Les glycosaminoglycanes (GAGs) sont des polysaccharides sulfatés qui participent à de nombreux processus physiologiques, via leurs interactions avec un large panel de médiateurs extracellulaires. Parmi ces facteurs, l’interleukine-4 (IL-4) joue un rôle central dans la polarisation M2 des macrophages. Bien que cette cytokine soit un ligand des GAGs, peu d’études ont été réalisées sur le rôle de ces interactions. Dans ce contexte, la première partie de ma thèse a porté sur le rôle des GAGs dans la polarisation M2 des macrophages. Nous avons montré que les activités de l’IL-4 sont dépendantes de sa fixation sur des GAGs 6-O-sulfatés. La sulfatation des GAGs est finement régulée par plusieurs familles d’enzymes, dont l’expression varie en fonction du type cellulaire et de l’environnement. Nous avons alors analysé les variations d’expression de ces enzymes de sulfatation au cours de la polarisation des macrophages. Nos résultats montrent qu’en fonction de leur phénotype, les macrophages expriment des GAGs différents, ce qui peut leur conférer des fonctions spécifiques. En effet, contrairement aux M1 pro-inflammatoires, les M2 sécrètent des facteurs de remodelage tissulaire et de tolérance immunitaire, ce qui peut favoriser la progression des tumeurs. Nous avons vérifié que les GAGs des M2 présentent le FGF-2 aux cellules cancéreuses, suggérant leur participation dans les mécanismes pro-tumoraux. Finalement, le dernier volet de ma thèse a porté sur le rôle des GAGs dans le cancer du sein. Nous avons confirmé que les GAGs sont de bons ligands pour les sélectines et qu’ils pourraient favoriser la promotion des métastases distantes. / Glycosaminoglycans (GAGs) are sulfated polysaccharides involved in many physiological processes, through their interactions with a wide range of extracellular mediators. Among these factors, interleukin-4 (IL-4) has a central role in the M2 polarization of macrophages. Although this cytokine is a ligand of GAGs, only a few studies have been performed on the physiological role of these interactions. In this context, the first part of my thesis focused on the role of GAGs in M2 macrophage polarization. We showed that the responses induced by IL-4 are dependent on the binding to 6-O-sulfated GAGs. The reactions of GAG sulfation are tightly regulated by several families of enzymes, for which the expression is dependent on cell type and environment. Then we analyzed changes in the expression of these sulfating enzymes during macrophage polarization. Our results showed that macrophages produced different GAGs based on their phenotype, which may give them specific functions. In contrast to pro-inflammatory M1 cells, M2 macrophages release factors involved in tissue remodeling and immune tolerance, which may enhance tumor progression. We verified that GAGs are capable of presenting FGF-2 to cancer cells, thus suggesting their involvement in pro-tumoral mechanisms. Finally, the role of GAGs in breast cancer was investigated in the last part of my thesis. Our results confirmed that GAGs are good ligands for selectins, so that they could promote distant metastasis.

The αVβ5 integrin is a member of integrin family that binds to different ligands such as vitronectin, fibronectin and soluble CD23 in order to mediate different biological responses such as cell growth, adhesion and metastasis. It is expressed by B cell precursors and by different acute lymphocytic leukaemia cell lines such as SMS-SB cells. This thesis is an attempt to explain how the sCD23- αVβ5 integrin interaction stimulates SMS-SB cell growth and to study the role of the αVβ5 integrin and other receptors such as PDGF receptor and CXCR4 in B cell development in the bone marrow. The maturation and differentiation of B-cells occur due to several factors that impact on gene expression in its development program. This program is divided into two main phases, the antigen-independent B-cell development phase and antigen-dependent B-cell development phase, respectively. The antigen - independent phase of B cell development starts from the pluripotent haemopoietic stem cell (PHSC) and progresses through several successive stages which are identified by somatic recombination and rearrangement of both heavy and light chain genes. Soluble CD23 and LP (a synthetic peptide derived from soluble CD23) significantly stimulate SMS-SB growth while a smaller growth stimulation is caused by either SDF1-α or PDGFAB. There are different signalling targets involved in the αVβ5 integrin-mediated proliferation due to its binding to either sCD23 or LP. These ligands enhance the association between the αVβ5 integrin and the PDGF receptor which promote the phosphorylation of both Jak2 and STAT5. Moreover, cell growth was reduced and the phosphorylation of Jak2 and STAT5 was also knocked down with using either PDGF receptor inhibitor (AG1295) or Jak2 inhibitor (AG490). Both soluble CD23 and LP activate the STAT5-DNA binding and strongly increase its transcriptional activity. In addition, both ligands induce the phosphorylation of other different substrates such as STAT2, c-Src, c-yes and AMPKα2 which might be related to cell growth stimulation. The αVβ5 integrin ligands also promote the phosphorylation of ERK1/2, p90RSK and activate a SRF transfected reporter gene. However, ERK1/2 and p90RSK phosphorylation was completely blocked by the specific MEK inhibitor (U0126). In similar context, SDF1-α stimulates the transcriptional activity of SRF but not STAT5 while PDGFAB does the opposite. Finally, soluble CD23 induces the proliferation of 697 and BAF03 which are other pre-B cell line models. These data suggest that the αVβ5 integrin-ligated ligands stimulate SMS-SB cell growth by promoting different signalling pathways, mainly Jak2/STAT5 and MEK/ERK1/2 pathway. Further work is required to determine the role of STAT5, p90RSK, c-Src and SRF in stimulating either the proliferation or apoptosis that promoted by the αVβ5 integrin-sCD23 interaction and to investigate the relationship between the activation of these targets.

CD31 mediates homophilic interactions between leukocytes and macrophages during inflammation, apoptotic cells remain attached and are engulfed whereas viable cells actively detach. We hypothesised that differential recruitment of signalling and adapter molecules to the cytoplasmic domain were responsible for the disengagement of the viable leukocyte from macrophages. Investigation with a static attachment assay using THP-1 as a macrophage model showed the ITIM of CD31 on the leukocyte was important for viable cell detachment. Our data also implicated a role for the recruitment of SHP-2 which we attempted to knock-down by siRNA delivered by lentivirus. SHP-2, in addition to its phosphatase activity, also acts as a docking protein. To examine for potential interacting partners we fused the cytoplasmic domain of CD31 to GST which we used in pulldown assays from lysates of viable and apoptotic leukocytes. We demonstrated that the recruitment of SHP-1 and SHP-2 were dependent on an intact ITIM (immunoreceptor tyrosine-based inhibitory motif). Interestingly, apoptotic cell lysates promoted dephosphorylation of the in vitro phosphorylated GST-CD31, suggesting an increase in phosphatase activity in aged neutrophils. We were unable to demonstrate an interaction between the cytoplasmic domain of CD31 with putative binding partners β-catenin, src, RasGAPp120, RhoGAPp190, talin or calmodulin. A proteomic approach by MALDI-TOF and MS/MS identified Hsp90 as a novel binding partner of CD31 irrespective of the phosphorylation state. In contrast, 14-3-3ε bound to phosphorylated CD31, whereas eIF3 specifically bound to an ITIM double tyrosine mutant. The binding of Hsp90 to CD31 was proposed to occur via a TPR motif within the cytoplasmic domain of CD31 which comprises a surface fold of basic amino acids complexing a highly acidic carboxy tail of Hsp90. Truncation and site directed mutagenesis of the cytoplasmic domain revealed multiple binding sites for Hsp90; specifically two regions containing the sequences (KAFYLRKAKAK), previously shown to be the calmodulin binding region, and a novel area (SNNEKMSDMEANSHY) which has significant homology with other TPR-containing proteins. Systematic mutagenesis of the putative basic charged amino acids within the cytoplasmic domain of CD31 which may mediate the interaction with Hsp90 also supports the presence of a TPR motif. The importance of Hsp90, 14-3-3ε and eIF3 is currently unknown, although it is interesting to note that CD31 was recently found within this laboratory to associate with the voltage-gated potassium channel HERG which also binds 14-3-3ε and Hsp90. eIF3 is an RNA helicase that may link CD31 and leukocyte motility to spreading initiation centres where motility can be viewed as a rapid turnover of focal adhesion complexes. Together, these studies have identified novel binding partners of CD31 may form a macromolecular complex to promote CD31-dependent leukocyte motility and detachment from macrophages.

The nematode Caenorhabditis elegans has been a powerful experimental organism for almost half a century. Over the past ten years, researchers have begun to exploit the power of C. elegans to investigate the biology of a number of human pathogens. This work continues to uncover mechanisms of host immunity and pathogen virulence that are either analogous to those involved during pathogenesis in alternative animal hosts or mechanisms which are, thus far, unique to the worm. In this thesis, we present data that describes an immunological balance in C. elegans, whereby heightened tolerance to one pathogen, the enteric bacteria Salmonella Typhimurium, comes at the cost of increased susceptibility to another, the fatal fungal human pathogen Cryptococcus neoformans. We find that this susceptibility trade-off is mediated by the reciprocal activity of two immune genes: the lysozyme lys-7 and the tyrosine kinase abl-1. We suggest that ABL-1 controls two different DAF-16-dependent pathways to regulate this balance. Both pathways are necessary for wild type resistance to C. neoformans, whilst the activity of only one pathway is a requirement for the tolerance phenotype to S. Typhimurium. We infer from sequence data that LYS-7 has an atypical mode of action in C. elegans, which we hypothesise to be detrimental to the worm during S. Typhimurium pathogenesis and thus a contributing factor to the tolerance phenotype. Furthermore, we find that this tolerance has a Salmonella-dependency which we propose to be under the control of the alternative sigma factor, RpoS. Taken together, we describe an immunological balance in C. elegans for the first time, one that is mediated by both host and pathogen factors. We therefore suggest that the innate immune response of C. elegans has a higher level of immune complexity than previously believed, and that such trade-offs are evolutionarily ancient mechanisms.

Cellular senescence is a state of permanent cell cycle arrest in which a cell is unable to further divide in response to normal growth stimuli. Telomere shortening, believed to be a mechanism of cellular senescence, is a result of numerous rounds of cell division and can be accelerated by heightened levels of oxidative stress. An accumulation of senescent T lymphocytes in blood and tissues has been suggested to contribute to the general immunosuppression seen in the elderly. Obese individuals and athletes taking part in regular high intensity exercise are subjected to heightened states of oxidative stress which appears to result in immunosuppression and increased susceptibility to infection. It was hypothesised that there would be an accumulation of senescent T lymphocytes with ageing, with obesity and during six months training for an Iron man competition. Senescent CD4+ and CD8+ T peripheral blood lymphocytes were identified by 4 colour flow cytometry, based on their cell surface expression of CD57, the killer cell lectin-like receptor G1 (KLRG1) and the lack of cell surface expression of CD28. Where telomere length measurements were performed, CD4+ and CD8+ T lymphocyte subsets were isolated and telomere lengths were compared to the proportions of KLRG1+, CD57+ and CD28+ cells in each subset. Older subjects and obese subjects had significantly larger proportions of senescent cells (KLRG1+/CD57+) in the CD8+ but not the CD4+ peripheral blood T lymphocyte subset. Furthermore, during six months training for an Iron Man competition, compared to the first month, club level athletes showed a 92% increase in the frequency of senescent cells (KLRG1+/CD57+) in the CD4+ but not the CD8+ T lymphocyte subset. Telomere length was negatively correlated with the proportion of KLRG1+ and CD57+ cells and positively correlated with the proportion of CD28+ cells in the CD4+ but not the CD8+ T lymphocyte subset. It is concluded from this work that KLRG1+/CD57+ senescent T lymphocytes accumulate with age, obesity and regular high intensity training, which may contribute to the increased immunosuppression associated with these states.

Neutrophils are phagocytic white blood cells which act as the first line of defence against entry of foreign microorganisms. Neutrophils are recruited to their target site through the process of spreading, extravasation and phagocytosis involving complex signal transduction within the cells, which might include the activation of the cytosolic Ca2+ activated protease, calpain-1. The work described here investigates the role of calpain-1 in regulating neutrophil functions such as spreading, trans-endothelial migration, chemotaxis, phagocytosis and Ca2+ signalling. Through the work done at European Mutant Mouse Archive (EMMA), Oxford, and by using intracellular sperm injection (ICSI) of calpain-1 deleted gene from mice generated in the USA, and with a selective genotype breeding programme, a colony of homozygous calpain-1 KO mouse has been generated in Cardiff. Homozygous calpain-1 KO neutrophils appeared to have a smaller surface spreading area and their recruitment into the peritoneal cavity of the mouse in vivo was disrupted. In vitro experiments showed significant defects in their ability to cross the ICAM-1 expressing endothelial cells in trans-endothelial migration assay. Disruption in this transmigration was only evident with ICAM-1 upregulated (TNF-treated) endothelial cells, suggesting a specific defect in the β2 integrin-ICAM-1 signalling process. Calpain-1 absence did not affect signal transduction as neutrophils were able to signal cytosolic Ca2+ in response to β2 integrin engagement (C3bi-opsonised zymosan) and also to release intracellular Ca2+ store upon IP3 uncaging. This showed that the IP3 pathway in the cells was not affected by knocking-out calpain-1 and continued to be functional. The key signalling mechanisms from β2 integrin also remained intact and this is consistent with calpain-1 activation by Ca2+ being an important event in trans-endothelial migration. In conclusion, calpain-1 absence has significantly affected the ability of neutrophils to undergo trans-endothelial migration and this effect is directed towards the event which happens downstream to the increase in cytosolic free Ca2+ concentration.

Les lymphocytes T régulateurs CD4+Foxp3+ (Treg) sont indispensables au contrôle des cellules T auto-réactives et au maintien de la tolérance immune en périphérie. Ils sont sélectionnés positivement dans le thymus, possèdent un répertoire TCR extrêmement diversifié suggérant une reconnaissance d’antigènes variés, requièrent une signalisation continue via leur TCR pour maintenir leurs fonctions en périphérie, fonctions qu’ils exercent avec une certaine spécificité de tissu. Ils contrôleraient donc spécifiquement les lymphocytes T auto-réactives en reconnaissant comme eux des antigènes du soi. Mon travail de thèse s’est articulé autour de deux axes visant à revisiter ce paradigme de l’autoréactivité des Treg, un point essentiel qui concerne les propriétés de reconnaissance antigénique de ces cellules. Afin de ne pas biaiser nos résultats avec des TCR artificiels, non issus de Treg, nous avons choisi d’étudier les Treg polyclonaux de souris normales. Dans une première série d’expériences, nous avons pu montrer que l’activation de lymphocytes T auto-réactifs anti-mâle dans une souris mâle conduisait à un remodelage du répertoire TCR des Treg, avec un usage préférentiel des Vβ5 et Vβ12. L’absence d’amplification clonale ainsi que des données récentes de la littérature démontrant l’amplification de sous-populations de Treg par des rétrovirus endogènes au cours de réponses immunitaires chroniques, nous ont conduit à conclure que l’activation de lymphocytes T auto-réactifs pourrait réactiver des rétrovirus endogènes et donc l’expression de super-antigènes capables d’amplifier les Treg portant certains Vβ. Dans le second volet de mon travail de thèse, nous avons caractérisé le niveau d’autoréactivité des Treg in vivo. En procédant à une comparaison systématique avec des Treg ayant des niveaux d’allo-réactivité différents et connus, nous avons montré que, de manière inattendue, les Treg polyclonaux possèdent un niveau étonnamment faible d’autoréactivité. Seule une fraction d’entre eux était capable de recevoir un signal TCR et des expériences de quantification ont révélé que cette fraction de cellules auto-réactives ne devait pas excéder 1% du répertoire total des Treg. L’ensemble de nos résultats suggèrent donc que les Treg sont faiblement réactifs en périphérie et/ou sont dirigés contre des antigènes endogènes rarement représentés. / No abstract available

The thymus is organised into distinct microenvironments, and trafficking through these regions enables thymocytes to receive essential signals for the generation of a diverse and self-tolerant T-cell repertoire. Thymic epithelial cells (TEC) represent a key stromal cell type during defined stages in T cell development, yet the mechanisms regulating their development are only partly understood. An ontogenetic approach was employed to study stages of cortical thymic epithelial cell (cTEC) development. This study identifies a previously unreported population of cTEC progenitors expressing CD205 and 5T and has defined distinct checkpoints in the development of the cTEC lineage. Furthermore, the importance of thymic crosstalk during specific stages of cTEC development and also the requirement for RANK-RANKL signalling for the development of various medullary thymic epithelial cell (mTEC) subsets has also been defined. Additionally, the importance of chemokine-mediated signalling for the establishment and compartmentalisation of the thymus has been highlighted by employing laser capture microdissection and studying thymus microenvironments in mice deficient for particular chemokine related signalling pathways. Overall this study has provided novel insight into the development of the thymic cortex and will help to understand how these cells become specialised in their ability to support positive selection of developing T cells.

Killer immunoglobulin-like receptors (KIRs) are a family of proteins expressed on human natural killer cells and a subset of T cells. Several inhibitory KIRs have been shown to recognise MHC class I molecules (predominantly HLA-C), with their engagement preventing target cell lysis. The ligand(s) and function(s) of activating KIRs, however, are less well characterised. Genetic studies of the association of KIRs with disease have identified an association with viral infections and autoimmune disease and this implicates that these proteins are important in human health. This thesis was concerned with an investigation of the factors that determine KIR expression on lymphocytes, and how this might influence the cellular functional response. In my initial work I produced soluble recombinant forms of activating and inhibitory KIRs and studied the biophysical interaction of these proteins with HLA-C molecules. I saw some evidence that KIR2DS2 binds to the HLA-C group 1 allele HLA-Cw*0702, supporting the idea that HLA-C alleles are a true ligand for stimulatory KIRs. I then went on to make a detailed 11 colour flow cytometric analysis of the expression of KIR proteins in healthy individuals. I was able to show that total, and individual, KIR protein expression was correlated and defined a pattern of dominance on lymphoid subsets. I then went on to study the distribution of KIR expression on discrete memory T cell subsets and showed that they were found predominantly on late differentiating CD45RA+ T cells. Interestingly there was also considerable expression on central memory CD8+ T cells although the biological basis for this is unclear. I demonstrated that age and CMV infection have a marked effect on KIR expression and I speculate on the reason for this. Finally I studied KIR expression on CMV-specific T cell clones in order to undertake a functional analysis of the consequence of KIR expression. I observed that KIR expression increased when cells were cultured in vitro but I could not detect any difference in cytokine production or cytotoxicity between KIR+ and KIR- cells. My work has contributed to the literature on KIR biology in relation to lymphoid cells and will have direct relevance to a number of clinical studies.

The interaction between the nervous and immune system is well documented, although is still not fully understood - particularly the impact of neurotransmitter receptors on immune cell function. 5-HT\(_3\)A receptor expression was identified on activated regulatory T cells (Treg) but not on effector T cells. Incubation of human peripheral blood mononuclear cells (PBMC) with the 5-HT\(_3\) receptor agonist DDP733 and the positive allosteric modulator 5-chloroindole increased the percentage of CD25+FoxP3+ lymphoyctes (Treg phenotype). Proliferation of PBMC was inhibited by DDP733 plus 5-chloroindole indicating functional impact by the 5-HT\(_3\) receptor. The GPR55 receptor was also expressed by human T cells. The GPR55 agonist lysophosphatidylinositol increased cell viability by preventing apoptosis. However, the induced response was not blocked by the GPR55 receptor antagonist cannabidiol casting doubt over the GPR55 receptor mediating the response. Cannabidiol was demonstrated to have a pro-apoptotic effect in its own right, although whether this effect is mediated by GPR55 or the CB2 cannabinoid receptor is unknown. Further experiments are required to elucidate the role of the 5-HT\(_3\)A receptor in lymphocyte function and the mechanism responsible for the immunoprotective role of lysophosphatidylinositol.