title: The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.
abstract: 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.
authors.affiliation: Department of Clinical Oncology, Building 1, K1-P, Leiden University Medical Center, PO box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Satwinder Kaur
authors.initials: SK
authors.lastname: Singh
authors.forename: Maaike
authors.initials: M
authors.lastname: Meyering
authors.forename: Tamara H
authors.initials: TH
authors.lastname: Ramwadhdoebe
authors.forename: Linda F M
authors.initials: LF
authors.lastname: Stynenbosch
authors.forename: Anke
authors.initials: A
authors.lastname: Redeker
authors.forename: Peter J K
authors.initials: PJ
authors.lastname: Kuppen
authors.forename: Cornelis J M
authors.initials: CJ
authors.lastname: Melief
authors.forename: Marij J P
authors.initials: MJ
authors.lastname: Welters
authors.forename: Sjoerd H
authors.initials: SH
authors.lastname: van der Burg
mesh_terms: D017951:Antigen Presentation / Q000276:immunology; D000941:Antigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D058501:Enzyme-Linked Immunospot Assay; D018984:Epitopes, T-Lymphocyte / Q000032:analysis* / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D010455:Peptides / Q000276:immunology*; D011070:Poly I-C / Q000276:immunology
chemical_list: D000941:Antigens; D018984:Epitopes, T-Lymphocyte; D010455:Peptides; D007371:Interferon-gamma; D011070:Poly I-C