Foxp3转染小鼠CD4+CD25-T细胞抑制NK细胞活性

目的： 通过逆转录病毒载体转染Foxp3基因到小鼠CD4+CD25-T细胞，以研究体外诱导获得的调节性T细胞对NK细胞免疫活性的调节作用及其机制。方法： 携带Foxp3基因的逆转录病毒转染初始CD4+CD25-T细胞，以获得持续性高表达Foxp3的CD4+ T细胞模型。CD4+Foxp3+ T细胞与NK细胞共培养后，用［51Cr］标记的YAC-1细胞检测NK细胞的杀伤毒性。在TGF-β阻断实验中，通过Transwell共培养实验以及向细胞共培养体系加入抗TGF-β抗体，并检测NK细胞杀伤毒性。结果：逆转录病毒转染初始CD4+CD25-T细胞，成功建立了表达Foxp3的CD4+T细胞模型，转染后1周Foxp3阳性表达的T细胞比例为38.0%。CD4+Foxp3+ T细胞在与NK细胞共培养的24 h和48 h后，对NK细胞的细胞毒性杀伤效应的抑制率分别为42.9%和22.7%。在CD4+Foxp3+ T细胞与NK细胞的共培养体系中加入抗TGF-β抗体后，其抑制率分别由原来的42.9%(24 h)、22.7%(48 h)变为3.2%(24 h)、2.1%(48 h)。在Transwell共培养实验中与NK细胞直接接触的Foxp3+CD4+T细胞可以诱导NK细胞免疫抑制，而没有直接接触的Foxp3+CD4+T细胞则不能抑制NK细胞的杀伤作用。结论：强制性表达Foxp3的CD4+CD25-T细胞可以在体外发挥免疫抑制作用，可以抑制NK细胞的细胞毒性杀伤作用。转染 Foxp3的CD4+CD25-T细胞对NK细胞发挥作用依赖于细胞之间的直接接触，与转染后T细胞表面表达TGF-β有关。

Foxp3-transduced polyclonal regulatory T cells suppress cytotoxicity of NK cells

AIM: To investigate the effect and mechanism of Foxp3-transduced CD4+CD25-T cells on the cytotoxicity of NK cells. METHODS: Retroviral Foxp3 gene transfection was applied to nave CD4+CD25-T cells. Fresh transduced CD4+Foxp3+ T cells were co-cultured with NK cells. ［51Cr］ labeled YAC-1 cells were used to detect NK cells cytotoxicity. The anti-TGF-β antibody was added into the co-culture system to detect the TGF-β blocking effect. Also the transwell co-culture system was used to investigate the regulatory effect of Treg cells on NK cells. RESULTS: One week after transduction, 38.0% of Foxp3-transduced T cells showed GFP expression by flow cytometry. Foxp3-transduced CD4+CD25-T cells suppressed function of NK cells. The inhibition rates of Foxp3 transduced CD4+CD25- T cells were 42.9% at 24 h and 22.7% at 48 h. When anti-TGF-β antibody was added to the co-culture system, the inhibition rate of CD4+Foxp3+ T cells was 3.2% and 2.1%, respectively. CONCLUSION: CD4+Foxp3+ T cells significantly inhibit the cytolytic function of NK cells. TGF-β plays different roles on this action in different inhibition systems. The inhibitory effect of Treg cells on NK cells is cell-to-cell contact dependent and associates with TGF-β expression.