Human BH3-only protein, Noxa, is a major contributor to proliferative metabolism and apoptosis in the CD8+T cell immune response

Abstract Human Noxa, the smallest BH3-only Bcl-2 protein, was originally identified as pro-apoptotic, but has since also been shown to promote cell growth in both malignant and activated normal T cells by facilitating proliferative metabolism (Lowman et al., 2010, Mol Cell, 40, 823–33; Hanse et al., 2017, Oncogene, 36, 3915–3924). To investigate the contribution of Noxa to the T cell immune response, we tracked its expression in primary human CD8+T cells in an in vitro anti-CD3/CD28 co-stimulation model. Noxa is undetectable in naïve CD8+T cells but is induced upon co-stimulation and remains highly expressed during the differentiation phase and through the onset of apoptosis. The viable cells that remain following apoptosis at the end of the immune response are ‘memory-like’, as determined by flow cytometry, harboring large mitochondria with robust spare respiratory capacity. These cells no longer express Noxa, but rapidly upregulate it upon re-stimulation. We observe that Noxa protein expression in CD8+T cells, upon stimulation, is directly dependent on glutamine and unaffected by glutaminase inhibitors, while the entry of glutamine carbons into the TCA cycle, in turn, requires Noxa. CD8+T Noxa KO cells, moreover, show reduced dependence on glutamine and significantly higher viability at the end of the immune response. CD8+T cells expressing an apoptosis deficient Noxa BH3 domain point mutant similarly presented an extended effector phase. The fundamental contributions of human Noxa to both activation-associated metabolic reprogramming and apoptosis in a prototypic immune response, makes this protein an attractive tool to manipulate the expansion, effector function, and longevity of CD8+T cells for immunotherapy. R21 AI148876