Nicholas R. J. Gascoigne

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling

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.

Hijacking and exploitation of IL-10 by intracellular pathogens

Macrophages play a central role in infections, as a target for pathogens and in activation of the immune system. Interleukin-10 (IL-10), a cytokine produced by macrophages, is a potent immunosuppressive factor. Some intracellular pathogens specifically target macrophages for infection and use IL-10 to dampen the host immune response and stall their elimination from the host. Certain viruses induce production of cellular IL-10 by macrophages, whereas other viruses encode their own viral IL-10 homologs. Additionally, specific bacteria, including several Mycobacteria spp. and Listeria monocytogenes, can survive and replicate in macrophages while inducing cellular IL-10, highlighting a potential role for IL-10 of macrophage origin in the immunosuppressive etiology of these pathogens. Thus, the exploitation of IL-10 appears to be a common mechanism of immunosuppression by a diverse group of intracellular pathogens that can infect macrophages.

The Impact of Duration versus Extent of TCR Occupancy on T Cell Activation: A Revision of the Kinetic Proofreading Model

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.

Qualitative and quantitative differences in T cell receptor binding of agonist and antagonist ligands.

The kinetics of interaction between TCR and MHC-peptide show a general relationship between affinity and the biological response, but the reported kinetic differences between antigenic and antagonistic peptides are very small. Here, we show a remarkable difference in the kinetics of TCR interactions with strong agonist ligands at 37 degrees C compared to 25 degrees C. This difference is not seen with antagonist/positive selecting ligands. The interaction at 37 degrees C shows biphasic binding kinetics best described by a model of TCR dimerization. The altered kinetics greatly increase the stability of complexes with agonist ligands, accounting for the large differences in biological response compared to other ligands. Thus, there may be an allosteric, as well as a kinetic, component to the discrimination between agonists and antagonists.

CD28 plays a critical role in the segregation of PKCθ within the immunologic synapse

The signaling pathways that lead to the localization of cellular protein to the area of interaction between T cell and antigen-presenting cell and the mechanism by which these molecules are further sorted to the peripheral supramolecular activation cluster or central supramolecular activation cluster regions of the immunologic synapse are poorly understood. In this study, we investigated the functional involvement of CD28 costimulation in the T cell receptor (TCR)-mediated immunologic synapse formation with respect to protein kinase C (PKC)θ localization. We showed that CD3 crosslinking alone was sufficient to induce PKCθ capping in naïve CD4+ T cells. Studies with pharmacologic inhibitors and knockout mice showed that the TCR-derived signaling that drives PKCθ membrane translocation requires the Src family kinase, Lck, but not Fyn. In addition, a time course study of the persistence of T cell molecules to the immunologic synapse indicated that PKCθ, unlike TCR, persisted in the synapse for at least 4 h, a time that is sufficient for commitment of a T cell to cell division. Finally, by using TCR-transgenic T cells from either wild-type or CD28-deficient mice, we showed that CD28 expression was required for the formation of the mature immunologic synapse, because antigen stimulation of CD28− T cells led to a diffuse pattern of localization of PKCθ and lymphocyte function-associated antigen-1 in the immunologic synapse, in contrast to the central supramolecular activation cluster localization of PKCθ in CD28+ T cells.

TCR affinity and negative regulation limit autoimmunity

Autoimmune diseases are often mediated by self-reactive T cells, which must be activated to cause immunopathology. One mechanism, known as molecular mimicry, proposes that self-reactive T cells may be activated by pathogens expressing crossreactive ligands. Here we have developed a model to investigate how the affinity of the T-cell receptor (TCR) for the activating agent influences autoimmunity. Our model shows that an approximately fivefold difference in the TCR affinity for the activating ligand results in a 50% reduction in the incidence of autoimmunity. A reduction in TCR-ligand affinity to approximately 20 times lower than normal does not induce autoimmunity despite the unexpected induction of cytotoxic T lymphocytes (CTLs) and insulitis. Furthermore, in the absence of a key negative regulatory molecule, Cbl-b, 100% of mice develop autoimmunity upon infection with viruses encoding the lower-affinity ligand. Therefore, autoimmune disease is sensitive both to the affinity of the activating ligand and to normal mechanisms that negatively regulate the immune response.

Control of MHC Restriction by TCR Vα CDR1 and CDR2

Individual T cell receptor (TCR) Vα elements are expressed preferentially in CD4 or CD8 peripheral T cell subsets. The closely related Vα3.1 and Vα3.2 elements show reciprocal selection into CD4 and CD8 subsets, respectively. Transgenic mice expressing site-directed mutants of a Vα3.1 gene were used to show that individual residues in either the complementarity-determining region 1 (CDR1) or CDR2 were sufficient to change selection from the CD4 subset to the CD8 subset. Thus, the germline-encoded Vα elements are a major influence on major histocompatibility class complex (MHC) restriction, most likely by a preferential interaction with one or the other class of MHC molecule.

The Vα14 NKT Cell TCR Exhibits High-Affinity Binding to a Glycolipid/CD1d Complex

Most CD1d-dependent NKT cells in mice have a canonical Vα14Jα18 TCR rearrangement. However, relatively little is known concerning the molecular basis for their reactivity to glycolipid Ags presented by CD1d. Using glycolipid Ags, soluble forms of a Vα14 NKT cell-derived TCR, and mutant and wild-type CD1d molecules, we probed the TCR/CD1d interaction by surface plasmon resonance, tetramer equilibrium staining, and tetramer staining decay experiments. By these methods, several CD1d α-helical amino acids could be defined that do not greatly alter lipid binding, but that affect the interaction with the TCR. Binding of the Vα14+ TCR to CD1d requires the agonist α-galactosylceramide (α-GalCer), as opposed to the nonantigenic β-galactosylceramide, although both Ags bind to CD1d, indicating that the carbohydrate moiety of the CD1d-bound Ag plays a major role in the TCR interaction. The TCR has a relatively high-affinity binding to the α-GalCer/CD1d complex, with a particularly slow off rate. These unique properties are consistent with the coreceptor-independent action of the Vα14 TCR and may be related to the intense response to α-GalCer by NKT cells in vivo.

Inhibition of T cell receptor-coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T-hybridoma immunological synapse.

The diverse effects of TCR agonists and antagonists on T cell activation are believed to be modified by the differential recruitment of CD4 or CD8 coreceptors to the TCR-MHCp complex. We used three-dimensional live cell imaging of fluorescence resonance energy transfer (FRET) between CD3zeta and CD4 fused to variants of the green fluorescent protein to investigate TCR-CD4 interactions during T cell activation. We demonstrate that recognition of agonist MHCp complexes triggers intermolecular interaction between CD4 and TCR, detectable across the T-hybridoma-APC contact area. This interaction is blocked by the presence of antagonist ligands without decreasing the recruitment of zeta and CD4 or preventing their partial colocalization in the immunological synapse.

Nonstimulatory peptides contribute to antigen-induced CD8–T cell receptor interaction at the immunological synapse

It is unclear if the interaction between CD8 and the T cell receptor (TCR)–CD3 complex is constitutive or antigen induced. Here, fluorescence resonance energy transfer microscopy between fluorescent chimeras of CD3ζ and CD8β showed that this interaction was induced by antigen recognition in the immunological synapse. Nonstimulatory endogenous or exogenous peptides presented simultaneously with antigenic peptides increased the CD8-TCR interaction. This finding indicates that the interaction between the intracellular regions of a TCR-CD3 complex recognizing its cognate peptide–major histocompatibility complex (MHC) antigen, and CD8 (plus the kinase Lck), is enhanced by a noncognate CD8-MHC interaction. Thus, the interaction of CD8 with a nonstimulatory peptide-MHC complex helps mediate T cell recognition of antigen, improving the coreceptor function of CD8.