The APC capture: rapid identification of target CD8+ T cell epitopes by flow cytometry

Abstract Identifying immunoreactive CTLs by current technologies (cytokine secretion, intracellular cytokine, cytolytic release, ELISPOT, and MHC tetramer assays) is often difficult when probing multiple target antigens. We developed a rapid flow cytometry assay to detect targeted IFN-γ secretion by CD8+ T cells using commonly available reagents. PBMCs were obtained from HLA-A2+ healthy donors, and CD8+ and CD8− fractions were purified. FluMI-specific T cells were confirmed by HLA-A2/M1 tetramer, and pooled CEF peptides (a mixture of CMV-, EBV-, and Flu-specific HLA class I epitopes) immunoreactivity was shown by IFN-γ ELISPOT. The CD8+ fraction was rested for 5 days in IL-2 (20 U/ml) and IL-7 (5 ng/ml). In parallel, autologous APCs were generated from the CD4− CD8− fraction using recombinant CD40L (100 ng/ml), IL-4 (5ng/mL), and cyclosporin A (1 μg/ml). After 5 days, the APC culture was >70% CD19+ CD86+ HLA-DR+ activated B cells. The APCs were pulsed with DMSO control or CEF peptide pool (10ug/mL) overnight, labeled with commercial IFN-γ catch (Miltenyi Biotec), washed, and co-cultured for 6–8 hours with purified CD8+ T cells. APC-surface-bound IFN-γ was detected by flow cytometry. CEF-pulsed APCs showed robust presence of surface-captured IFN-γ (ranging from 14%–56.8%) compared to DMSO-pulsed APCs (ranging from 1.6%–4.5% IFN-γ+). APC IFN-γ capture was sensitive even at low effector-target ratios (as low as 1:1) and was applicable to the artificial APC K562/A2. In conclusion, we have demonstrated epitope-reactive CD8+ T cell detection of target cells by capturing IFN-γ onto APCs. We are evaluating capture of other cytokines and cytolytic effectors on target APCs in response to CD8+ recognition.

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