CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) involved in the recruitment of a subset of leukocytes to sites of inflammation. In addition, CCR5 functions as a co-receptor for several strains of the human immunodeficiency virus (HIV). The finding that agonist-induced internalisation of CCR5 can protect susceptible cells from infection in vitro has stimulated research demonstrating that activated receptors are phosphorylated on C-tail serine residues and bind p-arrestins, which couple them to the clathrin-mediated endocytic machinery. After internalisation, receptors are recycled to the cell surface via a perinuclear recycling compartment, identified as the recycling endosome. Here, I extend these findings, demonstrating that CCR5 also traffics through the trans- Golgi network (TGN), and thus, the perinuclear recycling compartment can be considered to comprise both recycling endosome and TGN elements. Moreover, I show that Rabll regulates trafficking through this compartment and that dynamin inhibition blocks the recycling of CCR5. In combination with other electron microscopy studies, my data support the notion that clathrin and dynamin are involved in the recycling of CCR5 from the recycling compartment. Through morphological and biochemical analysis, I also show that p-arrestins maintain an interaction with CCR5 into the recycling compartment. Kinetic analysis of receptor recycling suggests that p-arrestin2 acts a negative regulator of CCR5 recycling but my data do not discount the possibility that p-arrestins couple CCR5 to clathrin to effect recycling. In keeping with the sustained p-arrestin interaction, CCR5 molecules remain phosphorylated as they traffic to the recycling compartment, but contrary to previous reports showing that receptor C-tail phosphorylation is required for high-affinity p-arrestin binding, I show that a phosphorylation-deficient CCR5 mutant undergoes p-arrestin-dependent agonist-induced internalisation. In addition to its relevance for HIV biology and inflammation, this study contributes to an understanding of GPCR trafficking in general.

As part of our ongoing efforts to solve a complete structure of factor H, the structure of module 13 (fH^~13) has been solved by NMR spectroscopy. Recombinant fH^~13 protein was produced in <i>Pichia pastoris</i> in our laboratory and we have prepared unlabelled and ¹⁵N, ¹³C labelled samples of this module. Among the 88-indivdual CCP-modules of the regulators of complement activation, module sequence-lengths range from 51 to 67 amino acid residues and fH^~13 possesses the shortest sequence. The solved solution structure of fH^~13, reflects this short primary sequence and is unusual amongst the complement control proteins (CCP modules). fH^~13 possess the expected disulphide-bonding pattern and consensus tryptophan, but lacks many overall 3D-structural features that characterise a “typical” CCP-module. fH^~13 possesses only two <i>β</i>-strands out of a maximum of eight. The most similar structure of fH^~13 is fH^~15, while the most dissimilar CCP module is CR1^~16. One side of the fH^~13 domain reveals a highly localised positively charged patch composed of eight residues. To recognize host from non-host cell membranes, factor H binds to polyanions such as sialic acid or heparin sulphate which are bound on to the surface of host cells. There are three putative polyanion binding sites located in modules 7, 13 and 20, whose involvement in this process is, to various extend, supported by experimental evidence. The one in module 13 is the most disputed of the three polyanion binding sites. We have performed binding studies using gel mobility shift assay and tested building of fH^~13 to a range of heparin derived oligosaccharides from disaccharide to dodecasaccharide with negative results. Similarly, NMR titrations using a fully sulphated heparin-derived tetrasaccharide yielded a negative result. These results point to the importance of an adequate distribution of positively charged residues for the binding of polyanions.

Recently, a population of embryonic thymic epithelial cells has been isolated in the mouse, identified by the monoclonal antibodies MTS20 and MTS24, which can differentiate into all thymic epithelial cell types and is sufficient to generate a functional thymus in vivo. This strongly suggests that a common, MTS20⁺24⁺ progenitor gives rise to all mature sub-populations of TEC, and opens up the possibility of supporting thymopoiesis in vitro using a single TEPC line. The aim of this thesis was therefore to generate cell lines of TEPC’s that retain the capacity to differentiate and form a functional thymic microenvironment. To allow the derivation of cell lines that correspond to the progenitor phenotype, the developmental capacity of TEPC’s must be suspended at a stage prior to the onset of differentiation. However, the developmental block must be reversible, so that the ability to form a functional thymic microenvironment is retained. The differentiation of TEPC’s is dependant on the transcription factor <i>Foxn1, </i>which is expressed specifically in epithelial cells of the thymus and epidermis. Nude mice, which harbour a loss of function mutation in <i>Foxn1, </i>contain an alymphoid thymus consisting of immature, MTS20⁺24⁺ epithelial cells. Thus, the TEPC population forms independently of <i>Foxn1, </i>but requires expression of this transcription factor to form a mature thymic microenvironment. This thesis therefore describes the generation of transgenic mice in which <i>Foxn1 </i>expression is reversibly down-regulated. In addition, as a consequence of Foxnl driven SV40TAg expression, these mice also result in the conditional immortalisation of TEC’s. A phenotypic analysis of these mice is described, as is the derivation and characterisation of TEC lines.

No description available.

When grafted <i>in vivo</i> a population of fetal thymic epithelial cells, marked by the monoclonal antibodies MTS20 and MTS24, can generate all major thymic epithelial cell subtypes. Furthermore, the resultant thymic organoid can recruit T cell precursors and support their differentiation into mature CD4⁺ and CD8⁺ T cells. The work presented here evaluates the potential of MTS20⁺ thymic epithelial progenitor cells to form the basis of a thymus equivalent <i>in vitro</i>. To assess the ability of the MTS20⁺ thymic epithelial progenitor cell (TEPC) population to support T cell differentiation <i>in vitro</i>, improvements were made to the established reaggregate fetal thymic organ culture (RFTOC) method that permitted the reliable generation and culture of TEPC-based RFTOC (TEPOC). Subsequent analysis demonstrated that both MTS20⁺ and MTS20⁺ fetal thymic epithelial cells were able to support the differentiation of αβ and γδ T cells and also supported the differentiation of several other haematopoietic populations including dendritic cells and thymic macrophages. However, while immunofluorescence analysis showed that MTS20⁺ cells were able to differentiate <i>in vitro</i> to generate all major thymic epithelial cell populations, only limited differentiation was observed in MTS20⁺cell-based cultures. Strikingly, the TEPOCs were able to form organised epithelial structures <i>in vitro</i>, characterised by clearly distinguished adjacent medullary and cortical areas. No evidence of organisation was seen in MTS20⁺ cell-based cultures. Together, these data establish that MTS20⁺ but not MTS20⁺ thymic epithelial cells can generate functional <i>in vitro</i> thymus-equivalents that recapitulate the epithelial and haematopoietic landscape of the wild-type thymus. Furthermore, the unique organisational ability inherent within the TEPOCs may be useful as an <i>in vitro</i> model of the processes governing thymus organisation.

No description available.

Codon-optimized recombinant HBD2 was expressed in <i>E.coli</i> cells as an insoluble mutated ketosteroid isomerase (KSI) fusion protein. Efficient cyanogens bromide chemical cleavage released a soluble HBD2 peptide that upon RP-HPLC purification resulted in a highly pure reduced peptide. Oxidation using the redox partners cysteine/cystine resulted in a properly folded, functionally active BD2 as assessed by both antimicrobial killing assay and chemotactic assay using CCR6 expressing human embryonic kidney (HEK) 293 cells. Subsequent purification of HBD1 and HBD2 single disulfide bond mutants revealed that this method could be used for the future production of other ß-defensins. Single disulfide mutant HBD2 peptides were analysed to investigate the effects that removing two disulfide bonds had on <i>ß</i>–defensins functions. It was clearly shown that although still retaining antimicrobial activity in an in vitro assay the concentration of single disulfide mutants needed to kill an equivalent number of <i>E.coli</i> colonies was higher as compared to wild-type HBD2. All three structurally constraining disulfide bonds are necessary for full antimicrobial activity and chemotactic ability. CCR6 was expressed in the HEK 293 cell line and expressed with an N-terminal FLAG tag and a C-terminal decahistidine tag. CCR6 expressed on the outer surface of these mammalian cells was solubilised in mild detergents, and its purification was optimized using various chromatographic techniques. Sufficient quantity was purified to analyze the receptors glycosylation state. A gel-shift western blot assay resulted in CCR6 being the third chemokine receptor in the family to be identified as N-glycosylated. N-terminal CCR6 truncation, resulting in removal of the first 27 amino acids, was expressed in the same cell line. Although western blot analysis revealed transfection had been successful, surface expression of the mutated receptor could not be detected and as such the N-terminus has been implicated as necessary for ell surface trafficking.

The stromal cells of the thymus provide the microenvironments necessary for T-cell maturation and repertoire selection. The thymic stroma consists of distinct subpopulations of epithelial and non-epithelial cells which regulate the development of thymocytes. This work evaluates the hypothesis that the mature epithelial subpopulations present in the adult thymus derive from a progenitor cell population in the thymic primordium marked by two monoclonal antibodies. MTS2O and MTS24. Analysis of cells in the embryonic day 12.5 (E12.5) thymic primordium showed that MTS2O and MTS24 were expressed by approximately 50% of epithelial cells and these cells did not express markers associated with terminally differentiated thymic epithelium Furthermore MTS2O and MTS24 were strongly down regulated during thymus ontogeny, and in the adult, reacted only with rare medullary epithelial cells. The MTS20/24⁺ population purified from E12.5 thymi expressed <i>whn, Pax1, </i> <i>Pax9 </i>and <i>Hoxa3, </i>transcription factors required during early thymus development. To assess the functional potential of MTS20/24⁺ cells, purified E12.5 thymus cell populations were grafted under the kidney capsule of <i>nude </i>mice. Immunohistochemical analysis of MTS2O/24⁺cell grafts demonstrated the presence of mature cortical and medullary thymic epithelium. These grafts were also capable of attracting lymphoid progenitors and of supporting thymocyte differentiation. Furthermore, mature single positive T-cells were present in the periphery of <i>nude </i>mice grafted with MTS20/24⁺ cells indicating that these cells can confer thymus function to athymic recipients. The MTS2O/24^- cells from E12.5 thymi could fulfil none of these functions. These data demonstrate that the MTS2O/24⁺ population from the thymic primordium contains progenitor cells that can give rise to the major epithelial populations and generate a functional thymic microenvironment.

The current literature describes two conflicting hypotheses regarding the embryonic origin of the TE. The first aim of this thesis was to clarify the role of the pharyngeal ectoderm in thymus organogenesis using two independent experimental approaches. Firstly, direct assessment via a lineage analysis found no evidence for physical ectodermal contribution to the TE. Secondly, an ectopic grafting model indicated that pharyngeal endoderm was sufficient to form a normal thymus. The molecular mechanisms underlying thymus development were investigated via <i>in situ</i> hybridisation of candidate genes. These studies demonstrated i) <i>Bmp2</i> and <i>Bmp4</i> expression at the ventral tip of the third pharyngeal pouch, suggesting a role in the initiation of TE morphogenesis ii) <i>Foxn1 </i>and <i>Gcm2 </i>in specific regions within the common primordium, indicating delineation of thymus and parathyroid domains prior to organ formation, iii) restricted expression of <i>Ehox, </i>a novel paired-like homeobox-containing gene, in the pharyngeal endoderm prior to the onset of <i>Foxn1 </i>expression, suggesting it may identify prospective thymic epithelial cells. To test this, a technique for the rapid assay of gene function during thymus organogenesis was established, which utilised a novel whole embryo culture system in conjunction with <i>in vivo </i>electroporation of morpholino oligonucleotides. Initial proof-of-concept experiments using this technique achieved successful knockdown of <i>Fox1 </i>gene function in cells of the third pharyngeal pouch. In conclusion, the work presented here provides definitive evidence for a single endodermal origin for the thymic epithelium and demonstrates the existence of predetermined organ domains within the third pharyngeal pouch. It also establishes a novel technique for further investigation of the molecular mechanisms underlying early thymus organogenesis.

How memory cells form and how they survive are pertinent, unanswered questions in the field of immunology. T cell memory develops following an adaptive immune response, which is initiated in secondary lymphoid organs. This response involves T cell proliferation, effector cell differentiation and the generation of memory cells. It has proved difficult to study CD4 T cell responses due to the problems of tracking the small number of antigen specific T cells. MHC class II tetramers, which bind to the T cell receptor of responding T cells, enable the identification of antigen specific CD4 T cells and, therefore, can be used to track memory cells. The importance of this system lies in the physiological number of precursor cells, providing a more realistic system to study CD4 T cell immunology than T cell receptor transgenic studies. In this PhD, MHC class II tetramers have been used to track endogenously generated, antigen specific CD4 T cells <i>ex vivo</i>. This has enabled the tracking of a CD4 T cell response from early activation, into memory, and during recall responses. Long-term studies have been carried out to determine the survival of memory cells in both the absence and presence of antigen, and to describe the phenotype of these cells. Furthermore, this system has been used to answer questions about the requirements of both naïve and memory cells for the costimulatory signals necessary for activation and proliferation. Specifically, the requirements for the interactions between the costimulatory molecules, CD40L-CD40 and ICOS-B7h, have been examined by comparing T cell priming, memory cell generation and recall responses in wild-type and knockout mice.