Classical immunoglobulin transgenic (Ig-Tg) mouse models such as the MD4 anti-hen egg lysozyme (-HEL) Ig-Tg line have been used extensively to study B cell responses in tolerance and immunity. This thesis describes a new generation of gene-targeted mice (designated SWHEL mice) whereby the VH10 Ig variable gene encoding the HyHEL-10 specificity of the original anti-HEL Ig-Tg mouse was targeted to the Ig heavy chain locus. B cells in the SWHEL mouse are therefore capable of undergoing class switch recombination (CSR) and somatic hypermutation (SHM), representing a major advance on the original MD4 mouse model. SWHEL mice were found to not only contain a large population of HEL-specific (HEL+) B cells but also a significant population of non-HEL-binding (HEL-) B cells generated by VH gene replacement. HEL+ SWHEL B cells were found to belong to the B2 lineage and displayed high levels of surface IgM. Nevertheless, they matured normally and colonised the primary B cell follicle and marginal zone (MZ) of the spleen. The SWHEL model thus provided an opportunity to re-examine some of the original observations made in the MD4 system and also to extend these observations, particularly with regard to the regulation of CSR by self-reactive B cells. As expected, analysis of SWHEL B cells exposed to high avidity membrane-bound HEL revealed that they underwent clonal deletion in the bone marrow (BM). More interestingly, analysis of HEL+ B cells exposed to low avidity soluble HEL revealed that they were able to emigrate from the BM to the spleen as anergic B cells. However, unlike anergic MD4 B cells, anergic SWHEL B cells were reduced in frequency, displayed an immature B cell phenotype, were excluded from the follicle and had a reduced lifespan. Direct measurement of B cell antigen receptor (BCR) occupancy by HEL and the frequency of HEL- competitor B cells was combined with mixed BM irradiation chimeras to demonstrate unequivocally that the difference in phenotype and fate of HEL+ B cells in the two systems was due solely to competition from HEL- B cells. In addition, the SWHEL model of B cell self-tolerance was used to show that while self-reactive B cells were hypo-responsive to BCR stimulation, BCR-independent signals delivered via anti-CD40 plus IL-4 or lipopolysaccharide could trigger them to undergo CSR and secretion of potentially pathogenic isotype-switched autoantibodies. Finally, the SWHEL model was used to study the responses of adoptively transferred follicular (Fo) and MZ B cells to in vivo activation with HEL conjugated to sheep red blood cells (HEL-SRBC). These studies revealed that both HEL+ MZ and Fo B cells were capable of mounting a robust T cell-dependent IgG1 antibody response to HEL-SRBC. However, HEL+ MZ B cells did not efficiently localise to the T cell-B cell border following antigen engagement and preferentially migrated to the bridging channels and red pulp. In contrast, HEL+ Fo B cells rapidly localised to the T cell-B cell border and subsequently colonised numerous germinal centres. As a result, the rate and pattern of SHM by HEL+ Fo and MZ B cells was shown to be distinct, with preferential targeting of mutations to the second complementarity-determining region in the former and to the second framework region in the latter. Together these data indicate illustrate the value of the SWHEL model and its potential to greatly advance the current understanding of B cell responses in tolerance and immunity.

Classical immunoglobulin transgenic (Ig-Tg) mouse models such as the MD4 anti-hen egg lysozyme (-HEL) Ig-Tg line have been used extensively to study B cell responses in tolerance and immunity. This thesis describes a new generation of gene-targeted mice (designated SWHEL mice) whereby the VH10 Ig variable gene encoding the HyHEL-10 specificity of the original anti-HEL Ig-Tg mouse was targeted to the Ig heavy chain locus. B cells in the SWHEL mouse are therefore capable of undergoing class switch recombination (CSR) and somatic hypermutation (SHM), representing a major advance on the original MD4 mouse model. SWHEL mice were found to not only contain a large population of HEL-specific (HEL+) B cells but also a significant population of non-HEL-binding (HEL-) B cells generated by VH gene replacement. HEL+ SWHEL B cells were found to belong to the B2 lineage and displayed high levels of surface IgM. Nevertheless, they matured normally and colonised the primary B cell follicle and marginal zone (MZ) of the spleen. The SWHEL model thus provided an opportunity to re-examine some of the original observations made in the MD4 system and also to extend these observations, particularly with regard to the regulation of CSR by self-reactive B cells. As expected, analysis of SWHEL B cells exposed to high avidity membrane-bound HEL revealed that they underwent clonal deletion in the bone marrow (BM). More interestingly, analysis of HEL+ B cells exposed to low avidity soluble HEL revealed that they were able to emigrate from the BM to the spleen as anergic B cells. However, unlike anergic MD4 B cells, anergic SWHEL B cells were reduced in frequency, displayed an immature B cell phenotype, were excluded from the follicle and had a reduced lifespan. Direct measurement of B cell antigen receptor (BCR) occupancy by HEL and the frequency of HEL- competitor B cells was combined with mixed BM irradiation chimeras to demonstrate unequivocally that the difference in phenotype and fate of HEL+ B cells in the two systems was due solely to competition from HEL- B cells. In addition, the SWHEL model of B cell self-tolerance was used to show that while self-reactive B cells were hypo-responsive to BCR stimulation, BCR-independent signals delivered via anti-CD40 plus IL-4 or lipopolysaccharide could trigger them to undergo CSR and secretion of potentially pathogenic isotype-switched autoantibodies. Finally, the SWHEL model was used to study the responses of adoptively transferred follicular (Fo) and MZ B cells to in vivo activation with HEL conjugated to sheep red blood cells (HEL-SRBC). These studies revealed that both HEL+ MZ and Fo B cells were capable of mounting a robust T cell-dependent IgG1 antibody response to HEL-SRBC. However, HEL+ MZ B cells did not efficiently localise to the T cell-B cell border following antigen engagement and preferentially migrated to the bridging channels and red pulp. In contrast, HEL+ Fo B cells rapidly localised to the T cell-B cell border and subsequently colonised numerous germinal centres. As a result, the rate and pattern of SHM by HEL+ Fo and MZ B cells was shown to be distinct, with preferential targeting of mutations to the second complementarity-determining region in the former and to the second framework region in the latter. Together these data indicate illustrate the value of the SWHEL model and its potential to greatly advance the current understanding of B cell responses in tolerance and immunity.

No description available.

Follicular dendritic cells (FDCs) reside in primary B-cell follicles and in the light zones of germinal centers (GCs) in secondary follicles, where their dendrites interdigitate forming extensive networks intimately interacting with B-cells. In GCs, FDCs can be found at the edges attached to the supporting reticular fibers. They trap and arrange immune complexes (ICs) in vivo and in vitro in a periodic manner with 200–500Å spacing and provide both antigen-specific and non-specific accessory signals to B-cells. FDCs exist in resting and activated states, with two characteristically different phenotypes. In their activated state, FDCs upregulate the expression of accessory molecules and cytokines important in the FDC-B cell interaction in GCs. We sought to determine the mechanisms influencing the transition of FDCs from a resting to an activated state in GCs and their impact on T-cell dependent (TD) and independent (TI)-GC reactions (GCRs). We found that IC-FDC interactions via FDC-FcgammaRIIB induce the upregulation of FDC-FcgammaRIIB, -ICAM-1, and -VCAM-1, at both the protein and mRNA levels. We also reported for the first time the expression of TLR-4 on FDCs. Moreover, engagement of FDC-TLR4 with LPS activated NF-kappaB, up-regulated expression of important FDC-accessory molecules, including FcgammaRIIB, ICAM-1, and VCAM-1, and enhanced FDC accessory activity in promoting recall IgG responses. Moreover, IC-activated FDCs produced IL-6 and FDC-IL-6 promoted GCRs, somatic hypermutation (SHM) and IgG production. Further, we reported that binding of FDCs to collagen coated surfaces induced restoration of their dendritic processes and networks in vitro. In addition, we designed an FDC-supported in vitro model capable of induction and assessment of primary human antibody responses to protein antigens characterized by class-switching and affinity maturation. Uniquely, we generated TI immune responses to TD protein Ags in the complete absence of T cell help in vivo and in vitro. In the presence of FDC-associated second signals such as BAFF and C4BP, FDC- FcgammaRIIB-periodically trapped-ICs induced the production of Ag-specific IgM, GC-development and plasmablast-differentiation in anti-Thy-1-pretreated nude mice. Purified murine and human B cells cultured in vitro with IC-bearing FDCs also showed the production of antigen–specific IgM within just 48 h.