Lupus-prone (NZBxNZW)F1 mice deficient for the FcR γ chain, lacking functional activating FcγR, do not develop IC-mediated severe glomerulonephritis (GN), despite high autoantibody titers (36). This suggests that FcγR play a dominant role in the efferent phase of Ab-driven diseases including lupuslike disease and therefore FcγRIIb might have a strong protective role in such a disease. In addition, FcγRIIb might also inhibit an ongoing auto-Ab response by suppressing the activating FcγR dependent, IgG-IC-triggered release of inflammatory mediators and other immune regulatory molecules by myeloid effector cells.

### Dendritic Cells

DCs are central regulators of immunity determining whether tolerance is induced, or an effective adaptive immune response is generated, bridging innate and adaptive immunity (37–39). DCs have the unique capacity to take up exogenous Ag via a variety of mechanisms and surface molecules, including FcγR, and subsequently process and present the Ag-derived peptides in their MHC molecules to prime naïve T cells. Three main subsets of DCs can be recognized, cDC, moDC and pDC. Their ontogeny and functions have been reviewed extensively (40, 41).

A series of observations suggest that FcγR on cDCs and moDCs can play a role in priming and regulation of adaptive immunity (16). Ag-specific IgG enhances Ab responses to soluble protein Ag via activating FcγRs, probably by increasing Ag presentation by dendritic cells to Th cells (42). Many laboratories have shown that soluble IgG-ICs strongly enhance cross-presentation by using either in vitro assays (43–45), or in vivo assays with in vitro loaded DCs from WT and FcγR KO mice (46–50). Signaling through the activating FcγRs results in lysosomal targeting of the Ag and importantly activation and maturation of the DCs (44), required for their migration to the lymph node and their presentation of Ag-derived peptides in MHC class I to CD8+ T cells (49, 51). In mouse bone marrowderived DCs (BMDCs), activating FcγRs modulate the expression of many genes, associated with T cell response induction, upon crosslinking by IgG-ICs. This is strongly regulated by FcγRIIb, setting a threshold for DC activation and maturation (52). FcγRIIb−/− mice showed an increased upregulation of costimulatory molecules, resulting in an enhanced capacity to generate antigen-specific T cell responses upon injection of IgG-ICs (52–54). However, in vivo, in mice, the role of FcγR in the presentation of soluble IgG-IC derived Ag is redundant (55, 56). In mice, cDCs consist of two main subsets, type 1 cDC or cDC1 and Type 2 cDC or cDC2 (41). In vivo IgG-IC improve strongly cross-presentation of the cDC2 but not the cDC1 DCs. Only cDC2 mediated cross-presentation is FcγR dependent (57). Moreover, FcγRs are dispensable for the in vivo uptake of IgG-IC by cDC1 and cDC2 (56, 57). The in vivo cross-presentation of IgG-IC derived Ag by cDC1 is completely and by cDC2 partially dependent on C1q (56).

Because it has been shown that treatment with FCGR2B blocking antibodies results in spontaneous maturation of human DCs (58) it has been hypothesized that FCGR2B does not only regulate DC activation but also actively prevents unwanted spontaneous DC maturation by small amounts of circulating IC present in serum under non-inflammatory steady-state conditions (2).

IgG-ICs endocytosed by activating FcγR on DCs ends up in a degradative Lamp-1 positive compartment where it is slowly degraded into peptides (59). In contrast, antigen, endocytosed in the periphery via FcγRIIb on DCs, enters preferentially in a non-degradative Lamp-1 negative intracellular vesicular compartment, that recycles to the cell surface to transfer the native antigen via interaction with the BCR to B cells in the lymphoid organs. This indicates that DCs, migrating into extrafollicular areas (60) and the splenic marginal zones (MZ) (61), are not only important for the production of B cell activating components but also for the delivery of Ag to the BCR (62).

The question is whether in an autoimmune disease selfantigen containing IgG-IC can trigger DCs to promote autoreactive immune responses by presenting autoantigens or to release B and T cell activating cytokines and other stimulating factors breaking tolerance and whether FcγRIIb on DCs negatively regulates these processes. That is any way at a stage of the disease that some autoantibodies are already produced.

pDCs produce type I IFN in response to viral nucleic acids sensed through TLR7 and TLR9 (63, 64). Their main function is to control tolerance in the steady state (65, 66). Mouse pDCs express exclusively FcγRIIb (67). Conflicting results have been published regarding FcγRIIb facilitated T cell priming by mouse pDCs (56, 67, 68). In vitro uptake of IgG-ICs by mouse pDC is FcγRIIb dependent but does not promote Ag presentation to T cells (67), similarly to what has been shown with FcγRIIb mediated IC uptake in cDCs (62). In contrast, it has been reported that subcutaneous (s.c.) injection of in vitro IgG1- IC loaded pDCs induces strong Ag-specific CD4+ and CD8+ T cell responses although with lower efficiency than cDCs. The IgG1-IC-loaded pDC mainly promoted a Th2/tolerogenic environment in vivo (68). Human pDCs express besides low levels of FCGR2B, the activating FCGR2A and FCGR3B (16) and show FCGR2A dependent IgG enhanced Ag presentation to T cells (69). SLE patients have circulating ICs, containing small nuclear RNA and anti-small nuclear RNA IgG. pDCs can acquire such IC via FCGR mediated uptake resulting in stimulation of TLR7 and 8 and production of IFNα (70), a cytokine that is believed to play a central role in SLE pathogenesis (71). However, this requires FCGR2A and not FCGR2B (72). Therefore, it is unlikely that such a pathogenic process plays a role in lupus-like disease in mice.

### B Cells and FDC

Primary B cells, developed and selected in the bone marrow, are recruited into GCs within the spleen and lymph nodes to undergo affinity maturation by Somatic Hypermutation (SHM). Three main mechanisms maintain self-tolerance in the primary B cell repertoire: central clonal deletion, receptor editing, clonal anergy induction (73). The first two effectively remove autoreactive B cells from the system. Clonal anergy occurs when self-reactive B cells interact with a self-Ag with relatively low avidity. The result is that BCR signaling is desensitized because of chronic exposure to self-antigens (74, 75) and differentiation into plasma cells is suppressed (76) resulting in the maintenance of anergic B cells with the potential to produce auto-Abs which can be recruited into GC (77). Anergic B cells can get T help if their BCR cross-reacts with foreign Ag but because of impaired BCR signaling FAS-mediated apoptosis is induced. However, extensive cross-linking by a foreign antigen can overcome the attenuated BCR signaling in anergic B cells inhibiting apoptosis (74). Autoreactive primary B cells can escape negative selection because of "clonal ignorance" when self-reactive B cells cannot encounter their self-Ag because it is hidden inside the cell.  The lack of T cell help after Ag contact induces apoptosis in ignorant self-reactive B cells in the periphery. However, it is striking that many auto-Abs are directed against intracellular Ags such as DNA. Therefore, it has been suggested that ignorant self-reactive B cells might be important for the development of SLE (77). So, the GC has to deal with three types of potential autoreactive B cells: anergic and ignorant, both recruited, and newly generated by somatic hypermutation in the GC reaction. Several mechanisms are in place in the GC to avoid the development of auto-Ab producing plasma cells. A very high concentration of self-Ag in the GC either overrules the binding of the BCR to foreign Ag presented by the FDC and apoptosis is induced, because of the lack of additional signals provided by the FDC (80), or/and blocks presentation of foreign Ag to follicular helper T cells (TFH), whose survival signals are required. Alternatively, self-reactive B cells can be maintained temporarily until their self-reactivity is abrogated by somatic hypermutation (SHM) (81). Ignorant self-reactive primary B cells, activated by cross-reactive foreign Ag, can enter the GC to get TFH help (82) and subsequently, receptor editing by SHM can destroy selfrecognition and improve specificity for foreign antigen. However, this appears not sufficient to prevent that autoreactive B cells escape negative selection in the GC and enter the AFC (antibodyforming cell) pathway. More downstream tolerance checkpoints are required.

In the GC Ag is presented to B cells on the cell surface of FDC, mainly in the form of CR1/2 bound C3d-coated ICs. FcγRIIb is expressed on both the GC B cell and the FDC. Although FcγRIIb is upregulated on FDC in GC compared to non-GC FDC, its expression is relatively low compared to CR2 expression. Therefore, it is unlikely that FcγRIIb plays a role in the capture and presentation of Ag early on in the GC response (83). It is unclear how a GC B cell becomes activated, because binding of its BCR to the Ag, within the FDC bound ICs, will also crosslink FcγRIIb on that B cell. It has been suggested that FcγRIIb expression on FDC competes with FcγRIIb expression on GC B cells by binding most of the Fc domains in the ICs (84). The outcome of co-engagement of BCR and FcγRIIb by ICs bound to FDC in GC might be dependent on the balance between concurrent activating and inhibiting signals, leading to stimulatory, inhibitory, or apoptotic responses (33, 85, 86). FcγRIIb might set a threshold for B-cell activation, that enables the selection of B cells with a BCR with sufficiently high affinity, to become activated. B cells with BCRs that have lost their affinity for the presented Ag during the process of affinity maturation by SHM will get only signals via crosslinking of FcγRIIb, which could result in induction of apoptosis as has been demonstrated in vitro (28, 33, 87, 88). In conclusion, the inhibitory FcγRIIb would be an important checkpoint for the deletion of potentially autoreactive B cells in the GC.

An additional apoptosis inducing mechanism in the bone marrow might also contribute to the control of autoreactive B cells (10). Long-lived plasma cells persist in the bone marrow. To provide room to newly generated plasma cells that migrate to the bone marrow after a new infection has occurred, a restricted number of plasma cells in the bone marrow has to be eliminated. Based on observations in vitro and in vivo in mice it has been hypothesized that plasma cells (which intrinsically lack BCR expression) are killed by apoptosis, induced by cross-linking of FcγRIIb highly expressed on these cells (10).

### Non-immune Cells

On LSEC FcγRIIb might function as an endocytic scavenger receptor removing small IgG-IC from circulation to prevent systemic IC triggered inflammation (25). FcγRIIb on renal mesangial cells might protect against IgG-IC induced inflammation in the kidney (89). Both mechanisms might protect against the pathogenesis of IC-driven autoimmune diseases such as glomerulonephritis in SLE in the efferent phase. Because of the lack of an endothelial cell-specific Cre expressing strain that is not transcriptionally active during early hematopoiesis, required to generate endothelium-specific FcγRIIb deficient mice, the specific role of FcγRIIb on LSEC should be studied by applying transplantation of bone marrow from WT mice into lethally irradiated Fcγ RIIb KO mice.

### FORWARD GENETICS: ASSOCIATION OF AUTOIMMUNITY AND FcγRIIb POLYMORPHISM

### In Mice

The association between autoimmunity and Fcγ RIIb polymorphism is extensively studied in NZW and NZB inbred stains. NZB mice show limited autoimmunity (90) while NZW mice are not autoimmune although their B cells have intrinsic defects sufficient to break tolerance to nuclear antigens (91, 92). However, the (NZBxNZW)F1 offspring of an accidental cross between NZW and NZB mice (93) showed a severe lupus-like

phenotype characterized by a gender-bias, expansion of activated B and CD4+ T cells, splenomegaly, elevated serum ANA and ICmediated GN causing renal failure and premature death at 10–12 months of age (94). By backcrossing (NZBxNZW)F1xNZW followed by brother-sister mating the NZM2410 recombinant inbred strain with a homozygous genome was generated (95–97). In this mouse four SLE susceptibility loci, Sle1-4, have been identified on different chromosomes. Sle1 is located on the telomeric region of Chr1 syntenic to human 1q23 that has shown strong linkage to SLE susceptibility in all human studies. The Fcγ R gene cluster maps in this region (**Figure 1**) and is from NZW origin in NZM2410 mice. From the NZM2410 strain, C57BL/6 strains have been developed congenic for a single SLE susceptibility locus. The presence of Sle1 appeared to be sufficient to break tolerance in C57BL/6 mice and to drive the production of high titers of anti-chromatin ANAs with a selective Ab reactivity to H2A/H2B/DNA sub-nucleosomes (99, 100). Importantly, this step appears to be necessary for the induction of disease (100) making Sle1 a key locus in the initiation of SLE. Transplantation of hematopoietic stem cells from C57BL/6 Sle1 congenic mice into C57BL/6 recipient mice showed that Sle1 causes independent B and T cell-intrinsic effects on the B cell response (101, 102).

Three Fcγ RIIb haplotypes [numbered I-III according to Jiang et al. (103), **Table 1**] have been recognized in inbred strains of mice and wild mice with variation in the promoter region and intron 3 (**Table 1**). Haplotype I with 2 deletions in the promoter region and one in intron 3 is found in autoimmune-prone strains and most wild mice and is associated with decreased expression of FcγRIIb on Mϕ, activated B cells and GC B cells (11, 103– 105). By using C7BL/6 congenic strains with the NZW (106) and NZB (107) allelic variants of Fcγ RIIb the effect of the deletions in haplotype I and II on B cell expression was studied. When immunized with KLH, FcγRIIb expression on splenic non-GC B

Verbeek et al. FcγRIIb and Autoimmunity

the Sle1NZW locus, Fcγ RIIbNZB was identified as an autoimmune susceptibility gene (114), in another it was not (115). Sle1 can be divided in four non-overlapping sub-loci: Sle1a, -b, -c, and -d. Sle1b is far the most potent autoimmune susceptibility locus causing almost the same phenotype as the whole Sle1 locus: gender-biased spontaneous loss of immune tolerance to chromatin, the production of high titers of IgG auto-Abs with a penetrance of 90% at 9 months of age and increase of total IgM and B7-2 expression on B cells (116). This suggests that Sle1b mainly affects B cells. The genomic location of Sle1b was determined by phenotypic analysis (e.g., ANA production) of a series of C57BL/6 congenic strains carrying truncated Sle1 intervals. C57BL/6 congenic mice with an NZW derived genomic fragment, containing the Fcγ R cluster, did not develop ANA whereas C57BL/6 mice, containing an adjacent 900 kb congenic NZW fragment expressing 24 genes including seven members of the highly polymorphic signaling lymphocytic activation molecules (Slam) cluster, did. This positions the Fcγ R cluster just outside the Sle1b locus (117) and confirms previous observations that Fcγ RIIb is located in between the Sle1a and Sle1b loci (113) (**Figure 1**). Together these data suggest that in C57BL/6 Sle1 congenic mice the Fcγ RIIbNZW allele is not required for the development of an autoimmune phenotype, whereas the adjacent Slam cluster is. Because of these puzzling results, the questions remain why FcγRIIb is upregulated on GC B cells in non-autoimmune inbred strains such as C57BL/6 and BALB/c and why this is impaired in autoimmune-prone mouse strains and how does that contribute to the autoimmune phenotype of these mice.

Slam family (Slamf) member genes encode cell surface glycoproteins with extracellular binding domains that mediate stimulatory and/or inhibitory signaling via associations with members of the Slam-associated protein (SAP) family of signaling adaptors during cell-cell interactions between many hematopoietic cell types (118–120). They are the only genes within the Sle1b interval with obvious immunological functions (117). Most Slamf members act as self-ligand and are expressed on many lymphoid and myeloid cell subsets, platelets, and hematopoietic stem and progenitor cells. Slamf plays a role in the interaction of CD4+ T cells with cognate B cells, recruitment and retention of T cells within the emerging GCs (121–123), long-lasting T cell:B-cell contact, optimal TFH function, T cell activation (124, 125), stabilization of B–T cell conjugates and sustaining effective delivery of T cell help required for GC formation (126, 127).

The Slamf genes show extensive polymorphisms (117) but only two haplotypes of the Slamf locus have been identified in laboratory mouse strains. Haplotype 1 is represented by C57BL/6 and related strains and haplotype 2 by all autoimmuneprone mouse strains, as well as many non-autoimmune mouse strains including BALB/c and 129. The polymorphism in Slamf member Ly108 affects the expression of two alternatively spliced isoforms, Ly108-1 and, Ly108-2, which differ exclusively in their cytoplasmic region (117). Ly108-1 is dominantly expressed in T and B lymphocytes of mice with haplotype 2, whereas Ly108-2 is dominantly expressed in T and B cells of mice with haplotype 1. Modulation of the BCR signaling by Ly108-1 results in the impaired negative selection of B cells (128). Overexpression of

cells was high and similar in C7BL/6 and C57BL/6 Fcγ RIIbNZB congenic mice. In contrast, the expression on activated GC B cells was markedly down-regulated in C57BL/6 congenic Fcγ RIIbNZB mice and up-regulated in control C57BL/6 mice, in comparison with the expression levels on non-GC B cells. The downregulation of FcγRIIb expression on activated GC B cells was associated with an increase of IgG anti-KLH Ab titers. C57BL/6 FcgRIIbNZB congenic mice also showed lower FcγRIIb expression on Mϕ compared with WT C57BL/6 mice (107). In a C57BL/6 knockin (KI) mouse model of the 5' region of the haplotype I Fcγ RIIb gene (Fcγ RIIbNZB), FcγRIIb failed to be upregulated on activated and GC B cells resulting in enhanced early GC responses and low auto-Ab production without kidney disease as discussed later in more detail (11).

As mentioned earlier, in vitro cross-linking of FcγRIIb on B cells from C57BL/6 mice can induce apoptosis. However, plasma cells from autoimmune-prone NZB or MRL mice could not be killed in vitro by FcγRIIb cross-linking because of too little expression of the receptor (10). This might partially explain why these autoimmune-prone mice have larger numbers of plasma cells and might contribute to the autoimmune phenotype of these mice.

Similarly, to the Fcγ RIIbNZB allele, the Fcγ RIIbNZW allele in the C57BL/6 Sle1 congenic strain did not upregulate its expression on GC B cells and plasma cells, as did the C57BL/6 allele, when immunized with SRBCs. However, in the absence of its Sle1 flanking regions, Fcγ RIIbNZW did not induce an autoimmune phenotype but was associated with an increased number of class-switched plasma cells (108). This might indicate that the decreased expression of the Fcγ RIIbNZW allele is not sufficient for the development of autoreactive B cells but can result in the increase of the number of autoreactive B cells, induced by other lupus-susceptibility loci, by enhancing the production of class-switched plasma cells. This suggests that the Fcγ RIIbNZB (haplotype I) allele has a stronger impact on susceptibility to autoimmunity than the Fcγ RIIbNZW (haplotype II) allele (**Figure 2**). However, in one study comparing the phenotypes of C57BL/6 strains congenic for different intervals of the Nba2 locus, a region on Chr1 of NZB mice corresponding to

FIGURE 2 | Epistasis between the FcγRIIb KO alleles and the Sle16 (Slam129) and Yaa autoimmune susceptibility loci resulting in lupus-like disease in C57BL/6 mice. Epistatic interactions are indicated as dotted arrows. The FcγRIIb flanking Sle16 genomic region contains the autoimmunity associated Slamf129 haplotype 2 gene cluster (see Figure 1). (1) Rahman et al. (108); (2) Espéli et al. (11); (3) Boross et al. (109); (4) Li et al. (110); (5) Bygrave et al. (111); (6) Bolland and Ravetch (112); (7) Bolland et al. (113); (8) Kanari et al. (98). The increasing severity of autoimmune disease in the different mouse models is depicted on top.

both C57BL/6 derived non-autoimmune Ly108 and CD84 Slamf members was required to restore tolerance in autoimmune-prone C57BL/6 Sle1 congenic mice (129), indicating that polymorphism in both Slamf genes contributes to the autoimmune phenotype of C57BL/6 Sle1 congenic mice.

In the NZM2410 model four NZW-derived SLE suppressor loci have been identified (130). The presence of such suppressor loci might explain why NZW and also129 and BALB/c mice do not develop autoimmune disease, although they carry the type 2 Slamf haplotype.

### In Humans

The reported copy number variation (CNV) in human FCGR genes does not involve FCGR2B (131–134). A series of single nucleotide polymorphisms (SNPs) have been reported to be located both in the promoter and the encoding region of the human FCGR2B gene (135). Two SNPs are located in the promoter region at nucleotide positions−386 and −120 (−386G>C; rs3219018 and −120A>T; rs34701572) (136) resulting in four haplotypes:−386G−120T (named FCGR2B.1), −386C−120T (FCGR2B.2),−386G−120A (FCGR2B.3), and −386C−120A (FCGR2B.4). The rare FCGR2B.4 haplotype increased the transcription of FCGR2B in vitro and resulted in increased FCGR2B expression on EBV transformed B cells and primary B cells (137) and myeloid cells (138), compared to the more frequent FCGR2B.1 haplotype. However, independently, others have shown that homozygosity of the −386C genotype decreases the transcription and surface expression of FCGR2B in peripheral B cells compared to the −386G homozygote genotype (139). Up till now, there is no explanation for these contradictory results.

In the transmembrane encoding fifth exon a non-synonymous C to T transition was identified, rs1050501, resulting in the substitution of isoleucine with threonine at position 232 (140), excluding the receptor from lipid rafts. This prevents interaction of FCGR2B with ITAM containing receptors such as the activating FCGR and the BCR (141, 142). Mϕs from individuals homozygous for FCGR2BT232 showed a stronger phagocytic capacity of IgG-IC while the B cells of these individuals showed reduced FCGR2B-mediated inhibition of BCR-triggered proliferation (142).

GWAS analyses have shown an association between rs1050501 and SLE (140, 143–147). Three meta-analyses confirmed these associations (147–149). The FCGR2BT232 homozygosity is associated with an odds ratio of 1.73, one of the strongest associations in SLE (147). Association of rs1050501 with Rheumatoid Arthritis (RA) has been reported for a Taiwanese cohort (150).

The frequency of homozygosity of the FCGR2BT232 allele is only 1% in Caucasians and in contrast 5–11% in African and South-East Asian populations (151). This might be one of the explanations for the ethnic differences in SLE susceptibility. Malaria is endemic in Africa and South-East Asia. An association was found between decreased susceptibility for severe malaria and homozygosity for the FCGR2BT232 allele (135). So, increased protection against malaria by down-regulation of FCGR2B expression goes along with increased risk to develop SLE.

A significant but weak association has been observed between SLAMF and susceptibility to SLE. The weakness of the association might be explained by the limited size of the cohorts studied (152). An association study of UK and Canadian families with SLE has revealed multiple polymorphisms in several SLAMF genes (153). However, the strongest association with a non-synonymous SNP could not be replicated in independent Japanese and European cohorts of SLE patients (154, 155). Instead, another SNP was significantly associated with the susceptibility to SLE in another Japanese cohort (156). One large-scale case-control association study showed an association of two SNPs with increased susceptibility to RA, in two independent Japanese cohorts (155). In conclusion, these observations indicate that also in human's polymorphisms of SLAMF contribute to the susceptibility to autoimmune disease.

Overall, a model emerges from both studies with C57BL/6 Sle congenic mouse strains and human SLE (157), in which disease susceptibility arises through the co-expression of multiple genetic variants that have weak individual effects (152, 158). According to the "threshold liability" model, the severity of the autoimmune phenotype increases with the increasing number of autoimmunity associated allelic variants of autoimmune susceptibility genes in the genome. However, epistatic interactions might result in a more complex nonadditive inheritance of the autoimmune phenotype (**Figure 2**). According to this "multiplicative model" the interactions of all susceptibility and suppressor alleles in the genome determine the susceptibility for autoimmune diseases of an individual (159). Importantly this means that the contribution of an individual gene to the autoimmune phenotype can vary depending on the presence of other susceptibility and suppressor genes in the genome (the genomic context). This might explain the puzzling and contradictory results with the Fcγ RIIbNZW and Fcγ RIIBNZB haplotypes. To uncover the polygenic effects associated with a complex disease such as SLE not a single gene association approach but gene set analysis (GSA) is required (160). However, a reverse genetic approach might offer the opportunity to reconstruct an autoimmune phenotype by modifying a combination of a limited number of candidate genes in a well-defined genetic background.

### REVERSE GENETICS

So far three Fcγ RIIb KO mouse models have been published. The first published KO was generated by gene targeting in 129 derived ES cells (161) and subsequently backcrossed into the C57BL/6 background, here called Fcγ RIIb−/− 129 mouse. This mouse on a not well-defined mixed genetic background was during 15 years (between 1996 and 2011) the only Fcγ RIIb KO model available and has been extensively used resulting in an overwhelming amount of literature concerning the role of FcγRIIb in immune tolerance. Subsequently, independently, in two different laboratories Fcγ RIIb KO mice were generated by gene targeting in C57BL/6 derived ES cells, here called Fcγ RIIb−/− B6 mice (109, 110). The published data regarding ANA titers of one of these mouse strains are inconsistent (162, 163) as are the autoimmune phenotypes of both C57BL/6 strains (109, 110). Moreover, it is still under debate to what extent the autoimmune phenotypes of the Fcγ RIIb−/− B6 mice differ from the autoimmune phenotype of the Fcγ RIIb−/− 129 mice. Therefore, we discuss in chronological order these different models.

### The Fcγ RIIb KO Mouse on Mixed 129/C57BL/6 Background

The Fcγ RIIb−/− 129 mouse develops elevated immunoglobulin levels in response to both T cell-dependent and T cellindependent Ags (161), have more plasma cells (10), and show an enhanced passive cutaneous anaphylaxis compared to WT controls (161). They develop arthritis (164) and Good pasture's syndrome-like disease (165) upon immunization with bovine collagen type II and type IV, respectively when backcrossed into the non-permissive (H-2b haplotype) C57BL/6 background. When backcrossed more than 7 generations into C57BL/6, but not BALB/c background, the Fcγ RIIb−/− 129 mice started to develop spontaneously with high penetrance lupus-like disease. This autoimmune disease is characterized by gender bias, splenomegaly, increase of the proportion of different subsets of activated lymphocytes with age, high titers of ANA, IC-mediated GN and vasculitis in different organs resulting in proteinuria and premature death (112) very similar to the phenotype of the NZM2410 mouse we discussed earlier. This is surprising because, as we have seen, genetic studies revealed that lupus susceptibility is a multigenic phenotype. Monogenic autoimmune diseases are rare (158). However, the strong autoimmune phenotype of the Fcγ RIIb−/− 129 mouse cannot be attributed exclusively to the deletion of the FcgRIIb alleles. This mouse has been generated by gene targeting in 129 derived ES cells and subsequently backcrossed into C57BL/6 background. Such a mouse is, even after 10–12 generations, not fully C57BL/6 but congenic for the 129 derived flanking regions of the targeted allele, containing still hundreds of genes of 129 origin (**Figure 1**). The 129 genome contains more than 1,000 non-synonymous mutations compared to the C57BL/6 genome (166). This is only one part of the problem. Epistasis between 129 derived loci and the C57BL/6 genome also occurs. It has been shown that mice without targeted alleles but congenic for the 129 derived distal-region of Chr1 (Sle16), a lupus-associated region including the autoimmuneprone haplotype 2 of the Slamf genes and the haplotype I of the Fcγ RIIb gene, develop a similar autoimmune phenotype as C57BL/6 Sle1 congenic mice (111). That might explain why several mouse strains generated by targeting genes in the proximity of the Slamf locus in 129 derived ES cells, and backcrossed into C57BL/6 background, develop autoimmunity.

Strikingly, the Fcγ RIIb−/− 129 mouse backcrossed more than seven generations into C57BL/6 background develops ANA with similar selective reactivity to H2A/H2B/DNA sub-nucleosomes as C57BL/6 Sle1 congenic mice, however, with earlier onset, stronger penetrance, and higher titers. Irradiated Rag−/− C57BL/6 or IgH−/− C57BL/6 mice adoptively transferred with bone marrow from Fcγ RIIb−/− 129 mice backcrossed more than seven generations into C57BL/6 background developed antichromatin antibodies and proteinuria, indicating that the disease is fully transferable, dependent on B cells. Myeloid Fcγ RIIb −/− cells are not required (112). This is in keeping with experiments, mentioned earlier, that show that the autoimmune phenotype of C57BL/6 Sle1 congenic mice is completely reconstituted in C57BL/6 irradiated mice that received bone marrow from C57BL/6 Sle1 congenic mice but not by the reciprocal reconstitution. This demonstrates that Sle1 is functionally expressed in B cells (101) although impaired FcγRIIb expression seems to play a minor role in that model (113, 117). Taken together these data all point in the same direction: the strong lupus-like phenotype of the Fcγ RIIb−/− 129 mice backcrossed more than seven generations into C57BL/6 background is caused by epistatic interaction between the Slamf129 locus, the C57BL/6 genome, and Fcγ RIIb−/− (**Figure 2**), similar to the epistatic interactions between Fcγ RIIbNZB (haplotype I), SlamfNZB (haplotype 2) and the C57BL/6 genome in C57BL/6 Nba2 congenic mice (114). As a consequence, the Fcγ RIIb−/− 129 mouse suffers from the confounding effect that the Fcγ RIIb129KO alleles are closely linked to the Slamf129 locus associated with autoimmunity. This means that in most experimental conditions, no distinction can be made between Fcγ RIIb−/− and Slamf129 mediated effects in these mice.

Ig gene analysis of ANA suggests that ANA develop in GCs (167–172). Therefore, analysis of the loss of tolerance in Fcγ RIIb−/− 129 mice focused on GC (173). The role of FcγRIIb as an immune tolerance checkpoint has been studied in a transgenic mouse model in which the variable heavy chain (VH) 3H9H-56R, derived from a dsDNA specific hybridoma, or its variant 56RVH, with higher affinity binding to dsDNA, were inserted in the Igh locus (IgMa allele) (174). Receptor editing, based on the use of specific light chains that abrogates the dsDNA binding, is the main mechanism to maintain tolerance in these mice (175–177). The Ab selection process was compared between WT C57BL/6 and Fcγ RIIb−/− 129 mice carrying the VH transgenes (178). C57BL/6 mice expressing the high-affinity 56R allele (B6.56R) developed low but significant anti-DNA titers, indicating that tolerance was broken, whereas C57BL/6 mice with the low-affinity 3H9 allele (B6.3H9) did not. Tolerance was also maintained in Fcγ RIIb−/− 129 mice carrying the low-affinity 3H9 allele (Fcγ RIIb−/− 129 .3H9). The development of IgM-positive autoreactive B cells was similar in Fcγ RIIb−/− 129 mice carrying the high-affinity 56R allele (Fcγ RIIb−/− 129 .56R) and B6.56R mice. Moreover, Fcγ RIIb−/− 129 .3H9 mice and Fcγ RIIb−/− 129 .56R mice did not show differences in the populations of activated and GC B cells or T cells compared to B6.3H9 and B6.56R control mice. However, Fcγ RIIb−/− 129 .56R mice developed higher IgG anti-DNA titers compared to B6.56R mice. Taking together these observations suggest that the function of FcγRIIb in B6.56R mice is limiting the production of serum IgG anti-dsDNA. Analysis of hybridomas derived from these different mouse strains showed that a much higher percentage of hybridomas from Fcγ RIIb−/− 129 .56R mice secreted IgG antibodies compared to the hybridomas from B6.56R mice. Moreover, Fcγ RIIb−/− 129 .56R mice had a higher percentage of splenocytes with a plasma cell phenotype compared to B6.56R mice. The cross of the Fcγ RIIb−/− 129 mice with autoimmune B cell receptor transgenic mice most likely bypasses the involvement of Slamf129 (which is mainly responsible for the spontaneous development of autoreactive B cells in a C57BL/6 Slamf129 congenic strain, as we will see later). So, in this case, the phenotype of the Fcγ RIIb−/− 129 . 56R mouse can be completely attributed to the absence of FcγRIIb. From these results, it was concluded that the main function of FcγRIIb in the GC reaction is to control, as one of the latest checkpoints, the development of autoreactive IgG-secreting plasma cells and that most likely FcγRIIb deficiency modifies autoimmunity rather than initiates loss of tolerance (178). This was confirmed independently, in an experimental model with two VH chain knockin strains, HKI65 and HKIR, with specificity for the hapten arsonate and a weak and strong specificity for DNA respectively (179). No indications for a role of FcγRIIb in primary or GC tolerance checkpoints were found. Only an increased number of plasma cells was detected in mice that received C57BL/6 HKIR/Fcγ RIIb−/− 129 B cells. FcγRIIb seems to prevent autoimmunity by suppressing the production of autoreactive IgG from B cells that escaped negative selection in GC and enter the AFC pathway (179). This is also in agreement with observations in C57BL/6 Fcγ RIIBNZW congenic mice mentioned earlier (108). However, more recently it has been shown that the number of spontaneous (Spt) GC B cells is increased in 6–7 months old Fcγ RIIb−/− mice on a pure C57BL/6 background, suggesting that FcγRIIb deficiency dysregulates the Spt-GC B cell response [(163); **Table 3**] as will be discussed later.

The view that FcγRIIb acts as a suppressor of autoimmunity caused by other loci is supported by the observed synergism between Fcγ RIIb−/− and several autoimmune susceptibility loci. Just like the Sle1 locus (100), Fcγ RIIb−/− interacts synergistically with the autoimmune susceptibility Yaa locus from BXSB autoimmune-prone mice, containing the Tlr7 gene translocated from the X chromosome to the Y chromosome, resulting in strong acceleration of lupus-like disease in Yaa+Fcγ RIIb−/− 129 male mice (113) (**Figure 2**). MRL/Faslpr/lpr mice develop lupus-like disease whereas C57BL/6 Faslpr/lpr mice do not, likely due to suppressor activity of the C57BL/6 genome. However, C57BL/6 Faslpr/lprFcγ RIIb−/− 129 mice develop systemic autoimmune disease (180). This is consistent with the presence of the haplotype I allelic variant of Fcγ RIIb in MRL mice with an impaired expression on B cell subsets. Mice deficient for both, deoxyribonuclease 1 like 3 (DNASE1L3) and FcγRIIb exhibit at the age of 10 weeks an IgG anti-dsDNA production higher than in 9 months old (NZBxNZW)F1 mice (181). The presence of either the Yaa locus or homozygosity for the Faslpr or Dnase1l3 KO alleles is most likely sufficient to break tolerance. However, FcγRIIb prevents strong autoimmunity by suppressing the production of autoreactive IgG from B cells that have escaped negative selection and enter the AFC pathway. Because Fcγ RIIb−/− 129 mice were used in the crosses mentioned a role for Slamf129 cannot be excluded in these models as indicated by the much milder phenotype of the Yaa+ Fcγ RIIb−/− B6 mouse on pure C57BL/6 background discussed later (109) compared to the severe lupus phenotype of the Yaa+Fcγ RIIb−/− 129 mouse (**Figure 2**). Nevertheless, these observations underscore the crucial role of FcγRIIb in the protection against the development of spontaneous autoimmunity determined by other autoimmune susceptibility loci.

Because of allelic exclusion, Ig transgenic mice do not have a normal B cell repertoire. Therefore, the development of selfreactive GC B cells and plasma cells was studied in Fcγ RIIb−/− 129 mice by large scale Ig cloning from single isolated B cells to determine how loss of FcγRIIb influences the frequency at which autoreactive ANA-expressing B cells participate in GC reactions and develop in plasma cells under physiological conditions (173). In comparison with WT controls the following was observed in Fcγ RIIb−/− 129 mice: (a) No skewing of Ig gene repertoire but enrichment for IgGs with highly positively charged IgH CDR3s which is associated with antibody autoreactivity; (b) lower numbers of somatic mutation; (c) increased numbers of polyreactive IgG+ GC B cells and bone marrow plasma cells and (d) enrichment of nucleosome-reactive GC B cells and plasma cells. The overall frequency of ANAs was high in GC B cells but not in plasma cells. These results demonstrate that in Fcγ RIIb−/− 129 mice IgG autoantibodies including ANAs are expressed by GC B cells and that somatic mutations contribute to the generation of high-affinity IgG antibodies suggesting that the Fcγ RIIb−/−/Slamf129 combination plays an important role in the regulation of autoreactive IgG+ B cells which develop from non-self-reactive or low-self-reactive precursors by affinity maturation (173). It would be of great interest to repeat this analysis in Fcγ RIIb−/− B6 mice on pure C57BL6 background and C57BL/6 Slamf129 congenic mice to define the individual contribution of the Slamf129 locus and the Fcγ RIIb KO alleles in the loss of immune tolerance in the C57BL/6 background. Interestingly the frequency of high-affinity autoreactive IgG+ plasma cells was relatively low, given the high frequency of autoreactive IgG+ GC B cells. This can be explained by the existence of a tolerance checkpoint before GC B cells differentiate into spleen or bone marrow plasma cells, downstream of FcγRIIb and Slamf (173).

Complementation of the mutant phenotype of an organism by expression of a transduced WT gene is considered as the ultimate proof that the mutated gene is the cause of the phenotype. Irradiated autoimmune-prone BXSB, NZM2410, and Fcγ RIIb−/− 129 mice transplanted with autologous bone marrow transduced with a viral vector expressing FcγRIIb showed reduced autoantibody levels and as a consequence much milder disease symptoms compared to mice that received autologous bone marrow transduced with an empty vector (182). These results were confirmed by using a transgenic mouse with a stable 2-fold B cell-specific overexpression of FcγRIIb (183). These mice hardly developed a lupus-like disease when backcrossed into autoimmune-prone MRL/Faslpr/lpr background. The underlying mechanism of these strong effects of overexpression of FcγRIIb is not known. These experiments mainly demonstrate that overexpression of FcγRIIb on B cells inactivates these cells resulting in a strong decrease in autoantibody production. Although they confirm a role of FcγRIIb in autoimmune disease they don't answer the intriguing question whether FcγRIIb deficiency is a modifier of autoimmunity rather than a primary initiator of the loss of tolerance.

### Fcγ RIIb KO on a Pure C57BL/6 Background

To avoid the confounding effect of 129 derived flanking sequences (Sle16), independently, in two different laboratories Fcγ RIIb−/− mice were generated by gene targeting in C57BL/6 ES cells. To distinguish between these two models, one is called here LeFcγ RIIb−/− B6 (109) and the other NYFcγ RIIb−/− B6 (110). LeFcγ RIIb−/− B6 mice exhibit a hyperactive phenotype in the effector phase, although somewhat milder than Fcγ RIIb−/− 129 mice, suggesting a contribution of Sle16 to the phenotype of the Fcγ RIIb−/− 129 mouse in the effector phase (109). Both KO mice develop very mild lupus-like disease (**Table 2**). Total IgG ANA was not significantly increased in 10 months old female LeFcγ RIIb−/− B6 mice compared to C57BL/6 mice although serum of 5% of these mice showed some total IgG anti-dsDNA and anti-ssDNA antibody titers just above (C57BL/6) baseline. In contrast, in 40% of 10 months old NYFcγ RIIb−/− B6 mice total IgG anti-nuclear Abs was significantly increased compared to C57BL/6 mice (110). But only five percent of NYFcγ RIIb−/− B6 mice showed premature death whereas mortality was not increased in LeFcγ RIIb−/− B6 mice although proteinuria and kidney pathology were significantly higher in these mice compared to C57BL/6 mice. The kidney phenotype in the absence of detectable ANA in LeFcγ RIIb−/− B6 mice points to a protective role of FcγRIIb in the kidney, in the efferent phase, as has also been shown in a model TABLE 2 | Disease phenotypes of FcγRIIb−/− B6 , C57BL/6 FcγRIIB−/− 129 SlamfB6 congenic, C57BL/6 Slamf129 congenic and the original FcγRIIb−/− 129 mice compared to WT C57BL/6 control mice at the age of 6–8 months.

aKanari et al. (98).

bSoni et al. (163).

cBoross et al. (109).

dLi et al. (110).

n.d., not determined.

of antibody-induced nephrotoxic nephritis (NTN) that will be discussed later (89).

The production of autoantibodies by C57BL/6 mice in the absence of FcγRIIb suggests that FcγRIIb deficiency, besides modifying autoimmunity caused by other autoimmune susceptibility loci (e.g., Slamf129, Yaa), as discussed earlier, can result in loss of tolerance in the GC. However, it is tempting to speculate that the low titers of autoantibodies, that develop with low penetrance in Fcγ RIIb KO mice on a pure C57BL/6 background, reflect the natural occurring autoreactive B cells in the GC of a WT C57BL/6 mouse, as described earlier, that are prevented to enter the AFC pathway in the presence of FcγRIIb (178). There are indications that C57BL/6 mice are more autoimmune prone than BALB/c mice. For example, B cell receptor editing as a mechanism to maintain B cell tolerance is less effective in these mice compared to BALB/c mice (178).

The NYFcγ RIIb−/− B6 mouse seems to exhibit a stronger disease phenotype than the LeFcγ RIIb−/− B6 mouse (**Table 2**). There are several explanations for this discrepancy:

TABLE 3 | Characteristics of GC B and T cells in NY FcγRIIb−/− B6 , C57BL/6 Slamf129 congenic, and the original FcγRIIb−/− 129 mice compared with WT C57BL/6 control mice.

n.d., not determined (163).

### The Individual Contribution of FcγRIIb Deficiency and Slamf129 to the Phenotype of the FcγRIIb KO Mouse on Mixed 129/C57BL/6 Background

Independently, in two different laboratories congenic C57BL6 Slamf129 mice have been generated. One was generated by intensive backcrossing of the original Fcγ RIIb−/− 129 mouse (161) into C57BL/6 background and selection for offspring in which the Slamf locus and the Fcγ RIIb KO allele had been segregated (98) resulting in two congenic strains called here as C57BL/6 Slamf 129 congenic and C57BL/6 Fcγ RIIb−/− 129 Slamf B6 congenic, respectively. The other C57BL6 Slamf129 congenic mice were generated by a marker-assisted speed congenic approach (163) (**Figure 1**).

The development of autoimmunity was compared between C57BL/6 Fcγ RIIb−/− 129 SlamfB6 congenic, C57BL/6 Slamf129 congenic and the original Fcγ RIIb−/− 129 mice (98) or between C57BL/6 Slamf129 congenic, NYFcγ RIIb−/− B6 and the original Fcγ RIIb−/− 129 mice (163). Both C57BL/6 Fcγ RIIb−/− 129 SlamfB6 congenic and C57BL/6 Slamf 129 congenic mice developed very mild disease symptoms whereas the original Fcγ RIIb−/− 129 mice developed severe disease compared to WT C57BL/6 mice. Importantly, the phenotype of the C57BL/6 Fcγ RIIb−/− 129 Slamf B6 congenic mouse strain confirmed mainly the phenotype of the LeFcγ RIIb−/− B6 mouse [(98); **Table 2**] showing very low ANA titers and little kidney pathology compared to Fcγ RIIb−/− 129 mice.

The development of Spt-GC B cell and TFH responses in C57BL/6 Slamf129 congenic, NYFcγ RIIb−/− B6 and Fcγ RIIb−/− 129 mice were carefully compared [(163); **Table 3**]. C57Bl/6 Slamf129 congenic mice had significantly more GC B cells and TFH and GC TFH cells 12 days after immunization with OVA compared to WT C57BL/6 mice. B cells and DCs from Slamf129 congenic mice exhibited stronger antigen presentation in in vitro assays compared to B cells and DCs from WT C57BL/6 mice. By using a variety of in vivo and in vitro assays with naïve B cells it was found that B cell-intrinsic deficiency of FcγRIIb and expression of Slamf129 has no effect on proliferation but promotes differentiation of naïve B cells into GC B cells as indicated by increased expression of Aicda and GL-7. The percentage of apoptotic GC B cells was significantly lower in Fcγ RIIb−/− 129 mice compared to WT C57BL/6 mice whereas in C57BL/6 Slamf129 congenic and NYFcγ RIIb−/− B6 mice this decrease was not significant. This suggests that FcγRIIb deficiency and Slamf129 act synergistically to increase the survival of GC B cells in Fcγ RIIb−/− 129 mice. Naïve and activated B cells from NYFcγ RIIb−/− B6 and to a lower extent from C57BL/6 Slamf129 congenic mice showed an enhanced metabolic capacity compared to B cells from C57BL/6 mice. This enhancement was stronger in Fcγ RIIb−/− 129 mice.

Taken together these observations suggest that Slamf129 plays a predominant, and FcγRIIb deficiency a modest role in modulating the Spt-GC B cell and TFH responses. Some of their functions are synergistic others mutually exclusive. GC TFH cell responses are mainly affected by Slamf129 [(163); **Table 3**]. By using the experimental model of the VH chain knockin strain HKIR mentioned earlier (179) it was demonstrated that B cell-specific expression of Slamf129 is necessary for the autoreactive B cells to expand in the GC confirming previous observations in C57BL/6 Sle1 congenic mice (129).

The increased Spt-GC responses in NYFcγ RIIb−/− B6 and C57BL/6 Slamf129 congenic mice were associated with the production of autoantibodies. However, the titers were much lower than in Fcγ RIIb−/− 129 mice which had also the strongest increase in Spt-GC responses. C57BL/6 Slamf129 congenic mice developed higher ANA titers than NYFcγ RIIb−/− B6 mice, staining both cytoplasm and nucleus of Hep-2 cells, whereas sera from NYFcγ RIIb−/− B6 mice show only cytoplasmic staining patterns (163) confirming previous results with the C57BL/6 Fcγ RIIb−/− 129 SlamfB6 congenic mouse strain (98). IgG2b and IgG2c ANA were significantly increased in C57BL/6 Slamf129 congenic mice whereas only IgG2c ANA were significantly increased in NYFcγ RIIb−/− B6 mice. With an autoantigen array, it was shown that Fcγ RIIb−/− 129 mice develop high titers of IgG antibodies against a large variety of autoantigens. Several of these antibodies were also present in the serum of NYFcγ RIIb−/− B6 mice but their titers were much lower than in Fcγ RIIb−/− 129 mice (163). Unfortunately, sera from C57BL/6 Slamf129 congenic mice were not tested in the autoantigen array.

Kidney pathology was absent (98) or very mild, with higher complement deposition than NYFcγ RIIb−/− B6 mice (163), in C57BL/6 Slamf129 congenic mice, mild in NYFcγ RIIb−/− B6 or C57BL/6 Fcγ RIIb−/− 129 SlamfB6 congenic mice with higher IgG deposition than in C57BL/6 Slamf129 congenic mice, and severe, with highest C3 and IgG deposition compared to the other genotypes, in Fcγ RIIb−/− 129 mice (98, 163). In conclusion, the deficiency of FcγRIIb together with the presence of Slamf129 results in a phenotype of the Fcγ RIIb−/− 129 mouse with increased Spt-GC B cell responses characterized by an increase of the following parameters: metabolic activity in B cells, differentiation of B cells into a GC B cell phenotype and GC B cell survival. This is associated with loss of immune tolerance resulting in ANA production and the development of severe lupus-like disease (163). However, the underlying cellular and molecular mechanisms of these associations are not well-understood and the subject of speculation and debate with respect to the role of FcγRIIb in GC (185). This can be illustrated with the surprising observation in the Fcγ RIIbNZB KI mouse model mentioned earlier, in which FcγRIIb failed to be upregulated on activated and GC B cells resulting in enhanced early GC responses (11). Upon immunization, these KI mice showed an early and sustained increased affinity maturation of Ag-specific GC B cells. Previous models suggest that low expression of FcγRIIb reduces the BCR activation threshold resulting in less affinity maturation. However, an alternative explanation might be that low FcγRIIb expression increases the survival of bystander Ag non-specific GC B cells and, as a consequence, increases competition for TFH help between Ag-specific and non-antigen specific B cells, resulting in increased affinity maturation (11).

### Cell-Type-Specific Fcγ RIIb KO Mouse Models

To determine on what B cell subset(s) and on what myeloid cells FcγRIIb might be involved in a checkpoint for immune tolerance, cell-type-specific Fcγ RIIb−/− mice were generated, independently, in two different laboratories. Both the LeFcγ RIIb−/− B6 and NYFcγ RIIb−/− B6 mouse models, on a pure C57BL/6 background, were originally generated as floxed Fcγ RIIb mice (Fcγ RIIbfl/fl B6 ) and subsequently crossed with a Cre deleter transgenic mouse to generate the germline Fcγ RIIb−/− B6 mice discussed earlier. In addition, the Fcγ RIIbfl/fl B6 mice were also crossed with a variety of cell type-specific Cre transgenic mice (**Table 4**) to generate cell-type-specific Fcγ RIIb−/− B6 strains that were analyzed in the following models of diseases for which germline Fcγ RIIb KOs are highly susceptible: (a) the induced autoimmune diseases CIA, both on permissive (immunization with chicken collagen type II) and non-permissive (immunization with bovine collagen type II) background and (b) anti-glomerular basement membrane antibody (anti-GBM) disease, (c) the spontaneous autoimmune disease lupus-like disease and (d) the non-autoimmune disease antibody-induced NTN.

Deletion of Fcγ RIIb in all B cells of the LeFcγ RIIbfl/fl B6 mouse by CD19Cre did not increase the susceptibility of this mouse for any of the mentioned disease models. Moreover, deletion of Fcγ RIIb on a subset of monocytes (LysMCre) had no effect on susceptibility for anti-GBM disease. Therefore, it was concluded that FcγRIIb deficiency on B cells or a subset of myeloid cells alone is not sufficient to increase susceptibility to anti-GBM (186). Only pan-myeloid deletion (cEBPαCre) of FcγRIIb increased the susceptibility of LeFcγ RIIbfl/fl B6 mice for CIA on the permissive background (187) and for the non-autoimmune disease NTN (89). These results suggest that for the protection against induced auto-Ab driven diseases, such as CIA, the role of FcγRIIb on B cells, as a checkpoint for immune tolerance, is less important than its role on myeloid effector cells, controlling downstream antibody effector mechanisms (187). However, it cannot be excluded that in the CIA model FcγRIIb on myeloid cells also plays a role in controlling the afferent phase of the disease, as was recently shown in Yaa+ LeFcγ RIIb−/− B6 mice that will be discussed later (188).

In contrast to the results with LeFcγ IIbfl/fl B6 mice, deletion of FcγRIIb in all B cells (Mb1Cre) or in GC and post GC B cells (Cg1Cre) in NYFcγ RIIbfl/fl B6 mice resulted in increased susceptibility for CIA on the non-permissive background and permissive background, respectively. Moreover, susceptibility to CIA was also increased in DC-specific CD11cCre/ NYFcγ RIIbfl/fl B6 mice indicating that FcγRIIb is involved in distinct immune tolerance controlling mechanisms (110). The reason for the discrepancy between the phenotypes of the B cell- and DCspecific NYFcγ RIIb−/− B6 and LeFcγ IIb−/− B6 mice is not known but, given the weak phenotype of the germline LeFcγ RIIb−/− B6 mouse, most likely the phenotype of a single cell-type-specific LeFcγ RIIb−/− B6 mouse is too weak to be detected with a small cohort of mice. Another partial explanation might be that the B-cell-specific Cre lines used are different. In addition, GC and post GC B cell (Cg1Cre) specific NYFcγ RIIb−/− B6 mice developed spontaneously ANA, similar to ANA in germline NYFcγ RIIb−/− B6 mice, whereas a deficiency in other cell types has no effect. This confirms previous results with transplantation of bone marrow from Fcγ RIIb−/− 129 mice that the role of FcγRIIb in the spontaneous development of ANA is B cell-specific (112) and suggests that FcγRIIb on GC or post GC B cells is a checkpoint for the maintenance of immune tolerance (110) (**Table 4**).

Upon immunization with the NP-CGG model antigen NYFcγ RIIb− B6 /− and Mb1Cre/NYFcγ RIIbfl/fl B6 mice developed similar increased primary IgG NP-specific Ab responses compared to NYFcγ RIIbfl/fl B6 mice and all other cell type-specific

TABLE 4 | Disease susceptibility of cell-type-specific FcγRIIb KO mice.

Germline FcγRIIb KO mice showed increased susceptibility to all diseases listed in the table compared with C57BL/6 mice.

aSharp et al. (186).

bSharp et al. (89).

cYilmaz-Elis et al. (187).

dLi et al. (110).

n.d., not determined.

NYFcγ RIIb−/− B6 mice. In contrast, secondary IgG Ab responses were increased in both Mb1Cre/NYFcγ RIIbfl/fl B6 and Cg1Cre/NYFcγ RIIbfl/fl B6 mice compared with NYFcγ RIIbfl/fl B6 mice. This suggests that FcγRIIb is a B cell-intrinsic negative regulator of both primary and secondary IgG responses (110).

Although individually not sufficient to induce substantial autoimmunity, epistasis between the Yaa locus, the LeFcγ RIIb−/− B6 alleles and the C57BL/6 genome results in severe lupuslike disease (109) (**Figure 1**). The cell-type-specific role of FcγRIIb in this genetic disease model was studied (188). The Yaa +/CD19Cre/LeFcγ RIIbfl/fl B6 mice developed milder lupus-like disease than Yaa+/ LeFcγ RIIb−/− B6 mice similar to the disease in Yaa+/C/EBPα Cre/LeFcγ RIIbfl/fl B6 mice whereas Yaa+/CD11cCre/LeFcγ RIIbfl/fl B6 mice stayed disease free, like Yaa+/ LeFcγ RIIbfl/fl B6 mice. This suggests that besides on B cells FcγRIIb on myeloid cells, but surprisingly not on DCs, contributes to the protection against spontaneous loss of immune tolerance in this mouse model. This confirms the observation with CIA in mice (110), discussed earlier, that FcγRIIb can be involved in different immune tolerance controlling mechanisms.

Strikingly, in the two strains with FcγRIIb deficient myeloid cells (Yaa+/ LeFcγ RIIb−/− B6 and Yaa+/C/EBPα Cre/LeFcγ RIIbfl/fl B6 ) but not in the strain with B cell-specific FcγRIIb deficiency (Yaa+/CD19Cre/LeFcγ RIIbfl/fl B6 ) the frequency of peripheral Ly6C−, but not Ly6C+ monocytes was increased. Monocytosis, an FcRγ dependent expansion of the monocyte compartment consisting mainly of Ly6C− monocytes, is associated with the development of lupus nephritis in Yaa+ lupus-prone mice. It has been reported that Ly6C+ monocytes mature in the circulation and are the precursors for Ly6C− monocytes (189). Deficiency of FcγRIIb most likely accelerates the maturation of monocytes in Yaa+/ LeFcγ RIIb−/− B6 mice. Compared to Ly6C+ monocytes, mature Ly6C− monocytes express significantly higher B cellstimulating cytokines such as BSF-3, IL-10, and IL-1β, DC markers including CD11c, CD83, Adamdec1, and the antiapoptotic factors Bcl2 and Bcl6. This makes monocytes the most promising FcγRIIb expressing candidate myeloid cells to modulate B cell tolerance (188, 190). The transcriptome of Ly6C− monocytes suggests that they are long-lived and committed to developing into DCs.

Whether this monocyte-dependent tolerance breaking mechanism is unique for Yaa+/Fcγ RIIb−/− B6 mice is not known but it is striking that also in SLE patients the serum levels of anti-dsDNA Abs highly correlate with the percentage of non-classical monocytes (191). Like mouse Ly6C− monocytes, the human counterpart CD14lowCD16+ monocytes secrete high amounts of IL-1β in a TLR7-TLR8-MyD88–dependent manner (192).

### CONCLUDING REMARKS

Forward and reverse genetics have provided convincing evidence that FcγRIIb is an important autoimmune susceptibility gene, involved in the maintenance of peripheral tolerance both in human and mice. In humans, a number of GWAS studies showed an association between a SNP (rs1050501) in the FCGR2B gene, causing a missense mutation (FCGR2BT232) resulting in impaired FCGR2B function, and susceptibility to SLE. Metaanalyses confirmed that FCGR2BT232 homozygosity is one of the strongest associations in SLE. Association of rs1050501with RA has also been reported.

In mice, the situation is more diffuse. Analysis of a variety of C57BL/6 mice congenic for the NZW and NZB haplotypes of Fcγ RIIb, with decreased expression, did not reveal clear unambiguous results with respect to the contribution of these haplotypes to the autoimmune phenotypes of these mice. The mechanism by which natural FcγRIIb variants contribute to autoimmunity is not well-understood.

The first Fcγ RIIb−/− mouse, generated by gene targeting in 129 derived ES cells and backcrossed into C57BL/6 background (Fcγ RIIb−/− 129 mice), exhibited a surprisingly strong spontaneous autoimmune phenotype suggesting that FcγRIIb deficiency initiates loss of immune tolerance. However, independent studies with Fcγ RIIb−/− 129 autoimmune VH chain knockin mice pointed to a central role of FcγRIIb in a late immune tolerance checkpoint, that prevents autoimmunity by suppressing the production of autoreactive IgG from B cells, that escape negative selection in the GC and enter the AFC pathway. This should mean that FcγRIIb deficiency is mainly an amplifier of autoimmunity caused by other autoimmune susceptibility loci, rather than a primary initiator of the loss of immune tolerance. That was confirmed by the observation that Fcγ RIIb−/− mice on a pure C57BL/6 background (Fcγ RIIb−/− B6 ) have a much milder autoimmune phenotype than Fcγ RIIb−/− 129 mice but when backcrossed into a mouse strain carrying the autoimmune susceptibility Yaa locus succumb to lupus-like disease. The strong autoimmune phenotype of the Fcγ RIIb−/− 129 mouse could be explained by epistatic interactions between the C57BL/6 genome, the FcγRIIb KO allele and the 129 derived sequences (Sle16) flanking the Fcγ RIIb KO allele, containing the autoimmunity associated Slamf129 (haplotype 2) gene cluster.

Spt-GC B and TFH cells are activated, modestly (mainly B cells) in Fcγ RIIb−/− B6 mice, moderately in C57BL/6 Slamf129 congenic mice and strongly in Fcγ RIIb−/− 129 mice compared to Spt-GC B and TFH cells in WT C57BL/6 mice. This was associated with a corresponding increase in ANA production, suggesting that FcγRIIb deficiency, besides enhancing autoimmunity caused

### REFERENCES

by other autoimmune susceptibility loci, might play a modest role in the induction of the loss of immune tolerance in the GC, explaining the development with low penetrance of low ANA titers in Fcγ RIIb−/− B6 mice. An alternative explanation is that the low ANA titers in Fcγ RIIb−/− B6 mice reflect the natural background of autoreactive B cells in the GC that are prevented to enter the AFC pathway in the presence of FcγRIIb. The analysis of the development of self-reactive GC B cells and plasma cells by large scale Ig cloning from single isolated B cells, as performed with Fcγ RIIb−/− 129 mice, should be repeated in Fcγ RIIb−/− B6 mice, to determine how FcγRIIb deficiency influences the frequency at which autoreactive ANA-expressing B cells participate in GC reactions, and develop in plasma cells, under physiological conditions, without the confounding effect of Slamf129 expression.

Studies with cell-type-specific FcγRIIb deficient mice revealed that besides on B cells, FcγRIIb on DCs and monocytes can also contribute to the maintenance of immune tolerance, indicating that FcγRIIb is involved in different immune tolerance maintaining mechanisms. Series of observations suggest that on B cells impaired FcγRIIb function effects not only antibody titers but also affinity maturation and memory responses of B cells and plasma cell homeostasis associated with an increase in the production of autoantibodies. However, the underlying cellular and molecular mechanisms are not well-understood. Most likely new model systems including adoptive cell transfer and tools such as cell type-specific KO mice, to study the GC reaction, are required to answer these questions.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

JV was supported by Japan Society for the Promotion of Science (FY2017 JSPS invitational Fellowship for Research in Japan, L17559).

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Verbeek, Hirose and Nishimura. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# There Is (Scientific) Strength in Numbers: A Comprehensive Quantitation of Fc Gamma Receptor Numbers on Human and Murine Peripheral Blood Leukocytes

Christina Kerntke1 , Falk Nimmerjahn1,2 \* and Markus Biburger 1,2 \*

1 Division of Genetics, Department of Biology, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany, 2 Medical Immunology Campus Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany

#### Edited by:

Latha P. Ganesan, The Ohio State University, United States

#### Reviewed by:

Friederike Jönsson, Institut Pasteur, France Wanli Liu, Tsinghua University, China Margaret E. Ackerman, Dartmouth College, United States

#### \*Correspondence:

Falk Nimmerjahn<EMAIL_ADDRESS>Markus Biburger<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

Received: 02 August 2019 Accepted: 16 January 2020 Published: 05 February 2020

#### Citation:

Kerntke C, Nimmerjahn F and Biburger M (2020) There Is (Scientific) Strength in Numbers: A Comprehensive Quantitation of Fc Gamma Receptor Numbers on Human and Murine Peripheral Blood Leukocytes. Front. Immunol. 11:118. doi: 10.3389/fimmu.2020.00118 Antibodies are essential mediators of immunological defense mechanisms, are clinically used as therapeutic agents, but are also functionally involved in various immune-mediated disorders. Whereas IgG antibodies accomplish some of their biological tasks autonomously, many functions depend on their binding to activating and inhibitory Fcγ receptors (FcγR). From a qualitative point of view expression patterns of FcγR on immunologically relevant cell types are well-characterized both for mice and humans. Surprisingly, however, there is only quite limited information available on actual quantities of FcγR expressed by the different leukocyte populations. In this study we provide a comprehensive data set assessing quantitatively how many individual human and mouse FcγRs are expressed on B cells, NK cells, eosinophils, neutrophils, basophils and both classical, and non-classical monocytes under steady state conditions. Moreover, among human donors we found two groups with different expression levels of the inhibitory FcγRIIb on monocytes which appears to correlate with haplotypes of the activating FcγRIIIa.

Keywords: Fc receptors, antibodies, human leukocytes, murine leukocytes, quantification, receptor numbers, neutrophils, monocytes

### INTRODUCTION

Antibodies of the immunoglobulin G (IgG) isotype are essential mediators of immunological defense mechanisms. Whereas they carry out some of their biological functions autonomously, such as blocking of cell surface receptors, most of their functions depend on the binding of the antibody Fc-domain to specific receptors, the Fc gamma receptors (FcγR). Whereas binding of immune complexes (IC) is possible to all FcγR including the low/medium receptors FcγRIIa and FcγRIIb, FcγRIIIa and FcγRIIIb in man and FcγRIIb, FcγRIII and FcγRIV in mice, monomeric IgG molecules can only bind to human or murine high affinity receptor FcγRI [reviewed e.g., in (1–3)].

The repertoire of murine and human FcγR includes the inhibitory FcγRIIb, which contains an intracellular immunomodulatory tyrosine based inhibitory motif (ITIM) whereas most other FcγR carry an activating ITAM motif. These motifs are either located in the same polypeptide chain as the ligand binding domain (FcγRIIb and FcγRIIIb) or on an accessory Fc receptor gamma chain. One exception is the human FcγRIIIb which contains no transmembrane or intracellular polypeptide domain, but is rather linked to the cell membrane by a GPI anchor (4).

Immune cell populations are characterized by a typical expression pattern of FcγR. Whereas most of them express both activating and inhibitory FcγR, B cells only express the inhibitory FcγRIIb, which regulates the activity of the B cell receptor. Natural Killer (NK) cells on the other side selectively express the activating FcγRIII. Thus, from a qualitative point of view expression patterns of FcγR on different immune cell subsets are well-characterized both, for mice and man (1–3, 5–12).

For many cell types, their functional response to external signals is accompanied by pronounced up- or down-regulation of various surface receptors. This clearly shows that the amount of any given surface receptor on a cell is an important characteristic for its physiological functionality. The outcome of immune complex binding to a cell is determined by the sum of activating and inhibitory signals triggered through the respective activating and inhibitory FcγRs. One factor influencing these signals is the affinity of the respective IgG isotype to the distinct FcγRs present on the cell surface. This led to the development of the concept of the so called A/I ratio as a prediction of the outcome of binding of IgG molecules of a given isotype based on its affinities to the inhibitory and activating receptors, respectively (13–15). Based on the A/I ratio concept mathematical models have been developed predicting IgG activity (16). However, it is to be expected that other factors such as the avidity of the immune complex which is influenced by the number of immunoglobulins bound to the antigen and the number of activating and inhibitory receptors on the surface of the respective effector cell may influence IgG effector functions (17). Thus, knowledge of the quantities of the various FcγR on the cell surface of immune cell subsets may help to develop more precise models to understand how IgG antibodies trigger cellular responses.

Whereas FcγR expression is rather well-described qualitatively, there is only quite limited information available on actual numbers of FcγR expressed by the different leukocyte populations (18–35). To our knowledge there is no publication, where such numbers have been determined for the main leukocyte populations in parallel, even though Antal-Szalmas et al. (18) and the group of Guyre (19, 22–25) provided data on human neutrophils and monocytes. In addition, work on FcγR cell surface numbers often dates back to times, where certain cell subpopulations with an entirely different FcγR repertoire (such as classical and non-classical monocytes), had not been distinguished.

Flow cytometry is a common method for the analysis of cell surface receptors. Qualitative comparison of the relative expression of a given cell surface receptor on different cell populations based on the fluorescence intensities mediated by binding of fluorochrome-labeled antibodies specific for this receptor is rather straightforward. In contrast, quantitative comparison of different receptors on the same or different cell populations by simple comparison of fluorescence intensities is not possible since there are inherent variations between the different anti-receptor antibody conjugates with respect to their specific fluorescence (i.e., fluorescence intensity per molecule). This is obvious when conjugates with different fluorochromes are used. However, even antibodies conjugated with the same fluorochrome may differ in the stoichiometry of fluorophores per antibody molecule. This variability can be partially reduced by using conjugates with very large fluorophores like R-Phycoerythrin (PE), where usually not more than one fluorochrome can be linked to each antibody molecule (36). In spite of this advantage of PE studies by David et al. suggest that there is also a variation in the quantum yield of PE from various organisms (37). The information, from which species the used PE is derived from, is usually not available from the antibody companies or providers of labeling kits. In addition, also for PE the stoichiometry of fluorophores per antibody molecule for any lot of antibody conjugate may be <1 due to partial degradation or incomplete coupling.

To achieve a more definitive estimate of receptor numbers per cell the establishment of reference curves comparing the observed fluorescence to the fluorescence of well-defined reference beads appears to be better suited. Thus, we used sets of commercially available beads with distinct numbers of binding sites for anti-FcγR antibodies, respectively, and established reference curves for all anti-FcR monoclonal antibody (mAb) conjugates used in this study. Based on these references we present here the ABC values (i.e., the antibody-binding capacity for respective anti-FcγR antibodies) for all activating and inhibitory FcγR on the main leukocyte populations in peripheral blood of C57BL/6 and FcγR knockout mice and in healthy human volunteers under steady state conditions. This includes Natural Killer (NK) cells, B cells, T cells, neutrophils, eosinophils and basophils as well as classical and non-classical monocytes. These monocyte subsets have comparable population size in murine blood. In contrast, in humans the classical monocytes represent the vast majority of monocytes whereas non-classical monocytes represent only a small fraction of the monocyte population but nonetheless exert important biological functions. In both species these subsets have differential FcγR repertoires and, thus, specific effector functions upon engagement with immune complexes [see e.g., (38)].

### MATERIALS AND METHODS

### Mice

Female mice at 8–16 weeks of age on C57BL/6 background were used in all experiments. C57BL/6J mice (JAX strain 000664) were purchased from Janvier (Le Genest-Saint-Isle, France). FcγRI deficient mice (39) were originally provided by M. Hogarth (JAX number: not available (N/A)), FcγRIIb deficient [(40), JAX number: N/A], FcγRIII deficient (JAX number N/A) and FcγRIV deficient [(41), JAX number: N/A] mice by J. Ravetch. Mice were kept in the animal facilities of Friedrich-Alexander-University Erlangen-Nürnberg under specific pathogen-free conditions in individually ventilated cages according to the guidelines of the National Institutes of Health and the legal requirements in Germany. Animal experiments conducted in the animal facility of the FAU were approved by government of lower Franconia.

### Human Donors

For the characterization of human leukocytes venous blood of male and female healthy adults was used. The use of human material for scientific purposes was carried out in accordance with the recommendations of and approved by the ethics committee of the Friedrich-Alexander University Erlangen-Nürnberg. All subjects gave written informed consent regarding usage of their biological material for the scientific research presented here.

### Preparation of Murine and Human Peripheral Blood Leukocytes

For isolation of murine PBLs blood was drawn from the retro-orbital plexus using anti-coagulant micro hematocrit capillaries. Human PBLs were isolated from venous blood of male and female healthy human adults using anticoagulant EDTA Monovettes. Erythrocytes from both murine and human blood were lysed using deionized H2O and subsequent restoration of iso-osmolality. After repeated washing in cold FACS buffer containing sodium azide to inhibit changes in the surface presentation of proteins, cells were continued processing for flow-cytometric analysis.

### Flow Cytometry

### Characterization of Murine PBLs

Single cell suspensions with typically 1–2 × 105 cells per sample were usually incubated for 15 min on ice with Fc-block antibodies to minimize unspecific binding to Fc receptors, followed by staining with fluorochrome-coupled antibodies for ∼20 min. Since single FcγRs were to be stained specifically, full Fc block was inconvenient. We, thus, pretreated cells with anti-CD16/32 clone 2.4G2 to block FcγRII and III only when FcγRIV was quantified. This Fc block was not used in analyses of FcγRI, since 2.4G2 may also block high affinity receptor FcγRI via its Fc-part on cells were the antibody is bound in cis to FcγRII or III (42). Since we have recently shown that also medium-affinity receptor FcγRIV can bind the Fc-part of several rat and mouse IgG subclasses and cause false positive results in flow cytometry (43), FcγRIV was blocked by clone 9E9 in all analyses where other receptors than FcγRIV were to be analyzed.

For the identification of cell populations we used antibodies against the following antigens: B220 (clone RA3-6B2, APC conjugated, BD Biosciences or FITC conjugated Biolegend), CD3e (clone 145-2C11, FITC conjugated, Biosciences and Biolegend or BV510 or AlexaFluor 647 conjugated, Biolegend), CD11b (clone M1/70, PerCP-Cy5.5 conjugated), CD19 (clone 6D5, BV510 conjugated, Biolegend), CD45 (clone 30-F11, APC-Cy7 conjugated BD Biosciences and Biolegend or APC-Fire750 conjugated, Biolegend), CD49b (clone DX5, APC conjugated, BD Biosciences), CD62L (clone MEL-14, PE-Cy7 conjugated, Biolegend), Gr1 (clone RB6-8C5, APC- or AlexaFluor 647 conjugated, BD Biosciences and Biolegend or BV510 conjugated, Biolegend), IgE (clone R35-72, FITC conjugated, BD Biosciences), Ly6G (clone 1A8, FITC-conjugated, provided by BD Biosciences and Biolegend), NK1.1 (clone PK136, FITC conjugated, Southern Biotech, BD Biosciences and Biolegend), and TCRß (clone H57-597, FITC conjugated, Biolegend). Flow cytometric measurements were carried out on a FACSCanto II (BD Bioscience). Briefly, cell aggregates were excluded by their light scatter characteristics. Dead cells were excluded from analysis using 4′ ,6- diamidino-2-phenylindole (DAPI). Single viable CD45+ leukocytes were divided into SSChigh granulocytes and further distinguished into neutrophils and eosinophils by Ly6G. Among SSClow cells NK cells were characterized as NK1.1+ CD11bintermediate and monocytes as NK1.1− CD11bhigh . Monocytes were further distinguished by expression of CD62L and Gr1 (high for classical monocytes and negative-low for non-classical monocytes). Among SSClow CD11b negative cell B cells were characterized by binding of anti-B220 whereas T cells were B220 negative and, in addition, in some experiments were characterized as positive for TCRß or CD3e. Murine basophils were identified as cells with low side-scatter characteristics which were negative for lineage markers CD19, CD3 and Gr-1 but were CD49b+ and positively stained for IgE. A representative gating for murine leukocytes is shown in **Supplementary Figure 1**.

### Characterization of Human PBLs

Antibodies detecting the following antigens were used for the characterization of human leukocyte populations: CD3 (clone SK7, PE-Cy conjugated, BD Biosciences), CD14 (clone HCD14, FITC conjugated and clone M5E2, PerCP-Cy5.5 conjugated, Biolegend), CD16 (clone 3G8, PE-Cy7 conjugated, BD Biosciences), CD19 (clone HIB19, APC conjugated, BD Biosciences and Biolegend), CD33 (clone WM53, BV510 conjugated, Biolegend), CD45 (clone HI30, APC-Fire750 or APC-H7 conjugated, Biolegend), CD56 (clone MEM188, PerCP-Cy5.5 conjugated and FITC conjugated, Biolegend), CD123 (clone 6H6, PE-Cy7 conjugated, Biolegend), FcεRIA (clone AER-37 (CRA-1), PerCP-Cy5.5 conjugated, Biolegend), HLA-DR (clone L243, APC conjugated, Biolegend). Per sample typically 1–2 × 105 human leukocytes were used.

To avoid unspecific binding of antibodies for the quantification of human Fc receptors to any other FcγR via their Fc part, we utilized the Human TruStain FcXTM Fc Receptor Blocking Solution (Biolegend). This Fc-block protects from Fc-mediated binding to FcγRs by pre-occupying their Fc binding sites, but does not inhibit antigen-specific detection of anti-huFcγR antibodies which takes place with higher affinity. In pretests we verified that this reagent did not affect antigen-specific FcγR-detection, prior to its employment in quantification experiments (**Supplementary Figure 2**). FcγRIIb quantification took additional advantage from the fact that the anti-FcγRIIb antibody is a 2B6-variant where N-glycosylation is prevented. Since binding to FcγRs via the Fc-part is impaired for a-glycosylated antibodies (44, 45), undesirable Fc-mediated binding of 2B6 to other Fc receptors is prevented. Flow cytometric measurements were carried out on a FACSCanto II (BD Bioscience). Briefly, cell aggregates were excluded by their light scatter characteristics and dead cells were excluded from analysis by DAPI staining.

Among CD45+ leukocytes, neutrophils and eosinophils were identified by their high granularity resulting in their distinct light scatter characteristics (SSChigh) and distinguished by the CD16 expression of neutrophils and/or by the intrinsic autofluorescence of the eosinophils. Among SCClow cells NK cells were gated as being CD56 positive but negative for CD14 and CD33. Monocytes were identified in the CD56− population by the expression of CD33 and CD14. They were further distinguished as classical CD14high CD16low and much less frequent non-classical CD14low CD16+ monocytes. Within the CD33- CD14- population B cells were identified by their expression of CD19 and T cells by the expression of CD3 in absence of CD56. Basophils were identified as SSClow CD45dim CD123+ HLA-DR negative cells, which were positively stained for IgE receptor FcεR1. In addition to leukocytes we analyzed human platelets, which were characterized by their small size as reflected by low light scatter and by their expression of CD41a. A representative gating strategy for human leukocytes and platelets is shown in **Supplementary Figure 3**.

### Quantification of Fc Receptors

Fcγ receptors on leukocytes were quantified by measuring their Antibody Binding Capacity (ABC) for antibodies specific for the respective FcγR. ABC values on leukocytes were calculated using a specific reference curve for the correlation between fluorescence intensity of a cell upon binding by the respective fluorochrome-conjugated anti-FcγR antibody and the number of antibody binding sites. These reference curves were generated using sets of Quantum Simply Cellular (QSC) microspheres (Bangs Laboratories Ltd.) with known numbers of antibody binding sites as provided by the manufacturer. Beads and cells were stained with the same concentration of the respective anti-FcγR antibodies. Reference curves were established in each experiment for the analyzed anti-FcγR antibodies. According to manufacturer's instructions a titration curve should be prepared for every quantitating antibody using the QSC beads to determine its saturating concentration. However, earlier experiments suggested that this concentration might not be sufficient to saturate all binding sites on cells for all antibodies. Insufficient saturation of binding sites on cells with full saturation on reference beads will lead to an underestimation of the number of binding sites on the target cells. The difference between saturating concentrations for cells and QSC beads is easily conceivable since binding to cells is achieved via antigenspecific binding domains in the Fab regions whereas binding to beads is achieved via other antibody domains probably via the Fc-region and binding to both entities may take place with very different binding affinities. We, thus, suggest titrating quantitating antibodies both for binding to cells and to QSC beads in order to aim at saturating binding to both entities.

The following PE-conjugated anti-FcγR antibodies were used for mouse receptor quantification: anti-msFcγRI/CD64 mouse IgG1 clone X54-5/7.1 (BD Biosciences), anti-msFcγRIIb/CD32b mouse IgG2a clone Ly17.2 (in-house production and labeling), anti-msFcγRIII/CD16 rat IgG2a clone 275003 (R&D Systems), anti-msFcγRIV Arm. hamster IgG clone 9E9 (in-house production and labeling or Biolegend).

PE-labeled antibodies specific for human FcγR were: antihuFcγRI/CD64 mouse IgG1 clone 10.1 (BD Biosciences), anti-huFcγRIIb/CD32B humanized IgG1 clone 2B6 (in-house production and labeling) and anti-huFcγRIII/CD16 mouse IgG1 clone 3G8 (Biolegend). Since no fully FcγRIIa/CD32A-specific mAb was at hand, expression of this receptor was analyzed by staining with a anti-CD32 antibody (clone IV.3, antibodiesonline GmbH) after pre-blocking of CD32B with clone 2B6 N297Q which recognizes specifically FcγRIIb but not FcγRIIa (46). Among anti-human FcγRII antibodies IV.3 appears to be one of the clones with the most preferential binding to FcγRIIa over FcγRIIb. However, under conditions of saturating binding to FcγRIIa expressing leukocytes, which are important for reliable quantification of antibody-binding sites by this method, even IV.3 reveals pronounced binding to FcγRIIb e.g., on B cells (**Supplementary Figure 4**). Binding of IV.3 to FcγRIIb can be efficiently blocked by pre-treatment with anti-FcγRIIb antibody 2B6 (10µg/ml) without affecting binding to highly FcγRIIa-expressing monocytes (**Supplementary Figure 4**). Thus, pre-blocking of FcγRIIb is a versatile and necessary step for reliable quantification of FcγRIIa expression by IV.3.

Human FcγRs I and III could be detected directly by antibody clones 10.1 and 3G8, followed by using anti-mouse IgG QSC beads. In a similar manner, human FcγRIIb was detected with the antibody clone 2B6 and by using anti-human IgG QSC beads. To quantify FcγRIIb we used a PE-conjugated recombinant 2B6 whose Fc part is of human origin. Thus, we employed QuantumTM Simply Cellular R (QSC) anti-human beads to establish a reference curve for 2B6 binding sites.

Anti-FcγR antibodies were purchased from BD Biosciences, BioLegend, R&D Systems Europe or prepared in house. To minimize potential systematic variations in the quantification of different receptors by engagement of different fluorophores we used a single type of fluorophore. We chose Phycoerythrin (PE) for this purpose, since due to its size there is typically one fluorochrome conjugated to each antibody molecule, thereby minimizing variations in specific fluorescence and it lacks the pronounced self-quenching capacity of fluorochromes like FITC (47). Anti-FcγR antibodies were either purchased pre-labeled or were conjugated in-house. According to the host species of the respective anti-FcγR antibody, we used anti-mouse IgG, antirat IgG or anti-human IgG QSC beads following manufacturer's instructions. Since anti-FcγRIV is derived from Armenian hamster and no QSC beads specifically binding antibodies of this species are available, we performed a sandwich-assay where anti-mouse IgG beads were pre-coated with mouse antihamster antibody. The formulation of the latter is a commercially available equal mixture (BD Biosciences) of two murine antibody clones specific for either hamster IgG1 or hamster IgG2-3, respectively. The anti-FcγRIV clone 9E9 is an Armenian hamster IgG not further characterized regarding the IgG subtype. It should, thus bind to one of these two mouse IgG clones. Assuming that upon loading QSC microspheres with this mixture half of the anti-mouse IgG binding sites on the microspheres are loaded with the antibody clone which binds 9E9 and each of these antibody molecules has two binding sites for 9E9 we assumed the capacity of the QSC microspheres for 9E9 binding to be equal to the mouse IgG binding capacity of these microspheres as provided by the manufacturer.

To enable subtraction of ABC-background values based on background fluorescence of the respective cells, we used FMO ("fluorescence-minus-one") controls in each experiment, where cells were stained with all antibodies except the anti-FcγR antibody. Flow cytometric analysis was done on a FACS Canto II (BD Biosciences, Heidelberg). Data were analyzed with FACSDiva Software (BD). For ABC calculation we used QuickCal R software provided by Bangs Laboratories.

An example for the quantification procedure from flow cytometric analysis to calculation of ABC values is provided in **Supplementary Figure 5**.

### Identification of Allelic Variants for Human Donors

For the identification of FcγR haplotypes of human donors, genomic DNA was isolated from peripheral blood and stored at −20◦C. For genotyping of FcγRIIb232I/T und FcγRIIbG/C−386/A/T−120 alleic variants a nested PCR was carried out with an initial long-range PCR (93◦C 15′′, 68◦C 17′ for 10 cycles; 93◦C 15′′, 68◦C 28′ for 28 cycles; PCR product ∼17 kbp) using primers LR-FOR (5′ ctccacaggttactcgtttctaccttatcttac 3 ′ and LR-REVERSE (5′ gcttgcgtggcccctggttctca 3′ ) PCR products were extracted from Agarose gels and used as template for a second PCR, either using primer pair PP-FOR (5′ caatttaccgagagcaagacagc 3′ ) and PP-REVERSE (5′ gcagtcagcccagtcactctcagt 3′ ) for amplification of promoter polymorphism alleles (95◦C 30′′, 58◦ 30′′, 72◦C 90′′ for 35 cycles; final elongation 72◦C 5′ ; PCR product 1946 bp) or primer pair I232T-FOR (5′ cctgcctgctcacaaatgta 3′ ) and I232T-REVERSE (5′ cactgctctccccaagac 3′ ) for amplification of alleles for the I232T transmembrane polymorphisms (98◦C 15′′ , 58◦C 20′′, 72◦C 30′′ for 35 cycles; final elongation 72◦C 7′ ; PCR product ∼750 bp). Products of both PCRs were eluted from agarose gels and sequenced by GATC Biotech using primer PPseq (5′ tgacatacctccttgtccttgtt 3′ ) for the promoter polymorphism and I232T-FOR for the polymorphism in the transmembrane region.

Polymorphisms of FcγRIIa131H/R and FcγRIIIa158V/F were characterized by nested allele-specific PCR. For FcγRIIa131H/R an initial PCR (94◦C 5′ , 56◦C 5′ , 72◦C 5′ for 10 cycles, followed by 94◦C 60′′, 56◦C 60′′, 72◦C 2′ for 30 cycles, final elongation 72◦C, 10′ ) with primer pairs IIa-1st FORWARD (5′ ggagaaaccatcatgctgag 3′ ) and IIa-1st REVERSE (5′ gaagagctgcccatgctg 3′ ) was performed to amplify a fcgr2a specific DNA fragment containing the polymorphism. This was followed by an allele-specific PCR with common IIa131-REVERSE primer (5′ caattttgctgctatgggc 3′ ) and either FORWARD primer IIa131H (5′ gaaaatcccagaaatttttcca 3′ ) or IIa131R (5′ gaaaatcccagaaatttttccg 3′ ) which could only amplify the histidine or arginine variant, respectively. Successful PCR reactions provided a 249 bp fragment. For FcγRIIIa158V/F an initial PCR (94◦C 5′ ; followed by 94◦C 60′′, 58◦C 30′′, 72◦C 2.5′ for 30 cycles, final elongation 72◦C, 10′ ) with primer pairs IIIa-1st FORWARD (5′ gtgtctttcaggctggctg 3′ ) and IIIa-1st REVERSE (5′ gaccagaatagtttaatctcg 3′ ) was performed to amplify a fcgr3a specific DNA fragment containing the polymorphism. This was followed by an allele-specific PCR with common IIIa158- FORWARD primer (5′ tcacatatttacagaatggcaaagg 3′ ) and either REVERSE primer IIIa158V (5′ tctctgaagacacatttctactccctac 3′ ) or IIIa158F (5′ tctctgaagacacatttctactccctaa 3′ ) which could only amply the histidine or arginine variant, respectively. Successful PCR reactions provided a 138 bp fragment.

### Statistics

Panels with data sets for analysis of statistical significance are depicted as bar charts. Data in bar charts are expressed as mean + standard deviation if all data sets are normally distributed and as median ± interquartile range (IR) if at least one data set in the panel is not normally distributed according to Shapiro-Wilk test. Otherwise, sets data are provided either in box plots or as individual data points. Statistical significance of differences between medians of two sets of data was analyzed by Mann-Whitney test. Data were analyzed and plotted with Graph Pad Prism software (GraphPad Software Inc., San Diego, CA).

## RESULTS

In the present work we analyzed the expression of Fcγ receptors I, IIb, III, and IV on murine peripheral blood leukocytes and Fcγ receptors I, IIa, IIb, and IIIa/b on human peripheral blood leukocytes by flow cytometric analysis with fluorochromeconjugated antibodies against the various Fcγ receptors (anti-FcR). FcγR expression on cells is referred to as their antibody binding capacity (ABC, depicted as antibody-binding sites per cell) for the respective anti-FcR antibodies. To achieve this, fluorescence intensity of anti-FcR antibody bound cells and cells from FMO controls without anti-FcR antibody was translated into ABC, using reference curves that were established in each experiment for all tested anti-FcR antibodies using QuantumTM Simply Cellular R (QSC) microspheres. Anti-mouse, anti-rat or anti-human QSC microspheres (Bangs Laboratories Ltd.) were used according to the host species of the respective anti-FcγR antibody.

### Quantification of Mouse Fcγ Receptors

Using this methodology, we quantified the ABC for murine Fcγ receptors I (CD64), IIb (CD32b), III (CD16), and IV using PElabeled anti-FcγR antibodies on peripheral blood leukocytes of C57BL/6 mice under steady state conditions. Quantification of mouse FcγRs was done repeatedly over several years by different researchers and by using different lots of QSC beads, antibodies and antibody providers. The summary of these measurements are depicted in **Figure 1**. In contrast to merely qualitative analyses our quantitative approach also allows direct comparison of ABC values for activating vs. inhibitory receptors on the different cell types that co-express both receptors.

As shown in **Figure 1**, expression of the high affinity FcγRI was restricted to monocytes, with classical monocytes expression roughly twice as much FcγRI than non-classical monocytes. With 24,000 ABC per cell, the inhibitory FcγRIIb was expressed most strongly on B cells, closely followed by eosinophils with 20,000 ABCs. On eosinophils FcγRIIb expression levels were matched by a nearly similar expression of activating FcγRIII (1.9 × 104 binding sites). Co-expression of inhibitory and activating receptors was also found on mouse monocytes. On classical monocytes FcγRIIb (1.6 × 104 binding sites) expression levels faced a slightly higher number of activating receptors comprising mainly of FcγRIII (1.7 × 104 binding sites) and FcγRI (1 × 104 binding sites), but also some FcγRIV (2 × 103 binding sites). This FcγRIV expression may be due to some classical monocytes

experiments.

upregulating FcγRIV in the process differentiation into nonclassical monocytes (48, 49). For FcγRIV on non-classical monocytes, we calculated the highest expression of all murine FcγRs (8.6 × 104 binding sites). In contrast to this high FcγRIV expression, moderate levels of activating FcγRs FcγRI (5 × 103 binding sites) and FcγRIII (7 × 103 binding sites) were noted. The inhibitory FcγRIIb was only expressed at intermediate levels on this monocyte subset (1 × 104 binding sites) and remained about one order of magnitude below activating FcγR numbers.

Low values of anti-FcγRIIb binding sites per cell have been measured on NK cells and neutrophils (both 2 × 103 binding sites), as well as FcγR-lacking T-cells (<1 × 103 binding sites)

non-classical monocytes and neutrophils. n = 5–6; median ± IR; Mann-Whitney test for significance; \*\*p < 0.01.

upon staining with self-labeled anti-FcγRIIb-PE. However, in comparison to cell subsets known to express the inhibitory FcγRIIb, such as monocytes, eosinophils, and B-cells these values—especially for T cells—appear negligible. At this point, however, we cannot explicitly distinguish whether there is in fact very low FcγRIIb expression at least on NK cells and neutrophils, or whether for example some free PE molecules from the Ly-17.2 in-house labeling were capable of binding to the cells. Even if the low expression of the inhibitory FcγRIIb on neutrophils was indeed real under steady state conditions, it faces pronounced expression of activating FcγRIV (1.5 × 104 binding sites) and moderate levels of FcγRIII (9 × 103 binding sites) on these cells. Finally, we could verify our previously published observation that murine NK cells express only low levels of FcγRIII (50) (2 × 10 3 binding sites) during the steady state.

### FcγR Expression by FcγR Knockout Mice

It has been reported previously that deficiencies in FcγR expression may modify the expression of other FcγRs. For example FcγRIV expression on neutrophils was increased in

FcγRIII knockout mice (51–53), but not in FcγRI deficient mice (51, 53). In addition we had observed that deletion of FcγRIV resulted in a slight up-regulation of FcγRIII on neutrophils (52). These results were corroborated in the present work where we analyzed potential compensatory effects upon deficiency of single activating receptors in a parallel analysis of C57BL/6, FcγRI-, FcγRIII- and FcγRIV- knockout mice. **Figure 2** depicts anti-FcR binding sites for FcγRI on monocytes (**Figure 2A**), and FcγRIV on non-classical monocytes and neutrophils (**Figure 2B**) (**Supplementary Figure 6** for other cell types also) as well as FcγRIII on monocytes, NK cells, neutrophils and eosinophils (**Figure 2C**) for C57BL/6 mice and knockout mice lacking FcγRI, FcγRIII, or FcγRIV. This analysis revealed that FcγRIV expression was significantly increased by nearly a factor of two on non-classical monocytes of FcγRIII deficient mice (2.8 × 105 antibody binding sites per cell) compared to C57BL/6 mice (1.4 × 105 binding sites) (**Figure 2D**). On neutrophils FcγRIII deficiency increased FcγRIV expression even about 5-fold (9.3 × 104 in FcγRIII deficient mice vs. 1.8 × 104 in wt mice). Among FcγRIII expressing cells lack of FcγRIV resulted in a significant increase of FcγRIII on neutrophils (2.2 × 104 in FcγRIV deficient mice vs. 1.6 × 104 in wt mice) and 3-fold higher ABC values on non-classical monocytes (3.5 × 104 in FcγRIV deficient mice vs. 1.2 × 104 in wt mice) (**Figure 2E**).

In FcγRIIb deficient mice the activating FcγRIII was moderately down regulated compared to wild type mice on most cell subsets (**Figure 3**). On non-classical monocytes, however, FcγRIII expression was reduced by about one half according to the combined results of two independent experiments (**Figure 3**). FcγRI and FcγRIV appeared unaffected by the FcγRIIb knockout (**Supplementary Figure 7**).

### Quantification of Human Fcγ Receptors

In addition to quantifying mouse FcγR-expression, we also assessed human FcγR expression. Thus, we quantified expression levels for inhibitory FcγRIIb and the closely related but ITAMbearing activating FcγRIIa on human leukocytes. We also studied but did not experimentally distinguish both variants of human FcγRIII, i.e., FcγRIIIa with a canonical polypeptide transmembrane domain and the GPI-anchored FcγRIIIb. As the latter receptor is exclusively expressed on neutrophils, and at low levels on basophils (54), however, this should not impact on the reported numbers. The calculated anti-FcR binding sites for the human Fc receptors are depicted in **Figure 4**. In the literature, FcγR expression has been mainly studied on human neutrophils and monocytes by various methods (18–34). According to our results, FcγRI is barely expressed by neutrophils in steady-state, consistent with observations by others (18–29). On monocytes, FcγRI expression was described with numbers ranging from about 1–4 × 104 anti-FcγRI or monomeric IgG binding sites (18, 19, 22, 23, 27, 30–33), but in contrast to our work these studies did not distinguish between monocyte subsets. As shown in **Figure 4A** non-classical monocytes have low to moderate FcγRI expression (6 × 103 anti-FcγRI binding sites). In contrast, classical monocytes (∼8 × 104 binding sites) expressed considerably higher numbers of FcγRI. The same was true for FcγRII where numbers between 2 and 4.7 × 104 molecules per cell have been published (18, 19, 22, 23), without differentiating between activating and inhibitory FcγRII receptors or between classical and non-classical monocytes. In our study, we calculated for classical monocytes ∼1 × 105 binding sites for FcγRIIa and 4 × 103 binding sites for FcγRIIb whereas non-classical monocytes had ABC values of ∼8 × 104 binding sites for FcγRIIa and 4 × 103 binding sites for FcγRIIb. For neutrophils, where we calculated 1.6 × 105 anti-FcγRIIa binding sites but barely any FcγRIIb expression (2 × 103 binding sites), published pan-FcγRII values are in the range of ∼1–4.5 × 104 (18, 19, 22–25, 34). The ABC values for the inhibitory FcγRIIb were the highest on B cells (5.7 × 104 ). For FcγRIIIa on NK cells we calculated 2.2 × 105 binding sites whereas 4.4 × 104 (22) and 7.9 × 104 (35) receptor numbers were reported in other studies. Eosinophils had an ABC for FcγRIIIa of 8 × 103 binding sites according to our data compared to a receptor number of 1.2 × 104 reported by others (35). Our data on FcγRIIIa expression on non-classical monocytes revealed 2.3 × 105 sites per cell whereas numbers of

∼3.5 × 104 -1.2 × 105 have been published by others (18, 19, 22). For neutrophils we extrapolated an ABC for FcγRIII (i.e., mainly FcγRIIIb) of 1.4 × 106 binding sites per cell which is as well higher than the values provided by others (18, 19, 22–26, 34, 35) (∼6 × 104 -4 × 105 ) using antibody clones CLB-Gran/1, ION16, and mostly 3G8. As the anti-FcγRIII-PE fluorescence was beyond the fluorescence of the reference beads with respect to the highest number of antibody binding sites, the ABC values for neutrophils are being extrapolated by extending the calculated reference line (linear correlation in a log/log scheme) beyond the highest reference point. This reduces the accuracy of the calculation of anti-FcγRIII binding sites on human neutrophils, but in every experiment performed with human leukocytes so far the fluorescence of neutrophils upon anti-FcγRIII staining had been higher than that of the respectively used reference beads with the highest binding capacity. This was also true in an assay where we used one lot of beads with ∼620,000 sites, the highest number of binding sites we noted so far.

### Differential Expression of FcγRIIb on Human Monocytes

Upon quantification of FcγRIIb expression for a cohort of 10 healthy donors we found a pronounced variability in ABC values for FcγRIIb mainly on monocytes, which has also been observed by others (55, 56). Four of the donors revealed pronounced FcγRIIb expression on monocytes and were grouped as FcγRIIb+, whereas the others had significantly lower FcγRIIb expression (FcγRIIblow) (**Figure 5A**). Consistent with the published data, the variation was especially pronounced on CD16+ CD14low non-classical monocytes. Whereas on classical monocytes the mean number of anti-FcγRIIb antibody binding sites was 1 × 104 for FcγRIIb+ donors vs. 3 × 103 for the FcγRIIblow group, non-classical monocytes also revealed an ABC of 3 × 103 binding sites for FcγRIIblow donors but even 1.7 × 104 for FcγRIIb+ donors (**Figure 5B**). Having identified the haplotypes of all donors regarding (i) the promoter polymorphism with either guanine at position −386 and thymine at position −120 or cytosine at −386 and adenine at −120 which affects transcription of the gene (57) and (ii) the transmembrane polymorphism of FcγRIIb which excludes the receptor from membrane rafts (58), we tried to correlate these haplotypes with the variances in FcγRIIb expression (**Figures 5C,D**). Among the 10 donors only two were heterozygous with respect to the promotor polymorphism carrying one allele of the−386C−120A haplotype whereas eight donors were homozygous "wild type" with the common−386G−120T (59). However, this promotor haplotype did not correlate with the observed dichotomy in expression on monocytes (**Figure 5C**). With respect to the FcγRIIb-232T variant, again no correlation with FcγRIIb expression level became apparent (58) (**Figure 5D**).

Since neither FcγRIIb haplotype accounted for the observed dichotomy in expression, we extended the analysis to known polymorphisms of FcγRIIa and IIIa. This includes the histidine vs. arginine polymorphism at position 131 of FcγRIIa (60) (referred to as position 133 in the original reference) as well as the valine vs. phenylalanine polymorphism at amino acid position 158 (or 176 if the leader sequence is included) of FcγRIIIa (61), which both affect IgG binding. We compared the respective high-affinity and low-affinity variants with the FcγRIIb expression (**Figures 5E,F**). The high affinity haplotypes for FcγRIIa carry at least one allele with a histidine at position 131 of FcγRIIa, whereas presence of two alleles encoding an arginine at this position represent the FcγRIIa low affinity haplotype (60). With respect to FcγRIIb expression, all three homozygous donors with 131R/R low affinity receptor (which, by chance, also carried the FcγRIIIa low affinity haplotype (see below)) revealed low expression of FcγRIIb on monocytes and heterogeneous expression on B cells. However, the high affinity haplotypes were heterogeneous for FcγRIIb expression on both monocytes and B cells (**Figure 5E**) (61). Whereas all donors with 158F/F low affinity receptors had rather low FcγRIIb expression, all those with pronounced expression of FcγRIIb carried the FcγRIIIa high affinity haplotypes (**Figure 5F**). For B cells we could not identify any single haplotype of those analyzed here or combinations thereof (not shown) that would correlate with the somewhat divergent expression of FcγRIIb on these cells (**Figures 5C–F**). Results on FcγRIIb expression from a second experiment performed for parallel quantification of activating and inhibitory receptors from six individuals (selected for containing two donors with FcγRIIIa 158 F/F, 158 V/F, and 158 V/V haplotype, respectively) were included in this data set. With the exception of a single sample with the homozygous FcγRIIIa high affinity haplotype but low FcγRIIb expression on monocytes—both datasets revealed corresponding results (**Supplementary Figure 8**). The individual data on age—which revealed no correlation with FcγRIIblow and FcγRIIb+ phenotype—and FcγRIIa/FcγRIIIa haplotype from both experiments are depicted in **Supplementary Figures 9A,B**, respectively.

### FcγR Expression on Basophils and Platelets

In addition to the major leukocyte populations in the peripheral blood we also quantified Fc receptor expression on murine and human basophils as well as human platelets (**Figure 6**). In full accordance with previous results (62) both murine and human basophils co-express activating and inhibitory Fcγ receptors. We verified that both human and murine basophils lack FcγRI expression (not shown). With 5.6 × 104 anti-FcγRIIb binding sites per cell, expression of the inhibitory receptor by murine basophils even exceeds that of B cells. This is contrasted by an even higher expression of the activating FcγRIII (7.1 × 104 binding sites per cell) whereas no FcγRIV expression was detected. Also human basophils revealed high FcγRIIb expression (9.5 × 104 binding sites). In contrast to murine basophils, expression of activating Fcγ receptors on human basophils is much smaller. As shown in **Figure 6B**, using the anti-FcγRII antibody IV.3 with pre-blocking FcγRIIb verified that the weak binding of IV.3 to basophils is not caused by low-affinity binding to FcγRIIb but indeed reflects moderate expression of FcγRIIa (8 × 103 binding sites per cell). With ∼4 × 103 anti-FcγRIII binding sites per cell, FcγRIII is expressed at very low levels. Of note, results of Meknache et al. suggest that this weakly expressed FcγRIII might in fact be GPI-anchored FcγRIIIb rather than signaling competent FcγRIIIa (54).

Finally, we studied human FcγR expression on platelets. In contrast to murine thrombocytes, human platelets are known to express Fcγ receptors, namely FcγRIIa. Our quantification of FcγRIIa on human platelets revealed low ABC values with ∼1 × 103 binding sites per cell. Relatively low expression of FcγRIIa by human platelets in comparison to the FcγR expressing leukocytes—has also been reported by others (1.5–4.7 × 103 binding sites) (63–65).

### Individual FcγR Expression Repertoires

Since for a cohort of six human donors the expression of FcγRI, FcγRIIa, FcγRIIb, and FcγRIII has been quantified in

transmembrane region of FcγRIIb for homozygous I/I, T/T or heterozygous I/T donors, (E) the low affinity haplotype of FcγRIIa being homozygous with arginine at position 131 vs. the high affinity haplotype with at least one histidine allele and (F) the low affinity haplotype of FcγRIIIa, homozygous with phenylalanine at position 158 or the high affinity haplotype bearing at least one valine allele.

parallel, the respective expression pattern of the different Fcγ receptors can be assessed for each individual donor. Thus, for peripheral blood leukocytes effectively expressing more than one signaling-competent Fcγ receptor, i.e., monocytes, eosinophils, and basophils—both the relative fraction of its receptor repertoire as well as the absolute amount is shown in **Supplementary Figure 10** for each individual donor. These data show that in spite of individual differences the principal Arec/Irec with respect to predominance of either the activating Fcγ receptors (on monocytes and eosinophils) or the inhibitory receptor (on basophils) is conserved for the respective cell populations among the various donors.

### DISCUSSION

The main purpose of our study was to quantify mouse and human FcγR expression on immune cells. From a qualitative point of view our results regarding the expression pattern of the different activating and the inhibitory FcγR on human and murine peripheral blood leukocytes correspond well with previously

published data (1, 9–11, 66). However, the main purpose of the current study was to add a quantitative aspect to these relative expression values. Apart from allowing to assign numerical values for FcR expression for various cell types, this also allows to estimate ratios of activating and inhibitory receptors on a given cell population and e.g., quantitative comparison of receptor expression on murine and human leukocytes.

### Experimental Caveats

The methodology used in this study to estimate and compare numbers of binding sites for antibodies for antigens of interest is better suited than qualitative statements on differences in fluorescence intensities. However, since the binding to the respective reference beads requires complete antibody molecules it should be noted that due to the bivalent nature of antibodies one anti-FcγR antibody may theoretically bind two receptor molecules if they are in close proximity and proper orientation to one another and, thus, numbers of antibody binding sites may under-represent the numbers of respective antigens as was observed for CD4 molecules on human leukocytes (37). However, experiments performed with the immobilized antigen indicated that bivalent binding takes place only if mobility of antigens in plasma membranes is sufficiently high. Thus, in spite of staining at low temperatures in our experiments to reduce membrane mobility, there is a theoretical possibility of systemic underrepresentation of the actual number of respective Fcγ receptor molecules by the ABC up to a factor of two at most. We, thus, strictly referred to ABC/anti-FcγR binding sites per cell in the context of this work. Antibodies could also bind to Fc receptors via their Fc portion, once the Fab binds with high affinity to its antigen. However, this could only affect the quantification (leading to an underestimation of the actual receptor numbers) if the Fc-binding receptor were the Fcγ receptor of interest in the respective measurement. In this case, the lower affinity binding via an Fc part competes with the specific high affinity binding via the Fab of the anti-FcγR detection antibody. Due to the anti-FcγR antibodies being present in saturating amounts in the experiments presented here, they will probably replace virtually all Fc domains bound to the respective receptor of interest and contribute normally to the total anti-FcγR-PE mediated fluorescence of the cell. Conversely, binding of anti-FcγR antibodies via their Fc part to other Fcγ receptors than the receptor of interest would represent unspecific binding and cause overestimation of actual receptor numbers. To avoid such unspecific Fc-mediated binding in the mouse system we used well-established blocking antibodies directed against Fc receptors that were not to be quantified. For experiments with human samples we used FcXTM Fc Receptor Blocking Solution which limits Fc-mediated binding to FcγRs by pre-occupying their Fc binding sites, but does not affect antigen-specific FcγRdetection. In physiologically active cells, pretreatment with FcγR binding antibodies can mediate receptor crosslinking, and, thus, induce signaling and changes in surface receptor numbers. Staining at low temperatures and in presence of sodium azide renders the cells physiologically inactive to avoid such potential influences of Fc block.

In most cases, where quantitative expression data of Fcγ receptors on distinct cell populations are available in the literature, these values are exceeded by the ABC values presented in this work. Besides the possibility that such differences may be inherent to the different methodologies being used for receptor quantification, conceivable reasons for the higher values presented here may be that (i) we use freshly isolated biological samples, and (ii) we aim at saturating conditions for anti-FcγR antibody binding to cells, which is critical for assays based on antibody-binding reference beads.

## A/I Ratios of Murine Fc Receptors

Based on the different affinities of the various IgG isotypes to the activating and inhibitory Fc receptors the concept of the so called A/I ratio has been developed as a prediction of the outcome of binding of IgG molecules of a given isotype (13). The numerical data of the present work enable a corresponding concept for the ratio of activating (Arec) and inhibitory receptors (Irec) on immune cell subsets to better comprehend the roles of the distinct cell types in antibody-mediated effector functions. For example, based on our ABC values for the activating receptors FcγRI, FcγRIII and FcγRIV and inhibitory FcγRIIb, classical monocytes have an overall Arec/Irec ratio of ∼2 with Fcγ receptor III providing a somewhat higher contribution to this Arec/Irec ratio than FcγRI. Taking into account the different affinities of these FcγRs to the various IgG isotypes, one may speculate that IgG1 or IgG2b may act mainly via FcγRIII and IgG2a may act via FcγRI. According to their respective Arec/Irec ratios either may be controlled by comparable amounts of FcγRIIb. According to our ABC values, non-classical murine monocytes have a total Arec/Irec ratio of ∼10 with an individual contribution of ∼1 by FcγRIII and ∼9 by FcγRIV. Thus, due to the very low affinity of IgG1 to FcγRIV—IgG1-mediated effects may be expected to induce signaling mainly via FcγRIII, whereas IgG2a and IgG2b responses should be dominated by FcγRIV. In models where non-classical monocytes play an important role, the lack of inhibitory receptors by using FcγRIIb knockout mice should affect mainly IgG1-mediated responses but have a much weaker effect on IgG2a and IgG2b responses, especially when taking into account (and, thus, implementing the classical A/I concept) the significantly higher affinity of IgG2a to FcγRIV compared to FcγRIIb. This may e.g., be one reason for earlier observations of isotype specific effects in mice lacking inhibitory Fcγ-receptor expression in anti-tumor responses (13, 67).

This concept of cell specific Arec/Irec ratios may be especially useful for a better understanding of effector functions mediated by subclasses with intermediate classical A/I ratio such as murine IgG2b as discussed in Nimmerjahn and Ravetch (13), or various murine or human glyco-variants, or by immune-complexes of mixed isotypes.

## FcγR Knockout Mice

It is common practice to use knockout mice lacking one or several FcγRs to study the role of these receptors in IgG dependent immune responses. However, a compensatory upregulation of other Fcγ-receptors may complicate the interpretation of the results. In mice this may be of special relevance for activating Fc-receptors as all FcγRs and the high affinity FcεRI require the common FcRγ-chain for cell surface expression and signaling function (41, 68). Indeed, this seems to be the case for activating receptors FcγRIII and FcγRIV on cells which express both molecules (**Figures 2B,C**). As has been suggested earlier in analogy to increased FcγRIII expression on mast cells that lack the high affinity IgE receptor FcεRI (69) this may be due to the fact that the activating receptors compete for association with the common FcRγ-chain, which is essential for expression of the activating Fc receptor αchains on the cell surface (51, 52). The relatively low expression of FcγRI (**Figure 1**), however, might be the reason why its knockout does not influence the expression of FcγRIV on nonclassical monocytes or neutrophils (**Figure 2B**) or FcγRIII on any population (**Figure 2C**).

### Functional Role Human and Murine NK Cells in ADCC

Both, human and murine NK cells, express only one activating receptor (FcγRIII in mice and FcγRIIIa in humans). However, in contrast to human NK cells, murine NK cells show the weakest expression of all tested FcγRIII-expressing leukocytes. This may have pronounced functional consequences: In humans, NK cells have been suggested to be key effector cells for antibody dependent cell mediated cytotoxicity (ADCC) (70). In contrast, due to their very low number of activating FcγRIII receptor molecules, a relevant role of NK cells in ADCC is unlikely in mice. This is in line with various experimental observations: (i) crosslinking of FcγRIII poorly activates murine NK cells in absence of additional stimulation (71); (ii) anti-CD20 mediated B cell depletion in mouse models is independent of NK cells but rather depends on FcγR expressing mononuclear phagocytic cells (72); (iii) anti-CD25 rat IgG1-mediated depletion of regulatory T cells is fully dependent on FcγRIII-expressing mononuclear phagocytic cells but is efficient in mice lacking NK cells (73, 74); (iv) the human anti-CCR4 monoclonal antibody KM2760 has anti-tumor activity both in murine and human experimental systems. However, whereas NK cell-mediated ADCC was suggested as the major anti-tumor effector mechanism in humans (75), in mice not NK cells but rather myeloid cells seem to be responsible for the anti-CCR4 dependent tumor cell depletion (75).

### Variability in Human FcγRIIb Expression

Upon quantification of human FcγRIIb on leukocytes we found that one group of donors showed low ABC for FcγRIIb on monocytes whereas another group showed pronounced expression. This observation is consistent with published data (i) by Bruhns et al. showing that only a fraction of human donors revealed significant FcγRIIb expression on monocytes (55) and here especially on CD16+ CD14low non-classical monocytes [referred to as patrolling monocytes in (55)] and (ii) by Glennie et al. where variable expression of FcγRIIb was found on nonclassical CD14lo monocytes with lower expression on CD14hi monocytes (56). Since the dichotomy of FcγRIIb ABC values was so obvious and careful assessment of FcγRIIb in the blood of patients receiving a therapeutic mAb was suggested to be an important marker of prognostic value (56) we investigated whether this variation correlated to any FcγR-related factor. Among all four haplotypes, interestingly, only FcγRIIIa variants revealed a correlation with FcγRIIb expression on monocytes: The high-affinity FcγRIIIa was present in all individuals with pronounced FcγRIIb expression, whereas all individuals with the low affinity haplotype revealed low ABC for FcγRIIb on monocytes. More studies with larger cohorts of human donors will be necessary to verify and understand this correlation in more detail in the future. Regarding the frequency and linkage of FcγRIIa and FcγRIIIa our data from the first random cohort with 10 donors, corresponds to published data on Caucasian populations. In accordance e.g., with van der Pol et al. (76) the two allelic variants of FcγRIIa are equally represented (ten 131R alleles and ten 131H alleles) among the 20 FcγRIIa alleles present in this cohort of 10 donors, whereas the 158F variant of FcγRIIIa is more common than the 158V variant (14 158F alleles and six 158V alleles). In addition, a linkage disequilibrium between FcγRIIa and FcγRIIIa became apparent: For nine of the 10 donors the allelic combinations of FcγRIIa and FcγRIIIa can be unequivocally determined since they are homozygous for at least one allele. Among these 18 allelic combinations the FcγRIIa131H FcγRIIIa158V (131H/158V) combination is present four times, the 131H/158F combination five times. 131R/158V and 131R/158F are present 1 and 8 times, respectively. This is in accordance with the enhanced co-occurrence of FcγRIIa131R with FcγRIIIa158F which has been described e.g., by Niederer et al. where a moderate/strong linkage disequilibrium between FcγRIIa and FcγRIIIa was found in a UK and Swedish Caucasian as well as a Kenyan cohort (77).

With respect to counterbalancing activating signals, it would be reasonable that cells with higher affinity activating Fcγreceptor alleles would require a higher level of FcγRIIb expression to counter regulate this lower threshold for cell activation. Inversely, cells with low affinity activating receptor haplotypes require less pronounced counter regulation. This is in line with the low FcγRIIb expression on monocytes, consistently (but not exclusively) detectable in individuals with low affinity FcγRIIa/FcγRIIIa haplotypes. Following this line of argument, the modulation of inhibitory receptor expression by activating receptor haplotypes may well be cell type dependent, since this is relevant mainly for cell types that co-express activating and inhibitory receptors, such as monocytes. Accordingly, we found no obvious association of FcγRIIb expression with the activating receptor haplotypes. For the FcγRIIa131H/R haplotypes it was recently shown also by others that the corresponding SNP (SNP rs1801274) does not affect FcγRIIb expression on B cells (78).

How genetic regulation of FcγRIIb expression by activating receptor haplotypes might be achieved on a cellular and molecular level is not yet clear. However, one might envision that the association of FcγRIIb expression and the activating Fcγreceptor affinity haplotype could be via a promoter/enhancer SNP that modulates expression of FcγRIIb in linkage disequilibrium with coding SNPs of activating Fc receptors as has been suggested by Roederer et al. (78).

### FcγRIIIb Expression on Human Neutrophils