Abstract: We conclude that classic pathway activation but none of the activators (IgM, SIGN-R1, CRP, and SAP) are required for antibody responses. This is particularly interesting, because complement activation by natural IgM has been thought to explain the requirement for classic pathway activation in primary antibody responses. These observations imply that spontaneous C1 cleavage is sufficient, an unknown factor is involved, or the factors tested here act redundantly. Autoimmune diseases, such as systemic lupus erythematosus, are associated with deficiencies in complement components. Therefore, understanding how complement regulates immune responses may have implications for the understanding of autoimmunity. The classic pathway is primarily activated by IgM and IgG antibodies, which acquire the ability to bind C1q after they form an immune complex with their specific antigen. In naive mice, very little specific antibody is present; therefore, immune complex formation is presumably rare. Thus, it appears paradoxical that the activation of the classic pathway would be crucial for primary antibody responses. A possible explanation has been suggested by two sets of observations. First, specific IgM administered with an antigen enhances the antibody response to that antigen; moreover, the enhancement is dependent on the ability of the IgM molecule to activate complement ( 2 , 3 ). Second, mice lacking secretory IgM have lower antibody responses than WT mice, and the responses can be restored by the transfer of IgM from normal mouse serum ( 4 , 5 ). These findings suggested that natural IgM likely forms an immune complex with the antigen in naive mice (albeit with inefficient binding), activates complement, and initiates the classic pathway. Subsequently, early specific IgM formation would further up-regulate the antibody response. We have sought to test this specific explanation by ascertaining whether the ability of natural IgM to activate complement is a prerequisite for normal antibody responses. For this purpose, we generated the Cμ13 mice, which carry a point mutation that renders IgM unable to activate complement. The mutation is identical to the one in a previous study ( 2 ). For confirming that IgM from Cμ13 mice did not activate complement, the number of single B cells producing an antibody to sheep red blood cells (SRBCs), IgM anti-SRBC, was tested with two sensitive assays: the direct hemolytic plaque-forming cell (PFC) assay, which only detects B cells producing complement-activating IgM, and the enzyme-linked immunospot (ELISPOT) assay, which detects all IgM-producing B cells. When Cμ13 mice were immunized with a high dose of SRBCs, they produced ∼70,000 ELISPOTs per spleen but only background numbers of PFCs (115 per spleen). Thus, Cμ13 B cells were able to produce IgM anti-SRBC, but this IgM could not activate complement. Having established this, we asked the crucial question of whether Cμ13 mice had an impaired antibody response to antigens known to depend on a functioning complement system. Cμ13 and WT controls were immunized with various doses of SRBCs or keyhole limpet hemocyanin (a large protein antigen). As determined by the sensitive ELISA, primary IgG and IgM and secondary IgG responses were normal in Cμ13 mice. In parallel, antibody responses in mice lacking C1q (C1qA −/− ) were tested. As expected, these responses were severely impaired. Thus, although secreted IgM ( 4 , 5 ) and classic pathway activation are both required for the normal primary antibody response, this does not require that IgM activate complement. These observations suggested that something other than IgM initiates the classic pathway. Although IgG antibodies can activate this pathway, they suppress antibody responses to SRBCs. Hence, it is unlikely that IgG-mediated classic pathway activation explains the positive effect of complement on antibody responses. However, three other endogenous activators of the classic pathway have been described: SIGN-R1, SAP, and CRP. We therefore immunized animals lacking any one of these components with SRBCs and tested their sera in ELISAs. As before, the IgG anti-SRBC responses were normal. Complement is primarily known as an immune defense system that leads to the lysis or rupturing of pathogens, increased phagocytosis (i.e., the engulfment of invading organisms or foreign material by white blood cells), and inflammation. However, complement also plays a lesser known but crucial role in the generation of antibody responses to many antigens ( 1 ). Complement can be activated by three different routes: the classic pathway, the alternative pathway, and the lectin pathway. All these pathways lead to the cleavage of a protein, known as C3, into various products, some of which are the ligands (i.e., free-moving molecules that bind to receptors and thereby activate or suppress different cellular pathways) for complement receptors 1 and 2 (CR1/2) ( Fig. P1 ). Animals lacking complement factors C1q, C2, C3, or C4, or CR1/2, but not mannan-binding lectin (MBL), factor B, or C5 have severely impaired primary and secondary antibody responses; that is, both the initial antibody response and the long-term one are impaired. In general, IgG responses (i.e., secondary antibody responses) are more affected than IgM ones (i.e., initial antibody responses). C1q is involved in the classic pathway, whereas MBL and factor B are important to the lectin pathway and the alternative pathway, respectively. Hence, these observations imply that activation of the classic pathway is crucial for antibody responses. This is an apparent paradox because ( i ) the classic pathway is activated mainly by antibodies, which form a complex with the antigen, and ( ii ) presumably, extremely low amounts of specific antibodies are present in a naive animal (i.e., when the primary response is initiated). A possible explanation is that the classic pathway is initiated by natural IgM, present also in naive mice. To test this, we generated a mouse strain, Cμ13, in which IgM cannot activate complement owing to a small genetic change (a point mutation) in a gene encoding a particular IgM region. Unexpectedly, the antibody responses in these animals were normal. Moreover, antibody responses were normal in mice lacking three other activators of the classic pathway: specific intracellular adhesion molecule-grabbing nonintegrin R1 (SIGN-R1), serum amyloid P component (SAP), and C-reactive protein (CRP). Thus, although C1q is required for antibody responses, none of the known classic pathway activators are.

Abstract: γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αβ T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4–producing T cells (both residual γδ T cells and αβ T cells) and in systemic IL-4 levels. Its B cells expressed IL-4–regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4–inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.

Abstract: B cells play multiple important roles in the pathophysiology of autoimmune disease. Beyond producing pathogenic autoantibodies, B cells can act as antigen-presenting cells and producers of cytokines, including both proinflammatory and anti-inflammatory cytokines. Here we review our current understanding of the non-antibody-secreting roles that B cells may play during development of autoimmunity, as learned primarily from reductionist preclinical models. Attention is also given to concepts emerging from clinical studies using B cell depletion therapy, which shed light on the roles of these mechanisms in human autoimmune disease.