Monoclonal IgM Rheumatoid Factor Research Articles

A rheumatoid factor paradox: inhibition of rituximab effector function

IntroductionRituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC).MethodsWe developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated.ResultsUsing human sera, addition of RTX resulted in rapid and profound (> 50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis.ConclusionsContrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.

Rheumatoid Factor, Complement, and Mixed Cryoglobulinemia

Low serum level of complement component 4 (C4) that occurs in mixed cryoglobulinemia (MC) may be due to in vivo or ex vivo activation of complement by the classical pathway. Potential activators include monoclonal IgM rheumatoid factor (RF), IgG antibodies, and the complexing of the two in the cold, perhaps modulated by the rheology and stoichiometry of cryocomplexes in specific microcirculations. There is also the potential for activation of complement by the alternative and lectin pathways, particularly in the setting of chronic infection and immune stimulation caused by hepatitis C virus (HCV). Engagement of C1q and interaction with specific cell surface receptors serve to localize immune complexes (ICs) to the sites of pathology, notably the cutaneous and glomerular microcirculations. Defective or saturated clearance of ICs by CR1and/or Fc receptors may explain persistence in the circulation. The phlogistic potential of cryoprecipitable ICs depends upon the cleavage of complement components to generate fragments with anaphylatoxin or leukocyte mobilizing activity, and the assembly of the membrane attack complex (C5b-9) on cell surfaces. A research agenda would include further characterization of the effector arm of complement activation in MC, and elucidation of activation mechanisms due to virus and viral antigens in HCV infection.

An experimental type II mixed cryoglobulinemia with renal glomerulopathy in ICR mice triggered by Capillaria hepatica infection.

Type II mixed cryoglobulinemia is characterized by systemic vasculitis with deposition of cryoprecipitatable-immunoglobulins containing rheumatoid factor. Pathogenesis of type II mixed cryoglobulinemia has not yet been completely clarified because of the lack of an experimental animal. Here, we report an animal model of type II mixed cryoglobulinemia that is induced by experimental infection with Capillaria hepatica in ICR mice. Capillaria hepatica is a nematode that causes necrotic hepatitis in several mammals. In this study, mice experimentally infected with C. hepatica eggs developed cryoglobulinemia at 20 and 30 days post injection. Using immunological analysis, cryoglobulinemia in infected mice was classified as type II mixed cryoglobulinemia by detection of monoclonal IgM rheumatoid factor and IgA in the cryoprecipitate of serum. Using immunofluorescence, we observed an increase in the number of double-positive cells for µ heavy and κ light chains of immunoglobulin in the spleens of infected mice. Histopathologically, this model was characterized by glomerulopathy associated with intense deposition of IgM and IgA filling in capillary lumina. Ultrastructural analysis showed that glomerular deposits consisted of stacks of twisted microtubular structures. These serological and histological features resembled those of type II mixed cryoglobulinemia in human. This is the first experimental animal model of type II mixed cryoglobulinemia that will enable detailed studies on the pathogenesis of cryoglobulinemia.

Cytokine profile of macrophages in vitro stimulated by ACPA immune complexes in the presence or absence of IgM rheumatoid factor

BackgroundAutoantibodies to citrullinated proteins (ACPA) are suspected to play a central role in the pathophysiology of rheumatoid arthritis (RA). By stimulating monocyte-derived macrophages from healthy donors with ACPA-containing immune complexes...

Crystal Structure of a Human Autoimmune Complex between IgM Rheumatoid Factor RF61 and IgG1 Fc Reveals a Novel Epitope and Evidence for Affinity Maturation

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 Å resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cγ3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,− 2, and − 3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V H:V L β-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V H residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.

Cryoglobulinemia

Mixed cryoglobulinemia (MC) type II is a disorder characterized by circulating cold-precipitable immune complexes composed of polyclonal immunoglobulin IgG and monoclonal IgM rheumatoid factor (RF). The systemic vasculitis which characterizes the disease is caused by the deposition of immune complexes on the walls of small vessels, and by the subsequent activation of the complement cascade. MC may be asymptomatic or lead to clinical manifestations characterized by a typical triad--purpura, arthralgia, and weakness--and in some cases by a more serious vasculitis with neurologic and renal involvement. In the vast majority (more than 90%) of patients, MC is associated with hepatitis C virus (HCV) infection, which is considered the triggering factor of the disease. The association between cryoglobulinemia and HCV infection, and the possible evolution to B-cell lymphomas usually after long-term follow-up, suggest the role of HCV infection both in the pathogenesis of MC and in lymphomagenesis. In fact, the virus chronically stimulates the polyclonal proliferation of B cells from which a monoclonal population may emerge. This paper also reviews the treatment strategies for MC syndrome, emphasizing the issue of the eradication of HCV, and the clinical and biological activity of rituximab for selective B-cell control.

A Rheumatoid Factor Specific Mimotope Identified by a Peptide Display Library

We screened a 10 amino acid peptide display library in filamentous phage with B'20, a monoclonal high affinity IgM rheumatoid factor (RF) expressing the Vk111a-dependent 4C9 idiotype. Using direct and indirect selection techniques, 12 B'20 reactive peptides were identified, 9 of which belonged to one of two motifs. Binding of B'20 to phage-bearing peptides was inhibited by both IgG and 4C9 antiidiotype. Synthetic peptides corresponding to the two motifs inhibited the Fc binding of a low avidity IgA B'20 construct. Purified IgM from 6/8 RF-positive RA patients and 8/11 monoclonal RFs with Vk111-encoded light chains bound to the phage, whereas none of the four monoclonal RFs with Vk1 or Vk11 encoded light chains bound. Phage binding appeared to be RF specific. Three 4C9 positive/RF negative cell lines from RA patients did not bind to phage nor did three B'20 mutants that had lost RF specificity, whereas two mutants that retained RF specificity also retained phage binding. We propose that there is a common epitope(s) recognized by Vk11 I encoded RFs that is mimicked by the structure of these peptides. Such mimotopes might be exploited to design novel agents that interfere with autoantibody binding.

Mapping IgG Epitopes Bound by Rheumatoid Factors from Immunized Controls Identifies Disease-Specific Rheumatoid Factors Produced by Patients with Rheumatoid Arthritis

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.

Heterogeneous RF structures between and within healthy individuals are not related to HLA DRB1 ∗0401

To study variations in Rheumatoid Factor (RF) autoantibodies between and within healthy individuals, we have produced and analysed the variable heavy chain (V H) regions of 18 new monoclonal IgM RFs from the peripheral blood of two healthy subjects before and after immunization with tetanus toxoid (TT). The majority of these RFs used germline genes of the V H3 family, but RFs of the V H1, V H2 and V H7 families were also found. No RF of the V H4 RF is found. Fourteen different V H germline (GL) genes encoded the RFs, suggesting an extraordinary heterogeneity in structure. Consequently, changes in RF V region structures following immunization were difficult to identify. There were, however, structural differences between RFs from the two donors. RFs from one donor (IP) used more λ light chains than RFs from the other donor and previously described RFs. In addition, 50% of the RFs from donor IP were encoded by GL genes frequently found to encode RFs from patients with Rheumatoid Arthritis (RA) but not described in RFs from other healthy subjects. The predominant use of V H3 RFs in the two healthy donors contrasts with the over-expression and expansion of V H1 RFs in one previously described healthy immunized donor. There are thus large individual differences in RF structures, which might be related to the immunological status, environment or genetic background of the donors. However, since these three donors are all HLA DRB1 ∗0401, it is unlikely that this HLA type, associated with seropositive RA, accounts for the individual differences.

Antigenic determinants reacting with rheumatoid factor: Epitopes with different primary sequences share similar conformation

Polyclonal or monoclonal human IgM rheumatoid factors (RF) react with eight antigenic sites on the CH3 IgG domain, four sites on CH2 and two on human β 2-microglobulin. All 14 of these RF-reactive epitopes are linear 7–11 amino acid peptides with different primary sequence. We questioned whether RF reactivity with such a variety of epitopes showing no obvious sequence homology might result from conformational similarities shared by various RF-reactive regions. Strong support for this concept was obtained using rabbit antisera as well as mouse mAbs to individual CH3, CH2 or β 2m RF-reactive peptides. Major cross-reactivity was demonstrated between most of the 14 different CH3, CH2, or β 2m RF-reactive peptides using individual anti-epitope antibodies. Molecular modelling studies of these peptides showed striking similarities in three-dimensional shape among many RF-reactive peptides. Main-chain atoms rather than side chains seemed to contribute most directly to conformational similarity. Molecular simulation studies on control peptides showed no conformational similarities with RF-reactive peptides. Our studies indicate that autoantibodies such as RF recognize main-chain conformations of reactive epitopes and react with a number of antigenic determinants of quite different primary sequence but similar main chain conformations.

High binding of immunoglobulin Mκ rheumatoid factor from type II cryoglobulins to cellular fibronectin: A mechanism for induction of in situ immune complex glomerulonephritis?

In our previous experimental work we suggested that the frequent nephritogenicity of type II cryoglobulins could depend on a particular affinity of the immunoglobulin (Ig) Mκ rheumatoid factor (RF) component for mesangial matrix. Since cellular fibronectin (cFN) in the human kidney is mainly represented in glomerular mesangium, we studied the binding capacity of cFN of IgMκ RFs from type II cryoglobulins compared with other different monoclonal and polyclonal IgM and IgM RFs. We purified 13 IgMκ from human IgMκ/IgG cryoglobulins, eight monoclonal IgM from patients with Waldenström's macroglobulinemia, nine polyclonal IgM from normal donors, and eight polyclonal IgM RFs from patients with rheumatoid arthritis. Purified IgM were used at the same concentration in enzyme- linked immunosorbent assay (ELISA) on cFN-coated plates. All the cryoglobulin IgM showed high specific binding to cFN while IgM from Waldenström's macroglobulinemia, normal IgM, and polyclonal IgM RFs had low or absent binding. These data were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cFN followed by Western blot analysis with purified IgM. The IgMκ binding to cFN persisted using IgMκ monomers, and was inhibited by cFN but not by plasma FN in a specific inhibition test. Further enzyme-linked immunosorbent assay studies showed that cryoglobulin IgMκ RFs are still able to bind IgG in a dose-dependent manner once linked to solid-phase cFN. The data suggest that the affinity of cryoglobulin IgMκ RFs for immobilized cFN could be involved in the particular high nephritogenicity of type II cryoglobulins and might lead to in situ immune complex formation.

IgM rheumatoid factors react with human class I HLA molecules.

Half of 30 human polyclonal IgM rheumatoid factors (RF) showed positive ELISA reactions with affinity-isolated human class I molecules (A2 and B7). Positive RF reactivity with isolated class I heavy chains indicated that anti-class I RF interaction did not merely reflect RF anti-beta 2m specificity. Cross-reactions between antigenic determinants on human IgG, class I molecules, and beta 2m reacting with RF could be demonstrated by ELISA inhibition. When gene products of A2 alpha 2 exons 2, 3, and 4 were synthesized as overlapping heptamers on polypropylene pins, six RF-reactive epitopes within solvent accessible class I HLA regions were identified: EPRAPWI, QEGPEYW, QTHRVDL (second A2 exon), EQLRAYL, GTCVEWL, and WLRRYLE (third A2 exon). Glycine-alanine substitution for each residue within these RF-reactive sites identified R48, W51, E55, Y107, R108, W147, Q155, and E172 as immunodominant residues for RF reactivity. Many of these RF-reactive class I regions also showed positive ELISA reactions with monoclonal human IgM RFs derived from rheumatoid arthritis synovial B cells. Whole serum from patients with rheumatoid arthritis showed a spectrum of IgM, IgA, and IgG Abs that adsorbed to and could be eluted from monomeric IgG or HLA A2 affinity columns. Normal serum similarly analyzed showed only trace reactions with IgG, A2, or beta 2m. Flow cytometry using RF incubated with A2 cell lines showed definite immunofluorescence reactivity with cell membranes not observed with normal non-RF IgM. Reactivity of human IgM RF with determinants on class I molecules may reflect antigenic overlap within several members of Ig gene superfamily products.

Monoclonal IgM rheumatoid factor secreted by CD5-negative B cells during mixed cryoglobulinemia. Evidence for somatic mutations and intraclonal diversity of the expressed VH region gene.

Mixed cryoglobulinemia is usually considered to be a nonmalignant human B cell proliferation that produces a monoclonal IgM rheumatoid factor (RF). Important immunologic similarities and differences were described between the monoclonal B cells during mixed cryoglobulinemia and during malignant chronic lymphocytic leukemia (CLL):high frequency of the same VH and V kappa gene usage by both types of monoclonal B cells producing IgM with RF activity, apparent intraclonal homogeneity, but different expression of the pan T cell CD5 Ag. The description of an unusual CD5-negative B cell CLL case secreting a mutated IgM RF led the authors to suggest that the usage of non-mutated germline Ig genes is a property of cells derived from the CD5 lineage or stage of differentiation, rather than an intrinsic property of CLL or of IgM RF-producing cells in general. Because mixed cryoglobulinemia cells are usually CD5-negative, it was of interest to test for the existence of mutations in the VH and V kappa regions, as well as for the intraclonal homogeneity of the expressed Ig genes. In this study, we used the PCR technique to analyze the monoclonal rheumatoid factor (mRF) V genes from a patient with mixed cryoglobulinemia. We show that the CD5-negative monoclonal B cells express a slightly mutated V kappa 3 gene, but a more mutated VH1 gene whose genomic counterpart was shown to be the 51p1 germline gene. The sequence analysis of several independent clones shows some degree of intraclonal diversity, suggesting the existence of a clonal filiation. These results are discussed in terms of the origin of the monoclonal B cell during mixed cryoglobulinemia and CLL.

Rheumatoid-factor-reactive sites on CH 3 established by overlapping 7-mer peptide epitope analysis

Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis and six monoclonal IgM RF were isolated from monomeric IgG affinity columns and studied for their reactivity with the entire CH 3 domain of IgG synthesized as overlapping 7-mers using a pin-ELISA assay. All ten polyclonal IgM RF showed similar profiles of reactivity which included peptides with solvent accessible residues PREPQVY (residues 343–349), PQVYTLP (residues 346–352), TLPPRSE (350–356), DGSFFLY (401–407), WQQGNVF (417–423), CSVMHEG (425–430), EGLHNHY (430–436) and KSLSLSP (439–446) of the CH 3 domain. Substitution of a neutral glycine or alanine for each residue within these RF-reactive epitopes indicated that tyrosine at position 349, prolines at 343, 346 and 352, glutamine 347, valine 348, threonine 350, leucine 351, arginine 354, aspartic acid 401, tyrosine 407, serine 426, histidine 429, leucine 432, tyrosine 436 and lysine 439 represented important single amino acids within CH 3 for RF reactivity. Regions of CH 3 primary sequence with and without the single allotype-specific amino acid substitutions of glycine for alanine 431 (Gmx) or aspartic acid for glutamic acid (356) and leucine for methionine (358) (Gma) often showed considerable differences in reactivity with individual polyclonal and monoclonal RF. However, these differences in RF reactivity did not correlate with the individual anti-Gm RF specificity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a much more restricted spectrum of reactive CH 3 epitopes. 7-mer peptides representing RF-reactive sites on CH 3 preincubated with polyclonal IgM RF showed strong inhibition (55–66%) of RF binding to whole IgG on the ELISA plate. These studies indicate that it is possible to define portions of the IgG CH 3 domain participating in the reaction with IgM RF using reactive epitope-mapping with sequential linear peptides derived from the primary IgG CH 3 sequence.

A subgroup of human VH3 germline genes that encode a high-avidity synovial rheumatoid factor.

We have previously derived and identified a highly avid monoclonal IgM rheumatoid factor (mRF), C6, from unstimulated rheumatoid synovial cells (RSC). At the time, the closet VH germline gene, VH26, demonstrated only 88% homology with C6. To identify the germline counterpart of C6, genomic DNA from the same rheumatoid arthritis (RA) patient from whom C6 was derived was used in the polymerase chain reaction (PCR). Four of the six closely related germline genes that we sequenced had exonic regions that were identical with the VH region of C6 cDNA. These six germline sequences differed in their intronic regions, suggesting that they were distinct, but closely related genomic sequences. To further evaluate the extent of these related genes we identified nine additional germline genes having VH-encoding exons that were 86-97% identical to the C6 cDNA sequence. Furthermore, we examined the polymorphic nature of the C6 VH gene using single strand conformation polymorphism (SSCP), and identified two peaks, confirming the existence of highly homologous genes. The sequence and polymorphism data suggest that: (1) the VH region of the high avidity mRF C6 was derived from an unmutated germline gene; (2) C6 was encoded by a VH gene belonging to a set of homologous genes within the larger VH3 family; and (3) in addition to somatic rearrangements of B-cell genes and antigen-driven somatic mutation, gene duplication and conversion events of germline genes could be important in generating diversity and polyclonality among high-affinity pathogenic autoantibodies.

Control of autoantibody affinity by selection against amino acid replacements in the complementarity-determining regions.

Rheumatoid factor (RF) autoantibodies can be produced in healthy individuals after infections or immunizations and thus escape normal tolerization mechanisms. It has not been clear whether such autoantibodies can undergo somatic hypermutation and affinity maturation similar to antibodies to exogenous antigens. We have investigated how these autoantibodies are regulated in normal individuals by analyzing the sequences of monoclonal IgM RFs obtained as hybridomas from donors after immunization. The variable regions undergo extensive hypermutation, but in contrast to antibodies against exogenous antigens, there is a strong selection against mutations that result in replacement of amino acids in the hypervariable, or complementarity-determining, regions. Furthermore, we found no increase in affinity of these RFs with the accumulation of mutations. This suggests that high-affinity variants are tolerized during the hypermutation process and there is a peripheral mechanism operating on certain autoreactive B cells that, while not deleting or anergizing all autoreactive cells, prevents the generation of high-affinity autoantibodies. Comparison of RFs by using the VH1 DP-10 heavy chain variable region segment from both normal individuals and rheumatoid arthritis (RA) patients suggests that RF from RA patients may not be subject to such a controlling mechanism.

Crystallization and Preliminary X-ray Analysis of the Fab Fragment of a Human Monoclonal IgM Rheumatoid Factor (2A2)

Crystals of the Fab fragment of a human monoclonal IgM rheumatoid factor have been obtained and are suitable for X-ray structure determination. This molecule, derived from the synovial B cells of a patient with rheumatoid arthritis, is an autoantibody with specificity for IgG Fc. The crystals have space group P21, cell dimensions a = 69·0 Å, b = 76·6 Å, c = 98·8 Å and β = 90·6°, and diffract to a resolution of at least 2·8 Å.

Rheumatoid-factor-reactive sites on CH2 established by analysis of overlapping peptides of primary sequence.

Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis (RA) isolated on IgG affinity columns were studied for their reactivity with overlapping 7-mer peptides representative of solvent accessible regions of the CH2 domains of IgG using a pin-ELISA assay. A panel of nine monoclonal RF from RA patients' B cells were studied in parallel. Four peptides SVFLFPP (239-245), KFNWYVD (274-280), NSTYRVVSV (297-305) and VLTVLHQNWL (305-314) reacted with most RF. Glycine substitution showed that tryptophanes at 277 and 313, tyrosine at 278, valines at 279 and 305, and leucine 314 represented important residues for RF reactivity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a restricted spectrum of reactivity for CH2 epitopes, which often were identical to those binding to polyclonal IgM RF. Combinations of synthetic 7-mer peptides representing RF-reactive CH2 or CH3 epitopes produced as much as 60-90% inhibition of RF binding to IgG when peptides were preincubated with RF in free solution before completion of the reaction of RF with IgG on the ELISA plate.

Changes in Rheumatoid Factor and Monoclonal IgG Antibody Specificity after Site-Specific Mutations in Antigenic Region of β 2-Microglobulin

Antigenic epitopes on the SKDWSFY region (positions 57-63) of human β 2-microglobulin (β 2m) reacting in ELISA with polyclonaI and monoclonal human IgM rheumatoid factors (RF) as well as with monoclonal mouse IgG anti-β 2m antibodies were studied after site-specific mutation of single or double β 2m amino acid residues. Single site-specific mutations at lysine 58 and serine 61 produced a major decrease (50-100%) in reactivity of polyclonal IgM RF with β 2m. Mutations at serine 57, tryptophane 60, and phenylalanine 62 also induced substantial decrements (25-49%) in IgM RF reactivity. β 2m mutants containing double amino acid mutations including one residue outside the SKDWSFY region along with four different mutations at tryptophane 60 also often showed marked decrements in IgM RF anti-β 2m reactivity. Site-specific mutations at virtually all residues within the SKDWSFY β 2m region produced a major decrease in reactivity of two of three monoclonal human IgM RF. Results with nine murine IgG anti-β 2m mAbs often showed an entirely different protile. Site-specific mutation of aspartic acid at position 59 produced a 50-100% decrease in binding of nine mAbs. Serine at position 61 and tyrosine at 63 also appeared to represent important residues for antigenic determinants reacting with anti-β 2m mAbs. Results of mapping discrete epitopes using glycine substitution within overlapping 7-mers of β 2m linear sequence compared with site-specific mutation of single β 2m residues were similar in many instances, but also often showed marked differences. A marked change in antigenic contribution of single amino acid residues to β 2m antigenic epitopes frequently depended on what residues were mutated to. Our findings emphasize that single residues within a small seven amino acid, solvent-accessible antigenic stretch of primary β 2m sequence vary considerably in their contribution to the reactive antigenic structures for IgM RF as well as monoclonal IgG antibodies.

Preferential utilization of a novel V lambda 3 gene in monoclonal rheumatoid factors derived from the synovial cells of rheumatoid arthritis patients.

To further our understanding about the molecular genetics of rheumatoid factor (RF) in rheumatoid arthritis (RA). The heavy and light chain variable region (V) genes of 5 new human monoclonal IgM RFs were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide termination method. The results reveal the recurrent usage in two RA patients of a novel V lambda 3 germline gene, designated Humlv3c93. Specifically, in 2 of 3 RFs (C93 and D53) from one patient, the light chains in the V lambda gene-encoded region were identical to each other and to the light chain of an RF (H4) from another patient. Serologically, the light chains of these 3 RFs were classified as members of the V lambda 3b sub-subgroup. Each of the RFs was encoded by a different VH gene. Both C93 and D53 bound specifically with human and rabbit IgG, whereas H4 was monospecific for rabbit IgG. Since the lv3c93 gene is not homologous to any reported V lambda sequence from natural autoantibodies, it is possible that lv3c93 may represent a disease-specific RF-related V lambda gene. Moreover, the amino acid sequence CSGGSCY in the third complementarity-determining regions of 2 of the RF heavy chains is encoded by the DLR2 gene segment and has been found previously in 2 other RA-derived RFs, and thus may play a significant role in antigen binding.