The crucial role of the thymus in immunological tolerance has been demonstrated by establishing that T cells are positively selected to express a specificity for self major histocompatibility complex (MHC), and that those T cells bearing receptors potentially reactive to self antigen fragments, presumably presented by thymic MHC, are selected against. The precise mechanism by which tolerance is induced and the stage of T-cell development at which it occurs are not known. We have now studied T-cell tolerance in transgenic mice expressing a T-cell receptor with double specificities for lymphocytic choriomeningitis virus (LCMV)-H-2Db and for the mixed-lymphocyte stimulatory (MIsa) antigen. We report that alpha beta TCR transgenic mice tolerant to LCMV have drastically reduced numbers of CD4+CD8+ thymocytes and of peripheral T cells carrying the CD8 antigen. By contrast, tolerance to MIsa antigen in the same alpha beta TCR transgenic MIsa mice leads to deletion of only mature thymocytes and peripheral T cells and does not affect CD4+CD8+ thymocytes. Thus the same transgenic TCR-expressing T cells may be tolerized at different stages of their maturation and at different locations in the thymus depending on the antigen involved.

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.

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An alloreactive cytotoxic T cell clone (433) possessing the L3T4-, Lyt-2+ phenotype is described that shows a double specificity. It has been derived from unprimed BALB/c (H-2d) spleen cells by repetitive in vitro restimulation with C57BL/6(H-2b) cells. The specificity of clone 433 was determined in cytotoxicity and proliferation experiments. One specificity was for the class I antigen H-2Db and the second was for the class II antigen I-Ek. Inhibition of cytotoxicity with monoclonal antibodies confirmed these results. Cold target competition experiments demonstrated that the two specificities were mediated by the same cell population. Anti-Lyt-2 antibodies inhibited only the H-2Db- but not the I-Ek-specific lysis, suggesting a higher affinity of the antigen receptor for I-Ek than for Db. To our knowledge, this is the first description of a T cell clone that is specific for a class I antigen and cross-reacts heteroclitically with a class II antigen.