Doctor of Philosophy

dissertationAutoreactive CD4+ T cells initiate the autoimmune disease Type-1 diabetes (T1D). Expression of DQ2, DQ8, and DQ2/8 trans-dimers are highly prevalent in T1D. However, the underlying molecular mechanisms are poorly understood. HLA-DM is essential for editing peptides bound to Major Histocompatibility Complex class II (MHCII) on antigen presenting cells, thus influencing the repertoire of peptides mediating selection and activation of CD4+ T cells. Here we explore the structural characteristics of DQ and the role of DM function, as well as the potential molecular mechanism of DM editing, as they may contribute to an understanding of autoreactive CD4+ T cell development in T1D. Cells coexpressing DM with these DQ molecules were observed to express elevated levels of class II-associated invariant chain peptides (CLIP), consistent with HPLC-MS/MS analysis of eluted peptides. Assays with purified recombinant soluble proteins further confirmed that T1D-associated DQ2 and DQ8 are resistant to DM editing. DM sensitivity was enhanced in mutant DQ8 with disruption of hydrogen bonds (H-bonds) that stabilize DQ8 near the DM-binding region. Compared to T1D nonassociated DQ1 and DQ6, the percentages of shared peptides among T1D-associated DQ molecules were significantly higher. Predicted peptide binding motifs of T1D-associated DQ molecules shared charged anchor residues, iv while hydrophobic anchors were present in DQ6 peptides. Competition binding assays and peptide dissociation rate measurements indicated that peptide with high affinity is necessary but not sufficient for DM editing resistance. DM editing dominates the stability of MHCII/peptide complexes in the cellular environment. DM catalyzes peptide exchange in MHCII through a mechanism that has been proposed to involve the disruption of specific conserved H-bond in MHCII/peptide complexes. HLA-DR1 molecules with alanine substitutions at each of the six conserved H-bonding positions were expressed in cells, and susceptibility to DM editing was evaluated by measuring the release of CLIP. Our results support the conclusion that no individual component of the conserved H-bond network plays an essential role in the DM catalytic mechanism. Taken together, our data support that the relative DM resistance in T1Dassociated DQ molecules affects the peptide repertoires and preferential presentation of T1D-associated autoantigenic peptides may contribute to the pathogenesis of T1D

Processing and presentation of the rheumatoid arthritis candidate autoantigen aggrecan, by antigen-specific B cells

The proteoglycan aggrecan, which is a major structural component of cartilage, has been identified as a candidate autoantigen in rheumatoid arthritis (RA). This is principally due to its degradation early in the disease pathology, its ability to induce an RA-like disease in mouse models and the presence of elevated numbers of reactive T and B cells in RA patients. Studies have also defined an essential requirement for autoantigen-specific B cells as antigen presenting cells (APC) in RA although the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by B cells remains unknown. To investigate the role of autoreactive B cells as APC in RA, I have used two complimentary approaches to generate B cells expressing an aggrecan-specific B cell receptor (BCR). The first was based on a modified monoclonal antibody production protocol and the second involved the transfection of B lymphoma cells with newly generated plasmids encoding an aggrecan-specific BCR. Using the second approach, I have successfully generated aggrecan-specific B cell lines (C71-4C5 and C71-5F10). I have shown that these B cells specifically bind aggrecan leading to efficient processing and the generation of the immunogenic T cell epitope 84-103 that is recognised by both aggrecan-specific T cell hybridomas and CD4+ T cells isolated from an aggrecan-specific TCR transgenic mouse. The aggrecan-specific B cells are able to present aggrecan at least 104 fold more efficiently than non-specific B cells, 102 fold more efficiently than macrophages and comparable to that seen by dendritic cells. By using a panel of inhibitors, I have also shown that the generation of the 84-103/MHC complex by aggrecan-specific B cells requires an acidic environment, proteolysis by aspartic, serine and metalloproteinases and the “classical” pathway of MHC class II biosynthesis. During this PhD, I have highlighted a novel role for aggrecan-specific B cells as important APC involved in aggrecan-presentation, as well as elucidating a role for metalloproteinases in aggrecan processing and presentation by APC.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Immunogenetic analysis of HLA Class II in premalignant disease of the cervix and correlation with HPV status

The human papilloma virus (HPY) infection has a causal association with cervical intraepithelial neoplasia (CIN) and cervical cancer. However, pre-malignant or malignant transformation is not always observed with HPY infection. lILA molecules are important in the regulation of the immune response to foreign antigens. The role of genetic variation at the HLA class II loci (DR and DQ) in CIN was investigated in 176 British Caucasian patients and 420 controls (normal cervical cytology and negative for HPY 16, 18, 31 and 33). HLA DQB 1 *03 typing was performed by a novel polymerase chain reactionrestriction fragment length polymorphism method (A-RFLP). The technique uses PCR to mutate the first base of codon 40 (DQ alleles) from T to G to create an artificial restriction site for an enzyme, MluI, which distinguishes DQB 1 *03 from other alleles and is confirmed by digestion of amplified DNA with MluI. Further HLA DR-DQ typing was performed by PCR DNA amplification and oligonucleotide probe typing. HPY types (16, 18, 31 & 33) were detected by using type-specific oligonucleotide primers and PCR. The alleles of the DQB 1 *03, DRB 1 *04 and DRB 1 * 11 groups were strongly associated with susceptibility to CIN. Specifically the haplotypes DRB 1 *040 I-DQB 1 *0301 and DRBl*1101-DQB1*0301 were significant and indicated susceptibility. The DQBl*03 locus was more contributory to this association than the DRB 1 loci. A weak protective effect was shown for the haplotype DRB 1 *0 10 I-DQB 1 *0501. Positive correlation was also observed for HPY-positive CIN, suggesting that specific HLA alleles may be important in determining the immune response to HPY antigens and the risk for CIN after HPY infection. Immunoaffinity purification of the susceptibility and protective HLA ~ molecules was performed and the naturally processed peptides were eluted and sequenced by Edman degradation. The data obtained was used for motif prediction of HPY 16 E6, E7, Ll and L2 sequences that may be capable of binding to these HLA molecules. Motif prediction as well as the binding affinity of predicted peptide motifs for HLA D RB 1 *0401 and DRB 1 *0 10 1 was accomplished using the published data' on the naturally bound peptide sequences bound to these HLA molecules. The results revealed significant differences in both the number and binding affinity of the HPV 16 derived peptides to the protective and susceptibility HLA molecules. These results should help in the rational design of vaccines against HPV