T cell activation and proliferation requires a reducing microenvironment that is provided by antigen presenting cells especially dendritic cells (DCs). Naturally occurring CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) suppress proliferation of CD4⁺CD25 ^- effector T cells (Teffs) by mechanisms that are not well understood. Here, we have demonstrated that inhibition by Tregs of DC-induced extracellular redox remodeling is a component of the Treg immunosuppressive mechanism. We showed that the mechanism of redox remodeling during T cell activation involved secretion of glutathione (GSH) by dendritic cells and its subsequent cleavage to cysteine. Extracellular cysteine accumulation resulted in a lower redox potential, which is conducive to proliferation, and changed the net redox status of exofacial protein domains. Suppression of DC-dependent Teff cell proliferation by Tregs was correlated with a significant diminution in extracellular cysteine concentration and was abrogated by addition of exogenous cysteine. We demonstrated that Treg-mediated redox perturbation was antigen-dependent, antigen-nonspecific and cytotoxic T-lymphocyte antigen 4-dependent. Tregs used multiple strategies for extracellular redox remodeling including modulation of GSH metabolism in DCs and competitive uptake of cysteine. By interfering with the extracellular cysteine pool, Tregs not only decreased the intracellular GSH levels in Teffs, but also blocked GSH relocalization into the cytoplasm, thus inhibiting T cell activation and proliferation.

The synthesis of GSH, a major cellular antioxidant with a critical role in T cell proliferation, is limited by cysteine. We have evaluated the contributions of the x_c^- cystine transporter and the transsulfuration pathway to cysteine provision for GSH synthesis and antioxidant defense in naïve versus activated T cells and in the immortalized T lymphocyte cell line, Jurkat. We showed that the x_c^- transporter while absent in naïve T cells, was induced after activation. We also demonstrated the existence of an intact transsulfuration pathway in naïve and activated T cells and in Jurkat cells. The flux through the transsulfuration pathway increased in primary T cells in response to oxidative challenge by peroxide. Inhibition of the transsulfuration pathway in both primary and transformed T cells decreased cell viability under oxidative stress conditions.