Kenneth Harder
Relevant Thesis-Based Degree Programs
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Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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Immunotherapy has improved patient survival in a subset of human malignancies. However, many patients do not respond to current therapies highlighting the need to better understand the interactions between tumours and the immune system. Phagocytes, such as dendritic cells (DCs), macrophages, and myeloid-derived suppressor cells (MDSCs), are critical in orchestrating or antagonizing innate and adaptive immune responses against tumours. In turn, phagocyte activity and development are dysregulated by tumour-derived factors. We previously identified granulocyte colony-stimulating factor (G-CSF) as a potent tumour-derived cytokine that alters phagocyte development and function. Using Cytometry by Time-of-Flight (CyTOF), we found that growth of G-CSF-expressing tumours led to a systemic accumulation of a complex mixture of myeloid cells and their progenitors, and perturbations in a wide variety of signalling pathways throughout the immune system. G-CSF significantly blocked the development of CD103⁺ classic dendritic cells (cDC1s), which are associated with improved cancer treatment outcomes and strong CD8⁺ cytotoxic T cell activity. Correspondingly, loss of tumour secreted-G-CSF improved adoptive T cell therapy of breast cancer tumours. In addition, treatment of colon cancer-bearing mice with neutralizing anti-G-CSF antibodies reduced MDSC accumulation, normalized colonic immune cell composition, and diminished tumour burden. Further, better patient survival was found to correlate with low G-CSF and neut/MDSC gene expression through analysis of patient CRC gene expression data. Altogether, our data suggest that G-CSF can induce myeloid lineage perturbations and immunosuppression and that modulating G-CSF bioactivity could improve immunotherapy efficacy in cancers associated with over-expression of G-CSF. Finally, we provide evidence that neutralization of human G-CSF delays tumour growth in humanized mouse models systems.
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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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The immune system is critical for host survival by providing protection against infectious organisms. However, the immune system also plays a major pathological role in many human diseases through inappropriate inflammatory responses that can lead to tissue damage and death. Therefore, understanding the mechanisms that drive and regulate these responses has broad implications for the development of therapies for numerous diseases including inflammatory bowel disease and atherosclerosis. Through its involvement in key signalling pathways, the Lyn tyrosine kinase is an important regulator of immune cell development and function. Using models of chemically induced colitis and enteric infection, we show that Lyn plays a critical role in regulating gastro-intestinal inflammation and in protection from enteric pathogens, by regulating host responses to intestinal microbes. Lyn-/- mice were highly susceptible to dextran sulfate sodium-induced colitis, whereas Lyn gain-of-function (LynUP) mice exhibited attenuated colitis. Protection in LynUP mice was independent of the adaptive immune system and involved hypersensitivity to microbial products such as LPS, leading to enhanced production of protective factors including IL-22. Increased DSS susceptibility in Lyn-/- mice correlated with dysbiosis and altered T cell responses. This dysbiosis was characterized by an expansion of segmented filamentous bacteria, which was associated with altered production of IL-22 and IgA. Furthermore, increased Lyn activity in dendritic cells was sufficient to drive increased IL-22 production by innate lymphoid cells. This extended beyond the gut to the spleen, which added to previous findings by our lab and others, identifying Lyn as an important regulator of systemic mononuclear phagocytes and their inflammatory responses. Finally, we identified Lyn as a negative regulator of Ly6C- patrolling monocytes (pMo). Lyn-/- mice displayed increased frequency and numbers of pMos whereas LynUP mice were skewed towards increased conventional monocytes (cMo). The increase in pMos in Lyn-/- mice was independent of the adaptive immune system and the autoimmune and myeloproliferative disorders that develop in these mice with age. Furthermore, these monocyte differences correlated with a pathological role for Lyn in a model of atherosclerosis. Together, these studies identify Lyn as an important regulator of innate immune responses involved in sterile and microbial-driven inflammatory diseases.
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Previous studies have revealed that perturbations in myelopoiesis can lead to the emergence of immature cells, which can facilitate tumorigenesis and metastasis. Our studies showed that mammary tumours led to a myeloproliferative-like disease, characterized by anemia, leukocytosis, expansion of immature myeloid cells, and defects in the hematopoietic stem and progenitor cell (HSPC) compartment in tumour-bearing mice. Furthermore, mammary tumours impaired DC development resulting in the accumulation of DC progenitors and the emergence of immunosuppressive DCs with impaired ability to activate NK and T cells. Mammary tumour-bearing mice exhibited a shift in hematopoiesis from the bone marrow to the spleen, with large numbers of primitive and committed progenitors accumulating in the spleen. Mammary tumour development was also associated with epigenetic modifications that facilitated the expression of key hematopoiesis and leukemia regulatory genes, such as Hoxa9, a gene that critically controls HSPC differentiation. Using in vitro assays, we identified mammary tumour derived G-CSF as the factor mediating the dysregulation of the HSPC compartment and suppressing DC development. DCs are critical for the priming/activation of NK cells and cytotoxic T lymphocytes, which together are critical components of the anti-tumour immune response. Therefore, proper regulation of signaling thresholds is critical for DC activation and function. Lyn tyrosine kinase is important in regulating signaling thresholds in immune cells, including DCs, and Lyn gain-of-function (Lynup/up) mice show increased sensitivity to PAMPs (i.e. LPS) characterized by high levels of proinflammatory cytokines and increased susceptibility to endotoxin-mediated death. Our studies demonstrated that DCs were necessary for the enhanced LPS-induced inflammation in Lynup/up mice, by priming excessive IFN-γ production by NK cells. As such, we hypothesized that enhanced Lyn activity, and the associated changes in DC development and activation will evoke improved anti-tumour immune responses and preliminary data from our lab demonstrates that Lynup/up mice develop smaller mammary tumours than wild type mice. Taken together, our results suggest that targeting mammary tumour derived G-CSF may reverse mammary tumour-induced anemia, leukocytosis, and DC defects, conversely enhancing DC function by increasing Lyn activity may result in in a more robust anti-tumour immune response, leading to increased survival of breast cancer patients.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Tumours are often associated with defects in hematopoiesis and DC function. Leukocytosis, anemia, development of immunosuppressive cells such as MDSCs and tolerogenic DCs are all part of tumour development. In breast cancer, although these hematopoietic and DC defects may serve as prognostic indicators, little is known about their origin. Our studies show that mammary tumours affect the hematopoietic system leading to myeloproliferative-like disease characterized by neutrophilia, anemia, and defects in the HSPC compartment. These defects were associated with changes in global epigenetic regulation, and specifically in the upregulation and histone methylation status of Hoxa genes, most notably Hoxa9, a gene that critically controls HSPC differentiation. These changes in gene regulation and HSPC defects were found to be driven by tumour-secreted G-CSF. Additionally, our research shows that the generation of immunosuppressive DCs in mammary tumour bearing mice was associated with impaired DC differentiation, where DCs that develop in the presence of mammary tumours and their factors acquire immunosuppressive features. G-CSF played an important role in the suppression of DC development. Tumour derived factors induced FLT3L and GMCSF DCs to expression Arginase-I, an enzyme known to suppress T cell proliferation and induce tolerance. Our data revealed the expression of Arg1 in these DCs was associated with enabling histone modifications in the Arg1 locus. Targeting of G-CSF may alleviate tumourinduced symptoms of anemia, leukocytosis, and DC defects, resulting in better patient survival and preventing DC immunosuppression.
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