Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens

J Immunol Methods. 1996 May 27;191(2):131-42. doi: 10.1016/0022-1759(96)00007-5.


We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8+ T cells recognizing peptide antigens presented by HLA-A2.1. CD8+ T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 x CEM.T2 cells. Tumor necrosis factor alpha (TNF-alpha) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-alpha antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1. CD8+ lymphocytes responsive to a known immunogenic HLA-A2.1-binding peptide from reverse transcriptase of the human immunodeficiency virus (HIV) were only detectable in HIV-infected patients, but not in anti-HIV-negative donors. T cells reacting with a peptide derived from a mutated cyclin-dependent kinase 4 (CDK4-R24C) were exclusively detected among CD8+ lymphocytes isolated from blood of the patient, whose melanoma had previously been found to carry the CDK4-R24C allele. T cells responding to HLA-A2.1-associated peptides of normal melanocyte differentiation antigens tyrosinase and Melan-A/MART-1 were found at low frequencies in almost all donors tested, which might reflect a natural autoimmunity to these antigens. However, in a melanoma patient we found a few days after surgery of melanoma metastases high frequencies of T cells against Melan-A/MART-1 and tyrosinase peptides (up to 38 per 10(5) CD8+ T cells), which gradually decreased during the following months. In an HIV-infected patient with progressive disease we observed a loss of T cells reactive with the HIV reverse transcriptase peptide. These observations provide evidence that peptide-dependent TNF-alpha spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-alpha ELISPOT assay might be useful in monitoring antigen-specific T lymphocyte responses during the natural course of diseases as well as during therapeutic interventions aiming at the induction of protective T cell immunity. In addition, it might help to identify immunodominant T cell epitopes.

MeSH terms

  • Amino Acid Sequence
  • Antigens, Neoplasm / chemistry
  • Antigens, Neoplasm / immunology*
  • CD8-Positive T-Lymphocytes / chemistry
  • CD8-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / metabolism*
  • Enzyme-Linked Immunosorbent Assay / methods
  • HIV Antigens / chemistry
  • HIV Antigens / immunology*
  • HLA-A2 Antigen / chemistry*
  • HLA-A2 Antigen / immunology
  • Humans
  • Kinetics
  • Lymphocyte Activation
  • Lymphocyte Count
  • Melanoma / immunology*
  • Molecular Sequence Data
  • Peptide Fragments / immunology*
  • Protein Binding / immunology
  • Tumor Cells, Cultured
  • Tumor Necrosis Factor-alpha / metabolism*


  • Antigens, Neoplasm
  • HIV Antigens
  • HLA-A2 Antigen
  • Peptide Fragments
  • Tumor Necrosis Factor-alpha