Cutting edge: in vivo trogocytosis as a mechanism of double negative regulatory T cell-mediated antigen-specific suppression

Recent data have demonstrated that treatment with alphabeta-TCR(+)CD3(+)CD4(-)CD8(-)NK1.1(-) double negative (DN) regulatory T cells (Tregs) inhibits autoimmune diabetes and enhances allotransplant and xenotransplant survival in an Ag-specific fashion. However, the mechanisms whereby DN Tregs suppress Ag-specific immune responses remain largely unknown. In this study, we demonstrate that murine DN Tregs acquire alloantigen in vivo via trogocytosis and express it on their cell surface. Trogocytosis requires specific interaction of MHC-peptide on APCs and Ag-specific TCR on DN Tregs, as blocking this interaction prevents DN Treg-mediated trogocytosis. Acquisition of alloantigen by DN Tregs was required for their ability to kill syngeneic CD8(+) T cells. Importantly, DN Tregs that had acquired alloantigen were cytotoxic toward Ag-specific, but not Ag-nonspecific, syngeneic CD8(+) T cells. These data provide new insight into how Tregs mediate Ag-specific T cell suppression and may enhance our ability to use DN Tregs as a therapy for transplant rejection and autoimmune diseases.     

Dual effects of TRAIL in suppression of autoimmunity: the inhibition of Th1 cells and the promotion of regulatory T cells

TRAIL is known to play a pivotal role in the inhibition of autoimmune disease. We previously demonstrated that administration of dendritic cells engineered to express TRAIL and myelin-oligodendrocyte glycoprotein reduced the severity of experimental autoimmune encephalomyelitis and suggested that CD4(+)CD25(+) regulatory T cells (Tregs) were involved in mediating this preventive effect. In the current study, we investigated the effect of TRAIL on Tregs, as well as conventional T cells, using TRAIL-deficient mice. Upon induction of experimental autoimmune encephalomyelitis, TRAIL-deficient mice showed more severe clinical symptoms, a greater frequency of IFN-γ-producing CD4(+) T (Th1) cells, and a lower frequency of CD4(+)Foxp3(+) Tregs than did wild-type mice. In vitro, conventional T cells stimulated by bone marrow-derived dendritic cells (BM-DCs) from TRAIL-deficient mice showed a greater magnitude of proliferation than did those stimulated by BM-DCs from wild-type mice. In contrast, TRAIL expressed on the stimulator BM-DCs enhanced the proliferative response of CD4(+)CD25(+) Tregs in the culture. The functional TRAILR, mouse death receptor 5 (mDR5), was expressed in conventional T cells and Tregs upon stimulation. In contrast, the decoy receptor, mDc-TRAILR1, was slightly expressed only on CD4(+)CD25(+) Tregs. Therefore, the distinct effects of TRAIL may be due to differences in the mDc-TRAILR1 expression or the signaling pathways downstream of mouse death receptor 5 between the two T cell subsets. Our data suggest that TRAIL suppresses autoimmunity by two mechanisms: the inhibition of Th1 cells and the promotion of Tregs.     

Regulatory T cells selectively preserve immune privilege of self-antigens during viral central nervous system infection

Regulatory T cells (Tregs) are important for the attenuation of immune reactions. During viral CNS infections, however, an indiscriminate maintenance of CNS immune privilege through Treg-mediated negative regulation could prevent autoimmune sequelae but impair the control of viral replication. We analyzed in this study the impact of Tregs on the development of acute viral encephalomyelitis, T cell-mediated antiviral protection, and prevention of CNS autoimmunity following intranasal infection with the gliatropic mouse hepatitis virus strain A59. To assess the contribution of Tregs in vivo, we specifically depleted CD4(+)Foxp3(+) T cells in a diphtheria toxin-dependent manner. We found that depletion of Tregs had no impact on viral distribution and clearance and did not significantly alter virus-specific CD4(+) and CD8(+) T cell responses. However, Treg depletion led to a more severe CNS inflammation associated with neuronal damage. Dissection of the underlying immunopathological mechanisms revealed the elaborate Treg-dependent regulation of self-reactive CD4(+) T cell proliferation within the CNS-draining lymph node and downtuning of CXCR3 expression on T cells. Taken together, these results suggest that Tregs preserve CNS immune privilege through selective control of CNS-specific Th cells while keeping protective antiviral immunity fully operative.     

Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR

Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4(+) Tregs on CD8(+) cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4(+)CD127(-)CD25(+)FOXP3(+) Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified (51)Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4(+)CD127(-)CD25(+)FOXP3(+) cells were generated after a 7 day MLR. After immunoselection for CD4(+)CD127(-)CD25(+) cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8(+) responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8(+) responders or even with CD8(+) responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4(+) T cells were added to the CD8(+) responders. Proliferation of CD8(+) cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8(+) cells. Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells. CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.     

CD8+CD45RA+CCR7+FOXP3+ T Cells with Immunosuppressive Properties: A Novel Subset of Inducible Human Regulatory T Cells

CD8 T cells stimulated with a suboptimal dose of anti-CD3 Abs (100 pg/ml) in the presence of IL-15 retain a naive phenotype with expression of CD45RA, CD28, CD27, and CCR7 but acquire new functions and differentiate into immunosuppressive T cells. CD8(+)CCR7(+) regulatory T cells (Tregs) express FOXP3 and prevent CD4 T cells from responding to TCR stimulation and entering the cell cycle. Naive CD4 T cells are more susceptible to inhibition than memory cells. The suppressive activity of CD8(+)CCR7(+) Tregs is not mediated by IL-10, TGF-β, CTLA-4, CCL4, or adenosine and relies on interference with very early steps of the TCR signaling cascade. Specifically, CD8(+)CCR7(+) Tregs prevent TCR-induced phosphorylation of ZAP70 and dampen the rise of intracellular calcium in CD4 T cells. The inducibility of CD8(+)CCR7(+) Tregs is correlated with the age of the individual with PBLs of donors older than 60 y yielding low numbers of FOXP3(low) CD8 Tregs. Loss of CD8(+)CCR7(+) Tregs in the elderly host may be of relevance in the aging immune system as immunosenescence is associated with a state of chronic smoldering inflammation.     

OX40 controls functionally different T cell subsets and their resistance to depletion therapy

T cell depletion is a widely used approach in clinical transplantation. However, not all T cells are equally sensitive to depletion therapies and a significant fraction of T cells persists even after aggressive treatment. The functional attributes of such T cells and the mechanisms responsible for their resistance to depletion are poorly studied. In the present study, we showed that CD4(+) T cells that are resistant to polyclonal anti-lymphocyte serum (ALS) mediated depletion exhibit phenotypic features of memory cells and uniformly express OX40 on the cell surface. Studies using the foxp3gfp knockin mice revealed that the remaining CD4(+)OX40(+) cells consist of Foxp3(+) Tregs and Foxp3(-) T effector/memory cells. The ALS-resistant CD4(+)OX40(+) cells failed to mediate skin allograft rejection upon adoptive transferring into congenic Rag(-/-) mice, but removal of Foxp3(+) Tregs from the OX40(+) cells resulted in prompt skin allograft rejection. Importantly, OX40 is critical to survival of both Foxp3(+) Tregs and T effector/memory cells. However, OX40 exhibits opposing effects on the functional status of Foxp3(+) Tregs and T effector/memory cells, as stimulation of OX40 on T effector cells induced amplified cell proliferation but stimulation of OX40 on the Foxp3(+) Tregs impaired their suppressor functions. Our study demonstrates that OX40 is a critical molecule in regulating survival and functions of depletion-resistant T cells; and these findings may have important clinical implications.     

Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis

Regulatory CD4(+)CD25(+) T cells (Tregs) are defective numerically and functionally in autoimmune hepatitis (AIH). We have investigated and compared the mechanism of action of Tregs in healthy subjects and in AIH patients using Transwell experiments, where Tregs are cultured either in direct contact with or separated from their targets by a semipermeable membrane. We also studied Treg FOXP3 expression and effect on apoptosis. Direct contact is necessary for Tregs to suppress proliferation and IFN-gamma production by CD4(+)CD25(-) and CD8(+) T cells in patients and controls. Moreover, in both, direct contact of Tregs with their targets leads to increased secretion of regulatory cytokines IL-4, IL-10, and TGF-beta, suggesting a mechanism of linked immunosuppression. Tregs/CD4(+)CD25(-) T cell cocultures lead to similar changes in IFN-gamma and IL-10 secretion in patients and controls, whereas increased TGF-beta secretion is significantly lower in patients. In contrast, in patients, Tregs/CD8(+) T cell cocultures lead to a higher increase of IL-4 secretion. In AIH, Treg FOXP3 expression is lower than in normal subjects. Both in patients and controls, FOXP3 expression is present also in CD4(+)CD25(-) T cells, although at a low level and not associated to suppressive function. Both in patients and controls, addition of Tregs does not influence target cell apoptosis, but in AIH, spontaneous apoptosis of CD4(+)CD25(-) T cells is reduced. In conclusion, Tregs act through a direct contact with their targets by modifying the cytokine profile and not inducing apoptosis. Deficient CD4(+)CD25(-) T cell spontaneous apoptosis may contribute to the development of autoimmunity.     

Foxp3(+)CD4(+)CD25(+) regulatory T cells are increased in patients with Coxiella burnetii endocarditis

Chronic Q fever, which principally manifests as endocarditis, is characterized by Coxiella burnetii persistence and an impaired cell-mediated immune response. The long-term persistence of pathogens has been associated with the expansion of regulatory T cells (Tregs), the CD4(+) T-cell subset that is characterized by the expression of CD25 and Foxp3. We investigated the presence of Tregs in patients with acute Q fever (n?=?17), known to exhibit an efficient immune response, patients with Q fever endocarditis (n?=?54) and controls (n?=?27) by flow cytometry. The proportion of CD3(+) , CD4(+) and CD8(+) T cells was similar in controls and patients with Q fever. The percentage of CD4(+) T cells that expressed CD25 was similar in controls and patients with Q fever. The population of CD4(+) T cells that expressed both CD25 and Foxp3 was significantly (P?相似文献   

Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus

CD4(+)CD25(+) T regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoimmunity. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear components. We hypothesized that altered function of CD4(+)CD25(high) Tregs might play a role in the breakdown of immunologic self-tolerance in patients with SLE. In this study, we report a significant decrease in the suppressive function of CD4(+)CD25(high) Tregs from peripheral blood of patients with active SLE as compared with normal donors and patients with inactive SLE. Notably, CD4(+)CD25(high) Tregs isolated from patients with active SLE expressed reduced levels of FoxP3 mRNA and protein and poorly suppressed the proliferation and cytokine secretion of CD4(+) effector T cells in vitro. In contrast, the expression of FoxP3 mRNA and protein and in vitro suppression of the proliferation of CD4(+) effector T cells by Tregs isolated from inactive SLE patients, was comparable to that of normal individuals. In vitro activation of CD4(+)CD25(high) Tregs from patients with active SLE increased FoxP3 mRNA and protein expression and restored their suppressive function. These data are the first to demonstrate a reversible defect in CD4(+)CD25(high) Treg function in patients with active SLE, and suggest that strategies to enhance the function of these cells might benefit patients with this autoimmune disease.     

Endogenous regulatory T cells adhere in inflamed dermal vessels via ICAM-1: association with regulation of effector leukocyte adhesion

Regulatory T cells (Tregs) must express appropriate skin-homing adhesion molecules to exert suppressive effects on dermal inflammation. However, the mechanisms whereby they control local inflammation remain unclear. In this study we used confocal intravital microscopy in wild-type and Foxp3-GFP mice to examine adhesion of effector T cells and Tregs in dermal venules. These experiments examined a two-challenge model of contact sensitivity (CS) in which Treg abundance in the skin progressively increases during the course of the response. Adhesion of CD4(+) T cells increased during CS, peaking 8-24 h after an initial hapten challenge, and within 4 h of a second challenge. At these time points, 40% of adherent CD4(+) T cells were Foxp3(+) Tregs. CD4(+) T cell adhesion was highly dependent on ICAM-1, and consistent with this finding, anti-ICAM-1 prevented Treg adhesion. Skin TGF-β levels were elevated in skin during both challenges, in parallel with Treg adhesion. In the two-challenge CS model, inhibition of ICAM-1 eliminated Treg adhesion, an effect associated with a significant increase in neutrophil adhesion. Similarly, total CD4(+) T cell depletion caused an increase in adhesion of CD8(+) T cells. Because Treg adhesion was restricted by both of these treatments, these experiments suggest that adherent Tregs can control adhesion of proinflammatory leukocytes in vivo. Moreover, the critical role of ICAM-1 in Treg adhesion provides a potential explanation for the exacerbation of inflammation reported in some studies of ICAM-1-deficient mice.     

Comprehensive analysis of frequency and phenotype of T regulatory cells in HIV infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease

There are conflicting data about the frequency and role of regulatory T cells (Tregs) during the course of HIV infection. Peripheral blood of a large cohort of HIV-infected patients (n = 131) at different stages of disease, including 15 long-term nonprogressors and 21 elite controllers, was analyzed to determine the frequency and phenotype of Tregs, defined as CD4(+), CD25(high), CD127(low), FoxP3(high) cells. A significantly increased relative frequency of Tregs within the CD4(+) compartment of HIV(+) patients compared to that of healthy controls (P < 0.0001) was observed. Additionally, the relative frequency of Tregs directly correlated with HIV viral load and inversely with CD4(+) counts. However, the absolute Treg number was reduced in HIV-infected patients versus healthy controls (P < 0.0001), with the exception of elite controllers (P > 0.05). The loss of absolute Treg numbers coincided with rising markers of immune activation (P < 0.0006). The initiation of antiviral therapy significantly increased absolute Treg numbers (P < 0.0031). We find that the expression of CD39, a newly defined ectonucleotidase with immunomodulatory functions on Tregs, correlated with progressive HIV disease, HIV viral load, and immune activation. Of note, when tested in peripheral blood mononuclear cells of healthy volunteers, the in vitro capacity to suppress T-cell proliferation was limited to CD4(+), CD25(high), CD39(+) T cells. Interestingly, Tregs of elite controllers exhibited not only the highest expression of CCR5, CTLA-4, and ICOS but also the lowest level of CD39. The data presented here reconcile the seemingly contradictory results of previous studies looking at Tregs in HIV and highlight the complexity of Treg-mediated immunoregulation during human viral infections.     

T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.     

CD8+ Foxp3+ regulatory T cells are induced during graft-versus-host disease and mitigate disease severity

Regulatory T cells (Tregs), in particular CD4(+) Foxp3(+) T cells, have been shown to play an important role in the maintenance of tolerance after allogeneic stem cell transplantation. In the current study, we have identified a population of CD8(+) Foxp3(+) T cells that are induced early during graft-versus-host disease (GVHD), constitute a significant percentage of the entire Treg population, and are present in all major GVHD target organs. These cells expressed many of the same cell surface molecules as found on CD4(+) Tregs and potently suppressed in vitro alloreactive T cell responses. Induction of these cells correlated positively with the degree of MHC disparity between donor and recipient and was significantly greater than that observed for CD4(+)-induced Tregs (iTregs) in nearly all tissue sites. Mice that lacked the ability to make both CD8(+) and CD4(+) iTregs had accelerated GVHD mortality compared with animals that were competent to make both iTreg populations. The absence of both iTreg populations was associated with significantly greater expansion of activated donor T cells and increased numbers of CD4(+) and CD8(+) T cells that secreted IFN-γ and IL-17. The presence of CD8(+) iTregs, however, was sufficient to prevent increased GVHD mortality in the complete absence of CD4(+) Tregs, indicating at least one functional iTreg population was sufficient to prevent an exacerbation in GVHD severity, and that CD8(+) iTregs could compensate for CD4(+) iTregs. These studies define a novel population of CD8(+) Tregs that play a role in mitigating the severity of GVHD after allogeneic stem cell transplantation.     

Frequency of circulating regulatory T cells increases during chronic HIV infection and is largely controlled by highly active antiretroviral therapy

Regulatory T cells (Tregs) act by suppressing the activation and effector functions of innate and adaptive immune responses. HIV infection impacts Treg proportion and phenotype, although discrepant results have been reported depending on the patient population and the way Tregs were characterized. The effects of highly active antiretroviral therapy (HAART) on Treg frequency have not been thoroughly documented. We performed a detailed longitudinal analysis of Treg frequency and phenotype in 11 HIV-infected individuals enrolled in a single, prospective clinical trial, in which all patients underwent the same treatment protocol and were sampled at the same time points. Tregs were characterized for their expression of molecules associated with activation, cell cycle, apoptosis, or function, and compared to circulating Tregs from a group of age-matched healthy individuals.Our results revealed increased proportions, but reduced absolute numbers of circulating CD3(+)CD4(+)FOXP3(+) Tregs in chronically infected HIV-infected patients. Treg frequency was largely normalized by HAART. Importantly, we show that similar conclusions were drawn regardless of the combination of markers used to define Tregs. Our results also showed increased expression of cell cycle markers (Ki67 and cyclin B) in Tregs from untreated infected individuals, which were decreased by HAART. However, the Treg phenotype in untreated patients was not consistent with a higher level of generalized activation, as they expressed very low levels of CD69, slightly elevated levels of HLA-DR and similar levels of GARP compared to Tregs from uninfected donors. Moreover, none of these markers was significantly changed by HAART. Treg expression of CTLA-4 and cytotoxic molecules was identical between patients and controls. The most striking difference in terms of functional molecules was the high expression of CD39 by Tregs in untreated patients, which HAART only partially controlled.     

CD8+CD122+ regulatory T cells (Tregs) and CD4+ Tregs cooperatively prevent and cure CD4+ cell-induced colitis

We identified CD8(+)CD122(+) regulatory T cells (Tregs) and demonstrated their importance in the maintenance of immune homeostasis and in the recovery from experimental autoimmune encephalomyelitis. In this paper, we show that CD8(+)CD122(+) Tregs effectively prevent and cure colitis in a mouse model. In our experiments, colitis was induced in lymphocyte-deficient RAG-2(-/-) mice by transferring CD4(+)CD45RB(high) cells that were excluded with CD4(+) Tregs. Cotransfer of CD8(+)CD122(+) cells clearly suppressed the development of colitis, and this suppressive effect was similar to that of CD4(+)CD45RB(low) cells that were mostly CD4(+) Tregs. CD8(+)CD122(+) cells obtained from IL-10(-/-) mice were unable to suppress colitis, indicating that IL-10 is an important effect-transmitting factor in the suppression of colitis. CD8(+)CD122(+) cells showed a suppressive effect when they were transferred 4 wk after CD4(+)CD45RB(high) cells, indicating the therapeutic potential of CD8(+)CD122(+) cells. A mixture of CD8(+)CD122(+) cells and CD4(+)CD45RB(low) cells was far more effective than single Tregs, indicating the synergistic effect of these Tregs. These overall findings demonstrate the potential role of CD8(+) Tregs, and possibly together with CD4(+) Tregs, in the medical care of inflammatory bowel disease patients.     

The indispensable role of CCR5 for in vivo suppressor function of tumor-derived CD103+ effector/memory regulatory T cells

CD103 is a marker for identification of effector/memory regulatory T cells (Tregs). CD103(+) Tregs are potent suppressors of tissue inflammation in several infectious diseases, autoimmune diseases, and cancers. However, the underlying mechanisms for this potent suppression ability remain unclear. The current study was designed to clarify this issue. Unexpectedly, we found both CD103(+) and CD103(-) Tregs had similar suppression capacity in vitro. We then chose a murine tumor model for investigation of the in vivo behavior of these Tregs. The suppression ability in vivo against the anti-tumor ability of CD8(+) T cells was restricted to CD103(+) Tregs although both Tregs had equal in vitro suppression ability. In addition, CD103(+) Tregs expressed significantly higher levels of CCR5 than those of CD103(-) Tregs and accumulated more in tumors than did CD103(-) Tregs. Furthermore, blockade of CCR5 signaling, either by CCR5(-/-)CD103(+) Tregs or by CCL5 knockdown tumor, could reduce the migration of CD103(+) Tregs into tumors and impair their in vivo suppression ability. In conclusion, these results indicate that the potent in vivo suppression ability of CD103(+) Tregs is due to the tissue-migration ability through CCR5 expression.     

IL-12R beta 2 promotes the development of CD4+CD25+ regulatory T cells

We have previously shown that mice lacking the IL-12-specific receptor subunit beta2 (IL-12Rbeta2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rbeta2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rbeta2(-/-) mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rbeta2-deficient mice to autoimmune diseases. T cells from IL-12Rbeta2(-/-) mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25(+)CD4(+) regulatory T cells (Tregs) in the thymus and spleen of IL-12Rbeta2(-/-) mice were comparable to those of WT mice. However, IL-12Rbeta2(-/-) mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-beta, as shown by significantly lower numbers of CD25(+)CD4(+) T cells that expressed Foxp3. Functionally, CD25(+)CD4(+) Tregs derived from IL-12Rbeta2(-/-) mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rbeta2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rbeta2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway.     

Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity

A number of tumor studies have indicated a link between CD4 help and the magnitude and persistence of CTL activity; however, the mechanisms underlying this have been largely unclear. To evaluate and determine the mechanisms by which CD4(+) T cells synergize with CD8(+) T cells to prevent tumor growth, we used the novel technique of monitoring in vivo CTL by labeling target cells with CFSE. This approach was supported by the direct visualization of CTL using peptide-MHC tetramers to follow tumor-specific T cells. The data presented demonstrate that while cotransfer of Ag-specific CD4(+) T cells was not required for the generation of CTLs, because adoptive transfer of CD8(+) T cells alone was sufficient, CD4(+) T cells were required for the maintenance of CD8(+) T cell numbers. Our data suggest that there is a correlation among the number of CD8(+) T cells, in vivo CTL function, and IFN-gamma production, with no evidence of a partial or nonresponsive phenotype among tetramer-positive cells. We also show that CD4(+) T cells are required for CD8(+) T cell infiltration of the tumor.     

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

Th3 cells in peripheral tolerance. I. Induction of Foxp3-positive regulatory T cells by Th3 cells derived from TGF-beta T cell-transgenic mice

TGF-beta has been shown to be critical in the generation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Because Th3 cells produce large amounts of TGF-beta, we asked whether induction of Th3 cells in the periphery was a mechanism by which CD4(+)CD25(+) Tregs were induced in the peripheral immune compartment. To address this issue, we generated a TGF-beta1-transgenic (Tg) mouse in which TGF-beta is linked to the IL-2 promoter and T cells transiently overexpress TGF-beta upon TCR stimulation but produce little or no IL-2, IL-4, IL-10, IL-13, or IFN-gamma. Naive TGF-beta-Tg mice are phenotypically normal with comparable numbers of lymphocytes and thymic-derived Tregs. We found that repeated antigenic stimulation of pathogenic myelin oligodendrocyte glycoprotein (MOG)-specific CD4(+)CD25(-) T cells from TGF-beta Tg mice crossed to MOG TCR-Tg mice induced Foxp3 expression in both CD25(+) and CD25(-) populations. Both CD25 subsets were anergic and had potent suppressive properties in vitro and in vivo. Furthermore, adoptive transfer of these induced regulatory CD25(+/-) T cells suppressed experimental autoimmune encephalomyelitis when administrated before disease induction or during ongoing experimental autoimmune encephalomyelitis. The suppressive effect of TGF-beta on T cell responses was due to the induction of Tregs and not to the direct inhibition of cell proliferation. The differentiation of Th3 cells in vitro was TGF-beta dependent as anti-TGF-beta abrogated their development. Thus, Ag-specific TGF-beta-producing Th3 cells play a crucial role in inducing and maintaining peripheral tolerance by driving the differentiation of Ag-specific Foxp3(+) regulatory cells in the periphery.