Studies On The Mechanism Of Jurkat-spceific Glycosylated Antigens Target MGL And DC-SIGN To Induce DC Immune Activation

Dendritic cells(DCs), the most potent and professional antigen-presenting cells(APCs), are key regulators which connect innate and adaptive immunity. DCs express a large repertoire of pattern recognition receptors(PRRs) on their surfaces, including Toll-like receptors(TLRs) and C-type lectin receptors(CLRs), play an important role in the delicate immunoregulation. In general, TLRs recognize conserved pathogen-associated compounds such as LPS and flagellin. CLRs recognize both exogenous and endogenous carbohydrate moieties. It is becoming evident that some pathogens, include HIV virus, Mycobacterium tubculosis and Schistosoma mansoni, imped DC function to escape immune surveillance by targeting CLRs expressed on DCs. Recently, several human tumour cell lines have been shown to interact with CLRs expressed on DCs. However, the mechanisms and the pathological function remain unclear.In the present study, we focused on two types of CLRs, MGL and DC-SIGN, and selected three kinds of human T lymphocytic leukemia cell lines, Jurkat, CCRF-HSB-2 and CCRF-CEM. We analyzed the mechanism of interaction between MGL, DC-SIGN and leukemia cells, the mechanism of the formation of MGL and DC-SIGN glycosylated ligands, and the effect of interaction between leukemia-specific glycosylated ligands and MGL, DC-SIGN on DC function.For MGL study, we firstly demonstrated that MGL highly binds to Jurkat, CCRF-HSB-2 and CCRF-CEM, but barely binds to T cells from healthy peripheral blood, indicating leukemia cells specific highly express MGL ligands. Next, we identified that CD45, CD43 and MUC1 expressed on Jurkat cells are MGL ligands, and Tn antigens, exist in CD45, CD43 and MUC1, conduces to the binding specificity of MGL to Jurkat cells. To gain more insight in the mechanism of abnormal formation of Tn antigens on Jurkat cells, we performed high-throughput transcription sequencing. Compared to peripheral blood T cells, Jurkat cells displayed increased expression of glycosyltransferase GalNAc-T2, 7, 13 and 14 which are responsible for Tn synthesis, accompanied by decreased expression of O-glycosylation extension-related synthase(B3GnT6 and ST6GalNAc-I) and molecular chaperone(Cosmc). Moreover, treatment with siRNA targeting indicated GalNAc-T apparently decreased Tn antigens level on the surface of Jurkat cells, and subsequently reduced the binding of MGL to Jurkat cells. These results indicated that all of these changes in glycosylation contribute to Tn antigen exposure, which is essential for MGL recognition. Finally, we found the MGL-Tn antigen-based interaction augments the secretion of the pro-inflammatory cytokines IL-6 and IL-12 by DCs, promotes DC activation signalling which increase p38, ERK phosphorylation and NF-?B expression. Meanwhile, we found monocytes also express MGL, and the engagement of MGL on monocytes promotes differentiation of monocyte towards mature DCs. These above results indicate that Jutkat-specific Tn antigen could promote DC immune activation by targeting MGL.For DC-SIGN study, we firstly demonstrated that Jurkat, CCRF-HSB-2 and CCRF-CEM highly express DC-SIGN ligands, and then identified that ICAM-2 and ICAM-3 expressed on Jurkat cells are DC-SIGN ligands. Secondly, we characterized the structure of DC-SIGN carbohydrate ligands on Jurkat cells. The results showed that fucose residues in N-glycan involve in DC-SIGN binding. Meanwhile, we found Jurkat highly express Lewis antigens containing fucose residues. Lex, Ley and Lea exist in ICAM-2 and ICAM-3 mediated the binding to DC-SIGN. We next determined the difference of DC-SIGN binding to Jurkat cells and peripheral blood T cells. The results showed that the binding of DC-SIGN to Jurkat cellsis much higher than to peripheral blood T cells. Further analysis showed that Jurkat cells expresse more ICAM-2, ICAM-3, and Lex, Ley and Lea than that on peripheral blood T cells. To explore the molecular mechanism of the high Lewis antigen modification on DC-SIGN ligands, the high-throughput transcription sequencing analysis was performed again. In contrast to peripheral blood T cells, the expression of fucosyltransferase 4(Fut4) which catalyse Lewis antigen synthesis, was significantly increased in Jurkat cells. The interference of Fut4 decreased DC-SIGN binding to Jurkat cells. These results implyed that the high Fut4 expression catalyse the synthesis of Lex, Ley and Lea on ICAM-2 and ICAM-3, thereby resulting in the binding of Jurkat cells to DC-SIGN. Finally, the effects of the interaction between Jurkat cells and DC-SIGN on DC functions were evaluated. It turned out that, in the experiment of cellular interaction between immature DCs and Jurkat cells, the blockage of DC-SIGN produced more immunesuppressive factor IL-10 and less proinflamantory factor IL-6, and impaired DCs maturation. Besides, the blockage of DC-SIGN attenuated T cell differentiation toward Treg cell and promoted T cell differentiation toward Th1 cell. These results implied that DC-SIGN could promote DC functional maturation, increase polarization to Th1 cell. In addition, Lex targeting DC-SIGN promoted DCs secreting more proinflammatory factor IL-6 and IL-12. Above of all, Jutkat-specific Lewis antigens could promote DC immune activation by targeting DC-SIGN.In summary, our study demonstrated that the ligands of MGL and DC-SIGN were highly expressed on T lymphoblastic leukemia cells. Besides, we identified the ligands of MGL and DC-SIGN, and illustrated the molecular mechanisms of the formation of specific ligand glycosylation. Furthermore, we provided the evidences that the specific carbohydrate ligands of T lymphoblastic leukemia cells could promote DC immune activation by targeting MGL and DC-SIGN on DCs. The present study provided a valuable experimental basis for DC immunotherapy against T lymphoblastic leukemia by targeting MGL and DC-SIGN.