Opa1 Regulates iNKT Cell Differentiation Through Control of TCR Signaling 2260028

Abstract

Invariant natural killer T (iNKT) cells are innate-like T cells with rapid effector functions. Distinct metabolic programs support their differentiation and function. We found that iNKT cells exhibit higher mitochondrial content, activity, and a punctate morphology compared to conventional CD4+ T cells. Notably, the mitochondrial fusion regulator Opa1, but not the fission factor Drp1, is specifically required for iNKT cell differentiation: NKT1 and NKT17 subsets are markedly reduced in Opa1-deficient mice, whereas NKT2 cells remain unaffected. We used complementary approaches, including conditional genetic models, flow cytometry, thymic organ culture, confocal imaging, and transcriptomic analyses, to dissect how mitochondrial dynamics influence iNKT cell development and signaling. The differentiation defect is cell-intrinsic and not rescued by Bcl-XL overexpression. Opa1 loss leads to increased mitochondrial volume and decreased membrane potential, indicating dysfunction supported by transcriptional changes. Metabolic reprogramming accompanies this defect, with increased Glut1 expression and 2-NBDG uptake, while LDHA appears to compensate for Opa1 loss. TCR signaling is impaired: NF-κB, AP-1, and mTOR activities are reduced, whereas PLZF and Egr2, downstream of Ca²+/NFAT, are elevated. Consistently, TCR-induced Ca²+ flux is altered. Remarkably, simultaneous loss of Opa1 and Drp1, which induces mitochondrial stasis, partially restores iNKT cell homeostasis, proliferation, and TCR signaling in Opa1-deficient cells. These findings uncover a critical role for Opa1-mediated mitochondrial dynamics in coordinating TCR signaling and metabolic programs that shape iNKT cell differentiation. NIH Hematopoiesis and Immune System Development (HEM)