Effect of T-cell immunoglobulin and mucin domain-3 on TRP-2180-188 peptide-stimulated murine spleen lymphocytes co-cultured with B16F10 murine melanoma cells / 中华皮肤科杂志

ABSTRACT

Objective To evaluate the effect ofT-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-21180-188 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-γ192.96 γ 5.05 ng/L vs.216.44 ± 7.85 ng/L and 223.67 ±7.79 ng/L,both P＜ 0.05;TNF-α58.43 ± 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-γ54.95 ± 0.57 ng/L vs.230.06 ± 4.23 ng/L and 167.24 ± 3.33 ng/L,both P ＜ 0.05;TNF-α30.23 ±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-γand 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.