结肠癌细胞上清液对CD4^+FOXP3^+调节性T淋巴细胞形成影响

The role of colon cancer cells condition medium in the conversion of CD4^+CD25^- T cells into CD4^+FOXP3^+ T cells

目的观察结肠癌细胞上清液是否能促进正常CD4+T淋巴细胞转换成调节性T淋巴细胞,并且探讨TGFβ1在促细胞转换中的重要作用。方法应用流式细胞仪染色CD4+FOXP3+,ELISA检测结肠癌上清液中TGFβ1浓度。结果分选的CD4+CD25-细胞纯度高达96.58%±0.59%,这群细胞中表达CD4+FOXP3+较低,为0.34%±0.03%。健康正常人CD4+FOXP3-细胞分别与人结肠癌细胞colo320HSR及DLD-1上清液共培养后,CD4+FOXP3+细胞表达增高(colo320HSR组为4.78%±0.41%,DLD-1组为4.48%±0.29%);这两组与RPMI 1640培养液阴性对照组CD4+FOXP3+细胞2.54%±0.41%相比差异有统计学意义(P<0.05)。ELISA法检测colo320HSR细胞上清液TGFβ1浓度为(1 228.80±10.65)ng/mL,DLD-1细胞上清液TGFβ1浓度(624.50±12.26)ng/mL,而RPMI 1640无血清培养液中未检测到TGFβ1。在与结肠癌细胞上清液培养同时给与TGFβ抗体后,CD4+FOXP3+表达较未加入抗体组明显降低(colo320HSR组2.20%±0.09%,P=0.011;DLD-1组1.67%±0.34%,P=0.033);而RPMI1640阴性对照组CD4+FOXP3+细胞表达下降不显著(1.80%±0.58%,P=0.159)。结论人结肠癌细胞上清液可以促进正常CD4+T淋巴细胞(CD4+FOXP3-细胞)转换为调节性T淋巴细胞(CD4+FOXP3+),TGFβ1在肿瘤微环境中调节T淋巴细胞形成可能有一定的作用。识别人调节性T淋巴细胞(CD4+FOXP3+)的起源将为肿瘤治疗提供重要信息。

Objective To observe the conversion of naive CD4＋FOXP3-T cells into CD4＋FOXP3＋ T cells in vitro induced by colon cancer cells condition medium,and investigate the role of TGFβ in the conversion process.Methods The expression of CD4＋FOXP3＋ cells was detected by flow cytometry.The concentration of TGFβ1 in the colon cancer cells condition medium was determined by ELISA.Results The purity of CD4＋CD25-cells separated by MACS reached 96.58%±0.59%.The expression of CD4＋FOXP3＋ cells was 0.34%±0.03%.The amounts of CD4＋ FOXP3＋ cells significantly increased in the co-culture system with colon cancer cells condition medium（colo320HSR 4.78%±0.41%,DLD-1 4.48%±0.29%）,compared with that of cells cultured with RPMI 1640 medium alone（2.54%±0.41%,P0.05）.Secreted TGFβ1 protein was detected in colon cancer cells condition medium [colo320HSR（1228.80±10.65）ng/mL,DLD-1（624.50±12.26）ng/mL].However,no TGFβ was detected in RPMI medium alone.Interestingly,when TGFβ antibody was added in colon cancer cells condition medium,the conversion of CD4＋FOXP3＋ significantly decreased（colo320HSR 2.20%±0.09%,P=0.011;DLD-1 1.67%±0.34%,P=0.033）,whereas there was no change in cells cultured with RPMI 1640 medium alone（1.80%±0.58%,P=0.159）.Conclusion The results demonstrated that tumor cells condition medium can directly convert normal CD4＋T cells into CD4＋FOXP3＋ Treg cells,and TGFβ1 may play a role in tumor cells condition medium mediating cell conversion.Thus,the findings of this study provide useful information for the potential immunotherapy of human colon cancer.