人膜型LIGHT分子对Mo-DC成熟和T细胞激活的调节作用

利用我们建立的表达人膜型LIGHT分子的基因转染细胞(L929/LIGHT)探讨LIGHT/HVEM信号体外对Mo-DC诱导分化的影响,并进一步研究其对T细胞活化和抗凋亡的共刺激作用。从健康人外周血中分离的单核细胞经GM-CSF联合IL-4诱导形成Mo-DC,流式细胞术分析Mo-DC诱导过程中HVEM和LIGHT的表达;基因转染细胞L929/mock、L929/LIGHT、L929/CD40L或L929/LIGHT联合L929/CD40L,分别经丝裂霉素处理后,与GM-CSF联合IL-4诱导的Mo-DC共育,流式细胞术检测Mo-DC细胞表面成熟标志CD83和CD86的表达,利用FITC-Dextran分析Mo-DC对抗原的摄取能力;L929/LIGHT或L929/mock经丝裂霉素处理后,与抗人CD3单抗激发的T细胞共育,流式细胞术分析CD4+和CD8+T细胞表面活化标志CD25的表达,Annexin V和PI双标记分析T细胞的凋亡率。结果表明,高表达HVEM的单核细胞在诱导形成成熟Mo-D(iDC)的过程中下调了HVEM的表达,成熟Mo-DC(mDC)又上调表达HVEM,而LIGHT在Mo-DC分化过程中呈短暂的诱导性表达;基因转染细胞L929/LIGHT及其联合L929/CD40L能上调Mo-DC表面共刺激分子CD83和CD86的表达,并下调Mo-DC对FITC-Dextran的摄取能力;L929/LIGHT细胞能上调CD4+、CD8+T细胞CD25的表达,并增强T细胞抗凋亡能力。因此,基因转染细胞L929/LIGHT表面表达的人膜型LIGHT分子介导的LIGHT/HVEM共刺激信号对Mo-DC的诱导成熟和T细胞活化及抗凋亡能力具有促进作用。

Effect of human membrane-bound LIGHT expressed by the transfectant L929/LIGHT on the co-stimulation of monocyte-derived dendritic cells and T cells

Utilizing transfectant L929/LIGHT-expressing human membrane-bound LIGHT molecule,we investigated the influence of LIGHT/HVEM signal on induction of Mo-DC differentiation and maturation in vitro,and further studied its costimulatory effect on T cell activation and resistance to apoptosis.Monocytes isolated from human peripheral blood mononuclear cells(PBMC) were induced by GM-CSF and IL-4 to form Mo-DC,in which process the expressions of HVEM and LIGHT were analyzed by flow cytometry.The transfectant L929/mock,L929/LIGHT,L929/CD40L or L929/LIGHT combined with L929/CD40L were respectively treated with mitomycin,and were respectively co-cultured with Mo-DC.Then the expression of CD83 and CD86 on the surface of Mo-DC membrane were detected by flow cytometry,the capability of Mo-DC to uptake antigen was analyzed employing FITC-Dextran.The transfectant L929/LIGHT or L929/mock treated with mitomycin was co-cultured with T cells stimulated by anti-human CD3 mAb.Subsequently,the activation marker of CD25 on CD4+ as well as CD8+T cells surface was analyzed by flow cytometry and the apoptosis rate of T cells was assayed by Annexin V and PI double markers.The results showed that HVEM expression was down-regulated when monocytes were induced to differntiate into immature Mo-DC(iDC),then up-regulated on mature Mo-DC,nevertheless,LIGHT temporarily presented up-regulation during the procedure of Mo-DC differentiation.The transfectants of L929/LIGHT and L929/LIGHT combined with L929/CD40L were capable of up-regulating the co-stimulatory molecules CD83 and CD86 on Mo-DC and down-regulating the activity of Mo-DC to uptake FITC-Dextran.In addition,the transfectant of L929/LIGHT could up-regulate the expression of CD25 on CD4+ and CD8+ T cells and enhance T cell resistance to apoptosis.In summary,the LIGHT/HVEM costimulatory signal mediated by transfectant L929/LIGHT may potentially promote the maturation of Mo-DC and T cell activation as well as T cell resistance to apoptosis.