Malika Nurtleu, Zhansaya Adish, Kasym Mukanov, Kanat Tursunov, Yerlan Ramankulov, Kanatbek Mukantayev
Republican State Enterprise National Center for Biotechnology, Ministry of Healthcare of the Republic of Kazakhstan, Nur-Sultan, 010000, Kazakhstan.
L.N. Gumilyov Eurasian National University, Satpayev st., 2, Nur-Sultan, 010008, Kazakhstan.
Rep Biochem Mol Biol. 2023 Jan;11(4):694-701. doi: 10.52547/rbmb.11.4.694.
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 treatment of personalized macrophages with recombinant cytotoxic T-lymphocyte-associated protein 4 (rCTLA-4), programmed death-ligand 1 (rPD-L1), and programmed cell death protein 1 (rPD-1) proteins to block immune checkpoints.
We investigated the development of humoral immunity against CTLA-4, PD-L1, and PD-1 receptors by introducing macrophages treated 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.
The 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.
The treatment of macrophages with rCTLA-4, rPD-L1, and rPD-1 can help induce humoral immunity and develop new approaches for cancer immunotherapy.
巨噬细胞是各种身体组织和肿瘤微环境中的重要细胞成分。巨噬细胞大量浸润到肿瘤微环境中，这决定了用重组细胞毒性T淋巴细胞相关蛋白4（rCTLA - 4）、程序性死亡配体1（rPD - L1）和程序性细胞死亡蛋白1（rPD - 1）蛋白治疗个性化巨噬细胞以阻断免疫检查点的重要性。
我们通过将用相应蛋白处理过的巨噬细胞引入小鼠体内，研究了针对CTLA - 4、PD - L1和PD - 1受体的体液免疫的发展。从BALB/c小鼠获取的腹腔巨噬细胞在含有重组人CTLA - 4、PD - L1和PD - 1蛋白的培养基中培养。使用针对CTLA - 4、PD - L1和PD - 1的抗体，通过免疫荧光染色分析处理重组蛋白的巨噬细胞。将处理过的巨噬细胞腹腔注射给小鼠以诱导抗CTLA - 4、抗PD - L1和抗PD - 1抗体。通过酶联免疫吸附测定法测定接种疫苗小鼠体内的抗体滴度，随后对结果进行统计分析。通过在MCF7细胞中的免疫荧光染色确定抗体的特异性。
用rCTLA - 4、rPD - L1和rPD - 1处理巨噬细胞可诱导接种疫苗小鼠体内形成特异性抗体。用于处理巨噬细胞的各种rPD - L1和rPD - 1浓度对特异性抗体滴度没有显著影响，而抗rCTLA - 4滴度取决于培养基中的蛋白浓度。免疫荧光分析表明，抗CTLA - 4和PD - L1抗体与MCF7细胞发生反应。
用rCTLA - 4、rPD - L1和rPD - 1处理巨噬细胞有助于诱导体液免疫并开发癌症免疫治疗的新方法。
