Abstract: 【Abstract】 Objective To investigate the effect of mast cell-derived chemokine C-C motif ligand 5 （CCL5） on the migration of CD8+ T cells in vitiligo under oxidative stress conditions. Methods From January 2017 to January 2023, 10 patients with progressive segmental vitiligo, 10 patients with non-segmental vitiligo, and 10 healthy individuals were collected from the Department of Dermatology, Xijing Hospital, and toluidine blue staining was performed to analyze the characteristics of mast cells infiltrating the skin lesions. The effect of H2O2 treatment on mast cells was investigated in Transwell chambers. Peripheral blood mononuclear cells （PBMCs） from vitiligo patients were added to the upper chamber, while the human mast cell line LAD2 was added to the lower chamber and divided into 4 groups to receive different treatments: untreated group receiving no special treatment, H2O2 group pretreated with H2O2, H2O2 + neutralization antibody group pretreated with H2O2 followed by the treatment with CCL5/RANTES-neutralizing antibodies, and H2O2 + PF group pretreated with H2O2 followed by the treatment with a Janus kinase 3/non-receptor protein tyrosine kinase selective inhibitor PF-06651600. After 6 hours of co-incubation, cell suspensions were collected from the lower chamber. The number of CD8+ T cells was counted using flow cytometry, and the CCL5 level in the cell culture supernatant was detected using enzyme-linked immunosorbent assay. One-way analysis of variance was used to analyze differences among groups, and least significant difference-t test was used for multiple comparisons. Pearson correlation analysis was performed for correlation analysis. Results Under a 200-fold microscopic field of view, the number ofthe numbers of mast cells infiltrating the skin lesions of patients with segmental or non-segmental vitiligo, and the skin tissues of healthy individuals significantly differed （15.7 ± 3.3, 20.9 ± 3.9, 7.2 ± 2.9, respectively; F = 8.07, P = 0.002）; additionally, the number of infiltrating mast cells was significantly higher in the segmental or non-segmental vitiligo lesions than in the normal skin tissues （LSD-t = 3.50, 5.70, P = 0.047, 0.001, respectively）, but there was no significant difference between the segmental and non-segmental vitiligo lesions （LSD-t = 2.20, P = 0.293）. A significant positive correlation was observed between the number of infiltrating mast cells and that of CD8+ T cells in the vitiligo lesions （r = 0.82, P = 0.004）. The numbers of CD8+ T cells and CCL5 levels significantly differed among the untreated group, H2O2 group, H2O2 + neutralization antibody group, and H2O2 + PF group （CD8+ T cells: 197.0 ± 45.9, 580.4 ± 62.6, 296.0 ± 43.2, 398.6 ± 62.8, respectively; CCL5: 2.2 ± 0.6 pg/ml, 9.9 ± 1.3 pg/ml, 3.4 ± 0.4 pg/ml, 6.33 ± 0.7 pg/ml, respectively; F = 11.03, 17.77, respectively, both P < 0.001）; additionally, the H2O2 group showed significantly increased number of CD8+ T cells and CCL5 levels compared with the untreated group, H2O2 + neutralization antibody group, and H2O2 + PF group （all P < 0.05）. Conclusion Mast cells may be involved in the pathogenesis of vitiligo under oxidative stress, and mast cell-derived CCL5 appears to contribute to the occurrence and development of vitiligo by affecting CD8+ T cell migration.

Key words: Vitiligo, Mast cells, Leukocytes, mononuclear, Chemokine CCL5, CD8-positive T-lymphocytes