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 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 NIH 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