Abstract
Background: Macrophages are essential cellular components in various body tissues and tumor microenvironments.The high infiltration of macrophages into the tumor microenvironment determines the importance of ex vivo treatment of personalized macrophages with recombinant cytotoxic T-lymphocyteassociated protein 4 (rCTLA-4), programmed death-ligand 1 (rPD-L1), and programmed cell death protein 1 (rPD-1) proteins to block immune checkpoints.Methods: We investigated the development of humoral immunity against CTLA-4, PD-L1, and PD-1 receptors by introducing macrophages treated ex vivo with the corresponding proteins into mice.Peritoneal macrophages from BALB/c mice were cultured in medium containing recombinant human CTLA-4, PD-L1, and PD-1 proteins.Macrophages processing recombinant proteins were analyzed via immunofluorescence staining using antibodies against CTLA-4, PD-L1, and PD-1.The treated macrophages were administered intraperitoneally to mice to induce anti-CTLA-4, anti-PD-L1, and anti-PD-1 antibodies.The antibody titer in vaccinated mice was determined via enzyme-linked immunosorbent assays, followed by statistical analysis of the results.The specificity of the antibodies was determined via immunofluorescence staining in MCF7 cells. Results:The ex vivo treatment of macrophages with rCTLA-4, rPD-L1, and rPD-1 induced the formation of specific antibodies in vaccinated mice.The various rPD-L1 and rPD-1 concentrations used to treat macrophages had no significant effect on the specific antibody titers, while the anti-rCTLA-4 titer was dependent on the protein concentration in the culture medium.Immunofluorescence analysis revealed that anti-CTLA-4 and PD-L1 antibodies reacted with MCF7 cells. Conclusions:The ex vivo treatment of macrophages with rCTLA-4, rPD-L1, and rPD-1 can help induce humoral immunity and develop new approaches for cancer immunotherapy.