Abstract
Aims: Reactive oxygen species (ROS) are critical in driving the onset of type 1 diabetes (T1D). Ablation of ROS derived from phagocytic NADPH oxidase 2 is protective against autoimmune diabetes in non-obese diabetic (NOD) mice. However, the mechanisms of NADPH oxidase 2-derived ROS in T1D pathogenesis need to be elucidated. Here, we have examined the role of Ncf1 (the regulatory subunit of NADPH oxidase 2) in dendritic cells (DC).Results: Ncf1-mutant DCs exhibit reduced ability to activate autoreactive CD8+ T cells despite no difference in co-stimulatory molecule expression or pro-inflammatory cytokine production. When provided with exogenous whole-protein antigen, Ncf1-mutant NOD DCs showed strong phagosome acidification and rapid antigen degradation, which lead to an absence of protein translocation into the cytoplasm and deficient antigenic peptide loading on MHC Class I molecules.Innovation: This study demonstrates that Ncf1 (p47phox) is required for activation and effector function of CD8+ T cells by acting both intrinsically within the T cell as well as within professional antigen presenting cells.Conclusion: ROS promote CD8+ T cell activation by facilitating autoantigen cross-presentation by DCs. ROS scavengers could potentially represent an important component of therapies aiming to disrupt autoantigen presentation and activation of CD8+ T cells in individuals at-risk for developing T1D.
Highlights
We explored the capacity of dendritic cells (DC) isolated from non-obese diabetic (NOD)-Ncf1m1J mice to activate autoreactive CD8+ T cells in the NOD mouse model of type 1 diabetes (T1D)
PCR were performed with pooled bone marrow-derived DCs (BMDC) from three mice and results were compiled from three independent experiments done in triplicate
To assess defects in the diabetogenicity of Cytotoxic CD8+ T cell (CTL) and antigen presenting cells (APCs) lacking intracellular superoxide production, purified naïve CD8+ T cells collected from prediabetic NOD or NOD-Ncf1m1J donor mice were adoptively co-transferred with CD4+ T cells from prediabetic NOD into either NOD-Rag1−/− or NOD-Rag1−/−.Ncf1m1J recipient animals
Summary
NOD/ShiLtJ (NOD), NOD.Cg-Rag1tm1Mom (NOD-Rag1−/−), NOD.Cg-Rag1tm1MomTg (TcraAI4)1Dvs/DvsJ (NOD.AI4αRag1−/−), and NOD.Cg-Rag1tm1MomTg (TcrbAI4)1Dvs/DvsJ (NOD.AI4β-Rag1−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Phagocytosis by BMDC was determined using beads coupled with the fluorescent pH insensitive indicator dye AF647. BMDC were incubated at 37◦C for 20 min in the presence of fluorescent beads, washed extensively with cold PBS, and immediately analyzed by flow cytometry. After an 8min incubation, these cells are immediately analyzed by flow cytometry and the emission ratio of the two fluorescent probes of BMDC incubated in HBSS at each pH was collected. PCR were performed with pooled BMDC from three mice and results were compiled from three independent experiments done in triplicate. To measure the cross-presentation of autoantigen, insulin reactive G9C8 and AI4 T cells were purified and incubated with BMDCs from NOD or NOD-Ncf1m1J at a ratio of 5:1. Except for the survival analysis, all data reported here are representative of at least three independent experiments performed in triplicate
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