Jurkat细胞特异性的糖基化抗原靶向MGL和DC-SIGN诱导DC免疫活化的机制研究
发布时间：2018-07-22 20:38
【摘要】：树突细胞(DCs)是体内功能最强大的专职抗原递呈细胞,也是连接先天性免疫和获得性免疫的关键调节者。在进行精细的免疫调节过程中,其表面大量表达的模式识别受体(PRRs)发挥了重要的作用,如Toll样受体(TLRs)和C-型凝集素受体(CLRs)。其中,TLRs主要识别保守的病原体相关复合物,如细菌脂多糖、鞭毛等。CLRs主要识别内源性及外源性的碳水化合物结构。有研究表明,某些病原体,如HIV、结核分枝杆菌和曼氏血吸虫等通过与DCs表面CLRs的相互作用,抑制DC的功能性成熟,从而逃避机体的免疫监视。也有研究表明,多种人肿瘤细胞系能够与DCs表面的CLRs相互作用,但其作用机制及病理功能并不清楚。在本研究中,我们以2种C-型凝集素MGL和DC-SIGN为研究对象,选取了3种人T淋巴细胞白血病细胞系Jurkat、CCRF-HSB-2和CCRF-CEM,系统研究了白血病细胞与MGL和DC-SIGN相互作用的机制、白血病细胞表面特异性的MGL和DC-SIGN糖基化配体形成的机制、以及白血病细胞特异性的糖基化配体与MGL和DC-SIGN的相互作用对DC免疫功能的影响。在MGL的相关研究中,我们首先证明了MGL可高结合于3种白血病细胞系Jurkat、CCRF-HSB-2和CCRF-CEM,但不结合或低结合于健康人外周血T细胞,表明了白血病细胞表面特异性地高表达MGL的配体。其次,我们鉴定了Jurkat细胞表面的CD45、CD43和MUC1为MGL的配体,且发现存在于CD45、CD43和MUC1上的高水平Tn糖抗原介导了其与MGL的特异性高结合。为探究MGL配体上高Tn糖抗原修饰的分子机制,我们使用了高通量测序及对比分析技术。分析结果显示,与健康人外周血T细胞相比,Jurkat细胞高表达4种负责Tn糖抗原形成的N-乙酰半乳糖胺基转移酶(GalNAc-T2、GalNAc-T7、GalNAc-T13和GalNAc-T14),同时低表达负责Tn糖抗原进一步延伸的相关酶(B3GnT6、ST6GalNAc-I)及其分子伴侣(Cosmc)。进一步的RNA干涉实验证明,对4种N-乙酰半乳糖胺基转移酶的抑制降低了Jurkat表面Tn抗原的表达,并因而抑制了MGL与Jurkat细胞的结合。以上结果提示,在T淋巴细胞恶性化的进程中,某些N-乙酰半乳糖胺基转移酶的表达增加及与Tn延伸相关酶的表达减弱,导致了Tn抗原的高水平形成,并作为凝集素的配体介导其与DCs表面MGL的识别。最后,我们系统分析了Tn抗原与MGL的相互作用对DC免疫功能的影响。结果显示,MGL的触发增加了DCs中促炎因子IL-6和IL-12的产生,诱发了DC免疫活化事件,即p38和ERK磷酸化水平及NF-?B的表达增加。我们同时意外地发现,单核细胞也表达MGL,对单核细胞MGL的触发有促进单核细胞向成熟DCs分化的趋势。以上结果表明,Jurkat细胞表面的Tn抗原能够通过靶向MGL促进DC的免疫活化。在DC-SIGN的相关研究中,我们首先证明了3种白血病细胞系Jurkat、CCRF-HSB-2和CCRF-CEM均高表达DC-SIGN的配体。经进一步鉴定发现Jurkat表面DC-SIGN的配体为ICAM-2和ICAM-3。其次,我们对Jurkat细胞表面DC-SIGN配体的糖基化修饰进行了结构表征,发现其表面N连接的岩藻糖型寡糖参与了同DC-SIGN的结合。进一步的实验结果显示,Jurkat细胞表面高表达含有岩藻糖基的N连接的Lewis糖抗原,且存在于ICAM-2和ICAM-3分子上的3种Lewis糖抗原(Lex、Ley和Lea)介导了其与DC-SIGN的高结合。为探究DC-SIGN与恶性T淋巴细胞结合的特异性,我们比较了DC-SIGN同Jurkat细胞和健康人外周血T细胞结合的差异,发现DC-SIGN与Jurkat细胞的结合明显高于其与外周血T细胞的结合。经进一步分析发现,Jurkat细胞表面ICAM-2、ICAM-3及Lewis抗原(Lex、Ley和Lea)的表达明显高于外周血T细胞。为探究DC-SIGN配体上高Lewis糖抗原修饰的分子机制,我们同样使用了高通量测序及对比分析技术。结果显示,与外周血T细胞相比,Jurkat细胞负责催化合成Lewis抗原的岩藻糖基转移酶4(Fut4)显著地高表达。对Jurkat细胞Fut4的RNA干涉明显降低了其同DC-SIGN的结合。以上结果提示,Jurkat细胞高表达的Fut4有利于DC-SIGN配体ICAM-2和ICAM-3上Lewis抗原(Lex、Ley和Lea)的合成,因而介导了同DC-SIGN的高结合。最后,我们系统分析了Jurkat细胞与DC-SIGN相互作用对DC免疫功能的影响。结果显示,在Jurkat细胞与DCs相互作用中,对DC-SIGN的阻断增加了DCs分泌IL-10的能力,同时减少了IL-6的分泌。另外,对DC-SIGN的阻断抑制了DCs成熟分子标记的表达、增加了DCs的内吞能力、产生了更多的Treg细胞,同时减少了分泌IFN-γ的Th1细胞的产生。这些结果表明,DC-SIGN能够促进DC的功能性成熟,并增加T细胞向Th1细胞的极化。在使用糖配体Lex靶向DC-SIGN的实验中,我们也发现DCs分泌了更多的促炎因子IL-6和IL-12。以上结果表明,Jurkat细胞表面的Lewis抗原能够通过靶向DC-SIGN促进DC的免疫活化。综上所述,本研究不但证明了T淋巴白血病细胞表面高表达了C-型凝集素MGL和DC-SIGN的配体,同时鉴定了其配体分子及阐明了其特异性糖基化修饰的分子机制,而且提供了白血病细胞以其特异性的糖配体靶向MGL和DC-SIGN促进DC免疫活化的实验证据。本研究为以MGL和DC-SIGN为靶点的T淋巴细胞白血病的DC免疫治疗提供了有价值的实验基础。
[Abstract]:Dendritic cells (DCs) are the most powerful specialized antigen presenting cells in the body, and are also the key regulators to connect innate and acquired immunity. In the process of fine immunomodulation, the pattern recognition receptor (PRRs), which is expressed on the surface, plays an important role, such as Toll like receptor (TLRs) and C- type lectin receptor (CLRs). Among them, TLRs mainly recognizes conserved pathogen related complexes, such as bacterial lipopolysaccharide, flagellum and other.CLRs mainly identify endogenous and exogenous carbohydrate structures. Some studies have shown that some pathogens, such as HIV, Mycobacterium tuberculosis and Schistosoma mansoni, can inhibit the functional maturity of DC by interacting with DCs surface CLRs. The immune surveillance of the body is avoided. There are also studies showing that a variety of human tumor cell lines can interact with CLRs on the DCs surface, but their mechanism and pathological function are not clear. In this study, we selected 2 kinds of C- type lectin MGL and DC-SIGN as the research objects, and selected 3 human T lymphocytic leukemia cell lines Jurkat, CCRF-HSB-2 and CCRF-. CEM, a systematic study of the mechanisms of leukemic cells interacting with MGL and DC-SIGN, the mechanism of the formation of specific MGL and DC-SIGN glycosylation ligands on the surface of leukemic cells, and the effect of the interaction of leukemic cell specific glycosylated ligands and MGL and DC-SIGN on the immune function of DC, we first demonstrated in the related study of MGL. MGL can be highly combined with 3 leukemia cell lines, Jurkat, CCRF-HSB-2 and CCRF-CEM, but not combined or low combined with T cells in healthy human peripheral blood. It shows that the ligand of MGL is highly expressed on the surface of the leukemia cells. Secondly, we identified the CD45 on the surface of the Jurkat cells, CD43 and MUC1 are MGL ligands. High levels of Tn sugar antigen on the high binding of MGL was mediated. In order to explore the molecular mechanism of high Tn carbohydrate modification on MGL ligands, we used high throughput sequencing and contrast analysis techniques. The results showed that Jurkat cells were more than 4 kinds of N- acetyl galactose that were responsible for the formation of Tn sugar antigen compared with the healthy human peripheral blood T cells. Aminotransferase (GalNAc-T2, GalNAc-T7, GalNAc-T13 and GalNAc-T14) and low expression of the related enzyme (B3GnT6, ST6GalNAc-I) and its molecular chaperone (Cosmc) responsible for the further extension of Tn sugar antigen. Further RNA interference experiments showed that the inhibition of the 4 N- acetyl galactaminotransferases reduced the expression of Tn antigen on the Jurkat surface and thus suppressed the expression of the Tn antigen. The combination of MGL and Jurkat cells was made. The above results suggest that in the process of T lymphocyte malignancy, the expression of some N- acetylgalactoaminotransferase and the expression of Tn extension related enzymes are weakened, leading to the high level formation of Tn antigen, and as ligands of the lectin mediated identification of the MGL of the DCs surface. Finally, our system The effect of the interaction of Tn antigen and MGL on the immune function of DC was analyzed. The results showed that the trigger of MGL increased the production of IL-6 and IL-12 in DCs, induced the immune activation events of DC, that is, p38 and ERK phosphorylation level and NF- B expression. There is a tendency to promote the differentiation of monocyte to mature DCs. The above results show that the Tn antigen on the surface of Jurkat cells can promote the immune activation of DC through target MGL. In the related study of DC-SIGN, we first demonstrated that the 3 leukemia cell lines, Jurkat, CCRF-HSB-2 and CCRF-CEM all expressed DC-SIGN ligands. The ligand of DC-SIGN on the surface of urkat is ICAM-2 and ICAM-3. next. We characterize the glycosylated modification of DC-SIGN ligand on the surface of Jurkat cells. It is found that the surface N connected fucose oligosaccharide oligosaccharide oligosaccharide oligosaccharides are involved in the binding with DC-SIGN. Further experimental results show that the Jurkat cell surface is highly expressed as a Lewis N connection containing the fucose base. The 3 Lewis carbohydrate antigens (Lex, Ley and Lea) that exist on the ICAM-2 and ICAM-3 molecules (Lex, Ley and Lea) mediate their high binding with DC-SIGN. In order to explore the specificity of the binding between DC-SIGN and malignant T lymphocytes, we compare the differences in the binding of DC-SIGN with Jurkat cells to the healthy human peripheral blood T cells. Further analysis showed that the expression of ICAM-2, ICAM-3 and Lewis antigens (Lex, Ley and Lea) on the surface of Jurkat cells was significantly higher than that of peripheral blood T cells. We also used high throughput sequencing and contrast analysis techniques to explore the molecular mechanism of high Lewis sugar antigen modification on the DC-SIGN ligand. Compared with the peripheral blood T cells, Jurkat cells are responsible for the significantly high expression of the fucoidase 4 (Fut4), which catalyzes the synthesis of Lewis antigens. The RNA interference of Fut4 in Jurkat cells significantly reduces the binding of its DC-SIGN. The above results suggest that Fut4 with high expression of Jurkat cells is beneficial to DC-SIGN ligand ICAM-2 and associated antigens. Synthesis, thus mediating a high combination with DC-SIGN. Finally, we systematically analyzed the effect of the interaction between Jurkat cells and DC-SIGN on the immune function of DC. The results showed that in the interaction of Jurkat cells with DCs, the blocking of DC-SIGN increased the ability of DCs to secrete IL-10 and reduced the secretion of IL-6. In addition, inhibition of DC-SIGN was inhibited. The expression of DCs matured molecular markers increases the endocytosis of DCs, produces more Treg cells, and reduces the production of Th1 cells that secrete IFN- gamma. These results suggest that DC-SIGN can promote functional maturation of DC and increase the polarization of T cells to Th1 cells. We also found that in the experiment of targeting the glucose ligand Lex to DC-SIGN, we also found that DCs secretes more pro-inflammatory factors IL-6 and IL-12. above the results that the Lewis antigen on the surface of Jurkat cells can promote the immune activation of DC through targeted DC-SIGN. To sum up, this study not only demonstrated that the surface of T lymphoblastic leukemia cells expressed the C- type agglutinin MGL and DC-SIGN ligands, but also identified its ligand molecules and clarifies. The molecular mechanism of its specific glycosylation modification provides experimental evidence for leukemic cells to promote DC immunization with its specific glucose ligand targeting MGL and DC-SIGN. This study provides an experimental basis for the DC immunotherapy of T lymphoblastic leukemia targeting MGL and DC-SIGN.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392
本文编号：2138501
[Abstract]:Dendritic cells (DCs) are the most powerful specialized antigen presenting cells in the body, and are also the key regulators to connect innate and acquired immunity. In the process of fine immunomodulation, the pattern recognition receptor (PRRs), which is expressed on the surface, plays an important role, such as Toll like receptor (TLRs) and C- type lectin receptor (CLRs). Among them, TLRs mainly recognizes conserved pathogen related complexes, such as bacterial lipopolysaccharide, flagellum and other.CLRs mainly identify endogenous and exogenous carbohydrate structures. Some studies have shown that some pathogens, such as HIV, Mycobacterium tuberculosis and Schistosoma mansoni, can inhibit the functional maturity of DC by interacting with DCs surface CLRs. The immune surveillance of the body is avoided. There are also studies showing that a variety of human tumor cell lines can interact with CLRs on the DCs surface, but their mechanism and pathological function are not clear. In this study, we selected 2 kinds of C- type lectin MGL and DC-SIGN as the research objects, and selected 3 human T lymphocytic leukemia cell lines Jurkat, CCRF-HSB-2 and CCRF-. CEM, a systematic study of the mechanisms of leukemic cells interacting with MGL and DC-SIGN, the mechanism of the formation of specific MGL and DC-SIGN glycosylation ligands on the surface of leukemic cells, and the effect of the interaction of leukemic cell specific glycosylated ligands and MGL and DC-SIGN on the immune function of DC, we first demonstrated in the related study of MGL. MGL can be highly combined with 3 leukemia cell lines, Jurkat, CCRF-HSB-2 and CCRF-CEM, but not combined or low combined with T cells in healthy human peripheral blood. It shows that the ligand of MGL is highly expressed on the surface of the leukemia cells. Secondly, we identified the CD45 on the surface of the Jurkat cells, CD43 and MUC1 are MGL ligands. High levels of Tn sugar antigen on the high binding of MGL was mediated. In order to explore the molecular mechanism of high Tn carbohydrate modification on MGL ligands, we used high throughput sequencing and contrast analysis techniques. The results showed that Jurkat cells were more than 4 kinds of N- acetyl galactose that were responsible for the formation of Tn sugar antigen compared with the healthy human peripheral blood T cells. Aminotransferase (GalNAc-T2, GalNAc-T7, GalNAc-T13 and GalNAc-T14) and low expression of the related enzyme (B3GnT6, ST6GalNAc-I) and its molecular chaperone (Cosmc) responsible for the further extension of Tn sugar antigen. Further RNA interference experiments showed that the inhibition of the 4 N- acetyl galactaminotransferases reduced the expression of Tn antigen on the Jurkat surface and thus suppressed the expression of the Tn antigen. The combination of MGL and Jurkat cells was made. The above results suggest that in the process of T lymphocyte malignancy, the expression of some N- acetylgalactoaminotransferase and the expression of Tn extension related enzymes are weakened, leading to the high level formation of Tn antigen, and as ligands of the lectin mediated identification of the MGL of the DCs surface. Finally, our system The effect of the interaction of Tn antigen and MGL on the immune function of DC was analyzed. The results showed that the trigger of MGL increased the production of IL-6 and IL-12 in DCs, induced the immune activation events of DC, that is, p38 and ERK phosphorylation level and NF- B expression. There is a tendency to promote the differentiation of monocyte to mature DCs. The above results show that the Tn antigen on the surface of Jurkat cells can promote the immune activation of DC through target MGL. In the related study of DC-SIGN, we first demonstrated that the 3 leukemia cell lines, Jurkat, CCRF-HSB-2 and CCRF-CEM all expressed DC-SIGN ligands. The ligand of DC-SIGN on the surface of urkat is ICAM-2 and ICAM-3. next. We characterize the glycosylated modification of DC-SIGN ligand on the surface of Jurkat cells. It is found that the surface N connected fucose oligosaccharide oligosaccharide oligosaccharide oligosaccharides are involved in the binding with DC-SIGN. Further experimental results show that the Jurkat cell surface is highly expressed as a Lewis N connection containing the fucose base. The 3 Lewis carbohydrate antigens (Lex, Ley and Lea) that exist on the ICAM-2 and ICAM-3 molecules (Lex, Ley and Lea) mediate their high binding with DC-SIGN. In order to explore the specificity of the binding between DC-SIGN and malignant T lymphocytes, we compare the differences in the binding of DC-SIGN with Jurkat cells to the healthy human peripheral blood T cells. Further analysis showed that the expression of ICAM-2, ICAM-3 and Lewis antigens (Lex, Ley and Lea) on the surface of Jurkat cells was significantly higher than that of peripheral blood T cells. We also used high throughput sequencing and contrast analysis techniques to explore the molecular mechanism of high Lewis sugar antigen modification on the DC-SIGN ligand. Compared with the peripheral blood T cells, Jurkat cells are responsible for the significantly high expression of the fucoidase 4 (Fut4), which catalyzes the synthesis of Lewis antigens. The RNA interference of Fut4 in Jurkat cells significantly reduces the binding of its DC-SIGN. The above results suggest that Fut4 with high expression of Jurkat cells is beneficial to DC-SIGN ligand ICAM-2 and associated antigens. Synthesis, thus mediating a high combination with DC-SIGN. Finally, we systematically analyzed the effect of the interaction between Jurkat cells and DC-SIGN on the immune function of DC. The results showed that in the interaction of Jurkat cells with DCs, the blocking of DC-SIGN increased the ability of DCs to secrete IL-10 and reduced the secretion of IL-6. In addition, inhibition of DC-SIGN was inhibited. The expression of DCs matured molecular markers increases the endocytosis of DCs, produces more Treg cells, and reduces the production of Th1 cells that secrete IFN- gamma. These results suggest that DC-SIGN can promote functional maturation of DC and increase the polarization of T cells to Th1 cells. We also found that in the experiment of targeting the glucose ligand Lex to DC-SIGN, we also found that DCs secretes more pro-inflammatory factors IL-6 and IL-12. above the results that the Lewis antigen on the surface of Jurkat cells can promote the immune activation of DC through targeted DC-SIGN. To sum up, this study not only demonstrated that the surface of T lymphoblastic leukemia cells expressed the C- type agglutinin MGL and DC-SIGN ligands, but also identified its ligand molecules and clarifies. The molecular mechanism of its specific glycosylation modification provides experimental evidence for leukemic cells to promote DC immunization with its specific glucose ligand targeting MGL and DC-SIGN. This study provides an experimental basis for the DC immunotherapy of T lymphoblastic leukemia targeting MGL and DC-SIGN.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 代红胜;曾彬;杨荣存;;肿瘤通过抑制树突状细胞功能逃避免疫监视的研究进展[J];国际免疫学杂志;2008年01期
2 ;DC-SIGN and Immunoregulation[J];Cellular & Molecular Immunology;2006年04期
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