Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein-Barr virus-specific CD8+ T cells

Kiyotaka Kuzushima, Naomi Hayashi, Ayumi Kudoh, Yoshiki Akatsuka, Kunio Tsujimura, Yasuo Morishima, Tatsuya Tsurumi

Research output: Contribution to journalArticle

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Abstract

We determined cytotoxic T lymphocyte (CTL) epitopes through screening with a computer-assisted algorithm and an enzyme-linked immunospot (ELISPOT) assay using in vitro-reactivated polyclonal Epstein-Barr virus (EBV)-specific CD8+ T cells as responders. In addition, to confirm that the epitopes were generated after endogenous processing and presentation of the EBV proteins, a novel T-cell receptor (TCR) down-regulation assay was introduced, in which a fluorescent tetrameric major histocompatibility complex (MHC)/peptide complex was employed for detecting TCR down-regulation after stimulation with the epitope presented on antigen-presenting cells. Through such screening, 3 HLA A*2402-restricted epitopes were identified: IYVLVMLVL, TYPVLEEMF, and DYNFVKQLF, derived from LMP2, BRLF1, and BMLF1 proteins, respectively. TCR down-regulation assays disclosed that, in contrast to the other 2 epitopes, IYVLVMLVL was not presented on HLA A24-positive fibroblast cells infected with recombinant vaccinia viruses expressing LMP2. Furthermore, ELISPOT assays with an epitope-specific CTL clone demonstrated that the presentation was partially restored by pretreatment of the fibroblast cells with interferon-γ. The epitope was presented on transporters associated with antigen processing (TAP)-negative T2 cells transfected with plasmids encoding HLA A*2402 and the minimal epitope, indicating that the presentation is TAP independent. In conclusion, the 3 epitopes thus defined could be useful for studying EBV-specific CD8+ T-cell responses among populations positive for HLA A*2402.

Original languageEnglish
Pages (from-to)1460-1468
Number of pages9
JournalBlood
Volume101
Issue number4
DOIs
Publication statusPublished - 15-02-2003

Fingerprint

HLA-A Antigens
T-cells
Human Herpesvirus 4
Viruses
Epitopes
T-Lymphocytes
Assays
T-Cell Antigen Receptor
Enzyme-Linked Immunospot Assay
Down-Regulation
Cytotoxic T-Lymphocytes
Fibroblasts
Screening
HLA-A24 Antigen
Cells
T-Lymphocyte Epitopes
Vaccinia virus
Antigen-Presenting Cells
Enzymes
Major Histocompatibility Complex

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

Cite this

Kuzushima, K., Hayashi, N., Kudoh, A., Akatsuka, Y., Tsujimura, K., Morishima, Y., & Tsurumi, T. (2003). Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein-Barr virus-specific CD8+ T cells. Blood, 101(4), 1460-1468. https://doi.org/10.1182/blood-2002-04-1240
Kuzushima, Kiyotaka ; Hayashi, Naomi ; Kudoh, Ayumi ; Akatsuka, Yoshiki ; Tsujimura, Kunio ; Morishima, Yasuo ; Tsurumi, Tatsuya. / Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein-Barr virus-specific CD8+ T cells. In: Blood. 2003 ; Vol. 101, No. 4. pp. 1460-1468.
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Kuzushima, K, Hayashi, N, Kudoh, A, Akatsuka, Y, Tsujimura, K, Morishima, Y & Tsurumi, T 2003, 'Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein-Barr virus-specific CD8+ T cells', Blood, vol. 101, no. 4, pp. 1460-1468. https://doi.org/10.1182/blood-2002-04-1240

Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein-Barr virus-specific CD8+ T cells. / Kuzushima, Kiyotaka; Hayashi, Naomi; Kudoh, Ayumi; Akatsuka, Yoshiki; Tsujimura, Kunio; Morishima, Yasuo; Tsurumi, Tatsuya.

In: Blood, Vol. 101, No. 4, 15.02.2003, p. 1460-1468.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein-Barr virus-specific CD8+ T cells

AU - Kuzushima, Kiyotaka

AU - Hayashi, Naomi

AU - Kudoh, Ayumi

AU - Akatsuka, Yoshiki

AU - Tsujimura, Kunio

AU - Morishima, Yasuo

AU - Tsurumi, Tatsuya

PY - 2003/2/15

Y1 - 2003/2/15

N2 - We determined cytotoxic T lymphocyte (CTL) epitopes through screening with a computer-assisted algorithm and an enzyme-linked immunospot (ELISPOT) assay using in vitro-reactivated polyclonal Epstein-Barr virus (EBV)-specific CD8+ T cells as responders. In addition, to confirm that the epitopes were generated after endogenous processing and presentation of the EBV proteins, a novel T-cell receptor (TCR) down-regulation assay was introduced, in which a fluorescent tetrameric major histocompatibility complex (MHC)/peptide complex was employed for detecting TCR down-regulation after stimulation with the epitope presented on antigen-presenting cells. Through such screening, 3 HLA A*2402-restricted epitopes were identified: IYVLVMLVL, TYPVLEEMF, and DYNFVKQLF, derived from LMP2, BRLF1, and BMLF1 proteins, respectively. TCR down-regulation assays disclosed that, in contrast to the other 2 epitopes, IYVLVMLVL was not presented on HLA A24-positive fibroblast cells infected with recombinant vaccinia viruses expressing LMP2. Furthermore, ELISPOT assays with an epitope-specific CTL clone demonstrated that the presentation was partially restored by pretreatment of the fibroblast cells with interferon-γ. The epitope was presented on transporters associated with antigen processing (TAP)-negative T2 cells transfected with plasmids encoding HLA A*2402 and the minimal epitope, indicating that the presentation is TAP independent. In conclusion, the 3 epitopes thus defined could be useful for studying EBV-specific CD8+ T-cell responses among populations positive for HLA A*2402.

AB - We determined cytotoxic T lymphocyte (CTL) epitopes through screening with a computer-assisted algorithm and an enzyme-linked immunospot (ELISPOT) assay using in vitro-reactivated polyclonal Epstein-Barr virus (EBV)-specific CD8+ T cells as responders. In addition, to confirm that the epitopes were generated after endogenous processing and presentation of the EBV proteins, a novel T-cell receptor (TCR) down-regulation assay was introduced, in which a fluorescent tetrameric major histocompatibility complex (MHC)/peptide complex was employed for detecting TCR down-regulation after stimulation with the epitope presented on antigen-presenting cells. Through such screening, 3 HLA A*2402-restricted epitopes were identified: IYVLVMLVL, TYPVLEEMF, and DYNFVKQLF, derived from LMP2, BRLF1, and BMLF1 proteins, respectively. TCR down-regulation assays disclosed that, in contrast to the other 2 epitopes, IYVLVMLVL was not presented on HLA A24-positive fibroblast cells infected with recombinant vaccinia viruses expressing LMP2. Furthermore, ELISPOT assays with an epitope-specific CTL clone demonstrated that the presentation was partially restored by pretreatment of the fibroblast cells with interferon-γ. The epitope was presented on transporters associated with antigen processing (TAP)-negative T2 cells transfected with plasmids encoding HLA A*2402 and the minimal epitope, indicating that the presentation is TAP independent. In conclusion, the 3 epitopes thus defined could be useful for studying EBV-specific CD8+ T-cell responses among populations positive for HLA A*2402.

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