Mast cells are granulocytes that are a key component of the innate and adaptive immune system, and contribute to allergic disorders. Mast cell activation following clustering of the high affinity IgE receptor (FcepsilonRI) by multivalent antigens requires reversible tyrosine phosphorylation of myriad signaling proteins. Activated mast cells rapidly release granule contents (eg. histamine and serine proteases) that cause vascular permeability, and in a more delayed manner they also synthesize and secrete eicosanoids and numerous cytokines (eg. IL-6 and TNFalpha) that recruit activated leukocytes. FcepsilonRI signaling is initiated by Lyn, a Src Family Kinase (SFK), that phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) found on the FcepsilonRI beta and gamma chains. This allows recruitment of Fyn SFK and Syk kinase that bind ITAMs and phosphorylate numerous downstream targets. Src Homology 2 domain-containing Phosphatase 2 (SHP2, encoded by ptpn11/shp2) is known to be recruited to several phosphorylated proteins following FcepsilonRI aggregation in mast cells, however attempts to define the role of SHP2 have been hampered by its essential role during embryonic development and hematopoiesis in mice. Recently, conditional SHP2 knock-out mice (shp2fl/fl) have been created allowing for shp2 inactivation in a tissue-specific manner by Cre recombinase. Here we describe the use of transgenic mice expressing a modified estrogen receptor-Cre Recombinase (TgCreER*) on a shp2fl/fl genetic background, that allows for maturation of bone marrow-derived mast cells (BMMCs) prior to shp2-inactivation using 4-hydroxytamoxifen (4OH-TM). SHP2-depleted BMMCs display reduced phosphorylation of the FcepsilonRI beta chain, but exhibit extended phosphorylation of Syk kinase. Additionally, SHP2-deficient cells display a defect in the activation of both Erk mitogen-activated protein kinase and Akt, which correlates with an observed defect in the production of TNFalpha and Leukotriene C4. Finally, we show that SHP2-deficient BMMCs display elevated FcepsilonRI-evoked phosphorylation of Csk-Binding Protein (Cbp or PAG) on residue Y317, which recruits C-terminal Src kinase (Csk) that phosphorylates SFKs on an inhibitory tyrosine. This hyperphosphorylation of Cbp correlates with elevated phosphorylation of the C-terminal inhibitory tyrosine on Fyn kinase. This study provides new insights into the role of SHP2 as a positive effector of FcepsilonRI signaling and cytokine production in mast cells. / Thesis (Master, Biochemistry) -- Queen's University, 2008-08-20 13:51:18.751

Regulatory T cells expressing the transcription factor Foxp3 have a critical role in the maintenance of tolerance to both self and innocuous exogenous antigens. Humans and mice die from overwhelming autoimmunity in the absence of Foxp3+ Treg whilst administration of regulatory T cells has shown promise therapeutically in ameliorating autoimmunity in several animal models. Regulatory T cells arise naturally in the thymus (nTreg) but may also be induced from naïve Foxp3- cells in the presence of TGF-β (iTreg), both in vitro and in vivo. This thesis focuses on in vitro generated mouse iTreg, testing the hypothesis that they are able to effect bystander suppression; iTreg activated by a given antigen are able to suppress other responding cells with different antigen reactivities. Chapter 3 details an in vitro assay system using iTreg and responder cells recognising different antigens (TCR transgenic cells). Evidence for bystander suppression is presented and that did not require the presence of iTreg-relevant antigen but did require iTreg-relevant MHC Class II. The kinetics of iTreg suppression are discussed, with evidence presented that iTreg exert their effects early in co-culture. Chapter 4 identifies the production of three pro-inflammatory cytokines by iTreg - IFN-γ, GM-CSF, and TNF. These were not involved in the in vitro suppressive mechanism, but early abrogation of TGF-β signalling did inhibit suppression. Chapter 5 describes the in vivo function of iTreg under various experimental protocols. iTreg did not limit initial proliferation of naïve T cells in response to antigen but did limit the development of effector cells producing pro-inflammatory cytokines. Exposure to a pro-inflammatory environment in vivo led to iTreg producing IFN-γ and TNF, but not GM-CSF. This could be replicated in vitro by exposure to IL-6, IL-12 or IL-27. Finally, evidence for bystander suppression by iTreg in vivo is presented, with a reduction in effector cells producing pro-inflammatory cytokines shown in an allergic airways diease model.

Immunomodulatory effects of lactic acid bacteria vary with strain and may vary with growth phase and medium. The ability of different lactobacilli strains (Lactobacillus helveticus R0052, L. rhamnosus R0011, L. rhamnosus GG) at different growth phases to modulate macrophage and intestinal epithelial cell cytokine production following a pro-inflammatory challenge was examined. Modulation of cytokine production by human macrophage cell lines (U-937) and intestinal epithelial cells (HT-29) induced by Tumor Necrosis Factor α was assayed by ELISA for interleukin-8 (IL-8). Granulocyte-macrophage colony stimulating factor (GM-CSF) production was assayed by ELISA in the HT-29 cell line. Strain-dependent differences were observed in the ability of viable bacteria and spent de Mann-Rogosa- Sharpe (MRS) broths from log versus stationary growth phase in HT-29 and U-937 cells. Overall, variation in the immunomodulatory activity of these lactic acid bacteria and spent broths reflects not only strain variation but potentially also differences in growth phase and substrate. / UOIT

Introduction: Patient outcome after trauma is influenced by their immune response to injury. How trauma activates the immune system and why this affects recovery is unclear. This investigation examined three aspects of the immune response after trauma: cytokine production, alarmin release and the innate immune cell populations. Methodology: Timed blood samples were drawn from trauma patients recruited to a prospective observational cohort study at a London Major Trauma Centre. The first sample was drawn at admission, within 2h of injury and prior to intervention in order to capture early inflammation events. Patients were observed until death or discharge for clinical outcomes. Results: Inflammation after traumatic tissue damage can be described in isolation. If haemorrhagic shock is also present, the inflammatory effects cannot be separated using the seven cytokines examined in this investigation. Within 2h of injury, isolated tissue damage is associated with systemic release of intracellular nuclear molecules. Tissue damage combined with shock, is associated with release of different nuclear materials. Low numbers of lymphocytes at 48h from injury are associated with poor clinical outcome. Patients who develop infections and multiple organ dysfunction syndrome during recovery, have high numbers of cytotoxic lymphocytes in their peripheral blood at admission. Conclusion: Inflammation is activated prior to arrival at hospital. Haemorrhagic shock augments the inflammatory response after isolated tissue damage. Tissue damage and blood loss may lead to the release of different alarmin substances. Lymphocytes are implicated in the pathogenesis of poor outcome. The molecular events which lead to poor clinical outcome are activated before hospital admission and prior to intervention. Greater understanding of the activation mechanism(s) may result in development of therapeutics for early delivery, in order to improve patient recovery.

Les troubles du spectre autistique (TSA) sont un ensemble de pathologies neurodéveloppementales dont la prévalence est en constante augmentation. Ils sont caractérisés par des déficits de la communication et des interactions sociales, et par des comportements répétitifs et stéréotypés. A l’origine d’un handicap sévère, ces troubles se manifestent dès la petite enfance et persistent chez l’adulte. Cette entité recouvre des profils cliniques très hétérogènes, tant par le spectre de sévérité des symptômes que par la variété des comorbidités psychiatriques et somatiques associées sous tendues, en partie, par des dysfonctionnements immunitaires. Dans ce contexte, nous nous sommes de ce fait intéressés à l’identification et à la caractérisation de biomarqueurs à valence immunogénétique et immunologique afin d’en étudier l’implication physiopathologique et d’en déterminer les corrélats cliniques.De manière plus précise, nous avons évalué l’implication de la diversité génétique de molécules intervenant dans l’immunité innée (PRR, CLR, Dectin-1) et l’immunité adaptative (système HLA) dans le but d’apprécier le poids du terrain immunogénétique sur le développement de ces troubles. Puis, nous avons analysé les caractéristiques phénotypiques et fonctionnelles des cellules Natural Killer de patients atteints de TSA afin d’en déterminer l’influence potentielle sur l’état inflammatoire permanent rapporté chez certains patients TSA.Sur le plan immunogénétique, nous avons montré que la diversité génétique de Dectin-1 (CLEC7A), candidat sélectionné en raison de son implication dans la modulation de pathologies microbiennes intestinales, était associé à une forme particulière de TSA, le syndrome d’Asperger. Nous avons observé que le génotype CLEC7A rs2078178 GG ainsi que l’haplotype rs2078178/rs16910631 GG/GG étaient non seulement plus fréquents chez les Asperger mais aussi associées aux scores de quotient intellectuel (QI). Dans le cadre de l’analyse de la diversité génétique du système HLA, nous avons identifié un haplotype à risque (HLA-DRB1 *11-DQB1*07) et un haplotype de protection (HLA-DRB1 *17-DQB1*02). L’haplotype à risque étant également associé avec la sévérité de la maladie, reflétée par des scores défavorables dans les échelles cliniques psychiatriques testées.Dans la seconde partie de cette thèse nous avons exploré les modifications phénotypiques et fonctionnelles des cellules NK CD3- CD56+ chez les patients atteints d’autisme de haut niveau. Nous avons observé un état d’activation cellulaire permanent concomitant avec une capacité de dégranulation spontanée, une production soutenue d’IFN-?, et un état hypofonctionnel/épuisement cellulaire après stimulation in vitro. De plus, nous avons identifié un cluster spécifique de cellules NK, basé sur les paramètres HLA-DR, NKG2C, et KIR2DL1, et nous avons observé une augmentation inattendue des cellules NK NKG2C+ chez les sujets TSA en dehors de toute piste infectieuse connue. Enfin, nous avons observé que l’expression de KIR2DL1 et de HLA-DR était respectivement corrélée aux scores de QI et à ceux évaluant les CCA-LS et SAWR.Pris dans leur ensemble, ces données pourraient permettre de contribuer à une meilleure connaissance des mécanismes physiopathologiques associés au système immunitaire dans les TSA et par conséquent à une meilleure catégorisation des groupes de patients susceptibles de bénéficier de stratégies thérapeutiques immunologiques ciblées. / Autism spectrum disorders (ASD) are severe neurodevelopmental conditions characterized by deficits in communication and social interactions, and by repetitive and stereotyped behaviors and exhibiting a constant increase in terms of prevalence. Affecting ages ranging from the early post-natal period to adulthood, ASD are clinically heterogeneous and often associated with psychiatric and somatic comorbidities underlying, in part, by immune dysfunctions. In this context, we thus focused our attention on the analysis of immunogenetic and immunological characteristics potentially implicated in the disease risk and/or in the modulation their clinical phenotype. More precisely, we evaluated the potential implication of the genetic diversity of molecules involved in innate (PRR, CLR, Dectin-1) and adaptive (HLA) immune responses in disease risk. We then analyzed the phenotypic and functional characteristics of Natural Killer cells in patients with ASD, investigating their influence on the permanent inflammatory state often reported in ASD settings.On the immunogenetic point of view, we found that the genetic diversity of Dectin-1 (CLEC7A), a candidate selected because of its involvement in the modulation of intestinal microbial disorders, was associated with Asperger syndrome, a clinical form of ASD. We observed that the CLEC7A genotype rs2078178 GG and the rs2078178 / rs16910631 GG /GG haplotype were not only more frequent in Asperger but also associated with IQ scores.In terms of HLA diversity, we identified a risk haplotype (HLA-DRB1 * 11-DQB1 * 07) and a protective haplotype (HLA-DRB1 * 17-DQB1 * 02). The risk haplotype was also found to be associated with disease’s severity as reflected by unfavorable scores in the psychiatric clinical scales tested.In the second part of this thesis, we explored the phenotypic and functional modifications of CD3-CD56 + NK cells in patients with high-functioning autism. We observed a permanent cell activation state concomitant with spontaneous degranulation capacity, sustained IFN-? production and cellular hypofunction /exhaustion after in vitro stimulation. In addition, we identified a specific cluster of NK cells, based on the HLA-DR, NKG2C, and KIR2DL1 parameters, and we observed an unexpected increase of NK NKG2C + cells in ASD subjects independent of CMV infection. Finally, we observed that the expression of KIR2DL1 and HLA-DR were respectively correlated with the scores of IQ and those evaluating the CCA-LS and SAWR scales.Taken together, these data could contribute to a better knowledge of the pathophysiological mechanisms associated with the immune system in ASD and consequently to a better categorization of the groups of patients likely to benefit from targeted immunological therapeutic strategies.

Thy-1 is a glycosylphosphatidylinositol-anchored protein that is expressed on murine T lymphocytes and is involved in T cell-mediated immune responses. In the presence of costimulatory signals, monoclonal antibody (mAb)-induced signaling through Thy-1 is associated with hallmarks of T cell activation, including IL-2 production and T cell proliferation. Thy-1-induced signaling promotes cytotoxic effector molecule expression, but is unable to trigger delivery of the lethal hit to target cells, suggesting that Thy-1 provides an incomplete T cell receptor (TcR)-like signal. However, the effect of Thy-1 signaling on cytokine production and the development of T helper (Th) cell phenotypes (Th1, Th2, Th17) remains unclear. The purpose of this work was to further our understanding of Thy-1-mediated signal transduction and the role that Thy-1 plays in the development of effector T cell responses. I found that, in the context of costimulatory signals, anti-Thy-1 mAb induced significantly less IL-2 production, CD25 expression and T cell proliferation than anti-TcR? mAb. Several key signaling molecules, including protein tyrosine kinases, zeta chain-associated protein-70 and extracellular signal-regulated kinase were activated with delayed kinetics during Thy-1-mediated T cell activation. The delayed signaling kinetics resulted in the delayed acquisition of cytotoxic effector function and also delayed delivery of the lethal hit to target cells. Interestingly, Thy-1-mediated signaling induced significantly more IL-17 and IL-4 synthesis and less IFN-? synthesis in comparison to TcR-mediated signaling. Moreover, Thy-1-activated CD4+ T cells produced high levels of IL-17 and IL-4 but minimal IFN? when restimulated with anti-Thy-1 mAb or anti-TcR? mAb with or without costimulatory signals. The unique ability of Thy-1 signaling to induce IL-17 production correlated with the expression of the Th17 lineage-specific transcription factor, retinoic orphan receptor gamma t. These observations show that Thy-1 signaling differs from TcR signaling in its ability to induce Th cell cytokines. Taken together, my findings show that Thy-1 signaling can provide the full TcR-like signal required for both the differentiation and triggering of Th cells and cytotoxic T lymphocytes, albeit with delayed kinetics in comparison to TcR signaling. They also suggest that Thy-1 signaling may be important in the development of Th2 and Th17 responses.

Auch unter physiologischen Bedingungen finden sich T-Zellen und andere Leukozyten nicht nur in den Sinusoiden, sondern auch im Parenchym der Leber. Da die Leber u. a. verschiedene Aufgaben für das Immunsystem übernimmt (z. B. Deletion aktivierter T Zellen, Induktion peripherer Toleranz), könnte die Akkumulation der T-Zellen in der Leber - neben der immunologischen Überwachung der Leber - Voraussetzung für ihre Modulation sein. In der vorliegenden Arbeit wurde der Einfluss von Leber-sinusoidalen Endothelzellen (LSEC), der Barriere zwischen Blut und Leber-Parenchym, auf CD4+ T-Zellen untersucht. Zum einen zeigte sich, dass die LSEC sowohl die spontane Transmigration der T-Zellen, als auch ihre Chemotaxis zu CXCL9 und CXCL12 effizienter unterstützen als andere Endothelien. Eine endotheliale Aktivierung durch die Chemokine wurde als Mechanismus ausgeschlossen. Dagegen schien eine effiziente Präsentation der Chemokine auf der luminalen LSEC-Oberfläche nach Aufnahme von abluminal für die gesteigerte Transmigration der T Zellen verantwortlich zu sein. Die LSEC könnten somit in vivo an der Rekrutierung von T-Zellen in die Leber beteiligt sein, indem sie eine rasche Wanderung der T-Zellen aus dem Blut ins Parenchym und möglicherweise auch zurück in die Zirkulation zulassen. Des Weiteren konnte gezeigt werden, dass die LSEC fähig sind, naive CD4+ T-Zellen in vitro Antigen-spezifisch zu aktivieren. Im Vergleich zu professionellen APZ war hierfür eine höhere Antigen-Dosis notwendig, die Expansion schwächer und es waren kaum Effektorzytokin-Produzenten detektierbar. Diese konnten jedoch durch Restimulierung mit professionellen APZ induziert werden (reversibler Phänotyp), was auf einen unreifen Differenzierungsstatus der T-Zellen schließen ließ. Es bleibt zu prüfen, in welchem Maße die Aktivierung naiver CD4+ T-Zellen durch LSEC in vivo stattfindet und diese durch LSEC aktivierten CD4+ T-Zellen funktionelle Bedeutung, z. B. regulatorische Kapazität, für das Immunsystem besitzen. / The liver plays a major role for the metabolism, but it is also of general importance for the immune system, e.g. for the deletion of activated T cells or the induction of peripheral tolerance. Under physiological conditions T cells and other leukocytes can be found in the liver, in the sinusoids as well as in the parenchyma. This hepatic accumulation of T cells might be due to immunosurveillance, but it would also be a prerequisite for modulation of T cells by hepatic cells. The present study investigated two different aspects of the interaction of liver sinusoidal endothelial cells (LSEC), the barrier between the sinusoidal lumen and the hepatic parenchyma, and CD4+ T cells. In the first part of the study it could be demonstrated that LSEC support the spontaneous transmigration of CD4+ T cells as well as their chemotaxis to CXCL12 and CXCL9 more efficiently than other endothelial cells. Whereas a direct endothelial activation by chemokines could be excluded the efficient chemokine presentation at the luminal LSEC surface (after abluminal uptake) might be responsible for the enhanced T cell transmigration. The findings suggest that LSEC might be involved in the recruitment of T cells by supporting a rapid transendothelial migration. The second part of the study focused on the characteristics of LSEC in the context of antigen presentation. LSEC were able to prime and expand naïve CD4+ T cells in vitro but less effective than professional APC as proven by weaker expansion of cells, a requirement for higher antigen concentration and the lack of cytokine producing T cells. The “immature effector” phenotype of the CD4+ T cells primed on LSEC was reversible since it could be overcome by restimulation on professional APC. In conclusion these data suggest that antigen presentation by LSEC results in activation but incomplete differentiation of CD4+ T cells.

<p> Virus-induced modulation of dendritic cell function is thought to be an effective mechanism for viral-immune evasion. The severe-acute respiratory syndrome coronavirus (SARS-CoV) has been shown to infected human myeloid dendritic cells (MDCs) and directly modulate the cellular cytokine production. The ability of other human coronaviruses to infect MDCs and impair cell immune function has not been assessed. </p> <p> This thesis describes the infection of human MDCs with coronavirus 229E, NL63, and OC43. 229E showed productive, but limited genomic replication, nucleocapsid protein synthesis and infectious progeny release in MDCs. 229E infection stimulated IFN-α, IL-6 and MCP-1 production in MDCs, but little to no IL-12, TNF-α, IL-8, IP-10, or RANTES . 229E-infected MDCs showed poor CD80 expression, down-regulated CD86 and HLA-DR expression and were poor stimulators of CD4+ T cell proliferation. In contrast to 229E, OC43 showed persistent and productive genomic replication, nucleocapsid protein synthesis and infectious progeny release in MDCs. OC43 infection stimulated IFN-α, IL-12, IP-10 and MCP-1 production in MDCs, but little to no TNF-α, IL-6, IL-8 or RANTES . The up-regulation of maturation molecules and CD4+ T cell stimulatory capacity in OC43-infected MDCs was donor cell-dependent. In contrast to 229E and OC43, NL63 infection of MDCs was non-productive, showing no viral genomic replication, protein production or infectious progeny release. NL63 infection stimulated strong cytokine (IFN-α, IL-12, TNF-β and IL-6) and chemokine (IL-8, IP-10, RANTES and MCP-1) responses in MDCs. NL63-infected MDCs showed up-regulated CD80, CD83, CD86 and HLA-DR expression and were efficient stimulators of CD4+ T cell proliferation. </p> <p> This study provides the first evidence that human coronaviruses other than SARSCo V can abrogate MDC immune effector function. It also provides the first side-by-side comparison of 229E, NL63 and OC43 and identifies the potential of 229E and OC43 to impair MDC cytokine production and T cell stimulation as a mechanism of immune response evasion. <p> / Thesis / Doctor of Philosophy (PhD)