However, lentivirus transduced Treg avatars likely offer greater potential for long-term efficacy in clearing islet infiltration/inflammation and leading to persistent engraftment. Importantly, adoptive cell therapy with polyclonal autologous peripheral blood Tregs has been demonstrated to be safe in Phase I clinical trials (26, 27). ability to alter antigen specificity by T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene transfer has facilitated personalized cellular immune therapies in cancer. Inversely, this approach can be harnessed in autoimmune settings to attenuate inflammation by redirecting the specificity of regulatory T cells (Tregs). Herein, we demonstrate efficient protocols for lentiviral gene transfer of TCRs that recognize type 1 diabetes-related autoantigens with the goal of tissue-targeted induction 3-Hydroxyisovaleric acid 3-Hydroxyisovaleric acid of antigen-specific tolerance to halt -cell destruction. We generated human Tregs expressing 3-Hydroxyisovaleric acid a high-affinity GAD555C567-reactive TCR (clone R164), as well as the lower affinity clone 4.13 specific for the same peptide. We demonstrated that Treg avatars potently suppress antigen-specific and bystander responder T-cell (Tresp) proliferation in a process that requires Treg activation (high-dose anti-thymocyte globulin (ATG) or fludarabine, plus immunomodulation 3-Hydroxyisovaleric acid with Rabbit polyclonal to BZW1 cyclosporine and granulocyte-colony stimulating factor (G-CSF) have been shown to preserve -cell function (2, 3), but the risks associated with these aggressive protocols preclude common clinical use. Comparatively, non-specific polyclonal immunotherapies, including immunoregulatory or depleting agents [e.g., alefacept (human LFA-3/IgG1-Fc 3-Hydroxyisovaleric acid fusion protein), teplizumab or otelixizumab (anti-CD3), and rituximab (anti-CD20)], have been better tolerated and offered some temporary efficacy but not long-term induction of tolerance (4C10). Until recently, most antigen-specific tolerance induction efforts have involved mucosal or peripheral administration of autoantigen(s), but thus far, such attempts have yielded limited efficacy in only a subset of patients, again with no indication for long-term tolerance induction (11, 12). Indeed, a safe treatment that controls persistent immune memory and induces long-term tolerance is needed. Islet cell antigen-reactive Tregs, isolated from BDC2.5 TCR transgenic mice, could be expanded but did not proliferate after transfer into recipient animals (14). Moreover, expression of cognate autoantigen is required for efficient trafficking of Tregs to the target organ and suppression of diabetes in NOD mice (15). These preclinical data support the notions that autoantigen-specific Tregs may offer an important therapy for type 1 diabetes, but also that intrinsic factors such as TCR specificity and/or avidity may play an important role in determining the capacity for immunomodulation and efficacy. The need for continued autoantigen expression by the host may render insulin-reactive TCRs less effective in patients with long-standing type 1 diabetes and support a need to investigate additional, potentially bystander, TCRs specific for additional/alternative autoantigen targets such as glutamic acid decarboxylase (GAD). Moreover, antigen localization, density, and persistence in -cells along with risk of effector cell reprogramming support the use of alternative TCRs (16). Genetically modified T cells with TCRs specific for tumor or viral antigens have become a valuable tool for the treatment of certain cancers or infections in humans (17C19). We previously demonstrated successful HLA class I-restricted TCR gene transfer in human Tregs using a high-affinity model receptor specific for the melanoma antigen tyrosinase presented by HLA-A*02:01 (20). We also generated a murine form of these tyrosinase-specific Tregs, and when transferred bystander suppression and infectious tolerance (14, 28). To expand on these efforts, we generated primary human Tregs expressing the two GAD555C567-reactive TCR clones (R164 and 4.13), and investigated the pre-transfer conditions needed to optimize suppressive activity for potential use in adoptive cell therapy. Research Design and Methods Design and Synthesis of Lentiviral Constructs Lentiviral vectors were generated to express TCR clones 4.13 and R164, both of which react to GAD555C567 (21, 25) (Table ?(Table1).1). Equimolar expression of TCR – and -chains was achieved by inclusion of a multicystronic P2A element, followed by a T2A element and the reporter, enhanced green fluorescent protein (eGFP). The constructs were cloned into pCNFW lentiviral vectors with expression driven by a cytomegalovirus promoter as previously described (25) (Figure ?(Figure1A).1A). Lentiviral vectors containing the Melan-A reactive TCR clone melanoma antigen recognized by T cells 1 (MART-1) were generated as previously described (29) (Table ?(Table11). Table 1 T-cell receptor (TCR) clone information..