title: The HLA-A,B "negative" mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon.
abstract: The HLA-A,B negative mutant cell line C1R is widely used as a transfection recipient in functional studies of class I MHC genes. It was derived from a normal B cell line, Hmy2, by three rounds of mutagenesis and immunoselection with anti-HLA mAb. Serology characterizes C1R to be negative for the HLA-A2, A3, B35, Bw62, and Cw3 Ag of the parental cell line while retaining expression of HLA-Cw4. We find, however, that CTL specific for HLA-B35 lyse C1R cells, suggesting that expression of HLA-B35 is also retained. To resolve this paradox we examined the expression of HLA-A,B,C genes and proteins in C1R cells. The results are consistent with deletion of the HLA-A3, Bw62, Cw3 haplotype and retention of the HLA-A2, B35, Cw4 haplotype in C1R. Although present, the HLA-A2 gene appears not to be transcribed. As expected, the HLA-Cw4 gene is transcribed and the protein expressed at normal levels. Transcription of the HLA-B35 gene is also normal and comparable to that of HLA-Cw4. However, expression of the HLA-B35 protein is reduced to a few percent of the parental level. Comparison of the nucleotide sequence of B35 alleles from C1R and Hmy2 revealed that reduced translation in C1R is caused by a point mutation (ATG to TTG) in the translation initiation codon. The HLA-B35 allele from C1R and Hmy2 represents a novel subtype, B*3503, differing from B*3501 by replacement of serine by phenylalanine at the peptide binding position 116. This study shows cell surface levels of a class I molecule which are insensitive to lysis by antibody and complement can be readily recognized by alloreactive T cells, further illustrating the relative sensitivity of Ag recognition by T cells.
authors.affiliation: Department of Cell Biology, Stanford University, CA 94305-5400.
authors.forename: J
authors.initials: J
authors.lastname: Zemmour
authors.forename: A M
authors.initials: AM
authors.lastname: Little
authors.forename: D J
authors.initials: DJ
authors.lastname: Schendel
authors.forename: P
authors.initials: P
authors.lastname: Parham
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D015870:Gene Expression; D005796:Genes; D015789:HLA-A2 Antigen / Q000235:genetics; D015797:HLA-B35 Antigen / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D012326:RNA Splicing; D016415:Sequence Alignment; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D015789:HLA-A2 Antigen; D015797:HLA-B35 Antigen