CD59分子在人T细胞抗原特异性活化中的作用研究
发布时间：2019-01-08 10:02
【摘要】：目的运用RNA干扰技术使正常人CD4+或CD8+T细胞的CD59基因表达发生沉默,然后观察其对负载肿瘤抗原的DCs或抗CD3/CD28/CD59(或IgG2a)包被珠的刺激反应,探讨CD59分子在T细胞抗原特异性活化中的作用及相关机制。 方法用含IL-4和GM-CSF的DC无血清培养基诱导培养健康供者的贴壁PBMCs,负载肿瘤抗原后,加入TNF-a促进DCs成熟。将CD59-siRNA表达质粒siCD59和空质粒pSRNG分别转染PA317细胞,包装出逆转录病毒,用于感染悬浮的PBMCs。G418筛选病毒感染细胞后,利用免疫磁珠纯化分选出CD4+T细胞和CD8+T细胞。利用FACS检测、补体溶解试验和PMA刺激CD4+T细胞活化试验评估CD59-siRNA对CD59、CD3、CD28表达以及T细胞非抗原特异性活化能力的影响。为探讨CD59分子在T细胞发生抗原特异性活化中的作用,利用负载肿瘤抗原的DCs刺激siCD59-T细胞(表达CD59-siRNA的T细胞)和cCD59-T细胞(对照细胞,空质粒来源的逆转录病毒感染的T细胞),分别检测IL-2+CD4+T细胞、IFN-γ+CD8+T细胞和CD4+CD25+FoxP3+调节性T细胞(Tregs)的频数以及CD4+或CD8+T细胞增殖强度。为了解CD59分子发挥抑制T细胞抗原特异性活化的机制,利用抗CD3/CD28/CD59(或其同型对照抗体IgG2a)包被微珠刺激CD4+或CD8+T细胞,在混合培养介质中加入(或不加)抗CD59单抗的情况下,检测细胞的增殖强度。 结果诱导培养的负载肿瘤抗原的DCs高表达CD40、CD83、CD86和HLA-DR等细胞成熟标志。在分选出的CD4+或CD8+T细胞中,GFP+细胞的比例95%。siCD59-T细胞CD59分子表达水平显著降低,对补体溶解的敏感性明显增强；对PMA刺激具有正常的活化反应能力,但对CD59分子交联的反应降低；CD3和CD28的表达没有明显变化。利用负载肿瘤抗原的DCs进行刺激时,CD4+或CD8+siCD59-T细胞的活化、增殖反应显著增强；CD4+siCD59-T细胞与CD4+cCD59-T细胞之间,CD4+CD25+FoxP3+Tregs增殖幅度无明显差别。用抗CD3/CD28/CD59包被珠进行刺激时,siCD59-T细胞表现出更强的增殖活性；在珠/T细胞培养介质中加入抗CD59单克隆抗体后,siCD59-T细胞和cCD59-T细胞的增殖反应均显著增强,同时,前者的增殖活性仍明显强于后者。用抗CD3/CD28/IgG2a包被珠进行刺激时,siCD59-T细胞和cCD59-T细胞的增殖反应没有明显差别；在珠/T细胞培养介质中加入抗CD59单克隆抗体后,二者的增殖反应无明显变化。 结论在TCR识别、结合抗原肽-MHC复合物时,CD59分子通过与抗原提呈细胞上的相应配体结合而抑制人CD4‘或CD8+T细胞发生抗原特异性活化。CD59分子对人T细胞抗原特异性活化的抑制效应与CD4+CD25+FoxP3+Tregs的作用无关。
[Abstract]:Objective to silence the expression of CD59 gene in normal CD4 or CD8 T cells by using RNA interference technique, and then to observe its stimulatory response to DCs or anti-CD3/CD28/CD59 (or IgG2a) coated beads loaded with tumor antigen. To investigate the role of CD59 molecule in T cell antigen specific activation and its related mechanism. Methods DC serum-free medium containing IL-4 and GM-CSF was used to induce adherent PBMCs, loaded with tumor antigen in healthy donors, then TNF-a was added to promote DCs maturation. The CD59-siRNA expression plasmid siCD59 and the empty plasmid pSRNG were transfected into PA317 cells respectively, and the retrovirus was packaged. After the suspension of PBMCs.G418 was used to screen the virus infected cells, the CD4 T cells and CD8 T cells were separated by immunomagnetic beads. The effects of CD59-siRNA on CD59,CD3,CD28 expression and non-antigen-specific activation of T cells were evaluated by FACS assay, complement lysis test and PMA stimulated CD4 T cell activation test. In order to investigate the role of CD59 molecules in antigen-specific activation of T cells, siCD59-T cells (T cells expressing CD59-siRNA) and cCD59-T cells (control cells) were stimulated by DCs loaded with tumor antigen. The frequency of (Tregs) in IL-2 CD4 T cells, IFN- 纬 CD8 T cells and CD4 CD25 FoxP3 regulatory T cells, and the proliferation intensity of CD4 or CD8 T cells were measured. In order to understand the mechanism by which CD59 molecules inhibit the specific activation of T cell antigens, CD4 or CD8 T cells were stimulated by microspheres coated with anti-CD3/CD28/CD59 (or IgG2a). The cell proliferation intensity was measured by adding (or without) anti CD59 McAb in mixed culture medium. Results DCs loaded with tumor antigen induced high expression of CD40,CD83,CD86 and HLA-DR and other cell maturation markers. In the selected CD4 or CD8 T cells, the ratio of GFP cells to 95%.siCD59-T cells significantly decreased the expression level of CD59 molecules, and the sensitivity to complement dissolution was significantly increased. The activation reaction to PMA stimulation was normal, but the reaction to CD59 molecular crosslinking was decreased, while the expression of CD3 and CD28 did not change significantly. When stimulated by DCs loaded with tumor antigen, the activation of CD4 or CD8 siCD59-T cells and the proliferation response of CD8 siCD59-T cells were significantly enhanced, but there was no significant difference in the range of CD4 CD25 FoxP3 Tregs proliferation between CD4 siCD59-T cells and CD4 cCD59-T cells. The siCD59-T cells showed stronger proliferative activity when treated with CD3/CD28/CD59 coated beads. The proliferation of siCD59-T cells and cCD59-T cells was significantly enhanced after the addition of monoclonal antibodies against CD59 in bead / T cell culture medium, and the proliferative activity of the former was still stronger than that of the latter. The proliferative response of siCD59-T cells and cCD59-T cells was not significantly different from that of cCD59-T cells stimulated by anti CD3/CD28/IgG2a coated beads, but the proliferation reaction of siCD59-T cells and cCD59-T cells did not change after the addition of anti CD59 monoclonal antibody in the medium of pearl / T cell culture. Conclusion when TCR recognizes and binds antigenic peptide-MHC complex, The antigen-specific activation of human CD4' or CD8 T cells was inhibited by the binding of CD59 molecules to the corresponding ligands on antigen-presenting cells. The inhibitory effect of CD59 molecules on antigen-specific activation of human T cells was not related to the effect of CD4 CD25 FoxP3 Tregs.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.11
本文编号：2404452
[Abstract]:Objective to silence the expression of CD59 gene in normal CD4 or CD8 T cells by using RNA interference technique, and then to observe its stimulatory response to DCs or anti-CD3/CD28/CD59 (or IgG2a) coated beads loaded with tumor antigen. To investigate the role of CD59 molecule in T cell antigen specific activation and its related mechanism. Methods DC serum-free medium containing IL-4 and GM-CSF was used to induce adherent PBMCs, loaded with tumor antigen in healthy donors, then TNF-a was added to promote DCs maturation. The CD59-siRNA expression plasmid siCD59 and the empty plasmid pSRNG were transfected into PA317 cells respectively, and the retrovirus was packaged. After the suspension of PBMCs.G418 was used to screen the virus infected cells, the CD4 T cells and CD8 T cells were separated by immunomagnetic beads. The effects of CD59-siRNA on CD59,CD3,CD28 expression and non-antigen-specific activation of T cells were evaluated by FACS assay, complement lysis test and PMA stimulated CD4 T cell activation test. In order to investigate the role of CD59 molecules in antigen-specific activation of T cells, siCD59-T cells (T cells expressing CD59-siRNA) and cCD59-T cells (control cells) were stimulated by DCs loaded with tumor antigen. The frequency of (Tregs) in IL-2 CD4 T cells, IFN- 纬 CD8 T cells and CD4 CD25 FoxP3 regulatory T cells, and the proliferation intensity of CD4 or CD8 T cells were measured. In order to understand the mechanism by which CD59 molecules inhibit the specific activation of T cell antigens, CD4 or CD8 T cells were stimulated by microspheres coated with anti-CD3/CD28/CD59 (or IgG2a). The cell proliferation intensity was measured by adding (or without) anti CD59 McAb in mixed culture medium. Results DCs loaded with tumor antigen induced high expression of CD40,CD83,CD86 and HLA-DR and other cell maturation markers. In the selected CD4 or CD8 T cells, the ratio of GFP cells to 95%.siCD59-T cells significantly decreased the expression level of CD59 molecules, and the sensitivity to complement dissolution was significantly increased. The activation reaction to PMA stimulation was normal, but the reaction to CD59 molecular crosslinking was decreased, while the expression of CD3 and CD28 did not change significantly. When stimulated by DCs loaded with tumor antigen, the activation of CD4 or CD8 siCD59-T cells and the proliferation response of CD8 siCD59-T cells were significantly enhanced, but there was no significant difference in the range of CD4 CD25 FoxP3 Tregs proliferation between CD4 siCD59-T cells and CD4 cCD59-T cells. The siCD59-T cells showed stronger proliferative activity when treated with CD3/CD28/CD59 coated beads. The proliferation of siCD59-T cells and cCD59-T cells was significantly enhanced after the addition of monoclonal antibodies against CD59 in bead / T cell culture medium, and the proliferative activity of the former was still stronger than that of the latter. The proliferative response of siCD59-T cells and cCD59-T cells was not significantly different from that of cCD59-T cells stimulated by anti CD3/CD28/IgG2a coated beads, but the proliferation reaction of siCD59-T cells and cCD59-T cells did not change after the addition of anti CD59 monoclonal antibody in the medium of pearl / T cell culture. Conclusion when TCR recognizes and binds antigenic peptide-MHC complex, The antigen-specific activation of human CD4' or CD8 T cells was inhibited by the binding of CD59 molecules to the corresponding ligands on antigen-presenting cells. The inhibitory effect of CD59 molecules on antigen-specific activation of human T cells was not related to the effect of CD4 CD25 FoxP3 Tregs.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.11
【参考文献】
相关期刊论文 前1条
1 ;CD59 Silencing via Retrovirus-Mediated RNA Interference Enhanced Complement-Mediated Cell Damage in Ovary Cancer[J];Cellular & Molecular Immunology;2009年01期
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