Background:  Energy metabolism of immune cells has been identified as critical in driving alloimmunity. While metabolic capacities change with aging, aspects linking age, immunosuppression, and T-cell metabolism remain unclear. We submit that age-specific immunosuppressive capacities of Tacrolimus (TAC) are operative through specific changes in T-cell metabolism.
Methods:  Full thickness skin grafts were transplanted from young (DBA/2 mice) to either young or old C57BL/6 recipients; animals were treated with TAC and graft survival was tested. To test the impact of T-cell metabolism, aging and TAC treatment, naïve CD4+ T cells were collected from recipient animals and activated with anti-CD3 and anti-CD28. Oxidative phosphorylation (OXPHOS) and aerobic glycolysis were assessed by oxygen consumption (OCR) and extracellular acidification rate (ECAR) using a XFe24 extracellular flux analyzer.
Results: Old and young recipients received weight-adapted doses of TAC. The majority of older recipients survived until the end of the observation period (day 30) while young animals rejected allografts by day 11 (mean survival time; n = 7; p < 0.0004;).  Old but not young CD4+ T cells demonstrated compromised metabolic rates with significantly lower OCR and ECAR (p<0.0001). Next, we co-cultured naïve CD4+ T cells with TAC at different concentrations and measured OCR and ECAR by 24 hrs. Dose-response curves of ECAR to TAC revealed a significantly reduced IC50 of old CD4+ T cells (p<0.0001), suggesting an age-specific effect of TAC on glycolysis. Next, we tested if energy metabolism in CD4+ T cells impacts IL-2 cytokine production in an age-specific manner. Here, we used glyceraldehyde 3- phosphate (G3P) to induce aerobic glycolysis in-vitro. G3P is the sole substrate of GAPDH, the pivotal enzyme in aerobic glycolysis. Activation of aerobic glycolysis through G3P increased IL-2 significantly in old, but not young TAC treated CD4+ T cells. Moreover, IL-2 expression increased dramatically (by 82%) subsequent to the introduction of G3P in old TAC treated T cells; notably, only a minor (26%) increase of G3P was observed in old naïve T cells suggesting a compromised aerobic glycolysis in old CD4+ T cells, an effect that is exacerbated subsequent to the treatment with TAC.
Conclusions: TAC appears significantly effective in older recipients leading to specific prolongation of graft survival in old recipients. Those effects are linked to age-specific metabolic changes in CD-4 T-cell metabolism with a compromised glycolysis of old CD4+ T cells that is exacerbated age-specifically subsequent to TAC treatment. Moreover, IL-2 expression increased dramatically in old CD4+ T cells following the activation of glycolysis. Taken together, our results suggest that age-specific aspects of TAC are linked to a compromised glycolysis of CD4+ T cells Those findings provide a novel concept to refine immunosuppression in and beyond aging.