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The pre-T cell receptor (preTCR) is required for αβ T cell development and hence efficient adaptive immunity. Composed of a rearranged TCRβ chain paired with the invariant preTCR α chain (pTα), it drives immature thymocytes through the “β-selection” checkpoint, promoting differentiation, proliferation and survival. Although two functional splice isoforms of pTα exist, non-redundant roles for pTαa and pTαb have yet to be ascribed. This thesis demonstrates that pTαa and pTαb display non-identical expression patterns in immature thymocyte subsets. Moreover, it demonstrates that preTCRa and preTCRb promote divergent T cell development; preTCRa drives prolonged expansion of post-β-selection thymocytes, while preTCRb drives rapid maturation of TCRαβ(+) cells. Importantly, by mutating charged residues in the extracellular domain of pTα, it is shown that pTα oligomerization is not required for preTCR signalling per se, as had previously been proposed. Instead, the capacity for oligomerization regulates surface preTCR levels, which in turn dictates subsequent developmental potential. This thesis also presents preliminary evidence for a novel role for the preTCR; that preTCR signalling sensitises the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway that in turn regulates the signalling thresholds for positive and negative selection in CD4(+)CD8(+) double positive (DP) cells. Indeed, it is suggested that this process may be critical for central tolerance in the thymus. Finally, this thesis describes the generation and initial characterisation of pTαa and pTαb BAC transgenic mice that will facilitate the separate investigation of pTαa and pTαb in a pTα-deficient background in vivo. Collectively, the results presented in this thesis ascribe non-redundant roles for the two isoforms of pTα and necessitate a fresh examination of the mechanism by which the preTCR initiates signalling. The implications of these results for αβ T cell development and the immune system as a whole are discussed.

Recent research has revealed a role of NF-B in the resolution of inflammation. Using Cre-lox mediated gene targeting, IKK was selectively deleted in macrophages (IKKβ∆Mye). From in vitro studies, LPS stimulated IKKMye macrophages increased STAT1 phosphorylation, iNOS, MHC II and IL-12 production, suggesting negative cross talk between NF-B and STAT1 signalling pathways. Since IKK is required for TNF gene expression and TNF signalling, I investigated the hypothesis that TNF inhibits ‘classical’ macrophage activation through IKK activation. Macrophages from p55-/- and mice treated with anti-TNF antibody show increased STAT1 activation and IL-12 expression after LPS and IFN stimulation. BMDM infected with adenovirus expressing IKKβ dominant negative rescued the inhibitory effect of TNFα on IL-12p40 production, indicating TNFα inhibits IL-12p40 via IKKβ activation. Macrophages are antigen presenting cells while IL-12 and MHC II are critical factors for TH1 cell development. I thus investigate the inhibitory effects of IKKβ∆Mye macrophages in TH1 responses. FACS analysis showed higher MHC II, costimulatory molecules expression on IKKβ∆Mye macrophages after LPS stimulation. In a DTH model, recall assay has shown increased antigen-specific IFN production from IKKMye splenocytes compared to IKKβF/F splenocytes. Furthermore, IFN production was greatly enhanced by CD4+ OTII T cells co-cultured with IKKMye macrophages. Further analysis of CD4+ OTII T cells with qRT-PCR showed increased TH1 genes including IRF1, IFN, IL-12R1 and IL-12R2 and reduced TH2 marker IL-4. In addition to the enhanced antigen-specific T cell responses, IKKMye macrophages also increased anti-tumour immunity. Injection of H-Y positive MB49 tumour cells into IKKF/F and IKKMye female mice has shown tumour rejection, but no tumours were rejected after CD8+ T cells depletion, suggesting tumour rejection is associated with enhanced CTL activity. Taken together, these studies demonstrated the negative regulatory roles of IKK in macrophage activation and their impact to the innate and adaptive immunity.

The immunological characteristics of the mucosae of rainbow trout, <i>Oncorhynchus mykiss</i>, were investigated. A technique for the isolation of viable cells from the intestinal and cutaneous mucosae was developed which enabled differential cell counts to be performed on suspensions of cells liberated from these sites. This was achieved using light and electron microscopical, enzyme histochemical and flow cytometric techniques. In cell suspensions derived from the intestinal mucosa, lymphocytes, macrophages and eosinophilic granulocytes (EGC) were present, lymphocytes being by far the most numerous. Goblet cells and epithelial cells were also present but neutrophils were not apparent. In cell suspensions derived from the cutaneous mucosa, lymphocytes, macrophages, neutrophils and several types of cell with hitherto undescribed morphologies were identified. In addition, goblet cells and epithelial cells were present. A small proportion of the cells from the cutaneous mucosa were leucocytes compared with cells from the intestinal mucosa. A small number (about 4%) of lymphocytes from the intestinal mucosa reacted with a monoclonal anti-trout immunoglobulin antibody, suggesting that these cells may be B cells. In the case of the intestinal mucosa it was found to be possible to enrich for certain cell types using Percoll gradients. Functional studies were performed using cells from the intestinal mucosa. These cells were capable of phagocytosing latex microspheres and releasing reactive oxygen species, although only in small quantities compared with cells from the head kidney. Intestinally-derived cells secreted a factor(s) with the ability to induce head kidney cells to migrate and this could be enhanced by treatment of the gut cells with calcium ionophore. Intestinal cells were unable to migrate themselves under the conditions investigated. In addition, intestinal cells were able to proliferate in response to stimulation by the T-cell mitogen phytohaemagglutinin, as determined by incorporation of triated thymidine. Cells isolated from the intestinal mucosa were also able to secrete a macrophage-activating factor (MAF) with the ability to upregulate the production of reactive oxygen species in normal head kidney macrophages.

Les macrophages sont des cellules du système immunitaire inné qui régulent la réponse inflammatoire, tout en contribuant à la mise en place de l’immunité adaptative. La glycosylation de la membrane cellulaire est impliquée dans les différents processus physiopathologiques, tels que la défense de l’hôte ou la réponse inflammatoire. Suite à l’exposition à un pathogène, la machinerie de glycosylation peut être perturbée par cet environnement inflammatoire. Des études transcriptomiques ont démontré que l’infection de macrophages humains par des mycobactéries module l’expression de gènes impliqués dans les processus de glycosylation. L’hypothèse qui en découle est que, lors d’une infection mycobactérienne, la glycosylation des macrophages est régulée soit par l'environnement cellulaire soit par le pathogène. L’objectif de notre étude est de déterminer les modifications structurales de la glycosylation suite à une infection mycobactérienne. Pour ce faire, les macrophages issus de la lignée THP-1 et de monocytes sanguins humains sont infectés par Mycobacterium bovis BCG. Les glycannes libérés des macrophages sont analysés par spectrométrie de masse MALDI-TOF. À la suite de l'infection par M. Bovis BCG, les macrophages présentent des différences de N-glycosylation. L'analyse des résultats obtenus montre une modification de la ramification et une augmentation des niveaux de fucosylation des N-glycannes de type complexe. Ces différences de fucosylation sont confirmées par l’utilisation en cytométrie en flux de lectines végétales fluorescentes. L’ensemble de ces résultats nous indiquent que la glycosylation des cellules hôte subit un remodelage suite à l’infection mycobactérienne. / Macrophages mediate innate immune system through the initiation and regulation of inflammation and contribute to adaptive immunity via antigen processing. Cell surface glycosylation has been widely described to be involved in different physiological or pathological processes, such as host defense, immunological and inflammatory responses. Following pathogens infection, glycosylation machinery can be also disturbed by the cellular environnement. In recent years, several studies have been shown that infection or stimulation of human macrophages with mycobacteria could modulate gene expression related to glycometabolism. These data establish that during mycobacterial infection, glycosylation of macrophages is finely regulated by the cellular environment or by the pathogen. The purpose of our study is to determining the structural modifcations of glycosylation after mycobacterial infection.To accomplish this, macrophages derived from THP-1 and from PBMC were infected with mycobacterium bovis BCG. Once the macrophages are infected, glycans were released by chemical extraction and enzymatic digestion. After purifcation, differents glycans were analyzed by MALDI-TOF spectrometry. To correlate our results, we have also performed FACS analysis using plant lectins. Following M. bovis BCG infection, macrophages present different pattern of N-glycosylation. Analysis of data obtained by mass spectrometry shows antennarisation modification of complex type N-glycans and variations of fucosylation level.

CCX-CKR is an atypical chemokine receptor for the homeostatic chemokines CCL19, CCL21 and CCL25. CCL19 and CCL21 are also ligands for CCR7 and are crucial for the induction of antigen specific immunity and tolerance, whereas CCL25 is the sole ligand for CCR9 and is involved in the recruitment of immune effector cells to the small intestine. CCX-CKR does not signal after binding its ligands, as determined by a failure to induce the rapid increase in intracellular calcium that is typical of G-protein mediated signalling. CCX-CKR also does not become desensitised to chemokine binding and therefore is proposed to act as a scavenger receptor that can regulate the activity of CCR7 and CCR9 by affecting the availability of their ligands in vivo. At the time of starting my project, there were no published reports describing the biological function of CCX-CKR in vivo and the principal aim of my thesis was to characterise the immune system of the recently generated CCX-CKR KO mouse with particular focus on the intestinal immune compartment where all three of the chemokine ligands are expressed. Firstly, as described in Chapter 3, I analysed the cellular composition of the secondary lymphoid organs of CCX-CKR KO mice. These studies revealed normal proportions and absolute numbers of lymphocytes, CD11c+ dendritic cells (DC), macrophages and natural killer (NK) cells in the absence of CCX-CKR. The proliferative responses of lymphocytes to mitogenic or TCR stimulation in vitro were also normal, although there was a decreased production of IFNγ by CD4+ T cells from CCX-CKR KO mice. Although most of the phenotypic subsets of conventional DC were present in comparable numbers in the mesenteric lymph nodes (MLN) of CCX-CKR KO and WT mice, there was a consistent and dramatic reduction in the numbers of CD11cloPDCA-1+ plasmacytoid DC (pDC) in CCX-CKR KO MLN. In parallel, fewer CD11cloB220+ cells from CCX-CKR KO MLN than WT expressed CCR9, despite this marker being expressed normally by lymphocytes in these mice. The proportions of pDC in CCX-CKR KO inguinal lymph nodes (ILN) were also significantly reduced compared to WT and pDC from CCX-CKR KO MLN, ILN and spleen all appeared to express higher levels of class II MHC than WT pDC. These data suggest that CCX-CKR may play an important role in the recruitment and/or survival of pDC in the LN and that in its absence, pDC in secondary lymphoid organs may have a more mature phenotype. In Chapter 4, I examined the cellularity of the intestinal immune compartment in resting CCX-CKR KO mice, as well as the effects of Flt3L administration in vivo. Although CCX-CKR KO mice displayed no histological abnormalities in their small intestinal architecture and had normal numbers of T cells in the lamina propria, they did have significantly reduced numbers of intra-epithelial lymphocytes (IEL) as well as decreased proportions of CD19+ B cells and increased proportions of CD11c+ cells in the lamina propria compared with WT mice. The proportions and absolute numbers of CD103+ DC were normal in the lamina propria and MLN of CCX-CKR KO mice, suggesting that CCX-CKR has little to no role in regulating DC migration from the lamina propria to the MLN, a process that is critically dependent on CCR7. Although the proportions and absolute numbers of B cells and CD11c+ cells were normal in CCX-CKR KO Peyer’s patches (PP), there were significantly decreased proportions of pDC compared with WT PP. In vivo treatment of CCX-CKR KO mice with the DC differentiation factor Flt3L, expanded CD11c+ DC numbers dramatically in both CCX-CKR KO and WT small intestinal lamina propria, ILN, spleen, MLN and PP. Although Flt3L abolished the apparent defects in pDC populations in the ILN and PP, this was not the case for CCX-CKR KO MLN, which remained significantly deficient in pDC compared to WT MLN. Work in Chapter 6 examined parallel effects in the blood and bone marrow. As the CCX-CKR ligands CCL19 and CCL21 are involved in the development of all adaptive immune responses, and together with the other CCX-CKR ligand, CCL25, orchestrate immune responses to antigen encountered in the gut, I next investigated the development of antigen specific immunity and tolerance in CCX-CKR KO mice. There were no significant differences in systemic immune responses to subcutaneous immunisation with ovalbumin (OVA) emulsified in complete Freunds adjuvant (CFA) between CCX-CKR KO and WT mice when assessed in vivo or in vitro. However, the development of oral tolerance in CCX-CKR KO mice was impaired, with no suppression of OVA specific delayed type hypersensitivity (DTH) responses or of serum OVA specific IgG2a as was seen in WT mice. In parallel with defective systemic tolerance after feeding OVA, CCX-CKR KO mice appeared to be more susceptible to priming of systemic and local antibody responses after feeding OVA with cholera toxin (CT) as a mucosal adjuvant. In addition, there was some evidence of priming of OVA specific antibody responses in CCX-CKR KO mice fed OVA alone, which was not seen in WT mice. Despite this evidence of abnormal mucosal immunity, CCX-CKR KO mice developed DSS colitis normally, with all indices of disease being identical in CCX-CKR KO and WT animals. Together, these data suggest that there are selective defects in the regulation of antigen specific mucosal immune responses in the small intestine of CCX-CKR KO mice that may predispose these animals towards exaggerated active immune responses. Finally, I performed some preliminary experiments to try and relate DC function to the immune dysregulation I observed in CCX-CKR KO mice and to explore the basis of the pDC defect in the MLN. Bone marrow derived and splenic DC from CCX-CKR KO mice showed a reduced ability to process and present intact protein antigens although endocytic activity was normal. MLN DC from normal and Flt3L treated CCX-CKR KO mice responded similarly to in vitro stimulation with the synthetic TLR7 agonist R848, showing an expansion in the numbers of CD11chiPDCA-1+ cells that nearly all expressed CD40 and CD86. In addition, in vivo administration of R848 triggered identical migration of CD11chiclassIIMHChiCD103+ DC into the MLN of CCX-CKR KO and WT mice, suggesting that lamina propria pDC can effectively mobilise local DC to the MLN in the absence of CCX-CKR. There were no differences in the proportions or absolute numbers of pDC in the liver of CCX-CKR KO and WT mice despite the fact that liver pDC have been implicated in other studies of oral tolerance. Although the proportions of pDC were normal in the bone marrow of resting and Flt3L treated CCX-CKR KO mice, there did appear to be a defective recruitment of Flt3L expanded pDC into the blood of these animals. I used an adoptive transfer approach to study the in vivo localisation of pDC into lymphoid organs and although these experiments were not entirely conclusive, they indicated that WT pDC could enter the MLN and other lymphoid tissues of CCX-CKR KO mice normally and that pDC from CCX-CKR KO mice may have a defect in their ability to enter WT MLN. Taken together, my data suggest that CCX-CKR is involved in the entry and/or survival of pDC in secondary lymphoid organs, a process that normally involves migration across high endothelial venules (HEV). This was associated with impaired oral tolerance induction and heightened immune responses to antigen delivered orally, indicating that CCX-CKR contributes to the regulation of mucosal immunity and tolerance by an as yet unclear mechanism. Further study of these animals will hopefully better define the relationship between pDC and the regulation of mucosal immune responses.

The integrin αVβ5 is a receptor for sCD23 molecule and the αVβ5-CD23 interaction sustains proliferation of the pre-B cell line SMS-SB. This thesis describes further investigation into the role of αVβ5 integrin during human B cell development. B cell development in the bone marrow involves stepwise maturation of progenitor cells through different defined stages. A tight regulation of proliferation and differentiation mediates the progression of progenitor cells through this developmental pathway. A variety of signals from soluble molecules and adhesive interactions regulate this balance of proliferation and differentiation. The main aim of this work was to assess the importance of the integrin αVβ5 during B cell development by defining its expression and function during specific stages of B cell development in the bone marrow. The αVβ5 integrin was expressed by B cell precursors in the bone marrow, by different pre-B cell lines and the αVβ5-CD23 interaction sustained proliferation of some pre-B cell lines. The pre-B cell lines SMS-SB, RS4;11 and 697 showed a significant proliferative response to αVβ5 stimulation by sCD23, a sCD23-derived long peptide and anti-αVβ5 MAb 15F11. Transitional and more mature B cell lines down-regulated αVβ5 expression and did not show a proliferative response. Both αVβ5 and αVβ3 integrin could be detected on normal bone marrow B cell precursor populations, though αVβ5 was the more highly expressed integrin. In preliminary functional experiments, stimulation of CD19+/κ- cells with sCD23 induced cell proliferation whereas equivalent treatment of CD19+/κ+ cells did not. The data are consistent with the interpretation that αVβ5 integrin is expressed in B cell precursors but that its ability to sustain growth of these cells wanes as the cells mature towards a membrane immunoglobulin-positive state. The αVβ5-mediated proliferation was enhanced by the chemokine SDF-1 and PDGF but not by the cytokines IL-3, IL-4, IL-7 or IL-11. This effect was apparently restricted to earlier B cell precursors and was independent of levels of expression of both αVβ5 and CXCR4. Stimulation of SMS-SB cells by αVβ5 ligands provoked ERK phosphorylation and co-stimulation with SDF-1 promoted a more rapid and sustained ERK activation. PDGF induced a similar effect on αVβ5-mediated activation of ERKphosphorylation. These data suggest that ligation of αVβ5 by soluble, adhesion-independent stimuli activates ERK phosphorylation and this pathway can be modulated by inputs from G-protein-coupled and tyrosine kinase receptors. The murine pro-B cell line BAF03 also displayed αVβ5-mediated proliferation in response to human CD23. Preliminary experiments showed that human CD23 sustained growth of murine bone marrow B cell precursors. Therefore, these data suggest that murine B cells can also use αVβ5 integrin to sustain their growth and the studies described here in human cell lines could be translated into in vivo murine models. Further work is needed to confirm the proliferative response due to the αVβ5-CD23 interaction in normal B cell precursors in the bone marrow and to define the exact stage of development where this interaction is critical. In addition to its expression in the bone marrow B cell precursors, previous work has also demonstrated the expression of αVβ5 integrin in B cells from patients with ALL. Therefore, the αVβ5-CD23 interaction could have important implications not only in proliferation of normal B cell precursors but also in proliferation of neoplastic B cells. These data identify the αVβ5-CD23 interaction as a potentially important interaction during early B cell development, as αVβ5 expression and function is stage-specific, regulated by other molecules and can be demonstrated in both human and murine cell lines.

Evidence suggesting that Hsp72 is actively participating in cellular signalling as well interacting with immune system dynamics has been increasing. This is true in healthy, stressed and diseased cells but to different degrees. Modulation of the plasma membrane association and secretion in the extracellular environment by different types of stressors is the key event that leads to different degrees of immune system activation. Hence a better understanding of the mechanisms of Hsp72 secretion and association with plasma membrane is crucial. This thesis investigated the tissue source and mechanism of Hsp72 surface presentation to plasma membrane structures and release in relation with different cellular and physiological stressors. In vivo models confirmed that different tissue types determine specific Hsp72 responses following the same stress and increase serum Hsp72 dependant on intensity and duration of the stress. Diseases models confirm that Hsp72 responses in specific cell populations is related to disease progression, while in vitro models clearly showed that there are multiple mechanisms of secretion and surface presentation, dependent on the nature of the stressor as well as the intensity and duration. This observations clearly change the view of extracellular Hsp72 as a danger signal and lead to a revision of the original danger model. It also suggests that manipulation of Hsp72 translocation through the different pathways involved may prove effective therapeutically.

The intention of the work described in this thesis was to identify whether the Notch signalling pathway is utilized by antigen presenting cells in order to influence CD4+ adaptive immune responses. The notion that Notch proteins may be involved in polarising CD4+ T cells is relatively recent and most of the work that had been done in this area so far has concentrated on the consequences of Notch signalling within T cells. In contrast, the work that I have done has focussed on Notch ligand expression by antigen presenting cells and addresses the question whether Notch signalling is a redundant, necessary or irrelevant tool in the arsenal of antigen presentation.