Abstract: 【Abstract】 Objective To investigate changes in the peripheral interleukin-35 （IL-35）level in patients with alopecia areata, and to assess its modulatory effect on regulatory T （Treg） cell activities. Methods Totally, 81 patients with alopecia areata （alopecia areata group） and 27 healthy volunteers （control group） were enrolled from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Sera and peripheral blood mononuclear cells （PBMCs） were isolated. Enzyme-linked immunosorbent assay （ELISA） was performed to detect the serum IL-35 level, real-time fluorescence-based quantitative PCR to determine the mRNA expression of IL-35 subunits EBI3 and IL-12p35, and flow cytometry to determine the proportion of CD4+CD25+CD127dim/- Treg cells. Sorted Treg cells were stimulated by recombinant human IL-35, ELISA was performed to detect levels of perforin and granzyme B in the culture supernatant, and real-time fluorescence-based quantitative PCR to determine the mRNA expression of EBI3, IL-12p35, and immune checkpoint molecules, such as programmed death protein 1 （PD-1）, T cell immunoglobulin and mucin protein-3 （Tim-3）, cytotoxic T lymphocyte-associated antigen 4 （CTLA-4） and lymphocyte activation gene-3 （LAG-3） in Treg cells. IL-35-stimulated or unstimulated Treg cells were co-cultured with autologous PBMCs, and cell counting kit-8 （CCK8） assay was conducted to assess cellular proliferative activity. Measurement data were compared between 2 groups by using t test, comparisons among multiple groups were carried out by using one-way analysis of variance, correlation analysis was carried out by using Pearson correlation analysis, and enumeration data were compared by using chi-square test. Results Compared with the control group, the alopecia areata group showed significantly decreased IL-35 levels （90.10 ± 11.98 ng/L vs. 100.74 ± 28.71 ng/L, t = 2.71, P = 0.008）, mRNA expression of EBI3 and IL-12p35 in PBMCs （EBI3: 1.06 ± 0.15 vs. 1.25 ± 0.11, t = 6.09, P < 0.001; IL-12p35: 1.00 ± 0.15 vs. 1.38 ± 0.22, t = 10.16, P < 0.001）, and proportions of Treg cells （5.91% ± 1.17% vs. 6.85% ± 1.23%, t = 3.54, P = 0.001）. In the alopecia areata group, the proportion of Treg cells was positively correlated with the serum IL-35 level （r = 0.25, P = 0.026）, and the mRNA expression of EBI3 and IL-12p35 in PBMCs （r = 0.31, 0.24, P = 0.004, 0.032, respectively）. Compared with the control group, the unstimulated Treg cells from the alopecia areata group showed significantly decreased supernatant levels of perforin and granzyme B, mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules （P < 0.05 or 0.001）, as well as weakened inhibitory effect on the proliferative activity of PBMCs （P = 0.013）. There was no significant difference in the level of perforin or granzyme B between the recombinant human IL-35-stimulated and unstimulated Treg cells from the patients with alopecia areata （both P > 0.05）. However, the mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules was significantly higher in the IL-35-stimulated Treg cells than in the unstimulated Treg cells in the alopecia areata group （P < 0.05 or 0.001）, and the inhibitory effect on the proliferative activity of PBMCs was also significantly enhanced in the IL-35-stimulated Treg cells compared with the unstimulated Treg cells （P = 0.037）. Conclusion The peripheral IL-35 level was significantly decreased in the patients with alopecia areata, which was closely associated with reduced activities of Treg cells, and IL-35 may be involved in the occurrence of alopecia areata.

Key words: Alopecia areata, Interleukins, T-lymphocytes, regulatory, Perforin, Granzymes, Interleukin-35, Immune checkpoint