Mutations in the alpha 2 helix of HLA-A2 affect presentation but do not inhibit binding of influenza virus matrix peptide.

Abstract

Previous studies have suggested that MHC class I molecules bind and present peptides to CTL in a manner that is analogous to the presentation of peptides by class II molecules to Th. Crystallographic studies of HLA-A2 have led to the assignment of a putative peptide binding site that is bordered by two alpha helices consisting of residues 50-84 and 138-180. In this study, we have investigated whether residues in the alpha 2 helix are involved in the binding and/or presentation of a peptide to CTL. We have generated CTL to type A influenza virus by stimulation of human PBL with a synthetic peptide from the influenza A virus matrix protein (M1 residues 57-68) in the presence of rIL-2. Such HLA-A2.1-restricted influenza virus-immune CTL do not recognize infected HLA-A2.3+ targets. A2.1 and A2.3 differ by three amino acids in the alpha 2 domain: Ala vs. Thr at position 149, Val vs. Glu at position 152, and Leu vs. Trp at position 156. Site-directed mutants of the A2.1 gene that encode A2 molecules that resemble A2.3 at positions 149, 152, and 156 have been constructed, transfected into human cells, and assayed for their ability to present the M1 peptide. The results demonstrate that most, but not all, A2.1-restricted M1-peptide-specific CTL fail to recognize M1 peptide-exposed transfectants with certain single amino acid substitutions at positions 152 and 156. In contrast, M1 peptide-exposed transfectants that express A2 molecules with an Ala----Thr substitution at position 149 were recognized by all CTL tested, but they exhibited an apparent difference in the kinetics of peptide binding. These results indicate that amino acid substitutions at positions 152 and 156 of the putative peptide binding site of the A2 molecule can affect presentation without eliminating binding, and indicate that the failure to recognize complexes between the peptide and the mutant A2 molecules is due to different TCR specificities and not to the failure to bind the peptide.

Analyses of Recombinant Vaccinia and Fowlpox Vaccine Vectors Expressing Transgenes for Two Human Tumor Antigens and Three Human Costimulatory Molecules

“…The cultures were free of mycoplasma and were maintained in complete medium [RPMI 1640 (Invitrogen Life Technologies Inc., Carlsbad, CA) supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 Ag/mL streptomycin (Invitrogen Life Technologies)]. The C1R cell line is a human plasma leukemia cell line that does not express endogenous HLA-A or B antigens (46). C1R-A2 cells are C1R cells that express a transfected genomic clone of HLA-A2.1 (47).…”

Autoreactive, Cytotoxic T Lymphocytes Specific for Peptides Derived from Normal B-Cell Differentiation Antigens in Healthy Individuals and Patients with B-Cell Malignancies

“…They expresses very low levels of cell surface HLA-A 2.1 and are unable to present endogenous antigens because of the lack of most of the MHC class II region including the known TAP (transporter proteins for antigenic peptide) and proteasome genes (37). C1R-A2 cells are a MHC-class I-defective LCL cell line, that expresses a transfected genomic clone of HLA-A2.1 (38,39) and were kindly provided by the laboratory of Dr. Jeffrey Schlom (National Cancer Institute, NIH, Bethesda, MD). The cells were maintained in CM supplemented with 10% FCS.…”

Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation.

“…Although preliminary fine mapping of the epitope specificity of CTL clone 35D45 was performed (see below), the limited ability to propagate clone 35D45 precluded definitive fine mapping of the epitope specificity of this clone. Attempts to produce a CTL line specific for peptide env/128 from subject 010-035i by in vitro stimulation of PBMC with synthetic peptide as previously described for influenza (20) were unsuccessful (data not shown). To fine map the HLA-B8-restricted CTL epitope, an envelope-specific CTL line was therefore derived from subject 010-035i using an autologous, envelope peptide-sensitized CD4 + T lymphocyte clone.…”