Ectopically Expressed HMSH2 Mediates Recognition Of Carcinoma And Epstein-barr Virus-transformed B Cells By Human Gamma Delta T Cells

Human y8 T cells account for a minor fraction of human peripheral T lymphocytes. The innate-like feature and unique major histocompatibility complex (MHC)-independent antigen recognition pattern define these cells a special status in tumor surveillance and pathogen defense. However, the molecular mechanisms underlying tumor/infected cells recognition byγδT cells are poorly understood, due to the limited number of antigens/ligands recognized byγδT cells that have been identified so far.Human MutS homologue 2 (hMSH2) is a critical element of the highly conserved DNA mismatch repair system, normally located in the nucleus and dimerized with hMSH3 or hMSH6 to form complexes essential for the maintenance of genome integrity. Inherited and acquired defects in hMSH2 are closely related to the pathogenesis of hereditary nonpolyposis colon cancer as well as various sporadic cancers. Previously, with a CDR3δpeptide-based affinity screening system, we identified hMSH2 as a putative tumor-associated protein ligand for Vδ2 TCRs. In addition, we observed an unusual surface expression of hMSH2 on SKOV3 cells that was seemingly correlated with an enhanced cytotoxicity ofγδT cells.Based on our previous study, we hypothesized that hMSH2 may be a tumor-associated ligand recognized by Vδ2 TCRs. To test this possibility, we first examined the surface expression of hMSH2 on HeLa. SKOV3, HO8910, ES-2,803, HR8348 and NCI-H520 cell lines. Flow cytometric analysis with specific anti-hMSH2 antibody (H-300) revealed that 10-70% of the tested tumor lines expressed hMSH2, whereas normal control cells rarely expressed hMSH2 on their surface. Confocal microscopy and immunohistochemistry analyses suggested an aberrant subcellular distribution of hMSH2 in carcinoma cells and tissues as well. The broad cell-surface expression of hMSH2 on carcinoma cells raised the possibility of its being a tumor-associated ligand forγδT cells.The second issue of this study was to verify the recognition of hMSH2 by yS T cells and to explore the significance of ectopically expressed hMSH2 inγδT cell-mediated anti-tumor immunity. With anti-hMSH2 antibody blocking, we found that the ectopic hMSH2 expression was correlated with an enhancedγδT cell-mediated cytolysis of HeLa,803 and NCI-H520 cells. The hMSH2-mediated specific recognition byγδT cells was further validate using small RNA interference technology to knockdown HMSH2 expression in HeLa,803 and NCI-H520 cell lines. At various E:T ratios, down-regulation of hMSH2 expression with siRNAⅠorⅡinterference resulted in significantly reduced cytolysis of HeLa,803 and NCI-H520 cells byγδT cells. The above results from antibody blocking or siRNAs interference suggest that ectopic hMSH2 mediates the recognition of carcinoma cells by humanγδT cells and contributes toγδT cell-mediated tumor cytolysis. The specific subset that recognizes the surface-expressed hMSH2 on tumor cells was Vδ2 T cells, which could be activated to proliferate and produce IFN-y by rhMSH2 stimulation. Besides, the cell surface-expressed hMSH2 on carcinoma cells was charaterized as a full-length protein with a m.w. of 105 kDa by blotting purified membrane proteins with anti-hMSH2 antibody.The third issue of this study was to determine whether both TCRγδand NKG2D were involved in the recognition of surface hMSH2. Firstly, the expandedγδT effector cells (or NK-92 cells) were pretreated with anti-TCRγδand/or anti-NKG2D Ab before incubation with siRNA I-treated HeLa cells or mock control cells. The ectopic hMSH2-mediated recognition byγδT cells was significantly inhibited by anti-TCRγδ, anti-NKG2D or both mAbs. Secondly, affinity-purified rhMSH2 was used to confirm the specific binding of hMSH2 by TCRγδor NKG2D receptors with flow cytometry. Decreased fluorescence intensity of TCRγδor NKG2D was observed in rhMSH2-blocking groups, suggesting a competitive binding of hMSH2 to both receptors. Furthermore, surface plasmon resonance analysis suggested a direct binding of rhMSH2 to NKG2D-Fc. These results indicate that both TCRγδand NKG2D participate in the ectopic hMSH2-mediated recognition and cytolysis of carcinoma cells by humanγδT cells.The final issue of this study was to determine the inducible expression of hMSH2 on EBV-transformed human PBMCs and the role of hMSH2 inγδT cell-mediated anti-viral immunity. We infected PBMCs isolated from healthy donor with EBV and analyzed hMSH2 expression. We found that both mRNA transcription and cell-surface expression of hMSH2 were upregulated by EBV infection, leading to a substantial enhancement ofγδT cell-mediated responses towards EBV-transformed cells. The sensitivity of freshly generated EBV-transformed B cells to autologous or allogenicγδT cell-mediated cytolysis was 2-3 folds greater than that of normal B cells. Blocking with anti-hMSH2 antibody significantly downregulated the specific cytolysis of these EBV-infected cells. The ability of induced hMSH2 to facilitate the killing of EBV-transformed cells byγδT cells was followingly confirmed by specific siRNAs interferencing in lymphoblastoid cell line (LCL) 3D5. Consistent with that observed in carcinoma cells, specific cytolysis of siRNAs-treated 3D5 cells byγδT cells was significantly reduced. These results suggest that the cell surface-expressed hMSH2 is inducible and may mediate the specific clearance of EBV-infected cells by humanγδT cells.To sum up, we concluded that the ectopic surface expression of hMSH2 is broad on human carcinoma cells. The ectopically expressed hMSH2 interacts with both TCRγδand NKG2D and contributes to Vδ2 T cell mediated cytotoxicity towards hMSH2 bearing tumor cells. Moreover, the surface expression of hMSH2 is induced on EBV-transformed LCLs, which dramatically increases the sensitivity of these LCLs toγδT cell-mediated cytolysis. Our data suggest that hMSH2 functions as a tumor-associated or virus infection-related antigen recognized by both TCRγδand NKG2D, and plays a role in eliciting the immune responses ofγδT cells against tumor and virus-infected cells. The recognition of ectopically surface expressing endogenous antigen by TCRγδand NKG2D may be an important mechanism of innate immune response to carcinogenesis and viral infection.