Proximal TCR signaling in self tolerance

This thesis investigates the molecular mechanisms involved in T‐cell receptor (TCR)
signaling during thymocyte selection. The T‐cell receptor of developing T‐cells interacts
with antigen‐ presenting cells (APCs) that display peptide‐MHC ligands (p‐MHC) of
different nature on their surface. The TCR interacts with these ligands and translates
the binding affinity for different p‐MHC (characterized by the dissociation constant, KD)
into a quantitative readout, thereby providing the basis for downstream signaling. How
the TCR distinguishes between high affinity ligands that induce apoptosis of individual
thymocytes (negative selection) and low affinity ligands that induce differentiation of
thymocytes into single‐positive immature T‐cells (positive selection) has fascinated
immunologists and biochemists for many years. This mechanism is critical to establish a
self‐MHC restricted, self‐tolerant T‐cell repertoire (central tolerance).
The first part of this thesis investigates the molecular interaction between the TCR and
the CD8 co‐ receptor in thymic selection. By tagging both molecules with variants of the
green fluorescent protein (GFP) and assessing their molecular approximation in the
immunological synapse by FRET microscopy (developed by P. Yachi and N. Gascoigne at the
Scripps Institute, LaJolla, USA), we found that negative‐selecting p‐MHC ligands induced
strong and sustained TCR/CD8 association. In contrast, positive‐selecting ligands induce
weak and delayed TCR/CD8 association in the synapse of T‐cell hybridomas with
antigen‐presenting cells (APCs). We found that the TCR/CD8 interaction in response to
positive‐ or negative‐selecting ligands was reflected in the phosphorylation of the ζ‐
chain. Therefore, the ability of the TCR to tightly associate with the co‐receptor is the
critical parameter that determines whether a p‐MHC ligand mediates strong intracellular
tyrosine phosphorylation and subsequently induces negative selection signaling. The
α‐chain connecting peptide motif (α‐CPM) is a region of 8 conserved amino acids in the
membrane‐proximal part of the constant region of the TCR α‐chain. Mutating the α‐CPM did
not affect ligand binding since α‐CPM mutant TCRs had similar p‐MHC affinities like
wild‐type TCRs. However, TCR/CD8 interaction as measured by FRET microscopy, changed
substantially in α‐CPM mutant TCRs. In response to negative‐selecting ligands, TCR/CD8
association was reduced in α‐CPM mutant cells, which was also reflected in decreased ζ
phosphorylation. Remarkably, in response to positive‐selecting ligands, α‐ CPM mutant
cells displayed no detectable TCR/CD8 interactions and failed to induce ζ
phosphorylation. Therefore, the α‐CPM is responsible for the molecular approximation of
the CD8 co‐receptor to the TCR complex, allowing efficient signaling initiation. We
hypothesize that the TCR and the co‐receptor may act like a molecular zipper. By binding
to the same p‐MHC molecule the zippering mechanism allows the two molecules to become
tightly associated via the α‐CPM towards the plasma membrane. Inside the cell, the
co‐receptor carries the Src kinase, Lck and shuffles it efficiently to the CD3 complex
once the zipper is fully closed. Only the zippered configuration allows efficient
signaling initiation, emphasizing the importance of the α‐CPM to functionally link the
TCR and CD8.
In the second part of this thesis we investigated TCR proximal signaling downstream of
the TCR complex. The ζ‐chain associated protein of 70 kDa (ZAP‐70) plays a central role
in transmitting the TCR‐generated signal to downstream signaling molecules. ZAP‐70 binds
to phosphorylated immunoreceptor tyrosine activation motifs (ITAMs) located on the ζ or
CD3 molecules of the TCR complex. The tyrosine kinase activity of ZAP‐70 is triggered if
the molecule binds to doubly phosphorylated ITAMs via its tandem SH2‐domain and
subsequently becomes phosphorylated at several tyrosine residues. We wondered whether
ZAP‐70 would function as molecular switch in TCR signaling, converting varying TCR inputs
(by binding p‐MHC ligands of different binding affinity) into discrete signaling
responses by generating distinct levels of ZAP‐70 kinase activity. In response to
negative‐selecting ligands, ZAP‐70 was efficiently recruited to the immunological
synapse. In the synapse, ZAP‐70 became phosphorylated at critical tyrosine residues,
which induced its kinase activity. In vitro kinase assays revealed a discrete 2‐fold
increase in ZAP‐70 kinase activity precisely at the negative selection threshold. In
contrast, ZAP‐70 recruitment to the synapse and its kinase activity remained low in
response to positive‐selecting ligands. Therefore, we speculate that a discrete elevation
of ZAP‐70 activity occurs at the threshold of positive and negative selection. Further
evidence for such a mechanism came from fetal thymic organ cultures (FTOCs), where
negative selection was converted into partial positive selection by reducing ZAP‐70
kinase activity with a specific inhibitior. We also asked whether the increased ZAP‐70
kinase activity in negative selection is generated by an increase in the ratio of ZAP‐70
/ TCR in the synapse. This idea seamed reasonable since multiple ITAMs and therefore
potential ZAP‐70 binding sites exist among the CD3 molecules. However, we did not detect
an increase in the ZAP‐70 / TCR ratio. Relative to positive selecting ligands, negative
selectors induced a 2‐fold increase in the amount of TCR and ZAP‐70 recruited to the
immunological synapse. However, the ZAP‐70 / TCR ratio was similar in both forms of
selection and therefore, the number of TCR molecules recruited to the synapse determines
the selection outcome. We postulate a model of TCR‐proximal signaling, where
TCR‐associated ZAP‐70 is recruited into the synapse proportionally to the TCR’s ability
to bind p‐MHC ligands and recruit the co‐receptor. According to the zipper model, only
negative‐selecting ligands mediate efficient co‐ receptor association and therefore,
increased ζ phosphorylation. ZAP‐70 becomes phosphorylated accordingly, which initiates a
2‐fold increase in its kinase activity in response to p‐MHC ligands above the negative
selection threshold. This step‐wise increase in ZAP‐70 kinase activity is sufficient to
mediate higher levels of LAT phosphorylation, which assembles a negative selection
signaling complex