Prenatal genotyping of RHD and SRY using maternal blood.

BACKGROUND AND OBJECTIVES: The aim of the study was to perform fetal RHD genotyping in maternal plasma using a fluorescent polymerase chain reaction (PCR) technique. Duplex PCR, amplifying RHD and SRY in the same tube, was undertaken. The effect of varying storage temperatures on the concentration of fetal DNA was investigated in a separate study involving 10 RhD-negative pregnant women. MATERIALS AND METHODS: Primers and probes for the RHD gene's exon 7 and the sex-determining region, Y, were designed, and monoplex and duplex PCR were performed. Blood samples from 10 RhD-negative women were split into four and treated in four different ways before measuring the concentration of fetal DNA by quantitative PCR. RESULTS: DNA extracted from the plasma of 114 RhD-negative pregnant women was tested for the presence of fetal RHD. The discrepancy between genotyping and serological RhD typing of the babies postpartum was 8% when counting one positive replicate as a positive result. Duplex PCR, amplifying RHD and SRY in the same tube, showed a reduced sensitivity for amplification of the SRY gene segment. There was a statistically significant reduction of fetal DNA in blood samples stored at room temperature for 48 h compared with the same sample stored at a temperature of <10 degrees C for the same length of time. CONCLUSIONS: This method is not suitable for routine analysis because of the lack of a positive control for RHD-negative female fetuses and a decrease in PCR sensitivity when performing duplex PCR. Fetal DNA in maternal plasma is better preserved when the blood sample is kept cool.

Structural restriction in the heavy chain CDR3 of human rheumatoid factors.

We have compared the variable regions of 14 new IgM rheumatoid factors (RFs), produced in healthy human immunized donors (HIDs) with RFs originating from patients with rheumatoid arthritis (RA) and monoclonal Ig RFs (paraproteins or M-components, MC). Two groups with very restricted variable region structures were found. Twelve RFs (3 HID, 3 MC, and 6 RA) encoded by variable heavy (VH) chain germ-line genes with closest homology to DP-10 co-express the Kv325 variable light (VL) chain germ-line gene. These RFs have a remarkable restriction in the length (12-14 amino acids) and structure of the CDRH3. One HID RF has a CDRH3 only two amino acids different from the CDRH3 of a MC RF. Two sets of clonally related RFs, one from an RA patient and one from an HID, have CDRH3s that differ by only three amino acids. Five RFs (3 HID, 1 MC, and 1 RA) encoded by VH germ-line gene segments with closest homology to DP-54 all use the Kv328 VL germ-line gene combined to J kappa 1. Four are rearranged to the D21/9 D segment in the same reading frame, with CDRH3s of 16 to 17 amino acids. Three RFs (1 HID, 1 RA, and 1 MC) have CDRH3s differing by only three amino acids. The highly homologous V-regions in RFs from these two groups imply an initial selection to very similar, if not identical, epitopes. However, it remains to be seen whether somatic hypermutation alters the fine specificity of these autoantibodies.

V-gene repertoire and hypermutation of rheumatoid factors produced in rheumatoid synovial inflammation and immunized healthy donors.

We have compared RF from normal, immunized donors and RF derived from the synovial tissues of RA patients. We have found a difference in the preferential use of VL and VH gene families. In both conditions, RFs were found to have accumulated somatic mutations. However, there was a striking difference in the patterns of mutation. RFs from normals were characterized by a very low R:S ratio in the CDR1+2, considerably lower than seen among the RARFs. In addition, there was little increase in affinity with increasing numbers of mutations in a group of clonally related RFs from an immunized normal. This contrasts with RF from RA, where there is evidence of both affinity maturation and class switching. Together these data suggest that in healthy persons there is a controlling mechanism to limit the affinity of RF autoantibodies, and that this is lost in RA. The higher affinity of the RA-derived RF may be of significance in the pathology of the disease.

The identification of germinal centres and follicular dendritic cell networks in rheumatoid synovial tissue.

We document here the identification of germinal centres with dark and light zones, a follicular dendritic cell network and clonal expansion in the synovium of rheumatoid arthritis patients. Synovial tissue from 24 patients suffering from rheumatoid arthritis or the polyarticular form of juvenile rheumatoid arthritis were screened for the presence of lymphoid follicles. The synovial tissues of 14 patients contained follicles and four of these had germinal centres and a follicular dendritic cell network. There was a statistically significant association between follicles in the synovium and the presence of rheumatoid factor autoantibodies in the patients' serum indicating a link between local germinal centre formation and the presence of pathological rheumatoid factors. Nucleotide sequencing of monoclonal rheumatoid factors from one of the patients' synovial tissue which contained germinal centres clearly supports the possibility that these rheumatoid factors have gone through a germinal centre reaction. While rheumatoid factors from healthy immunized donors are regulated through a tolerization mechanism which selects against replacement mutations and does not allow affinity maturation, synovial rheumatoid factors seem to lack this tolerization mechanism. The formation of germinal centres where B cells affinity mature and expand at the central site of disease in rheumatoid arthritis may explain why rheumatoid factors in rheumatoid arthritis develop into auto-aggressive antibodies.

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.

Three new cross-reacting idiotopes as markers for the products of two distinct human VH3 genes expressed in the early repertoire.

This article describes characterization of three new cross-reacting idiotopes, as recognized by mouse MoAbs, on human antibodies utilizing VH3 genes that are expressed in the early repertoire. Two of the mouse MoAbs (3H7 and 3H1) were raised against a human MoAb utilizing the DP47 (VH26) VH3 gene, whilst the third (7B4) was raised against a DP46 (GLSJ2) gene product. Evidence for the anti-idiotypic specificity of the mouse MoAbs was provided by their reactivity with the immunizing IgM, but not with Fc mu, and by their specific inhibition of the binding between each immunizing antibody and its antigen. The three anti-idiotypic MoAbs were shown to be VH-specific reagents by the independence of their reactivity upon the L-chain type, or the antigenic specificity of the human MoAbs tested. Specificity of each mouse MoAb for VH3 gene-products was demonstrated by its sole cross-reactivity with VH3 proteins. Each anti-Id had a different reactivity pattern with a panel of MoAbs utilizing different VH3 genes. By relating the VH sequences of the tested VH3 proteins to their germline counterparts, 3H7 and 3H1 appeared to be specific for DP47-encoded proteins, although 3H1 had weak cross-reactivities with a few other VH3 gene-products. 7B4 appeared to be specific for antibodies utilizing DP46-related genes. Both 3H7 and 3H1 were also completely different to B6 and D12, two previously described MoAbs that also recognize VH3 proteins. Although 7B4 was similar to B6 and D12 in its binding to DP46-related gene products, B6 and D12 additionally recognized non DP46-related proteins and were thus different to 7B4.

Human monoclonal rheumatoid factors: incidence of cross-reactions with tissue components and correlation with VH gene usage.

Human monoclonal antibodies with rheumatoid factor (RF) activity, derived from lymphocytes from the synovial tissue of rheumatoid arthritis (RA) patients and the peripheral blood of healthy individuals were examined for cross-reactivity with tissue and cellular antigens. The majority of IgM RF from RA patients (68%) showed reactivity with at least one component, and were frequently multispecific. A very significantly smaller proportion (28%) of the RF derived from healthy individuals demonstrated reactivities against tissue/cellular antigens (P = 0.004). RF from RA patients most commonly reacted with gastric glands (61%), nuclei (50%) and smooth muscle (50%), whereas RF from healthy donors most commonly reacted with gastric glands (20%), smooth muscle (16%), endothelium (16%) and glomeruli (16%). The most striking difference between the two groups was the reactivity with nuclear components, demonstrated by 50% of the RA RF, but by none of the healthy donor RF. As the two groups of antibodies share the same specificity for IgG Fc, but show differences in variable region segment usage, we investigated the relationship between VH gene usage and tissue/cell cross-reactivity using these antibodies and anti-blood group antibodies. Antibodies using VH3 or VH4 gene segments showed a very significantly greater frequency of tissue/cell reactions than those using VH1 (P = 0.0095 and 0.0004 respectively).

Variable region gene usage of human monoclonal rheumatoid factors derived from healthy donors following immunization.

A study was performed to compare the use of immunoglobulin V gene segments by rheumatoid factors (RF) produced in physiological responses following a defined antigenic stimulus, with RF produced in rheumatoid arthritis (RA) and RF produced as monoclonal (M)-components in certain lympho-proliferative diseases. A panel of 46 monoclonal RF was produced, using hybridoma techniques, from healthy individuals following immunization with foreign antigens (mis-matched red blood cells). A panel of previously characterized monoclonal RF from RA synovial tissues was extended to a total of 24 and included in the study. The variable heavy (VH) and variable light (VL) chain gene families used by these RF were determined using idiotypic markers and polymerase chain reaction amplification with VH-specific primers. The frequencies of expression of the various gene families was compared between the two groups, and compared with the published expression frequencies seen amongst M-component RF. The majority (87%) of RF from healthy donors were found with light chains using V gene segments of the V chi 3 family, in conjunction with VH gene segments belonging to the VH1, VH3 and VH4 families. The over-expression of V chi 3, together with the distribution of VH families, demonstrates close similarities with RF found as M-components in lympho-proliferative diseases. In contrast, RF from RA patients showed a predominant use of VH3 gene segments (82%) and an unbiased expression of V chi 3 segments (29% of the chi light chains). These data suggest that RF found as M-components are representative of RF used in normal physiological responses, but have undergone neoplastic or other transformation. RF found in the synovial tissue of RA patients appear to be driven by different mechanisms than RF seen in physiological responses in healthy individuals.

The binding of synovial tissue-derived human monoclonal immunoglobulin M rheumatoid factor to immunoglobulin G preparations of differing galactose content.

There are several sites on IgG Fc that have been reported to be the epitopes for binding rheumatoid factors (RF). It is now established that there are alterations in the oligosaccharides on IgG from patients with rheumatoid arthritis and it has been suggested that these changes may enhance immune complex and cryoglobulin formation. We have used a series of IgG preparations differing in their content of oligosaccharide chains lacking galactose from 18 to 86% to determine whether changes in sugar content affect the binding of rheumatoid factor. Five of 16 monoclonal rheumatoid factors prepared from synovial tissue, from patients with juvenile or adult rheumatoid arthritis, bound better to IgG which was deficient in galactose. Six of the 16 rheumatoid factors from the same patients bound independently of the galactose content. Four of the 16 rheumatoid factors could not be absolutely grouped in this manner but seemed to demonstrate a preference for agalactosyl IgG. One rheumatoid factor bound better to fully galactosylated IgG. There was an association between enhanced binding to galactose-deficient IgG and monoreactivity and a very strong association between the functional affinity of the rheumatoid factors and the dependent binding.

Mapping studies reveal unique epitopes on IgG recognized by rheumatoid arthritis-derived monoclonal rheumatoid factors.

We have used chimeric IgG antibodies and their genetically engineered variants prepared by a combination of site-directed mutagenesis and exon exchange to define the structure(s) on IgG recognized by monoclonal rheumatoid factor (RF) autoantibodies from rheumatoid arthritis (RA) patients. Nineteen RF produced by EBV-transformed cell lines from the synovium or blood of RA patients were analyzed. Their binding patterns differ significantly from those seen with RF obtained from patients with Waldenstrom's macroglobulinemia (WMac). Half of the RA-derived RF bound IgG1, 2, and 4, but not 3 (Ga specificity), the common pattern in WMac. However, heterogeneity in fine specificity within the Ga reactivity pattern was observed. Moreover, seven others bound all four IgG subclasses, a pattern observed for only one WMac-derived RF from a patient who also had RA. Three RF had subclass specificities unlike any observed with WMac-derived RF. Most RA-derived RF bound IgG at a discontinuous epitope comprised of residues from both the CH2 and CH3 H chain constant regions. However, unlike any WMac-derived RF, one RA-derived RF bound IgG in CH2, another in CH3, and a third at an undetermined site outside of the CH2-CH3 interface. Some RA-derived RF bound aglycosylated IgG4 less well than glycosylated IgG4, suggesting that the carbohydrate moiety was important in establishing their binding epitope in CH2. These studies demonstrate that the repertoire of RF expressed by RA patients contains some unique binding specificities for IgG epitopes not found among our panel of WMac-derived RF. Our results therefore call into question whether WMac-derived RF with their limited diversity are appropriate models for disease-related RF. In addition, RF with their multiple specificities can serve as probes of antibody structure.

Complementarity-determining region 2 is implicated in the binding of staphylococcal protein A to human immunoglobulin VHIII variable regions.

Staphylococcal protein A (SPA) has two distinct binding sites on human immunoglobulins. In addition to binding to the Fc region of most IgG molecules, an "alternative" binding site has been localized to the Fab region of human immunoglobulins encoded by heavy chain variable gene segments belonging to the VHIII family. Comparison of amino acid sequences of closely related SPA-binding and -non-binding proteins suggested that VHIII-specific residues in the second complementarity-determining region (CDR2) were likely responsible for SPA binding activity. Site-directed mutagenesis of a single amino acid residue in CDR2 converted an IgM rheumatoid factor which did not bind SPA to an SPA binder. These findings, therefore, locate a critical site involved in SPA binding to the CDR2 of human immunoglobulins encoded by VHIII family gene segments.

Differentiation of human lambda I variable regions with a monoclonal antibody to a defined, germ-line-encoded idiotope correlates with V lambda Ia chemical structure.

This article describes the characterization of a mouse monoclonal anti-idiotypic antibody (1H7B) prepared against a human monoclonal rheumatoid factor (RFSJ2) whose L chain utilized a V lambda I subgroup gene. The mAb 1H7B reacted with 6 of the tested 12 human V lambda I proteins, as well as with a newly produced lambda mAb whose V lambda gene usage has not as yet been determined. Because six of the 1H7B-positive mAb were heterogeneous with respect to both their VH gene utilization and antigenic specificity, and because mAb 1H7B did not react with any of the tested 43 kappa or 12 lambda proteins belonging to various subgroups other than V lambda I, mAb 1H7B appeared to be a V lambda I subgroup-specific reagent. The L chain specificity of mAb 1H7B was confirmed by Western blotting, and the inhibition of RFSJ2 binding to human Fc gamma by 1H7B provided additional evidence for the V region specificity of mAb 1H7B. The 11 sequenced V lambda I proteins used in this study were assigned to sub-subgroups by comparison with the previously published germ-line V lambda Ia, V lambda Ib, and V lambda Ic sequences. The mAb 1H7B only appeared to recognize V lambda Ia proteins as it reacted with five of the seven V lambda Ia, but not with three V lambda Ib or with one V lambda Ic protein. Because mAb 1H7B reacted with at least one V lambda Ia sequence in germ-line configuration, it appeared to be a marker for V lambda Ia sub-subgroup germ-line gene(s). The idiotope recognized by 1H7B was localized to the first framework region by inhibiting its binding to RFSJ2 with a synthetic peptide to the 11-24 amino acid region of V lambda Ia L chains. Comparison of the V lambda 1-24 region sequence of RFSJ2 with those of the two 1H7B-negative V lambda Ia mAb revealed a single amino acid difference at position 17, suggesting that the idiotope recognized by 1H7B encompassed this position.

Synovial IgG rheumatoid factors show evidence of an antigen-driven immune response and a shift in the V gene repertoire compared to IgM rheumatoid factors.

We have established IgG rheumatoid factor (RF)-secreting hybridoma cell lines from the synovial tissues of three patients from whom we have previously characterized several IgM RF. The IgG RF bind human and rabbit IgG and form intracellular complement-fixing complexes indicative of a self association process in vivo. Nucleotide sequence analysis revealed that two IgG RF used VHIII gene segments, while one used a VHI gene segment. The VL gene usage consisted of a V kappa 1, a V lambda 2 and a V kappa 4/V kappa 6 hybrid, confirming our previous findings that many different VL genes can contribute to RF specificity. Although the IgG RF used VH genes from the same families that dominated the IgM RF response, two of the actual gene segments employed were not found among the IgM RF. In contrast to IgM RF, which in general were not very mutated, the IgG RF showed somatic mutations characteristic of an antigen-driven immune response.

Human monoclonal IgG rheumatoid factors from the synovial tissue of patients with rheumatoid arthritis.

We have established IgG rheumatoid factor (RF)-secreting hybridoma cell lines from the synovial tissues of two patients (TS and SJ) with rheumatoid arthritis (RA) and one (KL) with the polyarticular form of juvenile rheumatoid arthritis (JRA). The IgG RF bind human and rabbit IgG and all except one form intracellular complement-fixing complexes indicative of a self-association process. The possibility of IgG RF for self association and immune complex formation is a feature thought to be important for the inflammatory processes in RA. Of the IgG RF-secreting cell lines established, three clones from patient SJ and one from patient KL are of the IgG1 kappa isotype while five clones from patient TS are of the IgG2 lambda isotype.

Nucleotide sequence analysis of rheumatoid factors and polyreactive antibodies derived from patients with rheumatoid arthritis reveals diverse use of VH and VL gene segments and extensive variability in CDR-3.

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of VH and VL gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain 'restrictions' do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of VH3 genes beyond that one would expect based on random utilization.

Clonally related IgM rheumatoid factors undergo affinity maturation in the rheumatoid synovial tissue.

Using hybridoma technology we established a panel of human monoclonal rheumatoid factors (RF) from the synovial tissues of two patients with rheumatoid arthritis (RA), and one patient with polyarticular juvenile RA. Nucleotide sequence analysis of the V regions of these RF indicates that two independently derived antibodies from one of the RA patients are clonally related. One of these antibodies appears to be close to germ-line configuration, whereas the other has accumulated a total of 36 substitutions in both H and L chains. Measurements of the affinity for human IgG of the two RF show that the extensively mutated RF has 100-fold higher affinity for IgG than the RF close to germline. These findings indicate that IgM RF in RA can undergo affinity maturation and suggest that certain RF may be the product of an Ag-driven immune response.

IgM rheumatoid factors in patients with rheumatoid arthritis derive from a diverse array of germline immunoglobulin genes and display little evidence of somatic variation.

Rheumatoid factors (RF) are present in the plasma of patients with rheumatoid arthritis (RA) although the site of synthesis of most of these antibodies is within the synovium. This report primary concerns RF of the IgM isotype. While a few of the RF derive from patients with systemic lupus erythematosus or from normal individuals, the remaining derive from the inflamed synovial tissue of patients with RA. Two RF are encoded by members of the VH1 gene family, 8 from the VH3 family and 2 from the VH4 family. Two polyreactive antibodies derive from the VH3 family and 2 come from the VH4 family. This distribution is not fundamentally different from the distributions seen in a large array of autoantibodies and antibodies to external antigens. Similarly, the light chains derive from most of the known kappa and lambda VL families. It is hard to escape the preliminary conclusion that gene segments from virtually any light chain variable region can contribute to RF or polyreactive antibody structures. Most IgM RF and polyreactive antibodies are direct copies of germline genes in one of their polypeptide chains or at most are 2 nucleotides away in one of their chains from a known germline gene.

Human monoclonal antibodies against blood group antigens preferentially express a VH4-21 variable region gene-associated epitope.

An anti-idiotypic antibody has been raised which recognizes human immunoglobulins with cold agglutinin activity of anti-I/i specificity. The pattern of reactivity of the antibody indicates that the structural basis for the epitope is located in the VH4-21 gene segment of the VHIV family, which is preferentially utilized by these cold reactive antibodies. Using this antibody, epitope expression was investigated in a panel of 72 human monoclonal allo-antibodies specific for human blood group antigens, as compared with a control panel of 39 randomly selected human monoclonal IgM antibodies of unknown specificities. The anti-blood group panel included 44 IgM and 28 IgG monoclonal antibodies against a variety of blood group antigens including the A antigen, Rh C, c, D, E, e, G antigens, and the Kidd antigens Jka and Jkb. The epitope was expressed by 64% (28/44) of the IgM anti-blood group antibodies and by 21% (6/28) of the IgG antibodies, but by only 7.7% (3/39) of the control IgM antibodies. These data indicate that the human alloimmune response to blood group antigens is biased in the use of VH gene families, with a preference for the VH4-21 gene segment of the VHIV family, or closely related gene segments. The fact that this mirrors the findings for the autoimmune cold agglutinins suggests a link in immunoglobulin gene usage between antibodies against structurally diverse antigens on the red cell surface.