Kawasaki disease (KD) is a multisystem vasculitis leading to coronary artery aneurysm formation. In a superantigen-mediated murine model of KD, the development of coronary arteritis is mediated by T-cells through the production of TNFα. TNFα localizes to the coronary arteries, where it induces the expression of MMP-9, resulting in the breakdown of elastin and the formation of aneurysms. Statins have been recently shown to have anti-inflammatory and immunomodulatory properties as a result of the inhibition of small GTPases. In our murine model of KD, atorvastatin treatment inhibits superantigen mediated T- cell proliferation and cytokine production, including IL-2 and TNFα. Additionally, statin treatment inhibits TNFα-mediated MMP-9 production by vascular smooth muscle cells, through inhibition of the MEK/ERK pathway. Thus, statins modulate each of the critical steps in the pathogenesis of KD in a disease model, suggesting that statin use could alter the outcome and prognosis of children suffering with this disease.

Viral hepatitis infection represents a significant epidemiological and economic burden on society. Following infection, some patients mount a blunted immune response to the virus, which ultimately can result in chronic infection. FGL2, a member of the fibrinogen-related protein superfamily, has been implicated in vitro in suppressing both innate and adaptive immune responses. In a murine model of acute viral hepatitis caused by Lymphocytic Choriomeningitis Virus strain WE, we demonstrate that FGL2 expressed by reticuloendothelial cells limits viral spread. When expressed by Treg cells FGL2 binds to FCγRIIB and prevents DC maturation and suppresses virus-specific T and B cell responses. We provide compelling evidence to suggest that hepatitis viruses utilize the FGL2-FCγRIIB pathway to evade immune detection. Inhibition of this pathway restores effective cellular and humoral antiviral immune responses towards hepatitis viruses.

Lactobacillus casei cell wall extract (LCWE)- induced coronary arteritis in mice models Kawasaki disease (KD). LCWE injections consist of T-cell dependent factors that expand superantigen (SAg)-activated T-cell receptor (TCR) Vβ6+ cells, and T-cell independent factors (i.e. TLR2 activity) that localize and sustain the immune response. TLR2 can upregulate costimulatory molecules to rescue SAg-activated T-cells from apoptosis. Accordingly, SAg-activated costimulation-rescued TCRVβ6+ cells are predicted to express activation markers, produce cytokines and be able to induce coronary arteritis. MAM was identified as a SAg able to activate TCRVβ6+ cells in a manner similar to LCWE; however a combination of MAM and TLR2 agonist Pam3Cys could not induce coronary arteritis. As another marker of disease, leukocyte recruitment molecule expression in the hearts of MAM+Pam3Cys- injected mice was found to be lower than in LCWE- injected mice. Therefore, LCWE contains unique features beyond TCRVβ6 stimulation and TLR2 activity that are important for disease induction.

Both CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice. A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2). A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3). Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.

B lymphocytes develop from pluripotent stem cells, and differentiate to plasma cells (PCs) in reaction to signals from the supportive microenvironment. Different sets of signals, which are derived from multiple sources such as soluble cytokines and cell-cell contacts, are required at different stages of development. For instance, murine B cell progenitors require the action of interleukin-7 (IL-7) in the early phase of their development in the bone marrow (BM). The necessity for IL-7 decreases as the cell matures, and this event is correlated with the appearance of CD22. The first two chapters of this thesis focus on the early stages of B cell development that take place in the BM. In chapter 1, I examine the IL-7 response and, although I do not show a specific role for CD22 in the loss of sensitivity to IL-7, my data suggest that cis interactions involving sialic acids might modulate the IL-7 response. This section is followed by the analysis of the effect of IL-21 on B cell progenitors in the BM. IL-21 is known to regulate the terminal stages of B cell differentiation. In collaboration with Dr. Danijela Konforte, I present evidence that B cell progenitors in the BM also express a functional IL-21 receptor and that stimulation of this receptor with IL-21 accelerates the maturation pace of B cells. I further demonstrate that proB cells stimulated with IL-21 and anti-CD40 can differentiate into immunoglobulin (Ig)-secreting cells, and discuss the possibility that IL-21 plays a role during inflammation for the development of B cell progenitors in peripheral lymphatic organs. Finally, in the last chapter, in collaboration with the laboratory of Dr. Gommerman, I investigate how the microenvironment can shape the development of B cells. It has been demonstrated by my collaborators that IgA+ PCs present in the gut produce iNOS and display traits commonly associated to the myeloid lineage, and in this chapter, I describe a co-culture system with BM and gut stroma to study the conditions that sustain the generation of IgA+iNOS+ cells. In particular, I show that the presence of microbial products is one of the key factors required for their development.

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating numerous biological processes to establish an antiviral state and influencing the activation of various immune cells. During influenza A infection, the NS1 encoded by the virus genome disrupts many cellular processes to block type I IFN responses. We show that expression of H5N1 NS1 in HeLa cells reduces IFN-inducible activation of STAT proteins and its subsequent binding to DNA complexes. Subsequent analysis suggests NS1 blocks IFN signaling by inhibiting expression of type I IFN receptor subunit, IFNAR1, as well as up-regulating SOCS1 expression. Finally, we demonstrate that pretreatment of primary human lung tissue with IFN alfacon-1 inhibits H5N1 viral replication by up-regulating a number of interferon-stimulated genes. The data suggest that NS1 can directly interfere with Type I IFN signaling, and that pretreatment with IFN can inhibit H5N1 infection in primary human lung tissue.

FK506 binding protein 5 (FKBP5) is a peptidyl propyl cis-trans isomerase that has been shown to interact with cellular immune pathways such as calcineurin and NF-κB. During an influenza infection, FKBP5 is up-regulated at the lung in an in vivo ferret infection model, yet the effect of FKBP5 on influenza replication and immune response is not understood. An in vitro model of human alveolar epithelial cell line A549 was established to study the cause and the function of FKBP5 up-regulation during an influenza infection. In this in vitro model, FKBP5 was not up-regulated by influenza replication, but instead it was up-regulated when A549 cells were treated with glucocorticoid. FKBP5 up-regulation did not have any effect on rate of influenza replication. However, FKBP5 up-regulation mediated the suppressive effect of glucocorticoid on pro-inflammatory cytokine production, since FKBP5 knock-down by siRNA increased cytokine production in the presence of glucocorticoid. Overall, the results suggested that the up-regulation of FKBP5 is a physiological response of lung cells to the increase of glucocorticoid during influenza infections, which facilitates the suppressive effect of glucocorticoid on pro-inflammatory cytokine production.

Conventional DC (cDC) arise from circulating immediate precursors (pre-cDC), and are currently thought to be terminally differentiated. Here we show that cDC are capable of generating progeny that lost all characteristic features of cDC and aquired regulatory properties. Sorted bone marrow pre-cDCs were cultured on a stromal monolayer in the presence or absence of granulocyte-macrophage colony stimulating factor (GM-CSF). In the absence of GM-CSF, pre-cDC derived DCs gave rise to a homogeneous population of CD11clow MHClow cells (DC-regs) on day 8-10 of culture. DC-regs failed to up-regulate major histocompatibility complex class II (MHCII) and co-stimulatory molecules in response to DC maturation stimuli, were poor stimulators in T cell proliferation assays and suppressed T cell proliferation in cultures containing immuno-stimulatory DC. Co-transfer of DC-regs with DCs in vivo did not inhibit proliferation of T cells. These findings reveal the potential of DCs to generate a regulatory DC population with immunosuppressive properties.

Both CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice. A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2). A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3). Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.

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.