Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of regulatory T cells (Treg). We have demonstrated that regulatory T cells can be easily identified in normal lymphoid tissues by expression of CD4, the interleukin-2 receptor alpha chain (CD25), and the transcription factor, FoxP3. Transfer of CD4+CD25-Foxp3- T cells to immunoincompetent mice results in the development of autoimmune disease that can be prevented by co-transfer of CD4+CD25+Foxp3+ T cells. The major goals of this project are to define the function and mechanism of action of Treg cells in vivo. We have used both polyclonal Treg and Treg that have been induced in vitro by stimulation of naive T cells in the presence of TGF-beta. TGF-beta induced Tregs (iTregs) have many of the phenotypic features of thymic-derived Tregs (nTregs), as they are anergic, suppressive, and can prevent the development of autoimmune disease. Furthermore, they can be generated from any naive antigen specific CD4+Foxp3- cell in vitro in unlimited numbers. We have analyzed the in vivo dynamics of the interaction between polyclonal Foxp3+ Treg, effector T cells (Teff), and DC in order to further our understanding of the mechanisms of Treg-mediated suppression. Co-transfer of polyclonal activated Treg into normal mice attenuated the induction of experimental autoimmune encephalomyelitis. Suppression of disease strongly correlated with a reduced number of Teff cells in the spinal cord, but not with Treg-mediated inhibition of Th1/Th17 differentiation. Co-transfer of Treg with TCR transgenic Teff cells followed by immunization by multiple routes resulted in an enhanced number of Teff cells in the lymph nodes draining the site of immunization without an inhibition of Teff differentiation. Fewer Teff cells could be detected in the blood in the presence of Tregs and fewer T cells could access a site of antigen exposure in a modified delayed type hypersensitivity assay. Teff cells recovered from LN in the presence of Tregs expressed decreased levels of CCR4, syndecan, and the sphingosine phosphate receptor, S1P1. Thus, polyclonal Tregs influence the Teff cell responses by targeting trafficking pathways, thus allowing immunity to develop in lymphoid organs, but limiting the number of potentially auto-aggressive cells that are allowed to enter tissues. The efficacy of vaccines can be greatly improved by adjuvants that enhance and modify the magnitude and the duration of the immune response. Several approaches to design rational adjuvants are based on suppression of Treg function. We evaluated whether removal or addition of Treg at the time of vaccination with tetanus toxoid and the mucosal adjuvant cholera toxin (CT), would affect immune responses. We found that depletion/inactivation of CD4+CD25+ Treg, either by treatment of BALB/c mice with anti-CD25 mAb, or by adoptive transfer of CD4+CD25- T lymphocytes depleted of CD4+CD25+ Treg into nu/nu mice, impaired antibody production after mucosal immunization in the presence of CT. Conversely, transfer of polyclonal, but not Agspecific, CD4+CD25+Foxp3+ Treg to normal BALB/c mice enhanced CT-induced antibody responses. Recipients of polyclonal Treg that had been immunized with CT had an increased number of Ag-specific CD4+ T cells with an activated phenotype (CD44hi) in the draining lymph nodes. This accumulation of antigen-specific CD4+ T lymphocytes could favour germinal centre formation and may promote T-dependent B cell responses. Overall, these studies indicate that Foxp3+ Treg can not only act as suppressor cells, but also as helper T cells, depending on the type of immune response being evaluated and the microenvironment in which the response is generated. In contrast to the results obtained by co-transfer of polyclonal Treg cells, dramatically different results were observed when we co-transferred antigen-specific iTregs together with naive antigen-specific T cells. Antigen-specific iTregs markedly inhibited the activation, expansion, and differentiation of co-transferred effector T cells specific for the same antigen. iTregs specific for an antigen distinct from the one recognized by the effector T cells could also inhibit effector cell priming when both antigens were presented by the same DC. DCs isolated from iTreg treated mice were markedly impaired in their capacity to activate nave T cells, but expressed normal levels of CD80/CD86. These results are most consistent with a model in which the primary target for the antigen-specific iTreg is the antigen-presenting DC. Although iTregs induced in vitro in the presence of TGF-beta have proven to be useful tools for the analysis of some aspects of Treg function,a number of studies have suggested that Foxp3 expression in iTregs is unstable upon transfer in vivo raising the possibility that iTregs would not be useful for long term treatment of autoimmune disease. To analyze the factors that control the stability of Foxp3 expression in iTregs, we generated OVA-specific iTregs from OT-II Foxp3-GFP knock-in (KI) mice. Following transfer to normal C57Bl/6 mice, sorted OT-II GFP+ cells maintained high levels of Foxp3 expression for 8 days. However, they rapidly lost Foxp3 expression upon stimulation with OVA in incomplete adjuvant in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region (TSDR) within the Foxp3 locus. As previous studies have shown that administration of IL-2 in the form of an anti-IL-2/IL-2 complex can markedly expand the numbers of Foxp3+ thymic-derived Treg, we analyzed the effects of IL-2 on iTreg. IL-2 treatment of recipient mice resulted in a marked expansion of transferred iTreg in vivo in the absence of antigen challenge. The expanded iTreg displayed an activated phenotype. Furthermore, administration of IL-2 also enhanced the expansion of the iTreg following antigen challenge. The expanded iTreg maintained Foxp3 expression accompanied by enhanced demethylation of the TSDR region of the Foxp3 locus. Taken together, our data suggest that stimulation with TGF-βin vitro is not sufficient for imprinting T cells with stable expression of Foxp3. Administration of IL-2 in vivo results in stabilization of Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.

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Akkaya, Billur; Holstein, Amanda H; Isaac, Christopher et al. (2017) Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies. J Immunol Methods 441:67-71
Shevach, Ethan M (2017) Garp as a therapeutic target for modulation of T regulatory cell function. Expert Opin Ther Targets 21:191-200
Vermeersch, Elien; Denorme, Frederik; Maes, Wim et al. (2017) The role of platelet and endothelial GARP in thrombosis and hemostasis. PLoS One 12:e0173329
Akkaya, Billur; Miozzo, Pietro; Holstein, Amanda H et al. (2016) A Simple, Versatile Antibody-Based Barcoding Method for Flow Cytometry. J Immunol 197:2027-38
Edwards, Justin P; Hand, Timothy W; Morais da Fonseca, Denise et al. (2016) The GARP/Latent TGF-?1 complex on Treg cells modulates the induction of peripherally derived Treg cells during oral tolerance. Eur J Immunol 46:1480-9
Ujiie, Hideyuki; Shevach, Ethan M (2016) ?? T Cells Protect the Liver and Lungs of Mice from Autoimmunity Induced by Scurfy Lymphocytes. J Immunol 196:1517-28
Edwards, Justin P; Thornton, Angela M; Shevach, Ethan M (2014) Release of active TGF-?1 from the latent TGF-?1/GARP complex on T regulatory cells is mediated by integrin ?8. J Immunol 193:2843-9
Chattopadhyay, Gouri; Shevach, Ethan M (2013) Antigen-specific induced T regulatory cells impair dendritic cell function via an IL-10/MARCH1-dependent mechanism. J Immunol 191:5875-84
Edwards, Justin P; Fujii, Hodaka; Zhou, Angela X et al. (2013) Regulation of the expression of GARP/latent TGF-?1 complexes on mouse T cells and their role in regulatory T cell and Th17 differentiation. J Immunol 190:5506-15
Davidson, Todd S; Shevach, Ethan M (2011) Polyclonal Treg cells modulate T effector cell trafficking. Eur J Immunol 41:2862-70

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