A sensitive ELISPOT assay for detection of CD8+ T lymphocytes specific for HLA class I-binding peptide epitopes derived from influenza proteins in the blood of healthy donors and melanoma patients

Clin Cancer Res. 1997 Feb;3(2):221-6.

Abstract

An enzyme-linked immunospot (ELISPOT) assay was adapted to detect peptide-specific CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs). In HLA-A1-, HLA-A2-, and/or HLA-A3-positive individuals, we determined the release of IFN-gamma on a single cell level in response to three different peptide epitopes derived from the influenza matrix protein and nuclear protein containing the HLA-A2.1- and HLA-A1- or HLA-A3-binding motif, respectively. Comparison of the ELISPOT assay with the standard chromium release assay revealed a close correlation between the number of peptide-specific IFN-gamma-releasing T cells in PBMCs and the level of specific cytotoxicity after 14 days of in vitro expansion. The ELISPOT assay detected T cells with specificity for the HLA-A2. 1-binding epitope derived from the matrix protein in 76% of HLA-A2-positive healthy individuals (n = 25); the median frequency was 41 in 10(6) PBMCs. We also detected peptide-specific T cells in 10 of 12 HLA-A2-positive patients with metastatic melanoma with a median frequency of 20.5 in 10(6) PBMCs. In 10 of 24 HLA-A3-positive individuals and in 2 of 14 HLA-A1-positive individuals, peptide-specific T cells for a HLA-A3- and a HLA-A1-binding epitope derived from the nucleoprotein, respectively, were present. In conclusion, the ELISPOT assay may be suitable to monitor a peptide-specific T-cell response in vaccination protocols using peptides derived from tumor or viral antigens.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD8-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / metabolism
  • Cell Separation / methods
  • Epitopes, T-Lymphocyte / immunology
  • HLA-A1 Antigen / metabolism
  • HLA-A3 Antigen / metabolism
  • Histocompatibility Antigens Class I / immunology*
  • Histocompatibility Antigens Class I / metabolism
  • Humans
  • Immunoassay / methods*
  • Interferon-gamma / metabolism
  • Melanoma / blood
  • Melanoma / immunology*
  • Nucleoproteins / blood
  • Nucleoproteins / immunology
  • Orthomyxoviridae / immunology*
  • Orthomyxoviridae / metabolism
  • Peptides / immunology
  • Peptides / metabolism
  • T-Lymphocyte Subsets / immunology
  • T-Lymphocyte Subsets / metabolism
  • Viral Proteins / immunology*

Substances

  • Epitopes, T-Lymphocyte
  • HLA-A1 Antigen
  • HLA-A3 Antigen
  • Histocompatibility Antigens Class I
  • Nucleoproteins
  • Peptides
  • Viral Proteins
  • Interferon-gamma