Studies on the affinity control of T cell development

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.