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, maintain expression of Foxp3 in vivo, 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. Co-transfer of congenically marked, labeled TCR transgenic naive effector T cells along with antigen-specific iTregs followed by immunization results in dramatic suppression of effector cell activation, proliferation and differentiation. In contrast, co-transfer of naive effector cells in the presence of polyclonal nTregs does not result in suppression of activation, proliferation or differentiation, even in the presence of 50-fold more nTregs than effector cells. Unexpectedly, effector cells are retained in the draining lymph node in the presence of nTregs, and prevented from entering the blood. This ultimately results in fewer effector cells entering peripheral tissues. Thus, polyclonal nTregs do not inhibit effector cell activation in vivo, but alter the migratory capacity of these cells thereby limiting pathology in peripheral tissues. In collaboration with Dr. S. Vendetti, we have extended these studies to analyze the effects of both polyclonal nTregs and antigen-specific iTregs on the modulation of antibody production. 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 adjuvants are based on suppression of Treg function. We evaluated whether the 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 ablation 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 BALB/c nu/nu mice, impaired the adjuvant effect of CT on antigen-specific antibody production after mucosal immunization. Transfer of low numbers of polyclonal, but not antigen-specific, CD4+CD25+ Treg to normal mice also enhanced CT-induced antibody responses. Recipients of polyclonal Treg that had been CT-treated and immunized had an increased number of antigen-specific CD4+ T cells with an activated phenotype in the draining lymph nodes. This accumulation of antigen-specific CD4+ T lymphocytes appears to favor germinal center formation and may promote T-dependent B cell responses. Taken together, these studies indicate that Foxp3+ Treg can not only act as suppressor cells by preventing the priming of effector T cells, or by preventing the migration of effector T cells to their target organ, but can also potentiate the function of helper T cells and augment antibody production depending on the class of the immune response being evaluated and the microenvironment in which the response is generated. The Scurfy mouse lacks functional Treg due to disruption of the Foxp3 gene by a 2bp insertion and as a result Scurfy mice develop a lymphoproliferative disease with multiorgan inflammation.Autoreactive CD4+ effector T cells infiltrate tissues, secrete cytokines, recruit other inflammatory cells and ultimately lead to the destructive pathology present in liver, lung and skin. B cells are also activated in the Scurfy mouse and high antibody titers are present in the serum.However, none of the antigenic targets recognized by the autoreactive T cells and autoantibodies have thus far been identified. We asked if an autoimmune response directed against skin antigens exists in Scurfy mice. We screened Scurfy sera for reactivity to skin proteins and identified several keratins as antigenic targets. In addition, we showed by adoptive transfer experiments involving nu/nu mice that Scurfy CD4+ T cells provided T cell help to autoreactive B cells present in the normal B cell pool, leading to the generation of autoantibodies similar to the ones present in Scurfy serum. For one of the target antigens, keratin 14, we localized the predominant epitope to the C-terminal part of the protein.Since B cells need help from activated T cells with the same antigen specificity, it is tempting to speculate that the Scurfy mouse also harbours autoreactive T cells, which recognize keratin 14. Indeed, CD4+ T cells from both skin and draining lymph nodes proliferated specifically when stimulated with the bulk preparation of keratins. Patients with the immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which resembles the disease in Scurfy mice and is also caused by mutations in the FOXP3 gene , often present with severe skin disease.We tested sera from IPEX patients with and without skin disease by western blot for their reactivity to skin antigens and identified strong reactivity in one of the patients with eczema. Taken together, these data identify keratins as antigenic targets in autoimmune disease and should facilitate the investigation of the specificity of autoreactive T cells in autoimmune skin diseases.our identification of keratin 14 as an antigenic target should facilitate the analysis of the specificity of autoreactive T cells in human skin diseases and thereby provide potential impact on future therapeutic strategies.

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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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