Animals deficient in complement components C1q, C4, C3, and CR1/2 have severely impaired antibody responses. C1q is primarily activated by antibody-antigen complexes. Antigen-specific IgM in complex with an antigen is able to enhance the antibody response against that antigen. This is dependent on the ability of IgM to activate complement. Naïve mice have very low amounts of specific antibodies and therefore it is surprising that classical pathway activation plays a role for primary antibody responses. It was hypothesized that natural IgM, present in naïve mice, would bind an antigen with enough affinity to activate C1q. To test this, a knock-in mouse strain, Cm13, with a point mutation in m heavy chain, making its IgM unable to activate complement was constructed. Surprisingly, the antibody responses in Cm13 were normal. Puzzled by the finding that the ability of IgM to activate complement was required only for some effects, the immunization protocol was changed to mimic an infectious scenario. With this regime, Cm13 mice had an impaired antibody response compared to wildtype (WT) mice. The antibody response in WT mice to these repeated low-dose immunizations was also enhanced. These observations suggest that IgM-mediated enhancement indeed plays a physiological role in initiation of early antibody responses. IgM-mediated enhancement cannot however compensate for the dependecy of T-cell help. Although IgM from WT mice enhanced the antibody response, the T-cell response was not enhanced. The connection between classical pathway activation and CR1/2 is thought to be generation of ligands for CR1/2. In mice, CR1/2 are expressed on B cells and follicular dendritic cells (FDC). Although CR1/2 are crucial for a normal antibody response, the molecular mechanism(s) are not understood. To investigate whether CR1/2 must be expressed on B-cells or FDC to generate a normal antibody response, chimeric mice between WT and CR1/2-deficient mice were constructed. The results show that CR1/2+ FDC were crucial for the generation of antibody responses. In the presence of CR1/2+ FDC, both CR1/2+ and CR1/2- B cells were equally good antibody producers. However, for an optimally enhanced antibody response against IgM-antigen complexes, both B cells and FDC needed to express CR1/2.

Antibodies can enhance or suppress the immune responses against their specific antigens. This phenomenon is known as antibody-mediated feedback regulation. We have studied the mechanisms underlying IgE-, IgM-, and IgG3-mediated enhancement of immune responses in mouse models using intravenous immunization. We attempted to answer the following questions: 1) Which cell type presents IgE-complexed antigens to CD4+ T cells? 2) Is complement activation required for specific IgM to enhance antibody responses? 3) Does IgM enhance CD4+ T-cell responses? 4) How are IgG3-antigen complexes transported into B-cell follicles? We found that CD23+ B cells transporting IgE-antigen complexes into B-cell follicles were not required to prime the antigen-specific CD4+ T cells in vivo, whereas CD11c+ cells were indispensable. After examining the three most common subpopulations of CD11c+ cells in the spleen, we determined that it was CD8α- conventional dendritic cells migrating into the T-cell zone following immunization that presented IgE-complexed antigens to CD4+ T cells. Next, we showed that specific IgM from Cµ13 mice, which is unable to activate complement, failed to enhance either antibody or germinal center responses whereas wild-type IgM enhanced both responses. Therefore, specific IgM must activate complement to enhance humoral responses. In addition, wild-type IgM did not up-regulate CD4+ T-cell responses. Finally, we showed that IgG3-antigen complexes were transported by marginal zone B cells into B-cell follicles via binding to complement receptors 1 and 2 (CR1/2) on those cells. The immune complexes were captured by follicular dendritic cells as early as 2 h after immunization. Germinal center responses were also enhanced by IgG3. Using bone marrow chimeric mice, we found that CR1/2 expression was required on both marginal zone B cells and follicular dendritic cells to provide an optimal enhancement of antibody responses.

An intact complement system is essential for the initiation of a normal antibody response. Antibodies can regulate their own production against the antigens that they are specific for. Both IgG3 and IgM are able to enhance the antibody response via complement. Here, we have compared the fate of OVA-TNP (ovalbumin-2,4,6-trinitrophenyl) administered intravenously to mice either alone or in complex with monoclonal IgG3 anti-TNP. IgG3-antigen complexes bind to marginal zone (MZ) B cells via complement receptors 1 and 2 (CR1/2) and are transported into splenic follicles. The majority (50% - 90%) of the antigens is deposited on follicular dendritic cells (FDC) and the antigen distribution pattern is strikingly similar to peripheral dendrites/processes of FDC already 2 h after immunization. The development of germinal centers (GC) induced by IgG3-antigen complexes is impaired in mice lacking CR1/2. Experiments on bone marrow chimeric mice show that CR1/2 expression on both MZ B cells and FDC is required for optimal IgG3-mediated enhancement of antibody responses. Complement factors C3 and C1q are essential for OVA-TNP delivery and deposition on splenic FDC. The production of IgG anti-OVA is abrogated in mice lacking CR1/2, C1q, and C3. Further, IgG3-antigen complexes dramatically upregulate the memory response against OVA-TNP by inducing OVA-specific memory cells. Besides small protein OVA, IgG3 can also upregulate humoral responses against large soluble keyhole limpet hemocyanin. To further study the role of MZ B-cells and CR1/2 in enhancement of antibody responses, a knock-in mouse strain, Cμ13, was used. IgM in this mouse strain is unable to activate complement due to a point mutation in the constant µ-heavy chain. Cμ13 mice have a higher proportion of MZ B cells, with higher CR1/2 expression, than wild-type mice. More IgG3-immune complexes are captured by MZ B cells and deposited on FDC in Cμ13 than in WT mice. In spite of this, IgG3 did not enhance the primary antibody response more efficiently in Cμ13 mice. The existence of endogenous IgM-mediated feedback regulation was suggested by the observation that GC development and antibody responses, after priming and boosting with suboptimal doses of SRBC, was lower in Cμ13 than in WT mice.

B lymphocytes play a significant role in autoimmune arthritis, with their function stretching beyond autoantibody production to cytokine secretion and presentation of autoantigen. However, the involvement and activation of different B-cell subset in the autoimmune response is not fully clear. The main focus of this thesis has been to understand the contribution of marginal zone (MZ) B cells in the induction of collagen-induced arthritis (CIA), a mouse model for rheumatoid arthritis (RA). We show that MZ B cells in the spleen of naïve mice display a natural self-reactivity to collagen type II (CII), the autoantigen used for immunization of CIA. The CII-reactive MZ B cells expand rapidly following immunization with CII, and produce IgM and IgG antibodies to CII. They also very efficiently present CII to cognate T cells in vitro and in vivo. Moreover, absence of regulatory receptors such as CR1/2 or FcγRIIb on the MZ B cells increases their proliferation and cytokine production in response to toll-like receptor, but not B-cell receptor, activation. Further, FcγRIIb-deficient MZ B cells present CII to T cells more efficiently than wild-type MZ B cells. We additionally demonstrate for the first time the existence of a small population of nodal MZ B cells in mouse lymph nodes. Similar to splenic MZ B cells, the nodal MZ B cells expand after CIA induction, secrete IgM anti-CII antibodies and can present CII to cognate T cells. Finally, we show that mast cells, associated with ectopic B cell follicles in inflamed RA joints, in coculture with B cells promote their expansion, production of IgM and IgG antibodies as well as upregulation of CD19 and L-selectin. Coculture with mast cells further causes the B cells to upregulate costimulators and class II MHC, important molecules for antigen-presenting function. In summary, my findings suggest that splenic and nodal self-reactive MZ B cells participate in breaking T-cell tolerance to CII in CIA. B-cell intrinsic regulation is needed to keep such autoreactive B cells quiescent. Mast cells can potentiate B-cell responses locally in the arthritic joint, thus feeding the autoimmune reaction.