Oestrogen treatment of experimental autoimmune encephalomyelitis requires 17β‐oestradiol‐receptor‐positive B cells that up‐regulate PD‐1 on CD4+ Foxp3+ regulatory T cells

It is now well accepted that sex hormones have immunoregulatory activity and may prevent exacerbations in multiple sclerosis during pregnancy. Our previous studies demonstrated that oestrogen (17β-oestradiol; E₂) protection against experimental autoimmune encephalomyelitis (EAE) is mediated mainly through oestrogen receptor-α (ERα) and the membrane receptor G-protein-coupled receptor 30 (GPR30) and is abrogated in the absence of B cells and the co-inhibitory receptor, Programmed Death-1 (PD-1). To critically evaluate the cell source of the E₂ and PD-1 co-inhibitory pathways in EAE regulation, we assessed the requirement for ERs on transferred B cells and downstream effects on expression of PD-1/PD-ligand on CD4⁺ Foxp3⁺ regulatory T (Treg) cells in B-cell-replenished, E₂-treated B-cell-deficient (μMT^−/−) mice with EAE. The results clearly demonstrated involvement of ERα and GPR30 on transferred B cells that mediated the protective E₂ treatment effect on EAE and further showed an E₂-mediated B-cell-dependent up-regulation of PD-1 on CD4⁺ Foxp3⁺ Treg cells. These findings identify regulatory B-cell populations as key players in potentiating Treg-cell activity during E₂-mediated protection against EAE.     

Novel CD8+ Treg suppress EAE by TGF‐β‐ and IFN‐γ‐dependent mechanisms

Although CD8⁺ Treg‐mediated suppression has been described, CD8⁺ Treg remain poorly characterized. Here we identify a novel subset of CD8⁺ Treg that express latency‐associated peptide (LAP) on their cell surface (CD8⁺LAP⁺ cells) and exhibit regulatory activity in vitro and in vivo. Only a small fraction of CD8⁺LAP⁺ cells express Foxp3 or CD25, although the expression levels of Foxp3 for these cells are higher than their LAP^? counterparts. In addition to TGF‐β, CD8⁺LAP⁺ cells produce IFN‐γ, and these cells suppress EAE that is dependent on both TGF‐β and IFN‐γ. In an adoptive co‐transfer model, CD8⁺LAP⁺ cells suppress myelin oligodendrocyte glycoprotein (MOG)‐specific immune responses by inducing or expanding Foxp3⁺ cells and by inhibiting proliferation and IFN‐γ production in vivo. Furthermore, in vivo neutralization of IFN‐γ and studies with IFN‐γ‐deficient mice demonstrate an important role for IFN‐γ production in the function of CD8⁺LAP⁺ cells. Our findings identify the underlying mechanisms that account for the immunoregulatory activity of CD8⁺ T cells and suggest that induction or amplification of CD8⁺LAP⁺ cells may be a therapeutic strategy to help control autoimmune processes.     

TGF‐β regulation of encephalitogenic and regulatory T cells in multiple sclerosis

Transforming growth factor beta (TGF‐β) is a pleiotropic cytokine that has been shown to influence the differentiation and function of T cells. The role that TGF‐β plays in immune‐mediated disease, such as multiple sclerosis (MS), has become a major area of investigation since CD4⁺ T cells appear to be a major mediator of autoimmunity. This review provides an analysis of the literature on the role that TGF‐β plays in the generation and regulation of encephalitogenic and regulatory T cells (Treg) in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as well as in T cells of MS patients. Since TGF‐β plays a major role in the development and function of both CD4⁺ effector and Treg, which are defective in MS patients, recent studies have found potential mechanisms to explain the basis for these T‐cell defects to establish a foundation for potentially modulating TGF‐β signaling to restore normal T‐cell function in MS patients.     

Curing CNS autoimmune disease with myelin‐reactive Foxp3+ Treg

The potential use of CD4⁺Foxp3⁺ Treg as a cellular therapy for autoimmune disease is of great interest. For clinical translation, the key objective is to reverse established disease. Here we demonstrate that myelin basic protein (MBP)‐reactive CD4⁺CD25⁺ Treg from TCR Tg mice, but not polyclonal (non‐MBP‐reactive) Treg, can transfer efficient protection against MBP‐induced EAE when used either directly from donor mice, or after in vitro expansion. MBP‐reactive Treg transfer also showed some ability to improve recovery from EAE initiated by T cells recognizing a distinct CNS autoantigen, proteolipid protein. Importantly, we also demonstrate for the first time in the context of EAE that in vitro‐expanded naturally occurring MBP‐reactive Treg can prevent disease relapse when given after the onset of clinical EAE. Our study also contains data pertaining to the most effective Treg sub‐population in vivo (CD4⁺CD25⁺CD62L^hi) and shows that their potent suppressive effects reflect stable expression of Foxp3, although not CD25 or CD62L. Our data provide proof of the principle that Treg‐based therapies can cure CNS autoimmune disease, highlight the challenges for clinical translation and open new avenues for assessing how changing immune function via Treg activity might impact on neurodegeneration and repair.     

1,25‐dihydroxyvitamin D3‐induced dendritic cells suppress experimental autoimmune encephalomyelitis by increasing proportions of the regulatory lymphocytes and reducing T helper type 1 and type 17 cells

Dendritic cells (DCs), a bridge for innate and adaptive immune responses, play a key role in the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Administration of tolerogenic DCs has been used as an immunotherapy in autoimmune diseases. Deficiency of vitamin D is an environmental risk factor of MS. In this study, we induced tolerogenic DCs by 1,25‐dihydroxyvitamin D₃ and transferred the tolerogenic DCs (VD₃‐DCs) into EAE mice by adoptive transfer. We found that VD₃‐DCs inhibited the infiltrations of T helper type 1 (Th1) and Th17 cells into spinal cord and increased the proportions of regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺), CD4⁺ IL‐10⁺ T cells and regulatory B cells (CD19⁺ CD5⁺ CD1d⁺) in peripheral immune organs, which resulted in attenuated EAE. However, the proportions of T helper type 1 (Th1) and Th17 cells in spleen and lymph nodes and the levels of pro‐inflammatory cytokines and IgG in serum also increased after transfer of VD₃‐DCs. We conclude that transfer of VD₃‐DCs suppressed EAE by increasing proportions of regulatory T cells, CD4⁺ IL‐10⁺ T cells and regulatory B cells in spleen and reducing infiltration of Th1 and Th17 cells into spinal cord, which suggests a possible immunotherapy method using VD₃‐DCs in MS.     

Expansion of regulatory T cells via IL‐2/anti‐IL‐2 mAb complexes suppresses experimental myasthenia

Human autoimmune diseases are often characterized by a relative deficiency in CD4⁺CD25⁺ regulatory T cells (Treg). We therefore hypothesized that expansion of Treg can ameliorate autoimmune pathology. We tested this hypothesis in an experimental model for autoimmune myasthenia gravis (MG), a B‐cell‐mediated disease characterized by auto‐Ab directed against the acetylcholine receptor within neuromuscular junctions. We showed that injection of immune complexes composed of the cytokine IL‐2 and anti‐IL‐2 mAb (JES6‐1A12) induced an effective and sustained expansion of Treg, via peripheral proliferation of CD4⁺CD25⁺Foxp3⁺ cells and peripheral conversion of CD4⁺CD25^?Foxp3^? cells. The expanded Treg potently suppressed autoreactive T‐ and B‐cell responses to acetylcholine receptor and attenuated the muscular weakness that is characteristic of MG. Thus, IL‐2/anti‐IL‐2 mAb complexes can expand functional Treg in vivo, providing a potential clinical application of this modality for treatment of MG and other autoimmune disorders.     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

Synergistic effect of TGF‐β superfamily members on the induction of Foxp3+ Treg

TGF‐β plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF‐β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF‐β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP‐2 and ‐4, are unable to induce non‐regulatory T cells to become Foxp3⁺ Treg. Neutralization studies with Noggin have revealed that BMP‐2/4 and the BMP receptor signaling pathway is not required for TGF‐β to induce naïve CD4⁺CD25^? cells to express Foxp3; however, BMP‐2/4 and TGF‐β have a synergistic effect on the induction of Foxp3⁺ Treg. BMP‐2/4 affects non‐Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3⁺ Treg induced by TGF‐β. Data further advocate that TGF‐β is a key signaling factor for Foxp3⁺ Treg development. In addition, the synergistic effect of BMP‐2/4 and TGF‐β indicates that the simultaneous manipulation of TGF‐β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.     

Diversification and senescence of Foxp3+ regulatory T cells during experimental autoimmune encephalomyelitis

The fate of Foxp3⁺ regulatory T (Treg) cells responding during autoimmunity is not well defined. We observed a marked elevation in KLRG1⁺ (where KLRG1 stands for killer cell lectin‐like receptor G1) CNS‐infiltrating Treg cells in experimental autoimmune encephalomyelitis (EAE), and assessed their origin and properties. KLRG1⁺ Treg cells showed increased activation marker expression, Foxp3 and CD25 levels, and more rapid cell cycling than KLRG1^? cells. KLRG1^? Treg cells converted into KLRG1⁺ cells and this was increased in autoimmune inflammation. Conversion was unidirectional; KLRG1⁺ Treg cells did not revert to a KLRG1^? state. KLRG1⁺ but notKLRG1^?Treg cells survived poorly, indicative of terminal differentiation. This was associated with diminished BCL2 and increased apoptosis of isolated cells. KLRG1 was also upregulated on iTreg cells after transfer and EAE induction or on iTreg cells developing spontaneously during EAE. KLRG1⁺ Treg cells produced more IL‐10 and had altered effector cytokine production compared with their KLRG1^? counterparts. Despite their differences, KLRG1⁺ and KLRG1^? Treg cells proved similarly potent in suppressing EAE. KLRG1⁺ and KLRG1^? populations were phenotypically heterogeneous, with the extent and pattern of activation marker expression dependent both on cellular location and inflammation. Our results support an extensive diversification of Treg cells during EAE, and associate KLRG1 with altered Treg‐cell function and senescence.     

Membrane‐bound Dickkopf‐1 in Foxp3+ regulatory T cells suppresses T‐cell‐mediated autoimmune colitis

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3⁺ regulatory T (Treg) cells use Dickkopf‐1 (DKK‐1) to regulate T‐cell‐mediated tolerance in the T‐cell‐mediated autoimmune colitis model. Treg cells from DKK‐1 hypomorphic doubleridge mice failed to control CD4⁺ T‐cell proliferation, resulting in CD4 T‐cell‐mediated autoimmune colitis. Thymus‐derived Treg cells showed a robust expression of DKK‐1 but not in naive or effector CD4 T cells. DKK‐1 expression in Foxp3⁺ Treg cells was further increased upon T‐cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3⁺ Treg cells expressed DKK‐1 in the cell membrane and the functional inhibition of DKK‐1 using DKK‐1 monoclonal antibody abrogated the suppressor function of Foxp3⁺ Treg cells. DKK‐1 expression was dependent on de novo protein synthesis and regulated by the mitogen‐activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane‐bound DKK‐1 as a novel Treg‐derived mediator to maintain immunological tolerance in T‐cell‐mediated autoimmune colitis.     

Foxp3+ regulatory T cells are activated in spite of B7‐CD28 and CD40‐CD40L blockade

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)‐cell activity. We previously reported that the blockade of the B7‐CD28 and CD40L‐CD40 interaction efficiently suppresses allogeneic T‐cell activation in vivo. This was characterized by an initial rise in Foxp3⁺ cells, followed by depletion of host‐reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4⁺ cell activation. When CTLA‐4Ig and anti‐CD40L mAb (MR1) were added to the cultures, T‐cell proliferation and IL‐2 production were strongly reduced. However, Foxp3⁺ cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4⁺ cells much more efficiently than did freshly isolated Treg cells. CD4⁺ cells activated by allogeneic cells in the presence of MR1 and CTLA‐4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4⁺ cells when Foxp3⁺ Treg cells were removed. We conclude that natural Treg cells are less dependent on B7‐CD28 or CD40‐CD40L costimulation compared with Foxp3^? T cells. Reduced costimulation therefore alters the balance between Teff and Treg‐cell activation in favor of Treg‐cell activity.     

‘Default’ generated neonatal regulatory T cells are hypomethylated at conserved non‐coding sequence 2 and promote long‐term cardiac allograft survival

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 Foxp3^gfp 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.     

Asthma Prevention by Lactobacillus Rhamnosus in a Mouse Model is Associated With CD4+CD25+Foxp3+ T Cells

Purpose

Probiotic bacteria can induce immune regulation or immune tolerance in allergic diseases. The underlying mechanisms have been recently investigated, but are still unclear. The aim of this study was to evaluate the protective effects of the probiotic Lactobacillus rhamnosus (Lcr35) in a mouse model of asthma and to identify its mechanism of action.

Methods

Lcr35 was administered daily by the oral route at a dosage of 1×10⁹ CFU/mouse in BALB/c mice for 7 days before the first sensitization. Clinical parameters and regulatory T (Treg) cells were examined. The role of CD4⁺CD25⁺Foxp3⁺ Treg cells was analyzed using a Treg cell-depleting anti-CD25 monoclonal antibody (mAb).

Results

Airway hyperresponsiveness, total IgE production, pulmonary eosinophilic inflammation, and splenic lymphocyte proliferation were suppressed after Lcr35 treatment. Th1 (IFN-γ) and Th2 (IL-4, IL-5, and IL-13) cytokines in the serum were suppressed, and the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the spleen was significantly increased in the Lcr35 treatment group. Anti-CD25 mAb administration abolished the protective effects of Lcr35, indicating that CD4⁺ CD25⁺Foxp3⁺ Treg cells are essential in mediating the activity of Lcr35.

Conclusions

Oral administration of Lcr35 attenuated the features of allergic asthma in a mouse model and induced immune regulation by a CD4⁺CD25⁺Foxp3⁺ Treg cell-mediated mechanism.     

The role of 1α,25‐dihydroxyvitamin D3 and cytokines in the promotion of distinct Foxp3+and IL‐10+ CD4+ T cells

1α,25‐Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL‐10‐secreting CD4⁺ T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3⁺ regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3⁺ and IL‐10⁺ CD4⁺T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL‐10 at more moderate levels, with little coexpression of these molecules. The Foxp3⁺ and IL‐10⁺ T‐cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL‐10. 1α25VitD3 enables the selective expansion of Foxp3⁺ Treg cells over their Foxp3^? T‐cell counterparts. Equally, 1α25VitD3 maintains Foxp3⁺ expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4⁺Foxp3⁺ T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL‐10⁺ and Foxp3⁺ Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.     

Exogenous interleukin‐33 targets myeloid‐derived suppressor cells and generates periphery‐induced Foxp3+ regulatory T cells in skin‐transplanted mice

Interleukin-33 (IL-33) has been a focus of study because of its variety of functions shaping CD4⁺ T-cell biology. In the present work, we evaluated the modulatory effect of IL-33 on suppressor cells in an in vivo transplantation model. C57BL/6 wild-type mice were grafted with syngeneic or allogeneic skin transplants and treated with exogenous IL-33 daily. After 10 days of treatment, we analysed draining lymph node cellularity and found in allogeneic animals an increment in myeloid-derived suppressor cells, which co-express MHC-II, and become enriched upon IL-33 treatment. In line with this observation, inducible nitric oxide synthase and arginase 1 expression were also increased in allogeneic animals upon IL-33 administration. In addition, IL-33 treatment up-regulated the number of Foxp3⁺ regulatory T (Treg) cells in the allogeneic group, complementing the healthier integrity of the allografts and the increased allograft survival. Moreover, we demonstrate that IL-33 promotes CD4⁺ T-cell expansion and conversion of CD4⁺ Foxp3⁻ T cells into CD4⁺ Foxp3⁺ Treg cells in the periphery. Lastly, the cytokine pattern of ex vivo-stimulated draining lymph nodes indicates that IL-33 dampens interferon-γ and IL-17 production, stimulating IL-10 secretion. Altogether, our work complements previous studies on the immune-modulatory activity of IL-33, showing that this cytokine affects myeloid-derived suppressor cells at the cell number and gene expression levels. More importantly, our research demonstrates for the first time that IL-33 allows for in vivo Foxp3⁺ Treg cell conversion and favours an anti-inflammatory or tolerogenic state by skewing cytokine production. Therefore, our data suggest a potential use of IL-33 to prevent allograft rejection, bringing new therapeutics to the transplantation field.     

Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3− T cells in complete DiGeorge anomaly

The development of T cells with a regulatory phenotype after thymus transplantation has not been examined previously in complete DiGeorge anomaly (cDGA). Seven athymic infants with cDGA and non-maternal pretransplantation T cell clones were assessed. Pretransplantation forkhead box protein 3 (Foxp3)⁺ T cells were detected in five of the subjects. Two subjects were studied in greater depth. T cell receptor variable β chain (TCR-Vβ) expression was assessed by flow cytometry. In both subjects, pretransplantation FoxP3⁺ and total CD4⁺ T cells showed restricted TCR-Vβ expression. The development of naive T cells and diverse CD4⁺ TCR-Vβ repertoires following thymic transplantation indicated successful thymopoiesis from the thymic tissue grafts. Infants with atypical cDGA develop rashes and autoimmune phenomena before transplantation, requiring treatment with immunosuppression, which was discontinued successfully subsequent to the observed thymopoiesis. Post-transplantation, diverse TCR-Vβ family expression was also observed in FoxP3⁺ CD4⁺ T cells. Interestingly, the percentages of each of the TCR-Vβ families expressed on FoxP3⁺ and total CD4⁺ T cells differed significantly between these T lymphocyte subpopulations before transplantation. By 16 months post-transplantation, however, the percentages of expression of each TCR-Vβ family became significantly similar between FoxP3⁺ and total CD4⁺ T cells. Sequencing of TCRBV DNA confirmed the presence of clonally amplified pretransplantation FoxP3⁺ and FoxP3⁻ T cells. After thymus transplantation, increased polyclonality was observed for both FoxP3⁺ and FoxP3⁻ cells, and pretransplantation FoxP3⁺ and FoxP3⁻ clonotypes essentially disappeared. Thus, post-transplantation thymic function was associated with the development of a diverse repertoire of FoxP3⁺ T cells in cDGA, corresponding with immunological and clinical recovery.     

Beta2‐adrenergic receptor signaling in CD4+ Foxp3+ regulatory T cells enhances their suppressive function in a PKA‐dependent manner

Beta2‐adrenergic receptor (B2AR) signaling is known to impair Th1‐cell differentiation and function in a cAMP‐dependent way, leading to inhibition of cell proliferation and decreased production of IL‐2 and IFN‐γ. CD4⁺ Foxp3⁺ Treg cells play a key role in the regulation of immune responses and are essential for maintenance of self‐tolerance. Nevertheless, very little is known about adrenergic receptor expression in Treg cells or the influence of noradrenaline on their function. Here we show that Foxp3⁺ Treg cells express functional B2AR. B2AR activation in Treg cells leads to increased intracellular cAMP levels and to protein kinase A (PKA)‐dependent CREB phosphorylation. We also found that signaling via B2AR enhances the in vitro suppressive activity of Treg cells. B2AR‐mediated increase in Treg‐cell suppressive function was associated with decreased IL‐2 mRNA levels in responder CD4⁺ T cells and improved Treg‐cell‐induced conversion of CD4⁺ Foxp3^? cells into Foxp3⁺ induced Treg cells. Moreover, B2AR signaling increased CTLA‐4 expression in Treg cells in a PKA‐dependent way. Finally, we found that PKA inhibition totally prevented the B2AR‐mediated increase in Treg‐cell suppressive function. Our data suggest that sympathetic fibers are able to regulate Treg‐cell suppressive activity in a positive manner through B2AR signaling.     

CD8α+ dendritic cells prime TCR‐peptide‐reactive regulatory CD4+FOXP3− T cells

CD4⁺ T cells with immune regulatory function can be either FOXP3⁺ or FOXP3^?. We have previously shown that priming of naturally occurring TCR‐peptide‐reactive CD4⁺FOXP3^? Treg specifically controls Vβ8.2⁺CD4⁺ T cells mediating EAE. However, the mechanism by which these Treg are primed to recognize their cognate antigenic determinant, which is derived from the TCRVβ8.2‐chain, is not known. In this study we show that APC derived from splenocytes of naïve mice are able to stimulate cloned CD4⁺ Treg in the absence of exogenous antigen, and their stimulation capacity is augmented during EAE. Among the APC populations, DC were the most efficient in stimulating the Treg. Stimulation of CD4⁺ Treg was dependent upon processing and presentation of TCR peptides from ingested Vβ8.2TCR⁺CD4⁺ T cells. Additionally, DC pulsed with TCR peptide or apoptotic Vβ8.2⁺ T cells were able to prime Treg in vivo and mediate protection from disease in a CD8‐dependent fashion. These data highlight a novel mechanism for the priming of CD4⁺ Treg by CD8α⁺ DC and suggest a pathway that can be exploited to prime antigen‐specific regulation of T‐cell‐mediated inflammatory disease.