The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools

Cancer Immunol Immunother. 2012 Nov;61(11):1953-63. doi: 10.1007/s00262-012-1251-3. Epub 2012 Apr 11.


The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigen Presentation / immunology
  • Antigens / immunology*
  • CD4-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / immunology*
  • Cells, Cultured
  • Enzyme-Linked Immunospot Assay*
  • Epitopes, T-Lymphocyte / analysis*
  • Epitopes, T-Lymphocyte / immunology
  • Flow Cytometry / methods*
  • Humans
  • Interferon-gamma / immunology
  • Lymphocyte Activation / immunology
  • Peptides / immunology*
  • Poly I-C / immunology


  • Antigens
  • Epitopes, T-Lymphocyte
  • Peptides
  • Interferon-gamma
  • Poly I-C