A Novel Image Analysis Approach Reveals a Role for Complement Receptors 1 and 2 in Follicular Dendritic Cell Organization in Germinal Centers.

Follicular dendritic cells (FDCs) are rare and enigmatic cells that mainly reside in germinal centers (GCs). They are capable of capturing immune complexes, via their 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.

Importance of antibody isotypes in antitumor immunity by monocytes and complement using human-immune tumor models.

Monoclonal antibodies (mAbs) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a preclinical human immune - three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer cultures, of human CD20+ B-cell lymphomas. We demonstrate that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, and when combined with apoptosis-inducing RTX-IgG2 isotype the therapeutic effect can be substantially enhanced. The results show further that the treatment outcome by RTX isotypes is influenced by tumor morphology and expression of the complement inhibitor CD59. Hence, the human immune-3D tumor model is a clinical relevant and attractive ex vivo system to predict mAbs for best efficacy in cancer immunotherapy.

IgG Suppresses Antibody Responses to Sheep Red Blood Cells in Double Knock-Out Mice Lacking Complement Factor C3 and Activating Fcγ-Receptors.

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.

Harnessing the immune system via FcγR function in immune therapy: a pathway to next-gen mAbs.

The human fragment crystallizable (Fc)γ receptor (R) interacts with antigen-complexed immunoglobulin (Ig)G ligands to both activate and modulate a powerful network of inflammatory host-protective effector functions that are key to the normal physiology of immune resistance to pathogens. More than 100 therapeutic monoclonal antibodies (mAbs) are approved or in late stage clinical trials, many of which harness the potent FcγR-mediated effector systems to varying degrees. This is most evident for antibodies targeting cancer cells inducing antibody-dependent killing or phagocytosis but is also true to some degree for the mAbs that neutralize or remove small macromolecules such as cytokines or other Igs. The use of mAb therapeutics has also revealed a "scaffolding" role for FcγR which, in different contexts, may either underpin the therapeutic mAb action such as immune agonism or trigger catastrophic adverse effects. The still unmet therapeutic need in many cancers, inflammatory diseases or emerging infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires increased effort on the development of improved and novel mAbs. A more mature appreciation of the immunobiology of individual FcγR function and the complexity of the relationships between FcγRs and antibodies is fueling efforts to develop more potent "next-gen" therapeutic antibodies. Such development strategies now include focused glycan or protein engineering of the Fc to increase affinity and/or tailor specificity for selective engagement of individual activating FcγRs or the inhibitory FcγRIIb or alternatively, for the ablation of FcγR interaction altogether. This review touches on recent aspects of FcγR and IgG immunobiology and its relationship with the present and future actions of therapeutic mAbs.

The Human FcγRII (CD32) Family of Leukocyte FcR in Health and Disease.

FcγRs have been the focus of extensive research due to their key role linking innate and humoral immunity and their implication in both inflammatory and infectious disease. Within the human FcγR family FcγRII (activatory FcγRIIa and FcγRIIc, and inhibitory FcγRIIb) are unique in their ability to signal independent of the common γ chain. Through improved understanding of the structure of these receptors and how this affects their function we may be able to better understand how to target FcγR specific immune activation or inhibition, which will facilitate in the development of therapeutic monoclonal antibodies in patients where FcγRII activity may be desirable for efficacy. This review is focused on roles of the human FcγRII family members and their link to immunoregulation in healthy individuals and infection, autoimmunity and cancer.

The Rare Anaphylaxis-Associated FcγRIIa3 Exhibits Distinct Characteristics From the Canonical FcγRIIa1.

FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.

The FcγR of humans and non-human primates and their interaction with IgG: implications for induction of inflammation, resistance to infection and the use of therapeutic monoclonal antibodies.

Considerable effort has focused on the roles of the individual members of the FcγR receptor (FcγR) family in inflammatory diseases and humoral immunity. Recent work has revealed major roles in infection and in particular HIV pathogenesis and immunity. In addition, FcγR functions underpin the action of many of the successful therapeutic monoclonal antibodies. This emphasises the need for a greater understanding of FcγR function in humans and in the NHP which provides a key model for human immunity and preclinical testing of antibodies. We discuss recent key aspects of the human FcγR receptor biology and structure to define differences and similarities in activity between the human and macaque Fc receptors. These differences and similarities nuance the interpretation of infection and vaccine studies in the macaque. Indeed passive IgG antibody protection in lentivirus infection models in the macaque provided early evidence for the role of Fc receptors in anti-HIV immunity that have subsequently gained support from human vaccine trials. None-the-less the diverse functions and cellular contexts of FcγR receptor expression ensure there is much still to understand of the protective and deleterious effects of FcγRs in HIV infection. Careful comparative studies of human and non-human primate FcγRs will facilitate our appreciation of what attributes of HIV specific IgG antibodies, either acquired naturally or via vaccination, are most important for protection.