Multiparameter cytokine-specific affinity matrix assay for the determination of frequencies and phenotype of antigen-reactive T cells

Abstract

A major challenge in Phase I clinical trials of cancer vaccines is how to identify the optimal regimen and/or dosing for Phase II and III trials. Immunologic responses to tumor-associated antigens (TAA) could potentially serve as surrogate markers. Assays which detect cytokine production and/or secretion by antigen (Ag)-reactive T cells are increasingly being used to monitor such responses in clinical trials. A potential problem of these assays is high non-specific background and/or inability to easily identify the T-cell phenotype. We designed an assay, termed cytokine-specific affinity matrix (Multi-CSAM) assay,(1) which can determine the frequencies of phenotypically identifiable antigen (Ag)-reactive T cells. The assay relies on the tightly regulated production and secretion of cytokine by Ag-activated cells, capture of secreted cytokine by an artificial cytokine-specific cell-surface affinity matrix and flow-cytometric detection of bound cytokine with a fluorochrome-labeled anti-cytokine mAb. In an attempt to minimize non-specific background, the assay excludes non-viable cells by using the cell-impermeant DNA-binding dye TO-PRO-3, and selects activated cells on the basis of CD69 expression. Furthermore, the phenotype of the Ag-reactive T cells is identified using the CD4 and CD8 T-cell markers. Thus, using an appropriately modified two-laser benchtop instrument, a single sample can be evaluated by five-color multiparametric flow-cytometry that employs a combination of appropriately labeled mAbs. The five colors selected are anti-IFNgamma-PE, the dye TO-PRO-3, anti-CD69-PerCP, anti-CD4-FITC and anti-CD8-PE/Texas Red. Although our ultimate goal is to identify T cells reactive to TAA, as a first step this report demonstrates that Multi-CSAM can be used to detect the reactivity to TT and Candida.