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

Free Radic Biol Med. 2015 Jun;83:77-88. doi: 10.1016/j.freeradbiomed.2015.02.004. Epub 2015 Feb 14.

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.

Keywords: BMDMΦ; CD8(+) T cells; Cysteine; Cytotoxicity; Free radicals; Glutathione; Nrf2; T cell tolerance; xCT; γ-GCS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antioxidants / metabolism
  • Apoptosis
  • Blotting, Western
  • Bone Marrow / immunology*
  • Bone Marrow / metabolism
  • CD8-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / metabolism
  • Cell Proliferation
  • Cells, Cultured
  • Cystine / metabolism*
  • Flow Cytometry
  • Glutathione / metabolism*
  • Histocompatibility Antigens Class I / immunology*
  • Histocompatibility Antigens Class I / metabolism
  • Immunoenzyme Techniques
  • Macrophages / immunology*
  • Macrophages / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mice, Transgenic
  • NF-E2-Related Factor 2 / physiology*
  • Ovalbumin / immunology
  • Ovalbumin / metabolism
  • Oxidative Stress
  • RNA, Messenger / genetics
  • Reactive Oxygen Species / metabolism
  • Real-Time Polymerase Chain Reaction
  • Receptors, Antigen, T-Cell / immunology
  • Receptors, Antigen, T-Cell / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction

Substances

  • Antioxidants
  • Histocompatibility Antigens Class I
  • NF-E2-Related Factor 2
  • Nfe2l2 protein, mouse
  • RNA, Messenger
  • Reactive Oxygen Species
  • Receptors, Antigen, T-Cell
  • Cystine
  • Ovalbumin
  • Glutathione