The Complex Association of FcγRIIb With Autoimmune Susceptibility

FcγRIIb is the only inhibitory Fc receptor and controls many aspects of immune and inflammatory responses. The observation 19 years ago that FcγRIIb−/− mice generated by gene targeting in 129 derived ES cells developed severe lupus like disease when backcrossed more than 7 generations into C57BL/6 background initiated extensive research on the functional understanding of this strong autoimmune phenotype. The genomic region in the distal part of Chr1 both in human and mice in which the FcγR gene cluster is located shows a high level of complexity in relation to the susceptibility to SLE. Specific haplotypes of closely linked genes including the FcγRIIb and Slamf genes are associated with increased susceptibility to SLE both in mice and human. Using forward and reverse genetic approaches including in human GWAS and in mice congenic strains, KO mice (germline and cell type specific, on different genetic background), knockin mice, overexpressing transgenic mice combined with immunological models such as adoptive transfer of B cells from Ig transgenic mice the involved genes and the causal mutations and their associated functional alterations were analyzed. In this review the results of this 19 years extensive research are discussed with a focus on (genetically modified) mouse models.


INTRODUCTION
Antibodies (Ab) form immune complexes (IC) with their cognate antigen (Ag). IgG-ICs are potent activators of the immune system via cross-linking of receptors for the Fc part of IgG, FcγR, mainly expressed on the surface of cells of the innate immune system.
FcγRs belong to the Ig supergene family of leukocyte FcR and are transmembrane glycoproteins containing a ligand-binding α subunit with two or three extracellular Ig-like domains, a transmembrane and a cytoplasmic domain. In mice, the high-affinity FcγRI, binding monomeric IgG, and the low-affinity receptors for complexed IgG, FcγRIII, and FcγRIV are activating receptors. The α subunits of the activating receptors form a multi-subunit complex with a dimer of the common γ-chain (FcRγ) (1,2) with an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking activating FcRs by IC initiates signal transduction via recruitment and subsequent activation of intracellular tyrosine kinases (3), switching on a large variety of effector mechanisms activating inflammatory cascades.
In humans, there are four activating FCGRs. The high-affinity FCGR1 (CD64) and the low-affinity FCGR3A (CD16A) are associated with the common γ chain whereas the low-affinity FCGR2A (CD32A), containing an ITAM in its cytoplasmic domain, and the low-affinity FCGR3B (CD16B), with a glycosylphosphatidylinositol (GPI) anchor, are single-chain receptors. All human FCGR genes are clustered at the distal end of Chr1, a region associated with susceptibility to autoimmune diseases such as Systemic Lupus Erythematosus (SLE) (4). In mice the FcγRII, -III, and -IV genes are clustered at the distal end of Chr1, a region orthologous with SLE associated genomic intervals on human Chr1 and associated also with susceptibility to autoimmune disease (Lupus-like disease). Fcγ RI is located on Chr3 due to a translocation during evolution after mouse and human had diverged.
In both humans and mice, the activating FcγRs are counterbalanced by one inhibitory single-chain low-affinity receptor FcγRIIb (FCGR2B or CD32B) with an inhibitory motif named immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic domain. In addition, co-engagement of FcγRIIb and the ITAM containing B-cell receptor (BCR) on B cells forms an important negative feedback mechanism to control antibody production. This regulatory mechanism of cellular activation by the ITAM-ITIM motif pair, observed originally with FcγR, has been described for many other receptors in the immune system e.g., T cell receptors and B cell receptors (5,6). This review focuses on the important but still puzzling immune regulatory role of the inhibitory FcγRIIb and the complex association of its impaired function with autoimmunity as studied extensively in mice.

GENERAL CHARACTERISTICS OF FcγRIIb Isoforms
In humans and mice, there are two membrane-bound isoforms of FcγRIIb identified: FcγRIIb1 and b2 (7) resulting from alternative splicing. The cytoplasmic domain is encoded by three exons whose 5 ′ exon encodes a 47 amino acid motif that prevents coated pit localization, which inhibits FcγRIIb mediated endocytosis of soluble immune complexes. This exon is present in the mRNA that encodes the b1 isoform, the only isoform expressed on B cells, but absent in the mRNA that encodes the b2 isoform (8,9) expressed on most innate immune cells. The ITIM dependent inhibition of cell activation is the same for both isoforms. Therefore, the name FcγRIIb is used in this review without making a distinction between the b1 and the b2 isoform.

Expression
In mice FcγRIIb is expressed on all innate immune cells and is the only FcγR expressed on B cells, including pre-, pro-, and mature B cells, memory B cells, plasma cells (10,11) and B1 cells (12). Unlike many other B cell surface receptors, expression of FcgRIIb is not downregulated during plasma cell differentiation (10). FcγRIIb expression is modulated on different B cell subsets (11) and increases when the B cells become activated (11,13). T cells do not intrinsically express FcγRs (14). However, it has been reported that expression of FcγRIIb but not any other FcγR, is upregulated in memory CD8 + T cells after Listeria monocytogenes infection and tempers the function of these cells in vivo (15). Guilliams et al. showed that according to the microarray expression values extracted from public data sets the mRNA expression of FcγRIIb in mice is from high to low as follows: Inflammatory macrophages (Mϕ), Ly6C hi classical monocyte, inflammatory monocyte-derived dendritic cell (moDC), lung CD11b + conventional or classical DC (cDC), Ly6C lo patrolling monocyte, alveolar Mϕ, follicular B cell, GC B cell, skin-draining lymph node CD11b + cDC, spleen CD8 + XCR1 + cDC, spleen plasmacytoid DC (pDC), spleen CD11b + cDC, neutrophils, spleen Mϕ, and NK cells (16). The overall FcγRIIb expression pattern is similar in mouse and human. In mouse cDCs the relatively low expression of FcγRIIb is higher than that of any activating FcγR.

Cellular Function
Co-aggregation of the inhibiting ITIM containing FcγRIIb with activating ITAM containing FcRs results in the recruitment of the inositol polyphosphate-5-phosphatase SHIP1 that counteracts the signals mediated by activating FcRs (3,28). Therefore, FcγRIIb has a strong regulatory role in all the processes in which activating FcγR are involved. The ratio between activating and inhibiting signals determines the outcome of the cellular response to IgG-ICs. This ratio depends mainly on the following factors: (a) the relative affinities of the different antibody isotypes involved for the different FcγR, (b) the level of opsonization, and (c) the relative expression level of inhibitory and activating FcγR, which is partially determined by the cytokine milieu. The binding of FcγRIIb for IgG-IC is strongest for IgG1 and weakest for IgG2a. So, FcγRIIb expression has the highest impact on IgG1-IC. In addition, FcγRIIb can inhibit complement-mediated inflammation when co-engaged with Dectin-1 by galactosylated IgG1-ICs (29) indicating that its immune-modulatory function in the efferent response is not restricted to the regulation of activating FcγRs.
In B cells co-crosslinking of the BCR and FcγRIIb results in the inhibition of activation, proliferation, Ag internalization and Ab secretion (30)(31)(32). Moreover, in vitro studies have shown that FcγRIIb on B cells can induce apoptosis upon clustering (10,12,28,33,34).
FcγRIIb can also function as an endocytic receptor of small ICs. The endocytic properties of FcγRIIb depend on the presence of a di-leucine motif in the intracellular domain (8) and are independent of the ITIM.

Myeloid Effector Cells
In the efferent phase, FcγRIIb sets a threshold for the activation by IgG-IC of myeloid effector cells, e.g., monocytes, Mϕs, and neutrophils. Crosslinking of activating FcγR by IgG-ICs induces effector mechanisms of these cells e.g., soluble IC clearance, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), the release of inflammatory mediators, degranulation, superoxide production, enhancement of Ag presentation, and cell maturation and proliferation. This includes also the regulation of high-affinity IgE receptor-mediated mast cell activation (35).
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)(38)(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)(44)(45), or in vivo assays with in vitro loaded DCs from WT and FcγR KO mice (46)(47)(48)(49)(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. Development, responsiveness, and lifespan of ignorant cells is normal (76,78,79). 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 (T FH ), 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 T FH 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.

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)(96)(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)(104)(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  (107). In a C57BL/6 knockin (KI) mouse model of the 5' region of the haplotype I Fcγ RIIb gene (Fcγ RIIb NZB ), 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 RIIb NZB allele, the Fcγ RIIb NZW 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 RIIb NZW 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 RIIb NZW 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 RIIb NZB (haplotype I) allele has a stronger impact on susceptibility to autoimmunity than the Fcγ RIIb NZW (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 the Sle1 NZW locus, Fcγ RIIb NZB 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 RIIb NZW 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)(119)(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 T FH 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 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)(132)(133)(134). A series of single nucleotide polymorphisms (SNPs) have been reported to be located both in the promoter and the encoding region of  (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 FCGR2B T232 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).
The frequency of homozygosity of the FCGR2B T232 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 FCGR2B T232 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 RIIb NZW and Fcγ RIIB NZB 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  (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-2 b 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 Slamf 129 locus, the C57BL/6 genome, and Fcγ RIIb −/− (Figure 2), similar to the epistatic interactions between Fcγ RIIb NZB (haplotype I), Slamf NZB (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 RIIb 129 KO alleles are closely linked to the Slamf 129 locus associated with autoimmunity. This means that in most experimental conditions, no distinction can be made between Fcγ RIIb −/− and Slamf 129 mediated effects in these mice.
Ig gene analysis of ANA suggests that ANA develop in GCs (167)(168)(169)(170)(171)(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 (V H ) 3H9H-56R, derived from a dsDNA specific hybridoma, or its variant 56RV H , with higher affinity binding to dsDNA, were inserted in the Igh locus (IgM a 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)(176)(177). The Ab selection process was compared between WT C57BL/6 and Fcγ RIIb −/− 129 mice carrying the V H 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 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 V H 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 RIIB NZW 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/Fas lpr/lpr mice develop lupus-like disease whereas C57BL/6 Fas lpr/lpr mice do not, likely due to suppressor activity of the C57BL/6 genome. However, C57BL/6 Fas lpr/lpr Fcγ 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 Fas lpr 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 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/Fas lpr/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 Le Fcγ RIIb  Table 2). Total IgG ANA was not significantly increased in 10 months old female Le Fcγ 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 NY Fcγ RIIb −/− B6 mice total IgG anti-nuclear Abs was significantly increased compared to C57BL/6 mice (110). But only five percent of NY Fcγ RIIb −/− B6 mice showed premature death whereas mortality was not increased in Le Fcγ 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 Le Fcγ 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 Slightly enlarged (♀) c Slightly enlarged (♀) a n.d. Slightly enlarged (♀) a Splenomegaly a,b,c Spt-GC formation n.d.
Augmented 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., Slamf 129 , 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 NY Fcγ RIIb −/− B6 mouse seems to exhibit a stronger disease phenotype than the Le Fcγ RIIb −/− B6 mouse ( Table 2). There are several explanations for this discrepancy: a. The strains are generated with different ES cell lines. There might be relevant genomic differences between the C57BL/6 derived ES cell lines used. This question can be answered by sequencing the Fcγ RIIb flanking genomic regions in both mouse strains. b. The mice have been backcrossed several generations into different C57BL/6 mouse strains. There are substantial genetic variations between the different C57BL/6 strains used in different laboratories (184). c. Environmental factors (immune status, microbiome) play a role. The incidence of lethal disease in Fcγ RIIb −/− 129 mice varies between different laboratories from 0% to more than 60% (98,109,112,173). d. Differences in the methods used to measure ANA. In the Le Fcγ RIIb Slamf B6 congenic, respectively. The other C57BL6 Slamf 129 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  Table 3]. By using the experimental model of the V H chain knockin strain HKIR mentioned earlier (179) it was demonstrated that B cell-specific expression of Slamf 129 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 NY Fcγ RIIb −/− B6 and C57BL/6 Slamf 129 congenic mice were associated with the production of autoantibodies. However, the titers were much lower than in Fcγ RIIb  (163). Unfortunately, sera from C57BL/6 Slamf 129 congenic mice were not tested in the autoantigen array.
Kidney pathology was absent (98) or very mild, with higher complement deposition than NY Fcγ RIIb 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 RIIb NZB 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 T FH 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 Le Fcγ RIIb  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 Le Fcγ RIIb fl/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 Le Fcγ RIIb fl/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 + Le Fcγ RIIb −/− B6 mice that will be discussed later (188).
In   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 Le Fcγ 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  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 + / Le Fcγ 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 CD14 low CD16 + 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 (FCGR2B T232 ) resulting in impaired FCGR2B function, and susceptibility to SLE. Metaanalyses confirmed that FCGR2B T232 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 V H 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 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.