Toll-like receptors (TLRs) are a group of trans-membrane proteins playing important roles as pattern-recognition receptors (PRRs) of innate immunity that are mainly expressed on professional antigen presenting cells (APCs). Stimulation of APC with TLR ligands, a variety of Jnolecular components derived from microorganisms, triggers innate immunity and establishes adaptive immune responses. I studied the impact of TLR specific for bacterial components on antigen processing and presentation of protective antigen (PA) of B. anthracis by bone marrow macrophages from mice.

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Although studies using B cell deficient mice have been useful in understanding the importance of B cells under different conditions, it is difficult to then dissect exactly how B cells could be regulating T cell responses. By transferring OT-II transgenic T cells into either B cell deficient (μMT) or C57BL/6 mice, expansion and contraction of T cells can be tracked <i>ex vivo. </i>Expansion of OT-II cells is reduced in μMT mice compared to C57BL/6 mice. Thus, B cells can provide costimulatory signals, secrete cytokines and influence the lymphoid microarchitecture. To dissect which B cell factor(s) are involved in enhancing OT-II T cell expansion, a model system was used where one molecule on the B cells is depleted at one time. This was achieved by creating bone-marrow chimeras using a combination of μMT bone-marrow and wildtype or deficient bone-marrow. Thus, all the B cells are either wildtype or deficient for a particular molecule. The molecules examined were MHC-II, which is required for antigen presentation, CD40, due to its costimulatory role, and lymphotoxin-alpha, for its role in maintenance of splenic architecture. Using the OT-II adoptive transfer system, we have shown a requirement for MHC-II but not CD40 on B cells for efficient T cell expansion. In light of these observations, the role of B cell-derived MHC-II for T cell memory generation was examined. To do this, I used MHC-II tetramers to track a polyclonal population of T cells in the host.  Using this technique, I have shown that T cell memory is also diminished when the B cells do not express MHC-II. Thus, a cognate interaction with B cells is required for both efficient expansion and memory generation of CD4⁺ T cells.

This thesis describes a method using an object-oriented database, P/FDM, to build 3D homology models of fish β-trefoil cytokines such as Interleukin-1 (IL-1), by exploiting existing structural data from known structures of the same fold.  This β-trefoil modelling procedure is based on the powerful querying capabilities of P/FDM that allow data access, navigation, and computation to be mixed freely and easily in order to formulate complex structural queries.  The structural part of the database contains objects for the description of proteins, as well as β-trefoil structure-specific entities.  Data are organised according to a global β-trefoil structural alignment based on superposition of the 18 core residues of the fold.  Using the P/FDM β-trefoil database, the method allows the “cutting-and-pasting” of fragments from known β-trefoil structures to model loops and to use torsion angles from structurally equivalent trefoil positions to guide side chain placement. The β-trefoil modelling method was applied to model trout (<i>Oncorhynchus mykiss</i>) IL-1β1, IL-1β2, IL-1FX, IL-1RA, IL-18 and catshark (<i>Scyliorhinus canicula</i>) IL-1β cytokines, while protein models for trout IL-1R1, TGFβ1 and TNFα1 and TNFα2 were generated using Modeller.  Superposition of the modelled trout IL-1 and receptor molecules to the human homologue complexes allowed their direct comparison and revealed several regions of ligand-receptor interactions.  Similar to the human complex, trout IL-1RA forms more extensive interactions with receptor binding site A than site B, in contrast to IL-1β which interacts with both sites.  Although there is a high level of variability in regions involved in receptor binding between fish and human homologues, the mode of binding and overall shape of the ligand-receptor complex is probably maintained, suggesting that each species has evolved its own unique interleukin-1 signalling system through ligand-receptor co-evolution.

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As part of a study investigating fundamental cellular immunology in the sheep, this thesis describes the characterisation and expression of the genes of the sheep MHC class II region. Cosmid libraries prepared from DNA from three unrelated sheep were screened with probes from the <I>DP, DQ</I> and <I>DR</I> sub-regions of the human and mouse MHC class II regions. Cosmids were used because they facilitate the cloning of relatively large genomic inserts. Restriction maps of the cosmids have been produced showing that some of the clones overlapped. The MHC <I>A</I> and <I>B</I> genes within the clones have been sequenced and assigned to a specific sub-type. Functional genes have been identified by the reaction of their products with anti-sheep class II monoclonal antibodies following DNA-mediated transfection into the mouse L cell, a fibroblast cell line which does not express endogenous mouse class II genes. Transcription of some of the genes has been demonstrated by Northern blots and reverse transcription polymerase chain reaction. A restriction map of the sheep class II <I>DQ</I> sub-region has been constructed and shown to contain two distinct <I>DQA</I> loci with associated <I>DQB</I> genes. The <I>DQ1</I> <I>A/B</I> gene pair was expressed in the mouse L cell. The sheep class II <I>DQ1</I> product at the cell surface reacted with a sub-set of the available anti-sheep class II monoclonal antibodies. The <I>DQ2</I> genes were transcribed and some evidence for their cell surface expression was obtained, although this was not formally proved.

Little is known about the importance of mycoplasmas in equine respiratory disease, and this project was undertaken to investigate the occurrence of mycoplasma antibody in horses. Two mycoplasmas, M. equirhinis and an unnamed species, N3, were selected from 13 strains recently isolated from the nasopharynx of horses. Immunization of rabbits and horses resulted in the development of specific antibody. Complement-fixation (CF) was found to be more sensitive than metabolic-inhibition and growth-inhibition for the detection of antibody in equine hyperimmune sera, and this method was used for the survey of antibody in other horses. Six ponies were chosen which were apparently free of mycoplasmas, without CF antibody to M. equirhinis and with low levels to N3. Two were inoculated intranasally with M. equirhinis, two with N3 and two remained as controls. N3 was not recovered from any pony, but within five days M. equirhinis was recovered from every animal, indicating that it had spread naturally from the inoculated ponies to the others. After six weeks it became difficult to isolate the organisms, and levels of CF antibody rose first in the nasal secretions and later in the sera. After several weeks antibody declined to indetectable levels. No clinical signs of infection were seen. In a survey of 817 sera taken over one year from 85 racehorses in seven stables, every horse was found to have CF antibody to M. equirhinis and N3 at some time during the sampling period. In five stables most horses had no M. equirhinis antibody when they entered the stable, but within two months they invariably developed significant titres, implying that they had been infected. When compared with a survey of 256 sera from 81 horses completed two years previously, it appeared that antibody to both mycoplasmas was more prevalent in the recent survey.