Signalling of E3 ubiquitin-protein ligases in the regulation of priming and tolerance of T cells

An effective immune system is essential for constant surveillance of potential threats to the organism, its’ primary role being to provide resistance to infection. Thus, the immune system must be able to discriminate between pathogenic and self or harmless foreign antigens in order to not only achieve a productive immune response but also to avert unwarranted hyperreactivity and the potential development of autoimmunity. A delicate balance between these two fundamental requirements of the immune system must therefore exist. T cells take central roles in the orchestration of the immune response. They are responsible for adaptive cell-mediated immunity and as such they can eliminate infected cells, produce cytokines that help in the resolution of the infection, stimulate other cells of the immune system to participate in the immune response and turn into long-lived memory cells. It is not surprising then that a wealth of mechanisms exist in order to ensure that T cells mediate immunity against pathogenic antigens but not to self or harmless ones. Indeed, various checkpoints in T cell development exist in the thymus to enable, on the one hand a large pool of diverse and functional T cells capable of recognising virtually all antigens while, on the other hand, eliminating self-reactive T cells. As some self-reactive T cells manage to leave the thymus, additional mechanisms have evolved to continually protect against auto-reactive and allergic inflammatory responses in the periphery. These mechanisms of peripheral tolerance include cell-mediated suppression of T cell responses, T cell apoptosis and T cell anergy. Anergy is a long-lived, cell-intrinsic mechanism that is characterised by cell cycle progression arrest and reduced IL-2 production. While T cell priming requires recognition of the cognate antigen in the context of MHC and co-stimulation via the interaction of its CD28 receptor with CD80/86 on an APC, recognition of the cognate antigen in the context of MHC without co-stimulation, such as when antigen is presented by immature APCs in the absence of inflammation, leads to anergy. These anergic T cells will also be hyporresponsive to re-challenge, even under immunogenic conditions. The acquisition of the anergic phenotype is an active process, with negative regulators of T cell signalling being induced. Among these are some E3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosomes. While a protein targeted to the proteasome is degraded by it, proteins in the endosomes can either be recycled for further signalling or directed to the lysosomes, where they are also degraded; in any case, ubiquitination leads to downregulation of the activity of signalling elements, whether permanently (degradation) or temporarily (recycling). Therefore a stable difference in the gene expression profile between anergic and primed T cells could be achieved through ubiquitin modification translating into an increase in turnover of signalling mediators involved in the T cell response, thereby establishing a persistent unresponsive state. In fact, some key signalling mediators involved in T cell activation, such as PLC and PKC, have been shown to be downregulated by the ubiquitin pathway in the context of anergy induction. Conversely, the E3 ubiquitin-protein ligases Cbl-b, Itch and Grail have been shown to be upregulated during anergy. These and other E3-ubiquitin-protein ligases have also been shown to ubiquitinate and downregulate TCR signalling elements. However, many of the studies leading to these findings have relied upon biochemical assessment of signalling in T cell lines or clones, at the population level following pharmacological stimulation in vitro. The data generated in this way is sometimes conflicting with that of studies which focused on the knockout of a single gene, followed by analysis of the phenotype. Moreover, as in vitro pharmacological stimulation data represents the responses of all cell types in the sample population at any one time, it does not necessarily reflect the responses of individual antigen-specific T cells within their environmental niche within lymphoid tissue. Information relating to differential kinetics or subcellular localisation of signals generated by functionally distinct subgroups within a population could help assess the full picture regarding the role of E3 ubiquitin-protein ligases during the induction and maintenance phases of T cell anergy. The main objectives of this thesis were therefore to determine the expression of Cbl-b, Itch and Grail in individual antigen-specific CD4+ T cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. In order to accomplish these objectives, induction of priming or tolerance in ovalbumin (OVA323-339 peptide)-specific T cells from DO11.10 TCR transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo, was carried out. Functional outcome, measured in terms of cell cycle progression, proliferation, cytokine readout assays, antibody production and T cell migration was correlated with E3 ubiquitin-protein ligases expression and the ubiquitination status of the TCR signalling machinery. Cbl-b, Itch and Grail protein expression in LN tissue, antigen-specific CD4+ T cells and subcellular compartments was assessed. Moreover, quantitative analysis at the single cell level was carried out by tracking the antigen-specific CD4+ T cells in vitro and in vivo by using Laser Scanning Cytometry. This relatively new technology combines the quantitative capabilities of flow cytometric analysis of cells with the ability to analyse them within a tissue in situ within their microenvironmental niche, thus more within physiological parameters. This study shows Cbl-b expression to be upregulated in CD4+ T cells undergoing induction of both anergy and priming, when compared to naive cells. This is a departure from the view that states Cbl-b is a differential factor promoting anergy. In the maintenance phase, Cbl-b levels also appear upregulated in both anergic and primed antigen-specific T cells. Interestingly, Cbl-b concentrates in the periphery of anergic cells immediately after re-stimulation and this concentration of Cbl-b in the periphery of the cells was also observed for primed T cells after re-stimulation but at later time points. These data may therefore implicate Cbl-b in the modulation of T cell activation in the context of CD28 co-stimulation, rather than acting solely as a selective agent in the promotion of anergy. Itch was similarly found to be upregulated in anergising and priming CD4+ T cells, when compared to naive cells. In fact, cells undergoing priming were found to express more Itch than those undergoing anergy. Likewise, in the maintenance phase, re-stimulation of primed cells resulted in higher Itch expression than re-stimulation of anergic antigen-specific T cells. Itch appears therefore to not play an exclusive role in anergy but instead acting in the general context of TCR signalling. Moreover, Grail expression was found to be upregulated in priming CD4+ T cells while there is also evidence it was transiently expressed at high levels during the anergy induction phase. Intriguingly, in the maintenance phase, Grail is associated with the migration of antigen-specific T cells into the follicles. In summary, the data presented here indicate that upregulation of Cbl-b, Itch and Grail is not exclusive to T cells undergoing or maintaining anergy, as priming and primed T cells also exhibit expression and upregulation of these signals. While these data therefore show that these molecules cannot be used as markers for T cell anergy, and while the search for a bona fide T cell anergy marker continues, it also opens up new possibilities for their role(s) in modulation of T cell activation. By advancing knowledge of the key signalling events that take place during antigen recognition, more targeted approaches for enhancing or inhibiting immunity or tolerance can be devised.