Summary In the thymus, high affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck. Coreceptor scanning is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (~0.2s) to initiate negative selection compared to MHCI restricted TCRs (~0.9s) because more CD4 coreceptors are Lck-loaded compared to CD8. Based on experimental data and mathematical analysis, we generated a model (Lck come&stay/signal duration) that accurately predicts the experimentally observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance.

T lymphocytes are generated in the thymus, where developing thymocytes must accept one of two fates: They either differentiate or they die. These fates are chiefly determined by signals that originate from the T cell receptor (TCR), a single receptor complex with a remarkable capacity to decide between distinct cell fates. This review explores TCR signaling in thymocytes and focuses on the kinetic aspects of ligand binding, coreceptor involvement, protein phosphorylation, and mitogen-activated protein kinase (MAPK) activation. Understanding the logic of TCR signaling may eventually explain how thymocytes and T cells distinguish self from nonself, a phenomenon that has fascinated immunologists for 50 years.

T cell tolerance depends on the T cell receptor's affinity for peptide/major histocompatibility complex (MHC) ligand; this critical parameter determines whether a thymocyte will be included (positive selection) or excluded (negative selection) from the T cell repertoire. A quantitative analysis of ligand binding was performed using an experimental system permitting receptor–coreceptor interactions on live cells under physiological conditions. Using three transgenic mouse strains expressing distinct class I MHC–restricted T cell receptors, we determined the affinity that defines the threshold for negative selection. The affinity threshold for self-tolerance appears to be a constant for cytotoxic T lymphocytes.

The affinity of the T-cell receptor (TCR) for self antigen is the basis for the selection of a useful (MHC-restricted) and safe (self-tolerant) T-cell repertoire. However, it has been difficult to understand how thymocytes measure ligand affinity and translate this signal into a cellular response. In this Opinion article, we propose a new model that describes how the TCR discriminates between low- and high-affinity ligands, which is based on the duration of TCR-ligand interactions and a 'zipper' mechanism that mediates the interaction of the TCR and co-receptor molecules to initiate negative-selection signalling.

T cells expressing T cell receptor (TCR) complexes that lack CD3␦, either due to deletion of the CD3␦ gene, or by replacement of the connecting peptide of the TCR␣ chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8␤ or tailless CD8␤, we examined whether CD3␦ serves to couple TCR⅐CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K d . We report that, in thymocytes and hybridomas expressing the T1 TCR⅐CD3 complex, CD8␣␤ associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8␣␣ or a CD3␦؊ variant of the T1 TCR. CD3␦ was selectively coimmunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3␦. Because CD8␣␤ is a raft constituent, due to this association a fraction of TCR⅐CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3␦ couples TCR⅐CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8 ؉ T cells.The differentiation of CD4 CD8 double-positive (DP) 1 thymocytes into CD8 single-positive (SP) T cells requires appropriate signals from the TCR and the coreceptor CD8 (1, 2). DP thymocytes and CD8 SP peripheral T cells express TCR␣␤ that are associated with three signal-transducing units, namely CD3␦⑀ and CD3␥⑀ heterodimers and a disulfide-linked chain homodimer (3-5). The CD3␥␦ and ⑀ subunits contain in their cytoplasmic tail a single immunoreceptor tyrosine-based activation motif, whereas the tail of the chain harbors three immunoreceptor tyrosine-based activation motifs. For surface expression of TCR␣␤, their association with CD3⑀, ␥, and but not with CD3␦ is required (6 -9). Accordingly, knockout of CD3⑀, ␥, and chain arrests T cell development at early stages (6 -11). By contrast, in CD3␦ knockout mice T cell development proceeds to the DP stage, but positive selection of CD8 (and CD4) SP T cells is severely compromised (9, 12). During TCR␣␤ assembly the TCR␤ chain first associates with CD3⑀␥ and the TCR␣ chain with CD3⑀␦, and the resulting trimers then associate and the TCR␣␤ disulfide bond is formed (13). Although CD3␦ is physically associated with the pre-TCR complex, it is not required for pre-TCR signaling, which is essential for the transition of double-negative (DN) to DP thymocytes (10,14,15).A conserved motif in the TCR␣ chain-connecting peptide domain, which connects the transmembrane and the Ig domains, referred to as ␣CPM, plays a crucial role in positive selection of CD8 and CD4 SP T cells (16 -18). The ␣CPM consists of seven highly conserved amino acids (FETDXNLN) and is present in TCR␣␤ but not in TCR␥␦ (16). In mice expressing TCR in which the ␣CPM is replaced by the corresponding sequence of the TCR␦ chain, po...

Recognition by CD8؉ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that ␤ 1 and ␤ 3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC⅐peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling. CD8ϩ cytotoxic T lymphocytes (CTLs) 1 are activated upon engagement of their T cell receptor (TCR) by major histocompatibility (MHC)⅐peptide complexes on antigen-presenting cells (APCs) (1,2). This interaction results in the formation of the immunological synapse, harboring in its center TCR, tyrosine kinases, CD8, and CD2 and in its periphery the ␤ 2 integrin LFA-1 and the cytoskeletal linker talin (1,(3)(4)(5)(6)(7). Upon TCR triggering, LFA-1 dramatically increases its binding to ICAMs on APCs, which promotes conjugate formation and T cell activation (8). Antigen-specific T cell activation is initiated in the immunological synapse in detergent-insoluble glycolipid-enriched complexes, which, by including kinases (e.g. Lck and Fyn) and their substrates (e.g. LAT) and excluding phosphatases (e.g. CD45), are privileged sites for TCR signal induction (5, 6, 9 -12).Cloned CTLs are propagated by periodic re-stimulation, and hence are activated T cells, which express high levels of LFA-1 and ␤ 1 and ␤ 3 integrins. The latter interact with extracellular matrix (ECM) proteins, like fibronectin, vitronectin, and collagen, as well as with counter receptors (e.g. vasular cell adhesion molecule) on other cells (13)(14)(15)(16)(17). Whereas LFA-1-mediated adhesion requires TCR triggering, ␤ 1 and ␤ 3 integrin-mediated adhesion of activated T cells, although enhanced upon TCR triggering, also takes place spontaneously (16,18,19). Therefore these integrins can sense changes in the extracellular environment, e.g. when T cells leave the vasculature and enter secondary lymphoid organs or inflamed tissues, where they become strongly exposed to ECM proteins (16). Integrin-mediated adhesion to ECM proteins results in activation and recruitment at the contact sites of the focal adhesion kinases FAK (20, 21), Itk (22),, which promotes their association with the cytoskeleton linkers paxillin and talin (20,23,30,31) and the Src kinases Fyn (29) and Lck (28,30). Pyk2 is translocated to the T cell-target cell contact site after TCR triggering and plays an important role in degranulation of CTLs and natural killer cells (4,32). Although the avidity and the redistribution of in...

Significance The adaptive immune system has the potential to generate a self-reactive response, which can eventually lead to an autoimmune disease. To avoid this outcome, T lymphocytes with high-affinity, self-reactive antigen receptors are blocked from entering the mature T-cell pool (negative selection). Given this mechanism for removing dangerous high-affinity T cells, we wondered whether autoimmunity is more likely to be caused by chronic stimulation of low-affinity T cells or by stimulation of a few high-affinity T cells that escaped negative selection. In this paper, we show that T cells with an affinity just above the selection threshold can bypass negative selection and have the highest potential to cause an experimental autoimmune disease.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.