Plasmacytoid dendritic cells (pDCs) are members of the innate immune system and are particularly important in anti-viral immunity since they are the most potent producers of type I interferon (IFN). The objective of this research project is to analyze the phenotype of knockout mice for Ly49Q, a cell surface receptor expressed on pDCs. In vivo, KO mice with PGK-Neor insertion control early murine cytomegalovirus (MCMV) infection better, but no difference could be detected in mice with recombined LoxP sites. Thus, we observed differential immune response in KO mice with the PGK-Neor insertion and recombined LoxP sites. In mice with PGK-Neor insertion, we found enhanced hematopoiesis of some populations of the myeloid lineage and lower activation of pDCs following treatment with particular Toll-like receptors (TLR) ligands, which translates into diminished T cell stimulatory capacity. This suggests a role for Ly49Q in regulating hematopoiesis and pDC activation potential.

Asthma is a common inflammatory airway disease affecting over 2.5-3 million Canadians. This disease involves a Th2-driven immune response. Th2-type cytokines, (interleukin (IL)-4, IL-5, and IL-13) are upregulated in human asthmatics and in animals models of allergic airway inflammation. A recently characterized cytokine, IL-17E (IL-25), expressed in vitro by Th2 lymphocytes, macrophages and mast cells, generates a Th2 phenotype in mice. Overexpression of IL-17E promotes asthma-like features in animal models, namely, elevated levels of IL-4, IL-5 and IL-13, increased airway hyperreactivity, eosinophilia, increased IgE, airway epithelial cell hypertrophy/hyperplasia, and airway mucus hypersecretion. IL-17E binds the interleukin-17B receptor (IL-17BR), which is expressed in a variety of tissues and is upregulated under inflammatory conditions. We have investigated the expression of this receptor on structural cells of the airways in addition to assessing the effect of IL-17E on these cells. We hypothesized that airway smooth muscle cells (ASMC), lung epithelial cells and lung fibroblasts express IL-17BR and that proinflammatory cytokines could modulate its expression. The three cell types constitutively express IL-17BR mRNA and protein, and its expression was consistently upregulated by the proinflammatory cytokine TNFalpha. We also observed that the Th1 type cytokine IFNgamma decreased IL-17BR expression in ASMC, we observed a similar decrease by TGFbeta in fibroblasts. IL-17E appears to have profibrotic properties, as it upregulates the expression of pro-collagen alpha1 mRNA in ASMC, this could suggest that it could increase basement membrane thickening in vivo, a characteristic feature of airway remodelling. Pro-inflammatory and pro-eosinophilic cytokines such as RANTES, eotaxin, IL-8 and GM-CSF are increased in lung fibroblasts treated with IL-17E. In A549 lung epithelial cells, IL-17E also acts synergistically with TNFalpha to induce GROalpha mRNA and this effect can be inhibited by recombinant IL-17BR. In addition, IL-17E promoted the proliferation of bronchial epithelial cells isolated from biopsies taken from control and mild asthmatic donors. Collectively, our results show that the IL-17E receptor is consistutively present in structural cells of the airways and increases in the presence of TNFalpha. Our results also suggests a potential pro-remodelling and pro-inflammatory role for IL-17E on these cells.

Viral vector-based vaccines represent an effective means of in vivo antigen expression and the ensuing generation of a sustained immune response in the host. These new generation vaccines are deemed promising against pathogens for which researchers have so far failed to put forth preventive strategies and/or effective, accessible, treatment modalities. HIV-1 stands at the foremost of this list. In the current study, we have evaluated the use of two different viral vector-based vaccines against Clade A of HIV-1, namely recombinant modified vesicular stomatitis virus (VSV-AV3) and Adenovirus serotype 5 (Ad5) expressing the Gag protein from subtype A. These viral vectors, which are also inherently endowed with adjuvant properties, were delivered via a mucosal immunization strategy in a humanized transgenic mouse model. Transgenic mice expressing both HLA-A*0201 and HLA-DR*0101 represent a versatile model in which HIV-specific immunogenic epitopes and the resulting T cell receptor (TCR) specificity can be determined. We show that following mucosal delivery of vaccine, there is induction of antigen-specific systemic T cells against epitopes which were previously shown to be immunogenic in humans. We next developed novel multimeric reagents for the detection of CD4 + T cells, namely dodecameric HLA-DR1 molecules using a murine immunoglobulin M (IgM) scaffold. These reagents aim at increasing the overall avidity of peptide-MHC Class II complexes to detect low-affinity TCRs. These multimers were able to activate in vitro a Jurkat T cell line in an antigen-specific manner. The identification and characterization of the molecular requirements boosting the qualitative and quantitative features of the immune response to HIV vaccines, in addition to the development of novel state-of-the-art immune monitoring tools, will be crucial in better understanding of the mechanisms of interaction between the virus and the host immune system, leading to rational strategies in the fight against the AIDS epidemic.

A number of reports examining the role of CEACAM1 (Carcinoembryonic Antigen-related Cell Adhesion Molecule 1) in T lymphocytes have defined for it both stimulatory or inhibitory co-receptor functions. Using our unique Ceacaml1-/- mouse model, we previously observed that CEACAM1 is not involved in the development nor in the migration of T cells, but that it exerts a co-inhibitory effect on their activation and proliferation. Here, we report that its loss does not affect the ratios of naive and memory helper or cytotoxic T cells. Moreover, cytotoxic T cells from OT-1:Ceacam1-/- mice, resulting from breeding OT-1 TCR transgenic mice with Ceacam1-/- mice, are hyperproliferative and secrete more interleukin-2 and interferon-7 than their OT-1 counterparts. Finally, studies involving CD2-CCl-L transgenic mice whose T cells overexpress CEACAM1 did not consistently yield the hypoproliferative behaviour expected. Together, these findings suggest that CEACAM1 is an inhibitory co-receptor in T cells.

Natural killer (NK) cells possess the ability to destroy abnormal cells such as cancerous and virally-infected cells. A common feature of these cells is the downregulation of class I MHC molecules in an attempt to evade recognition by CD8+ T lymphocytes. "Missing-self" recognition allows NK cells to identify and destroy cells with decreased MHC-I expression. MHC-I receptors are expressed by NK cells, therefore allowing "missing-self" recognition. The major MHC-I receptors on human NK cells are KIR while mouse NK cells express Ly49 receptors. In addition to the recognition of MHC-I on target cells, Ly49 receptors are also involved in the education of NK cells, a process in which developing NK cells acquire effector functions.In human, KIR haplotypes vary greatly between individuals and the same phenomenon is observed in mice; Ly49 haplotypes differ in various mouse strains. Sequencing of the non-obese diabetic (NOD) Ly49 haplotype revealed that this strain encodes for the largest Ly49 haplotype known to date. The NOD Ly49 cluster is characterized by the largest number of genes coding for activating Ly49 receptors. The presence of a large number of Ly49 activating receptors may affect NK cell activation status and influence the development of diabetes in NOD mice.To gain insight in the in vivo role of Ly49 receptors in the control of NK cell activity, a NKC-knockdown mouse was generated. NK cells from these mice show greatly decreased expression of NKC-encoded receptors such as Ly49. NKC-knockdown NK cells have reduced lysis of MHC-I-negative target cells in vitro and in vivo. The ability of NK cells to destroy MHC-I-negative cells is restored by the re-introduction of a single Ly49 receptor. These results provide the first direct evidence that Ly49 receptors are involved in the in vivo surveillance of MHC-I molecules. Interestingly, the absence of Ly49 receptors results in defective "missing-self" recognition but activation of NK cells by MHC-I-independent mechanisms is not impaired.NKR-P1 receptors, which bind to Clr proteins, are members of another receptor family expressed by NK cells. Interestingly, the genes coding for NKR-P1 and Clr ligands are located in the same cluster and are therefore inherited together. Previous analyses demonstrated that the Nkrp1-Clr gene cluster is conserved in B6 and BALB/c mice. Mapping of the 129-strain cluster confirmed the high degree of conservation of the Nkrp1-Clr gene order in 129-strain mice. Gene sequencing revealed conservation of 129 and BALB/c Nkrp1 and Clr genes and divergence from the B6 genes, suggesting that the 129 cluster is more closely related to the BALB/c cluster than to the B6 cluster.Taken together, the Ly49 receptor family varies greatly in contrast with the highly conserved NKR-P1 receptors. Loss of Ly49 receptor expression reveals that Ly49 receptors are required for the acquisition of "missing-self" responses by NK cells. / Les cellules 'natural killer' (NK) possèdent l'habileté de détruire des cellules anormales telles que des cellules cancéreuses ou infectées par des virus. Un trait distinctif de ces cellules est la perte d'expression de molécules CMH de type I dans une tentative d'éviter la reconnaissance par les lymphocytes T CD8+. La reconnaissance de la 'perte de soi' permet aux cellules NK d'identifier et de détruire les cellules ayant une diminution de l'expression des molécules CMH-I. Des récepteurs pour les CMH-I sont exprimés par les cellules NK permettant la reconnaissance de la 'perte de soi'. Les principaux récepteurs de CMH-I pour les cellules NK humaines sont les KIR tandis que les cellules NK de souris expriment les récepteurs Ly49. En plus de la reconnaissance de CMH-I à la surface de cellules cibles, les Ly49 sont également impliqués dans l'éducation des cellules NK, un procédé par lequel les cellules NK en développement font l'acquisition de leurs fonctions. Chez l'humain, les haplotypes de gènes KIR varient grandement entre individus et le même phénomène est observé chez les souris; les haplotypes de gènes Ly49 diffèrent entre plusieurs souches de souris. Le séquençage de l'haplotype Ly49 des souris non-obèses diabétiques (NOD) a révélé que cette souche possède le plus grand haplotype de gènes Ly49 connu à ce jour. L'haplotype Ly49 des souris NOD est caractérisé par le plus grand nombre de gènes codant pour des récepteurs Ly49 activateurs. La présence d'un grand nombre de récepteurs Ly49 activateurs pourrait influencer le niveau d'activation des cellules NK et le développement du diabète chez les souris NOD.Afin de déduire le rôle in vivo des récepteurs Ly49 dans le contrôle de l'activité des cellules NK, une souris 'NKC-knockdown' a été créée. Les cellules NK de ces souris ont une expression grandement diminuée des récepteurs encodés dans le NKC dont les Ly49. Les cellules NK 'NKC-knockdown' possèdent une lyse réduite des cellules négative pour les CMH-I autant in vitro que in vivo. La capacité des cellules NK à détruire des cellules négatives pour les CMH-I est restaurée par la réintroduction d'un seul récepteur Ly49. Ces résultats fournissent la première preuve directe que les récepteurs Ly49 sont impliqués dans la surveillance des molécules CMH-I in vivo. Étonnamment, l'absence des récepteurs Ly49 entraine une reconnaissance de la 'perte de soi' défectueuse mais l'activation des cellules NK par des mécanismes n'impliquant pas des molécules CMH-I n'est pas diminuée. Les récepteurs NKR-P1, qui se lient aux protéines Clr, sont membres d'une autre famille de récepteurs exprimés par les cellules NK. Il est intéressant de noter que les gènes codant pour les NKR-P1 et leurs ligands Clr sont regroupés et sont donc hérités ensemble. Des analyses précédentes ont démontré que le groupe de gènes Nkrp1-Clr est conservé chez les souris B6 et BALB/c. La cartographie du groupe des souris de souche 129 a confirmé le haut degré de conservation de l'ordre des gènes dans le groupe Nkrp1-Clr des souris de souche 129. Le séquençage des gènes a démontré une conservation des gènes Nkrp et Clr chez les souris 129 et BALB/c tandis que ces gènes ont divergé chez les souris B6, suggérant que le groupe Nkrp1-Clr des souris de souche 129 est plus apparenté au groupe BALB/c qu'au groupe B6.En résumé, la famille de récepteurs Ly49 varie grandement contrairement au faible degré de diversité observé chez les récepteurs NKR-P1. La perte d'expression des récepteurs Ly49 révèle qu'ils sont requis pour l'acquisition de la réponse à la 'perte de soi' par les cellules NK.

Innate responses in the central nervous system (CNS) provide first-line defense against infection and injury. Microglia and astrocytes may direct leukocyte infiltration to the injured CNS. The signaling mechanisms that orchestrate this response are ill-defined. Innate roles for microglia and astrocytes are supported by reports demonstrating glial expression of Toll-like receptors (TLRs). TLRs recognize conserved pathogen-associated motifs and generate innate immune responses, usually by signaling through the adaptor protein, MyD88. TLRs may also generate innate responses to tissue damage. The data in this thesis implicate TLR2/MyD88 as key mediators of innate response to brain injury in mice. Transection of axons in the entorhinal cortex causes tissue damage analogous to a stab injury at the lesion site, and axonal degeneration distal from the wound, in the denervated, lesion-reactive hippocampus. A significant increase in leukocyte proportions was detected by 3h in the stab-injured entorhinal cortex, but not until 12h in the denervated hippocampus. This identified a window of CNS-directed innate response in the denervated hippocampus, without influence of infiltrating cells. Expression of numerous cytokines and chemokines was induced during this time. Microglia and astrocytes were identified as major sources of the chemokine CCL2. Macrophage infiltration to the stab-injured entorhinal cortex and the denervated hippocampus was dependent on MyD88-dependent CCL2/CCR2 signaling, but not TLR2-signalled response. T cell entry to the denervated hippocampus was regulated by TLR2 signaling and required MyD88-mediated response, whereas T cell recruitment to the stabinjured entorhinal cortex was only partially dependent on MyD88 signaling and did not require TLR2-mediated response. TLR2 signaling also regulated expansion of the hippocampal microglial population 5 days after lesion. Microglia were supplemented by circulating bone marrow-derived precursors, but this occurred predominantly at later times post-injury. No parameter measured was dependent on TLR4. These data identify novel signaling pathways that link glial responses to brain injury with subsequent neuroinflammation.

Two models of HIV infection have been studied extensively with the goal of identifying immune correlate(s) of protection against HIV: (1) the simian immunodeficiency virus (SIV) infection of rhesus macaque monkeys and (2) individuals with repeated exposure to HIV who remain uninfected by the virus (EUs). Both paradigms suggest that T cell-mediated immunity plays an important role in controlling HIV replication. Evidence from the SIV/macaque system, however, predicts that HIV vaccines aimed at eliciting T cell responses will fail to induce sterilizing immunity against HIV. The aim of the work presented in this thesis was to correlate HIV-specific T cell immunity in EUs with protection from HIV infection. The study cohort was comprised of (a) men who have unprotected sexual intercourse with HIV-infected men (MSM), (b) intravenous drug users (IVDU) who share syringes with HIV-infected peers, and (c) heterosexual partners of HIV-infected subjects. EUs were shown to exhibit HIV-specific IFN-gamma secretion, IL-2 production, and T cell proliferation, whereas low-risk negative controls did not. Furthermore, HIV-specific IFN-gamma secretion and T cell proliferation were not observed among a population of EUs who seroconverted soon after the tested time point. HIV-specific IL-2 secretion and T cell proliferation were shown to be correlated in our cohort of EUs. These effector functions are considered hallmark properties of central memory T cells (TCM), the subset of memory T cells that has been shown to mediate sterilizing immunity in mouse models of viral infection. The presence of TCM in EUs implies the development of immunity against HIV that is either fully protective or partially so, by increasing the threshold for HIV infection. Since these HIV-specific effector functions were absent in EUs who eventually seroconverted, it can be inferred that EUs in our cohort develop HIV-specific T cell immunity that mediates protection from HIV infection. Thus, our contribution to the EU paradigm is that sterilizing immunity is an attainable goal of prospective HIV vaccines.

Immune modulation is one treatment modality which is being explored in the context of human diseases and disorders. Methods of such manipulation which use gene therapy have an advantage in the treatment of chronic diseases such as autoimmunity because they are less invasive and more persistent. Furthermore, naked plasmid has advantages as a vector over other methods: it is more persistent and less immunogenic and cytotoxic than viral vectors, and simpler than DNA-conjugate vectors. Thus, naked plasmid is a viable alternative treatment to study in the context of an autoimmune disease such as diabetes mellitus, despite it's disadvantages of low transfection and expression rates. Here, we demonstrate that treatment of an autoimmune model, the non-obese diabetic (NOD) mouse, with an autoantigen to which a signal sequence had been added was protective, even in the apparent absence of secretion of that gene product. In contrast, treatment with the native cDNA of the same antigen was not protective. Furthermore, we show by immunohistochemistry that gene expression is still detectable in the muscle 22 weeks after injection. Other experiments demonstrate that multiple vaccinations with the altered form of the antigen were essentially as protective as a single vaccination following by multiple injections of blank plasmid, suggesting an important role for immunostimulatory sequences in bacterial DNA in causing surveying dendritic cells to migrate out of the tissue and present antigen in draining lymph nodes. Attempts to study the results of DNA vaccination by comparing immunization via different routes were inconclusive. / We have demonstrated that DNA vaccination of an autoimmune model with a autoantigen can delay disease. The simplicity and economy of such vectors and the benefits they have for the treatment of chronic disease in contrast to more inflammatory viral vectors, support future research into their use in the treatment of autoimmune diseases.

Tumor necrosis factor alpha (TNFalpha) is a key proinflammatory cytokine which is produced primarily by macrophages. Although this cytokine is very beneficial to the host when released in small amounts or in localized fashion, abnormally high levels of TNFalpha can however be very detrimental. The biological effects of this cyokine is thus dependent on its timing, location and extent of release. In recent years major interest has been placed on the AU rich elements (ARE) present in the 3' untranslated region of the TNFalpha mRNA as it plays a pivitol role in the posttranscriptional control of TNFalpha protein production. / We have previously shown that a protein binding site within this main ARE (positioned between bases 1291 and 1320) has the ability to bind to three protein complexes (A,B,C). In this study we describe a second AU rich element which also has protein binding capabilities. This second protein binding site (located 158 bases downstream from the first ARE) is 31 nucleotides long and contains a single AUAUUUAU sequence. Fractionation experiments have enabled us to show that a protein complex (complex D) present in both nuclear and cytoplasmic cell compartments can interact with this second binding region. The binding of this second ARE to this complex is readily competed for by other AU rich elements present in the mRNA of certain protooncogenes and cytokines such as c-fos and IL-1beta. The importance of binding of macrophage proteins to the ARE in the posttranscriptional regulation of TNFalpha was evidenced by the fact that polymorphisms (GAU trinucleotide insertional mutation) in the main ARE (positioned between bases 1291 and 1320 of the 3 ' untranslated region) of TNFalpha mRNA results in the decreased binding of macrophage protein complexes to the element. The GAU insertional mutation in the main ARE hinders the binding of a protein named HuR to the region. This protein is a nucleo-cytoplamic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing ARE. In order in elucidate and further our understanding of signalling mechanisms which regulate the function of proteins binding to ARE we verified if priming of macrophages with IFNgamma is important in the posttranscriptional regulation of TNFalpha mRNA. We show that priming of macrophages with IFNgamma prior to LPS treatment posttranscriptionally upregulates TNFalpha production by increasing the stability of the message and the levels of mRNA present on translationally active polyribosomes. The involvement of IFNgamma in the posttranscriptional regulation of / Overall data presented in the thesis will further our understanding of how ARE in the 3'UTR of the TNFalpha mRNA posttranscriptionally regulates the expression of TNFalpha.

Following administration of a phlogistic agent, a low macrophage inflammatory response is observed in mice of the A/J strain, in comparison to mice of the C57BL/6 strain. In addition to a genetic defect in the fifth component of complement (C5) which is characteristic of A/J mice, defect(s) in monocytopoiesis and/or component(s) involved in this process could also lead to a low accumulation of macrophages at the inflammatory site. Defect(s) could exist at the level of (i) the total number of bone marrow (BM) nucleated cells and/or macrophage progenitors, (ii) response of these cells to factor(s), (iii) the production of factor(s), or (iv) cell cycle time of the macrophage progenitors. The objective of this dissertation was to investigate the two former possibilities. The BM cellularity, as measured by the total number of BM nucleated cells and of granulocyte/macrophage-colony forming cells per femur, in mice of the C5-deficient A/J and C5-sufficient A/J.C5 congenic strains during normal steady state (NSS), were examined and both found to be significantly lower (2-fold) in comparison to that obtained from mice of the C57BL/6 strain. The significance of these results was shown using AxB/BxA recombinant inbred (RI) mouse strain system which determined that total BM nucleated cells observed in NSS did not influence the consequence of the macrophage inflammatory response in mice of the A/J and C57BL/6 strains. BM cells were also examined for their responsiveness to Interleukin-3 (IL-3), a multi-CSF, produced in response to injury or infection and which enhances the generation of myeloid cells. The BM cells of A/J mice were shown to be hyporesponsive to IL-3 treatment in vitro, but BM cells harvested from C57BL/6 mice strongly proliferated to the same treatment. The genetic control of the BM cell response to IL-3 was identified to a single gene. However, the type of BM cell response to IL-3 was shown not to be a major determinant factor in the magnitude of the macropha