3 research outputs found
Proximal TCR signaling in self tolerance
This thesis investigates the molecular mechanisms involved in Tâcell receptor (TCR)
signaling during thymocyte selection. The Tâcell receptor of developing Tâcells interacts
with antigenâ presenting cells (APCs) that display peptideâMHC ligands (pâMHC) of
different nature on their surface. The TCR interacts with these ligands and translates
the binding affinity for different pâMHC (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 singleâpositive immature Tâcells (positive selection) has fascinated
immunologists and biochemists for many years. This mechanism is critical to establish a
selfâMHC restricted, selfâtolerant Tâcell repertoire (central tolerance).
The first part of this thesis investigates the molecular interaction between the TCR and
the CD8 coâ 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 negativeâselecting pâMHC ligands induced
strong and sustained TCR/CD8 association. In contrast, positiveâselecting ligands induce
weak and delayed TCR/CD8 association in the synapse of Tâcell hybridomas with
antigenâpresenting cells (APCs). We found that the TCR/CD8 interaction in response to
positiveâ or negativeâselecting ligands was reflected in the phosphorylation of the ζâ
chain. Therefore, the ability of the TCR to tightly associate with the coâreceptor is the
critical parameter that determines whether a pâMHC ligand mediates strong intracellular
tyrosine phosphorylation and subsequently induces negative selection signaling. The
뱉chain connecting peptide motif (뱉CPM) is a region of 8 conserved amino acids in the
membraneâproximal part of the constant region of the TCR αâchain. Mutating the αâCPM did
not affect ligand binding since αâCPM mutant TCRs had similar pâMHC affinities like
wildâtype TCRs. However, TCR/CD8 interaction as measured by FRET microscopy, changed
substantially in αâCPM mutant TCRs. In response to negativeâselecting ligands, TCR/CD8
association was reduced in αâCPM mutant cells, which was also reflected in decreased ζ
phosphorylation. Remarkably, in response to positiveâselecting ligands, αâ CPM mutant
cells displayed no detectable TCR/CD8 interactions and failed to induce ζ
phosphorylation. Therefore, the 뱉CPM is responsible for the molecular approximation of
the CD8 coâreceptor to the TCR complex, allowing efficient signaling initiation. We
hypothesize that the TCR and the coâreceptor may act like a molecular zipper. By binding
to the same pâMHC molecule the zippering mechanism allows the two molecules to become
tightly associated via the 뱉CPM towards the plasma membrane. Inside the cell, the
coâreceptor 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 뱉CPM 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 ζâchain associated protein of 70 kDa (ZAPâ70) plays a central role
in transmitting the TCRâgenerated signal to downstream signaling molecules. ZAPâ70 binds
to phosphorylated immunoreceptor tyrosine activation motifs (ITAMs) located on the ζ or
CD3 molecules of the TCR complex. The tyrosine kinase activity of ZAPâ70 is triggered if
the molecule binds to doubly phosphorylated ITAMs via its tandem SH2âdomain and
subsequently becomes phosphorylated at several tyrosine residues. We wondered whether
ZAPâ70 would function as molecular switch in TCR signaling, converting varying TCR inputs
(by binding pâMHC ligands of different binding affinity) into discrete signaling
responses by generating distinct levels of ZAPâ70 kinase activity. In response to
negativeâselecting ligands, ZAPâ70 was efficiently recruited to the immunological
synapse. In the synapse, ZAPâ70 became phosphorylated at critical tyrosine residues,
which induced its kinase activity. In vitro kinase assays revealed a discrete 2âfold
increase in ZAPâ70 kinase activity precisely at the negative selection threshold. In
contrast, ZAPâ70 recruitment to the synapse and its kinase activity remained low in
response to positiveâselecting ligands. Therefore, we speculate that a discrete elevation
of ZAPâ70 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 ZAPâ70
kinase activity with a specific inhibitior. We also asked whether the increased ZAPâ70
kinase activity in negative selection is generated by an increase in the ratio of ZAPâ70
/ TCR in the synapse. This idea seamed reasonable since multiple ITAMs and therefore
potential ZAPâ70 binding sites exist among the CD3 molecules. However, we did not detect
an increase in the ZAPâ70 / TCR ratio. Relative to positive selecting ligands, negative
selectors induced a 2âfold increase in the amount of TCR and ZAPâ70 recruited to the
immunological synapse. However, the ZAPâ70 / 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 TCRâproximal signaling, where
TCRâassociated ZAPâ70 is recruited into the synapse proportionally to the TCRâs ability
to bind pâMHC ligands and recruit the coâreceptor. According to the zipper model, only
negativeâselecting ligands mediate efficient coâ receptor association and therefore,
increased ζ phosphorylation. ZAPâ70 becomes phosphorylated accordingly, which initiates a
2âfold increase in its kinase activity in response to pâMHC ligands above the negative
selection threshold. This stepâwise increase in ZAPâ70 kinase activity is sufficient to
mediate higher levels of LAT phosphorylation, which assembles a negative selection
signaling comple
Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover
To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIHÂ 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover