The adaptive immune response forms an essential part of the cancer immuno-editing process, whereby nascent malignant cells are detected and destroyed prior to forming tumours. The process is tightly controlled to minimise collateral damage to healthy tissue. One of the mechanisms evolved for this purpose and frequently co-opted by malignant cells is the creation of a microenvironment scarce in essential amino-acids through the use of catabolic enzymes such as Indoleamine 2,3-dioxygenase (IDO) , responsible for the rate-limiting step in tryptophan catabolism. The evolutionary conserved GCN2 and mTORC1 pathways respond to amino-acid starvation by triggering emergency homeostatic response programmes that aim to conserve nutrients by shutting down biosynthetic pathways, slowing cell cycle progression and facilitating autophagy. This research project focuses on elucidating the interaction between IDO activity and these pathways and its implications for the immune-editing process. The role of the mTOR kinase as a regulator of T cell fate following exposure to cognate antigen has recently become apparent. Experiments described herein confirm that in murine and human models of T cell activation exposure to tryptophan starvation results in significant mTORC1 inhibition and a modified phenotype with reduced Tbet expression, altered cytokine secretion profile, greatly impaired proliferative capability and expanded CD4⁺ FoxP3⁺ CD25^high subpopulations. Additional results confirmed that the action of IDO is sufficient to deplete tryptophan from the microenvironment to levels sufficient to depress the mTORC1 axis and trigger GCN2 activity even in tumour cell lines. Lower extracellular tryptophan levels were necessary to perturb these pathways In IDO expressing cell lines, suggesting that compensatory mechanisms allow continued proliferation of malignant cells in the face of conditions that severely impede an anti-cancer immune response. In conclusion, manipulation of the mTORC1 axis via IDO-induced tryptophan depletion is an important tumour immune-escape mechanism that can be a target for cancer immunotherapies.

The serine protease factor Xa (FXa) plays an integral role in the coagulation cascade and has recently been implicated in a variety of proinflammatory roles, establishing it as a link between coagulation and inflammatory processes. In this thesis, I elaborate on previous literature by characterizing further the response of primary human T lymphocytes to FXa. Building on previous literature that describes the effect of FXa on whole T cell populations, I describe here the effect of FXa on both antigen-independent and antigen-dependent proliferation and costimulation of primary CD4+ and CD8+ T cells, thereby establishing an immunological role for FXa. Further, I show that FXa elicits an immediate and direct effect on T cells demonstrated by the rapid upregulation of the signalling cascade kinases, ERK1 and ERK2. Lastly, I demonstrate that the protease activated receptor 2 (PAR2) is involved in the mediation of this direct FXa effect.

The serine protease factor Xa (FXa) plays an integral role in the coagulation cascade and has recently been implicated in a variety of proinflammatory roles, establishing it as a link between coagulation and inflammatory processes. In this thesis, I elaborate on previous literature by characterizing further the response of primary human T lymphocytes to FXa. Building on previous literature that describes the effect of FXa on whole T cell populations, I describe here the effect of FXa on both antigen-independent and antigen-dependent proliferation and costimulation of primary CD4+ and CD8+ T cells, thereby establishing an immunological role for FXa. Further, I show that FXa elicits an immediate and direct effect on T cells demonstrated by the rapid upregulation of the signalling cascade kinases, ERK1 and ERK2. Lastly, I demonstrate that the protease activated receptor 2 (PAR2) is involved in the mediation of this direct FXa effect.

<p>  </p> <p>IL-9-producing CD4+ T helper (Th9) cells contribute to inflammatory responses during infection, anti-cancer responses and autoimmune disease. Thus, elucidating the signals that regulate their differentiation is critical for understanding the roles of Th9 cells in protective immunity and disease. Th9 cells differentiate in response to IL-4, TGF-β and IL-2, where IL-2 signaling through STAT5 is crucial for transactivating <em>Il9</em> locus. While the roles of IL-4 and TGF- β-mediated signaling are relatively well understood, how IL-2 signaling contributes to Th9 cell differentiation outside of directly inducing the <em>Il9</em> locus remains less clear. I found that human allergen-induced Th9 cells exhibited a strong signature of STAT5-mediated gene repression that was associated with inhibition of a Th17-like transcriptional signature. Likewise, blockade of IL-2/STAT5 signaling increased IL-17 and RORγt expression in murine Th9 cells <em>in vitro</em>. Interestingly, development of this Th17-like phenotype was independent of STAT3. While STAT3 was not required for IL-17 expression, it was required for their long-term persistence. These results suggest that IL-2/STAT5 signaling controls the balance between Th9 and Th17-like cell differentiation in vitro and during allergy. Additionally, I found that murine Th9 cells cultured in a low IL-2 environment had reduced IL-9 production and a diminished NF-kB-associated transcriptional signature, suggesting that IL-2 signaling is associated with NF-kB activation in Th9 cells. Interestingly, NF-kB activation via IL-1β stimulation enhanced Th9 differentiation under IL-2 limiting conditions and promoted their inflammatory potential in a mouse model of Lung inflammation. Mechanistically, we found that IL-2- limiting conditions enhanced IL-1β receptor expression and that IL-1β/NF-kB signaling increased the sensitivity to IL-2 and silenced the expression of the anti-Th9 transcription factor BCL6. Together, these findings indicate that IL-1β /NF-kB signaling can promote Th9 cell differentiation in IL-2-limiting conditions and that this pathway may be targeted to enhance Th9 differentiation and their inflammatory function.  Collectively, these data revealed two novel roles for the IL-2/STAT5 axis in Th9 cells.</p> <p>The Thymocyte associated High Mobility Group (HMG) box, known as TOX has been previously described to have paramount functions in the development of all the lineages of CD4+ T cells during thymic selection, during CD8+ T cell exhaustion and in Tfh cell differentiation and function. However, the role of TOX in non-Tfh CD4+ T cells in the periphery has not been addressed. In these studies, I found that CD4+ T cells express TOX in the steady state in secondary lymphoid organs like spleen, lymph nodes, and Peyer’s patches. Specifically, TOX was expressed remarkably in Tfh, Th1, Treg cells, and other non-Tfh unidentified Th cells, as well as Th2 cells in the lungs. Transcriptomics analyses using bulk RNA-seq revealed that TOX minimally alters s gene expression, however it revealed for the first time, that TOX induced genes associated with cell migration i.e., <em>Xcl1</em> <em>Ccl3</em>, <em>Ccl4</em> and also the inhibitory cytokine <em>Il10</em>. The induction of IL-10 and CCL3 was validated at the protein levels, and mechanistic studies revealed that the induction of these molecules required the transcription factor BATF, indicating for the first time a mechanism of TOX-mediated functions. Together, these data shed light in novel roles of TOX in CD4+ T cell function and opens the door for future functional and mechanistic studies that may be relevant during health and disease.</p>