Abstract: 【Abstract】 Objective To investigate changes of natural killer （NK） cell subsets and interleukin-18 （IL-18） level in peripheral blood of patients with alopecia areata, and to assess the regulatory effect of IL-18 on NK cell activity. Methods A total of 67 patients with alopecia areata （alopecia areata group） and 25 healthy volunteers （control group） were collected from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Peripheral blood mononuclear cells （PBMCs） and plasma were isolated. The percentage of NK cell subsets was investigated by flow cytometry, and plasma IL-18 level was measured by enzyme-linked immunosorbent assay. PBMCs were stimulated with recombinant human IL-18, and co-culture systems of PBMCs with 721.221 cells, K562 cells and P815-Ab cells were established separately. NK cell function was assessed by determining the percentage of CD107a-expressing NK cells and fluorescence intensity of CD16+ NK cells. Comparisons between groups were performed using t test or paired t test. Results Compared with the control group, the alopecia areata group showed significantly decreased percentage of CD56+CD16－ NK cells（8.12% ± 3.14% vs. 10.78% ± 4.08%, t = 3.33, P = 0.001）, but significantly increased percentage of CD56+CD16+ NK cells （46.08% ± 15.21% vs. 32.14% ± 10.45%, t = 4.22, P < 0.001）, and there was no significant difference in the percentage of CD56-CD16+ NK cells between the alopecia areata group and control group （28.81% ± 8.65% vs. 27.09% ± 7.62%, t = 0.88, P = 0.383）. The plasma IL-18 level was significantly higher in the alopecia areata group than in the control group （112.0 ± 23.72 pg/ml vs. 99.34 ± 15.15 pg/ml, t = 2.48, P = 0.015）. After co-culture with 721.221 cells, K562 cells and P815-Ab cells, the percentage of CD107a-expressing NK cells was significantly higher in NK cells from the alopecia areata group （9.53% ± 1.70%, 5.15% ± 1.35%, 6.50% ± 1.64%, respectively） than in those from the control group （5.00% ± 1.17%, 4.40% ± 1.09%, 5.13% ± 1.36%, respectively, all P < 0.05）. After the stimulation with P815-Ab cells, the alopecia areata group showed significantly decreased fluorescence intensity of CD16+ NK cells （151.10% ± 59.30%） compared with the control group （221.90% ± 93.56%, t = 4.31, P < 0.001）. After IL-18 stimulation, the percentage of CD107a-expressing NK cells significantly increased in the co-culture system of NK cells with 721.221 cells compared with the unstimulated co-culture system （14.47% ± 2.67% vs. 9.93% ± 1.94%, t = 6.00, P < 0.001）, while there was no significant difference between the IL-8-stimulated co-culture system of NK cells with K562 cells or P815-Ab cells and the unstimulated co-culture systems （both P > 0.05）. Conclusion IL-18 could enhance NK cell activity in patients with alopecia areata, likely by promoting natural cytotoxicity receptor-mediated cytotoxicity.

Key words: Alopecia areata, Interleukin-18, Natural killer T-cells, Antibody-dependent cell cytotoxicity, Lysosomal-associated membrane protein 1