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V set and Ig domain containing 4 (VSIG4), a co-inhibitory molecule expressed by macrophages, is a B7 family-related protein. It can serve as the second signal of T cell activation and regulate the function of T cells. It has been found that VSIG4 is essential in maintaining immune tolerance. Moreover, VSIG4 can also act as a complement receptor, playing a role in recognizing pathogens, regulating the complement alternative pathway and inhibiting inflammatory response. This review summarized the expression of VSIG4 and its role in the immune system.
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Objective To analyze the infiltration abundance of macrophage M2 in breast cancer tissues and explore the correlation between VSIG4 and macrophage M2 and the potential mechanism of regulating the invasion and migration of breast cancer patients. Methods We downloaded the RNA-seq data of TCGA-BRCA and assessed the infiltration abundance of immune cells in the samples by CIBERSORT, and established a prognostic risk prediction model. Then, we analyzed the effect of macrophage M2 and VSIG4 on the prognosis of breast cancer patients. In addition, we analyzed the signaling pathway associated with VSIG4 by gene set enrichment analysis and predicted its upstream regulation of miRNA. Results The infiltration abundance of macrophage M2, age, PR status and pathological stage were involved in the establishment of risk prediction model, and the model had a good prediction performance (AUC=0.816). High infiltration of macrophage M2 (HR=1.35, P < 0.05) and high expression of VSIG4 (HR=1.4, P=0.039) suggested poor prognosis of breast cancer patients. VSIG4 could be regulated by upstream miR-29a-3p and significantly correlated with Toll-like receptor, cell adhesion, production and release of cytokine. Conclusion VSIG4 is significantly associated with breast cancer patients' prognosis and infiltration of macrophage M2, regulated by the upstream miR-29a-3p and promotes the invasion and migration of breast cancer cells. It can be used as a potential prognostic marker for breast cancer.
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ABSTRACT:Objective To construct V-set and immunoglobulin domain containing 4 (Vsig4)nanobodies (Nbs) as specific macrophage probes so as to use them as molecular probes of macrophagocytes.Methods A nanobody phage library was generated by using peripheral blood lymphocytes isolated from an alpaca immunized with recombinant Vsig4 protein.After three rounds of selection against recombinant Vsig4.The Nbs were subjected to sequencing and genome alignment to obtain VHH sequence.Nbs were isolated and tested for Vsig4 specificity in an ELISA using recombinant Vsig4.The affinity capacity of Nbs was verified by the cell line stably expressing Vsig4. Results A nanobody phage library with an estimated 7.27 × 107 clones with 70% insertion was successfully constructed.Totally 1 3 6 Vsig4-positive clones were sequenced and aligned according to different CDR3 sequences. In summary,1 5 Vsig4 nanobodies were obtained and grouped into 3 different CDR3 epitopes.The affinity of representing nanobody and Vsig4 was analyzed via ELISA;Nb1 1 9 showed the highest affinity against both recombinant and native Vsig4.Conclusion We successfully constructed and screened Vsig4 specific nanobody number 1 1 9 with high affinity and specificity.It can help with macrophage detection and in vivo monitoring.
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Objective: To prepare anti-VSIG4 monoclonal antibodies and characterize their biological functions.Methods: BALB/c mice were immunized with transfected cell line (L929/VSIG4L) as immunogen.The spleen B cells of the mice were fused with SP2/0 and hybridoma cells were screened with transfected cell line (L929/VSIG4) by FCM.After acquisition of the hybridomas secreting anti-VSIG4 mAb,their biological activities were investigated by indirect immunofluorescence,Western blot,competitive inhibition test,and MTT assay.Results:Two stable hybridomas,9A7 and 9D5 were obtained,which could continuously secrete specific anti-VSIG4 monoclonal antibodies.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 and H446.Furthermore,they could block the inhibitory effects of VSIG4 on proliferation of T cells in vitro.Conclusion: Two hybridomas secreting anti-VSIG4 monoclonal antibodies have been established.These monoclonal antibodies provide useful tools for further studying VSIG4's biological function and its unknown receptor.