Preparation Of Mouse Anti-human CXCR4 Monoclonal Antibody And Its Biological Effect On Tumor Cells And T Cell In Vitro

More attention was paid to CXCR4/SDF-1α,a pair of chemokine receptor and ligand currently.They are not only involved in cell growth,development,differentiation and proliferation,but also in a variety of pathological processes,such as cancer metastasis and inflammation.To be widely known,CXCR4/SDF-1αsignal regulates trafficking of leukocyte subsets to inflammatory sites through chemoattraction and by activating leukocyte integrins to bind their adhesion receptors on endothelial cells in the inflammatory response.Many studies had shown that tumor metastasis was similar with inflammatory cell infiltration,and cell rolling,adhesion and trans-endothelial migration are involved. More studies had reported that some tumor cells could express certain chemokines or chemokine receptors,and some chemokine signaling pathways were abnormal in tumor cells,suggesting that chemokine or its receptor may be involved in tumor metastasis by a similar mechanism of inflammatory cell infiltration.Currently CXCR4/SDF-1αsignal involved in cancer metastasis and proliferation,such as breast,lung,prostate cancer had been reported.Recently,it was found that in an effective immune response,CXCR4 /SDF-1αsignal not only chemotaxis T cells to the secondary lymphoid organs,but also had co-stimulatory effect on activation of T cells.This study aimed prepare mouse anti-human CXCR4 monoclonal antibody with CXCR4 gene transfected cell line(L929/CXCR4) as immunogen,and illustratedCXCR4/SDF-1αeffect on T cells as a co-stimulatory signal,and revealed biological significance of CXCR4/SDF-1αon glioma cells.PartⅠPreparation of mouse anti-human CXCR4 monoclonal antibodies and analysis of their biological characteristicsObjective:To prepare mouse anti-human CXCR4 monoclonal antibody for investigating CXCR4/SDF-1αbiological functions in vivo and the mechanisms.Methods:BALB/c mice were immunized with human CXCR4 transfectant L929/CXCR4 as an immungen.Mouse spleen B cells were fused with mouse plasmocytoma cells SP2/0.Hybridoma cells were screened with the transfectant L929/CXCR4.Fast-strip analysis was performed to identify Ig subclass of the generated monoclonal antibodies.The methods of immunophenotyping and western blot were designed to identify the specificity of generated mAbs.The epitopes recognized by mAbs were analyzed by competition assay.The expression of CXCR4 on subgroups of PBMCs was detected by FCM.Results:After multiple screening and subcloning,two monoclonal antibodies named 6H7 and 7D4 were obtained.Immunophenotyping showed that the two mAbs could bind to L929/CXCR4 transfected cells,but not to L929/mock,L929/CD133 and L929/HVEM cells,indicating that the two antibodies are specific for human CXCR4. The isotype of mAb 6H7 was proved to be IgG1 withκlight chain,while mAb 7D4 was IgG2a withκlight chain.Western blot showed mAb 7D4 could specifically bind to CXCR4 line-like molecule extracted from L929/CXCR4 cells,while 6H7 could not. The competition test conducted among the two mAbs evidenced that these mAbs were completely specific to different antigen binding sites of CXCR4.It was revealed that both mAbs could recognize CXCR4 in subgroups of PBMCs.Conclusion:Two anti-human CXCR4 monoclonal antibodies were generated, which recognize different epitopes.The successful preparation of mouse anti-human CXCR4 monoclonal antibody also provided materials to reveal CXCR4/SDF-1αbiological functions and the mechanisms. PartⅡProliferation and migration about glioma cell line mediated by CXCR4/SDF-1αsignal and blocking of monoclonal antibodyObjective:Preliminary analysis of CXCR4 expression on a few of tumor cell lines and glioma tissue and investigation about CXCR4/SDF-1αsignal biological function on glioma cell lines by monoclonal antibody against human CXCR4 preparedMethods:The expression of CXCR4 on tumor cell lines was analyzed by FCM with indirect immunofluorescence labeling.Immunohistochemistry was used to detect CXCR4 expression in glioma tissue.The proliferation of U251 cells stimulated with SDF-1αwas analyzed by MTT method.Blocking effect of 7D4 on proliferation of U251 cells was analyzed by MTT method.Migration of U251 glioma cells mediated by CXCR4/SDF-1αsignal was investigated with transwell chambers.Results:FCM analysis revealed that CXCR4 was expressed on the majority of tumor cell lines.In particular,CXCR4 expressed higher on tumor cell lines from hematopoietic system,while CXCR4 expressed relatively low on umor cell lines from epithelial-derived,such as M231,MCF-7 and 95D.Immunohistochemistry results showed CXCR4 expression on glioma tissue.Proliferation results of U251 cell line showed that,SDF-1αcould stimulate the proliferation of U251 with a concentration-dependent manner in vitro.CXCR4-specific monoclonal antibody 7D4 could block Proliferation of U251 cell line stimulated by SDF-1α.The transwell chambers results showed that monoclonal antibody 7D4 could block migration of U251 cells by SDF-1α-mediated.Conclusion:CXCR4 expression in a wide range of tumor cell lines and glioma tissue were analyzed and confirmed.CXCR4/SDF-1αsignals could affected glioma cell line U251 growth and metastasis in vitro,suggesting that the signal was related to the invasive growth pattern of glioma in vivoPartⅢCo-stimulatory function of human CXCR4 on T cells and the intervention effect of monoclonal antibodyObjective:To investigate CXCR4/SDF-1αco-stimulatory function on the by mAb and recombinant human SDF-1α,on the base of analysis of CXCR4 expression in immune cells.Methods:The expression of CXCR4 on subgroups of T cell stimulated with PHA was respectively analyzed by FCM.Human monocytes were cultured in the medium containing GM-CSF and IL-4 to be induced to imDCs and further to develop into mDCs driven by LPS.In this course CXCR4 expression was analyzed by FCM.With or without anti-CD28 mAb,human T cells were stimulated with anti-CD3 mAb in presence of SDF-1α,then the activation was detected by FCM,the proliferation of T cells was analyzed by MTT method,and the production of cytokines was measured by ELISA method.Furthermore,blocking function of monoclonal antibody 7D4 on co-stimulatory induced by SDF-1αwas detected.Results:When T cells were stimulated by PHA,CXCR4 expression on CD4~+T increased gently at the beginning and then decreased from 72 h.,while CXCR4 expression on CD8~+T showed no significant changes.In the process of monocytes being induced to DCs,CXCR4 expression was up-regulated.It was demonstrated that SDF-1αcould stimulate T cell proliferation and cytokines secretion,such as IL-2,IL-10 and IFN-γwith a dose dependent manner when anti-CD28 mAb was present,while it showed not without anti-CD28 mAb.With mAb 7D4, upregulation of proliferation and cytokine production mediated by SDF-1αwas suppressed.Even,CD25 and CD69 expression was decreased by mAb 7D4.Conclusion:By means of mAb 7D4,it was revealed that CXCR4 expression was regulated on immune cells.CXCR4/SDF-1αsignals together with CD28 signals on T cell activation had a co-stimulatory effect,mAb 7D4 could block CXCR4/SDF-1αsignals co-stimulatory effect.In summary,this paper has successfully accomplished the investigations as follows: Using the transfectant L929/CXCR4 as immungen,two specific anti-human CXCR4 monoclonal antibodies were generated.Further investigations indicated CXCR4 was expressed on the majority of tumor cell lines,and CXCR4/SDF-1αsignals could promoted glioma growth and migration in vitro,which suggested participation of the signal in the invasive growth pattern of glioma in vivo.Besides,Co-stimulatory function of human CXCR4 on T cells and the effect of monoclonal antibody were explored.It was proved that CXCR4/SDF-1αstimulated T cell proliferation and cytokines(IL-2, IL-10 and IFN-γ) secretion with a dose dependent manner when anti-CD28 mAb was present,while mAb 7D4 could block CXCR4/SDF-1αsignals co-stimulatory effect.