The Biological Characteristics Of Human VSIG4 And Its Co-inhibitory Function On T Cells

VSIG4 (V-set and Ig domain containing 4), also known as CRIg, is a typeⅠtransmembrane Ig superfamily member. VSIG4 contains typical features of B7 family members, suggesting that it is a B7 family–related protein. VSIG4 was initially cloned as an Ig superfamily gene (Z39Ig). The degree of sequence identity and the chromosomal location identify Z39Ig as the human VSIG4. Like many members of the Ig superfamily, VSIG4 has typical extracellular Ig domains. Human VSIG4 exists as two alternatively spliced isoforms. One is the longer form of human VSIG4 (VSIG4L), and there are 1200bp in its coding region that can encodes both IgV and IgC type terminal Ig domains in the extracellular region. The other is the short form(VSIG4S, 918bp), which encodes a single IgV-type Ig domain in the extracellular region, and whose other regions is identical with VSIG4L. Human VSIG4L is also known as Z39Ig. However, the murine VSIG4 exists only one spliced isoform, and lacks an extracellular IgC domain like human VSIG4S. Both human and murine VSIG4 are located on the X chromosome at position Xq12 between hephaestin and moesin. Human VSIG4 mRNA is mainly expressed in placenta, fetus tissue, adult liver, lung and many other tissues. VSIG4 molecule is principally found on monocyte-derived macrophages, but not on monocytes. And when macrophages were activated, the expression of VSIG4 on the macrophages were downregulated. This VSIG4 down regulation tendency also occur on the macrophages in inflammation tissues. VSIG4 expression can not be detected in B cells, T cells, NK cells and granulocytes in PBMC. Some prior studies had shown that VSIG4 was also a complement receptor on macrophages and could bind the C3 cleaved products, C3b or iC3b molecules, which is required for phagocytosis of circulating pathogens. And a recent study had identified that VSIG4 is also a new B7 family member. The experiments in vitro showed that VSIG4 is a strong negative regulator of T cell proliferation. As human VSIG4 is a newly recognized molecule, there are many unkown function and information about VSIG4 immunoregulation on T cells. In order to study VSIG4's biological effects and mechanism of its inhibition on T cells, our reseach works were carried out as follow: to clone the two alternatively spliced forms of human VSIG4 gene (VSIG4L and VSIG4S), establish VSIG4 transgenic cell lines, prepare the VSIG4-Fc fusion protein and aslo use the VSIG4 transgenic cell lines as an immunogen to prepare mouse anti-human VSIG4 monoclonal antibody. Then using above materials we planed to illustrate the biological characteristics of human VSIG4 molecule as well as its coinhibitory function on T cells.PartⅠCloning of human VSIG4 gene, establishment of transgenic cell lines and preliminary studying of the biological function of the transfectantsObjective: To clone human complementary VSIG4 gene (VSIG4L and VSIG4S), construct recombinant retrovirus vectors carrying human VSIG4 gene (VSIG4L and VSIG4S), which can be stably expressed in L929 and 293T cell lines, and study the transfectant cells'function on T cells.Methods: The full length human VSIG4 genes (VSIG4L and VSIG4S) were amplified by RT-PCR from the human normal lung. Digested with restriction endonuclease Pst I and EcoR I, each of the VSIG4 gene was inserted into retrovirus vector pEGZ-Term. The recombinant vector together with its two helper virus vectors was co-transfected into the package cell 293T with LipofectAMINE 2000TM. The supernatant of 293T was used to infect L929 cells. L929 cells and 293T cells stably expressing VSIG4L and VSIG4S protein were selected in the presence of zeocin, and then analyzed and identified by RT-PCR, Western-blot and FCM. The preliminary functions of VSIG4L and VSIG4S for proliferation and IL-2 secretion of T cells were studied by MTT and ELISA respectively.Results: Both of the full longth genes of human VSIG4L and VSIG4S were successfully cloned, and their recombinant retrovirus vectors were constructed. After identified by flow cytometry, RT-PCR and Western blot analysis, the transfectants stably expressing human VSIG4L and VSIG4S protein on the cells membrane were established successfully. In vitro, L929/VSIG4 cells had an inhibitory effect on the proliferation and IL-2 secretion of T cells stimulated by anti-CD3 mAb in the presence or absence of anti-CD28 mAb.Conclusion: The transgenic cell lines stably expressing human VSIG4L and VSIG4S molecule on the cells surface have been obtained and afford effective immunogen for preparing mouse anti-human VSIG4 monoclonal antibody, as well as the experimental materials for the following studies.PartⅡPreparation of VSIG4-Fc Fusion ProteinObjective: To construct CHO cell lines that can stably express and secrete human VSIG4L-Fc and VSIG4S-Fc fusion proteins, and purify these fusion proteins.Methods: Extracellular domain of human VSIG4L and VSIG4S genes were amplified by PCR from pMD19-T-VSIG4L and pMD19-T-VSIG4S, the crystalline fragment (Fc) of human IgG1 was obtained through PCR from pMD18-T-hIgG1Fc. Then, the genes of VSIG4 and Fc were inserted into retrovirus vector pEGZ-Term to construct the recombinant vector pGEZ-Term-VSIG4L-Fc and pGEZ-Term-VSIG4S-Fc. The recombinant vectors together with its 2 helper virus vectors were cotransfected into the package cell 293T. The supernatant of 293T containing intact virus granules was used to infect CHO cells. CHO cells stably expressing VSIG4-Fc protein were selected in the presence of Zeocin, and then analyzed by FCM, RT-PCR and Dot-blot. The condensed FCS free supernatant of infected CHO cells was harvested and purified through protein G affinity chromatography column. The purified fusion proteins were identified by western blot, and FCM was used to detect the binding of fusion proteins with T cells.Results: The recombinant retrovirus vectors pEGZ-Term-VSIG4L-Fc and pEGZ- Term-VSIG4S-Fc were constructed successfully. The selected transfected CHO cells can stably express and secrete VSIG4L-Fc or VSIG4S-Fc fusion protein that can bind the unknown receptor on the T cells. Conclusion: The transgenic CHO cell lines stably express and secrete human VSIG4L-Fc and VSIG4S-Fc fusion protein have been obtained. The fusion proteins were purified and provides experiment materials to the following studies.PartⅢPreparation and characterization of the monoclonal antibodies against human VSIG4Objective: To prepare mouse anti-human VSIG4 monoclonal antibodies applied to illuminate the regulatory expression and co-inhibitory function of human VSIG4 on T cells.Methods: BALB/c mice were immunized with human VSIG4L transfectant L929/VSIG4L as immunogen. Mouse spleen B cells were fused with mouse plasmocytoma cells SP2/0. Hybridoma cells were screened with human VSIG4L and VSIG4S transfected cell lines. Fast-strip analysis was performed to identify Ig subclass of the generated monoclonal antibodies. The methods of indirect immunofluorescence 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 VSIG4 on different human tumor cell lines, and macrophages was detected by FCM.Results: After multiple screening and subcloning, three monoclonal antibodies named 2H4, 9A7 and 9D5 were obtained. The isotype of the three mAbs were proved to be IgG1 withκlight chain. Indirect immunofluorescence showed that mAbs 2H4 and 9A7 could bind to VSIG4L and VSIG4S transfected cells specifically, and 9D5 could only bind to VSIG4L transfected cells, but not to VSIG4S. Western blot showed that mAbs 9A7 could specifically bind VSIG4L and VSIG4S, 9D5 could only bind to VSIG4L, and 2H4 could bind neither VSIG4L nor VSIG4S. The mAb competition test conducted among the three mAbs evidenced that these three mAbs were completely specific to different antigen binding sites. The following biological activity studies showed that these monoclonal antibodies could recognize the natural VSIG4 expressed on the macrophages and several cancer cell lines, such as Jurkat, THP-1, H446 and so on.Conclusion: Three anti-human VSIG4 monoclonal antibodies were generated, which recognize three epitopes. The successful preparation of mouse anti-human VSIG4 monoclonal antibody also provided materials to reveal the expression characteristics of human VSIG4 molecule on immunocytes and to investigate its functional mechanism on T cells.PartⅣStudy of inhibitory function of human VSIG4 on T cellsObjective: To analyze the co-inhibitory function of human VSIG4 on T cells by VSIG4 transfected cells, VSIG4 fusion protein and the monoclonal antibodies.Methods: The purified T cells or CD4+, CD8+ T cells, which were stimulated by fixed anti-CD3 in the presence or absence of anti-CD28, were employed as effector cells. Effect of the VSIG4L and VSIG4S transfected cells, VSIG4L-Fc and VSIG4S-Fc fusion proteins, mouse anti-human VSIG4 monoclonal antibodies on T cells'proliferation and IL-2 secretion in vitro was studied by means of FCM, MTT and ELISA.Results:1. VSIG4L-Fc and VSIG4S-Fc were a strong inhibitor of T cell proliferation and IL-2 secretion induced by fixed anti-CD3 in the presence or absence of anti-CD28(P<0.05), and the inhibition strength of VSIG4-Fc were similar to PD-L1-F(cP>0.05). And there was no statistics difference between the roles of VSIG4L-Fc and VSIG4S-Fc.2. The VSIG4L and VSIG4S expressing on the membrane of 293T cell were not only a strong inhibitor to T cells, but aslo to CD4+ and CD8+ T cells. IL-2 secretion of CD4+ T cells was also inhibited by the VSIG4 membrane protein. And there was no statistics difference between VSIG4L and VSIG4S transfected 293T cells groups.3. When T cells were stimulated by fixed anti-CD3 and inhibited by VSIG4L transfected cells, the mAbs 2H4, 9A7, 9D5 were added in order to block the VSIG4 inhibition effects. The result of MTT assay showed that mAb 9A7 can block the VSIG4L and VSIG4S inhibition effects, but the blocking effect of 2H4 and 9D5 were no statistics difference compared with IgG control group.4. T cells costimulation and co-inhibition assays were performed with a fixed concentration of anti-CD3 and VSIG4L-Fc, the inhibitory activity of VSIG4-Fc compared with IgG1-treated cells was significant (P<0.05), and could be abolished by the addition of IL-2. Although enhancing the concentration of VSIG4-Fc, its inhibitory activity could also be supressed in the presence of IL-2 (P>0.05).Conclusion: VSIG4 may play a negative role in regulation of T cell proliferation and IL-2 production. And there was not significant between VSIG4L and VSIG4S. But mAb 9A7 can block this inhibition of VSIG4 in a certain degree. And the inhibitory activity of VSIG4-Fc could also be abolished by the addition of IL-2.