The 1α,25-dihydroxyvitamin D3 modulates T cell activation and immune checkpoint pathways in human T cells

Background: Vitamin D is a pleiotropic steroid hormone with immunomodulatory properties. The role of the active metabolite, 1α,25-dihydroxyvitamin D3 (1,25(OH)2 vitamin D), in regulating human T-cell effector function and immune checkpoint (IC) pathways remains poorly defined. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured for five days in presence or absence of 1,25(OH)2 vitamin D (10 or 100 nM). IFN-γ production was assessed by ELISpot assay. Baseline plasma 1,25(OH)2 vitamin D levels were measured. IC-related gene expression was evaluated by RT-qPCR. Flow cytometry was used to assess the expression of IC, degranulation, and activation markers. FluoroSpot assays were performed to quantify interleukin secretion. Results: 1,25(OH)2 vitamin D reduced IFN-γ expression and secretion independently of baseline circulating 1,25(OH)2 vitamin D levels. Dose-specific modulation of IC genes was detected, with increased PDCD1 and CTLA4 mRNA expression. Flow cytometry showed selective modulation within the CD8+ T-cell compartment, with increased PD-1 expression at 10 nM and increased CTLA-4 expression at 100 nM, together with decreased degranulation and activation markers. No significant changes were detected for TIM-3 or TIGIT. In CD4+ T cells, no significant modulation of IC molecules was observed, whereas IL-22 secretion was reduced. Conclusions: These findings support an immunomodulatory effect of 1,25(OH)2 vitamin D on human CD8+ T cells, with attenuation of effector-associated responses and modulation of checkpoint-associated pathways in vitro. Our results suggest that vitamin D may contribute to the regulation of inflammatory T-cell responses under controlled culture conditions. The functional and therapeutic implications of these observations require further investigation.