Jurkat細(xì)胞特異性的糖基化抗原靶向MGL和DC-SIGN誘導(dǎo)DC免疫活化的機(jī)制研究
發(fā)布時間：2018-07-22 20:38
【摘要】：樹突細(xì)胞(DCs)是體內(nèi)功能最強(qiáng)大的專職抗原遞呈細(xì)胞,也是連接先天性免疫和獲得性免疫的關(guān)鍵調(diào)節(jié)者。在進(jìn)行精細(xì)的免疫調(diào)節(jié)過程中,其表面大量表達(dá)的模式識別受體(PRRs)發(fā)揮了重要的作用,如Toll樣受體(TLRs)和C-型凝集素受體(CLRs)。其中,TLRs主要識別保守的病原體相關(guān)復(fù)合物,如細(xì)菌脂多糖、鞭毛等。CLRs主要識別內(nèi)源性及外源性的碳水化合物結(jié)構(gòu)。有研究表明,某些病原體,如HIV、結(jié)核分枝桿菌和曼氏血吸蟲等通過與DCs表面CLRs的相互作用,抑制DC的功能性成熟,從而逃避機(jī)體的免疫監(jiān)視。也有研究表明,多種人腫瘤細(xì)胞系能夠與DCs表面的CLRs相互作用,但其作用機(jī)制及病理功能并不清楚。在本研究中,我們以2種C-型凝集素MGL和DC-SIGN為研究對象,選取了3種人T淋巴細(xì)胞白血病細(xì)胞系Jurkat、CCRF-HSB-2和CCRF-CEM,系統(tǒng)研究了白血病細(xì)胞與MGL和DC-SIGN相互作用的機(jī)制、白血病細(xì)胞表面特異性的MGL和DC-SIGN糖基化配體形成的機(jī)制、以及白血病細(xì)胞特異性的糖基化配體與MGL和DC-SIGN的相互作用對DC免疫功能的影響。在MGL的相關(guān)研究中,我們首先證明了MGL可高結(jié)合于3種白血病細(xì)胞系Jurkat、CCRF-HSB-2和CCRF-CEM,但不結(jié)合或低結(jié)合于健康人外周血T細(xì)胞,表明了白血病細(xì)胞表面特異性地高表達(dá)MGL的配體。其次,我們鑒定了Jurkat細(xì)胞表面的CD45、CD43和MUC1為MGL的配體,且發(fā)現(xiàn)存在于CD45、CD43和MUC1上的高水平Tn糖抗原介導(dǎo)了其與MGL的特異性高結(jié)合。為探究MGL配體上高Tn糖抗原修飾的分子機(jī)制,我們使用了高通量測序及對比分析技術(shù)。分析結(jié)果顯示,與健康人外周血T細(xì)胞相比,Jurkat細(xì)胞高表達(dá)4種負(fù)責(zé)Tn糖抗原形成的N-乙酰半乳糖胺基轉(zhuǎn)移酶(GalNAc-T2、GalNAc-T7、GalNAc-T13和GalNAc-T14),同時低表達(dá)負(fù)責(zé)Tn糖抗原進(jìn)一步延伸的相關(guān)酶(B3GnT6、ST6GalNAc-I)及其分子伴侶(Cosmc)。進(jìn)一步的RNA干涉實(shí)驗(yàn)證明,對4種N-乙酰半乳糖胺基轉(zhuǎn)移酶的抑制降低了Jurkat表面Tn抗原的表達(dá),并因而抑制了MGL與Jurkat細(xì)胞的結(jié)合。以上結(jié)果提示,在T淋巴細(xì)胞惡性化的進(jìn)程中,某些N-乙酰半乳糖胺基轉(zhuǎn)移酶的表達(dá)增加及與Tn延伸相關(guān)酶的表達(dá)減弱,導(dǎo)致了Tn抗原的高水平形成,并作為凝集素的配體介導(dǎo)其與DCs表面MGL的識別。最后,我們系統(tǒng)分析了Tn抗原與MGL的相互作用對DC免疫功能的影響。結(jié)果顯示,MGL的觸發(fā)增加了DCs中促炎因子IL-6和IL-12的產(chǎn)生,誘發(fā)了DC免疫活化事件,即p38和ERK磷酸化水平及NF-?B的表達(dá)增加。我們同時意外地發(fā)現(xiàn),單核細(xì)胞也表達(dá)MGL,對單核細(xì)胞MGL的觸發(fā)有促進(jìn)單核細(xì)胞向成熟DCs分化的趨勢。以上結(jié)果表明,Jurkat細(xì)胞表面的Tn抗原能夠通過靶向MGL促進(jìn)DC的免疫活化。在DC-SIGN的相關(guān)研究中,我們首先證明了3種白血病細(xì)胞系Jurkat、CCRF-HSB-2和CCRF-CEM均高表達(dá)DC-SIGN的配體。經(jīng)進(jìn)一步鑒定發(fā)現(xiàn)Jurkat表面DC-SIGN的配體為ICAM-2和ICAM-3。其次,我們對Jurkat細(xì)胞表面DC-SIGN配體的糖基化修飾進(jìn)行了結(jié)構(gòu)表征,發(fā)現(xiàn)其表面N連接的巖藻糖型寡糖參與了同DC-SIGN的結(jié)合。進(jìn)一步的實(shí)驗(yàn)結(jié)果顯示,Jurkat細(xì)胞表面高表達(dá)含有巖藻糖基的N連接的Lewis糖抗原,且存在于ICAM-2和ICAM-3分子上的3種Lewis糖抗原(Lex、Ley和Lea)介導(dǎo)了其與DC-SIGN的高結(jié)合。為探究DC-SIGN與惡性T淋巴細(xì)胞結(jié)合的特異性,我們比較了DC-SIGN同Jurkat細(xì)胞和健康人外周血T細(xì)胞結(jié)合的差異,發(fā)現(xiàn)DC-SIGN與Jurkat細(xì)胞的結(jié)合明顯高于其與外周血T細(xì)胞的結(jié)合。經(jīng)進(jìn)一步分析發(fā)現(xiàn),Jurkat細(xì)胞表面ICAM-2、ICAM-3及Lewis抗原(Lex、Ley和Lea)的表達(dá)明顯高于外周血T細(xì)胞。為探究DC-SIGN配體上高Lewis糖抗原修飾的分子機(jī)制,我們同樣使用了高通量測序及對比分析技術(shù)。結(jié)果顯示,與外周血T細(xì)胞相比,Jurkat細(xì)胞負(fù)責(zé)催化合成Lewis抗原的巖藻糖基轉(zhuǎn)移酶4(Fut4)顯著地高表達(dá)。對Jurkat細(xì)胞Fut4的RNA干涉明顯降低了其同DC-SIGN的結(jié)合。以上結(jié)果提示,Jurkat細(xì)胞高表達(dá)的Fut4有利于DC-SIGN配體ICAM-2和ICAM-3上Lewis抗原(Lex、Ley和Lea)的合成,因而介導(dǎo)了同DC-SIGN的高結(jié)合。最后,我們系統(tǒng)分析了Jurkat細(xì)胞與DC-SIGN相互作用對DC免疫功能的影響。結(jié)果顯示,在Jurkat細(xì)胞與DCs相互作用中,對DC-SIGN的阻斷增加了DCs分泌IL-10的能力,同時減少了IL-6的分泌。另外,對DC-SIGN的阻斷抑制了DCs成熟分子標(biāo)記的表達(dá)、增加了DCs的內(nèi)吞能力、產(chǎn)生了更多的Treg細(xì)胞,同時減少了分泌IFN-γ的Th1細(xì)胞的產(chǎn)生。這些結(jié)果表明,DC-SIGN能夠促進(jìn)DC的功能性成熟,并增加T細(xì)胞向Th1細(xì)胞的極化。在使用糖配體Lex靶向DC-SIGN的實(shí)驗(yàn)中,我們也發(fā)現(xiàn)DCs分泌了更多的促炎因子IL-6和IL-12。以上結(jié)果表明,Jurkat細(xì)胞表面的Lewis抗原能夠通過靶向DC-SIGN促進(jìn)DC的免疫活化。綜上所述,本研究不但證明了T淋巴白血病細(xì)胞表面高表達(dá)了C-型凝集素MGL和DC-SIGN的配體,同時鑒定了其配體分子及闡明了其特異性糖基化修飾的分子機(jī)制,而且提供了白血病細(xì)胞以其特異性的糖配體靶向MGL和DC-SIGN促進(jìn)DC免疫活化的實(shí)驗(yàn)證據(jù)。本研究為以MGL和DC-SIGN為靶點(diǎn)的T淋巴細(xì)胞白血病的DC免疫治療提供了有價值的實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 代紅勝;曾彬;楊榮存;;腫瘤通過抑制樹突狀細(xì)胞功能逃避免疫監(jiān)視的研究進(jìn)展[J];國際免疫學(xué)雜志;2008年01期
2 ;DC-SIGN and Immunoregulation[J];Cellular & Molecular Immunology;2006年04期
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