Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8(+) T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay.
Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are useful tools for studying the CTL responses exclusively among those who own the major histocompatibility complex (MHC) class I molecules that present the peptides. For widening the application, an efficient strategy to determine such epitopes in the context of a given MHC is highly desirable. A rapid and efficient strategy is presented for the determination of CTL epitopes in the context of given MHC molecules of interest through multiple screenings consisting of a computer-assisted algorithm and MHC stabilization and enzyme-linked immunospot assays. A major cytomegalovirus (CMV)-specific CTL epitope, QYDPVAALF, in the amino acid sequence of its lower matrix 65 kd phosphoprotein (pp65) presented by HLA-A*2402 molecules was identified from 83 candidate peptides. The results indicate that the CMV-specific CTL response is highly focused to pp65 in the context of HLA-A*2402. Endogenous processing and presentation was confirmed using a peptide-specific CD8(+) T-cell clone as the effectors and autologous fibroblast cells infected with recombinant vaccinia virus expressing pp65 gene or CMV as antigen-presenting cells. Flow cytometric analysis of intracellular interferon-gamma production revealed 0.04% to 0.27% of CD8(+) T cells in peripheral blood of HLA-A24(+) and CMV-seropositive donors to be specific for the peptide. The tetrameric MHC-peptide complexes specifically bound to the reactive T-cell clone and 0.79% of CD8(+) T cells in peripheral blood from a seropositive donor. The peptide could be a useful reagent to study CTL responses to CMV among populations positive for HLA-A*2402.
Kuzushima
K
K
Division of Virology, Aichi Cancer Center Research Institute, Nagoya, Japan. kkuzushi@aichi-cc.pref.aichi.jp
Hayashi
N
N
Kimura
H
H
Tsurumi
T
T
D000465:Algorithms; D000595:Amino Acid Sequence; D002460:Cell Line; D002999:Clone Cells; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D015234:HLA-A Antigens / Q000276:immunology*; D059871:HLA-A24 Antigen; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D009716:Numerical Analysis, Computer-Assisted; D010455:Peptides / Q000276:immunology; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014616:Vaccinia virus / Q000235:genetics; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D018984:Epitopes, T-Lymphocyte; D015234:HLA-A Antigens; D059871:HLA-A24 Antigen; D015395:Histocompatibility Antigens Class I; D010455:Peptides; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa