Fine tuning of TCR signaling via CD8αβ and PD-1 and the fate of autoreactive thymocytes during negative selection.

During thymocyte development, positive selection produces cells whose T cell receptors (TCRs) bind to self MHC. Then, negative selection culls most thymocytes whose TCRs have too high an affinity for self MHC+peptide. Signal transduction events control these processes. CD8-αβ (via CD8-β) recruits p56lck to the immunological synapse and promotes signaling through the TCR. Conversely, PD-1 attenuates TCR signal transduction. We examined the roles of CD8-β and PD-1 in the survival of thymocytes in H-2k haplotype mice expressing a transgenic BM3 TCR, which has high affinity for the allogeneic H-2Kb MHC I molecule. In transgenic mice expressing both H-2Kb in the thymic medulla and the BM3 TCR, apoptosis eliminates most (but not all) post-selection thymocytes. To analyze the roles of CD8-β and PD-1 in the survival of post-selection thymocytes, we devised a novel probabilistic gating strategy employing Gaussian mixture models and statistical methods using sliding windows and changepoint detection. We found that at high levels of CD8-β and therefore high levels of CD8-αβ), thymocytes are prone to apoptosis, regardless of the PD-1 level. At intermediate levels of CD8-β, thymocyte survival increases concordantly with increasing PD-1 levels. At low levels of CD8-β, thymocyte survival is high regardless of the PD-1 level. Surviving DPlo post-selection thymocytes give rise to PD-1+CCR7+DN and PD-1+CCR7-DN post-selection thymocytes, which appear to become DN T cells and IELs, respectively. Thus, PD-1 appears to promote the survival of both IEL precursors and thymocytes destined for other fates. More strikingly, downregulation of CD8-β is a hallmark of autoreactive MHC I-restricted thymocytes that survive negative selection.