Abstract: Lu Liangjun, Wang Suiquan, Xu Aie. Department of Dermatology, Affiliated Third People′s Hospital of Hangzhou, Zhejiang Chinese Medical University, Hangzhou 310009, China Corresponding author: Xu Aie, Email: xuaiehz@msn.com 【Abstract】 Objective To establish a model for studying CD8+ cytotoxic T lymphocyte proliferation in vitro and to screen traditional Chinese drugs （TCDs） with immunosuppressive effects. Methods Spleen tissue was isolated from mice, and made into single cell suspensions followed by separation of CD8+ T lymphocytes with specific antibodies. Then, the CD8+ T lymphocytes were seeded into anti-CD3/CD28 antibody-coated 96-well plates and cocultured with the extracts of 23 TCDs （100 mg/L） separately for 96 hours. Those cells cultured with and without the presence of anti-CD3/CD28 antibody alone served as the positive control and negative control respectively. The 3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium, inner salt （MTS） assay was performed to evaluate the proliferation of cells and to select the top four TCDs with the strongest inhibitory effect. The relationship between the inhibitory effect and TCD concentrations was further assessed for the four selected TCDs. Enzyme-linked immunospot （ELISPOT） assay was carried out to estimate the influence of the four TCDs on the secretion of interferon （IFN）-γ by CD8+ T lymphocytes induced by anti-CD3/CD28 antibodies. Statistical analysis was done by nonparametric rank sum test. Results Of the 23 TCDs, 14 significantly inhibited the proliferation of CD8+ T lymphocytes（all P < 0.05）, of which, Rhizoma Coptidis, Radix Scutellariae, Radix Aucklandiae and Rhizoma Curcumae Longae displayed the strongest inhibitory capacity with the 50% inhibitory concentration being 25, 35, 50 and 60 mg/L respectively, and the 100% inhibitory concentration being 200, 100, 200 and 200 mg/L respectively. The anti-CD3/CD28 antibody-induced secretion of IFN-γ by CD8+ T lymphocytes was markedly suppressed by Radix Scutellariae, Radix Aucklandiae and Rhizoma Curcumae Longae at the concentration of 100 mg/L, but not by Rhizoma Coptidis at this concentration. Conclusions A model for studying the proliferation of CD8+ T lymphocytes is successfully developed in vitro, and four TCDs with strong inhibitory effects on the proliferation of CD8+ T lymphocytes have been screened out with this model.

Key words: CD8-positive T-lymphocytes, Drug evaluation, preclinical, Drugs, Chinese herbal, Cell proliferation