Tacrolimus Exerts Age-Specific Immunosuppressive Effects Linked to Modifications of CD4+ T cells Metabolism

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Abstract

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.

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