HLA-G^＋调节性T细胞在肾移植受者外周血中的比例及其功能研究

Percentage and regulatory function of HLA-G^+ T cells in peripheral blood of kidney transplantation recipients

目的确定人类白细胞抗原-G(HLA-G)区别于CD4+CD25+FoxP3+调节性T细胞的细胞表型特征,观察HLA-G+T细胞在混合淋巴细胞培养中的免疫调节作用及其在移植免疫调节中的活性和意义。方法采用流式细胞术检测肾移植受者外周血CD4+HLA-G+、CD8+HLA-G+T淋巴细胞的含量,分选HLA-G+T淋巴细胞,分析细胞表型,采用RT-PCR检测调节性T细胞的特异性标志FoxP3在HLA-G+T淋巴细胞的表达情况,以淋巴细胞分离液分离的PBMC、CD4+CD25high、CD4+CD25-细胞作为对照。取5对活体肾移植供受者的淋巴细胞进行混合培养,分别加入流式细胞术分选纯化后的HLA-G+、HLA-G-T淋巴细胞,对照组只加入供、受者淋巴细胞,MTT法观察细胞增殖和抑制情况。结果肾移植受者外周血CD4+HLA-G+、CD8+HLA-G+T淋巴细胞表达率分别为1.95%±0.34%、3.13%±0.56%。流式细胞术分选HLA-G+T淋巴细胞后纯度可达55.0%～75.1%。RT-PCR结果证明上述细胞CD25、FoxP3均为阴性表达。与HLA-G-组、对照组比较,混合淋巴培养3d后HLA-G+组细胞增殖率明显受到抑制(P<0.05)。结论在肾移植受者外周血中存在CD4+HLA-G+、CD8+HLA-G+T淋巴细胞,这类细胞不同于CD4+CD25+FoxP3+调节性T细胞,它们不表达CD25和FoxP3而表达免疫耐受分子HLA-G,在混合淋巴培养体系中,能显著抑制反应性细胞增殖,具有免疫调节功能。

Objective To detect the expression and percentage of human leukocyte antigen-G （HLA-G） on T cells in peripheral blood of kidney transplant recipients,distinguish the distinction of HLA-G from CD4＋CD25＋FoxP3＋ regulation T-cells by cell phenotype analysis,and observe the immune regulatory role of HLA-G＋ T cells in mixed lymphocyte culture system,and to explore the activity and significance of HLA-G in the transplantation immunoregulation. Method Contents of CD4＋HLA-G＋ and HLA-G＋ T lymphocytes in peripheral blood were detected by flow cytometry,the HLA-G＋ T lymphocytes was then sorted out. The freshly isolated HLA-G＋ T cells were cultured in several mix lymphocyte reaction （MLR） systems. The expression of FoxP3 in HLA-G＋ T lymphocytes was analyzed by RT-PCR,and PBMC,CD4＋CD25high and CD4＋CD25-cells were used as control. The lymphocytes taken from the peripheral blood of five pairs of kidney transplant donors-recipients were mixedly cultured,the sorted HLA-G＋ and HLA-G-T lymphocytes were added into the mixed lymphocyte culture respectively,and only the donor-recipient lymphocytes were added into the control group. MTT was used to detect the cell proliferation. Results The expressions of CD4＋HLA-G＋ and CD8＋HLA-G＋ T lymphocytes in the peripheral blood of recipients were 1.95%±0.34% and 3.13±0.56%,respectively. The purity of HLA-G＋ T lymphocytes sorted by flow cytometry was 55%-75.1%,and RT-PCR revealed that no expression of CD25 or FoxP3 was found in those cells. The cell proliferation was remarkably inhibited in HLA-G＋ group 3 days after mixed culturing compared to that in HLA-G-group and control group （P0.05）. Conclusions CD4＋HLA-G＋ and CD8＋HLA-G＋ T lymphocytes are presented in the peripheral blood of renal transplant recipients,and different from the CD4＋CD25＋FoxP3＋ regulator T cells. In CD4＋HLA-G＋ and CD8＋HLA-G＋ T lymphocytes,there is no expression of CD25 and FoxP3,but the immune tolerance molecule HLA-G. The proliferation of reactive cells may be inhibited remarkably in MLR systems,implying that these cells have immunoregulatory function.