Detection of antigen-specific T cell interferon γ expression by ELISPOT and cytokine flow cytometry assays in rhesus macaques

Abstract

Both enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) methods have been developed for the detection of low-frequency, antigen-specific, cytokine-producing T cells following short-term in vitro stimulation. Peptide-based ELISPOT and CFC assays were compared for the quantitative detection of interferon γ-positive (IFN-γ+) antigen-specific T cells in rhesus macaques. Ten normal and nine simian immunodeficiency virus (SIV)-infected monkeys were tested for the detection of IFN-γ+ memory T cells specific for p27 gag peptides of SIV with both assays. The CFC assay detected more IFN-γ+ cells than the ELISPOT assay and this assay was more informative in identifying the phenotype of responding cells. Cryopreserved cells were as functional as fresh cells in heparinized blood samples and compared to EDTA, heparin was the better anticoagulant for yielding IFN-γ+ cells. Using overlapping peptide pools, 20-mer peptides were more efficient in stimulating CD4+ T cells than 15-mer peptides in the ELISPOT assay, but there was no significant difference between 20- and 15-mer peptides in detecting CD4 or CD8+, IFN-γ+ T cells in the CFC assay.