CD59分子在人T細胞抗原特異性活化中的作用研究
發(fā)布時間：2019-01-08 10:02
【摘要】：目的運用RNA干擾技術使正常人CD4+或CD8+T細胞的CD59基因表達發(fā)生沉默,然后觀察其對負載腫瘤抗原的DCs或抗CD3/CD28/CD59(或IgG2a)包被珠的刺激反應,探討CD59分子在T細胞抗原特異性活化中的作用及相關機制。 方法用含IL-4和GM-CSF的DC無血清培養(yǎng)基誘導培養(yǎng)健康供者的貼壁PBMCs,負載腫瘤抗原后,加入TNF-a促進DCs成熟。將CD59-siRNA表達質粒siCD59和空質粒pSRNG分別轉染PA317細胞,包裝出逆轉錄病毒,用于感染懸浮的PBMCs。G418篩選病毒感染細胞后,利用免疫磁珠純化分選出CD4+T細胞和CD8+T細胞。利用FACS檢測、補體溶解試驗和PMA刺激CD4+T細胞活化試驗評估CD59-siRNA對CD59、CD3、CD28表達以及T細胞非抗原特異性活化能力的影響。為探討CD59分子在T細胞發(fā)生抗原特異性活化中的作用,利用負載腫瘤抗原的DCs刺激siCD59-T細胞(表達CD59-siRNA的T細胞)和cCD59-T細胞(對照細胞,空質粒來源的逆轉錄病毒感染的T細胞),分別檢測IL-2+CD4+T細胞、IFN-γ+CD8+T細胞和CD4+CD25+FoxP3+調節(jié)性T細胞(Tregs)的頻數(shù)以及CD4+或CD8+T細胞增殖強度。為了解CD59分子發(fā)揮抑制T細胞抗原特異性活化的機制,利用抗CD3/CD28/CD59(或其同型對照抗體IgG2a)包被微珠刺激CD4+或CD8+T細胞,在混合培養(yǎng)介質中加入(或不加)抗CD59單抗的情況下,檢測細胞的增殖強度。 結果誘導培養(yǎng)的負載腫瘤抗原的DCs高表達CD40、CD83、CD86和HLA-DR等細胞成熟標志。在分選出的CD4+或CD8+T細胞中,GFP+細胞的比例95%。siCD59-T細胞CD59分子表達水平顯著降低,對補體溶解的敏感性明顯增強；對PMA刺激具有正常的活化反應能力,但對CD59分子交聯(lián)的反應降低；CD3和CD28的表達沒有明顯變化。利用負載腫瘤抗原的DCs進行刺激時,CD4+或CD8+siCD59-T細胞的活化、增殖反應顯著增強；CD4+siCD59-T細胞與CD4+cCD59-T細胞之間,CD4+CD25+FoxP3+Tregs增殖幅度無明顯差別。用抗CD3/CD28/CD59包被珠進行刺激時,siCD59-T細胞表現(xiàn)出更強的增殖活性；在珠/T細胞培養(yǎng)介質中加入抗CD59單克隆抗體后,siCD59-T細胞和cCD59-T細胞的增殖反應均顯著增強,同時,前者的增殖活性仍明顯強于后者。用抗CD3/CD28/IgG2a包被珠進行刺激時,siCD59-T細胞和cCD59-T細胞的增殖反應沒有明顯差別；在珠/T細胞培養(yǎng)介質中加入抗CD59單克隆抗體后,二者的增殖反應無明顯變化。 結論在TCR識別、結合抗原肽-MHC復合物時,CD59分子通過與抗原提呈細胞上的相應配體結合而抑制人CD4‘或CD8+T細胞發(fā)生抗原特異性活化。CD59分子對人T細胞抗原特異性活化的抑制效應與CD4+CD25+FoxP3+Tregs的作用無關。
[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- 緯 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.
【學位授予單位】：青島大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392.11
本文編號：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- 緯 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.
【學位授予單位】：青島大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392.11
【參考文獻】
相關期刊論文 前1條
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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