Fig. 5.

Multiplexed secretion-based profiling of prostate tissue antigen-specific T cells. (A) FACS analysis and sorting gates for identifying functional antigen-specific T cells transduced with TCR128 loaded on HLA-A*02:01 restricted PAP21 pMHC labeled nanovials. IFN-γ and TNF-α secretion signals were analyzed from the CD3+/CD8+/NGFR+ cells. Images of T cells on nanovials that were sorted reflecting each of the four quadrant gates including an IFN-γ and TNF-α polyfunctional population (Q2). (Scale bars represent 50 μm.) (B) The population distribution based on secretion phenotype is shown as a pie chart for TCR 128, 218, and 156 transduced cells. CD3+CD8+ cells with secretion signal below the background threshold were considered as nonsecretors. (C) Overview of multiplexed profiling of untransduced human primary T cells based on cytokine secretion and cell phenotype. T cells loaded on nanovials labeled with two cytokine capture antibodies (anti-IFN-γ and anti-TNF-α, or anti-IFN-γ and anti-IL-2) and anti-CD45 were activated under PMA/ionomycin stimulation. Secreted cytokines and cell surface markers (CD4, CD8) were stained with fluorescent detection antibodies, followed by analysis and sorting with a cell sorter. (D) The distribution of secretion phenotype for CD4+ and CD8+ human primary T cells based on IFN-γ and TNF-α secretion.