Abstract:

Lyu Yalin, Zhou Xiaowei, Hu Bin, Wu Qiong, Zeng Xuesi, Liu Yi, Sun Jianfang Department of Pathology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China（Lyu YL, Hu B, Wu Q, Zeng XS, Liu Y, Sun JF）; Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China （Zhou XW） Corresponding authors: Liu Yi, Email: dr.liuyi@gmail.com; Sun Jianfang, Email: sunjf57@163.com 【Abstract】 Objective To evaluate the effect of T-cell immunoglobulin and mucin domain-3 （TIM-3） on TRP-2180-188 peptide-stimulated murine spleen lymphocytes co-cultured with B16F10 murine melanoma cells. Methods A recombinant plasmid pFUSE-TIM-3-mIgG2Aae1-Fc2 encoding TIM-3 was constructed. Then, the recombinant plasmid and an empty plasmid pFUSE-mIgG2Aae1-Fc2 were transfected into human 293T epithelial cells followed by 48-hour culture for the preparation of supernatants containing TIM-3 and Ig-tail respectively. C57BL/6 mice were immunized with the TRP-2180-188 peptide vaccine for 4 sessions. One week after the last vaccination, C57BL/6 mice were sacrificed, and spleen lymphocytes were collected and then cultured with the TRP-2180-188 peptide and interleukin-2 （IL-2） for 5 days, with lymphocytes untreated with the TRP-2180-188 peptide or IL-2 serving as the control group. Mitomycin-treated B16F10 murine melanoma cells and TRP-2180-188 peptide-stimulated lymphocytes were co-cultured with the presence of supernatants of 293T cells that had been cultured for 48 hours（blank control group）, TIM-3-containing supernatants （TIM-3 group） and Ig-tail-containing supernatants （negative control group） separately. After 24 and 48 hours of co-culture, cell counting kit-8（CCK-8） assay was performed to estimate the proliferative activity of lymphocytes, enzyme-linked immunosorbent assay （ELISA） to determine the supernatant levels of interferon （INF）-γ and tumor necrosis factor （TNF）-α, flow cytometry to determine the percentage of CD8+ T cells in the co-culture system. Results Enzyme digestion and sequence analysis showed that the TIM-3 gene was successfully inserted into the eukaryotic expression plasmid. After 48-hour culture, TIM-3 and Ig-tail expressions were detected in the supernatants of 293T cells transfected with the recombinant plasmid and empty plasmid respectively. As CCK-8 assay showed, the proliferative activity of lymphocytes was significantly lower in the TIM-3 group than in the blank control group and negative control group after 24- and 48-hour culture （78.06% ± 6.37% vs. 100.00% ± 10.42% and 108.70% ± 9.90% at 24 hours, 42.93% ± 5.93% vs. 100.00% ± 6.24% and 168.00% ± 2.98% at 48 hours, all P < 0.05）, so was the ratio of cellular proliferative activity at 48 hours to that at 24 hours （all P < 0.05）. Compared with the blank control group and negative control group, the TIM-3 group showed significantly decreased supernatant levels of IFN-γ and TNF-α after 24-hour （IFN-γ: 192.96 ± 5.05 ng/L vs. 216.44 ± 7.85 ng/L and 223.67 ± 7.79 ng/L, both P < 0.05; TNF-α: 58.43 ± 0.26 ng/L vs. 26.43 ± 0.01 ng/L and 86.85 ± 1.12 ng/L, both P < 0.05） and 48-hour culture （IFN-γ: 54.95 ± 0.57 ng/L vs. 230.06 ± 4.23 ng/L and 167.24 ± 3.33 ng/L, both P < 0.05; TNF-α: 30.23 ± 0.26 ng/L vs. 26.84 ± 0.20 ng/L and 45.34 ± 0.22 ng/L, both P < 0.05）. In addition, the median percentage of CD8+ T cells was significantly increased in the TIM-3 group compared with the blank control group and negative control group after 24- and 48-hour culture （3.30% vs. 0.421% and 2.22% at 24 hours, 4.06% vs. 0.577% and 0.691% at 48 hours, all P < 0.05）. Conclusion TIM-3 in vitro can suppress the proliferative activity of and secretion of IFN-γ and TNF-α by lymphocytes, but increase the percentage of CD8+ T cells in the co-culture system of TRP-2180-188 peptide-stimulated lymphocytes and B16F10 cells.