阻断ICOS/B7h信号的供体特异性输血对移植受体CD4＋CD25＋调节性T细胞的影响

Influence of donor specific transfusion with impaired ICOS/B7h allorecognition on CD4 ＋ CD25＋regulatory T cells of cardiac allograft recipient

目的 观察阻断ICOS/B7h信号的供体特异性输血（DST）对异基因小鼠心脏移植术后体内CD4＋CD25＋调节性T细胞（Treg）的影响.方法 按陈氏方法建立小鼠颈部异位心脏移植模型,实验分3组,异基因组及同基因组：供心分别来源于BALB/C和C57BL/6小鼠,受体均为C57BL/6小鼠,未予治疗.治疗组：移植当天给予受体鼠（C57BL/6）尾静脉注射5×106 ICOS-Fc靶定的供体（BALB/C）脾B淋巴细胞,d0～6连续给予受体鼠尾静脉注射ICOS-Fc 200 μg/d.术后统计各组移植物的存活时间,通过流式细胞术检测受体鼠外周血中CD4＋CD25＋Treg的亚群比例,利用逆转录-聚合酶链反应（RT-PCR）检测移植物中FOXP3的mRNA表达,在混合淋巴细胞反应中检测CD4＋CD25＋Treg对CD4＋CD25-效应T细胞（Teff）的增殖抑制效率.结果 与异基因组比较,治疗组心脏移植物存活时间明显延长[（84.38±29.14）d比（7.00±0.76）d,P＜0.01].各组中,治疗组受体外周血中CD4＋CD25＋Treg亚群比例显著上调[（15.60±5.69）%,P＜0.01].与其他两组比较,治疗组心脏移植物中FOXP3 mRNA表达显著上调.以正常鼠为对照,耐受鼠脾脏中获取的CD4＋CD25＋Treg能够更高效地抑制CD4＋CD25-Teff在混合淋巴细胞培养中的增殖效应.结论 通过阻断ICOS/B7h信号的DST可以诱导异基因小鼠心脏移植耐受,CD4＋CD25＋Treg在耐受的形成与维持中均起着重要作用.

Objective To investigate the possibility of CD4＋CD25＋ regulatory T cells （Treg） enhancement, which induced by donor specific transfusion combined with blockade of ICOS/B7h costimulation following an allogenic murine heterotopic cardiac transplantation. Methods Recipient mice were assigned to 3 groups. In allogenic group, donor hearts were harvested from BALB/C mice, and allografts were heterotopically transplanted in the neck of C57BL/6 using Chen＇ s technique, without treatment. In isogeneic group, donor hearts were harvested from C57BL/6 mice, and isografts were heterotopically transplanted in the neck of C57BL/6, without treatment. In treatment group, a combination of 5 × 106 ICOS-Fcrecipient C57BL/6 mice following a heterotopic cardiac allograft transplantation. Graft function was assessed daily by palpation, with rejection defined as the absence of detectable beating, then survival of grafts was recorded. CD4 ＋ CD25 ＋ Treg subsets in the peripheral blood of allograft recipients were analyzed by flow cytometry. The expression of FOXP3 mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. To assess the suppressive activity of CD4 ＋ CD25 ＋ Treg from tolerant C57BL/6 mice on CD4 ＋ CD25- Teff from naive BALB/C mice, suppression allogeneic MLR assays were performed in vitro.Results In comparison with allogenic group, the survival of cardiac grafts could be prolonged by the comICOS-Fc on the day 0-6 [（84.38 ± 29. 14） days vs （7.00 ± 0. 76） days, P 〈 0. 01]. Among 3 groups, a considerable proportion of CD4 ＋ CD25 ＋ Treg could be observed in treatment group [（15.60 ± 5.69 ）% ,P 〈0. 01], meanwhile, high expression of FOXP3 mRNA was also detected within allografts in treatment group.Furthermore, in contrast to naive C57BL/6 mice, CD4＋ CD25 ＋ Treg harvested from tolerant C57BL/6 mice showed more potent suppressive effect on the proliferation of CD4 ＋ CD25 - Teff. Conclusion Allograft tolerance can be induced by donor specific transfusion with impaired ICOS/B7h allorecognition. CD4 ＋ CD25 ＋ Treg played a critical role in the establishment and the maintenance of this allograft tolerance.