The ELISPOT (angl. Enzyme-Linked ImmunoSpot) method is a highly sensitive and user-friendly method, commonly applied in different areas of research and diagnostics. The interferon (IFN)-γ ELISPOT assay is used to enumerate antigen (Ag)-specific T cells upon Ag-specific stimulation. The principal aim of our study was to establish an ELISPOT assay for the determination of Ag-specific immune response of CD8+ cytotoxic T cells (CTL). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples obtained from healthy human blood donors. PBMCs were used to run the mixed lymphocyte reaction (MLR) and were ex vivo stimulated with different peptide pools. Whole CD8+ T cells and monocytes were isolated from PBMC by positive immunomagnetic selection and monocytes were used for dendritic cell (DC) differentiation. The latter were used for in vitro stimulation of autologous CD8+ CTL. After its optimization, the ELISPOT assay was applied to determine the Ag-specific immune response of T cells. Our results show that the ELISPOT assay enables the detection of allo-Ag-specific T cells and can be used to determine virus-specific T cells in PBMCs upon 24 h ex vivo stimulation with peptide pool from cytomegalovirus (CMV), Epstein-Barr virus and influenza A virus (CEF) and peptide pool of the phospohoprotein 65 (pp65) from CMV. The ELISPOT method also enables the detection of rare clones of melanoma-specific CTL upon in vitro stimulation with autologous mature DC (αDC and zDC), pulsated with melanoma-associated peptides gp100, Melan-A and tyr. Finally, our results demonstrate zDC are superior in their capacity to induce melanoma-specific CTL compared to αDC.
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