both in TRAMP and wild-type animals. Hence, we conclude that Treg accrual is a phenomenon common to the sites of an ongoing immune response, and in TRAMP mice in particular, Treg are dispensable for induction of tumor-specific tolerance.

Problem: Pre-eclampsia seems to be related to the disturbance of immune tolerance regulation during pregnancy. Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) decidual level alterations were concomitant with changes in immune cell number and activity in decidua. As decidual immunomodulating activity participates in the development of immune tolerance during pregnancy, we aimed to evaluate the immunoreactivity level of decidual RCAS1 with respect to the presence and activity of immune cells., Method of Study: RCAS1, CD3, CD56, CD69, and CD25 immunoreactivity was assessed by immunohistochemistry in 30 decidual samples derived from patients with severe pre-eclampsia (sPE) and from a healthy control group., Results: RCAS1 immunoreactivity was statistically significantly higher in decidual tissue samples derived from patients with sPE tissue than in those derived from healthy patients in whom elective cesarean section at term was performed. A statistically significantly lower number of CD56(+) and CD3(+) cells and lower immunoreactivity level of CD69 were found in patients with sPE compared with those from the control group., Conclusion: The limited immune cells infiltration in decidua during sPE is associated with increase in RCAS1 decidual level.

Scurfy mice which lacks functional Foxp3 transcription factor and CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, spontaneously develop autoimmune responses against skin, lung, liver and tail. However, many organs/tissues are spared from autoimmune attack. Here, we demonstrate that scurfy mice contain dormant autoimmune T cells that induced new diseases such as sialoadenitis, dacryoadenitis, pancreatitis, gastritis, intestinal inflammation, colitis, and myositis in RAG-1 KO mice. Inflammation in as many as 12 organs/tissues was consistently induced in individual recipients with scurfy lymph node cells containing as few as 1.25 x 10(6) CD4(+) T cells. Moreover, transfer of the multiple organ autoimmune diseases could be suppressed by as little as 0.5 x 10(6) CD4(+)CD25(+) Treg cells, mediated by inhibiting autoimmune T-cell expansion. Our study provides evidence for the presence of a large repertoire of autoimmune lymphocytes against various organs/tissues in scurfy mice as well as Treg cells in B6 mice capable of suppressing the expansion of these autoimmune lymphocytes. Various conditions that control the expression of autoimmune T cells are discussed.

Background and Aims: Obtaining antigen-specific immune suppression is an important goal in developing treatments of autoimmune, inflammatory, and allergic gastrointestinal diseases. Oral tolerance is a powerful means for inducing tolerance to a particular antigen, but implementing this strategy in humans has been difficult. Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (L lactis) provides a novel therapeutic approach for inducing tolerance., Methods: We engineered the food grade bacterium L lactis to secrete ovalbumin (OVA) and evaluated its ability to induce OVA-specific tolerance in OVA T-cell receptor (TCR) transgenic mice (DO11.10). Tolerance induction was assessed by analysis of delayed-type hypersensitivity responses, measurement of cytokines and OVA-specific proliferation, phenotypic analysis, and adoptive transfer experiments., Results: Intragastric administration of OVA-secreting L lactis led to active delivery of OVA at the mucosa and suppression of local and systemic OVA-specific T-cell responses in DO11.10 mice. This suppression was mediated by induction of CD4(+)CD25(-) regulatory T cells that function through a transforming growth factor beta-dependent mechanism. Restimulation of splenocytes and gut-associated lymph node tissue from these mice resulted in a significant OVA-specific decrease in interferon gamma and a significant increase in interleukin-10 production. Furthermore, Foxp3 and CTLA-4 were significantly up-regulated in the CD4(+)CD25(-) population., Conclusions: Mucosal antigen delivery by oral administration of genetically engineered L lactis leads to antigen-specific tolerance. This approach can be used to develop effective therapeutics for systemic and intestinal immune-mediated inflammatory diseases.

Background and Aims: Autoimmune gastritis is one of the most common autoimmune diseases and is caused by a CD4(+) T-cell response to the gastric H(+)/K(+) ATPase encoded by Atp4a and Atp4b (H(+)/K(+) ATPase). Here, we have elucidated events that result in immunological tolerance to the H(+)/K(+) ATPase and thus the prevention of autoimmune gastritis., Methods: T cells from H(+)/K(+) ATPase-deficient mice and H(+)/K(+) ATPase-specific T-cell receptor transgenic mice were purified and transferred to wild-type (WT) or H(+)/K(+) ATPase-deficient recipients to assess the impact of exposure to antigen on pathogenicity., Results: The CD4(+) T-cell population from H(+)/K(+) ATPase-deficient mice was highly effective at inducing gastritis when compared with T cells from WT mice and, as a population, was comparatively resistant to the suppressive activity of regulatory T cells. Exposing T cells from H(+)/K(+) ATPase-deficient mice to H(+)/K(+) ATPase in WT mice decreased their ability to induce gastritis and resulted in a population that could be more easily suppressed by T(reg) cells. Transfer of clonotypic antigen-inexperienced H(+)/K(+) ATPase-specific T cells into WT mice resulted in extra-thymic clonal deletion., Conclusions: Prevention of autoimmune gastritis requires the extra-thymic purging of highly autoaggressive H(+)/K(+) ATPase-specific T cells to produce a T-cell repertoire that is more susceptible to the suppressive activity of regulatory T cells. Taken together with recent published data describing the role of T-cell receptor signalling in the maintenance of regulatory T-cell populations, we propose that exposure of T cells to antigen in the periphery is able to both delete autoaggressive specificities and maintain regulatory T-cell activity, establishing a balance between pathogenicity and regulation.

Background: This study addresses the question of the mechanism for maintaining tolerance to donor alloantigen in the absence of antigen and the role of secondary lymphoid tissues., Methods: Depleting anti-CD4 antibody administration in conjunction with allogeneic heart transplantation generates donor-specific operational tolerance. Primary C57BL/6 heart grafts were transplanted into the neck cavity of the anti-CD4 antibody pretreated C3H/He mice. At day 50, functioning heart grafts were removed from tolerant mice. At day 100, a secondary C57BL/6 or a third-party heart was transplanted into the abdomen., Results: Anti-CD4 antibody therapy induced CD4CD25 regulatory T cells by 50 days after transplantation, as depleting anti-CD25 treatment in tolerant mice abrogated graft prolongation when spleen leukocytes were adoptively transferred to syngeneic mice. Tolerance was maintained by CD4CD25 regulatory T cells via a CTLA-4 signal at 100 days, even after removal of the primary graft at day 50. Administration of anti-CD25 antibody immediately after removal of the primary graft did not break tolerance, as five out of six second allografts transplanted at day 100 were accepted. Anti-CD25 antibody therapy in conjunction with splenectomy, but not thymectomy, immediately after removal of primary heart grafts at day 50 broke tolerance at day 100; all allografts were rejected., Conclusion: The spleen is important in maintaining CD4CD25 regulatory T cells after primary allograft removal.

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.

IL-2 contributes to the production, function, and homeostasis of CD4+CD25+ T(reg) cells. However, it remains uncertain whether IL-2 is essential for the development of T(reg) cells in the thymus, their homeostasis in the periphery, or both. The present study was undertaken to investigate the contribution of IL-2 during thymic T(reg) cell development and its maintenance in peripheral immune tissue. Relying on genetic mouse models where IL-2R signaling was either completely blocked or selectively inhibited in peripheral CD4+CD25+ T(reg) cells, we show that the IL-2/IL-2R interaction is active in the thymus at the earliest stage of the development of T(reg) cells to promote their expansion and to up-regulate Foxp3 and CD25 to normal levels. Furthermore, CD4+CD25+Foxp3+ T(reg) cells with impaired IL-2-induced signaling persist in the periphery and control autoimmunity without constant thymic output. These peripheral T(reg) cells with poor responsiveness to IL-2 exhibited slower growth and extended survival in vivo, somewhat lower suppressive activity, and poor IL-2-dependent survival in vitro. Mixed thymic and bone marrow chimeric mice showed that wild-type-derived T(reg) cells were substantially more effective in populating peripheral immune tissue than T(reg) cells with impaired IL-2 signaling. Collectively, these data support the notion that normally IL-2 is a dominant mechanism controlling the number of thymic and peripheral T(reg) cells.

CD4(+) CD25(+) Foxp3(+) naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4(+) CD25(-) T cells from T-cell receptor (TCR) transgenic mice on a RAG deficient (RAG(-/-)) background in the presence of anti-CD3 and interleukin-4 (IL-4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non-responsive to restimulation with anti-CD3 or exogenous IL-2, and could not up-regulate IL-2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti-CD3 but responded to restimulation with IL-2. The undivided population was capable of suppressing the response of fresh CD4(+) CD25(-) T cells and CD8(+) T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti-transforming growth factor-beta partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4(+) CD25(-) responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive.

Three major mucosal systems exist in the body, the oral-gastrointestinal, the respiratory and the genitourinary systems. In particular, the gastrointestinal (GI) tract contains the largest mucosal surface in the body and is the major port of entry for foreign antigens. Therefore, the gut immune system has to differentiate to tolerate dietary antigens and expel infectious and harmful pathogens. During the complex but well-orchestrated immune responses in the mucosal system, T cells play a pivotal role in both immunity and tolerance. Of many T cell subpopulations, CD4(+)CD25(+) T regulatory cells (Tregs) are instrumental in regulation of immune responses in mucosea. Among the multitude of cytokines and factors that are produced in the gut, Transforming Growth Factor-beta (TGF-beta) is probably the most important one in influencing mucosal T cell responses. The interaction and mutual regulation between TGF-beta and CD4(+)CD25(+) Tregs may be the key in maintaining the balance between T cell immunity and tolerance in mucosal system. In this article, we attempt to discuss both beneficial and detrimental effects of TGF-beta and Tregs on oral tolerance, mucosal inflammation and autoimmunity, colon cancer and HIV infection in the gut.

The natural CD4+CD25+ T regulatory (Treg) lymphocyte has emerged as a critical cell for controlling immune responses to self, foreign proteins, and pathogens. Identified initially by the constitutive expression of CD4 and CD25, natural Tregs suppress a variety of immune cells and responses, including CD4+CD25- proliferation and IL-2 production, and CD8 cell proliferation, IFNgamma production and CTL activity. Although natural Tregs require activation with specific antigen to attain their suppressive phenotype, once activated they execute inhibition in an antigen specific as well as non-specific (bystander) fashion. Treg suppression depends on IL-2, CD25, and cell:cell contact. The use of live cell imaging in vivo and in vitro to visualize the dynamic cell:cell interactions involving natural Tregs as well as the CD4+CD25+ Treg inhibitory hybridoma RD6 has refined the mechanistic models of contact dependent Treg suppression.

Background: CD4CD25 regulatory T (Treg) cells are essential for the induction and maintenance of immunologic self-tolerance as well as transplant tolerance. The effects of cyclosporin A (CsA), a widely used immunosuppressive agent, on CD4CD25Treg cells in mice were investigated., Methods: Balb/c mice were injected with CsA or control solution for one month. The levels, phenotype, and function of CD4CD25Treg cells in these mice were then assayed., Results: The percentages and total cell numbers of CD4CD25Treg cells in the peripheral blood and spleen were significantly reduced after the treatment with CsA. The total numbers of CD4CD25Treg cells in the thymus of CsA-treated mice were markedly reduced as compared to the control mice. However, the percentage of CD4CD25Treg cells in the thymus of CsA-treated mice was markedly enhanced. More CD4CD25Treg cells expressing high levels of CD44 and CD45RB, and less CD4CD25Treg cells expressing CD62L were observed in CsA-treated mice, compared with the control mice. CD4CD25Treg cells expressed slightly lower levels of Foxp3 in CsA-treated mice. Furthermore, CsA markedly impaired the immunosuppressive function of CD4CD25Treg cells., Conclusions: CsA significantly impaired the development and function of CD4CD25Treg cells. The present studies suggest that CsA may block the potential induction of immune tolerance and increase the susceptibility to develop autoimmune diseases while preventing graft rejection.

Although it is known that septic shock induces immunosuppression, the mechanism for this phenomenon is not well understood. Monocytes play a central role in septic shock pathophysiology, which is also characterized by an increased proportion of natural regulatory T (Treg) cells. We therefore investigated whether Treg could be involved in the decreased monocyte expression of CD14 and HLA-DR observed during septic shock. We demonstrated that human Treg inhibit LPS-induced retention of monocyte CD14. Because loss of CD14 is a hallmark of monocyte apoptosis, this suggests that Treg inhibit monocyte survival. This effect was largely mediated through the release of a soluble mediator that was not identical with either IL-10 or IL-4. The Fas/FasL pathway participated in the effect as it was blocked by anti-FasL Abs and reproduced by Fas agonist and recombinant soluble FasL. Furthermore, expression of FasL was much higher on Treg than on their CD25(-) counterparts. Collectively, these results indicate that Treg act on monocytes by inhibiting their LPS-induced survival through a proapoptotic mechanism involving the Fas/FasL pathway. This may be an important mechanism for septic shock-induced immunosuppression and may offer new perspectives for the treatment of this deadly disease.

CD4+ CD25+ regulatory T cells (Treg) exert suppressive functions on effector T cells in vitro and in vivo. However, the exact cellular events that mediate this inhibitory action remain largely unclear. To elucidate these events, we used intravital microscopy in a model of contact hypersensitivity (CHS) and visualized the leukocyte-endothelium interaction at the site of antigen challenge in awake C57BL/6 mice. Injection of Treg i.v. into sensitized mice at the time of local hapten challenge significantly inhibited rolling and adhesion of endogenous leukocytes to the endothelium. A similar inhibition of leukocyte recruitment could be recorded after injection of Treg-derived tissue culture supernatant. Thus, these data indicate that soluble factors may account for the suppressive effects. Accordingly we found that IL-10, but not TGF-beta, was produced by Treg upon stimulation and that addition of anti-IL-10 antibodies abrogated the suppressive effects of Treg and tissue culture supernatant in CHS reactions. Moreover, CD4+ CD25+ T cells isolated from IL-10-/- mice were not able to suppress the immune response induced by hapten treatment in C57BL/6 mice. In conclusion, our data suggest that cytokine-dependent rather than cell-cell contact-dependent mechanisms play a pivotal role in the suppression of CHS reactions by Treg in vivo.

Background: Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis., Methods and Results: Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor-/- mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%; P<0.05) and progression (45%; P<0.05) of atherogenesis. Tolerance to oxLDL induced a significant increase in CD4+ CD25+ Foxp3+ cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-beta production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis., Conclusions: OxLDL-specific T cells, present in LDL receptor-/- mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4+ CD25+ Foxp3+ regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.