Molecular analysis of an HLA-DP mutant cell line selected for its resistance to killing by HLA-DPw2-specific T-cell clones.

Abstract

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.