Investigation of T cell signalling events regulating immunity and tolerance in vivo

Abstract

Summary The ability of the immune system to discriminate between pathogenic and self or food antigens is essential not only for the generation of a productive immune response against invasive pathogens, but also for a state of antigen-specific tolerance to be elicited against harmless antigens. A breakdown in such tolerance can result in the development of a variety of autoimmune diseases including rheumatoid arthritis, Type 1 diabetes, inflammatory bowel disease and coeliac disease. Despite a wealth of studies in this field, however, the mechanisms by which the immune system can distinguish harmless and pathogenic antigens remain to be fully elucidated. If these mechanisms were better understood, such information could be exploited to help develop better therapies for autoimmune diseases, improve the rate of successful transplantations and increase the efficacy of vaccines. The primary means of maintaining tolerance to self antigens is to prevent self-reactive T cells from exiting the thymus following their development therein (central tolerance). However, some self-reactive T cells escape thymic deletion and as such, central tolerance is incomplete. Indeed, peripheral tolerance is required for an individual to elicit tolerance to all self-antigens, developmental antigens and some food and environmental antigens which are not present in early life. Peripheral tolerance is defined as a state of antigen-specific hyporesponsiveness, which is induced by exposure of T cells to antigen under sub-optimal activating conditions. For a T cell to become fully activated, and therefore productively primed, it must recognise its cognate antigen in the context of MHC and receive co-stimulation via the interaction of its CD28 receptor with CD80/86 on an antigen-presenting cell (APC). Clonal anergy, one of the proposed mechanisms of peripheral tolerance, describes a state of long lasting unresponsiveness to antigen in the T cell. Such anergy is induced when the TcR is ligated in the absence of co-stimulation and can be evidenced, upon re-stimulation with antigen, by reduced IL-2 production, cell cycle progression and proliferation, relative to that observed in primed cells. It has been widely proposed that both qualitative and quantitative differences in T cell signalling may underlie the differential functional outcomes of priming and tolerance. However, the majority of these studies have relied upon biochemical assessment of signalling in T cell lines or clones, at the population level following polyclonal stimulation in vitro, and thus has led to the generation of conflicting data. Moreover, and most importantly, these data do not necessarily reflect the responses of individual antigen-specific T cells within their environmental niche within primary or secondary lymphoid tissue. In addition, as such data represents the responses of all cell types in the sample population at any one time, they do not provide any information pertaining to the differential kinetics, amplitude or subcellular localisation of signals generated by functionally distinct subgroups within the population. A relatively new technology, laser scanning cytometry (LSC), offers an attractive means of investigating such responses, as it essentially marries the quantitative capabilities of flow cytometric analysis of cells in suspension with the ability to analyse spatially the fluorescence of large numbers of individual cells, either in suspension or in tissue in a slide-based format. Moreover, the adoptive transfer system, in which limited numbers of TcR transgenic (Tg) T cells are distributed evenly throughout the thymus-dependent area of the lymph node, provides an attractive means of studying antigen-specific responses occurring at near physiological frequencies in situ. Such antigen-specific T cells can be readily distinguished from endogenous T cells by LSC, following fluorescent staining of their TcR, as they are sparsely situated amongst the endogenous T cell population within the lymph node. Use of the adoptive transfer system, in combination with LSC analysis, has therefore enabled the development, in this thesis, of a quantitative imaging technology with which to study T cell signalling in individual antigen-specific T cells in vitro and in situ. In T cells, the maintenance phase of anergy has been reported to reflect defective activation of transcription factors, such as c-Jun/c-Fos, that are involved in formation of the AP-1 complex, which is required for inducing transcription of the IL-2 gene and optimal activation and effector function of T cells. In turn, this appears to be determined by the lack of recruitment of the ERK, JNK and p38 MAPK signalling cascades. The small GTPase, Rap1, has long been implicated in such desensitisation of ERK, and the consequent reduced IL-2 production, observed in tolerised T cells. However, as noted above, the majority of these findings were obtained from in vitro studies of T cell lines or clones and as such are not necessarily representative of physiological responses of primary antigen-specific T cells in situ. This study therefore describes, for the first time, an inverse relationship between ERK activation (pERK) and Rap1 expression in individual primary antigen-specific T cells during the maintenance phases of tolerance and priming, both in vitro and in vivo. Analysis at the single cell level further revealed that the proportion of antigen-stimulated cells expressing pERK was lower in the anergic relative to primed groups in vitro and in vivo, and the few anergised T cells expressing pERK did so at a lower level than the primed cells in vitro. By contrast, Rap1 was found to be expressed in a greater proportion of anergic antigen-specific T cells, and at considerably higher levels, compared with primed T cells following re-stimulation with antigen both in vitro and in vivo. An additional inverse relationship was observed between pERK and Rap1, concerning their subcellular localisation, with pERK appearing to co-localise with lipid raft structures in primed but not anergic cells and Rap1 appearing to be targeted to lipid rafts in anergic but not primed cells. These data suggests that Rap1 may be up-regulated and recruited to the immunological synapse upon re-stimulation with Ag in anergic T cells and that such Rap1 localisation and expression may contribute to the downregulation of ERK recruitment and activation in these cells. It is important to note that this inverse relationship between the accumulation of Rap1 and antagonism of ERK activation was only observed during the maintenance, and not induction, phases of both systemic and oral tolerance in vivo. Furthermore, assessment of the activation status of downstream cell cycle modulators in priming and tolerance, revealed that downregulation of ERK activation and upregulation of p27kip1 might not be sufficient for maintenance of the anergic state, as indicated by G1 arrest, and hence, one or more additional negative signals may be required. Indeed, this study suggests that perhaps such a negative signal could be provided by the downregulation of p-Rb and/or increased expression of inactive cyclin dependent kinases (CDKs). In summary, defective ERK signalling correlates with the up-regulation of Rap1 expression in tolerised relative to primed antigen-specific CD4+ T cells during the maintenance phases of tolerance in vitro and in vivo. As this association occurs after the induction of both systemic and oral routes of tolerance, these data suggest that Rap1 antagonism of pERK signalling may play an important and general role in the maintenance of antigen-specific CD4+ T cell tolerance. Moreover, as oral tolerance induction has been proposed as a potential therapy for autoimmune disorders and, oral administration of compounds offers a more attractive route for drug delivery in humans, these findings may have potential clinical applications. By advancing our knowledge of these key signals in regulating tolerance and priming at the single cell level in vitro and in vivo, we will therefore increase our understanding of an important physiological process at the molecular level, ultimately leading to identification of potential targets for enhancing or inhibiting immunity and tolerance