Anti-Thy-1 antibody responses evoked by Thy-1 antigen expressed in transfected mouse mastocytoma cells and rat fibroblast.
The mouse genomic Thy-1.1 gene was isolated from a phage library constructed from AKR/J (Thy-1.1) mouse DNA. Partial nucleotide sequence analysis of the coding region showed that it has only a single nucleotide difference from the Thy-1.2 gene, namely that amino acid 89 reads CGA (Arg) in Thy-1.1 and CAA (Glu) in Thy-1.2, corresponding to the amino acid substitutions previously identified. It was subcloned into an SV-40 derived vector for transfection. Transient transfection into HeLa cells gave 2% positive staining by immunofluorescence. The gene in this vector was also co-transfected into L cells and mastocytoma cells (both of Thy-1.2 strain origin) together with the Agpt gene. L-cell clones selected for transformation proved almost negative for Thy-1.1 expression, and any positive clones gradually lost Thy-1.1 antigen expression in culture. On the contrary, all clones of mastocytoma transformants gave a high level of expression after more than 3 months in culture. The mastocytoma transformants were used to study the immunogenicity of Thy-1.1 molecules expressed on transfected cells. They evoked clear anti-Thy-1.1 plaque-forming cell (PFC) responses both in vivo and in vitro. The mastocytoma transformants also proved able to induce a T-dependent anti-Thy-1.1 antibody response in a cell transfer experiment. The immunogenicity of Thy-1.2 molecules on rat fibroblasts was also studied after transfection with a Thy-1.2 gene cosmid. Although Thy-1.2 expression was very low, these transfectants elicited a clear anti-Thy-1.2 PFC response from AKR spleen cells hyperimmunized against CBA thymocytes.
Isobe
K
K
Fortunato
A
A
Giguere
V
V
Grosveld
F
F
Mitchison
N A
NA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000912:Antibodies, Neoplasm / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology*; D001483:Base Sequence; D005347:Fibroblasts / Q000276:immunology; D005802:Genes, MHC Class II; D006367:HeLa Cells; D006462:Hemolytic Plaque Technique; D007518:Isoantibodies / Q000096:biosynthesis / Q000276:immunology*; D007739:L Cells; D012515:Mast-Cell Sarcoma / Q000276:immunology; D051379:Mice; D008806:Mice, Inbred AKR; D008808:Mice, Inbred CBA; D051381:Rats; D018800:Thy-1 Antigens; D014162:Transfection
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000954:Antigens, Surface; D007518:Isoantibodies; D018800:Thy-1 Antigens; C035678:anti-Thy antibody