The non-obese diabetic (NOD) mouse is widely used model Type 1 diabetes (T1D), a chronic inflammatory disease characterized by destruction of the insulin producing β cells in the islets of Langerhans by immune cells. The classical symptoms include increased glucose levels in urine and blood, frequent urination and enhanced thirst. The disease has a strong genetic component and is also influenced by the environment. NOD mice develop T1D spontaneously. The disease occurs in two phases; insulitis - the infiltration of immune cells in the islets of Langerhans and overt diabetes caused by the destruction of insulin producing β cells. Several disease associated gene regions or loci [termed insulin dependent diabetes (Idd) loci] have been associated with T1D development. Although, T1D is recognized as a T cell mediated disease in both mouse and man, many studies have shown the importance of B cells in the pathogenesis of the disease. Autoantibodies appear prior to islet infiltration and several molecular and cellular events precede this beta-cell autoimmunity. Although the pathogenesis of T1D is well characterized, less is known about the environmental and immunological factors that trigger the disease. In this thesis, we studied the contribution of B cell anomalies to the skewed immune response observed in the NOD mouse. In our studies covered in the thesis we observed that NOD mice display enhanced IgE in the serum already at one week of age. In addition, upon treatment of pre-diabetic NOD mice with anti-IgE antibodies, diabetes incidence was delayed. We hypothesize that the presence of IgE in the system may be explained due to enhanced class switching. Antibody feedback however, is an essential component of the immune response and can lead to either enhanced or dampened responses. Thus, increased IgE may provide positive feedback that might sustain an immune response. We also aimed to analyze the biological consequence of this feature. In vitro stimulation of B cells by the TACI ligand APRIL resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. In addition, TACI+ cells were observed in NOD germinal centers facilitating increased BAFF uptake and subsequent escape of low affinity antibody producing clones. NOD mice elicited an enhanced and prolonged immune response towards T-dependent antigens such as hen-egg lysozyme (HEL). Serum HEL-specific IgG level was significantly increased and was predominantly of the IgG1 isotype. Immunofluorescence analysis of NOD spleen revealed the presence of spontaneous germinal centers which others have perceived to provide a ready niche for the entry of naïve B cells that encountered novel antigen. Adoptive transfer experiments of purified B and T cells from NOD into NOD.Rag2-/- (NOD-RAG) mice illustrated the importance of B cell intrinsic defects in the reproduction of the original phenotype as observed in NOD.

Thesis advisor: Thomas C. Chiles / For ten weeks during the summer of 2005, I was a Summer Undergraduate Research Fellow in an immunology laboratory at the Mayo Clinic. My research focused on the BLyS/APRIL system and the receptor TACI on the surface of B cells. Going into my summer research, I had very little experience in immunology. Throughout the process of writing this thesis, I have sought to improve upon my knowledge of immunology, building a cohesive story that begins with basic biology and ends with the results of the summer's experiments. The first part of this thesis covers topics in general immunology, and narrow down in focus to cover the function and development of lymphocytes and B cell maturation and activation. In the second part of the thesis, the background for my research is described in more detail, and topics such as autoimmunity and cancer, lipid rafts, cell polarization, the BLyS/APRIL system for B cell survival, and TACI are covered extensively. The final portion of this thesis discusses the experimental logic, a background on materials and methods, and the results of the experiments I conducted over the summer. By reading this thesis, anyone with a background in biology should become familiar with basic subjects in immunology, advanced concepts in the study of lymphocytes, the ligands BLyS and APRIL, and the receptor TACI in B cells. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program.

The T cell function of a proliferation inducing ligand (APRIL or TNFSF13) remains unclear. By comparing APRIL-/- mice with wild type (WT) mice, we have investigated the roles of APRIL in Th1, Th2 and Th17 responses. With regard to APRIL in Th1 responses, cultured APRIL-/- CD4+ T cells showed increased IFN-gamma production under non-polarizing, but not under Th1 polarizing, conditions. No difference in antigen-specific IgG2a levels existed between APRIL-/- and WT mice immunized with ovalbumin (OVA) and complete Freund's adjuvant (CFA) which induces Th1 polarization. Our data indicate that APRIL represses Th1 responses only under non-polarizing conditions. As for APRIL in Th2 responses, cultured APRIL-/- CD4+ T cells exhibited enhanced Th2 cytokine production under non-polarizing conditions, and augmented IL-13 production under Th2 polarizing conditions. Upon immunization with OVA and aluminum potassium sulfate (alum) which induces Th2 polarization, APRIL-/- mice responded with an increased antigen-specific IgG1 response. In the OVA-induced allergic lung inflammation model which is mediated by Th2 responses, APRIL-/- mice had significantly aggravated allergic lung inflammation. Accordingly, a decoy receptor-Ig fusion protein, TACI-Ig, treatment to block APRIL in WT mice enhanced allergic lung inflammation. In agreement with the role of APRIL in CD4+ T cells, the transfer of APRIL sufficient, OVA-specific, TCR transgenic CD4+ T (OT-II) cells to APRIL-/- mice restored the suppressive effect of APRIL on allergic lung inflammation. Mechanistically, the expression of c-maf, the IL-4 gene transcription factor, was markedly enhanced in APRIL-/- CD4+ T cells under non-polarizing and Th2 polarizing conditions. Our data suggest that APRIL inhibits Th2 responses and allergic lung inflammation by suppressing IL-4 production in CD4+ T cells via diminished c-maf expression, and by suppressing IL-13 production in CD4+ T cells via an IL-4 independent, IL-13 specific pathway. Regarding APRIL in Th17 responses, the incidence of Th17-mediated collagen-induced arthritis (CIA) in APRIL-/- mice was reduced, in parallel with diminished levels of antigen-specific IgG2a autoantibody and IL-17 production. Our data indicate that APRIL promotes IL-17 production, and that APRIL-triggered signals contribute to arthritis. Our data clearly show that APRIL is important in T cell immunity, inhibitory in Th2 responses and costimulatory in Th17 responses.

Type 1 diabetes (T1D) is a chronic autoimmune disease in which the insulin producing β-cells in the pancreatic islets of Langerhans are selectively attacked by the immune system. The β-cells are destroyed resulting in a reduced or eliminated insulin production, which in turn lead to a high blood glucose level. The non-obese diabetic (NOD) mouse is the most commonly used animal model for human T1D. NOD mice develop diabetes spontaneously through a process that closely resembles the human pathogenesis. In both humans and the NOD mouse, disease is caused by a combination of genetic and environmental factors. In the NOD mouse, more than 30 insulin-dependent diabetes (Idd) loci on 15 chromosomes have been linked to disease susceptibility, however, most of the Idd-regions lack identification of a disease associated gene. B cells are required for T1D development, although the underlying mechanisms are not fully revealed. The aim of this thesis was to dissect B cell-related immune deviations in the NOD mouse, including the underlying genetics of these traits. The TACI receptor binds two ligands, i.e. the cytokines BAFF and APRIL.TACI ligation by APRIL mediates class switch, drives plasma cell differentiation and increases immunoglobulin production. In Paper I, a novel NOD-specific B cell-related trait was identified, i.e. the increased percentage of TACIhigh-expressing splenic B cells, by comparing NOD mice with non-autoimmune disease prone C57BL/6 mice. To investigate if the described TACI trait was controlled by genes linked to any Idd-region, an Idd-focused linkage analysis was performed. The TACI-trait mapped to regions on chromosome 1 and 8, more specifically to the vicinity of the Idd5.4 and Idd22. Interestingly, the linkage to Idd22 was explained by mice ≥61 days of age, suggesting a temporal genetic regulation of TACI expression possibly influenced by the ongoing autoimmune process. In Paper II, the linkage of the TACI trait to chromosome 1 and 8 was confirmed by analyzing the percentage of TACIhigh-expressing B cells in congenic NOD.C1/Idd22 mice. Moreover, the functional consequence of TACI upregulation was investigated, with the focus on plasma cell development and immunoglobulin production. NOD splenic B cells stimulated with APRIL displayed increased numbers of plasma cells and produced higher amounts of IgG and IgA compared to B cells from C57BL/6 mice. Thus, the TACI upregulation on NOD B cells possibly contribute to a B cell compartment which is more disposed to plasma cell differentiation and isotype switch. NOD mice display enhanced and prolonged immune response towards several antigens, including non-self immunoglobulins. In Paper III, the genetic factor(s) controlling the altered immune response against a BALB/c derived monoclonal antibody were dissected. Significant linkage to the Idd1/Idd24, Idd12, and Idd18.1 regions as well as to a proximal region on chromosome 2 (33.5 Mb) was detected. The linkage to Idd1/24 was verified by analyzing a set of H2-congenic NOD and C57BL/6 mice, and the linked region was narrowed down to ~8 Mb. Candidate gene analysis revealed a significant difference in the transcription of the H2-O/DO molecule. This suggests that multiple mechanisms contribute to the loss of immune response control, including an altered MHC class II peptide loading on NOD B cells. In Paper IV, a novel B cell intrinsic receptor for IgM and IgG was revealed. The receptor appeared to be more abundant in NOD mice compared to C57BL/6 mice, as the level of extramembranous IgG monomers and IgM pentamers on peripheral blood B cells from NOD mice was significantly higher compared to C57BL/6 mice. In addition, analysis of immune complex binding using IgG- or IgM-opsonized bacterial particles revealed a higher degree of binding in NOD mice compared with C57BL/6 mice. The enhanced capture of immunoglobulins and immune complexes could thus contribute to the development of T1D by altering normal B cell functions such as activation and immune complex transportation.

The idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of chronic muscle diseases with frequent extramuscular organ involvement that contributes to serious prognosis. The presence of autoantibodies and composition of muscle infiltrates both support autoimmune nature of the disease and pathogenic role of B lymphocytes. Besides the traditional diagnostic subgroups, autoantibody characterised phenotype subsets have been identified with presumed similar pathogenic mechanisms. The best known is the antisynthetase syndrome which is characterised by presence of myositis, antisynthetase autoantibodies (with anti-Jo-1 being the most frequent), interstitial lung disease and other extramuscular manifestations. BAFF (B cell-Activating Factor of the TNF Family) is a key factor in B cell homeostasis modulation. In high levels, it allows survival of autoreactive B cell clones and thus participates in the pathogenesis of autoimmune diseases. Its expression is induced by type I interferons (IFN-1). The aim of the PhD thesis was to explore the role of BAFF in pathogenesis of IIMs by analysis of its serum levels, the receptors for BAFF in muscle tissue, their associations to IFN-1 and expression of BAFF gene mRNA transcription variants in peripheral blood cells. Further aspect was to study a possible...