Biochemical and functional characteristics of soluble MHC molecules derived from H-2Ld/Q10d chimeric gene.
We have constructed a chimeric class I gene in which the 5' half of the H-2Ld gene is linked to the 3' half of Q10d. The resulting H-2Ld/Q10d protein is homologous to the native H-2Ld heavy chain for the three external domains except for an Arg to His substitution at position 260. The transmembrane and intracytoplasmic domains of the H-2Ld chain are replaced by the short low hydrophobic transmembrane-like domain of the Q10d chain. Following DNA-mediated gene transfer into mouse L cells, transformants were selected for the presence of specific mRNA. Radiolabelling and immunoprecipitation analysis revealed secretion of a 48-46 kd chain weakly associated with beta 2-microglobulin. This molecule reacts with H-2Ld-specific mAb that identify determinants on the first and second domains as well as with an anti-Q10 carboxyl-terminal peptide antiserum, but is not recognized by a mAb specific for a determinant of H-2Ld third domain. The integrity of antibody reactivity of the first and second domains together with beta 2-microglobulin association suggest that our molecule may be considered a good soluble counterpart of the native membrane H-2Ld molecule with which to perform functional studies. In order to analyze the immunogenic capacities and T-cell recognition of the soluble H-2Ld molecules, T-cell lines were produced from mice of various inbred strains immunized with supernatant from H-2Ld/Q10d-transfected fibroblasts. Characterization of these T cells revealed that they expressed a CD4+CD8- phenotype, and recognized H-2Ld/Q10d products in a class II-restricted manner.
Urbero
B
B
U93 INSERM, Hôpital Saint-Louis, Paris, France.
Gillet
D
D
Frangoulis
B
B
Pla
M
M
Kress
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D002460:Cell Line; D002678:Chimera / Q000235:genetics*; D004247:DNA / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D005805:Genes, MHC Class I / Q000235:genetics*; D008285:Major Histocompatibility Complex / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D013601:T-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D016827:CD8 Antigens; D012333:RNA, Messenger; D004247:DNA; D009439:Neuraminidase