We have mapped the specificity of 28 monoclonal IgM rheumatoid factors (RFs) produced by heterohybridomas derived from five healthy blood donors immunized with mismatched human red blood cells (HID). The HID-RFs did not differ in their binding specificity for IgG epitopes from RFs that we previously analyzed from patients with Waldenström’s macroglobulinemia. However, IgM RFs produced by HID differed in their specificity for IgG compared with RFs expressed by patients with rheumatoid arthritis (RA-RFs). Only 1 of 28 HID-RFs bound all IgG subclasses (pan binding pattern) compared with 7 of 19 RA-RFs (p = 0.006). Three HID-RFs bound IgG3 compared with 9 RA-RFs (p = 0.007). Fine specificity differences were also identified between HID- and RA-RFs. Therefore, some RA-RFs show novel specificities for IgG not found among RFs from HID or individuals with Waldenström’s macroglobulinemia who do not have joint disease. These Abs with unique specificities may represent disease-specific autoantibodies in patients with RA. Nine of the HID-RFs from the same individual were clonally related, and several contained somatic mutations. Even when the clonally related HID-RFs were considered as one RF for comparison, the reactivity of the HID-RFs differed significantly from RA-RFs in their inability to recognize all IgG subclasses (p = 0.044) and recognize IgG3 (p = 0.041). Interestingly, among the clonally related RFs, considerable differences in the specificity for IgG were also observed, with the RF containing the most somatic mutations in VH and VL showing the most distinctive specificity changes. Therefore, these studies also demonstrate a correlation between somatic mutation and binding specificity.

Rheumatoid factor (RF)3 autoantibodies bind epitopes within the constant regions of IgG, and they are expressed in the sera of almost all patients with rheumatoid arthritis (RA) (1, 2). However, RFs are also expressed during the normal immune response following antigenic challenge (3), and they are produced by patients with other diseases not associated with arthritis (4). Nevertheless, RF expression correlates with disease activity and severity in RA (5). RFs are produced by inflamed synovial tissue along with their cognate ligand IgG and complement (6, 7, 8), and they can activate complement by forming RF-IgG immune complexes in the joint fluid and synovial tissues of patients with RA (6, 7, 8, 9, 10). In this way, RFs have been implicated in the pathogenesis of synovial disease by their ability to induce inflammation (7, 8, 11, 12, 13). Within the synovia of RA patients there are disproportionately high concentrations of IgG3- and IgG3-binding RFs compared with those in the blood (14), suggesting that a select subgroup of RFs may be expressed uniquely in patients with synovial disease (14, 15). These RFs may recognize a limited repertoire of determinant(s) expressed on only some IgG subclasses, and by virtue of this restricted binding specificity, they may be identified as disease-specific autoantibodies (15).

To better understand the pathogenic role of RFs in RA, many investigators have studied the specific IgG epitopes bound by RFs from patients with RA (RA-RF) and RFs from individuals without arthritis. The specificity of RFs for IgG subclasses, allotypes and isoallotypes, constant domains, and specific residues within these domains have been described in detail (14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Indeed, when we characterized the specificities of monoclonal IgM RFs from patients with RA and Waldenström’s macroglobulinemia (Wmac) (15, 18, 23), using genetically engineered, chimeric IgG Abs bearing human γ constant regions, we observed that some RA-RFs recognized distinct epitopes not recognized by Wmac-RFs, including epitopes outside the CH2-CH3 interface, which is the major RF binding site of almost all the Wmac-RFs we studied (15, 23).

In this communication we report the binding specificity of a panel of 28 monoclonal IgM RFs derived from healthy immunized donors (HID). In addition, we observed changes in gross and fine specificities among the nine clonally related, somatically mutated RFs in the panel. We found that HID-RFs are similar to Wmac-RFs in their specificity for IgG. In contrast, a subgroup of RA-RFs exhibits significant differences in IgG binding specificity compared with HID-RFs. This subgroup may represent disease-specific autoantibodies associated with the immune dysregulation present in RA.

Human constant region genes of the four subclasses of IgG were cloned from a genomic library and were joined to the murine VH gene cloned from the antidansyl-specific murine hybridoma 27-44. The human κ light chain constant region gene was similarly joined to the murine antidansyl VK gene. The nucleotide sequencing of heavy chain constant regions showed the IgG3 heavy chain constant gene to be identical in amino acid sequence to the IgG3 heavy chain OMM, and the IgG4 gene to be identical with those previously reported (24). The allotypes and isoallotypes expected to be expressed on IgG3 and IgG4 have been described previously (18). The methods of expression, Ab production, assembly, m.w., and secretion of the chimeric IgG Abs used in these studies have also been described in detail (18, 23, 25, 26).

To facilitate exon shuffling, the heavy chain constant genes were subcloned into pBR322 as a SalI-BamHI cassette (18). Using oligonucleotide linkers, a family of genes was constructed with a PvuI site inserted in each intron. Because pBR322 contains a single PvuI site, digestion with PvuI allowed the isolation of two DNA fragments, each containing part of the heavy chain constant gene. These fragments, from either IgG3 or IgG4, were ligated to complementary fragments from IgG4 or IgG3, respectively, to create hybrid heavy chain genes composed of domains from different IgG subclasses.

Human constant region genes for IgG3, IgG4, and a hybrid IgG3/IgG4 gene were cloned into the SalI and BamHI sites of the M13 mp19 polylinker, and site-directed mutations were introduced either by the two-primer method (27) or by the uracil-containing template method (28). Mutations were verified by dideoxy sequencing (26), and mutated constant region genes were subcloned into the heavy chain vector for expression as previously described in detail (18).

Secreting clones were grown in culture for 3 to 4 days in Iscove’s modified Dulbecco’s medium (Life Technologies, Grand Island, NY) supplemented with 5% or 10% bovine calf serum (HyClone, Logan, UT) (18). The assembly, secretion, and m.w. of the chimeric IgG Abs were determined by labeling cells with [35S]methionine. The secreted IgG was immunoprecipitated with polyclonal rabbit anti-human IgG antiserum and fixed Staphylococcus aureus bacteria (Calbiochem, La Jolla, CA) and was analyzed by SDS-PAGE under both reducing and nonreducing conditions (18).

To quantitate RF binding to IgG, a direct binding ELISA was developed in which 96-well plates were coated with DNS-BSA and chimeric IgG as previously described in detail (18). Because most IgM RFs bound strongly to IgG4, RF binding to each chimeric IgG protein was usually expressed as a percentage of IgM RF binding to IgG4. In those instances where poor binding to IgG4 was observed, RF binding was expressed as a percentage relative to the IgG subclass that was bound best. The concentration of chimeric IgG used in these assays was always chosen to be in excess with regard to RF binding (18). In short, RF binding to immobilized chimeric IgG plateaued at 0.1 to 0.2 μg of IgG/well over a wide range of RF concentrations. To ensure that the amount of immobilized IgG was in Ag excess, 0.22 μg of IgG/well was routinely used for all ELISA performed.

SPA was used as a competitive inhibitor of RF binding to chimeric IgG Abs by ELISA as described above (15). RF binding to the various IgG subclasses in the presence of excess, fluid phase concentrations of SPA was expressed as the percent binding of RF to the same IgG subclass Ab in the absence of SPA (15). In short, serial dilutions, 0.1 to 100 μg/ml of SPA was incubated in fluid phase with the HID-RFs at concentrations optimal for IgG binding. The mixtures were then added to chimeric IgG bound to dansylated BSA-coated wells for 4 h at room temperature. Wells were washed, and the amount of RF bound was quantified as previously described (15). To determine whether any of the RFs bound SPA directly, SPA at a concentration of 0.5 μg/well was incubated overnight at 4°C in 96-well plates (Immunlon II, Dynatech, Chantilly, VA). Wells were then washed with PBS-Tween, and then blocked with 2.5% BSA at 37°C for 1 h. Various concentrations of each RF were added for 1 h at 37°C and then washed with PBS-Tween. Horseradish peroxidase-conjugated goat anti-μ-specific Abs were used to determine whether any RF bound SPA as previously reported (18). None of the RFs bound SPA directly.

Monoclonal RFs were obtained from a panel of five women volunteers immunized with mismatched human RBC Ags by fusing blood-derived B cells with heterohybridomas as previously described (30, 31, 32). The HID who provided the study RFs were enrolled in a licensed program for the production of polyclonal anti-blood group antisera for prophylactic and diagnostic purposes. Informed consent was obtained from all women, and the risks associated with immunization were explained (30).

Comparisons were made between HID-, Wmac-, and RA-RF binding to the same genetically engineered IgG Abs by two-tailed Fisher’s exact test.

The IgG subclass recognition patterns of 28 monoclonal RFs produced by B cell/heterohybrids derived from HID were determined using chimeric IgG Abs of the four subclasses (Table I). Twenty-one of twenty-eight (75%) HID-RFs (MR-1, -2, -3, -5, -12, -13, -14, -20, -25, -27, -28, -30, -33, -37, -39, and -41; DI-2; FO-3; and TT-3, -7, and -9) bound IgG1, -2, and -4 only, consistent with the Ga binding pattern (33). Only one of the HID-RFs (MR-24) bound all four IgG subclasses. Of the 28 HID-RFs, only 3 (11%) bound IgG3 well (MR-24, DI-4, and LN-11). Two HID-RFs bound IgG1 only (DI-1 and FO-2). In addition, three other HID-RFs showed various combinations of reactivity with the four IgG subclasses: DI-4 bound IgG1, -3, and -4 only; MR-16 bound IgG1 and -4 only; and LN-10 bound IgG1 and -2 only.

Table I.

Monoclonal healthy immunized donor-derived RF binding to IgG subclassesa

RFIgG1IgG2IgG3IgG4Binding Pattern
MR-1 93.1 ± 3.6 93.8 ± 2.2 11.5 ± 5.3 100% Ga 
MR-2b 97.2 ± 2.5 96.6 ± 6.2 4.7 ± 2.3 100% Ga 
MR-3 77.5 ± 16.4 70.8 ± 11.1 9.1 ± 5.1 100% Ga 
MR-5 76.4 ± 2.5 51.2 ± 12.3 15.4 ± 7.5 100% Ga 
MR-12 92.2 ± 3.0 91.0 ± 8.1 15.7 ± 8.0 100% Ga 
MR-13b 87.0 ± 0.3 78.5 ± 1.7 12.2 ± 1.0 100% Ga 
MR-14b 128.0 ± 12 37.0 ± 6.9 3.0 ± 0.6 100% Ga 
MR-20 92.0 ± 7.1 92.7 ± 7.7 7.7 ± 3.4 100% Ga 
MR-25b 97.0 ± 3.1 39.0 ± 8.2 4.3 ± 1.0 100% Ga 
MR-27 87.4 ± 2.4 86.6 ± 2.4 9.8 ± 1.1 100% Ga 
MR-28b 84.0 ± 3.4 67.8 ± 3.1 11.5 ± 3.1 100% Ga 
MR-30b 85.3 ± 8.2 52.0 ± 16.5 6.6 ± 3.9 100% Ga 
MR-33b 105.4 ± 3.7 84.9 ± 3.9 17.2 ± 1.0 100% Ga 
MR-37b 82.0 ± 8.4 77.3 ± 0.7 5.6 ± 5.9 100% Ga 
MR-39 98.0 ± 2.1 101.6 ± 4.6 15.2 ± 0.6 100% Ga 
MR-41 94.1 ± 13.4 44.1 ± 2.9 5.1 ± 6.5 100% Ga 
DI-2 90.5 ± 7.3 88.2 ± 6.8 10.5 ± 3.1 100% Ga 
FO-3 24.6 ± 3.4 108.2 ± 9.1 5.2 ± 1.0 100% Ga 
TT-3 78.0 ± 6.0 54.0 ± 4.4 2.1 ± 6.3 100% Ga 
TT-7 109.8 ± 6.0 100.7 ± 6.5 14.3 ± 2.4 100% Ga 
TT-9 99.0 ± 2.4 93.5 ± 1.1 12.4 ± 1.7 100% Ga 
MR-24 98.8 ± 2.4 121.5 ± 2.9 123.6 ± 0.1 100% pan 
DI-4 33 ± 0.4 2.7 ± 0.2 100% 48 ± 1.1 1, 3, 4 
LN-11 79.6 ± 6.3 90.8 ± 9.4 100% 17.9 ± 2.6 1, 2, 3 
MR-16b 90.7 ± 0.5 17.6 ± 12.6 9.7 ± 3.3 100% 1, 4 
LN-10 93.2 ± 1.7 100% 12.2 ± 1.9 5.7 ± 0.9 1, 2 
DI-1 100% 3.3 ± 0.4 6.7 ± 1.8 7.4 ± 2.0 1 only 
FO-2 100% 4.0 ± 0.2 7.7 ± 1.1 6.5 ± 0.5 1 only 
RFIgG1IgG2IgG3IgG4Binding Pattern
MR-1 93.1 ± 3.6 93.8 ± 2.2 11.5 ± 5.3 100% Ga 
MR-2b 97.2 ± 2.5 96.6 ± 6.2 4.7 ± 2.3 100% Ga 
MR-3 77.5 ± 16.4 70.8 ± 11.1 9.1 ± 5.1 100% Ga 
MR-5 76.4 ± 2.5 51.2 ± 12.3 15.4 ± 7.5 100% Ga 
MR-12 92.2 ± 3.0 91.0 ± 8.1 15.7 ± 8.0 100% Ga 
MR-13b 87.0 ± 0.3 78.5 ± 1.7 12.2 ± 1.0 100% Ga 
MR-14b 128.0 ± 12 37.0 ± 6.9 3.0 ± 0.6 100% Ga 
MR-20 92.0 ± 7.1 92.7 ± 7.7 7.7 ± 3.4 100% Ga 
MR-25b 97.0 ± 3.1 39.0 ± 8.2 4.3 ± 1.0 100% Ga 
MR-27 87.4 ± 2.4 86.6 ± 2.4 9.8 ± 1.1 100% Ga 
MR-28b 84.0 ± 3.4 67.8 ± 3.1 11.5 ± 3.1 100% Ga 
MR-30b 85.3 ± 8.2 52.0 ± 16.5 6.6 ± 3.9 100% Ga 
MR-33b 105.4 ± 3.7 84.9 ± 3.9 17.2 ± 1.0 100% Ga 
MR-37b 82.0 ± 8.4 77.3 ± 0.7 5.6 ± 5.9 100% Ga 
MR-39 98.0 ± 2.1 101.6 ± 4.6 15.2 ± 0.6 100% Ga 
MR-41 94.1 ± 13.4 44.1 ± 2.9 5.1 ± 6.5 100% Ga 
DI-2 90.5 ± 7.3 88.2 ± 6.8 10.5 ± 3.1 100% Ga 
FO-3 24.6 ± 3.4 108.2 ± 9.1 5.2 ± 1.0 100% Ga 
TT-3 78.0 ± 6.0 54.0 ± 4.4 2.1 ± 6.3 100% Ga 
TT-7 109.8 ± 6.0 100.7 ± 6.5 14.3 ± 2.4 100% Ga 
TT-9 99.0 ± 2.4 93.5 ± 1.1 12.4 ± 1.7 100% Ga 
MR-24 98.8 ± 2.4 121.5 ± 2.9 123.6 ± 0.1 100% pan 
DI-4 33 ± 0.4 2.7 ± 0.2 100% 48 ± 1.1 1, 3, 4 
LN-11 79.6 ± 6.3 90.8 ± 9.4 100% 17.9 ± 2.6 1, 2, 3 
MR-16b 90.7 ± 0.5 17.6 ± 12.6 9.7 ± 3.3 100% 1, 4 
LN-10 93.2 ± 1.7 100% 12.2 ± 1.9 5.7 ± 0.9 1, 2 
DI-1 100% 3.3 ± 0.4 6.7 ± 1.8 7.4 ± 2.0 1 only 
FO-2 100% 4.0 ± 0.2 7.7 ± 1.1 6.5 ± 0.5 1 only 
a

IgM RF binding to the four subclasses of chimeric IgG was measured using a RF ELISA. Chimeric antibodies were first bound to dansyl-BSA-coated plates, and then RF binding to the immobilized chimeric antibody was detected using an enzyme conjugated anti-μ chain specific antiserum. Results are the mean of six determinations from two separate experiments ± SEM for each RF expressed as a percentage binding to IgG4 defined as 100%. For RFs DI-4 and LN-11, the results are expressed as a percentage ± SEM of binding to IgG3 defined as 100%. For RFs DI-1 and FO-2, the results are expressed as a percentage ± SEM of binding to IgG1 defined as 100%. For RF LN-10, the results are expressed as a percentage ± SEM of binding to IgG2 defined as 100%.

b

Clonally related RFs.

Nine HID-RFs from one immunized control were produced by clonally related B cells (MR-2, -13, -14, -16, -25, -28, -30, -33, and -37) (30). Eight of these were reactive with the Ga epitope, while the ninth, MR-16, showed a different specificity for IgG; MR-16 failed to bind IgG2 and recognized only IgG1 and -4 (see Table I). Therefore, the IgG epitope bound by MR-16 must differ from the Ga epitope recognized by the eight other clonally-related RFs. All the clonally related RFs are encoded by the VH1 and the HumKv325 genes; however, MR-16 contains the most mutations in the VH domain compared with the other eight related HID-RFs: two replacement and two silent mutations in CDR1 and -2, and there are four replacement and two silent mutations in the framework (30). In addition, MR-16 contains two mutations, one replacement and one silent, in the framework of the HumKv325 gene (M. Børretzen et al., manuscript in preparation). These results, therefore, provide evidence that somatic mutation can lead to changes in RF binding specificity.

Previously we showed that the CH3 domain determines the preferential reactivity of RA- and Wmac-derived RFs that recognize the Ga epitope present on IgG1, -2, and -4, but not IgG3 (13, 18). We now find (Table II) that the CH3 domain determines the specificity of the 21 HID-RFs that also show this binding pattern; these RFs bind IgG3 bearing the CH3 domain of IgG4 (IgG3/4 hybrid Ab 2208), yet fail to recognize IgG4 bearing the CH3 domain of IgG3 (antibody 2204). Only LN-11, which binds IgG1, -2, and -3, was unable to bind any of the hybrid Abs that contained the CH3 domain of IgG4 (Table II), suggesting that at least one polymorphism that distinguishes IgG3 from IgG4 is contained within the epitope recognized by LN-11, and that the epitope is disrupted by domain switches between IgG3 and -4. As anticipated, HID-RFs LN-10, DI-1, and FO-2 that do not recognize IgG3 or -4 did not bind any of the IgG3/4 hybrids (data not shown).

Table II.

Monoclonal healthy immunized donor-derived RF binding to domain-shuffled IgGa

RFExchangesBinding Pattern
Wild-type IgG3CH1 ExchangesCH2-CH3 ExchangesCH3 Exchanges IgG3
2205 4-3-3-32203 3-4-4-42207 4-4-3-32206 3-3-4-42204 4-4-4-32208 3-3-3-4
MR-1 4.6 ± 2.3 6.6 ± 0.3 98.1 ± 3.3 4.0 ± 1.3 91.6 ± 4.3 2.3 ± 2.1 99.6 ± 8.3 Ga 
MR-2b 0.4 ± 0.1 2.3 ± 0.2 94.5 ± 8.2 0.7 ± 0.5 91.2 ± 4.3 5.7 ± 1.6 89.1 ± 13 Ga 
MR-3 5.6 ± 2.0 7.4 ± 1.7 78.5 ± 5.5 9.9 ± 2.6 89.1 ± 0.8 8.6 ± 3.4 92.0 ± 2.8 Ga 
MR-5 4.9 ± 5.2 7.0 ± 5.3 92.6 ± 1.0 8.6 ± 4.3 85.4 ± 2.1 9.6 ± 4.3 99.3 ± 4.0 Ga 
MR-12 5.3 ± 3.2 2.8 ± 3.3 88.0 ± 9.1 1.3 ± 0.8 85.4 ± 2.2 3.7 ± 2.1 91.2 ± 2.1 Ga 
MR-13b 9.8 ± 4.3 6.4 ± 3.3 96.7 ± 5.8 4.2 ± 2.7 89.7 ± 7 5.5 ± 5.1 91.6 ± 2.6 Ga 
MR-14b 3.6 ± 3.6 6.5 ± 3.5 83.7 ± 8.4 6.6 ± 2.9 85.9 ± 6.5 7.2 ± 2.9 96.9 ± 4.2 Ga 
MR-20 6.6 ± 3.4 9.1 ± 6.5 96.0 ± 9.6 6.7 ± 4.9 84.8 ± 4.5 5.9 ± 4.3 90.4 ± 7.3 Ga 
MR-25b 9.4 ± 4.7 7.8 ± 5.4 88.0 ± 5.1 5.7 ± 4.9 87.4 ± 9.9 8.6 ± 4.3 91.8 ± 0.8 Ga 
MR-27 11.3 ± 1.1 8.7 ± 2.1 88.5 ± 2.2 8.6 ± 0.4 90.8 ± 9.5 6.2 ± 6.6 92.3 ± 9.9 Ga 
MR-28b 6.1 ± 2.8 3.5 ± 0.8 85.7 ± 3.1 3.8 ± 4.0 92.0 ± 5.0 1.6 ± 0.3 95.3 ± 7.5 Ga 
MR-30b 6.4 ± 3.5 3.4 ± 3.3 87.6 ± 8.7 3.3 ± 0.1 88.8 ± 2.2 7.5 ± 1.1 90.1 ± 7.5 Ga 
MR-33b 4.7 ± 1.3 3.6 ± 0.3 82.8 ± 7.9 4.7 ± 0.2 89.3 ± 9.9 5.1 ± 0.3 86.9 ± 8.8 Ga 
MR-37b 6.3 ± 2.2 6.1 ± 0.6 81.6 ± 1.4 5.8 ± 0.1 80.5 ± 6.5 7.1 ± 1.5 89.9 ± 9.9 Ga 
MR-39 8.4 ± 5.5 8.2 ± 2.8 91.1 ± 0.3 6.4 ± 4.7 96.6 ± 3.5 8.6 ± 5.3 93.1 ± 9.3 Ga 
MR-41 9.6 ± 4.0 5.2 ± 0.7 82.2 ± 9.0 7.6 ± 2.8 80.0 ± 0.4 7.4 ± 3.1 86.8 ± 9.0 Ga 
DI-2 7.7 ± 0.5 9.8 ± 7.8 98.6 ± 9.9 7.5 ± 0.3 94.2 ± 8.2 5.5 ± 0.1 90.5 ± 8.2 Ga 
FO-3 5.2 ± 1.0 10.7 ± 0.4 81.1 ± 0.8 9.4 ± 0.3 83.4 ± 0.7 12.9 ± 0.7 72.4 ± 0.7 Ga 
TT-3 1.9 ± 3.4 8.3 ± 0.2 83.5 ± 1.0 8.0 ± 0.5 85.2 ± 1.2 9.1 ± 0.7 78.5 ± 1.0 Ga 
TT-7 8.4 ± 7.6 9.1 ± 3 96.6 ± 3.4 7.6 ± 5 92.8 ± 4.1 6.5 ± 5.0 89.7 ± 1.2 Ga 
TT-9 12.4 ± 1.7 12.3 ± 0.4 94.0 ± 5.5 9.0 ± 0.3 90.3 ± 0.7 8.6 ± 0.2 94.5 ± 1.4 Ga 
MR-24 123.6 ± 0.1 96.0 ± 0.9 74.2 ± 0.6 88.7 ± 2.2 87.4 ± 0.8 89.5 ± 2.5 89.7 ± 3.1 Pan 
DI-4 100% 100.0 ± 1.1 92.3 ± 5.3 69.1 ± 15 79.0 ± 13 83.0 ± 3.6 96.8 ± 1.1 1, 3, 4 
LN-11 100% 103.4 ± 0.8 7.2 ± 0.9 82.4 ± 1.2 16.4 ± 0.4 86.2 ± 0.6 7.7 ± 1.1 1, 2, 3 
MR-16b 8.1 ± 2.3 8.4 ± 3.9 96.7 ± 5.8 7.2 ± 6.3 89.7 ± 7.3 9.8 ± 4.3 91.6 ± 2.6 1, 4 
RFExchangesBinding Pattern
Wild-type IgG3CH1 ExchangesCH2-CH3 ExchangesCH3 Exchanges IgG3
2205 4-3-3-32203 3-4-4-42207 4-4-3-32206 3-3-4-42204 4-4-4-32208 3-3-3-4
MR-1 4.6 ± 2.3 6.6 ± 0.3 98.1 ± 3.3 4.0 ± 1.3 91.6 ± 4.3 2.3 ± 2.1 99.6 ± 8.3 Ga 
MR-2b 0.4 ± 0.1 2.3 ± 0.2 94.5 ± 8.2 0.7 ± 0.5 91.2 ± 4.3 5.7 ± 1.6 89.1 ± 13 Ga 
MR-3 5.6 ± 2.0 7.4 ± 1.7 78.5 ± 5.5 9.9 ± 2.6 89.1 ± 0.8 8.6 ± 3.4 92.0 ± 2.8 Ga 
MR-5 4.9 ± 5.2 7.0 ± 5.3 92.6 ± 1.0 8.6 ± 4.3 85.4 ± 2.1 9.6 ± 4.3 99.3 ± 4.0 Ga 
MR-12 5.3 ± 3.2 2.8 ± 3.3 88.0 ± 9.1 1.3 ± 0.8 85.4 ± 2.2 3.7 ± 2.1 91.2 ± 2.1 Ga 
MR-13b 9.8 ± 4.3 6.4 ± 3.3 96.7 ± 5.8 4.2 ± 2.7 89.7 ± 7 5.5 ± 5.1 91.6 ± 2.6 Ga 
MR-14b 3.6 ± 3.6 6.5 ± 3.5 83.7 ± 8.4 6.6 ± 2.9 85.9 ± 6.5 7.2 ± 2.9 96.9 ± 4.2 Ga 
MR-20 6.6 ± 3.4 9.1 ± 6.5 96.0 ± 9.6 6.7 ± 4.9 84.8 ± 4.5 5.9 ± 4.3 90.4 ± 7.3 Ga 
MR-25b 9.4 ± 4.7 7.8 ± 5.4 88.0 ± 5.1 5.7 ± 4.9 87.4 ± 9.9 8.6 ± 4.3 91.8 ± 0.8 Ga 
MR-27 11.3 ± 1.1 8.7 ± 2.1 88.5 ± 2.2 8.6 ± 0.4 90.8 ± 9.5 6.2 ± 6.6 92.3 ± 9.9 Ga 
MR-28b 6.1 ± 2.8 3.5 ± 0.8 85.7 ± 3.1 3.8 ± 4.0 92.0 ± 5.0 1.6 ± 0.3 95.3 ± 7.5 Ga 
MR-30b 6.4 ± 3.5 3.4 ± 3.3 87.6 ± 8.7 3.3 ± 0.1 88.8 ± 2.2 7.5 ± 1.1 90.1 ± 7.5 Ga 
MR-33b 4.7 ± 1.3 3.6 ± 0.3 82.8 ± 7.9 4.7 ± 0.2 89.3 ± 9.9 5.1 ± 0.3 86.9 ± 8.8 Ga 
MR-37b 6.3 ± 2.2 6.1 ± 0.6 81.6 ± 1.4 5.8 ± 0.1 80.5 ± 6.5 7.1 ± 1.5 89.9 ± 9.9 Ga 
MR-39 8.4 ± 5.5 8.2 ± 2.8 91.1 ± 0.3 6.4 ± 4.7 96.6 ± 3.5 8.6 ± 5.3 93.1 ± 9.3 Ga 
MR-41 9.6 ± 4.0 5.2 ± 0.7 82.2 ± 9.0 7.6 ± 2.8 80.0 ± 0.4 7.4 ± 3.1 86.8 ± 9.0 Ga 
DI-2 7.7 ± 0.5 9.8 ± 7.8 98.6 ± 9.9 7.5 ± 0.3 94.2 ± 8.2 5.5 ± 0.1 90.5 ± 8.2 Ga 
FO-3 5.2 ± 1.0 10.7 ± 0.4 81.1 ± 0.8 9.4 ± 0.3 83.4 ± 0.7 12.9 ± 0.7 72.4 ± 0.7 Ga 
TT-3 1.9 ± 3.4 8.3 ± 0.2 83.5 ± 1.0 8.0 ± 0.5 85.2 ± 1.2 9.1 ± 0.7 78.5 ± 1.0 Ga 
TT-7 8.4 ± 7.6 9.1 ± 3 96.6 ± 3.4 7.6 ± 5 92.8 ± 4.1 6.5 ± 5.0 89.7 ± 1.2 Ga 
TT-9 12.4 ± 1.7 12.3 ± 0.4 94.0 ± 5.5 9.0 ± 0.3 90.3 ± 0.7 8.6 ± 0.2 94.5 ± 1.4 Ga 
MR-24 123.6 ± 0.1 96.0 ± 0.9 74.2 ± 0.6 88.7 ± 2.2 87.4 ± 0.8 89.5 ± 2.5 89.7 ± 3.1 Pan 
DI-4 100% 100.0 ± 1.1 92.3 ± 5.3 69.1 ± 15 79.0 ± 13 83.0 ± 3.6 96.8 ± 1.1 1, 3, 4 
LN-11 100% 103.4 ± 0.8 7.2 ± 0.9 82.4 ± 1.2 16.4 ± 0.4 86.2 ± 0.6 7.7 ± 1.1 1, 2, 3 
MR-16b 8.1 ± 2.3 8.4 ± 3.9 96.7 ± 5.8 7.2 ± 6.3 89.7 ± 7.3 9.8 ± 4.3 91.6 ± 2.6 1, 4 
a

H chain gene exons were shuffled between IgG3 and IgG4 generating six hybrid antibodies. The domain structure of each hybrid antibody is represented schematically at the top of each column in the order CH1-hinge-CH2-CH3. The number 3 and 4 indicates whether the exon originated from the IgG3 or IgG4 heavy chain gene. Results are the mean of six determinations from two separate experiments ± SEM for each RF expressed as a percentage binding to IgG4 defined as 100%. For RFs DI-4 and LN-11, the results are expressed as a percentage ± SEM of binding to IgG3 defined as 100%.

b

Clonally related RFs.

To define the amino acids within CH3 that determine the specificity of RF binding to IgG, we reacted the panel of HID-RFs with Abs containing amino acid substitutions in the CH3 domain of IgG3 or IgG4 (Tables III and IV).

RFs that bind IgG1, -2, and -4 only (Ga reactivity).

We previously observed that the His/Arg polymorphism at position 435 in CH3 that distinguished IgG1, -2, and -4 (His) from IgG3 (Arg) was critical for Wmac- and RA-RFs that recognize the Ga epitope (13, 23). Similar results were seen with HID-RFs that also recognize IgG1, -2, and -4. An Arg→His change at position 435 in the CH3 domain of the IgG3/4 hybrid Ab 3601 (containing the CH1, hinge, and CH2 domains of IgG4 fused with the CH3 domain of IgG3 containing His435) supports RF binding for all these RFs. Substituting His→Ala at position 435 in IgG4 (construct 3612, Table III) prevented Ga-reactive, HID-RF binding to IgG4. While this reactivity demonstrates the importance of His435 in IgG recognition by these RFs, only one Ga-reactive HID-RF (FO-3) was able to bind construct 3603 (Table III) that contains the Arg→His change at 435 in the context of IgG3. However, many of the Ga-reactive, HID-RFs (14 of 21) bound IgG4 containing Arg at 435 (antibody 3605). Therefore, although the His/Arg polymorphism is important in determining the Ga epitope, additional residues present in IgG4, but absent in IgG3, are also required.

Table III.

Monoclonal healthy immunized donor-derived RF binding to chimeric IgG containing mutations in CH3 at positions 435-436a

Binding PatternRF3601 4-4-4-3 His-Tyr3603 3-3-3-3 His-Tyr3605 4-4-4-4 Arg-Tyr3612 4-4-4-4 Ala-TyrIgG3 3-3-3-3 Arg-TyrComments
Ga MR-1 86.7 ± 2.8 6.9 ± 0.2 99.8 ± 3.0 12.1 ± 0.9 11.5 ± 5.3 Polar435, CH2γ4 
Ga MR-2b 84.5 ± 7.6 2.5 ± 1.0 10.9 ± 9.4 12.5 ± 1.0 4.7 ± 2.3 His435, CH2γ4 
Ga MR-3 93.7 ± 8.6 2.6 ± 0.2 92.9 ± 4.1 11.3 ± 1.1 9.1 ± 5.1 Polar435, CH2γ4 
Ga MR-5 74.0 ± 9.1 11.6 ± 0.2 61.6 ± 8.9 21.0 ± 0.5 15.4 ± .5 Polar435, CH2γ4 
Ga MR-12 87.3 ± 4.1 9.6 ± 1.7 66.5 ± 9.4 24.2 ± 3.0 15.7 ± 8.0 Polar435, CH2γ4 
Ga MR-13b 73.7 ± 3.6 8.8 ± 4.5 59.3 ± 7.1 12.1 ± 0.3 12.2 ± 1.0 Polar435, CH2γ4 
Ga MR-14b 65.4 ± 4.4 10.1 ± 2.9 90.0 ± 6.3 13.5 ± 1.3 3.2 ± 2.8 Polar435, CH2γ4 
Ga MR-20 96.2 ± 3.5 13.9 ± 0.5 91.8 ± 3.1 12.5 ± 0.9 7.7 ± 3.4 Polar435, CH2γ4 
Ga MR-25b 73.1 ± 6.2 11.6 ± 5.5 79.3 ± 2.1 14.9 ± 2.1 8.6 ± 2.5 Polar435, CH2γ4 
Ga MR-27 87.8 ± 4.9 8.3 ± 3.3 6.3 ± 5.1 14.1 ± 2.4 9.8 ± 1.1 Polar435, CH2γ4 
Ga MR-28b 78.9 ± 2.3 6.8 ± 1.1 88.0 ± 1.0 13.1 ± 1.0 11.5 ± 3.1 Polar435, CH2γ4 
Ga MR-30b 74.3 ± 4.5 6.7 ± 0.7 99.8 ± 1.0 10.5 ± 1.2 6.6 ± 3.9 Polar435, CH2γ4 
Ga MR-33b 74.8 ± 9.6 11.2 ± 3.3 92.1 ± 1.4 31.4 ± 5.1 17.2 ± 1.0 Polar435, CH2γ4 
Ga MR-37b 74.1 ± 9.0 10.5 ± 3.2 93.8 ± 3.9 12.8 ± 1.0 5.6 ± 5.9 Polar435, CH2γ4 
Ga MR-39 97.1 ± 8.5 7.6 ± 2.0 6.5 ± 0.6 14.2 ± 2.1 15.2 ± 0.6 His435, CH2γ4 
Ga MR-41 68.7 ± 13 7.9 ± 0.7 7.7 ± 1.2 10.4 ± 1.1 5.1 ± 6.5 His435, CH2γ4 
Ga DI-2 78.0 ± 2.1 10.5 ± 2.7 12.0 ± 7.5 11.4 ± 1.6 10.5 ± 3.1 His435, CH2γ4 
Ga FO-3 73.3 ± 2.7 73.6 ± 2.2 42.4 ± 2.4 12.8 ± 0.6 5.2 ± 1.0 Polar435, CH3γ4 
Ga TT-3 86.9 ± 4.9 8.5 ± 1.9 8.6 ± 1.2 19.1 ± 0.7 2.1 ± 6.3 His435, CH2γ4 
Ga TT-7 80.9 ± 4.5 10.3 ± 1.1 6.8 ± 0.9 11.5 ± 2.0 14.3 ± 2.4 His435, CH2γ4 
Ga TT-9 60.8 ± 1.3 4.3 ± 0.7 92.7 ± 1.1 11.8 ± 0.3 12.4 ± 1.7 Polar435, CH2γ4 
Pan MR-24 77.5 ± 0.4 67.2 ± 0.5 44.0 ± 0.7 106.1 ± 1.9 123.6 ± 0.1 Outside 435 
1, 3, 4 DI-4 4.3 ± 0.6 93.9 ± 1.4 97.1 ± 1.1 5.7 ± 0.1 100% Polar435 
1, 2, 3 LN-11 76.8 ± 3.2 59.5 ± 2.2 38.2 ± 3.0 11.2 ± 0.2 100% Polar435, CH3γ3 
1, 4 MR-16b 77.4 ± 1.0 7.5 ± 3.7 5.0 ± 2.0 13.8 ± 2.6 9.7 ± 3.3 His435, CH2γ4 
Binding PatternRF3601 4-4-4-3 His-Tyr3603 3-3-3-3 His-Tyr3605 4-4-4-4 Arg-Tyr3612 4-4-4-4 Ala-TyrIgG3 3-3-3-3 Arg-TyrComments
Ga MR-1 86.7 ± 2.8 6.9 ± 0.2 99.8 ± 3.0 12.1 ± 0.9 11.5 ± 5.3 Polar435, CH2γ4 
Ga MR-2b 84.5 ± 7.6 2.5 ± 1.0 10.9 ± 9.4 12.5 ± 1.0 4.7 ± 2.3 His435, CH2γ4 
Ga MR-3 93.7 ± 8.6 2.6 ± 0.2 92.9 ± 4.1 11.3 ± 1.1 9.1 ± 5.1 Polar435, CH2γ4 
Ga MR-5 74.0 ± 9.1 11.6 ± 0.2 61.6 ± 8.9 21.0 ± 0.5 15.4 ± .5 Polar435, CH2γ4 
Ga MR-12 87.3 ± 4.1 9.6 ± 1.7 66.5 ± 9.4 24.2 ± 3.0 15.7 ± 8.0 Polar435, CH2γ4 
Ga MR-13b 73.7 ± 3.6 8.8 ± 4.5 59.3 ± 7.1 12.1 ± 0.3 12.2 ± 1.0 Polar435, CH2γ4 
Ga MR-14b 65.4 ± 4.4 10.1 ± 2.9 90.0 ± 6.3 13.5 ± 1.3 3.2 ± 2.8 Polar435, CH2γ4 
Ga MR-20 96.2 ± 3.5 13.9 ± 0.5 91.8 ± 3.1 12.5 ± 0.9 7.7 ± 3.4 Polar435, CH2γ4 
Ga MR-25b 73.1 ± 6.2 11.6 ± 5.5 79.3 ± 2.1 14.9 ± 2.1 8.6 ± 2.5 Polar435, CH2γ4 
Ga MR-27 87.8 ± 4.9 8.3 ± 3.3 6.3 ± 5.1 14.1 ± 2.4 9.8 ± 1.1 Polar435, CH2γ4 
Ga MR-28b 78.9 ± 2.3 6.8 ± 1.1 88.0 ± 1.0 13.1 ± 1.0 11.5 ± 3.1 Polar435, CH2γ4 
Ga MR-30b 74.3 ± 4.5 6.7 ± 0.7 99.8 ± 1.0 10.5 ± 1.2 6.6 ± 3.9 Polar435, CH2γ4 
Ga MR-33b 74.8 ± 9.6 11.2 ± 3.3 92.1 ± 1.4 31.4 ± 5.1 17.2 ± 1.0 Polar435, CH2γ4 
Ga MR-37b 74.1 ± 9.0 10.5 ± 3.2 93.8 ± 3.9 12.8 ± 1.0 5.6 ± 5.9 Polar435, CH2γ4 
Ga MR-39 97.1 ± 8.5 7.6 ± 2.0 6.5 ± 0.6 14.2 ± 2.1 15.2 ± 0.6 His435, CH2γ4 
Ga MR-41 68.7 ± 13 7.9 ± 0.7 7.7 ± 1.2 10.4 ± 1.1 5.1 ± 6.5 His435, CH2γ4 
Ga DI-2 78.0 ± 2.1 10.5 ± 2.7 12.0 ± 7.5 11.4 ± 1.6 10.5 ± 3.1 His435, CH2γ4 
Ga FO-3 73.3 ± 2.7 73.6 ± 2.2 42.4 ± 2.4 12.8 ± 0.6 5.2 ± 1.0 Polar435, CH3γ4 
Ga TT-3 86.9 ± 4.9 8.5 ± 1.9 8.6 ± 1.2 19.1 ± 0.7 2.1 ± 6.3 His435, CH2γ4 
Ga TT-7 80.9 ± 4.5 10.3 ± 1.1 6.8 ± 0.9 11.5 ± 2.0 14.3 ± 2.4 His435, CH2γ4 
Ga TT-9 60.8 ± 1.3 4.3 ± 0.7 92.7 ± 1.1 11.8 ± 0.3 12.4 ± 1.7 Polar435, CH2γ4 
Pan MR-24 77.5 ± 0.4 67.2 ± 0.5 44.0 ± 0.7 106.1 ± 1.9 123.6 ± 0.1 Outside 435 
1, 3, 4 DI-4 4.3 ± 0.6 93.9 ± 1.4 97.1 ± 1.1 5.7 ± 0.1 100% Polar435 
1, 2, 3 LN-11 76.8 ± 3.2 59.5 ± 2.2 38.2 ± 3.0 11.2 ± 0.2 100% Polar435, CH3γ3 
1, 4 MR-16b 77.4 ± 1.0 7.5 ± 3.7 5.0 ± 2.0 13.8 ± 2.6 9.7 ± 3.3 His435, CH2γ4 
a

Site-directed mutations at positions 435 and/or 436 in the CH3 of IgG3 or IgG4 were introduced in the CH3 domain of IgG3 or IgG4 as represented schematically at the top of each column. Mutated CH3 domains at these positions were also shuffled into hybrid proteins generated from the IgG3 and IgG4 genes. The number 3 or 4 indicates whether the exon originated from the IgG3 or IgG4 heavy chain gene. Results are the mean of six determinations from two separate experiments ± SEM for each RF expressed as a percentage binding to IgG4 defined as 100%. For RFs DI-4 and LN-11, the results are expressed as a percentage ± SEM of binding to IgG3 defined as 100%.

b

Clonally related RFs.

RFs that bind all IgG isotypes.

The only HID-RF that bound all IgG subclasses, MR-24, bound all IgG ligands bearing mutations in the CH3 domains of IgG3 or -4, indicating that the epitope bound by MR-24 is not dependent on the His/Arg polymorphism at 435.

RFs with unique specificities.

The genetically engineered IgG Abs bearing mutations in CH3 domains of IgG3 or IgG4 provided insights into the specificity of only three of the six HID-RFs that showed variable binding to IgG subclasses other than the Ga- or pan-reactive patterns (DI-1, DI-4, LN-10, LN-11, MR-16, and FO-2). For LN-11, the CH3 domain of IgG3 was critical for binding (Table II), and a polar residue at position 435 was also required (Table III). DI-4 also required a polar residue at position 435 in CH3. The CH3 of IgG3 was critical for LN-11 binding, which bound IgG3 but not IgG4 (Table II), suggesting that residues present in the CH3 domain of IgG1, -2, and -3, but absent in IgG4 are important in the epitope that LN-11 recognizes. DI-4, which binds IgG1, -3, and -4, but not IgG2, requires a polar residue at position 435 in CH3, since it will not bind IgG4 Ab 3612 bearing Ala435. MR-16 is of special interest because it also has a different fine specificity for IgG compared with the eight other related HID-RFs from patient MR. Although the epitope that MR-16 recognizes requires His435 in the context of CH2 of IgG3, this is not sufficient for binding. MR-16 requires His435 for binding IgG4 while the eight other related RFs can tolerate Arg at this position. HID-RFs DI-1, FO-2, and LN-10 could not be studied using the genetically engineered constructs of IgG3 and -4 because they do not bind these subclasses (data not shown).

RF reactivity with the IgG3 polymorphism (Tyr/Phe) at 436.

To determine the reactivity of HID-RFs for the naturally occurring polymorphism Tyr/Phe at 436, variants of our wild-type IgG3 (containing Tyr436) were engineered. Ab construct 3611 is IgG3 containing Arg435-Phe436, and Ab constructs 3602 (4-4-4-3 containing His435-Phe436) and 3604 (3-3-3-3 containing His435-Phe436) were used to determining RF specificity for this IgG polymorphism (Table IV).

Table IV.

Binding of monoclonal healthy immunized donor-derived RFs to chimeric IgG containing mutations in CH3 at positions 435-436: reactivity with the IgG3 polymorphism at 436a

Binding PatternRF:Chimeric IgG:Comments
3601 4-4-4-3 His-Tyr3602 4-4-4-3 His-Phe3603 3-3-3-3 His-Tyr3604 3-3-3-3 His-Phe3605 4-4-4-4 Arg-Tyr3611 3-3-3-3 Arg-Phe
Ga MR-1 86.7 ± 2.8 78.1 ± 4.5 6.9 ± 0.2 52.2 ± 3.6 99.8 ± 3.0 14.8 ± 1.2 Phe436 contributes 
Ga MR-2b 84.5 ± 7.6 42.1 ± 1.0 2.5 ± 1.0 19.1 ± 0.6 10.9 ± 9.4 18.4 ± 4.7  
Ga MR-3 93.7 ± 8.6 37.9 ± 0.7 2.6 ± 0.2 30.5 ± 1.1 92.9 ± 4.1 17.7 ± 0.4  
Ga MR-5 74.0 ± 9.1 61.4 ± 4.9 11.6 ± 0.2 20.1 ± 3.1 61.6 ± 8.9 16.4 ± 0.4  
Ga MR-12 87.3 ± 4.1 72.6 ± 1.7 9.6 ± 1.7 18.8 ± 1.1 66.5 ± 9.4 15.5 ± 1.5  
Ga MR-13b 73.7 ± 3.6 77.6 ± 1.0 8.8 ± 4.5 29.0 ± 3.5 59.3 ± 7.1 20.0 ± 0.3  
Ga MR-14b 65.4 ± 4.4 79.4 ± 2.9 10.1 ± 2.9 19.6 ± 2.2 90.0 ± 6.3 16.1 ± 0.2  
Ga MR-20 96.2 ± 3.5 66.3 ± 4.0 13.9 ± 0.5 25.6 ± 4.9 91.8 ± 3.1 24.1 ± 1.9  
Ga MR-25b 73.1 ± 6.2 48.2 ± 2.1 11.6 ± 5.5 23.1 ± 0.9 79.3 ± 2.1 19.1 ± 1.5  
Ga MR-27 87.8 ± 4.9 48.8 ± 0.9 8.3 ± 3.3 21.2 ± 0.4 6.3 ± 5.1 26.7 ± 0.1  
Ga MR-28b 78.9 ± 2.3 62.2 ± 3.6 6.8 ± 1.1 68.1 ± 1.3 88.0 ± 1.0 61.4 ± 2.2 Anti-allotype, Phe436 IgG3 
Ga MR-30b 74.3 ± 4.5 85.8 ± 0.3 6.7 ± 0.7 23.1 ± 1.2 99.8 ± 1.0 22.4 ± 0.1  
Ga MR-33b 74.8 ± 9.6 91.0 ± 1.4 11.2 ± 3.3 29.8 ± 6.5 92.1 ± 1.4 18.3 ± 1.9  
Ga MR-37b 74.1 ± 9.0 82.5 ± 3.9 10.5 ± 3.2 29.6 ± 3.6 93.8 ± 3.9 19.3 ± 0.3  
Ga MR-39 97.1 ± 8.5 35.5 ± 0.1 7.6 ± 2.0 19.2 ± 0.6 6.5 ± 0.6 17.0 ± 2.9  
Ga MR-41 68.7 ± 13 47.4 ± 1.4 7.9 ± 0.7 16.4 ± 0.3 7.7 ± 1.2 15.2 ± 0.7  
Ga D1-2 78.0 ± 2.1 41.0 ± 1.3 10.5 ± 2.7 17.1 ± 3.4 12.0 ± 7.5 9.6 ± 1.9  
Ga FO-3 73.3 ± 2.7 92.0 ± 2.1 73.6 ± 2.2 108.4 ± 6.8 42.4 ± 2.4 10.8 ± 1.8  
Ga TT-3 86.9 ± 4.9 46.5 ± 2.0 8.5 ± 1.9 43.3 ± 1.5 8.6 ± 1.2 52.0 ± 1.1 Anti-allotype, Phe436 IgG3 
Ga TT-7 80.9 ± 4.5 77.6 ± 4.0 10.3 ± 1.1 31.6 ± 0.9 6.8 ± 0.9 41.2 ± 2.6 Anti-allotype, Phe436 IgG3 
Ga TT-9 51.0 ± 3.7 83.5 ± 1.6 4.3 ± 0.7 90.4 ± 3.2 92.7 ± 1.1 100.4 ± 0.6 Anti-allotype, Phe436 IgG3 
Pan MR-24 77.5 ± 0.4 60.0 ± 1.2 67.2 ± 0.5 82.7 ± 1.4 44.0 ± 0.7 100.7 ± 0.5  
1, 3, 4 D1-4 86.0 ± 0.7 24.0 ± 0.6 93.7 ± 1.4 9.0 ± 0.3 103.1 ± 1.4 97.1 ± 1.1 Tyr436 important 
1, 2, 3 LN-11 74.9 ± 1.3 84.3 ± 3.5 49.3 ± 5.1 42.8 ± 0.9 24.7 ± 1.2 102.8 ± 0.6  
1, 4 MR-16b 77.4 ± 1.0 11.2 ± 1.4 7.5 ± 3.7 17.6 ± 1.5 5.0 ± 2.0 16.1 ± 0.8 Tyr436 important 
Binding PatternRF:Chimeric IgG:Comments
3601 4-4-4-3 His-Tyr3602 4-4-4-3 His-Phe3603 3-3-3-3 His-Tyr3604 3-3-3-3 His-Phe3605 4-4-4-4 Arg-Tyr3611 3-3-3-3 Arg-Phe
Ga MR-1 86.7 ± 2.8 78.1 ± 4.5 6.9 ± 0.2 52.2 ± 3.6 99.8 ± 3.0 14.8 ± 1.2 Phe436 contributes 
Ga MR-2b 84.5 ± 7.6 42.1 ± 1.0 2.5 ± 1.0 19.1 ± 0.6 10.9 ± 9.4 18.4 ± 4.7  
Ga MR-3 93.7 ± 8.6 37.9 ± 0.7 2.6 ± 0.2 30.5 ± 1.1 92.9 ± 4.1 17.7 ± 0.4  
Ga MR-5 74.0 ± 9.1 61.4 ± 4.9 11.6 ± 0.2 20.1 ± 3.1 61.6 ± 8.9 16.4 ± 0.4  
Ga MR-12 87.3 ± 4.1 72.6 ± 1.7 9.6 ± 1.7 18.8 ± 1.1 66.5 ± 9.4 15.5 ± 1.5  
Ga MR-13b 73.7 ± 3.6 77.6 ± 1.0 8.8 ± 4.5 29.0 ± 3.5 59.3 ± 7.1 20.0 ± 0.3  
Ga MR-14b 65.4 ± 4.4 79.4 ± 2.9 10.1 ± 2.9 19.6 ± 2.2 90.0 ± 6.3 16.1 ± 0.2  
Ga MR-20 96.2 ± 3.5 66.3 ± 4.0 13.9 ± 0.5 25.6 ± 4.9 91.8 ± 3.1 24.1 ± 1.9  
Ga MR-25b 73.1 ± 6.2 48.2 ± 2.1 11.6 ± 5.5 23.1 ± 0.9 79.3 ± 2.1 19.1 ± 1.5  
Ga MR-27 87.8 ± 4.9 48.8 ± 0.9 8.3 ± 3.3 21.2 ± 0.4 6.3 ± 5.1 26.7 ± 0.1  
Ga MR-28b 78.9 ± 2.3 62.2 ± 3.6 6.8 ± 1.1 68.1 ± 1.3 88.0 ± 1.0 61.4 ± 2.2 Anti-allotype, Phe436 IgG3 
Ga MR-30b 74.3 ± 4.5 85.8 ± 0.3 6.7 ± 0.7 23.1 ± 1.2 99.8 ± 1.0 22.4 ± 0.1  
Ga MR-33b 74.8 ± 9.6 91.0 ± 1.4 11.2 ± 3.3 29.8 ± 6.5 92.1 ± 1.4 18.3 ± 1.9  
Ga MR-37b 74.1 ± 9.0 82.5 ± 3.9 10.5 ± 3.2 29.6 ± 3.6 93.8 ± 3.9 19.3 ± 0.3  
Ga MR-39 97.1 ± 8.5 35.5 ± 0.1 7.6 ± 2.0 19.2 ± 0.6 6.5 ± 0.6 17.0 ± 2.9  
Ga MR-41 68.7 ± 13 47.4 ± 1.4 7.9 ± 0.7 16.4 ± 0.3 7.7 ± 1.2 15.2 ± 0.7  
Ga D1-2 78.0 ± 2.1 41.0 ± 1.3 10.5 ± 2.7 17.1 ± 3.4 12.0 ± 7.5 9.6 ± 1.9  
Ga FO-3 73.3 ± 2.7 92.0 ± 2.1 73.6 ± 2.2 108.4 ± 6.8 42.4 ± 2.4 10.8 ± 1.8  
Ga TT-3 86.9 ± 4.9 46.5 ± 2.0 8.5 ± 1.9 43.3 ± 1.5 8.6 ± 1.2 52.0 ± 1.1 Anti-allotype, Phe436 IgG3 
Ga TT-7 80.9 ± 4.5 77.6 ± 4.0 10.3 ± 1.1 31.6 ± 0.9 6.8 ± 0.9 41.2 ± 2.6 Anti-allotype, Phe436 IgG3 
Ga TT-9 51.0 ± 3.7 83.5 ± 1.6 4.3 ± 0.7 90.4 ± 3.2 92.7 ± 1.1 100.4 ± 0.6 Anti-allotype, Phe436 IgG3 
Pan MR-24 77.5 ± 0.4 60.0 ± 1.2 67.2 ± 0.5 82.7 ± 1.4 44.0 ± 0.7 100.7 ± 0.5  
1, 3, 4 D1-4 86.0 ± 0.7 24.0 ± 0.6 93.7 ± 1.4 9.0 ± 0.3 103.1 ± 1.4 97.1 ± 1.1 Tyr436 important 
1, 2, 3 LN-11 74.9 ± 1.3 84.3 ± 3.5 49.3 ± 5.1 42.8 ± 0.9 24.7 ± 1.2 102.8 ± 0.6  
1, 4 MR-16b 77.4 ± 1.0 11.2 ± 1.4 7.5 ± 3.7 17.6 ± 1.5 5.0 ± 2.0 16.1 ± 0.8 Tyr436 important 
a

Site-directed mutations at positions 435 and/or 436 in the CH3 of IgG3 or IgG4 were introduced in the CH3 domain of IgG3 or IgG4 as represented schematically at the top of each column. Mutated CH3 domains at these positions were also shuffled into hybrid proteins generated from the IgG3 and IgG4 genes. The number 3 or 4 indicates whether the exon originated from the IgG3 or IgG4 heavy chain gene. Results are the mean of six determinations from two separate experiments ± SEM for each RF expressed as a percentage binding to IgG4 defined as 100%. For RFs DI-4 and LN-11, the results are expressed as a percentage ± SEM of binding to IgG3 defined as 100%.

b

Clonally related RFs.

Construct MR-16 again differed from the eight other clonally related RFs by not binding the 4-4-4-3 hybrid Ab 3602 that contains Arg→His435 and Phe436 in the context of the CH3 domain of IgG3. However, MR-16 bound the related hybrid Ab 3601 with Arg→His435 and Tyr436. This requirement for both His435 and Tyr436 (Table IV) was not observed with any RA- or Wmac-RF we previously studied (15, 23). The IgG1-, IgG-3-, and IgG-4-reactive DI-4 also showed decreased reactivity with Abs 3602 and 3604 bearing His435-Phe436 in CH3. In addition the Ga-reactive HID-RFs, MR-28, TT-3, TT-7, and TT9, derived from two different individuals, preferentially recognized the naturally occurring Tyr/Phe polymorphism at position 436 in IgG3 by binding Ab 3611 (IgG3 bearing Phe436), but not wild-type IgG3 that contains Tyr436. Therefore, these HID-RFs are anti-allotypic autoantibodies.

Two loops of amino acids within CH2, residues 252 to 254 and 309 to 311, fold in proximity to CH3 and have been shown to be essential in most Ga-reactive RF binding to IgG. We replaced the residues of each of these loops either with three glycines, thus removing any existing side chains, or with three prolines. In addition, we also singly changed from Ile→Ala at 253, a known contact residue for SPA (34), to determine the importance of these loops in HID-RF recognition of IgG (15).

For all 21 Ga-reactive HID-RFs, substitution of either three Gly or Pro amino acids in the proximal loop of the CH2 domain of IgG4 eliminated binding (Table V). Ile253 is very important for Ga-reactive, HID-RF binding; of the 21 Ga-reactive HID-RFs, only MR-16, which does not bind IgG2, bound IgG4 bearing Ala253 (antibody 3614). This binding specificity again distinguishes MR-16 from the eight other clonally related RFs.

Table V.

Binding of healthy immunized donor-derived RFs to chimeric IgG4 containing mutations in CH2 at positions 252-254 or 309-311a

3606 252-254 P-P-P3609 252-254 G-G-G3614 253 A3607 309-311 P-P-P3610 309-311 G-G-GComments
Ga MR-1 8.0 ± 0.2 10.4 ± 1.0 17.6 ± 0.5 99.6 ± 4.5 66.0 ± 2.3 Proximal loop important 
Ga MR-2b 9.7 ± 1.6 12.0 ± 0.1 19.2 ± 0.4 95.2 ± 2.7 51.0 ± 1.6 Proximal loop important 
Ga MR-3 11.7 ± 0.4 9.8 ± 0.2 15.8 ± 0.6 92.9 ± 4.1 29.6 ± 2.3 Proximal loop important 
Ga MR-5 13.6 ± 7.1 10.8 ± 1.2 15.1 ± 4.7 14.7 ± 1.8 17.7 ± 1.7 Both loops important 
Ga MR-12 8.0 ± 0.3 10.3 ± 0.6 10.6 ± 0.2 66.5 ± 9.4 18.6 ± 0.6 Both loops important 
Ga MR-13b 9.4 ± 1.6 10.2 ± 0.3 10.6 ± 0.2 46.8 ± 2.0 5.4 ± 0.7 Both loops important 
Ga MR-14b 11.7 ± 0.5 11.3 ± 0.8 13.0 ± 1.3 28.1 ± 1.8 6.9 ± 0.4 Both loops important 
Ga MR-20 13.3 ± 2.6 11.1 ± 0.2 12.0 ± 0.6 91.8 ± 3.1 53.6 ± 1.4 Proximal loop important 
Ga MR-25b 12.2 ± 1.7 12.5 ± 0.5 13.2 ± 0.8 47.5 ± 1.7 4.6 ± 0.3 Both loops important 
Ga MR-27 11.7 ± 1.0 10.6 ± 1.1 12.6 ± 0.1 6.3 ± 5.1 30.4 ± 1.9 Both loops important 
Ga MR-28b 10.5 ± 1.5 13.1 ± 5.2 10.6 ± 0.3 88.0 ± 1.4 8.4 ± 0.7 Proximal loop important 
Ga MR-30b 11.4 ± 0.3 9.3 ± 0.8 23.8 ± 4.1 100.3 ± 1.0 5.5 ± 1.0 Proximal loop important 
Ga MR-33b 13.5 ± 0.2 10.9 ± 1.2 10.3 ± 0.2 92.1 ± 1.4 66.3 ± 2.4 Proximal loop important 
Ga MR-37b 15.0 ± 0.5 9.3 ± 0.1 10.6 ± 1.3 93.8 ± 1.1 5.0 ± 0.1 Proximal loop important 
Ga MR-39 9.6 ± 0.6 8.5 ± 0.1 14.7 ± 5.2 92.7 ± 0.3 80.0 ± 2.4 Proximal loop important 
Ga MR-41 9.8 ± 0.5 9.0 ± 0.5 7.4 ± 0.3 7.7 ± 1.2 12.3 ± 2.6 Both loops important 
Ga DI-2 11.9 ± 0.3 13.4 ± 0.8 16.8 ± 1.9 81.5 ± 0.4 76.7 ± 3.7 Proximal loop important 
Ga FO-3 14.0 ± 1.0 7.6 ± 5.6 10.2 ± 0.2 11.0 ± 0.9 12.4 ± 0.3 Both loops important 
Ga TT-3 16.2 ± 0.6 14.7 ± 5.5 92.5 ± 1.7 71.1 ± 1.7 38.5 ± 0.3 Proximal, not Ile253 
Ga TT-7 11.6 ± 1.0 13.5 ± 0.2 14.3 ± 7.7 65.3 ± 1.8 7.2 ± 1.5 Proximal loop important 
Ga TT-9 14.9 ± 0.8 11.2 ± 1.2 12.3 ± 0.6 19.5 ± 0.3 17.3 ± 0.9 Both loops important 
Pan MR-24 13.1 ± 0.7 10.6 ± 0.1 87.5 ± 1.0 105.0 ± 1.1 107.0 ± 1.2 Proximal, not Ile253 
1, 3, 4 DI-4 15.0 ± 2.2 13.8 ± 0.3 5.9 ± 0.5 14.9 ± 1.1 23.2 ± 1.5 Both loops important 
1, 4 MR-16b 12.0 ± 1.0 11.0 ± 0.3 88.7 ± 1.8 10.7 ± 0.1 6.8 ± 1.6 Both loops, not Ile253 
3606 252-254 P-P-P3609 252-254 G-G-G3614 253 A3607 309-311 P-P-P3610 309-311 G-G-GComments
Ga MR-1 8.0 ± 0.2 10.4 ± 1.0 17.6 ± 0.5 99.6 ± 4.5 66.0 ± 2.3 Proximal loop important 
Ga MR-2b 9.7 ± 1.6 12.0 ± 0.1 19.2 ± 0.4 95.2 ± 2.7 51.0 ± 1.6 Proximal loop important 
Ga MR-3 11.7 ± 0.4 9.8 ± 0.2 15.8 ± 0.6 92.9 ± 4.1 29.6 ± 2.3 Proximal loop important 
Ga MR-5 13.6 ± 7.1 10.8 ± 1.2 15.1 ± 4.7 14.7 ± 1.8 17.7 ± 1.7 Both loops important 
Ga MR-12 8.0 ± 0.3 10.3 ± 0.6 10.6 ± 0.2 66.5 ± 9.4 18.6 ± 0.6 Both loops important 
Ga MR-13b 9.4 ± 1.6 10.2 ± 0.3 10.6 ± 0.2 46.8 ± 2.0 5.4 ± 0.7 Both loops important 
Ga MR-14b 11.7 ± 0.5 11.3 ± 0.8 13.0 ± 1.3 28.1 ± 1.8 6.9 ± 0.4 Both loops important 
Ga MR-20 13.3 ± 2.6 11.1 ± 0.2 12.0 ± 0.6 91.8 ± 3.1 53.6 ± 1.4 Proximal loop important 
Ga MR-25b 12.2 ± 1.7 12.5 ± 0.5 13.2 ± 0.8 47.5 ± 1.7 4.6 ± 0.3 Both loops important 
Ga MR-27 11.7 ± 1.0 10.6 ± 1.1 12.6 ± 0.1 6.3 ± 5.1 30.4 ± 1.9 Both loops important 
Ga MR-28b 10.5 ± 1.5 13.1 ± 5.2 10.6 ± 0.3 88.0 ± 1.4 8.4 ± 0.7 Proximal loop important 
Ga MR-30b 11.4 ± 0.3 9.3 ± 0.8 23.8 ± 4.1 100.3 ± 1.0 5.5 ± 1.0 Proximal loop important 
Ga MR-33b 13.5 ± 0.2 10.9 ± 1.2 10.3 ± 0.2 92.1 ± 1.4 66.3 ± 2.4 Proximal loop important 
Ga MR-37b 15.0 ± 0.5 9.3 ± 0.1 10.6 ± 1.3 93.8 ± 1.1 5.0 ± 0.1 Proximal loop important 
Ga MR-39 9.6 ± 0.6 8.5 ± 0.1 14.7 ± 5.2 92.7 ± 0.3 80.0 ± 2.4 Proximal loop important 
Ga MR-41 9.8 ± 0.5 9.0 ± 0.5 7.4 ± 0.3 7.7 ± 1.2 12.3 ± 2.6 Both loops important 
Ga DI-2 11.9 ± 0.3 13.4 ± 0.8 16.8 ± 1.9 81.5 ± 0.4 76.7 ± 3.7 Proximal loop important 
Ga FO-3 14.0 ± 1.0 7.6 ± 5.6 10.2 ± 0.2 11.0 ± 0.9 12.4 ± 0.3 Both loops important 
Ga TT-3 16.2 ± 0.6 14.7 ± 5.5 92.5 ± 1.7 71.1 ± 1.7 38.5 ± 0.3 Proximal, not Ile253 
Ga TT-7 11.6 ± 1.0 13.5 ± 0.2 14.3 ± 7.7 65.3 ± 1.8 7.2 ± 1.5 Proximal loop important 
Ga TT-9 14.9 ± 0.8 11.2 ± 1.2 12.3 ± 0.6 19.5 ± 0.3 17.3 ± 0.9 Both loops important 
Pan MR-24 13.1 ± 0.7 10.6 ± 0.1 87.5 ± 1.0 105.0 ± 1.1 107.0 ± 1.2 Proximal, not Ile253 
1, 3, 4 DI-4 15.0 ± 2.2 13.8 ± 0.3 5.9 ± 0.5 14.9 ± 1.1 23.2 ± 1.5 Both loops important 
1, 4 MR-16b 12.0 ± 1.0 11.0 ± 0.3 88.7 ± 1.8 10.7 ± 0.1 6.8 ± 1.6 Both loops, not Ile253 
a

Site-directed mutations were introduced into IgG4 at positions 252-254, 309-311, or 253 only, within two amino acid loops in CH2 that project into the CH2-CH3 interface that are SPA contact residues. Shown at the top of each column are the three positions that were replaced with either proline (P), glycine (G), or alanine (A) residues. Results are the mean of six determinations from two separate experiments ± SEM for each RF expressed as a percentage binding to IgG4 defined as 100%.

b

Clonally related RFs.

Binding of the HID-RFs to IgG4 with three Gly or Pro residues in the distal loop of CH 2 (residues at 309–311) was more variable. As previously noted with RA- and Wmac-RFs, four separate patterns of binding were again observed. Four of the Ga-reactive HID-RFs (MR-5, MR-41, FO-3, and TT-9) and two others (MR-16 and DI-4), bound neither of the IgG4 Abs containing three Gly or three Pro residues in the distal loop of CH2 (Table V). Therefore, the proximal and distal loops in CH2 that project into the CH2-CH3 interface are important for these RFs to bind IgG4. MR-16 again differed from the eight other clonally related RFs in failing to bind IgG4 with Ala253 or three Pro residues in the distal loop of CH2.

Twelve HID-RFs (MR-1, -2, -3, -12, -20, -28, -30, -33, -37, and -39; TT-3; and TT-7) bound strongly (>50% of maximum binding) to the IgG4 distal loop mutant with three Pro residues at 309 to 311, but less well or not at all to the IgG4 mutant containing three Gly residues. Of these HID-RFs, those bearing HumKv325 gene products preferentially bound the IgG4 Ab containing three Pro residues at 309 to 311, consistent with the Ga epitope specificity we previously mapped with some RA- and Wmac-RFs (18, 23). None of the HID-RFs preferentially bound IgG4 bearing three Gly residues at 309 to 311 (Ab 3610). The pan-reactive HID-RF MR-24 bound strongly to both IgG4 mutants, indicating that the distal loop of CH2 is not important for this RF to bind IgG. As anticipated, the HID-RFs that did not bind IgG4 (LN-10, LN-11, DI-1, and FO-2) did not bind any of the IgG4 constructs (data not shown).

SPA and other bacterial Fc binding proteins bind IgG at the CH2-CH3 interface (17, 18, 23, 35). We previously showed that some RA-RFs and almost all Wmac-RFs bind IgG at this interface. However, the epitope recognized by RFs and SPA are not superimposable (18, 23, 35). SPA inhibited binding of 18 of 21 (86%) of the Ga-reactive HID-RFs, but only partially inhibited binding of the three others (MR-14, MR-41, and DI-2; Table VI). Fine specificity differences were again apparent among the clonally related HID-RF subgroup; MR-14 bound IgG4 in the presence of SPA, while the other eight in this group were completely inhibited by SPA. Mutations in VH1 of MR-14 may account for this fine specificity difference (30). Of the seven non-Ga-reactive HID-RFs, two (DI-1 and F0–2) showed little inhibition by SPA in binding IgG1, and DI-4 was only partially inhibited by SPA in binding IgG4.

Table VI.

Staph protein A inhibition of HID-RF binding to chimeric IgG antibodiesa

Binding PatternRFNo SPA addedaSPA addedComments:
Ga MR-1 100% 7.5 ± 1.3  
Ga MR-2b 100% 14.5 ± 1.3  
Ga MR-3 100% 9.7 ± 0.8  
Ga MR-5 100% 12.2 ± 1.4  
Ga MR-12 100% 17.5 ± 2.5  
Ga MR-13b 100% 18.2 ± 1.3  
Ga MR-14b 100% 39.8 ± 8.1 Partial inhibition 
Ga MR-20 100% 1.2 ± 1.2  
Ga MR-25b 100% 14.7 ± 2.6  
Ga MR-27 100% 21.1 ± 2.9  
Ga MR-28b 100% 25.9 ± 4.2  
Ga MR-30b 100% 20.6 ± 0.6  
Ga MR-33b 100% 13.6 ± 3.0  
Ga MR-37b 100% 18.0 ± 4.4  
Ga MR-39 100% 23.9 ± 5.1  
Ga MR-41 100% 78.6 ± 1.3 Partial inhibition 
Ga DI-2 100% 76.5 ± 5.1 Partial inhibition 
Ga FO-3 100% 9.6 ± 2.2  
Ga TT-3 100% 7.3 ± 1.8  
Ga TT-7 100% 16.7 ± 4.2  
Ga TT-9 100% 14.3 ± 2.5  
Pan MR-24 100% 8.5 ± 1.1  
1, 3, 4 DI-4 100% 63.2 ± 8.2 Partial inhibition 
1, 2, 3 LN-11 100%c 10.1 ± 1.3  
1, 4 MR-16b 100% 14.1 ± 2.4  
1, 2 LN-10 100%c 14.7 ± 1.6  
1 only DI-1 100%c 93.0 ± 3.7 No inhibition 
1 only FO-2 100%c 88.0 ± 1.3 No inhibition 
Binding PatternRFNo SPA addedaSPA addedComments:
Ga MR-1 100% 7.5 ± 1.3  
Ga MR-2b 100% 14.5 ± 1.3  
Ga MR-3 100% 9.7 ± 0.8  
Ga MR-5 100% 12.2 ± 1.4  
Ga MR-12 100% 17.5 ± 2.5  
Ga MR-13b 100% 18.2 ± 1.3  
Ga MR-14b 100% 39.8 ± 8.1 Partial inhibition 
Ga MR-20 100% 1.2 ± 1.2  
Ga MR-25b 100% 14.7 ± 2.6  
Ga MR-27 100% 21.1 ± 2.9  
Ga MR-28b 100% 25.9 ± 4.2  
Ga MR-30b 100% 20.6 ± 0.6  
Ga MR-33b 100% 13.6 ± 3.0  
Ga MR-37b 100% 18.0 ± 4.4  
Ga MR-39 100% 23.9 ± 5.1  
Ga MR-41 100% 78.6 ± 1.3 Partial inhibition 
Ga DI-2 100% 76.5 ± 5.1 Partial inhibition 
Ga FO-3 100% 9.6 ± 2.2  
Ga TT-3 100% 7.3 ± 1.8  
Ga TT-7 100% 16.7 ± 4.2  
Ga TT-9 100% 14.3 ± 2.5  
Pan MR-24 100% 8.5 ± 1.1  
1, 3, 4 DI-4 100% 63.2 ± 8.2 Partial inhibition 
1, 2, 3 LN-11 100%c 10.1 ± 1.3  
1, 4 MR-16b 100% 14.1 ± 2.4  
1, 2 LN-10 100%c 14.7 ± 1.6  
1 only DI-1 100%c 93.0 ± 3.7 No inhibition 
1 only FO-2 100%c 88.0 ± 1.3 No inhibition 
a

Monoclonal IgM RFs from health immunized donors were reacted with wild-type IgG bound to DNS-BSA with or without the addition of 100 μg/ml of SPA. Results are the mean of six determinations from three separate experiments ± SEM for each RF expressed as a percentage binding to IgG4 defined as 100%.

b

Clonally related RFs.

c

For RF LN-10 and LN-11, the results are expressed as a percentage ± SEM of binding to IgG2 defined as 100%. For RFs DI-1 and FO-2, the results are expressed as a percentage ± SEM of binding to IgG1 defined as 100%.

The N-linked carbohydrate at Asn297 in CH2 has a significant impact on the conformation of this domain and has been reported to influence RF binding to IgG (36, 37). We compared the HID-RFs with aglycosylated IgG3 and IgG4 Abs produced by mutating Asn297 to His (IgG4), Lys (IgG3), His (IgG2), or His (IgG1), see Table VII (18, 23). Two HID-RFs recognized epitopes that were influenced by glycosylation. DI-2, a Ga-reactive RF, bound aglycosylated IgG4 only half as well as glycosylated IgG4 (Table VII). In addition, LN-11, a RF that recognizes IgG1, -2, and -3 only, bound aglycosylated IgG3 less well than glycosylated IgG3.

Table VII.

Comparison of healthy immunized donor-derived RF binding to glycosylated and aglycosylated chimeric IgG antibodiesa

RFIgG4 Asn 297Agly IgG4 His 297IgG3 Asn 297Agly IgG3 Gln 297Comments
Ga MR-1 100% 97.7 ± 5.5 11.6 ± 2.3 14.9 ± 1.1  
Ga MR-2b 100% 94.8 ± 2.0 10.3 ± 0.2 10.5 ± 1.2  
Ga MR-3 100% 97.0 ± 0.7 8.2 ± 0.4 6.8 ± 1.1  
Ga MR-5 100% 88.1 ± 4.3 7.0 ± 1.3 6.9 ± 0.4  
Ga MR-12 100% 101.4 ± 0.4 11.6 ± 1.0 6.2 ± 0.4  
Ga MR-13b 100% 99.9 ± 1.0 3.0 ± 0.1 5.0 ± 1.8  
Ga MR-14b 100% 97.9 ± 5.8 4.2 ± 0.3 5.6 ± 1.0  
Ga MR-20b 100% 99.6 ± 3.0 2.2 ± 0.4 2.7 ± 0.3  
Ga MR-25 100% 93.5 ± 2.5 3.4 ± 0.1 3.6 ± 0.1  
Ga MR-27 100% 93.0 ± 1.6 19.0 ± 1.1 16.5 ± 0.8  
Ga MR-28b 100% 93.2 ± 6.4 3.7 ± 0.1 4.5 ± 0.3  
Ga MR-30b 100% 92.7 ± 2.0 3.8 ± 0.6 5.3 ± 0.4  
Ga MR-33b 100% 94.7 ± 2.2 2.3 ± 0.5 2.0 ± 2.2  
Ga MR-37b 100% 95.0 ± 0.6 4.5 ± 1.2 3.1 ± 0.1  
Ga MR-39 100% 101.7 ± 1.8 3.1 ± 0.2 3.6 ± 0.8  
Ga MR-41 100% 90.8 ± 0.7 10.5 ± 0.6 9.7 ± 0.5  
Ga DI-2 100% 56.8 ± 4.2 5.8 ± 0.2 7.2 ± 1.2 ↓ Agly γ4 
Ga FO-3 100% 98.6 ± 0.7 17.1 ± 1.0 14.8 ± 0.3  
Ga TT-3 100% 98.6 ± 0.5 3.5 ± 0.2 4.5 ± 0.3  
Ga TT-7 100% 97.6 ± 2.0 1.2 ± 0.3 0.7 ± 0.3  
Ga TT-9 100% 97.3 ± 2.9 1.2 ± 0.1 1.8 ± 0.8  
Pan MR-24 100% 98.2 ± 1.1 101.2 ± 0.8 99.2 ± 0.9  
1, 3, 4 DI-4 46.0 ± 0.8 45.3 ± 0.5 100% 101.3 ± 1.9  
1, 2, 3 LN-11 2.7 ± 0.7 4.5 ± 0.8 100% 28.3 ± 0.2 ↓ Agly γ3 
1, 4 MR-16b 100% 83.8 ± 3.6 4.5 ± 0.8 5.6 ± 0.1  
1, 2 LN-10 100%c 97.0 ± 3.6d 105.2 ± 0.4e 99.7 ± 0.9f  
1 only DI-1 100%c 99.6 ± 0.2d 3.6 ± 0.3e 2.9 ± 0.3f  
1 only FO-2 100%c 97.6 ± 0.5d 5.5 ± 1.2e 6.2 ± 0.6f  
RFIgG4 Asn 297Agly IgG4 His 297IgG3 Asn 297Agly IgG3 Gln 297Comments
Ga MR-1 100% 97.7 ± 5.5 11.6 ± 2.3 14.9 ± 1.1  
Ga MR-2b 100% 94.8 ± 2.0 10.3 ± 0.2 10.5 ± 1.2  
Ga MR-3 100% 97.0 ± 0.7 8.2 ± 0.4 6.8 ± 1.1  
Ga MR-5 100% 88.1 ± 4.3 7.0 ± 1.3 6.9 ± 0.4  
Ga MR-12 100% 101.4 ± 0.4 11.6 ± 1.0 6.2 ± 0.4  
Ga MR-13b 100% 99.9 ± 1.0 3.0 ± 0.1 5.0 ± 1.8  
Ga MR-14b 100% 97.9 ± 5.8 4.2 ± 0.3 5.6 ± 1.0  
Ga MR-20b 100% 99.6 ± 3.0 2.2 ± 0.4 2.7 ± 0.3  
Ga MR-25 100% 93.5 ± 2.5 3.4 ± 0.1 3.6 ± 0.1  
Ga MR-27 100% 93.0 ± 1.6 19.0 ± 1.1 16.5 ± 0.8  
Ga MR-28b 100% 93.2 ± 6.4 3.7 ± 0.1 4.5 ± 0.3  
Ga MR-30b 100% 92.7 ± 2.0 3.8 ± 0.6 5.3 ± 0.4  
Ga MR-33b 100% 94.7 ± 2.2 2.3 ± 0.5 2.0 ± 2.2  
Ga MR-37b 100% 95.0 ± 0.6 4.5 ± 1.2 3.1 ± 0.1  
Ga MR-39 100% 101.7 ± 1.8 3.1 ± 0.2 3.6 ± 0.8  
Ga MR-41 100% 90.8 ± 0.7 10.5 ± 0.6 9.7 ± 0.5  
Ga DI-2 100% 56.8 ± 4.2 5.8 ± 0.2 7.2 ± 1.2 ↓ Agly γ4 
Ga FO-3 100% 98.6 ± 0.7 17.1 ± 1.0 14.8 ± 0.3  
Ga TT-3 100% 98.6 ± 0.5 3.5 ± 0.2 4.5 ± 0.3  
Ga TT-7 100% 97.6 ± 2.0 1.2 ± 0.3 0.7 ± 0.3  
Ga TT-9 100% 97.3 ± 2.9 1.2 ± 0.1 1.8 ± 0.8  
Pan MR-24 100% 98.2 ± 1.1 101.2 ± 0.8 99.2 ± 0.9  
1, 3, 4 DI-4 46.0 ± 0.8 45.3 ± 0.5 100% 101.3 ± 1.9  
1, 2, 3 LN-11 2.7 ± 0.7 4.5 ± 0.8 100% 28.3 ± 0.2 ↓ Agly γ3 
1, 4 MR-16b 100% 83.8 ± 3.6 4.5 ± 0.8 5.6 ± 0.1  
1, 2 LN-10 100%c 97.0 ± 3.6d 105.2 ± 0.4e 99.7 ± 0.9f  
1 only DI-1 100%c 99.6 ± 0.2d 3.6 ± 0.3e 2.9 ± 0.3f  
1 only FO-2 100%c 97.6 ± 0.5d 5.5 ± 1.2e 6.2 ± 0.6f  
a

Aglycosylated (Agly) IgG antibodies were engineered by replacing the Asn glycosylation site at position 297 by site-directed mutagenesis. The aglycosylated antibodies are shown by the abbreviated name of the amino acid present at position 297. IgM RF binding to glycosylated and aglycosylated chimeric IgG was measured using a RF ELISA and the results are expressed as a percentage of the mean of six determinations from two separate experiments ± SEM with binding to glycosylated IgG4, IgG3, or IgG1 defined as 100% binding.

b

Clonally related RFs.

c

Glycosylated IgG1.

d

IgG1 with Gln297.

e

Glycosylated IgG2.

f

IgG2 with His297.

One common goal of previous investigations that mapped RF binding sites on IgG was to determine whether a subgroup of RF autoantibodies, associated with chronic articular inflammation, could be identified within the repertoire of RFs expressed by patients with RA by their novel specificity for IgG (11, 16, 17, 18, 19, 20, 21, 22, 23). To date, it has not been possible to document novel VH and VL gene usage by RFs expressed by patients with RA compared with RFs from other individuals without synovial disease (38, 39). We have instead approached this question by characterizing the binding specificities of RFs expressed by patients with RA and have compared them to those of RFs found in individuals without arthritis. Our first studies (15, 18, 23) compared RA-RFs with RFs from patients with Wmac, and indeed, we identified differences in binding specificities between these two groups. Presently, we extended these studies and have characterized the gross and fine specificities of a panel of 28 monoclonal IgM RFs from the blood of five immunized normal individuals. Comparisons of the gross and fine specificities of the panel of HID-RFs with our previously studied panels of RA-RFs (15) and Wmac-RFs (18, 19, 20, 21, 22, 23) were made, and they indicate that a different repertoire of RFs is present in individuals with arthritis. However, when the specificities of the HID-RFs were compared with those of Wmac-RFs without arthritis, no significant differences in RF recognition of IgG were observed.

The major differences in gross specificity among HID-, Wmac-, and RA-RFs were identified by comparing the HID-RF panel with the two previously studied RF panels (15, 18, 23).

Tables VIII and IX summarize the gross binding specificity differences observed between HID-RFs and RA-RFs (15) or Wmac-RFs (18, 23). These specificity differences include the ability of some RA-RFs to recognize IgG3 along with the three other IgG subclasses (pan reactivity), and IgG3 in combination with another IgG subclass. Recognition of all four IgG subclasses (p = 0.007) and IgG3 binding in combination with at least one other subclass (p = 0.005) were rare among HID-RFs compared with RA-RFs. However, the Ga-reactive pattern was similar among HID-, RA-, and Wmac-RFs: HID-RFs, 21 of 28 (75%); RA-RFs, 9 of 19 (47%; p = 0.069); and Wmac-RFs, 12 of 17 (75%; p = 0.743). In addition, IgG3 binding was rare and similar between the HID- and Wmac-RFs. When the clonally related RFs were considered as one RF for statistical comparison, these gross specificity differences remained significant: pan reactivity was 1 of 20 (5%) for HID-RFs and 7 of 19 (37%) for RA-RFs (p = 0.0197); reactivity with IgG3 was 3 of 20 for HID-RFs and 9 of 19 of the RA-RFs (p = 0.040). In addition, six HID-RFs differed from any previously studied RA- or Wmac-RF; they recognized the IgG3 polymorphism Tyr/Phe436 (MR-28 and TT-3, -7, and -9), or an epitope singly expressed on IgG1 (DI-1 and FO-2).

In general, three major fine specificity differences were identified. First, HID- and RA-RFs exhibit different requirements for Ile at position 253. When the HID-RFs that recognize IgG4 were reacted with the IgG4 Ab containing Ala235, only 3 of 24 (12.5%) bound strongly to this Ab compared with 11 of 17 (65%) of the RA-RFs (p = 0.0008). Taking all nine related RFs as one for statistical comparison with RA-RFs and recalling that 16 of 20 of the remaining HID-RFs bind IgG4, the difference in reactivity of the HID-RFs to IgG4 containing an Ile→Ala change at position 253 (antibody 3614) remains significant (HID-RFs, 3 of 16 (19%); RA-RF, 11 of 17 (65%); p = 0.013). Second, HID- and RA-RFs showed different requirements for His435 for binding. All 21 Ga-reactive HID-RFs bound well to the hybrid IgG3/4 Ab 3601 (4-4-4-3 with His435-Tyr436). In contrast, three of nine of the Ga-reactive RA-RFs (p = 0.04) failed to recognize this Ab. In addition, 7 of 14 (50%) of the HID-RFs could bind IgG4 with Arg435, while only two of seven RA-RFs bound this Ab well (p = 0.045). Third, unlike any Ga-reactive RA-RFs previously studied (15), three Ga-reactive HID-RFs recognized a Tyr/Phe polymorphism at position 436 in IgG3. Only 1 of 21 Ga-reactive HID-RFs could bind IgG4 with Ala253, while four of nine (44%) RA-RFs bound this Ab (p = 0.0019). However, when Ga-reactive, HID- and Wmac-RFs were compared, the fine specificities were similar.

Importantly, novel subgroups of IgM RFs from patients with RA can be identified by comparing their specificity with RFs derived from individuals without joint disease. Although the method of immortalization used to establish the panel of RF-producing B cells from HID (heterohybridoma fusion) was different from that used to generate the original panels of RA-RFs we studied (EBV transformation), it is unlikely that the differences in methods influenced the repertoire of RFs obtained in these different panels (40); we analyzed a second panel of RA-RFs derived by heterohybridoma fusion and showed similar specificity patterns for IgG compared with RA-RFs generated by EBV transformation (41). In addition, the frequency of RF-expressing B cells recovered from the blood or the synovia of RA patients using EBV transformation correlates with the number of B cells exposed to EBV, suggesting that EBV only expands the existing B cell repertoire (40).

It is clear from these and other studies of RF VH and VL gene usage that many VH and/or VL genes can produce RF activity (15, 38, 39). However, determining the specificity of in vivo-selected, RF-expressing B cells can readily identify RF subgroups. Of interest, the panels of RFs expressed by HID and patients with RA both contain autoantibodies with mutations in VH and/or VL suggestive of an Ag-driven process in the generation of these RFs. However, the differences in specificity in both groups suggest that RF V gene mutation itself does not necessarily produce a similar RF repertoire in different study populations.

The RF VH mutations that occurred among the nine clonally related RFs from patient MR produced both gross and fine specificity differences for IgG. Among the nine clonally related RFs, eight were Ga reactive, and among these eight, fine specificity differences for IgG were also observed. MR-2 failed to bind IgG4 3605 bearing Arg435-Try436 (Table IV), while the other seven RFs bound this IgG Ab well. Considerable variation in binding to IgG4 Ab 3607, bearing three Pro residues at 309 to 311 was also noted (Table V). MR-16 bound IgG4 containing an Ile→Ala change at position 235, while the eight others did not (Table V); unlike the eight others, MR-16 failed to recognize either of the 309 to 311 IgG4 mutants. In addition, MR-14 was only partially inhibited by SPA, while the seven others were completely inhibited in binding IgG by SPA (Table VI). Most of these HID-RFs contain VH and VL somatic mutations (30), and our results provide evidence, for the first time with in vivo selected RFs, that V gene mutations in RF autoantibodies (most extensive in MR-16 among the clonally related RFs) can change their gross and fine specificities.

Changes in autoantibody affinity have also been shown to be induced by V gene hypermutation in both RA-RFs and anti-DNA Abs (30, 42, 43). Small numbers of amino acid substitutions have been shown to effect the pathogenicity of some autoantibodies, while pathogenicity does not necessarily correlate with the affinity for cognate ligand (43, 44). KLH immunization of a nonautoimmune murine model (44) suggests that central deletion of autoreactive B cells may not always be sufficient to prevent Ab-induced autoimmunity during Ag challenge. A high frequency of the responding primary B cells was found to produce dual reactive Abs that recognize KLH and dsDNA (44). The pool of these cross-specific Abs contains mutations that appear 10 days after KLH challenge and only if apoptosis is overcome through constitutive expression of bcl-2 by the B cell fusion partner. Furthermore, these dual reactive Abs are deposited in the kidney in patterns similar to those observed in models of systemic lupus erythematosus (44). These experiments demonstrate that central clonal deletion of autoreactive B cells may not necessarily be sufficient to prevent the Ab-associated autoimmunity that develops during Ag challenge. A peripheral mechanism would therefore be necessary to prevent the emergence of frequently occurring, potentially pathogenic autoantibodies that can be generated by antigenic challenge.

It is possible that during the Ag-driven process that is hypothesized to occur in RA, a defect in peripheral immunoregulation might allow for the emergence of select RF autoantibodies that recognize this putative Ag(s) in addition to IgG. Support for this possibility is found in the recent crystallization of an RA-RF-IgG complex where the RF combining site is open for potential interaction with another ligand (35). Therefore, potentially pathogenic RFs generated following antigenic stimulation may exist in RA and would rarely be expressed in individuals without arthritis.

By defining the specificity of RFs from individuals with arthritis compared with those RF specificities from individuals without disease, insight may be gained into the relationship among specificity, function, and the disease process. Our results suggest that some RA-RFs do indeed appear to be disease specific. Others have shown that IgG3 binding, RA-RFs, and IgG3 are disproportionately expressed in the synovia compared with the blood (14), and IgG3 appears to be the pivotal subclass that distinguishes some RA-RFs as disease-specific autoantibodies from others expressed by individuals without joint disease (15, 17, 18, 23). Future studies of other panels of monoclonal RFs from individuals who express RFs with acute or chronic infection or autoimmunity without synovial disease should be helpful in further illuminating which RA-RFs are truly disease specific. These studies should help identify the role(s) of these RFs in synovial disease. In addition, experiments that examine the peripheral immunoregulation of RF expression may clarify the mechanism(s) responsible for disease-specific RF production in RA.

Table VIII.

Summary of RF binding specificitiesa, VH and VL usagea

Alab 4354443c His4353333 His435Alab 253Prob 252-4Glyb 252-4Prob 309-11Glyb 309-11Inhibitd by SPAVLVH
GA pattern            
MR-1 − − − − − 325 VH
MR-2e − − − − − 325 VH
MR-3 − − − − − +/− K3 VH
MR-5 − − − − − − − 328 VH
MR-12 − − − − − − K3 ND 
MR-13e − − − − − − 325 VH
MR-14e − − − − − +/− − +/− 325 VH
MR-20 − − − − − K3 VH
MR-25e − − − − − − 325 VH
MR-27 − − − − − − ND VH
MR-28e − − − − − − 325 VH
MR-30e − − − − − − 325 VH
MR-33e − − − − 325 VH
MR-37e − − − − − − 325 VH
MR-39 − − − − − K3 VH
MR-41 − − − − − − − − 328 VH
DI-2 − − − − − − K3 VH
FO-3 − − − − − − λ VH
TT-3 − − − − K3 VH
TT-7 − − − − − − K3 ND 
TT-9 − − − − − − − 328 VH
Bind all IgG isotypes            
MR-24 − − K3 VH
Bind IgG1,-3,-4 Only            
DI-4 − − − − − − − − K3 VH
Bind IgG1,-2,-3 Only            
LN-11 − − − − − − +f NDg ND 
Bind IgG1,-4 Only            
MR-16e − − − − − − 325 VH
Bind IgG1,-2 Only            
LN-10 ND ND ND ND ND ND ND ND +f ND ND 
Bind IgG1 Only            
DI-1 ND ND ND ND ND ND ND ND f λ VH
FO-2 ND ND ND ND ND ND ND ND f λ VH
Alab 4354443c His4353333 His435Alab 253Prob 252-4Glyb 252-4Prob 309-11Glyb 309-11Inhibitd by SPAVLVH
GA pattern            
MR-1 − − − − − 325 VH
MR-2e − − − − − 325 VH
MR-3 − − − − − +/− K3 VH
MR-5 − − − − − − − 328 VH
MR-12 − − − − − − K3 ND 
MR-13e − − − − − − 325 VH
MR-14e − − − − − +/− − +/− 325 VH
MR-20 − − − − − K3 VH
MR-25e − − − − − − 325 VH
MR-27 − − − − − − ND VH
MR-28e − − − − − − 325 VH
MR-30e − − − − − − 325 VH
MR-33e − − − − 325 VH
MR-37e − − − − − − 325 VH
MR-39 − − − − − K3 VH
MR-41 − − − − − − − − 328 VH
DI-2 − − − − − − K3 VH
FO-3 − − − − − − λ VH
TT-3 − − − − K3 VH
TT-7 − − − − − − K3 ND 
TT-9 − − − − − − − 328 VH
Bind all IgG isotypes            
MR-24 − − K3 VH
Bind IgG1,-3,-4 Only            
DI-4 − − − − − − − − K3 VH
Bind IgG1,-2,-3 Only            
LN-11 − − − − − − +f NDg ND 
Bind IgG1,-4 Only            
MR-16e − − − − − − 325 VH
Bind IgG1,-2 Only            
LN-10 ND ND ND ND ND ND ND ND +f ND ND 
Bind IgG1 Only            
DI-1 ND ND ND ND ND ND ND ND f λ VH
FO-2 ND ND ND ND ND ND ND ND f λ VH
a

Binding ≥25% of wild-type binding is considered positive.

b

RF binding to IgG4.

c

RF binding to hybrid antibody, CH3 of IgG3 contains Arg → His change at 435.

d

RF binding to IgG4 in the presence of Staph. protein A.

e

Clonally related RF.

f

RF binding to IgG1 in the presence of Staph. protein A.

g

Not done.

Table IX.

Summary of the specificity differences between RFs from healthy immunized donors and patients with RA or Wmaca

RF Gross SpecificityHID-RFRA-RF (Ref. 15)Wmac-RF (Refs. 18 and 23)
Pan (IgG1,-2,-3, -4) 1/28 (3.6%) 7/9 (37%) p = 0.007 0/17 
IgG3 reactive 3/28 (11%) 9/19 (47%) p = 0.005 2/17 (12%) p = 1.0 
Ga-reactive 21/28 (75%) 9/19 (47%) p = 0.069 12/17 (75%) p = 0.743 
RF fine specificity    
Ile254 important 3/24 (12.5%) 11/17 (65%) p = 0.0008 NDb 
His435 important 21/21 (100%) 6/9 (67%) p = 0.04 14/15 (93%) p = 0.42 
RF Gross SpecificityHID-RFRA-RF (Ref. 15)Wmac-RF (Refs. 18 and 23)
Pan (IgG1,-2,-3, -4) 1/28 (3.6%) 7/9 (37%) p = 0.007 0/17 
IgG3 reactive 3/28 (11%) 9/19 (47%) p = 0.005 2/17 (12%) p = 1.0 
Ga-reactive 21/28 (75%) 9/19 (47%) p = 0.069 12/17 (75%) p = 0.743 
RF fine specificity    
Ile254 important 3/24 (12.5%) 11/17 (65%) p = 0.0008 NDb 
His435 important 21/21 (100%) 6/9 (67%) p = 0.04 14/15 (93%) p = 0.42 
a

All comparisons were made by a two-tailed, Fisher’s exact test.

b

ND = not done.

1

This work was supported in part by a Basic Science Grant from the Arthritis Foundation and was presented in part at the American College of Rheumatology meeting in 1995.

3

Abbreviations used in this paper: RF, rheumatoid factor; RA, rheumatoid arthritis; Wmac, Waldenström’s macroglobulinemia; HID, healthy immunized donors; SPA, Staphylococcus aureus protein A; KLH, keyhole limpet hemocyanin.

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