A novel approach of direct ex vivo epitope mapping identifies dominant andsubdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes

We used a novel approach for the direct ex vivo identification and characte rization of T cell epitopes based on the screening of peptide spot librarie s with freshly isolated splenocytes in a sensitive enzyme-linked immunospot (ELISPOT) assay. This technique was applied for the analysis of splenocyte s from Listeria monocytogenes-infected BALB/c and C57BL/6 mice. The screeni ng of peptide spot libraries covering the whole listeriolysin O and p60 of L. monocytogenes confirmed all known CD4 and CD8 T cell epitopes of these p roteins and additionally revealed six new H-2(d) and six new H-2(b)-restric ted T cell epitopes. New epitopes were categorized into CD4 and CD8 T cell epitopes by ex vivo ELISPOT analysis with separated T cell populations. The quantitative analysis of cells reactive with these CD4 and CD8 T cell epit opes revealed the existence of dominant and subdominant CD4 and CD8 T cell populations during L. monocytogenes infection. As a consequence of these da ta we suggest that ELISPOT-based screening of peptide spot libraries could be a general approach for the rapid identification and characterization of pathogen-specific T cell populations during various infectious diseases.