In this thesis a number of features of hybridoma cells have been investigated experimentally to obtain a better understanding of their growth and antibody secretion <I>in vitro</I>, with a view to improving the efficiency of antibody production. Initially the effect of immune derived mediators on the growth and secretion of hybridomas was explored as some of these mediators have been shown to play an important role in the growth and antibody secretion of B-cells <I>in vivo</I>. In particular interleukin-2 and interleukin-6 were studied and were shown, in some cases, to improve the rate of cell growth, but they had little or no effect on the level of antibody secretion. Using flow cytometry, the expression of antibody on the surface of a hybridoma cell was investigated. The presence of antibody on the cell surface was correlated with overall antibody secretion and to the stage of the cell cycle. It was shown that antibody that is in the process of being secreted from the cell can be detected and that it is related not only to the overall secretion of antibody but also to the stage of the cell cycle. Attempts to synchronize hybridoma cells in one phase of the cell cycle by well documented chemical means were unsuccessful, and a technique was developed to isolate a synchronous population using flow cytometry. A synchronous cell population was cultured and samples were analysed for cell surface antibody, stage of the cell cycle and antibody secretion at various points in the cell cycle.

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T-lymphocytes are an essential component of the adaptive immune system and require a distinct anatomical location for their development, namely the thymus. This study focuses on early events during development of these cells in the adult mouse. The most immature compartment of murine thymocytes is subdivided by the cell surface expression of CD44 and CD25. The most primitive progenitor population resides in the CD44+CD25-double negative 1 (DN1) fraction and can be further subdivided according to expression of the receptor tyrosine kinase c-kit (CD 117). The DN1 CD117+ fraction is homogeneous, unlike the CD117-fraction, which constitutes a preponderant CD45R+CD127- population and a smaller CD45R-CD127+ population. Analysis of gene expression was employed to understand the relationship between the DN1 CD117+ (DN1 CD117) and the DN1 CD45R+ (DN1 CD45R) populations. Expression of genes associated with the T-cell lineage, such as Notch-1, Runx-1 and Rag-1 was observed in both populations. Furthermore, substantial expression levels of genes involved in T-cell commitment, such as pre-Ta, in the DN1 CD45R population could reflect the initiation of T-cell fate specification. In parallel, a Cre recombinase based lineage tracing approach was utilised to further dissect the earliest thymocyte pools. Upon expression of the Cre recombinase, all cells and their progeny are permanently marked by the expression of a fluorescent reporter protein. In this study, the human CD2 (hCD2) promoter and locus control regions drove expression of the Cre recombinase. Analysis of the DN1 population revealed that nearly all the DN1 CD117 fraction was reporter negative, whereas the DN1 CD45R subset was completely reporter positive. Since expression of hCD2 has so far been reported only in the lymphoid lineage, this observation further underlines the fundamental differences between the two progenitor fractions and indicates their alternate developmental origins. To address the developmental potential of these populations, the DN1 CD117 and the DN1 CD45R pools were functionally assessed in vitro and in vivo. The DN1 CD117 population showed a robust T-, B- and NK-cell potential in vitro and in vivo, in addition to a residual myeloid activity in vivo, whereas the DN1 CD45R fraction was strongly biased towards a CD8 NK-T cell lineage in vivo. In summary, these data indicate that developmentally distinct progenitor populations seed the adult murine thymus and can potentially contribute to discrete subsets of mature haematopoietic cells.

Dendritic Cells (DC) are a group of professional antigen presenting cells that act as sentinels for the body's defense system against microbial pathogens. DC respond to local signals from invading pathogens, by migrating to lymph nodes or spleen, and there activating T-cell dependent antigen-specific immune responses. Both quantitative and qualitative parameters of the immune response depend critically on signals given out by DC. These signals, in turn, depend on complex signal transduction pathways triggered in the DC by exposure to different types of microbes. These pathways are engaged by pattern recognition receptors such as Toll-like receptors (TLRs) and various sugar-binding receptors. The integration of DC signalling pathways, and how this controls the eventual type of T cell response remains a central question of immunology. The thesis focuses on the characterisation of the MUTZ-3 cell line as a DC model. The MUTZ-3 acute myeloid leukaemia cell line has previously been proposed as a useful in vitro model for DC. The cell line is not homogenous and contains a mixture of different differentiation stages. The first results chapter focuses on optimisation of DC differentiation and purification in this model, as well as the activation using different TLRs. The second focuses on the analysis and validation of base line array data comparing the MUTZ-3 cells to DC. Gene expression analysis revealed differences between M3DC and MDDC, amounting to over 5% of the transcriptome. The differentially expressed genes were enriched with functional cluster representing cellular responses to external stimuli. The role of mixed linage kinases (MLK) in DC signalling forms the final results chapter. It has been observed that a small molecular weight inhibitor of MLK blocks DC maturation in response to some ligands but not others. MLK may therefore be an interesting surrogate for DC activation. This chapter investigates the role of MLKs in MUTZ-3 using protein inhibitors, RNAi and dominant negative mutants.

The work presented in this thesis focusses on the sequence and structural analysis of antibodies and has fallen into three main areas. First I developed a method to assess how typical an antibody sequence is of the expressed human antibody repertoire. My hypothesis was that the more \humanlike" an antibody sequence is (in other words how typical it is of the expressed human repertoire), the less likely it is to elicit an immune response when used in vivo in humans. In practice, I found that, while the most and least-human sequences generated the lowest and highest anti-antibody reponses in the small available dataset, there was little correlation in between these extremes. Second, I examined the distribution of the packing angles between VH and VL domains of antibodies and whether residues in the interface in uence the packing angle angle. This is an important factor which has essentially been ignored in modelling antibody structures since the packing angle can have a signicant eect on the topography of the combining site. Finding out which interface residues have the greatest in uence is also important in protocols for `humanizing' mouse antibodies to make them more suitable for use in therapy in humans. Third, I developed a method to apply standard Kabat or Chothia numbering schemes to an antibody sequence automatically. In brief, the method uses proles to identify the ends of the framework regions and then lls in the numbers for each section. Benchmarking the performance of this algorithm against annotations in the Kabat database highlighted several errors in the manual annotations in the Kabat database. Based on structural analysis of insertions and deletions in the framework regions of antibodies, I have extended the Chothia numbering scheme to identify the structurally correct positions of insertions and deletions in the framework regions.

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