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

• Published in: Cancer Immunology, Immunotherapy Immunotherapy, 2012-11, Vol.61 (11), p.1953-1963
• Main Authors: Singh, Satwinder Kaur, Meyering, Maaike, Ramwadhdoebe, Tamara H., Stynenbosch, Linda F. M., Redeker, Anke, Kuppen, Peter J. K., Melief, Cornelis J. M., Welters, Marij J. P., van der Burg, Sjoerd H.
• Format: Article
• Language: English
• Subjects: adenomatous polyposis coli; alpha -Interferon; Amino acids; Antigen Presentation - immunology; Antigen processing; Antigen-presenting cells; Antigens; Antigens - immunology; Antineoplastic agents; Biological and medical sciences; Cancer; Cancer Research; CD4 antigen; CD4-Positive T-Lymphocytes - immunology; CD8 antigen; CD8-Positive T-Lymphocytes - immunology; Cells, Cultured; Cryopreservation; Cytokines; Cytotoxicity; Enzyme-Linked Immunospot Assay; Epitopes; Epitopes, T-Lymphocyte - analysis; Epitopes, T-Lymphocyte - immunology; Flow Cytometry - methods; Histocompatibility antigen HLA; Humans; Immunization; Immunology; Immunotherapy; Interferon-gamma - immunology; Lymphocyte Activation - immunology; Lymphocytes; Lymphocytes T; Medical sciences; Medicine; Medicine & Public Health; Monocytes; Oncology; Original; Original Article; Peptides; Peptides - immunology; Peripheral blood mononuclear cells; Pharmacology. Drug treatments; Poly I-C - immunology; Vaccination
• ISSN: 0340-7004; 1432-0851
• DOI: 10.1007/s00262-012-1251-3

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.