The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor alpha spots in response to peptide antigens

J Immunol Methods. 1997 Apr 25;203(2):141-52. doi: 10.1016/s0022-1759(97)00019-7.


Enzyme-linked immunospot (ELISPOT) analysis is a sensitive technique for the detection and quantification of single T lymphocytes forming cytokine spots after antigen contact in vitro. Herein computer-assisted video image analysis (CVIA) was applied to automatically determine the number and size of tumor necrosis factor alpha (TNF-alpha) spots formed by single blood-derived CD8+ T cells after contact with peptide-loaded target cells. With CVIA and TNF-alpha ELISPOT analysis we quantified CD8+ T cells responsive to HLA-A2.1-binding tyrosinase and influenza matrix peptides in healthy donors. We followed the course of the virus-specific T cell response in two HLA-A2-positive patients with reactivation of latent cytomegalovirus (CMV) infection during immunosuppressive therapy. The test proved sufficiently sensitive to detect in the blood of both patients a temporary expansion of CD8+ T lymphocytes reactive with a known immunogenic HLA-A2.1-binding peptide from glycoprotein B of CMV. Reactivity to peptide antigens was not only reflected by numeric increases of spot formation, but also by the appearance of larger spot areas, presumably formed by strongly peptide-reactive CD8+ T cells. We conclude that the combined use of the TNF-alpha ELISPOT assay and CVIA allows reliable monitoring of the T cell responsiveness to peptide antigens in peripheral blood.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • CD8-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / metabolism*
  • Cell Line
  • Cytomegalovirus / immunology
  • Cytomegalovirus Infections / immunology
  • Enzyme-Linked Immunosorbent Assay / methods
  • HLA-A2 Antigen / immunology
  • Humans
  • Image Processing, Computer-Assisted / methods*
  • Lymphocyte Count / methods
  • Microscopy, Video / methods
  • Middle Aged
  • Peptides / immunology*
  • Tumor Necrosis Factor-alpha / chemistry*
  • Viral Envelope Proteins / immunology


  • HLA-A2 Antigen
  • Peptides
  • Tumor Necrosis Factor-alpha
  • Viral Envelope Proteins
  • glycoprotein B, Simplexvirus