Molecular analysis of an HLA-DP mutant cell line selected for its resistance to killing by HLA-DPw2-specific T-cell clones.
A collection of HLA-DP mutants was generated, using ICR 191 as the mutagenic agent and resistance to lysis mediated by HLA-DPw2 allospecific cytotoxic T lymphocytes (CTLs) as the selection criterion. These mutants were derived from the HLA haploid lymphoblastoid cell line 45.1. Loss of HLA-DPw2 surface expression accounted for the lack of HLA-DPw2 CTL recognition in all the mutants. However, one of them, 45.EM19, binds to DPw2-specific monoclonal antibodies (mAb) after cell permeabilization. HLA-DPA1 and DPB1 mRNA expression studies permitted the classification of the mutants in four categories: 1) DPA1-negative, DPB1-positive; 2) DPA1-positive, DPB1-negative; 3) DPA1- and DPB1-negative, and 4) DPA1- and DPB1-positive mutants. Mutant 45.EM19 is included in the last group. The cloning and sequencing of the full-length DPA1 (DPA1*0103) and DPB1 (DPB1*02012) cDNAs from this mutant showed no changes in the DPA1 sequence compared to the wild-type sequence. However, a frame-shift mutation in the DPB1 gene exon coding for the transmembrane region was detected. The insertion of a guanine nucleotide provokes an extension of the open reading frame, increasing the length of the C-terminal domain and changing the hydropathicity pattern of the transmembrane domain. This change should be responsible for the phenotype of the 45.EM19 mutant.
Arroyo
J
J
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Díez-Orejas
R
R
Alvarez
A M
AM
Shaw
S
S
Sánchez-Pérez
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D005434:Flow Cytometry; D016368:Frameshift Mutation; D015870:Gene Expression; D006682:HLA-DP Antigens / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D059867:HLA-DP alpha-Chains; D059868:HLA-DP beta-Chains; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014158:Transcription, Genetic
D017931:DNA Primers; D006682:HLA-DP Antigens; D059867:HLA-DP alpha-Chains; D059868:HLA-DP beta-Chains; C071172:HLA-DPA1 antigen; C071170:HLA-DPB1 antigen; C083176:HLA-DPw2 antigen; D012333:RNA, Messenger