To further validate that the observed differences in mitochondrial proteome are not due to differences in cell cycle, we reanalyzed our proteomic dataset after applying the ccRemover algorithm to remove cell cycle effects (20). aged T cells). Data are representative of at least two independent experiments. To assess the AV412 proliferative capacity of aged na?ve T cells, we labeled na?ve CD4+ T cells with carboxyfluorescein succinimidyl ester (CFSE), a cell permeable dye, and assessed CFSE dilution at 48 h postactivation. Aged T cells had an increased percentage of nondividing cells (generation = 0) and a decreased percentage of cells that underwent two cell divisions (generation = 2; Fig. 1and and and 0.05, ** 0.01, *** 0.001 (Students test comparing young vs. aged T cells). Data are representative of at least two independent experiments. Analysis of oxygen consumption rate (a measure of mitochondrial respiration) in activated na?ve CD4+ T cells from young and aged mice revealed a significant reduction in basal respiration (Fig. 2and and and and Dataset S2) and included proteins associated with inflammation and immune regulation, such as Vnn1 (15), Nfkbid (16), and foxp4 (17). Interestingly, the majority of these proteins are not well studied in the context of immune cell function and may highlight pathways contributing to immunosenescence. We further identified 40 proteins AV412 that were elevated at least twofold more in aged T cells compared with young T cells (and Dataset S2), suggesting TNFA that the aged T cell phenotypes were not solely due to blunted activation. The proteins most induced in activated aged T cells included Gm16519, a predicted ribosomal protein; Iglc2, an immunoglobulin; and Bicd2, involved in Golgi trafficking (18). While immunoglobulins are generated by B lymphocytes, our proteomic data from sorted T cells did not detect other B cell markers such as CD19, ruling out a general B cell contamination. One possible explanation for detection of Iglc2 is attachment of the antibody to the T cells surface, that is not completely excluded by the wash. To identify functional patterns during activation, proteins were grouped based on the kinetics and magnitude of induction in young cells (Fig. 3and and and 0.01, *** 0.001 (Students test). Our analysis of young and aged T cells was performed AV412 at 24 h AV412 postactivation, before proliferation occurs. To further validate that the observed differences in mitochondrial proteome are not due to differences in cell cycle, we reanalyzed our proteomic dataset after applying the ccRemover algorithm to remove cell cycle effects (20). Mitochondrial proteins were then grouped into clusters based on kinetics and magnitude of activation (Fig. 4and and and and and 0.05, *** 0.001 (Students test comparing each treatment group to its untreated control, and the aged controls to young controls, when marked by a line). Data are representative of at least two independent experiments. Discussion In this study we performed a side-by-side comparison of mitochondrial biogenesis, intracellular metabolites, and quantitative proteomics in young versus aged T cells. We found cell-intrinsic defects in metabolism during the activation of aged na?ve CD4+ T cells, including evidence of lower glycolysis and attenuated induction of one-carbon metabolism. Importantly, addition of metabolites in one-carbon metabolism partially rescued defects in activation of aged CD4+ T cells. To investigate intrinsic deficits in aged na?ve CD4+ T cells, we purified na?ve CD4+ cells from aged mice and analyzed their activation ex vivo using anti-CD3/anti-CD28. This ex vivo approach eliminated the effect of other potential age-related factors, such as reduced efficiency of antigen uptake and/or presentation (21) and the increase in immune suppressor populations (e.g., regulatory T cells and myeloid derived suppressor cells) (22). We found that mitochondrial mass and activation are reduced in stimulated aged compared AV412 with young T cells. Reduced mitochondrial activation may impair functionality by dysregulating critical early signaling events. For.
