Protective effect of human CD40-Ig fusion protein in a murine model of acute graft-versus-host disease

Objective To investigate the protective effects of blocking CD40/CD40L interactions with h uman CD40-Ig fusion protein in a murine graft-versus-host disease model.Methods Human CD40 gene extracellular region was inserted into plasmid pIG1, which conta ins genomic human IgG1 Fc gene. A transient vector containing CD40-Fc fusion g ene was transfected into COS-7 cells. The CD40-Ig fusion protein was detected through enzyme-linked immunosorbent assay (ELISA). A constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1 and transfect ing it into CHO cells. CD40-Ig was purified by protein A affinity chromatograp hy. SDS-PAGE, Western blot and ligand binding assay were used to identify the qualities of CD40-Ig. Murine acute graft-versus-host disease (GVHD) was ind uced by intravenous injection of C57BL/6J (H-2(b)) spleen cells into sub-letha lly irradiated BALB/c (H-2(d)) mice. Protective effects against murine graft- versus-host disease by in vivo administration of CD40-Ig were evaluated. Results Mammalian expression vectors pIG/40Ig and p3.1/40Ig were constructed as describ ed above. Chimeric proteins were expressed in COS-7 and CHO cell culture super natant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion pr oteins had a disulfide-bonded dimeric structure and existed as homodimer. Puri fied CD40-Ig could bind to CD40L. In vivo administration of CD40-Ig could pre vent the development of GVHD and significantly prolong the mean survival time of mice with graft-versus-host disease.Conclusions These results demonstrate that CD40/CD40L interactions play an important role in the pathogenesis of graft-versus-host disease and suggest clinical potential for CD40-Ig in the prevention and treatment of human graft-versus-host disease.