Birgitta Heyman

Secretory IgM plays a pivotal role in promoting robust antigen‐specific IgG responses, yet the mechanisms underlying its immune‐enhancing effects remain incompletely understood. IgM functions through two distinct pathways: engagement of the IgM Fc receptor (FcµR) and activation of the classical complement pathway. However, the extent of redundancy between these pathways and their roles at different stages of B cell differentiation remains unclear. To address this, we utilized FcµR‐deficient mice and Cµ13 mice, which express mutant IgM incapable of activating complement. Both strains exhibited impaired T‐dependent immune responses to low‐dose 4‐hydroxy‐3‐nitrophenyl‐chicken γ globulin. Remarkably, FcµR ^−/− Cµ13 double‐mutant mice showed profound defects in antigen‐specific IgG production compared with either single mutant, revealing nonredundant, synergistic roles for FcµR and complement. Mechanistically, both pathways are required for early B cell activation and expansion, promoting efficient class switch recombination, germinal center (GC) formation, and plasma cell differentiation. During the GC response, IgM BCR‐mediated complement activation, but not FcµR, is required for GC B‐cell proliferation, survival, and affinity maturation. In contrast, FcµR primarily enhances BCR signaling in naïve B cells through downstream PI3K‐AKT and MAPK pathways. These findings define two cooperative yet distinct IgM‐mediated mechanisms that promote humoral immunity and regulate B cell differentiation in vivo.

Summary: It was at the turn of the 20th century, that immune serum was found both to save children dying from toxins of deadly pathogens, and to kill a dog within minutes following an injection of harmless doses of sea anemone toxins. This means that, before being formally identified in immune serum, antibodies were already known to be both protective and pathogenic. For this provocative finding, Charles Richet was awarded the 1913 Nobel Prize in Physiology or Medicine. Because, as its name said, anaphylaxis was understood as “the contrary of protection,” unique mechanisms were found to explain it. Because, as its name did not initially say but finally said, allergy was understood as a reaction “other” than immunity, its symptoms were explained by mechanisms similar to those that explained anaphylaxis. We examined here the intricate relationships between anaphylaxis, allergy and immunity. Progressively anaphylaxis became one among other pathological effects of an immune response, and allergy an inflammatory disease among others. Looking at antibodies back in the past enables us not only to learn where they come from, but also to follow trends that contributed to shape immunology, some of which may persist in today's immunological thinking and say something about the future.

Summary: Antibodies are able to up‐ or downregulate antibody responses to the antigen they bind. Two major mechanisms can be distinguished. Suppression is most likely caused by epitope masking and can be induced by all isotypes tested (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgE). Enhancement is often caused by the redistribution of antigen in a favorable way, either for presentation to B cells via follicular dendritic cells (IgM and IgG3) or to CD4⁺ T cells via dendritic cells (IgE, IgG1, IgG2a, and IgG2b). IgM and IgG3 complexes activate complement and are transported from the marginal zone to follicles by marginal zone B cells expressing complement receptors. IgE–antigen complexes are captured by CD23⁺ B cells in the blood and transported to follicles, delivered to CD8α⁺ conventional dendritic cells, and presented to CD4⁺ T cells. Enhancement of antibody responses by IgG1, IgG2a, and IgG2b in complex with proteins requires activating FcγRs. These immune complexes are captured by dendritic cells and presented to CD4⁺ T cells, subsequently helping cognate B cells. Endogenous feedback regulation influences the response to booster doses of vaccines and passive administration of anti‐RhD antibodies is used to prevent alloimmunization of RhD‐negative women carrying RhD‐positive fetuses.

Lack of complement factor C1q of the classical pathway results in severely impaired primary antibody responses. This is a paradox because antibodies, especially IgM, are the most efficient activators of the classical pathway and very little specific IgM will be present at priming. A possible explanation would be that natural IgM, binding with low affinity to the antigen, may suffice to activate complement. In support of this, mice lacking secretory IgM have an impaired antibody response, which can be rescued by transfer of non-immune IgM. Moreover, passive administration of specific IgM together with antigen enhances the antibody response in a complement-dependent fashion. To test the idea, we have used a knock-in mouse strain (Cμ13) carrying a point mutation in the IgM heavy chain, rendering the IgM unable to activate complement. Mutant mice backcrossed to BALB/c or C57BL/6 background were primed and boosted with a low dose of sheep red blood cells. Confirming earlier data, no impairment in early, primary IgM- or IgG-responses were seen in either of the Cμ13 strains. However, in one of the mutant strains, late primary IgG responses were impaired. A more pronounced effect was observed after boost, when the IgG response, the number of germinal center B cells and antibody secreting cells as well as the opsonization of antigen were impaired in mutant mice. We conclude that complement activation by natural IgM cannot explain the role of C1q in primary antibody responses, but that endogenous, specific, wildtype IgM generated after immunization feedback-enhances the response to a booster dose of antigen. Importantly, this mechanism can only partially explain the role of complement in the generation of antibody responses because the IgG response was much lower in C3- or complement receptor 1 and 2-deficient mice than in Cμ13 mice.

Follicular dendritic cells (FDCs) are rare and enigmatic cells that mainly reside in germinal centers (GCs). They are capable of capturing immune complexes,viatheir Fc (FcRs) and complement receptors (CRs) and storing them for long periods in non-degradative vesicles. Presentation of ICs on FDCs to B cells is believed to drive affinity maturation. CR1 and CR2 are expressed on B cells and FDCs. Cr2 knock out (KO) mice, lacking both receptors, have impaired antibody and GC responses. Utilizing a novel ImageJ macro to analyze confocal fluorescence microscopy images of spleen sections, we here investigate how FDCs in wild type (WT) and Cr2 KO mice behave during the first two weeks after immunization with sheep red blood cells (SRBC). Mice were immunized with SRBC i.v. and spleen and serum samples harvested at various time points. As expected, antibody and GC responses in Cr2 KO mice were impaired in comparison to WT mice. Fewer FDCs were identified in Cr2 KO mice, and these exhibited differential localization and organization in comparison to WT mice. WT FDCs were primarily located within GCs at the light zone/dark zone border. FDCs from WT but not Cr2 KO mice were actively dispersed in GCs, i.e. tended to move away from each other, presumably to increase their surface area for B cell interaction. FDCs from Cr2 KO mice were more often found on follicles outside of the GCs and those within the GCs were closer to the periphery in comparison to WT FDCs. Expression of CR1 and CR2, FcγRIIB, and FcµR increased in FDCs from WT mice during the course of immunization. The results suggest that decreased ability to capture ICs by FDCs lacking CR1 and CR2 may not be the only explanation for the impaired GC and antibody responses in Cr2 KO mice. Poor FDC organization in GCs and failure to increase receptor expression after immunization may further contribute to the inefficient immune responses observed.

Antigen-specific IgG antibodies, passively administered together with erythrocytes, prevent antibody responses against the erythrocytes. The mechanism behind the suppressive ability of IgG has been the subject of intensive studies, yet there is no consensus as to how it works. An important question is whether the Fc-region of IgG is required. Several laboratories have shown that IgG suppresses equally well in wildtype mice and mice lacking the inhibitory FcγIIB, activating FcγRs (FcγRI, III, and IV), or complement factor C3. These observations consistently suggest that IgG-mediated suppression does not rely on Fc-mediated antibody functions. However, it was recently shown that anti-KEL sera failed to suppress antibody responses to KEL-expressing transgenic mouse erythrocytes in double knock-out mice lacking both activating FcγRs and C3. Yet, in the same study, antibody-mediated suppression worked well in each single knock-out strain. This unexpected observation suggested Fc-dependence of IgG-mediated suppression and prompted us to investigate the issue in the classical experimental model using sheep red blood cells (SRBC) as antigen. SRBC alone or IgG anti-SRBC together with SRBC was administered to wildtype and double knock-out mice lacking C3 and activating FcγRs. IgG efficiently suppressed the IgM and IgG anti-SRBC responses in both mouse strains, thus supporting previous observations that suppression in this model is Fc-independent.

Antibodies forming a complex with antigen in vivo can dramatically change the antibody response to this antigen. In some situations, the response will be a 100‐fold stronger than in animals immunized with antigen alone and in other situations the response will be completely suppressed. IgG is known to suppress the antibody response, for example to erythrocytes, and this is used clinically in Rhesus prophylaxis. The mechanism behind IgG‐mediated immune suppression is still not understood. Here, we will review studies performed in experimental animal models and discuss the various hypotheses put forward to explain the profound suppressive effect of IgG. We conclude that an exclusive role for negative regulation of B cells through FcγRIIB, increased clearance of erythrocytes from the circulation, or complement‐mediated lysis, is unlikely. Epitope masking, where IgG hides the epitope from B cells, or trogocytosis, where IgG removes the epitope from the erythrocyte, are compatible with many observations. These two mechanisms are not mutually exclusive. Moreover, it cannot be ruled out that clearance, in combination with other mechanisms, plays a role.

Rheumatoid arthritis–associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody–induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain–deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain–deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain–deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics.

Specific IgG antibodies, passively administered together with erythrocytes, suppress antibody responses against the erythrocytes. Although used to prevent alloimmunization in Rhesus (Rh)D-negative women carrying RhD-positive fetuses, the mechanism behind is not understood. In mice, IgG suppresses efficiently in the absence of Fcγ-receptors and complement, suggesting an Fc-independent mechanism. In line with this, suppression is frequently restricted to the epitopes to which IgG binds. However, suppression of responses against epitopes not recognized by IgG has also been observed thus arguing against Fc-independence. Here, we explored the possibility that non-epitope specific suppression can be explained by steric hindrance when the suppressive IgG binds to an epitope present at high density. Mice were transfused with IgG anti-4-hydroxy-3-nitrophenylacetyl (NP) together with NP-conjugated sheep red blood cells (SRBC) with high, intermediate, or low NP-density. Antibody titers and the number of single antibody-forming cells were determined. As a rule, IgG suppressed NP- but not SRBC-specific responses (epitope specific suppression). However, there was one exception: suppression of both IgM anti-SRBC and IgM anti-NP responses occurred when high density SRBC-NP was administered (non-epitope specific suppression). These findings answer a longstanding question in antibody feedback regulation and are compatible with the hypothesis that epitope masking explains IgG-mediated immune suppression.

Antigen-specific IgG antibodies, passively administered together with large particulate antigens such as erythrocytes, can completely suppress the antigen-specific antibody response. However, the mechanism behind has been elusive. Herein, we made the surprising observation that mice immunized with IgG anti-SRBC (sheep red blood cells) and SRBC, in spite of a severely suppressed anti-SRBC response, have a strong germinal center (GC) response. This occurred regardless of whether the passively administered IgG was of the same allotype as that of the recipient or not. Six days after immunization, the GC size and the number of GC B cells were higher in mice immunized with SRBC alone than in mice immunized with IgG and SRBC, but at the other time points these parameters were similar. GCs in the IgG-groups had a slight shift towards dark zone B cells six days after immunization and towards light zone B cells ten days after immunization. The proportions of T follicular helper (TFH) and T follicular regulatory (TFR) cells were similar in the two groups. Interestingly, mice immunized with allogeneic IgG anti-SRBC together with SRBC mounted a vigorous antibody response against the passively administered suppressive IgG. Thus, although their anti-SRBC response was almost completely suppressed, an antibody response against allogeneic, and probably also syngeneic, IgG developed. This most likely explains the development of GCs in the absence of an anti-SRBC antibody response.