The localization of the binding site(s) on human IgG1 for the Fc receptors on homologous monocytes and heterologous mouse macrophages.

Two different methods, a rosette assay and a direct binding assay, have been employed in an examination of the binding of human IgG1 to mouse macrophages. In both cases, inhibition of IgG binding was demonstrated by Fc (CH2 + CH3 domains) and pFc' (CH3 domains) fragments of human IgG. In a homologous system, the binding of 125I-human IgG to human peripheral-blood monocytes was inhibited by the Fc fragment whereas the pFc' fragment was inactive. Scatchard plot analysis of the binding data from both the heterologous and homologous systems allowed association constants and numbers of receptors per cell to be calculated. A more thorough examination of the possible location of IgG Fc-receptor binding sites was made using less orthodox proteolytic cleavage fragments of IgG. The site on human IgG1 responsible for binding to mouse macrophage Fc receptors was confirmed to be within the CH3 domains. Human IgG1 binding to homologous monocytes was shown, using a dimeric C gamma 2 domain fragment, to be via the CH2 domains, and was dependent on the integrity of the covalent interaction between the C gamma 2 domains at the hinge region.     

Terminal sugars of Fc glycans influence antibody effector functions of IgGs

IgG molecules contain glycans in the CH(2) domain of the Fc fragment (N-glycosylation) which are highly heterogeneous, because of the presence of different terminal sugars. The heterogeneity of Fc glycans varies with species and expression system. Fc glycans influence the binding of IgG to Fc receptors and C1q, and are therefore important for IgG effector functions. Specifically, terminal sugars such as sialic acids, core fucose, bisecting N-acetylglucosamine, and mannose residues affect the binding of IgG to the FcgammaRIIIa receptor and thereby influence ADCC activity. By contrast, terminal galactose residues affect antibody binding to C1q and thereby modulate CDC activity. Structural studies indicate that the presence or absence of specific terminal sugars may affect hydrophilic and hydrophobic interactions between sugar residues and amino acid residues in the Fc fragment, which in turn may impact antibody effector functions.     

The heterogeneity of bovine IgG2--V. Differences in the primary structure of bovine IgG2 allotypes.

The partial amino acid sequences of the gamma chains of the bovine IgG2a(A1) and IgG2a(A2) allotypes were determined. Sequence differences were found in the CH1 domain, the hinge region, and the CH3 domain. The hinge regions displayed only 71.4% similarity and all of the differences were of a radical nature. The A2 hinge has isoleucine instead of serine at 229, histidine for asparagine at 235, proline for histidine at 238, and cysteine instead of proline in position 234; the latter has the potential for forming an additional interheavy chain disulphide bridge. The occurrence of such a bridge could explain the presence of a pepsin fragment consisting of the hinge region and the Fc. A corresponding fragment is not obtained with the A1 allotype. Both allotypes have a shortened hinge region and a truncated CH2 domain. This feature is characteristic of all reported sequences of IgG2 proteins but not IgG1 in cattle and the goat. This structural feature may be important in subclass-specific recognition by Fc gamma receptors in ruminants. A surprising discovery was the occurrence of five substitutions in the CH3 domain of the IgG2a(A2) in comparison with the A1, which are shared with the CH3 of IgG1. These permit the occurrence of isoallotypic determinants and can explain the difficulty encountered in preparing A2-specific antisera during which adsorption with IgG1 is a routine procedure. The primary sequence data we report confirm the presence of major structural differences between the A allotypes of cattle that was suggested by previous work. The sequence of the A1 allotype most closely agrees with the two IgG2 sequences deduced from their nucleotide sequences whereas the sequence differences in the hinge and C-terminal CH3 make IgG2a(A2) unique. The structural differences between allotypes could have major consequences for such biological activities as phagocytosis, transepithelial transport, lymphocyte and complement activation.     

The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding.

We have found that amino acid residues necessary for C1q and Fc gamma R binding of human IgG1 are located in the N-terminal region of the CH2 domain, residues 231-238, using a matched set of engineered antibodies based on the anti-HLA-DR antibody L243. Changing the leucine 235 in the CH2 region of IgG3 and IgG4 to glutamic acid was already known to abolish Fc gamma RI binding. We have confirmed this for IgG1 and also found a concomitant abolition of human complement lysis with retention of Fc gamma RIII-mediated function. Changing the glycine at 237 to alanine of IgG1 also abolished Fc gamma RI binding and reduced human complement lysis and Fc gamma RIII-mediated function. Exchanging the whole region 233-236 with the sequence found in human IgG2, abolished Fc gamma RI binding and human complement lysis and reduced Fc gamma RIII-mediated function of IgG1. In contrast, a change in the previously described C1q-binding motif, from lysine at 320 to alanine, had no effect on IgG1-mediated complement lysis.     

Catabolism of the Murine IgGl Molecule: Evidence that Both CH2-CH3 Domain Interfaces are Required for Persistence of IgGl in the Circulation of Mice

Site-directed mutagenesis of a recombinant Fc-hinge fragment has previously been used to identify a region of the murine IgG 1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains. In the current study the nature of this 'catabolic site' has been further analysed using recombinant techniques. Fc-hinge, CH2-hinge, CH2 and CH3 fragments have been expressed in Escherichia coli , purified and analysed in pharmacokinetic studies in mice. The CH2-hinge has been analysed as both a monomer and dimer, and the dimer has a longer β phase half-life (61.6 h) than the monomer (29.1 h). This suggests that two catabolic sites per Fc fragment are required for serum persistence. The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc-hinge fragment comprising a heterodimer of one Fc-hinge with the wild type (WT) IgGl sequence and a mutant Fc-hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434). This hybrid is cleared with a β phase half-life of 37.9 h and this is significantly shorter than that of the WT Fc-hinge fragment (82.9 h). In contrast to the CH2-hinge dimer, the CH3 domain is cleared rapidly (β phase half-life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment. The data extend our earlier observations concerning a region of the murine IgGl molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.     

Computer localization of some Gm markers on the surface of the Fc region of human immunoglobulin G.

The surface localization of some Gm markers on the Fc fragment of IgG has been identified from previously published amino acid sequences associated with known Gm markers using the atomic coordinates described by Deisenhofer, INSIGHT software and a Digital VAX 11/785 computer, which together permit a study of the three-dimensional structure of the Fc fragment. The G1m(x)-associated amino acid residue 431, the G3m(s)- and G3m(u)-associated residue 435 and the nG4m (a)- and (b)-associated residue 309 are all localized in the interface between the CH2 and CH3 domains. Furthermore, it is postulated that the G1m(a)-associated residue 356 (Asp, Glu) influences the interface formation through an ion pair interaction to Lys 439. Finally, G3m(b) and G3m(g) are associated with the interface via residues 435 and 436. The data explain why sera from patients with rheumatoid arthritis are useful tools for the detection of some Gm markers and support the view that rheumatoid factors from these patients are internal images of microbial Fc-binding proteins.     

Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn.

The analysis of the pharmacokinetics of wild-type and mutated Fc fragments derived from human IgG1 indicates that Ile253, His310 and His435 play a central role in regulating serum half-life in mice. Reduced serum half-life of the recombinant, mutated fragments correlates with decreased binding to the MHC class I-related neonatal Fc receptor, FcRn. In addition, the analysis of an Fc fragment in which His435 is mutated to Arg435 demonstrates that the sequence difference at this position between human IgG1 (His435) and IgG3 (Arg435) most likely accounts for the shorter serum half-life of IgG3 relative to IgG1. In contrast to His310 and His435, the data indicate that His433 does not play a role in regulating the serum half-life of human IgG1. Thus, the interaction site of mouse FcRn on human and mouse IgG1 involves the same conserved amino acids located at the CH2-CH3 domain interface of the IgG molecule. The sequence similarities between mouse and human FcRn suggest that these studies have direct relevance to understanding the factors that govern the pharmacokinetics of therapeutic IgG.     

The effect of synthetic peptides corresponding to Fc sequences in human IgG1 on various steps in the B cell activation pathway

The influence of synthetic peptides comprising sequences in the exposed positions of the Fc region of human IgG 1 was tested on B lymphocyte activation. CH 2 domain peptides having an inhibitory effect on antibody-dependent cellular cytotoxicity, as well as the whole Fc fragment, induced the appearance of the early signs of activation on resting B lymphocytes such as increase in cell volume and HLA-DR antigen expression or leukocyte migration inhibitory factor production. The peptides did not induce proliferation of resting B cells even when B cell growth factor (BCGF)-containing supernatants were added. Exposure to Fc fragment, however, induced a weak proliferation which was significantly enhanced by BCGF. On the other hand, both the Fc fragment and the CH 2 or CH 3 domain peptides enhanced the IgM synthesis of human blood mononuclear cells when a suboptimal dose of pokeweed mitogen was present. This effect was lost when Fc fragment or the peptides were added on the third day of culture. These results suggest that the early steps of B cell activation can be induced by Fc fragment and by small molecular weight Fc peptides, which are potential ligands of Fc receptors. The Fc fragment activates B cells to the state where they respond to BCGF, but the peptides do not possess this activity. Furthermore, both Fc fragment and Fc peptides are able to enhance the IgM synthesis, when accessory cells and the appropriate differentiating factors are present.     

Herpes simplex type 1-induced Fc receptor binds to the Cgamma2-Cgamma3 interface region of IgG in the area that binds staphylococcal protein A

The binding site of immunoglobulin G (IgG) to herpes simplex virus (HSV) type 1-induced Fc receptor was investigated using human IgG Fc intermediate (Fc(i)) fragments, fragment D of staphylococcal protein A (SPA) and chemically modified human IgG. Human IgG Fc(i) fragment composed of one Cgamma2 and two Cgamma3 domains, bound strongly to HSV-1-infected cells. Fragment D, a monovalent subunit of SPA, inhibited the binding of radiolabelled human IgG Fc fragments to the HSV Fc receptor. Reductively methylated human IgG reacted equally well to HSV-infected cells, as did chemically unmodified IgG in contrast to N-acetylimidazole-modified and diethylpyrocarbonate-modifed human IgG, which were unreactive. These results suggest a similar binding site on human IgG for SPA and the HSV-1 Fc receptor with involvement of the amino acid residues Tyr and His but not Lys. The similarities of binding sites on the IgG molecule for the HSV-1 Fc receptor and rheumatoid factors (RF) may be important for understanding the mechanism of RF production in rheumatoid arthritis or other disease states.     

Inhibition of Antibody-Dependent Human Lymphocyte-Mediated Cytotoxicity by Immunoglobulin Classes, IgG Subclasses, and IgG Fragments

The cytotoxicity of human peripheral blood lymphocytes against chicken erythrocytes sensitized by rabbit antibodies was inhibited by human immunoglobulin and immunoglobulin fragments. Myeloma proteins isolated in dimeric state or aggregated by heat treatment inhibited better than the corresponding monomeric proteins. Strong inhibition was observed with IgG1 and IgG3, and with IgG₂ after aggregation, while IgG₄ inhibited very little. No inhibition was found with IgM, IgA. IgD and IgE. The F(ab')₂. and Fab fragments of IgG inhibited poorly or not at all. While- considerable inhibition was observed with the Fc fragment, the pFc' fragment, which roughly corresponds to the C-terminal half of the Fc portion, showed little inhibitory capacity. A fragment isolated from IgG3, containing an extension of the N-terminal part of Fc (the Fch fragment), was an even better inhibitor than tin Fc fragment. The inhibitory capacity of the Fch and Fc fragments was greatly diminished following partial reduction and alkylation On the basis of the inhibitory pattern of IgG fragments, it is suggested that the region on the immunoglobulin molecule involved in binding to the Fc receptor of the effector lymphocytic cell may be located within the CH2 domain.     

Localization of the site of the murine IgG1 molecule that is involved in binding to the murine intestinal Fc receptor

Site-directed mutagenesis of a recombinant Fc hinge fragment has recently been used to localize the site of the murine IgG1 molecule that is involved in the control of catabolism (the “catabolic site”). In the current study, the effects of these CH2 and CH3 domain mutations (Ile 253 to Ala 253, His 310 to Ala 310, Gln 311 to Asn 311, His 433 to Ala 433 and Asn 434 to G1n 434) on intestinal transfer of Fc hinge fragments in neonatal mice have been analyzed. Studies using direct transfer and competition assays demonstrate that the mutations affect the transmission from intestinal lumen into serum in a way that correlates closely with the effects of the mutations on pharmacokinetics. Binding studies of several of the Fc hinge fragments to isolated neonatal brush borders have been used to confirm the in vivo transmission data. These analyses have resulted in the localization of the binding site for the intestinal transfer receptor, FcRn, to specific residues of the murine Fc hinge fragment. These residues are located at the CH2-CH3 domain interface and overlap with both the catabolic site and staphylococcal protein A (SpA) binding site. The pH dependence of IgG1 or Fc fragment binding to FcRn is consistent with the localization of the FcRn interaction site to a region of the Fc that encompasses two histidine residues (His 310 and His 433). To assess whether one or two FcRn binding sites per Fc hinge are required for intestinal transfer, a hybrid Fc hinge fragment comprising a heterodimer of one Fc hinge with the wild-type IgG1 sequence and a mutant Fc hinge with a defective catabolic site (mutated at His 310, G1n 311, His 433 and Asn 434) has been analyzed in direct and competition transmission assays. The studies demonstrate that the Fc hybrid is transferred with significantly reduced efficiency compared to the wild type Fc hinge homodimer and indicate that the binding to FcRn, and possibly subsequent transfer, is enhanced by the presence of two FcRn binding sites per Fc hinge fragment.     

Effect of zinc on human IgG1 and its FcγR interactions

In the present study, we show that histidines 310 and 435 at the CH2-CH3 interface of the Fc portion of human IgG1 can coordinate a Zn2+ and participate in the control of the CH2-CH2 interdomain opening. Structures obtained in the absence of Zn2+ have a reduced interdomain gap that likely hamper FcγR binding. This closed conformation of the Fc is stabilized by inter-CH2 domain sugar contacts. Zinc appears to counteract the sugar mediated constriction, suggesting that zinc could be an important control factor in IgG1/FcγR interactions. The results of binding studies performed in the presence of EDTA on FcγR expressing cells supports this hypothesis. When a mutated Fc fragment, in which histidines 310 and 435 have been substituted by lysines (Fc H/K), was compared with the wild-type Fc in crystallographic studies, we found that the mutations leave the interface unaltered but have a long-range effect on the CH2 interdomain separation. Moreover, these substitutions have a differential effect on the binding of IgG1 to Fcγ receptors and their functions. Interaction with the inhibitory FcγRIIB is strongly perturbed by the mutations and mutant IgG1 H/K only weakly engages this receptor. By contrast, higher affinity FcγR are mostly unaffected.     

Identification of epitopes recognized by a panel of six anti-human IgG2 monoclonal antibodies.

Human IgG2 contains several subclass specific amino acid residues or deletions in the CH1 and CH2 domains and also in the hinge region. These substituted residues are the structural correlates for IgG2 specific epitopes. Since human IgG2 has different biological properties from other subclasses, some IgG2 epitopes may be located in regions correlating with sites determining the biological functions. Previously, we produced three anti-IgG2 monoclonal antibodies (mAbs) with highly specific and interesting reactivities using improved immunization protocols. However, it has been almost impossible to identify epitopes conventionally, because human IgG2 is so resistant to proteolysis that various proteolytic fragments could not be isolated. In this study, we identified the epitopes recognized by anti-IgG2 mAbs by SDS-PAGE, Western blotting, amino acid sequence analysis and peptide/mAb binding ELISA, thus overcoming the need for fragment isolation. A panel of six anti-human IgG2 mAbs, including the current WHO/IUIS specificity standards (HP6002, HP6008, HP6014) and our own (HG2-6A, HG2-30F, HG2-56F), reacted with distinct epitopes. The residues essential to expression of the epitopes recognized by the mAbs were: Pro234, Val235 and Val309 for HG2-56F, HG2-30F and HP6008, respectively. HP6014 reacted with the epitope expressed by Thr214 and its neighboring residues. HG2-6A was reactive with the hinge region, and HP6002 was assumed to be directed against discontinuous epitopes requiring intact Fc for expression. Through these studies, the pepsin and papain cleavage sites of human IgG2 were also clarified.     

Specific cleavage sites on human IgG subclasses by cruzipain, the major cysteine proteinase from Trypanosoma cruzi

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi, might have other biological roles than its metabolic functions. In this report, we have explored the interaction of cruzipain with molecules of the immune system. The enzyme was used to digest all human IgG subclasses at different pH values and lengths of time. At pH 7.3, all subclasses were readily split at the hinge region. Immunoblot and amino acid sequence analysis showed fragments of IgG1 and IgG3 to be compatible with Fab and Fc, whereas IgG2 and IgG4 rendered Fab2 and Fc. In all cases the fragments produced might impair the binding capacities and the effector functions of specific IgG. At these cleavage sites cruzipain displays cathepsin L and/or cathepsin B activities and shows a clear preference for Pro at the P'2 position and polar residues at P1. Despite the activity of cruzipain within the hinge, the enzyme also cleaved all heavy chains between the CH2 and CH3 domains; producing Fc'-like-fragments of 14 kDa. These fragments are potential candidates to block or saturate Fc receptors on immunocompetent cells. At mild acidic pH cruzipain produced further degradation of the Fc of all subclasses, the Fd of IgG4 and partially the Fd of IgG1, with the consistent loss of any antibody activity. The L chains apparently were not affected. Thus, cruzipain should be able to modulate, depending on the subclass selected and the pH of the environment, the production and the length of different biologically active/inactive IgG fragments.     

Immunomodulatory effect of synthetic peptides corresponding to sequences within the CH2 and CH3 domain of human IgG1

The Fc region of IgG is known to be the source of small peptides possessing immunomodulatory function. Results are summarized showing the effect of synthetic peptides composed of surface exposed residues of C gamma 2 or C gamma 3 domains on different steps of human B lymphocyte activation cycle. Both the CH2 (289Thr-301Arg) and CH3 (407Tyr-416Arg) peptides as well as the whole Fc fragment enhanced the IgM synthesis of PWM or PMA + CaI activated lymphocytes. This effect was exerted at the early phase of B cell activation. The incubation of separated resting B cells with Fc fragments or CH2 peptides resulted in increase of cell volume and in expression of HLA-DR antigen. On the other hand, LIF production was induced both by CH2 and CH3 peptides. It was also shown that Fc peptides induce IL-1 release from monocytes. The results suggest that the CH2 and CH3 domain peptides exert their effect partly directly, by activating resting B cells, rendering the cells more susceptible to other stimuli; and moreover, by enhancing the humoral response by triggering the release of IL-1.     

Investigation of the binding site of mouse IgG subclasses to homologous peritoneal macrophages.

The binding of mouse myeloma IgG1, IgG2a, IgG2b, IgG1 Fc, IgG2b Fc and a pepsin produced C-terminal subfragment of IgG1 Fc and IgG2b Fc (provisionally identified as pFc') to mouse peritoneal macrophages was investigated. The high affinity cytophilic antibodies belonged to IgG2 subclasses and the binding site of these antibodies was located in the CH3 homology region.     

Structure of bovine immunoglobulin constant region heavy chain gamma 1 and gamma 2 genes

Two bovine immunoglobulin constant region gamma heavy chain germline gene sequences are described. A gamma 1 gene was cloned from a lambda 2001 calf liver library screened with a human gamma 4 (pBRH4.1) probe and is contained in a 5.8 kb BamH1 hybridizing fragment. The gamma 2 gene was from an EMBL4 lambda library and is in a 6.6 kb BamH1 fragment. Each of these genes is arranged in four exons corresponding to the three CH domains and the hinge of gamma heavy chain genes; normal RNA splice and polyadenylation sites are present. The translated C-terminal peptide sequences of the genes match exactly the equivalent peptide sequences of bovine IgG1 and IgG2 heavy chains, identifying them as gamma 1 and gamma 2. The derived protein sequences reveal 96, 80 and 83% identity of amino acid residues between their CH1, CH2 and CH3 domains. Two adjacent cysteine residues encoded in the CH1 exons suggest that, as in rabbit gamma, an extra intra-chain disulphide bond occurs in the bovine gamma heavy chains. Significant DNA rearrangement in the hinge-CH2 region is evident in the bovine gamma 2 gene, with resultant deletion and substitution of amino acid residues in the lower hinge and N-terminal portion of the CH2 domain. The Fab-Fc interface of bovine IgG2 is predicted to be sterically blocked, relative to IgG1, which has implications for effector differences between the bovine gamma subclasses.     

Crystallization of a complex between the Fab fragment of a human immunoglobulin M (IgM) rheumatoid factor (RF-AN) and the Fc fragment of human IgG4.

Rheumatoid factors (RF) are the characteristic autoantibodies found in patients with rheumatoid arthritis. They recognize epitopes in the Fc region of immunoglobulin G (IgG) and are often of the IgM isotype. In order to analyse the nature of RF-Fc interactions, we have crystallized a complex between the Fab fragment of a human monoclonal IgM rheumatoid factor (RF-AN) and the Fc fragment of human IgG4. The stoichiometry of the complex within the crystals was found to be 2:1 Fab:Fc. The crystals diffracted X-rays to 0.3 nm resolution, and the space group was C2, with cell dimensions a = 16.03 nm, b = 8.19 nm, c = 6.42 nm, beta = 98.3 degrees. We have also determined the sequence of the variable region of the RF-AN light chain, not hitherto reported. This belongs to the V lambda III-a subgroup and is closely related to the germline gene Humlv318, from which it differs in three amino acid residues. This is the first reported crystallized complex between a human autoantibody and its autoantigen.     

Biochemical analysis and crystallisation of Fc gamma RIIa, the low affinity receptor for IgG.

Fc gamma RIIa is one of a family of specific cell surface receptors for immunoglobulin. Fc gamma RIIa, which binds immune complexes of certain IgG isotypes, plays important roles in immune homeostasis. However, the precise characteristics of IgG binding and three-dimensional structure of Fc gamma RIIa have not been reported. This study describes the affinity of the Fc gamma RIIa:IgG interaction as well as biochemical characterisation of recombinant Fc gamma RIIa that has been used to generate high quality crystals. Equilibrium binding analysis of the Fc gamma RII:IgG interaction found, IgG3 binds with an affinity of K(D) = 0.6 microM, as expected. Unlike other Fc gamma R, IgG4 also bound to Fc gamma RIIa, K(D) = 3 microM, clearly establishing Fc gamma RIIa as an IgG4 receptor. Biochemical analysis of mammalian and insect cell derived Fc gamma RIIa established the genuine N-terminus with Q being the first amino acid in the sequence Q, A, A, A, P... extending the N-terminus further than previously thought. Furthermore, both potential N-linked glycosylation sites are occupied. Electrospray ionisation mass spectrometry (ESMS) indicate that the N-glycans of baculovirus derived Fc gamma RIIa are core mannose oligosaccharide side chains. Finally, we describe the first crystallisation of diffraction quality crystals of soluble Fc gamma RIIa. Orthorhombic crystals diffract X-rays beyond 2.1 A resolution in the space group P2(1)2(1)2 with cell dimensions a = 78.8 A, b = 100.5 A, c = 27.8 A. This marks a significant advance towards understanding the three-dimensional structure of Fc gamma RIIa and related FcR proteins that share high amino acid identity with Fc gamma RIIa.     

Complement activation is not required for IgG-mediated suppression of the antibody response

Feedback suppression of the antibody response by IgG is known to be dependent on intact Fc regions. However, it is not clear which of the Fc-mediated effector functions is required. In the present report we have studied whether ability or inability of the IgG antibodies to activate the complement system was of consequence for their immunosuppressive effect. First, a monoclonal IgG1-anti-2,4,6-trinitrophenyl (TNP) antibody, unable to activate complement via the classical or alternate pathway, was shown to be able to inhibit more than 90% of the in vivo sheep erythrocyte-specific antibody response in mice when TNP coupled to sheep erythrocytes was used as antigen. Second, we investigated the immunosuppressive ability of a non-complement-activating mutant IgG2a-anti-TNP monoclonal antibody. The mutant differs from the wild type by a single amino acid substitution in the CH2 domain leading to inability to fix complement factor C1q. However, the mutant has the same affinity for antigen and the same Fc receptor-binding capacity as the wild type antibody. It is demonstrated that the mutant was as efficient as the wild type antibody in inhibiting an in vitro antibody response to TNP-coupled sheep erythrocytes. These findings confirm the non-determinant specificity and Fc dependence of IgG-mediated suppression, and show that the Fc-mediated effector mechanism is independent of complement activation. The results instead suggest binding to Fc receptors as a necessary step in feedback immunosuppression and favor inactivation of B cells by cross-linking of Fc and antigen receptors on their surface rather than elimination of antigen by complement-dependent phagocytosis as the effector mechanism.