MHC I tetramer staining tends to overestimate the number of functionally relevant self-reactive CD8 T cells in the preimmune repertoire

Previous studies that used peptide-MHC (pMHC) tetramers (tet) to identify self-specific T cells have questioned the effectiveness of thymic-negative selection. Here, we used pMHCI tet to enumerate CD8 T cells specific for the immunodominant gp33 epitope of lymphocytic choriomeningitis virus glycoprotein (GP) in mice transgenically engineered to express high levels of GP as a self-antigen in the thymus. In GP-transgenic mice (GP(+)), monoclonal P14 TCR+ CD8 T cells that express a GP-specific TCR could not be detected by gp33/D-b-tet staining, indicative of their complete intrathymic deletion. By contrast, in the same GP(+) mice, substantial numbers of polyclonal CD8 T cells identifiable by gp33/D-b-tet were present. The gp33-tet staining profiles of polyclonal T cells from GP(+) and GP-negative (GP(-)) mice were overlapping, but mean fluorescence intensities were similar to 15% lower in cells from GP(+) mice. Remarkably, the gp33-tet(+) T cells in GP(+) mice failed to clonally expand after lymphocytic choriomeningitis virus infection, whereas those of GP(-) mice did so. In Nur77(GFP)-reporter mice, dose-dependent responses to gp33 peptide-induced TCR stimulation revealed that gp33-tet(+) T cells with high ligand sensitivity are lacking in GP(+) mice. Hence, pMHCI tet staining identifies self-specific CD8 T cells but tends to overestimate the number of truly self-reactive cells.