Vagaries of the ELISpot assay: Specific detection of antigen responsive cells requires purified CD8+ T cells and MHC class I expressing antigen presenting cell lines

•MHC class I restricted peptides can induce CD4+ T cell IFNγ responses.•Current ELISpot assays do not specifically measure CD8+ T cell response to antigen.•Specific measurement achieved with pure CD8+ T cells and engineered APCs.

Quantification of antigen-specific CD8+ T cells is important for monitoring infection, vaccination, and response to therapy in cancer and immune-mediated diseases. Cytokine enzyme-linked-immunospot (ELISpot) assays are often used for this purpose. We found that substantial spot formation in IFNγ ELISpot assays occurred independently of CD8+ T cells even when classical MHC class I restricted peptides are used for stimulation. Using fractionated cells and intracellular cytokine staining, the non-CD8+ T cell IFNγ production was attributed to the CD4+ T cell fraction. We therefore refined a cell line-based ELISpot assay combining HLA-A*0201 expressing K562 cells for antigen presentation with purified CD8+ T cells and demonstrated that it specifically detected CD8+ T cell responses with detection limits comparable to traditional ELISpot assays and dextramer-based quantification. The assay was further adapted to whole antigen responses with antigen (pre-proinsulin)-expressing HLA-A*0201K562 cells. Thus, we revealed and corrected a weak spot of the CD8+ ELISpot assay.