活化的Treg细胞对NKT细胞迁移的调节作用

Regulatory effect of activated Treg cells on migration of NKT cells in mice

目的探讨Treg细胞活化后对NKT细胞迁移的调控作用。方法分离纯化小鼠脾脏Treg细胞及NKT细胞后,通过病毒感染的方式制备Foxp3修饰的Treg细胞;验证Treg细胞的活化情况。体外共培养活化的Treg细胞和NKT细胞,流式细胞仪检测NKT的数量;RT-PCR及ELISA分析NKT细胞胞内因子的分泌水平(IL-4、IFN-γ)。制备卵清白蛋白(OVA)致敏哮喘小鼠模型,体内观察Foxp3修饰的Treg细胞对NKT细胞体内迁移的影响。结果经Foxp3修饰后的Treg细胞(Le-Foxp3组)中Foxp3的表达明显增加;CFSE标记法检测显示Le-Foxp3组细胞可显著抑制自体CD4+CD25-T效应T细胞的增殖;同时ELISA检测结果显示相较于空载体转染组(Le-scr组),Le-Foxp3组细胞上清液中IL-10及TGF-β的水平显著升高。经transwell培养板体外共培养Treg细胞和NKT细胞后,流式细胞仪检测下室中NKT细胞的数目结果显示Le-Foxp3组为(221.15±79.36),Le-scr组为(649.38±153.97);同时IL-4及IFN-γ的水平显著下降。在体内实验结果显示,相较于OVA组,OVA+Le-Foxp3组小鼠肺组织中NKT的数量显著降低,而外周血及脾脏中NKT细胞的数量显著升高。结论 Foxp3修饰可活化体外培养的Treg细胞,此外活化的Treg细胞可抑制NKT细胞的活化及体外迁移并影响OVA哮喘模型小鼠NKT细胞的体内分布。

Objective To investigate the regulatory effect of activated Treg cells on the migration of NKT cells in mice. Methods Treg cells and NKT cells were isolated and purified,then Treg cells were modified by the lentivirus carrying Foxp3 and the activation of Treg cells were verified. The activated Treg cells and NKT cells were co-cultured in vitro and flow cytometry was used to detect the number of NKT cells. The secretion of intracellular factors(IL-4 and IFN-γ) in the NKT cells was measured with reverse transcription polymerase chain reaction( RT-PCR) and enzyme-labeled immunosorbent assay( ELISA). Ovalbumin( OVA)-induced asthma mouse models were established and used to observe the effect of Treg cells modified by Foxp3 on the migration of NKT cells in vivo. Results The expression of Foxp3 was significantly up-regulated in the Treg cells modified by Foxp3( Le-Foxp3 group). CFSE assay showed that the Treg cells carrying Foxp3 significantly inhibited the proliferation of autologous CD4+CD25-T cells( Teff cells),and ELISA showed that the levels of IL-10 and TGF-β were increased markedly in the Le-Foxp3 group compared with the control group( the cells were infected with the lentivirus carrying scrambled sequence,Le-scr group). After co-culture of the Treg cells and NKT cells in a transwell culture plate,the flow cytometer showed that the NKT cells in the lower chamber were( 221.15±79.36) in the Le-Foxp3 group and( 649.38±153.97) in the Le-scr group. Meanwhile,the levels of IL-4 and IFN-γdecreased remarkably. The results from OVA-induced asthma model showed that the forced expression of Foxp3 remarkably increased the NKT cells in the spleen and the peripheral blood,while decreased the NKT cells in the lungs. Conclusion Infection of lentivirus carrying Foxp3 can activate Treg cells. The activated Treg cells can inhibit the activation and migration of NKT cells in vitro and affect the distribution of NKT cells in OVA-induced asthma mouse model.