Objective To investigate the characteristics and mechanisms of the immune tolerance of CD4 + CD25-T cells induced by recipient-derived immature dendritic cells (imDC) transfected with dominant negative form of IκB kinases 2 (IKK2dn) and loaded with donor antigens.Methods ImDC were obtained from recipient rats' (Lewis) bone marrow,transfected with IKK2dn at 200 multiplicities of infection (MOI) (200 MOI) for 2 days,loaded with donor antigens for 48 h,and cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR) for 72 h.Thereafter,the CD4 + CD25-T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes for another 72 h.The T lymphocyte proliferation in vitro was measured by MTT and the expression of serum interleukin (IL)-2,IL-10,transforming growth factor-β (TGF-β) and interferon (IFN)-γ was detected by using ELISA.Results Western blotting revealed high and stable expression of IKK2dn gene in imDC transfected with IKK2dn gene.In contrast to the untransfected imDC group (72.20 ±3.30)％ and the recipient Adv0 transfected imDC group (63.70 ± 1.80)％,the CD4 +T cells induced by recipient imDC transfected with IKK2dn contained a very low percentage of CD25 (18.50 ± 1.40) ％ (P ＜ 0.05).As compared with the CD4 +T cells induced by Adv0-DC (0.419 ± 0.014),the CD4 + CD25-T cells induced by Adv-IKK2dn-DC could inhibit recipient T cells proliferation (0.044 ± 0.006) (P ＜ 0.05).The CD4 + CD25-T cells were able to completely suppress the recipient T cells proliferation at high ratio (0.044 ± 0.006),but at low ratio the suppressive effect was no longer evident (0.106 ±0.006) (P ＜0.05).There was a stronger secretion of IL-10 and TGF-β (P ＜ 0.05) and a weaker secretion of IL-2 and IFN-γ(P ＜ 0.05) in the supematants in MLR.Conclusion Recipient-derived imDC transfected with IKK2dn and loaded with donor antigen could induce Lewis rat T lymphocytes into CD4 + CD25-T cells.This kind of CD4 + CD25-T cells could inhibit T-cell response and induce immune tolerance.%目的 探讨体外转染抑制性蛋白IκB激酶2的显性负性突变体(IKK2dn)基因的受者大鼠未成熟树突状细胞(imDC)诱导产生的CD4+ CD25-T细胞致耐受作用的特性及机制.方法 将含IKK2dn基因的腺病毒载体按1:200的感染复数(200 MOI)体外转染受者Lewis大鼠骨髓源性imDC,2d后负载供者BN大鼠抗原,48 h后与受者大鼠T细胞初次混合淋巴细胞反应(MLR),72 h后免疫磁珠法分离筛选混合淋巴细胞反应(MLR)中诱导产生的CD4+ CD25-T细胞,将获得的CD4+ CD25-T细胞与受者大鼠T细胞再次MLR 72h,检测其对T细胞增殖反应的影响,酶联免疫吸附试验(ELISA)测定MLR上清中白细胞介素(IL)-2、IL-10、转化生长因子(TGF)-β和干扰素(IFN)-γ的水平.结果 Western blot结果显示,转染IKK2dn基因的imDC稳定、高效表达IKK2dn.转染IKK2dn受者imDC诱导产生的CD4+T细胞低表达CD25(18.50±1.40)％,与未转染组(72.20±3.30)％及转染空载体组(63.70±1.80)％比较,差异有统计学意义(P＜0.05).转染IKK2dn诱导产生的CD4+CD25-T细胞能抑制异种抗原刺激引起的受者T细胞的增殖反应(0.044±0.006),与转染空载体所诱导产生的CD4+T细胞比较(0.419±0.014),差异有统计学意义(P＜0.05).MLR中高比例的CD4+CD25-T细胞能够明显抑制受者T细胞的增殖反应(0.044±0.006),与低比例组比较(0.106±0.006),差异有统计学意义(P＜0.05),且CD4+ CD25-T细胞介导的MLR上清液中IL-10、TGF-β水平明显升高(P＜0.05),IL-2、IFN-γ水平明显降低(P＜0.05).结论 转染IKK2dn基因并负载供者抗原的受者imDC可以诱导受者T细胞产生CD4+ CD25-T细胞,此种CD4+ CD25-T细胞能够引起受者T细胞针对供者抗原的特异性低反应,具有诱导免疫耐受的作用.
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