A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

The widely accepted kinetic proofreading theory proposes that rapid TCR dissociation from a peptide/MHC ligand allows for stimulation of early but not late T cell activation events, explaining why low-affinity TCR ligands are poor agonists. We identified a low-affinity TCR ligand which stimulated late T cell responses but, contrary to predictions from kinetic proofreading, inefficiently induced early activation events. Furthermore, responses induced by this ligand were kinetically delayed compared to its high-affinity counterpart. Using peptide/MHC tetramers, we showed that activation characteristics could be dissociated from TCR occupancy by the peptide/MHC ligands. Our data argue that T cell responses are triggered by a cumulative signal which is reached at different time points for different TCR ligands.

Costimulation of the T cell receptor (TCR) and CD28 is required for optimal interleukin-2 (IL-2) induction. These signals, which can be replaced by the pharmacological agents phorbol ester (PMA) and Ca 2⍣ ionophore, synergistically activate the mitogen-activated protein kinase (MAPK) JNK. Cyclosporin A, an inhibitor of the Ca 2⍣ -dependent phosphatase calcineurin which blocks IL-2 induction, abrogates Ca 2⍣ -triggered synergistic JNK activation. As protein kinase C (PKC) downregulation inhibits PMA⍣ionophore-induced JNK activation, we examined whether a particular PKC isoform is preferentially involved in this response. We found that PKC-θ but neither PKC-α nor PKC-ε participates in JNK activation, whereas all three PKCs lead to ERK MAPK activation. PKC-θ specifically cooperates with calcineurin, and together their signals converge on (or upstream of) Rac leading to potent JNK activation. Similarly, calcineurin and PKC-θ specifically synergize to induce transcription of reporters driven by the c-jun and IL-2 promoters. PKC-θ and calcineurin are also partially responsible for the synergistic activation of JNK following TCR and CD28 ligation. Preferential cooperation between PKC-θ and calcineurin is observed in Jurkat T cells but not in HeLa cells. These results indicate that PKC isozymes have distinct biological functions and suggest that synergistic JNK activation is an important function for PKC-θ in T-cell activation.

Positive selection allows thymocytes that recognize an individual's own major histocompatibility complex (self-MHC) molecules to survive and differentiate, whereas negative selection removes overtly self-reactive thymocytes. Although both forms of thymic selection are mediated by the alphabeta T-cell receptor (TCR) and require self-MHC recognition, an important question is whether they are controlled by distinct signalling cascades. We have shown that mutation of an essential motif within the TCR alpha-chain-connecting peptide domain (alpha-CPM) profoundly affects positive but not negative selection. Using transgenic mice expressing a mutant alpha-CPM TCR we examined the contribution of several mitogen-activated protein kinase (MAPK) cascades to thymic selection. Here we show that in thymocytes expressing a mutant alpha-CPM receptor, a positively selecting peptide failed to activate the extracellular signal-regulated kinase (ERK), although other MAPK cascades were induced normally. The defect in ERK activation was associated with impaired recruitment of the activated tyrosine kinases Lck and ZAP-70, phosphorylated forms of the TCR component CD3zeta and the adaptor protein LAT to detergent-insoluble glycolipid-enriched microdomains (DIGs). Therefore, an intact DIG-associated signalosome is essential for sustained ERK activation, which leads to positive selection.

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.