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 human CD40-Ig fusion protein in a murine graft-versus-host disease model.


METHODS
Human CD40 gene extracellular region was inserted into plasmid pIG1, which contains genomic human IgG1 Fc gene. A transient vector containing CD40-Fc fusion gene 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 transfecting it into CHO cells. CD40-Ig was purified by protein A affinity chromatography. 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 induced by intravenous injection of C57BL/6J (H-2b) spleen cells into sub-lethally irradiated BALB/c (H-2d) 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 described above. Chimeric proteins were expressed in COS-7 and CHO cell culture supernatant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion proteins had a disulfide-bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L. In vivo administration of CD40-Ig could prevent 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.
Liu
H
H
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Mao
N
N
Hou
C
C
Li
X
X
Shen
B
B
Tang
P H
PH
D000208:Acute Disease; D000818:Animals; D019013:CD40 Antigens / Q000627:therapeutic use*; D023201:CD40 Ligand / Q000378:metabolism; D005260:Female; D006086:Graft vs Host Disease / Q000276:immunology / Q000517:prevention & control*; D006801:Humans; D007074:Immunoglobulin G / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*; D013601:T-Lymphocytes / Q000276:immunology
D019013:CD40 Antigens; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand