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目的研究HVEM、CD160在不同CD4+T细胞亚群,不同功能状态下的表达以及HVEM-CD160通路对CD4+CD25+调节性T细胞(regulatory T cell,Treg)免疫调节作用的影响。方法采用流式细胞分析术分别检测3例不同个体CD4+CD25+Tregs及CD4+CD25-T细胞在1 000 IU/ml人重组IL-2、与细胞数量1∶1的抗CD3/CD28 mAb包被磁珠的刺激下第0、3、5天HVEM或CD160的表达情况。采用1.25、2.5、5μg/ml HSV1gD蛋白处理4例不同个体Treg,培养5 d后,将处理的Treg与Teff在体外以1∶1混合并加入300 IU/ml IL-2及等量抗CD3/CD28 mAb包被磁珠,行混合淋巴细胞共培养,于培养结束前18 h加入3H-TdR,共培养7 d后检测各组细胞CPM增殖情况。5μg/ml gD蛋白处理5例不同个体Treg后将其细胞浓度调整为5×106并接种于6孔培养板内,加入500 IU/ml IL-2共培养7 d,行RT-PCR(Realtime PCR)分析Treg Foxp3基因表达。结果 HVEM在初始CD4+CD25+Tregs表面呈中度表达,平均表达率为42%,且随着Treg的活化其表达HVEM上调;CD160在初始CD4+CD25-T细胞表面呈低度表达,平均表达率仅7%,但伴随其活化,表达CD160有所上调。采用不同浓度HSV1gD蛋白处理Treg后2.5μg/ml与5μg/ml浓度组抑制CD4+CD25-T细胞增殖作用与空白对照组(276±35)相比明显下降,CPM值分别为(2 014±202)、(6 535±531),差异具有统计学意义(P<0.05),且具有浓度依赖性;RT-PCR分析发现Treg经5μg/ml gD蛋白处理后Foxp3基因表达率下降,仅为空白对照组的48%,差异具有统计学意义(P<0.01)。结论机体接受免疫刺激后Treg通过上调HVEM表达与CD4+CD25-效应性T细胞(ef-fective Tcell,Teff)表面上调表达的受体CD160交联从而上调Treg Foxp3基因的表达,最终介导了Treg对Teff活化、增殖的抑制作用,HVEM-CD160通路对Treg免疫调节作用具有重要意义。
Objective To investigate the effect of HVEM-CD160 on the immunoregulation of CD4 + CD25 + regulatory T cell (Treg) in different CD4 + T cell subsets and in different functional states. Methods Flow cytometry was used to detect the percentage of CD4 + CD25 + Tregs and CD4 + CD25-T cells in 1000 IU / ml human recombinant IL-2 from 3 different individuals and 1: 1 anti-CD3 / CD28 mAb The expression of HVEM or CD160 on days 0, 3 and 5 stimulated by magnetic beads. Four Tregs from different individuals were treated with 1.25, 2.5 and 5μg / ml HSV1gD protein. After 5 days of culture, treated Tregs and Teffs were mixed in vitro by 1: 1 and added with 300 IU / ml IL-2 and an equal amount of anti-CD3 / CD28 mAb was coated with magnetic beads and mixed lymphocytes were co-cultured. 3H-TdR was added 18 h before the end of culture. After 7 days of co-culture, the proliferation of CPM cells in each group was detected. After 5 μg / ml gD protein was treated with 5 different Tregs, the cell concentration was adjusted to 5 × 106. The cells were inoculated into 6-well plates and cultured with 500 IU / ml IL-2 for 7 days. Real-time PCR ) Analysis of Treg Foxp3 gene expression. Results HVEM was moderately expressed on the surface of primary CD4 + CD25 + Tregs with an average expression rate of 42%. The expression of HVEM was up-regulated with the activation of Treg. The expression of CD160 was low on the surface of primary CD4 + CD25-T cells Only 7% of the rate, but with its activation, the expression of CD160 increased. Compared with blank control group (276 ± 35), the inhibitory effect of 2.5μg / ml and 5μg / ml concentrations of HSV1gD protein treated Treg on the proliferation of CD4 + CD25-T cells was significantly lower (2 014 ± 202 ) (6 535 ± 531), the difference was statistically significant (P <0.05), and in a concentration-dependent manner; RT-PCR analysis showed that the Foxp3 gene expression rate of Treg treated with 5μg / ml gD protein decreased only blank control 48% of the group, the difference was statistically significant (P <0.01). Conclusion Tregs can up-regulate Foxp3 expression by up-regulating the expression of HVEM and up-regulating the expression of CD160 on the surface of CD4 + CD25- effector T cells (Teff) The inhibition of Teff activation and proliferation, HVEM-CD160 pathway on Treg immune regulation has important significance.