转hCTLA4Ig树突状细胞诱导T细胞免疫耐受的实验研究

An experimental study on the role of CTLA4Ig-gene transfected DCs in the induction of immune tolerance

目的 通过逆转录病毒载体将人CTLA4Ig转染DCs ,探讨转人CTLA4Ig(hCTLA4Ig)树突状细胞 (DCsRev)诱导T细胞免疫耐受的可能性。方法 通过重组逆转录病毒将目的基因hCTLA4Ig转染到大鼠骨髓来源的DCs中 ,通过流式细胞检测目的基因hCTLA4Ig表达及DCs表面分子的改变 ;通过混合淋巴细胞反应 (MLR)检测DCsRev抑制T细胞免疫反应的能力。 结果 重组逆转录病毒转染DCs的最大效率为 91 2 5 % ;在功能上 ,DCsRev不但丧失了刺激MLR的能力 ,并且能够强烈抑制MLR中反应T细胞的增殖 ,而且抑制率与加入DCsRev的数量和DCsRev预处理反应T细胞的时间长短有关。具体来说 ,DCsRev数量在 10 3 ～ 10 4之间时 ,抑制率与剂量呈正相关 ,最高为 71 96%。而当DCsRev数量达到 5× 10 4抑制率下降为 5 9 2 %。在 12～ 48h之间 ,随着预处理时间的延长 ,抑制率却不断下降 ,预处理 12h抑制率最高 ,为 99 6%。但不做预处理 ,在反应开始时同时加入DCsRev ,则抑制率明显降低 ,仅为 5 9 2 %。对腹腔注射DCsRev大鼠脾T淋巴细胞体外分析表明 ,DCsRev也能在动物体内诱导耐受 ,但这种免疫耐受状态不能维持终身。结论 通过逆转录病毒载体将人CTLA4Ig转染DCs,不但DCs表面CD86分子被CTLA4Ig有效的封闭 。

Objective To investigate the possibility of DCs transfected with human CTLA4Ig(hCTLA4Ig) cDNA by retrovirus vector to induce antigen specific hyporesponsiveness.Methods The modified DCs(CTLA4Ig DCs) was prepared by transferring the DCs from cultured rat BM cells with the constructed retro virus hCTLA4Ig vector.The hCTLA4Ig expression and certain cell surface molecules were detected on the prepared DCs by FACS.The influence of the modified DCs on mixed lymphocyte reaction(MLR) intensity was determined by T cell proliferation.Results The efficiency of DCs transfection by retrovirus was 91 25%.As compared with control, DCsRev could significantly and antigen specifically inhibit MLR in vitro and this immune suppression was dose dependent and time dependent.That is, the number of DCRev from 10 3 to 10 4 could reach the maximal inhibition by 71 96%.However, if DCsRev's number was increased furthermore,i.e.,5×10 4, the inhibiton was interestingly reduced to 59 2%.On the other hand, the inhibition capacity of DCsRev increased from 48 hour to 12 hour prior to adding stimulating cells and the maximal inhibition was 99 6% at 12 hour, and interestingly only 59 2% at 0 hour (i.e., DCsRev and stimulating cells were added simultaneously).Analysis of T cell proliferation revealed that donor specific inhibition could be induced by DCsRev in an ex vivo model.But this kind of inhibition was not lifetime.Conclusion The B7 molecules on DCsRev surface could be effectively blocked by autocrined hCTLA4Ig.,which might be responsible for the antigen specific suppression induced by DCsRev.