Potential biological roles for two novel human glycan-binding proteins have been investigated. In the first part of the work, analysis of the scavenger receptor C-type lectin (SRCL) leads to the proposal that the primary function of this receptor is to clear glycoproteins released from neutrophils. The expression of SRCL was characterized by screening of cDNA libraries and by immunostaining of tissues. These results confirm and extend earlier reports that SRCL is found in a range of tissues and is expressed by cultured umbilical vein endothelial cells, and demonstrated that SRCL is localized primarily in endothelial cells. SRCL is thus positioned to interact with circulating glycoproteins and cells. Potential ligands were isolated from neutrophils by affinity chromatography and shown by mass spectrometry to comprise lactoferrin and other soluble granule proteins. The interaction between SRCL and lactoferrin was found to be dependent on fucose residues and analysis of lactoferrin N-glycans by mass spectrometry revealed multiple Lewisx epitopes borne on unsialylated triantennary glycans, consistent with selective binding of SRCL to the Lewisx trisaccharide. Transfected cells expressing SRCL were shown to internalize fluorescein-labelled lactoferrin. Taken together, these results provide support for the hypothesis that soluble glycoproteins in neutrophil secondary granules are tagged with the Lewisx epitope to direct their clearance by SRCL after release at sites of inflammation. Prolectin, a novel C-type lectin, was identified by screening of the human genome. Screening of cDNA libraries and immunostaining were used to ascertain that prolectin expression is restricted to dividing B cells in and around germinal centres of lymphoid tissues. This expression pattern, together with the presence of motifs in the cytoplasmic tail of prolectin that may interact with intracellular signalling molecules, leads to the suggestion that prolectin may have a role in the migration of B cells during the adaptive immune response.

Syndecan-4 (SDC-4), a transmembrane heparan sulphate proteoglycan and a co-receptor of integrins for fibronectin, has been reported to modulate the adhesion of cells to a range of extracellular matrix ligands. In addition to the modulation of integrins, SDC-4 has been recently reported to modify the interaction of some growth factors with their receptors. T cells are essential effectors of the adaptive immune response. When conjugating with antigen-presenting cells (APCs), T cells transform their shape to enable formation of a specialised morphological structure, called the immunological synapse (IS). The IS formation is associated with the polarisation of signalling and adhesive molecules towards the APC. The IS is formed and stabilized by similar adhesive forces and structures as in the motile fibroblasts - an extensively studied model of syndecan function. In this thesis I have investigated the role of SDC-4 in T cell - APC recognition and the IS. First, I confirmed the tight regulation of SDC-4 expression in T cells during the process of activation. Flow cytometry and PCR data demonstrate increased presence of SDC-4 in stimulated human T cells compared to their resting counterparts, indicating involvement of SDC-4 in the processes of T cell activation. Transient transfection of exogenous SDC-4 into Jurkat T cells with low endogenous SDC-4 expression was used as a model to study the effect of increased SDC-4 expression on T cell function. Using live cell imaging and advanced data image analysis I have quantitatively demonstrated that SDC-4 transfected Jurkat T cells are less likely to modify their shape during IS formation when compared to mock transfected Jurkat T cells. I also observed a delay in T cell antigenic response caused by SDC-4 over-expression. Moreover, I was able to visualise the exogenous SDC-4 in the IS using confocal microscopy and demonstrate the independence of this phenomenon on T cell receptor. In summary, my observations indicate a regulatory role for SDC-4 in T cell adhesion causing delays in the IS formation and reduced transformation of T cell shape during conjugate formation.

Allelic exclusion coupled to lineage and stage specific regulation ensure the Igk locus undergoes RAG mediated V-J recombination only at the small pre-BII stage of lymphocyte development. These mechanisms also ensure the final antigen receptor is monospecific and allow self-specific receptors to be recognised and altered at later stages of development. Allelic exclusion is controlled by mechanism involving either epigenetic based allele selection early in development or probabilistic activation of a single allele at the small pre-BII stage. Current models favour the probabilistic model based on an elegant GFP reporter system. We present a reappraisal of this model based on an absence of the originally detected probabilistic activation. We find the absence to be explained in part by an aberrant splice event generated by developmentally regulated Igk germline promoter usage. The activity of the promoter was investigated using in-vitro models of critical events during the B cell development but their role remains elusive. Intense investigation for the last 20 years has determined that chromatic regulation underpins the stage and lineage specification of rearrangement. The protein factors responsible for this regulation remain unknown. Using transgene reporters of cis-acting sequences and in-vitro model systems of B cell development, we find an involvement for members of the zinc finger family of Ikaros transcription factors, IRF4 and LEF1. We determined a bipartite role for Ikaros whereby its activity could both suppress rearrangement prior to pre-BCR signalling and promote rearrangement thereafter, possibly through an IRF4 based mechanism. Finally we present evidence for a role for Ikaros protein in later stages of B cell development. We find modulation of Ikaros activity directly influences the ability of cells to differentiate to the plasma cell fate. We used a transgenic model to allow genome-wide mapping of the transcriptional events regulated by Ikaros and find multiple small modulations of factors and pathways.

Natural Killer (NK) cells are lymphocytes of the innate immune system. However, there is increasing evidence that they can also play important roles in the adaptive immune system; as initiators, through antigen presentation; as effectors, via early release of IFN-y; and as immunoregulators, by eliminating over-activated macrophages. The functions of NK cells in these roles are intimately linked to their interactions with other cells during an immune response, for example recognition of target cells via activating receptors. The activating receptor NKG2D recognises proteins that are not normally expressed at the surface of most cells but are up-regulated during a cellular ‘stress’ response. However, NKG2D ligands can also be induced on human macrophages by TLR stimulation, leading to NK cell-mediated lysis. Here, I clarify that ligation of TLR4 preferentially up-regulates MICA but not MICB, TLR7/8 ligation up-regulated both MICA and MICB, while ligating TLR3, signalling through a MyD88-independent pathway, up-regulated neither. TLR4 stimulation decreased expression of microRNAs, miR-17-5, miR- 20a and miR-93, which target MICA, implicating a novel role for microRNAs in post-transcriptional regulation of NKG2D ligand expression. Moreover, the pathway involved IL-12/TNF- a-mediated autocrine signalling, thus incorporating an intrinsic mechanism for NK cell-mediated elimination of particularly activated macrophages. In addition to this immunoregulatory role, NK cell activity can shape a subsequent adaptive immune response. Subsets of NK cells can have distinct functions. Here, I demonstrate that following culture with IL-2, >25% of human peripheral blood NK cells express HLA-DR, due to an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLADR is upregulated on previously negative cells. HLA-DR-expressing NK cells exhibited enhanced degranulation to susceptible target cells and expression of the chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggestive of an immunological role, stimulation of PBMCs with Mycobacterium bovis BCG triggered dramatic expansion of HLADR- expressing NK cells. In addition, the magnitude of the NK cell IFN-y secretion response in PBMC triggered by BCG was associated with the proportion of HLA-DR-expressing NK cells ex vivo. A direct contribution to the immune response was determined by specifically enriching the HLA-DR-expressing NK cell compartment, which substantially augmented the total NK cell IFN-y secretion response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in peripheral blood but readily expanded by IL-2, that can play a significant role during immune responses.

The role of coagulation proteins (CPs) in systems other than haemostasis is now recognised. Many of these cellular effects are through protease activated receptors (PARs). This project investigates how CPs influence the adaptive immune response, firstly through the expression of tissue factor (TF) on DCs; secondly through the action of PARs on dendritic cells (DCs) and T cells; and thirdly by examining the direct effect of anti-thrombin (AT) on DCs. This work identified for the first time a subset of mouse DCs that expresses TF. The form of TF changed from cryptic to pro-coagulant as DCs matured. In addition it was found that blocking TF on immature but not mature DCs enhanced their stimulatory capacity, possibly through PAR-2 signalling. In vivo studies supported this finding and suggest that inhibiting TF breaks T cell tolerance. Thrombin enhanced the T cell response through an effect on DCs but not T cells. Both primary and secondary responses increased but there was no change when stimulated T cells were rechallenged with thrombin-incubated DCs. This mechanism was not through changes in MHCII, co-stimulatory molecules or cytokine production. Although DCs expressed PAR, individual PAR-1 or PAR-4 activation did not affect DC stimulatory capacity. Anti-thrombin was found to reduce T cell activation in vivo. When DCs were treated with AT alone there was no change in T cell response, whereas treatment with AT before LPS led to an increase in IL-4 and IL-10 from T cells suggesting induction of tolerance. Anergy and T cell suppressor assays, however, failed to demonstrate a tolerant phenotype. Overall the findings demonstrate that DCs have the ability to generate and respond to CPs and thus influence the T cell response. This work identifies potential new targets in the innate system which may be used to influence the adaptive immune response.

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