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• W1570890819 abstract "This thesis investigates the molecular mechanisms involved in T‐cell receptor (TCR)signaling during thymocyte selection. The T‐cell receptor of developing T‐cells interactswith antigen‐ presenting cells (APCs) that display peptide‐MHC ligands (p‐MHC) ofdifferent nature on their surface. The TCR interacts with these ligands and translatesthe binding affinity for different p‐MHC (characterized by the dissociation constant, KD)into a quantitative readout, thereby providing the basis for downstream signaling. Howthe TCR distinguishes between high affinity ligands that induce apoptosis of individualthymocytes (negative selection) and low affinity ligands that induce differentiation ofthymocytes into single‐positive immature T‐cells (positive selection) has fascinatedimmunologists and biochemists for many years. This mechanism is critical to establish aself‐MHC restricted, self‐tolerant T‐cell repertoire (central tolerance).The first part of this thesis investigates the molecular interaction between the TCR andthe CD8 co‐ receptor in thymic selection. By tagging both molecules with variants of thegreen fluorescent protein (GFP) and assessing their molecular approximation in theimmunological synapse by FRET microscopy (developed by P. Yachi and N. Gascoigne at theScripps Institute, LaJolla, USA), we found that negative‐selecting p‐MHC ligands inducedstrong and sustained TCR/CD8 association. In contrast, positive‐selecting ligands induceweak and delayed TCR/CD8 association in the synapse of T‐cell hybridomas withantigen‐presenting cells (APCs). We found that the TCR/CD8 interaction in response topositive‐ 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 thecritical parameter that determines whether a p‐MHC ligand mediates strong intracellulartyrosine phosphorylation and subsequently induces negative selection signaling. Theα‐chain connecting peptide motif (α‐CPM) is a region of 8 conserved amino acids in themembrane‐proximal part of the constant region of the TCR α‐chain. Mutating the α‐CPM didnot affect ligand binding since α‐CPM mutant TCRs had similar p‐MHC affinities likewild‐type TCRs. However, TCR/CD8 interaction as measured by FRET microscopy, changedsubstantially in α‐CPM mutant TCRs. In response to negative‐selecting ligands, TCR/CD8association was reduced in α‐CPM mutant cells, which was also reflected in decreased ζphosphorylation. Remarkably, in response to positive‐selecting ligands, α‐ CPM mutantcells displayed no detectable TCR/CD8 interactions and failed to induce ζphosphorylation. Therefore, the α‐CPM is responsible for the molecular approximation ofthe CD8 co‐receptor to the TCR complex, allowing efficient signaling initiation. Wehypothesize that the TCR and the co‐receptor may act like a molecular zipper. By bindingto the same p‐MHC molecule the zippering mechanism allows the two molecules to becometightly associated via the α‐CPM towards the plasma membrane. Inside the cell, theco‐receptor carries the Src kinase, Lck and shuffles it efficiently to the CD3 complexonce the zipper is fully closed. Only the zippered configuration allows efficientsignaling initiation, emphasizing the importance of the α‐CPM to functionally link theTCR and CD8.In the second part of this thesis we investigated TCR proximal signaling downstream ofthe TCR complex. The ζ‐chain associated protein of 70 kDa (ZAP‐70) plays a central rolein transmitting the TCR‐generated signal to downstream signaling molecules. ZAP‐70 bindsto phosphorylated immunoreceptor tyrosine activation motifs (ITAMs) located on the ζ orCD3 molecules of the TCR complex. The tyrosine kinase activity of ZAP‐70 is triggered ifthe molecule binds to doubly phosphorylated ITAMs via its tandem SH2‐domain andsubsequently becomes phosphorylated at several tyrosine residues. We wondered whetherZAP‐70 would function as molecular switch in TCR signaling, converting varying TCR inputs(by binding p‐MHC ligands of different binding affinity) into discrete signalingresponses by generating distinct levels of ZAP‐70 kinase activity. In response tonegative‐selecting ligands, ZAP‐70 was efficiently recruited to the immunologicalsynapse. In the synapse, ZAP‐70 became phosphorylated at critical tyrosine residues,which induced its kinase activity. In vitro kinase assays revealed a discrete 2‐foldincrease in ZAP‐70 kinase activity precisely at the negative selection threshold. Incontrast, ZAP‐70 recruitment to the synapse and its kinase activity remained low inresponse to positive‐selecting ligands. Therefore, we speculate that a discrete elevationof ZAP‐70 activity occurs at the threshold of positive and negative selection. Furtherevidence for such a mechanism came from fetal thymic organ cultures (FTOCs), wherenegative selection was converted into partial positive selection by reducing ZAP‐70kinase activity with a specific inhibitior. We also asked whether the increased ZAP‐70kinase 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 thereforepotential ZAP‐70 binding sites exist among the CD3 molecules. However, we did not detectan increase in the ZAP‐70 / TCR ratio. Relative to positive selecting ligands, negativeselectors induced a 2‐fold increase in the amount of TCR and ZAP‐70 recruited to theimmunological synapse. However, the ZAP‐70 / TCR ratio was similar in both forms ofselection and therefore, the number of TCR molecules recruited to the synapse determinesthe selection outcome. We postulate a model of TCR‐proximal signaling, whereTCR‐associated ZAP‐70 is recruited into the synapse proportionally to the TCR’s abilityto bind p‐MHC ligands and recruit the co‐receptor. According to the zipper model, onlynegative‐selecting ligands mediate efficient co‐ receptor association and therefore,increased ζ phosphorylation. ZAP‐70 becomes phosphorylated accordingly, which initiates a2‐fold increase in its kinase activity in response to p‐MHC ligands above the negativeselection threshold. This step‐wise increase in ZAP‐70 kinase activity is sufficient tomediate higher levels of LAT phosphorylation, which assembles a negative selectionsignaling complex" @default.
• W1570890819 created "2016-06-24" @default.
• W1570890819 creator A5085122879 @default.
• W1570890819 date "2008-01-01" @default.
• W1570890819 modified "2023-09-25" @default.
• W1570890819 title "Proximal TCR signaling in self tolerance" @default.
• W1570890819 doi "https://doi.org/10.5451/unibas-004911612" @default.
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