We examine an early model for the interaction of HIV with CD4+ T cells in vivo and define possible parameters and effects of said parameters on the model. We then examine a newer, more simplified model for the interaction of HIV with CD4+ T cells that also considers four populations: uninfected T cells, latently infected T cells, actively infected T cells, and free virus. The stability of both the disease free steady state and the endemically infected steady state are examined utilizing standard methods and the Routh-Hurwitz criteria. We show that if N, the number of infectious virions produced per actively infected T cell, is less than a critical value, , then the uninfected state is the only steady state in the non negative orthant, and this state is stable. We establish an expression for . If , then the uninfected steady state is unstable, and the endemically infected state can be stable or unstable, depending on the value of the parameters utilized.

The therapy suppressive tumour microenvironment (TME) continues to hinder anti-cancer therapies. Local delivery of therapeutic proteins, including potentially toxic factors, is increasingly needed to enhance immunotherapeutic bioactivities and minimize systemic toxicity. To this end, we are developing vehicles that immobilize to extracellular matrix (ECM) components upregulated in TME for localization of polymer-grafted bioactive cytokines with tunable degradation rates to control cytokine clearance. The grafted cytokine would be bioactive, and the length of the therapy would be governed by the degradation kinetics of the hydrolytic linker between the cytokine and polymer. The cytokines were expressed and purified, and their biological activity was confirmed. Click chemistry was used to graft the therapeutic proteins and collagen-binding peptides to the copolymer. Production of the therapeutic carriers was confirmed by SEC and fluorescent measurements. Biolayer interferometry and tracking immobilization inside collagen gel confirmed the binding affinity between carriers and collagen type 1. In vitro studies confirmed the bioactivity of the carriers in the presence of T-cells and macrophages. In summary, ECM binding vehicles for local sustained protein release will aid in the local delivery of therapeutic proteins to alter TME and promote immunotherapies. Screens will be conducted in multicellular spheroid models to identify bioactive formulations. / Thesis / Master of Science (MSc)

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Activating the immune system in the therapeutic treatment of cancer is rapidly growing and has demonstrated tremendous success. One such method is engineering T cells with chimeric antigen receptors (CARs) to specifically direct them in targeting tumours, however this has been associated with several toxicities that may be linked to the synthetic nature of the CAR. To address this, our laboratory created the T Cell Antigen Coupler (TAC), an alternative receptor that redirects T cells in a more natural TCR-dependent fashion. The TAC consists of three components: the antigen-binding domain that recognizes a tumour antigen, a TCR-recruitment domain that co-opts the native CD3-TCR complex and a CD4 co-receptor domain. The TAC displays unique biology, specifically in the increased antitumor infiltration and clearance of solid malignancies without any of the observed host toxicities seen with CARs. The functionality of the TAC was shown to be dependent on both the antigen binding and TCR-recruitment domains, however the co-receptor domain remains relatively uninvestigated despite evidence in the literature indicating its importance in endogenous T cell activation. This thesis seeks to better understand the biology of the TAC receptor by investigating the contributions of co-receptor domain. In Chapter 3, we replaced the CD4 co-receptor domain with CD8 variants and showed that the TAC retains functionality. In Chapter 4, we removed the cytosolic domain of the TAC in its entirety (creating a “tailless TAC”) and observed increased in vivo efficacy. In Chapter 5, we evaluated the tailless TAC in different cancer models and consistently observed increased in vivo efficacy compared to the full length TAC. These results demonstrate an increase in the in vivo functionality of the TAC receptor when the cytoplasmic tail is removed, giving us further insights into the mechanisms behind the unique biology of the TAC receptor. / Thesis / Doctor of Philosophy (PhD) / Cancer is the leading cause of death in Canada, and it is expected that 2 in 5 Canadians will develop some form of cancer in their lifetime. The immune system presents an intriguing alternative method to treat tumours since immune cells such as T cells can circulate through the body and seek and destroy harmful cells, including tumours. Here, we focus on the T cell Antigen Coupler (TAC), a genetically engineered receptor that our laboratory originally designed that directs T cells to recognize and destroy specific cancer cells. This thesis looks at the inner workings of the receptor, specifically a part called the inner tail, and how this feature contributes to how the TAC works. Our results show that removing the tail increases the T cell’s ability to safely clear different tumours in living organisms, bringing us a step closer in designing new and safe treatments for cancer patients.

<p>E and Id proteins are members of the basic helix-loop-helix (bHLH) transcription regulator family. These proteins control a broad range of lymphocyte biology, from the development of multiple lineages to execution of their effector functions. With the development of new experiment models, novel functions of E and Id proteins continued to be discovered. In this thesis, I focused my study on the role of Id2 in gamma delta T cells and CD4<super>+</super> alpha beta T cells, as well as the role of Id3 in B cells.</p><p> Id proteins have been shown to control gamma delta T cell development. Id3 knockout mice demonstrate a dramatic expansion of innate-like Vgamma1.1<super>+</super> Vdelta6.3<super>+</super> T cells in the neonatal stage, suggesting that Id3 is an inhibitor of their development. Interestingly, Id3 knockout mice with a B6/129 mix background have much less expansion of the Vgamma1.1<super>+</super> Vdelta6.3<super>+</super> T cells compared to mice with pure B6 background. Genetic studies showed that this difference is strongly influences by a chromosome region very close to the Id2 locus. Using the Id2<super>f/f</super> CD4Cre<super>+</super> mice, I found that Id2 is also an inhibitor of gamma delta T cell development. Deletion of Id2 alone is sufficient to enhance the maturation of these cells in the thymus and induce a moderate expansion of gamma delta T cells in the periphery. This study demonstrated the delicate balance of transcription control in cells of the immune system.</p><p> The Id2<super>f/f</super> CD4Cre<super>+</super> mice also enabled me to study the role of Id2 in peripheral CD4<super>+</super> alpha beta T cell functions, which was difficult in the past because Id2 knockout mice lack lymph node development. I found that CD4 T cells in these mice have a profound defect in mounting immune responses, demonstrated by a complete resistance to induction of experimental autoimmune encephalomyelitis (EAE). I found that Id2-deficient CD4 T cells fail to infiltrate the central nervous system, and the effector CD4 T cell population is smaller compared to that in control mice. Id2 is important for the survival and proliferation of effector CD4 T cells, and this phenotype was correlated with an increased expression of <italic>Bim</italic> and <italic>SOCS3</italic>. This study revealed a novel role of Id2 in the functioning of CD4<super>+ </super>alpha beta T cells.</p><p> Switching my focus to B cells, recent next generation sequencing of human Burkitt lymphoma samples revealed that a significant proportion of them have mutations of Id3. This finding suggests that Id3 may be a tumor suppressor gene in the lymphoid system. Utilizing various Id3 knockout and conditional knockout mouse models, I showed that Id3 deficiency can accelerate lymphoid tumor genesis driven by the over-expression of oncogene c-Myc. This work may lead to development of a more realistic mouse model of human Burkitt lymphoma, allowing more mechanistic studies and perhaps preclinical tests of new therapies.</p> / Dissertation