Expression analysis of PD-1 and Tim-3 immune checkpoint receptors in Vitiligo patients; positive association with disease activity.

The contribution of immune checkpoint receptors in the immunopathogenesis of various autoimmune diseases has been addressed in previous reports. In this study, the expression profile of T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) and programmed cell death-1 (PD-1) checkpoint molecules was investigated in CD8(+) T cells of Vitiligo patients. The association of Tim-3 and PD-1 expression with disease activity was also explored. The frequency of Tim-3(+)/PD-1(+)/CD8(+) T cells in 30 patients with vitiligo and 30 sex- and age-matched controls was determined by flow cytometry. CD8(+) T cells were then positively isolated by magnetic beads, and the mRNA expression of PD-1 and Tim-3 was determined by TaqMan-based real-time PCR. To measure the cytokines production, PBMCs were stimulated with PMA/ionomycin and concentrations of IL-4, IFN-gamma and TNF-alpha were measured in culture supernatants by ELISA. Disease activity of patients with vitiligo was determined using the Vitiligo Area Severity Index. Patients with vitiligo have significantly shown more expression of Tim-3 and PD-1 on their CD8(+) T cells compared with controls. Expression analysis of Tim-3 mRNA, but not PD-1, confirmed the results obtained from flow cytometry. While the production levels of TNF-alpha and IFN-gamma were found higher by patients with vitiligo, IL-4 production was lower in patients compared with controls. A direct association was observed between the Tim-3 and PD-1 expression and also the production of pro-inflammatory cytokines with disease activity of patients with vitiligo. Our results indicate that Tim-3 and PD-1 are involved in immune dysregulation mechanisms of CD8(+) T cells in vitiligo and may introduce as potential biomarkers for disease progression and targeted immunotherapy.