*Purpose: Metabolic reprogramming has been identified as a critical regulator of immunity. Little is known on changes of T-cell metabolism in aging and how to utilize metabolic targets for immunosuppression. Here, we show that T-cell metabolism changes drastically in aging. Most notably, targeting T-cell metabolism provides an age-specific and effective immunosuppression.

*Methods: Naïve CD4+ T cells were collected from C57BL/6 mice (3 and 18 mths) and activated with anti-CD3 and anti-CD28 for 24 hrs. Oxidative phosphorylation (OXPHOS) and aerobic glycolysis were assessed by oxygen consumption (OCR) and extracellular acidification rate (ECAR) using a Seahorse XFe96 extracellular flux analyzer. Full thickness skin grafts were transplanted from young (3 mth DBA/2 mice) to young and old C57BL/6 recipients (3 and 18 mths). Immune profiling was performed at sequential time intervals using a FACS Canton II flow cytometer.

*Results: Old, but not young CD4+ T cells demonstrated compromised metabolic rates with significantly lower OCR and ECAR rates (p<0.0001). Moreover, old CD4+ T cells demonstrated a significant mitochondrial impairment with compromised respiratory and glycolytic capacities (p<0.0001). These results indicated a limited capacity of old CD4+ T cells to respond to metabolic stress. Glutaminolysis has been identified as a critical metabolic pathway in T-cell immunity. 6-diazo-5-oxo-l-norleucine (DON) is an analog of glutamine that inhibits critical enzymes of glutaminolysis. DON treatment inhibited IL-2 production in both young and old CD4+ T. To test the immunosuppressive capacity in fully mismatched transplantation model from DBA/2 donor to either young or old C57BL/6 recipients, recipients of full-thickness skin allografts were treated with DON (1.6mg/kg every other day/x60). Most impressive were age-specific differences: DON improved graft survival times in young animals from 7 to 18 days; in old recipients DON prolonged graft survival to 43 days (n=7, p<0.001)). Of note, treatment with DON had been superior to TAC treatment (in young recipients: TAC treated vs control vs DON = 7 vs 11 vs 18 days; in old recipients old TAC treated vs control vs DON=10 vs 14 vs 43 days, n=7, p<0.001). Immune profiling by day 7 showed a 41% reduction of CD4+ T cell proliferation in the old but not young recipients. (n=5, p<0.001). However, DON induced an age-specific increase of in IL-10 production (44% in old, no significant difference in young, n=5, p<0.001). Moreover, DON inhibited the frequency of CD4+IL-2+ T cells more robustly in old recipients (34% vs 13%, young vs old, n=5, p<0.001). DON appeared to specifically targeting CD-4+ T-cells as CD8+ T cells numbers and function were not impacted by DON treatment.

*Conclusions: Inhibiting glutaminolysis demonstrates an effective immunosuppression targeting old CD4+ T-cells. Those experimental data provide a novel concept of immunosuppression with relevance in older recipients.

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