Ninan Abraham

The immune response to solid tumours involves a complex interplay of both tumour-promoting immunosuppression and tumour inhibition via targeted cell killing. As tumour development progresses, immunosuppression occurs in the tumour microenvironment (TME) and killer CD8⁺ T cell activity can become inhibited by checkpoint receptor engagement. Current immune checkpoint inhibitors (ICI) are only effective in a subset of lung cancer patients and alternate immune evasion strategies can render these therapeutics less successful. To better understand mechanisms behind alternate immune evasion, this thesis examined whether tumour cell-secreted chemokines, circulating immune cells, and CD4⁺ tissue resident memory T cells (CD4⁺ Trm) can shape immunosuppression in the TME. In Chapter 3, driver oncogenes in lung cancer cells were shown to induce production of the chemokine, CCL5. Mouse models with tumour cell CCL5 knockdown had decreased regulatory T cells (Tregs), display reduced evidence of T cell exhaustion and reduced lung tumour burden. This shows that tumour cell CCL5 production contributes to an immune suppressive environment in the lungs. Chapter 4 details a longitudinal, peripheral blood-based immunophenotyping study of stage IV NSCLC patients treated with anti-PD-1 ICI. This study identified several innate and adaptive immune cell populations, markers of interest, and cytokines that were altered pre-treatment or during ICI in a patient with durable clinical benefit compared to early progressors. These findings will help focus future, larger studies on peripheral blood biomarkers of response to ICI. Chapter 5 assessed CD4⁺ Trm cells presence, location, and potential function in lung cancer. We established that both genetically engineered and syngeneic mouse models of lung cancer had increased CD4⁺ Trm cells with distinct expression of checkpoint programmed death receptor-1 (PD-1), Th1 or Th2 transcriptional profiles, and type-1 cytokine production. In NSCLC patients, transcriptomic signatures of CD4⁺ Trm cells correlated with improved survival. In summation, the data details how tumour cell oncogenic signaling-induced CCL5 can contribute to immunosuppression in the TME, that longitudinal alteration to circulating immune cells and cytokines distinguished a patient with durable response to ICI and that populations of CD4⁺ Trm cells were a significant component of the immune response in mouse models of lung cancer.

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Interleukin-7 (IL-7) is a cytokine with well-established roles in lymphocyte development in the bone marrow and thymus. Recent discoveries have expanded IL-7’s contribution to enhancing the effector responses of CD8 T cells in chronic LCMV infection and tumor clearance. IL-7Rα is highly expressed by mature lymphocytes in the lungs, but how IL-7 directs their function in acute airway viral infections is unclear.Using multiple mouse models, I show in chapter 3 that loss of IL-7 signaling results in impaired production of IL-5 and IL-13 in lung group two innate lymphoid cells (ILC2s) following influenza infection. Conversely, mice treated with IL-7 have increased production of IL-5 and IL-13 by lung ILC2s. Moreover, I show that IL-7 regulates GATA3 and CD25 expression in lung and bone marrow ILC2s. However, IL-7 is non-essential for the expression of the anti-apoptotic protein Bcl-2 and survival of ILC2s. In chapter 4, I show that IL-7 signaling plays an important role in CD8 T cell responses to acute influenza infection. Specifically, IL-7Rα is required for a normal sized mediastinal lymph node and the expansion of influenza-specific CD8 T cells therein. Terminal differentiation of influenza-specific CD8 T cells requires normal IL-7 signaling as well. Interestingly, IL-7 also plays a selective role in enhancing the effector function of influenza-specific CD8 T cells depending on their antigen specificity. Finally, IL-7 is inducible in the lungs by multiple cellular sources following viral infection. These findings on IL-7 and its effects on lower respiratory diseases will be important for expanding the utility of therapeutics that are currently available.

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The cytokine Interleukin-7 (IL-7) is critical for T cell development and function. Mutations that block IL-7 signaling in humans cause severe combined immunodeficiency syndrome due to the failure of T cells to develop. Conversely, mutations that result in constitutive signaling through the IL-7 receptor can drive human leukemias, including Early Thymic Progenitor (ETP) acute lymphoblast leukemia. How IL-7 influences ETP development is still unclear since ETPs do not normally express IL-7Rα. We found that ETPs are highly dependent on and sensitive to changes in IL-7 signaling however, IL-7Rα expressing bone marrow progenitors are not. IL-7 does not regulate the survival or proliferation of ETPs, although it does at subsequent stages of T cell development. We hypothesize an instructive role of IL-7 signaling in the differentiation or migration of bone marrow precursors of ETPs that is distinct from its classic mechanisms regulating survival and proliferation.Adjuvant IL-7 has been shown to increase the quantity and quality of T cell responses to clear chronic viral infections, however, its physiological role in anti-viral T cells is unclear. We found that IL-7 signaling was required for efficient clearance of Influenza A virus (IAV) and protection from viral induced pathology. T cells require cell intrinsic IL-7 signaling for efficient primary CD4 and CD8 T cell responses to IAV. The requirement for IL-7 signaling in the anti-viral response, and the ability of exogenous IL-7 to boost T cell responses suggests that it may be clinically useful in therapy or vaccination against IAV. IAV remains an important global health challenge, with up to 10% of the global population infected annually. Current IAV vaccines do not generate universal cross-serotype immunity. T cell memory responses to IAV correlate with cross-serotype protection. Transcutaneous immunization has shown potential to induce memory T cell responses that can protect against infections at other mucosal sites, including the lung. We found that transcutaneous immunization generates a strong memory CD8 T cell response that can protect from challenge with IAV. Therefore, transcutaneous immunization may be a useful to produce cross-serotype IAV immunity.

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The cytokine interleukin (IL)-7 is necessary for human T cell development and murine B and T lymphopoiesis. Mice lacking IL-7, either of its receptor components, the common γ chain (γc) or IL-7 receptor α (IL-7Rα), or essential intracellular signaling molecules are severely lymphopenic. Due to the developmental block in early T and B progenitors, the requirement for IL-7Rα signaling in later T and B cell stages has been difficult to evaluate. To address this question, we characterized lymphopoiesis in IL-7Rα449F mice harboring a single point mutationin IL-7Rα, where a key signaling residue (tyrosine 449) is mutated to phenylalanine (F).Biochemical analysis revealed that IL-7Rα Y449 is essential for activation of STAT5 and that there are both Y449-dependent and independent contributions to cell survival through regulation of Bcl-2 family members. IL-7Rα449F T and B cells are able to overcome the characteristic developmental block of IL-7Rα-/- mice and develop appreciable numbers of peripheral T and B cells. This finding permitted evaluation of peripheral T cell function. These experiments demonstrated that IL-7Rα Y449 signals are required for naïve T cell homeostasis, generation of a primary CD4 T cell response and for maintenance of memory CD8 T cells following Listeria monocytogenes infection. In contrast to expectations, the CD8 memory T cell maintenance defect does not appear to be a direct result of decreased Bcl-2 expression.Dysregulated cytokine signaling can also contribute to development and/or maintenance of leukemia and lymphoma. Through genetic analysis of two distinct oncogenes, we were able to show that the IL-7Rα449F mutation was sufficient to protect mice from IL-7 mediated T and B cell transformation and significantly delay the emergence of B cell lymphomas in the Eμ-myc mouse model of Burkitt’s lymphoma. Disruption of STAT5 activation appears to play a significant role in both models of tumor protection. Collectively, the data demonstrate that IL-7Rα signaling has both Y449-dependent and independent modalities that cooperate to ensure optimal B and T cell development and response to infection. Further, the data show that these signaling pathways can contribute to lymphomagenesis, and may be attractive targets for immunotherapeutics of IL-7 responsive tumors.

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