Intravenous immunoglobulin is a human blood derived product that is used to treat immunodeficiencies and autoimmune disorders. An adverse side effect of IVIg therapy is hemolysis. Patients who experience hemolysis are mainly blood group A or AB. Clinical laboratory studies have demonstrated that IVIg contains ABO blood group antibodies, which can bind complement proteins. This study hypothesizes that anti-A/B in IVIg will bind to A/B antigens and activate complement in a dose dependant manner, which may lead to enhanced RBC phagocytosis. This study observed that the quantity of ABO antigens does not affect the in vitro binding of IVIg to RBCs. IVIg induced C3b deposition at high doses; however, the amount of complement deposition was insufficient to enhance phagocytosis of IVIg-sensitized RBCs by monocytic THP-1 cells in vitro. These studies emphasize that hemolytic reactions involve many factors in conjunction with antibodies and complement proteins.

Intravenous immunoglobulin is a human blood derived product that is used to treat immunodeficiencies and autoimmune disorders. An adverse side effect of IVIg therapy is hemolysis. Patients who experience hemolysis are mainly blood group A or AB. Clinical laboratory studies have demonstrated that IVIg contains ABO blood group antibodies, which can bind complement proteins. This study hypothesizes that anti-A/B in IVIg will bind to A/B antigens and activate complement in a dose dependant manner, which may lead to enhanced RBC phagocytosis. This study observed that the quantity of ABO antigens does not affect the in vitro binding of IVIg to RBCs. IVIg induced C3b deposition at high doses; however, the amount of complement deposition was insufficient to enhance phagocytosis of IVIg-sensitized RBCs by monocytic THP-1 cells in vitro. These studies emphasize that hemolytic reactions involve many factors in conjunction with antibodies and complement proteins.

The homeodomain-interacting protein kinase (HIPK) family is comprised of four evolutionarily conserved and highly related serine/threonine kinases originally identified as co-repressors for homeodomain-containing transcription factors. While the HIPKs are most noted for regulation of apoptosis, proliferation and differentiation, I report a pleiotropic function of HIPK1 within the B cell lineage. Although lymphocyte development was normal within the thymus and bone marrow of HIPK1-deficient (HIPK1-/-) mice, the spleen exhibited a reduced number of transitional and follicular (FO) B cells, but with an increase in the marginal zone (MZ) B cell population. HIPK1-/- B cells exhibited impaired proliferation in response to B cell receptor (BCR) cross-linking in vitro; and immunization of HIPK1-/- mice with T-independent type 2 (TI-2) antigen resulted in a significantly impaired humoral response despite the expanded MZ B cell population. Immunization with T-dependent (TD) antigen resulted in a kinetically delayed response, with impaired affinity maturation. Identification of a kinase-substrate interaction between HIPK1 and the B cell adaptor 3BP2 suggests a potential context for HIPK1 function in BCR signaling. HIPK1-/- B cells were uniquely resistant to reactive oxygen species (ROS)-induced apoptosis, but equally susceptible to UV- and γ-irradiation compared to controls. In vitro class-switch recombination (CSR) assays revealed that HIPK1 is required for the negative regulation of CSR. HIPK1-/- B cell cultures harbored more viable cells, more switched cells, and elevated AID mRNA levels. The findings presented in this thesis demonstrate that HIPK1 is required for splenic B cell homeostasis and optimal BCR-responsiveness. In contrast, HIPK1 is also required for the negative regulation of CSR, possibly by mediating CSR-induced apoptosis.

Systemic lupus erythematosus (SLE) is an autoimmune disorder that is characterized by the production of antibodies directed against self-antigens, such as nuclear components. Genetic analyses of lupus patients have consistently demonstrated a complex genetic basis for disease susceptibility that involves multiple genes. Identifying genes and pathways that promote disease genesis has been aided by murine studies. In particular, congenic mouse studies that examine the role of chromosomal intervals from inbred lupus-prone mouse strains on a non-autoimmune background have been useful in dissecting the genetic contribution of novel susceptibility loci in lupus pathogenesis. In this thesis, the role of New Zealand Black (NZB) chromosomes 4 and 13 are examined in non-lupus prone C57BL/6 (B6) congenic mouse strains, denoted B6.NZBc4 and B6.NZBc13, respectively. Although repeatedly mapped to contain disease augmenting alleles,NZBc4 alone was not sufficient to initiate disease, despite an expansion of NKT and B1a cells���both with controversial pathogenic roles in lupus. Instead, by crossing the B6.NZBc4 mouse with another congenic mouse strain that develops fatal lupus autoimmunity, NZBc4 was unexpectedly found to contain a suppressor locus; disease suppression was mediated by a shift away from pathogenic immunoglobulin isotypes and associated with changes in the NKT and B1a cell compartments. In contrast to the NZBc4 locus, the NZBc13 locus is sufficient to initiate polyclonal B cell activation, ANA production and mild GN, similar to NZB mice. A B cell intrinsic defect was found to be responsible for initiating the abnormal cellular phenotype in B6.NZBc13 mice. Functional analyses of the B cell subset in B6.NZBc13 mice revealed normal BCR-signaling responses and tolerance mechanisms; however, they were hyper-responsive to TLR3 stimulation, resulting in increased survival and proliferation. Thus, the study of these NZB congenic mouse strains has been instrumental in confirming the presence of loci on NZB chromosomes 4 and 13 that modulate the development of disease and highlights that disease onset is mediated by a balance of both susceptibility and suppressor alleles. Collectively, these findings contribute to the current field of lupus immunogenetics and confirm the power of congenic mouse models in understanding the genetic basis of SLE.

The homeodomain-interacting protein kinase (HIPK) family is comprised of four evolutionarily conserved and highly related serine/threonine kinases originally identified as co-repressors for homeodomain-containing transcription factors. While the HIPKs are most noted for regulation of apoptosis, proliferation and differentiation, I report a pleiotropic function of HIPK1 within the B cell lineage. Although lymphocyte development was normal within the thymus and bone marrow of HIPK1-deficient (HIPK1-/-) mice, the spleen exhibited a reduced number of transitional and follicular (FO) B cells, but with an increase in the marginal zone (MZ) B cell population. HIPK1-/- B cells exhibited impaired proliferation in response to B cell receptor (BCR) cross-linking in vitro; and immunization of HIPK1-/- mice with T-independent type 2 (TI-2) antigen resulted in a significantly impaired humoral response despite the expanded MZ B cell population. Immunization with T-dependent (TD) antigen resulted in a kinetically delayed response, with impaired affinity maturation. Identification of a kinase-substrate interaction between HIPK1 and the B cell adaptor 3BP2 suggests a potential context for HIPK1 function in BCR signaling. HIPK1-/- B cells were uniquely resistant to reactive oxygen species (ROS)-induced apoptosis, but equally susceptible to UV- and γ-irradiation compared to controls. In vitro class-switch recombination (CSR) assays revealed that HIPK1 is required for the negative regulation of CSR. HIPK1-/- B cell cultures harbored more viable cells, more switched cells, and elevated AID mRNA levels. The findings presented in this thesis demonstrate that HIPK1 is required for splenic B cell homeostasis and optimal BCR-responsiveness. In contrast, HIPK1 is also required for the negative regulation of CSR, possibly by mediating CSR-induced apoptosis.

Prostate cancer is a leading cause of cancer deaths in North American men. Current treatments are often not curative, particularly in cases of advanced metastatic disease. Immunotherapy is a promising approach to treating cancer as it harnesses the immune system’s ability to mount potent responses against tumor-associated antigens (TAAs). Dendritic cells (DCs) play a central role in mediating antigen-specific immunity and have been recently used with some success in clinical trials. The difficulties associated with obtaining sufficient quantities of DCs from cancer patients provided the rationale for developing low-dose DC-based immunotherapy approaches in my thesis project. DCs were genetically engineered using a lentiviral vector (LV) to express erbB2tr, a kinase-deficient version of erbB2. The human form of erbB2, HER2/neu, is overexpressed in 20% of primary prostate tumors and 80% of their metastases, making this TAA an attractive target. Using this LV system, efficient transgene delivery into DCs was achieved without compromising DC function or phenotype. Administering low prime and boost doses (2x10^5 or 2x10^3) of LV-transduced DCs to mice yielded potent and long-term anti-tumor responses against murine prostate tumors engineered to overexpress erbB2tr. The 2x10^5 DC dose yielded complete tumor protection and was associated with humoral and cellular responses. The 2x10^3 dose also offered complete protection in some mice, suggesting that we had reached a lower threshold DC dose. This novel finding prompted us to determine if co-transducing DCs with an additional LV carrying the cDNA for an immunomodulatory factor could augment the efficacy of our low-dose strategy. We chose to test both the DC survival-enhancing RANKL protein and DC function-enhancing IL-12 in combination with erbB2tr. Although DCs co-transduced with the LV/RANKL and LV/erbB2tr did not appear to offer enhanced anti-tumor benefits in a prophylactic setting, co-transduction with LV/IL-12 and LV/erbB2tr did. The incorporation of IL-12 into the low-dose immunization strategy led to robust long-term tumor protection and relatively high levels of Th1 immunity. This is the first demonstration of the efficacy of low-dose DC-mediated immunotherapy using lentiviral vectors as gene transfer tools. These studies establish a platform for DC-mediated therapies that can be realistically translated to the clinic.

Progressive obesity and its associated metabolic syndromes represent a globally growing challenge, yet mechanistic understanding and current therapeutics are unsatisfactory. We discovered that CD4+ T-lymphocytes, resident in visceral adipose tissue (VAT), control insulin-resistance in diet-induced obese (DIO) mice and likely humans. DIO VAT-associated T cells display biased TCR-Valpha/betarepertoires suggesting antigen-specific expansion. CD4+ T-lymphocyte control of glucose homeostasis is compromised in DIO when VAT accumulates pathogenic IFNgamma-secreting Th1 cells, overwhelming static numbers of Th2 (CD4+GATA-3+) and regulatory Foxp3+ T cells. CD4+ T cell transfer into DIO, lymphocyte-free RAGnull mice reversed weight gain and insulin resistance predominately through Th2 cells. Brief systemic treatment with anti-CD3 antibody or its F(ab’)2 fragment, restores the Th1/Foxp3+ balance and reverses insulin resistance for months, despite continuing high-fat diet. CD4+ T cells impact the progression of obesity-associated metabolic abnormalities and can be manipulated by immunotherapy.

B lymphopoiesis is regulated by cytokines, chemokines and cell surface proteins that initiate signal transduction pathways necessary for maturation to proceed. Many of these factors are expressed by cells in the surrounding bone marrow (BM) microenvironment, which also form the niches that support development. Interleukin-7 (IL-7) is an essential cytokine for progenitor B cells and is important in providing survival, proliferation and maturation signals. By growing BM B cells for extended periods of time in vitro with IL-7 it is possible to select for cells that possess the ability to grow indefinitely, and these cultures can be used to generate cell lines. Data presented herein describe the generation and characterization of IL-7-dependent B cell lines as well as their utility in investigating aspects of B cell development. As B cells mature they lose responsiveness to IL-7, yet retain IL-7 receptor expression. I demonstrate that a B cell’s ability to respond to IL-7 is controlled by the expression of suppressor of cytokine signaling (SOCS) proteins, which are regulated by a variety of signaling pathways including those initiated by IL-7. Development of progenitor B cells to mature immunoglobulin secreting B cells is mediated in part by surface proteins present on stromal cells as well as on B cells themselves. Heparan sulfate and CD19 play important roles in regulating this transition and I provide data that demonstrates how their ability to regulate Erk activation downstream of the pre-B cell receptor (pre-BCR) alters the proliferation and maturation of developing B cells.

The PTPN22 gene encoding the Lyp/Pep protein tyrosine phosphatase has recently been described as a negative regulator of T-cell receptor signalling. Little biological information is available on this protein, but a variant allele in this gene conferring a R620W change has been associated with rheumatoid arthritis and other autoimmune disease states. To gain further understanding into the roles of Lyp, this work is aimed at identifying and characterizing Lyp interactions, and elucidating the effect of the variant Lyp in immunological disease. Specifically, the interaction of Lyp with the ubiquitin ligase Cbl was further examined and characterized to uncover its role in T-cells. Furthermore, the biochemical and functional differences of the variant Lyp were examined by utilizing a murine model of the variant, Pep R619W. This work led to novel findings on the stability of the protein and its resulting dysfunction, leading to cell hyperresponsiveness. Finally, a new role for Lyp in controlling cell migration was uncovered through its interaction with GRK2. The inhibitory properties of Lyp on cell migration are disrupted in the presence of the Lyp R620W variant, leading to dysregulation of GRK2 function and altered migratory properties of cells, particularly in the collagen-antibody induced arthritis model. Understanding the normal function of Lyp, as well as dysfunction of the variant, will provide new insights into normal T-cell signalling and aid in the understanding of the processes of autoimmunity.

Although induction of innate immune responses during viral infection is essential, it can cause acute inflammation and lead to devastating results. The deleterious effect of innate immune responses has been demonstrated in gene therapy where administration of a replication deficient adenoviral vector (Ad) caused fatality during a clinical trial. Despite recent advances in our understanding of the innate immunity, there is a lack of understanding on how different cell types interact to mount inflammatory responses, which may play an important role in regulating immune responses in vivo. In this study, we investigated the interaction between macrophages and epithelial cells, the two major cell types capable of sensing and responding to viral infection in the airway, in induction of inflammatory responses against replication deficient Ads. We show in Chapter 2 that Ad infection of the macrophage-epithelial cell co-culture resulted in synergistic induction of inflammatory responses. Ad infection of the co-culture compared to macrophages alone resulted in higher cytotoxicity and induction of significantly higher levels of inflammatory mediators including pro-inflammatory cytokines, chemokines, nitric oxide, and reactive oxygen species. We found that these synergistic responses require macrophages and epithelial cells to be in close proximity suggesting that a novel mechanism regulates the inflammatory responses. In Chapter 3, we studied whether ATP plays a role in regulating inflammatory responses during acute Ad infection. Using the co-culture system, we found that ATP signaling through P2X7 receptor (P2X7R) is critical as inhibition or deficiency of P2X7R resulted in reduced inflammatory responses. We demonstrate that ATP-P2X7R signaling regulates inflammasome activation and IL-1β secretion. Furthermore, intranasal administration of Ad resulted in high mortality in mice but inhibition of ATP-P2X7R signaling enhanced survival and reduced inflammatory responses. These results suggest that ATP released by the infected cells plays an important role in regulating inflammatory responses during acute viral infection.