Loss of Nrf2 in bone marrow-derived macrophages impairs antigen-driven CD8(+) T cell function by limiting GSH and Cys availability.

Abstract NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8 + T cell activation by using an antigen-driven coculture model consisting of Nrf2 −/− and Nrf2 +/+ bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8 + T cells. OT-1 CD8 + T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257–264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257–264)-pulsed Nrf2 −/− BMDMΦ with transgenic OT-1 CD8 + T cells attenuated CD8 + T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8 + T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2 −/− cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2 −/− cocultures via addition of glutathione ester, N -acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2 +/+ BMDMΦ mimicked the effect of Nrf2 −/− BMDMΦ on CD8 + T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2 −/− BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8 + T cell function.