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In the thymus, high-affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen-engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck; this is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (0.2 s) to initiate negative selection compared to MHCI-restricted TCRs (0.9 s) because more CD4 coreceptors are Lck-loaded compared to CD8. We generated a model (Lck come&stay/signal duration) that accurately predicts the observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance., National Institutes of Health (U.S.) (Grant 1-P01-AI091580)

Defined interactions between thymocytes and thymic antigen presenting cells ensure that each T cell found in an individual is both self-restricted and self-tolerant. By interacting with pMHC ligands, T cell receptors (TCRs) expressed on developing T cells initiate intracellular signaling cascades which lead either to survival and differentiation, referred to as positive selection, or to apoptotic cell death, referred to as negative selection. This thesis is aimed to a better understanding of how thymocytes distinguish between pMHC ligands inducing positive selection from those inducing negative selection. Transgenic mice and hybridomas expressing TCRs of defined specificity, referred to as T1-TCRs, were produced to study the role of TCR-ligand affinity in thymic selection of developing T cells. Ligand binding studies were performed with the photoaffinity labeling system developed by Immanuel Lüscher (Ludwig Institute; Epalinges, Switzerland). By defining the selection properties of various T1-TCR ligands and comparing their potency in inducing positive and negative selection with their TCR affinity it is shown, that TCR affinity is a key parameter for thymocyte selection. High affinity ligands induced negative selection, while low affinity ligands induced positive selection. A ligand with a moderate affinity was shown to induce either positive or negative selection, depending on the dose of the peptide. It was further shown, that the reduced sensitivity observed for mature T cells compared to thymocytes is not mediated by developmental changes in the affinity of TCR-ligand interactions. All ligands tested bound the TCR expressed on naive, mature T cells with the same affinity as the TCR expressed on thymocytes. Therefore, the results presented in the first part of this thesis suggest, that positive and negative selection of T cells depends on TCR-ligand affinity and that this affinity is preserved through all stages of T cell development. In the second part of this thesis studies are presented analyzing the role of the evolutionarily conserved α-chain connecting peptide motif (α-CPM) in TCR-ligand binding and thymocyte development. Experiments performed with hybridomas and transgenic mice showed that α-CPM deficient TCRs are not properly cooperating with the coreceptor molecule, CD8 to elicit responses to low affinity ligands. Interestingly responses to high affinity ligands were less affected. Thymocytes of α-CPM mutant mice were specifically defective in undergoing positive selection but were still able to undergo negative selection. By comparing the TCR-ligand affinities on cells expressing either wildtype or α-CPM mutant T1-TCRs it was shown, that the absence of the α-CPM leads to a slight decrease in CD8 cooperativity for ligand binding. The data therefore suggest, that the α-CPM plays an important role for successful cooperation of TCR and coreceptor in generating signals to low affinity, positive selecting ligands.