T cell Signalling events in vivo during the induction of Immunity and Tolerance

The intestine is continually exposed to viruses, bacteria and parasites which threaten its function and against which we must defend ourselves. This is the role of the protective immune response and to combat infection, the intestine contains parts of the immune system. However, the majority of the food we eat is also ?foreign? to our immune system, as are the huge numbers of harmless bacteria (commensals) that live normally in our intestines and are essential for life. It is important that we do not attempt to make protective immune responses against these useful materials, as this can lead to severe intestinal disorders such as coeliac disease and Crohn?s disease. Indeed, the immune system normally becomes unresponsive (tolerant) to these materials as it has evolved mechanisms for distinguishing between dangerous and harmless materials. It is important to understand how this happens, not only to have a better chance of treating intestinal disease, but also because there is a great need for new vaccines to combat infections of the intestine. In addition, it may be possible to treat a number of inflammatory conditions such as diabetes and rheumatoid arthritis by exploiting this ability of immune system to become tolerant to ingested materials. A considerable amount is already known about the cellular processes involved and it now seems clear that the signals dictating how a particular population of T lymphocytes (those carrying the CD4 marker) responds may be very different in cells reacting to dangerous versus harmless materials. Thus, it may be possible to target some of these signals to deliberately switch on or off immune responses in the intestine. However, progress in this area has been limited by the fact that it is extremely difficult to identify directly those CD4+ T cells that respond. Moreover, study of the signals usually requires cells to be removed from the immune system and submitted to harsh biochemical processes. We have now developed new models that allow normal and mutant antigen specific T lymphocytes to be tracked and characterised in the intact immune system with highly sensitive laser-driven microscopy techniques that can assess such signals in individual cells. By analysing these events in situ, we hope to be able to identify precisely molecules that might prove useful in treatment of disease and in vaccine development.

The mucosal immune system has to discriminate between pathogenic antigens that require a strong protective immune response and those antigens that are harmless, or even beneficial. For example, aberrant T cell responses against foods or commensal bacteria can cause intestinal disease and so the ability of such antigens to induce immunological tolerance is of great physiological importance. In addition oral tolerance has also been exploited to deliver immunotherapy against systemic inflammatory diseases. Thus it is important to understand the molecular processes that determine whether antigen-specific T cells are primed or tolerised by antigen (Ag). One such mechanism of peripheral tolerance is the unresponsiveness of T cells to secondary antigenic stimulation as a result of the induction of anergy. Consistent with this unresponsiveness, it has been shown that primed and tolerised T cells display different signalling responses with tolerised cells exhibiting defective ErkMAPkinase activation. The physiological relevance of these differential Erk signals has not been clear, however, as such defects were identified by in vitro biochemical analysis of T cell clones or immortalized lines, which do not reflect the responses of primary Ag-specific T cells in vivo. We therefore recently developed techniques to quantify signaling events in Ag-specific T cells on an individual cell basis in vivo, using laser-scanning cytometry (LSC). This has now allowed us to show that there are indeed marked differences in the kinetics, amplitude and cellular localization of phosphorylated ERK1/2 MAPkinase signals when naive, primed and tolerised T cells are challenged with Ag both ex vivo and in vivo. Moreover, the reduced ability of tolerised T cells to activate ERK1/2 upon challenge reflects differential induction of the GTPase, Rap1 by primed and tolerised T cells. We now wish to capitalize on these novel technologies to further dissect the regulatory mechanisms involved and their potential roles in inducing T cell tolerance for example, by determining their relationship to the ubiquitin ligase-mediated proteolytic degradation of the signalling machinery required for productive T cell priming. We shall do this by infection of Adenovirus receptor-expressing Ag-specific T cells with signalling mutants of the Rap and RasERKMAPkinase cascade and following adoptive transfer, visualizing their responses in vivo by LSC and 2-photon microscopy. By defining the key signals regulating these different immunological outcomes in vivo, we will increase our understanding of an important physiological process at the molecular level ultimately leading to identification of novel targets for enhancing or inhibiting immunity and tolerance.