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Blood, 15 September 2001, Vol. 98, No. 6, pp. 1872-1881
IMMUNOBIOLOGY
Efficient identification of HLA-A*2402-restricted
cytomegalovirus-specific CD8+ T-cell epitopes by a computer
algorithm and an enzyme-linked immunospot assay
Kiyotaka Kuzushima,
Naomi Hayashi,
Hiroshi Kimura, and
Tatsuya Tsurumi
From the Division of Virology, Aichi Cancer Center
Research Institute, Nagoya, Japan; and the Department of Pediatrics,
Nagoya University School of Medicine, Japan.
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- 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.
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