No description available.

CD23 is a multi-functional protein which exists in membrane-bound and soluble forms. Its functions include acting as the low affinity receptor for IgE and generating pro-inflammatory cytokine release in monocytes. CD23 has been found to interact with αvβ5 and this interaction greatly enhances growth of the B cell precursor cell line SMS-SB. This interaction may have a role in the development of normal human B cells and in cancer as the integrin is expressed on both precursor and ALL cells but not on normal mature B cells. One of the aims of this investigation was to expand on the finding that CD23 peptides containing an RKC motif had the same positive growth effect on SMS-SB cells as CD23. Other B cell lines – representative of both precursor and mature stages – were studied to ascertain whether this proliferative effect was dependent upon cell differentiation stage and/or presence of the αvβ5 integrin. It was found that peptides containing the basic RKC motif were mitogenic only for precursor B cells which were expressing αvβ5. Details of these peptides and their varying effects on the different cell lines are in Chapter 4. Stimulation of SMS-SB cells, presumably via the αvβ5, results in signalling through PI3K and subsequent phosphorylation of Akt. The growth of SMS-SB cells observed following stimulation with peptides containing the RKC motif was abrogated by the PI3K inhibitor LY294002 and western blotting revealed that phosphorylation of Akt was enhanced by stimulation with RKS containing peptides. Among CD23’s receptors is the integrin αvβ3. This integrin can form a signalling complex with CD47. Ligation of CD47 by anti-CD47 antibodies induces apoptosis in some cell lines. To determine whether a pattern exists between response to this stimulation and expression of αvβ3 integrin, cell lines with and without the integrin were tested. It was found that the myeloma cell lines KMS11 and H929 were responsive to this stimulus. Since these cell lines differ in their expression of αvβ3 (H929 cells express αvβ3 whereas KMS11 do not) it does not appear that any connection between the presence of the integrin and response via CD47 exists and therefore this signalling mechanism would appear to occur independently of the complex formed by CD47 and αvβ3.

Palmitoylation variant GPCR-G protein fusion proteins were created between the porcine u2A-adrenoceptor or the human 5-HT1A-serotonin receptor and the pertussis toxin resistant, Cys35lIle, form of the rat Go1u protein. These palmitoylation-variant fusions were transiently expressed in HEK293T cells prior to analysis of the regulation of palmitoylation and the functional consequences of palmitoylation for both the GPCR and G protein parts of the fusions. When the regulation of palmitoylation was studied for u2A-adrenoceptor-GoluCys35IIle fusion proteins, dynamic palmitoylation and depalmitoylation of both the Cys442residue of the u2A-adrenoceptor and the Cys ' residue of the GoluCys351Ile protein were found to occur. However, only the GOluCys351Ileprotein part of the fusion was found to undergo adrenaline-stimulated regulation of palmitoylation and the effect of adrenaline required G protein activation. Adrenaline regulation proceeded in a concentration-dependent manner correlating with agonist occupancy of the u2A-adrenoceptor. Such agonist effects were found to be, at least in part, due to agonist-stimulation of GOluCys351Ile protein depalmitoylation. The requirements for palmitoylation of the u2A-adrenoceptor and GoluCys351Ile protein elements of the u2A-adrenoceptor-GoluCys35IUe fusion proteins were subsequently assessed for various functional properties. Palmitoylation of neither the U2Aadrenoceptor nor the GoluCys351Ile protein parts of the fusion determined fusion protein expression levels, affinity for the agonist adrenaline, affinity for the antagonist RS- 79948-197, ability to bind or to hydrolyse GTP or their ability to influence the efficiency of RGS 16 protein to accelerate the GTPase reaction. In regulation of palmitoylation studies for 5-HTIA-receptor-GoluCys35IIle fusion proteins, dynamic palmitoylation of the Cys' residue of the GoluCys351Ue protein and the Cys417 residue of the 5-HTIA-receptor was observed as well as a lack of incorporation of palmitate into Cys420 of the 5-HT1A-receptor. Dynamic depalmitoylation was only observed for the Cys' residue of the GoluCys351Ile protein, not for the 5-HT1A-receptor. In the latter case, palmitate once incorporated appeared to remain stably attached. Both the 5-HT1A-receptor and the GoluCys351Ile protein parts of the fusion were found to undergo 8-0H-DPAT-stimulated regulation ofpalmitoylation. 8-0H-DPAT was able to regulate palmitoylation levels of both proteins in a concentration-dependent manner. For the regulation of GoluCys351Ile protein palmitoylation such agonist effects were found likely to be, at least in part, due to an agonist-stimulated rate of depalmitoylation. For the regulation of 5-HT1A-receptor palmitoylation such agonist-stimulated increases in observed palmitoylation levels were only attributable to the addition of palmitate, given that no depalmitoylation of the 5- HT1A-receptor could be detected. The requirements for palmitoylation of the 5-HT1A-receptor and GoluCys351Ile protein elements of the 5-HT1A-receptor-GoluCys351Ile fusion proteins were also assessed for a selection of functional properties. Similar to the results obtained with Go1uCys351Ile protein constrained to the uZA-adrenoceptor, the palmitoylation of the GoluCys351Ile protein did not determine fusion protein expression levels, their affinity for the antagonist WAYI00635, or their ability to bind GTP. Palmitoylation of 5-HT1Areceptor did not alter fusion protein expression levels or their affinity for the antagonist WAYI00635. However, in contrast, it did cause enhanced levels of GTP binding to the 5-HT1A-receptor-GoluCys351Ile fusion proteins. The results of this investigation suggest that there are different requirements for regulation of GPCR and G protein palmitoylation dependent on the GPCR-G protein fusion in question. These requirements may be responsible for the specific functional properties displayed by such fusions. The current study also demonstrates that GPCR-G protein fusion proteins can be successfully used as tools to study both the regulation of palmitoylation and the functional consequences of this modification.

The objective of this thesis is to determine the role of specific PKC isotypes during B cell development and transformation. B cell generation systems were validated both in vitro and in vivo, by coculturing haematopoietic progenitor cells (HPCs) on the calvanial cell line, 0P9, or by adoptively transferring HPCs into recombinase-activating gene 1-deficient (RAG-1-/-) mice, respectively. In both cases, mature B cells were generated as determined by analysing surface B cell marker expression. Coupling of these in vitro and in vivo B cell generation systems with a retroviral gene transfer technique, plasmids-encoding PKC mutants in the retroviral vector MIEV were stably expressed in foetal liver (FL)-derived HPCs from wild type mice and cultured to assess the ability of individual PKC isoforms to modulate the development or transformation of B cells. Of note, expression of a plasmid-encoding dominant negative PKCalpha (PKCalpha-KR) in HPCs and placement in B cell generation system in vitro or in vivo resulted in the generation of a population of cells that displayed an enhanced proliferative capacity. Analysis of PKCalpha-KR-expressing cells in vitro revealed that these cells incorporated BrdU significantly more than the MIEV control, and unexpectedly upregulated cell cycle regulators, p21waf-1 and p27kip-1. Of surprise, PKCalpha-KR-expressing cells phenotypically resemble human B cell chronic lymphocytic leukaemia (CLL) cells. Expression of constitutively active PKCalpha, PKCalpha-CAT, or dominant negative PKCalpha, PKCalpha-KR in HPCs caused significant decrease in cell number. CLL is characterised by the accumulation of long-lived phenotypically mature B cells with the distinctive phenotype: CD19hi, CDS+, CD23+, IgMdim, which are deficient in apoptosis and have undergone cell cycle arrest in the G0/G1 phase. Closer analysis of PKCalpha-KR-expressing cells uncovered that these cells undergo cell cycle arrest in the absence of growth factors and stroma and consistent with their ability to escape growth factor withdrawal-induced apoptosis, exhibited elevated levels of Bcl-2 and Mcl-1 expression. Upon stimulation with IL-7, PKCalpha-KR-expressing cells showed explosive proliferation, suggesting that IL-7 is a proliferation factor for these cells. In accordance with this, IL-7R expression was upregulated in these cells, which may contribute to the increased sensitivity to IL-7. Mice injected with wildtype PKCalpha-KR-HPCs bore solid intraperitonial tumours at the injection site and the cells from both tumour and spleen showed CLL-like phenotype. Interestingly, splenocytes from these mice were cycling whereas the tumour cells were arrested at the G0/G1 stage, probably reflecting the two phases of this disease, a quiescent stage and an extensive proliferative stage, respectively. The expansion of the leukaemic cells was halted when they were cultured on 0P9-DL1, 0P9 cells with ectopic expression of Notch ligand, DL1, suggesting that Notch signalling mediates tumour suppression in CLL cells.

In metozoans, most of the key events that lead to cell activation and inhibition are controlled by tyrosine phosphorylation. Extracellular signals are transmitted by membrane bound receptors, which have intrinsic kinase activity or themselves recruit intracellular kinases to specialised inhibitory or activating phosphorylation motifs. In this way, the pattern of kinase activation creates its own turnover and can rapidly generate amplified signals by positive feedback, or recruit inhibitory proteins to counteract the signals. This process of inhibition is also constitutive since it requires continuous counter-inhibition by phosphatases at the cell surface and intracellularly even in the absence of ligands. The absence of phosphatase activity results in unbridled protein phosphorylation and form this and other data it has been proposed that the triggering of the T cell receptor and other co-receptors may result simply by physical exclusion of the large phosphatases such as CD45 from the vicinity of the receptors. Superagonist monoclonal antibodies may work in a similar way, by binding receptors close to the plasma membrane and excluding extracellular phosphatases. The work described in this thesis seeks to discover if antibody superagonists can be generated against the T cell inhibitory cell surface receptor PD-1 and test if this approach can attenuate the immune response. Using in vitro assays of lymphocyte activation and a mouse model expressing human PD-1, this study characterises a series of anti-PD-1 antibodies and shows how patterns of inhibitory activity varying according to binding sites. The inhibitory effects of the anti-PD1 antibodies are seen in the humoral, cellular and transplant immune responses. Agonistic anti-PD1 antibodies induce regulatory T cells and may have role in suppression of autoimmune disease. The thesis suggests that superagonism may be harnessed clinically to dampen the immune response, through activation of inhibitory receptors.

Counter current chromatography (CCC) is a form of liquid liquid chromatography, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. In this thesis, its application has been successfully extended to the rapid, scalable purification of monoclonal antibodies (mAb) from mammalian cell culture, using aqueous two-phase systems (ATPS) of inorganic salts and polymer. A polyethylene glycol (PEG) and sodium citrate system was found to be the most appropriate by robotic phase system selection. The search for an economical alternative to protein A HPLC is a substantial bioprocessing concern; in this work CCC has been investigated. Initial studies showed that unpredictably, despite separation from impurities being achieved, some loss in the IgG‘s ability to bind to Protein A was seen, as confirmed by Protein A BiaCore analysis. CCC machines were seen to adversely affect IgG functionality. This led to a systematic investigation of the effect of CCC phase mixing on IgG functionality in a number of different CCC instruments, allowing direct comparisons of modes of CCC (hydrodynamic and hydrostatic CCC) and their associated mixing (wave-like and cascade, respectively). The varying g forces produced within the CCC column were determined using a recently developed model to calculate g force range. The effect of interfacial tension was also studied using a custom built 'g' shaker. The optimum CCC mode was identified to be the non synchronous CCC, operated in a hydrodynamic mode but allowing bobbin to rotor speed (Pr ratio) to be controlled independently. In a normal synchronous J type centrifuge a Pr of 1 is fixed, this is where the bobbin and rotor speed are identical I.e. one bobbin rotation (where mixing occurs) to one rotor revolution (where settling occurs). Constraints were seen with this 1:1 ratio and the separation of mAb using ATPS. This work has shown with the use of the non synchronous CCC at a Pr of 0.33, mixing is reduced and rotor rotations increased. Consequently the associated g force range is decreased. Furthermore, by the extension of settling time, the clear separation of the mAb from impurities has been achieved with retention of biological activity. This thesis demonstrates the importance of settling time for ATPS in phase separation and documents the fundamental requirements for the successful separation of biologics. Purified non synchronous CCC samples have additionally undergone rigorous quality control testing at Lonza Biologics by their purification scientists. This work has ultimately showed that with optimisation, the non synchronous CCC can be used to produce biological samples that are of industry standard.

Les myosites sont des maladies auto-immunes, caractérisées par des atteintes musculaires et extra musculaires. Le diagnostic des myosites peut être difficile et nécessite de l’expertise, afin d’éviter l’administration de thérapie inapproprié. Les mécanismes impliqués au cours des myosites sont peu connus. Notre but était de décrire le profil immunitaire des patients, afin d’identifier des biomarqueurs. Nous avons utilisé un panel de 36 marqueurs pour caractériser les PBMC issus de patients actifs (MIs, SAS anti-Jo1, myopathies anti-SRP et anti-HMGCR) et de sujets sains par cytométrie de masse combiné au « barcoding ». Tout d’abord, nous avons mis au point une procédure technique pour la détection simultanée de cibles extracellulaires et intracellulaires. En utilisant différents outils bio-informatiques, nous avons isolé une fréquence de lymphocytes CD8+T-bet+ > 51.5% comme étant un biomarqueur spécifique de la MIs en comparaison aux autres myosites, avec une sensibilité de 94,74% et une spécificité de 88,46%. De plus, nous avons identifié un profil immunitaire CD8+T-bet+ CD57- activé, potentiellement capable de prolifération et de maintien de mécanismes auto-immuns chez les patients atteints de MIs. Chez les patients anti-Jo1, nous avons observé une dérégulation de l’homéostasie des lymphocytes B, caractérisée par une diminution des lymphocytes B mémoires circulants. La présence de ces derniers dans le muscle des patients suggère qu’ils se nichent dans le muscle afin d’éviter l’action des immunosuppresseurs. Ces travaux ont permis l’identification de biomarqueurs et de phénotypes cellulaires potentiellement impliqués au cours de la MIs et du SAS anti-Jo1. / Myositis is an autoimmune disease characterized by muscular and extra-muscular disorders. In the early stages of the disease, the diagnosis of myositis can be misleading and requires expertise, in order to avoid the administration of inappropriate treatment. The mechanisms involved in these diseases are poorly understood. Our aim was to describe the immune profile specific to each patient group, in order to identify biomarkers that may be useful for diagnosis and management of patients. We used a panel of 36 markers by mass cytometry to characterize PBMCs derived from active patients (sIBM, anti-Jo1 ASyS, anti-SRP and anti-HMGCR myopathies) and healthy subjects. First, we developed and optimized a technical procedure for the simultaneous detection of extracellular and intracellular targets by mass cytometry. Using different bioinformatics tools, we isolated a frequency of CD8 +T-bet + cells > 51.5% as a specific biomarker for sIBM compared to other myositis, with a sensitivity of 94.74% and a specificity of 88. , 46%. In addition, we identified an activated CD8 + T-bet + CD57- immune profile, potentially capable of proliferation and the maintenance of autoimmune mechanisms in patients with sIBM. In anti-Jo1 patients, we observed a dysregulation of B cell homeostasis, characterized by a decrease in circulating memory B cells. The presence of the latter in the muscle of patients suggests that they nest in the muscle to avoid immunosuppressants. This work allowed the identification of a biomarker that could enhance the diagnosis of MIs compared to other myositis and the identification of cells potentially involved during sIBM and anti-Jo1 ASyS.