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R Jefferis

Publications and source records attributed to R Jefferis.

At least 91 records · Page 5Linked to original sources
PubMed1991

Quantification of cross-reactive idiotype-positive rheumatoid factor produced in autoimmune rheumatic diseases. An indicator of clonality and B cell proliferative mechanisms.

The aetiology of sustained autoantibody production in human autoimmune diseases is unknown. Evidence for structural similarities and common clonal origin among autoantibodies have been demonstrated through the expression of cross-reactive idiotype (CRI). In the present study we use four monoclonal antibodies (MoAbs) with specificity for non-overlapping CRI on human rheumatoid factor (RF) autoantibodies to define the structural features of polyclonal RF characteristic of patients with autoimmune rheumatic diseases. The pattern of CRI expression in the serum of 12 patients with rheumatoid arthritis (RA), eight with systemic lupus erythematosus (SLE) and 20 with primary Sjögren's syndrome and 34 normal individuals were determined in parallel with the level of IgM RF, IgA RF and autoantibodies to the cellular antigens SS-A, SS-B, Sm, nRNP and dsDNA and cryoglobulins. The results demonstrate significant elevation in the level of IgM and IgA expressing VHI (G6 and G8) and VHIII (B6 and D12) associated CRI in the serum of patients with autoimmune rheumatic diseases compared with normal individuals. These increases paralleled, but did not equal the increase in the level of immunoglobulins and RF. However, when expressed as proportion of immunoglobulin, only the VHI-associated CRI were significantly elevated in patients compared with normal individuals. The proportion of IgM RF expressing the VHI-associated CRI was higher in patients with Sjögren's syndrome compared with SLE and RA. Furthermore, the proportion of IgA RF expressing the G6 CRI was higher than G6+ IgM RF. These findings imply that different mechanisms contribute to RF production in autoimmune diseases. It is suggested that polyconal B cell activation is likely to be a contributing mechanism. However, such polyclonal activation is unlikely to be random since a selective elevation in the level of specific autoantibodies and VHI-associated CRI is observed. Furthermore, the data demonstrate that a proportion of autoantibodies in autoimmune diseases are immunoglobulin germline gene encoded. This is more evident in some patients with primary Sjögren's syndrome, where RF is likely to be oligoclonal or monoclonal in individuals with lymphoproliferation.

Adult↗
PubMed1991

Structure-function relationships in human immunoglobulins.

The majority of monoclonal antibodies having potential for in vivo diagnostic and therapeutic applications, in man, are of mouse or rat origin. They need to be "humanized" to prevent rejection by the human immune system and, where indicated, to activate host effector mechanisms. Genetic engineering techniques have made all nine human immunoglobulin isotypes available, but we do not have a sufficient understanding of their functional activities to determine which isotype may be optimal for a given application. This review discusses the major effector systems identified and recent studies directed at defining the structural basis for immunoglobulin recognition by Fc receptors and complement. Site-directed mutagenesis has demonstrated that ligand-binding sites can be modulated by single amino acid substitutions. Additionally, it has been shown that glycosylation is essential for full functional activity. It appears that there is a subtle interplay between the polypeptide and oligosaccharide structures to generate these ligand binding sites. Taken together, these findings suggest that it will be possible to generate "customized" antibody molecules having a pre-determined profile of effector functions.

Complement C1↗
PubMed1990

A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins.

Quantitative oligosaccharide profiles were determined for each of 18 human IgG paraproteins representing the four subclasses. Each paraprotein exhibits a unique profile that may be substantially different from that observed for polyclonal IgG. The IgG2 and some IgG3 proteins analysed exhibit a predominance of oligosaccharide moieties having galactose on the Man(alpha 1----3) arm rather than the Man(alpha 1----6) arm; it was previously held that galactosylation of the Man(alpha 1----6) arm is preferred, as observed for IgG1, IgG4 and polyclonal IgG. An IgG4 protein is reported that has galactosylated Man(alpha 1----3) and Man(alpha 1----6) arms on both Fc-localized carbohydrate moieties; previous findings suggested that such fully glycosylated structures could not be accommodated within the internal space of the C gamma 2 domains. Unusual monoantennary oligosaccharides present in IgG2 and IgG3 proteins were isolated and their structures determined.

Carbohydrate Sequence↗
PubMed1990

Human monoclonal rheumatoid factors derived from the polyclonal repertoire of rheumatoid synovial tissue: incidence of cross-reactive idiotopes and expression of VH and V kappa subgroups.

A panel of 14 human monoclonal and monoreactive IgM rheumatoid factors (RF) derived from the synovial tissue of rheumatoid arthritis (RA) patients was studied for the expression of cross-reactive idiotopes (CRI) and variable heavy (VH) and variable kappa (V kappa) subgroups. Four of the twelve kappa RF expressed light chains of the V kappa III subgroup. Three of these were characterized as belonging to the V kappa IIIb sub-subgroup, and expressed the V kappa IIIb associated CRI, 17.109. None of the RF expressed the V kappa IIIa-associated CRI, 6B6.6. One of the fourteen RF expressed the VH-associated CRI, G6, and five expressed either or both the VHIII-associated CRI, B6 and D12. Seven RF bound to protein A (SpA), which indicates the expression of VHIII subgroup V regions. Together the data indicated that 9/11 RF express VHIII regions and 2/11 express VHI regions. There was no obvious correlation between V region usage or CRI expression and fine specificity of the RF for human IgG subclasses. These data indicate a difference in V gene usage by RF derived from RA patients compared with RF paraproteins derived from non-RA patients. There is not a bias towards variable chains of the V kappa III subgroup, but a marked preference for variable heavy chains of the VHIII subgroup is seen. Further studies may elucidate the pathological significance of these findings in RA.

Animals↗
PubMed1990

A highly conserved conformational idiotope on human IgM rheumatoid factor paraproteins of the Wa cross-reactive idiotype family defined by a monoclonal antibody.

Human IgM rheumatoid factors have been classified into major (Wa) and minor (Po) idiotypically cross-reactive families on the basis of reactivity with polyclonal anti-idiotypic antisera. Extensive structural studies have revealed that RFs from the Wa CRI family have homologous light chains. The structural basis of this CRI however is not known. In this study we have defined a conformational idiotope requiring the association of the VH and VL of a number of RF from the Wa CRI family for its expression and which thus comprise part of the Wa CRI. A murine monoclonal antibody, G8 specific for the idiotope reacted with 67% of RFs from the Wa CRI family. G8 also reacted with one IgG2K paraprotein with unknown specificity suggesting that the idiotope per se does not confer RF reactivity. Comparative studies of G8 expression relative to previously described sequence-dependent heavy and light chain associated idiotopes suggest that G8 recognises a separate determinant. Furthermore, the G8 idiotope is found on RFs expressing the VHI subgroup of heavy chain. Study of G8 expression in polyclonal IgM in normal sera demonstrated that the idiotope is widely expressed in the population. However, significantly higher levels of IgM bearing G8 were detected in the sera of patients with rheumatoid arthritis.

Amino Acid Sequence↗
PubMed1990

Molecular definition of interaction sites on human IgG for Fc receptors (huFc gamma R).

Evidence from several experimental approaches allows us to conclude that the primary amino acid sequence of the lower hinge region (residues 234-237) of human IgG molecules determines recognition by human Fc gamma RI, Fc gamma RII and Fc gamma RIII. Glycosylation of the CH2 domain is also essential, although the carbohydrate is not accessible for direct interaction with ligands. The role of the carbohydrate moiety may be to maintain a protein conformation that allows accessibility to amino acid side chains essential for ligand recognition and binding. It appears logical that the evolutionarily-related Fc gamma R molecules should interact with overlapping non-identical sites on the IgG molecule.

Amino Acids↗
PubMed1990

The incidence of a new human cross-reactive idiotype linked to subgroup VHIII heavy chains.

Cross-reactive idiotypes (CRI) on human rheumatoid factors (RF), which are identified by murine monoclonal antibodies (mAb), have proved useful in defining both the incidence and the structural characteristics of these autoantibodies. In this study, a new murine anti-idiotypic reagent, mAb B6, has been used to identify and define the expression of a distinct heavy chain CRI. The B6 CRI was found on 20% of monoclonal IgM (16 of 81), but on only 5% of monoclonal IgA (1 of 20) and on no monoclonal IgG. In addition, this CRI was expressed exclusively on a subset of Ig derived from the VHIII protein variable region subgroup. In immunoblotting experiments, the mAb B6 bound directly to the heavy (H) chains of CRI positive proteins. The B6 CRI was found frequently on monoclonal IgM-RF molecules, and the mAb B6 could inhibit the binding of the RF to its IgG antigen. It was also demonstrated that Staphylococcus aureus protein A (SpA), which has recently been shown to bind to the F(ab) region of VHIII molecules, could block the interaction of some B6 CRI positive IgM to the anti-CRI. These experiments suggest that the B6 CRI is a marker for one or a few VHIII genes and that it is expressed commonly on IgM paraproteins, many of which have RF activity.

Antibodies, Monoclonal↗
PubMed1990

A protein structural change in aglycosylated IgG3 correlates with loss of huFc gamma R1 and huFc gamma R111 binding and/or activation.

Glycosylated chimeric mouse-human anti-NIP IgG3 antibody produced by growth of the J558L mouse B cell plasmacytoma is characterised with respect to the single carbohydrate chain at Asn-297 in the CH2 domain indicating that the mouse cell glycosyl transferases dictate the pattern of glycosylation rather than the human CH region of the heavy chain. Additionally, three unusual alpha-galactose-containing oligosaccharides are reported. Only the Fc region has detectable carbohydrate. Aglycosylated anti-NIP IgG3 antibody has been produced by cell growth in the presence of the antibiotic tunicamycin. Functionally, whilst the glycosylated intact IgG3 interacts with human Fc gamma R111 expressed on human killer (K) cells to trigger antibody-dependent cellular cytotoxicity the aglycosylated intact IgG3 fails to trigger cell lysis, localising the site on IgG for triggering human Fc gamma R111 mediated functions to the CH2 domain. The monomeric aglycosylated trypsin Fc fragment inhibits human Fc gamma R1 recognition by U937 cells 115-fold less well (K50 = 2 microM) than does glycosylated Fc (K50 = 17 nM), confirming that aglycosylation disrupts the site for human Fc gamma R1 within the CH2 domain and indicating that the trypsin Fc fragments reflect the functional properties of the intact IgG glycoforms. Structurally, 1H NMR shows that the absence of carbohydrate at Asn-297 results in a small and localised protein structural change in the vicinity of the reporter group His-268 within the CH2 domain. The site on IgG for triggering human Fc gamma R111 mediated functions is then localised to the vicinity of His-268. The profound impact of aglycosylation on human Fc gamma R1 recognition implies structural disruption of the proposed site for human Fc gamma R1 in the lower hinge region of IgG (residues 234-239), proximal to His-268.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1990

Analysis of immunoglobulins secreted by hybridomas derived from rheumatoid synovia.

Twenty-six IgG-secreting and eight IgM-secreting hybridomas were derived from the synovia of two patients with rheumatoid arthritis (RA). Hybridomas were obtained by fusing a heteromyeloma cell line, SPAZ-4 with synovial mononuclear cells that were not deliberately stimulated in vitro. Over 96% of the IgG-secreting hybridomas produced antibodies which belonged to the IgG1 subclass and showed lambda light chain predominance; the latter was not seen in IgM antibodies, where kappa light chains dominated by 3:1. All IgG antibodies were cationic. Synovial B cells were not exposed to extrinsic stimuli prior to fusion, therefore these results reflect the state of B cell activation and differentiation in vivo. Our results indicate that IgG-secreting B cells in the RA joint are under a selective influence which is, as yet, unidentified. One out of eight IgM-secreting and two out of 26 IgG-secreting hybridomas produced rheumatoid factors (RF). The IgM-RF specificity for IgG heavy chain subclasses was determined and showed that the monoclonal bound to IgG1, IgG2 and IgG4 but not IgG3 with exception of IgG3 Goe of the G3m (st) allotype, a profile typical of specificity for the Ga epitope. This monoclonal also distinguished a determinant in the Fc region of human IgG which was not present in rabbit IgG. The overall frequency of RF-secreting hybridomas we observed indicates that B cells committed to RF production in the synovium of a seropositive and a seronegative RA patient is below 10%.

Adult↗
PubMed1990

Selective IgG subclass deficiency: quantification and clinical relevance.

Each of the four human IgG subclasses exhibits a unique profile of effector functions relevant to the clearance and elimination of infecting microorganisms. The quantitative response within each IgG subclass varies with the nature of the antigen, its route of entry and, presumably, the form in which it is presented to the immune system. This results in antibody responses to certain antigens being predominantly or exclusively of a single IgG subclass. An inability to produce antibody of the optimally protective isotype can result in a selective immunodeficiency state. This is particularly apparent for responses to certain bacterial carbohydrate antigens that are normally of IgG2 isotype. A failure to produce the appropriate specific antibody response may result in recurrent upper and/or lower respiratory tract infection. Careful patient investigation can identify such deficiencies and suggest appropriate clinical management. In this review we outline the biology and clinical relevance of the IgG subclasses and summarize current rational treatment approaches.

Antigens, Bacterial↗
PubMed1990

The antibody repertoire of early human B cells. III. Expression of cross-reactive idiotopes characteristic of certain rheumatoid factors and identifying V kappa III, VHI, and VHIII gene family products.

Our previous studies have shown that a high frequency of Epstein-Barr virus (EBV)-immortalized cord blood (CB) and fetal liver (FL) clones produce IgM antibodies which display extensive autoreactivity for IgG Fc (rheumatoid factor, RF). To investigate further the repertoire of these early B cells, we have examined the expression of CRI associated with RF paraproteins in relation to antibody specificity and polyreactivity. CRI were detected by ELISA and/or flow cytometry using a panel of well-characterized monoclonal antibodies defining idiotopes associated with particular V kappa and VH gene family products and raised against Fc-specific paraproteins. Many of the CRI were expressed by these clones, suggesting that they may be markers of early B cells. The presence of the CRI was not always associated with Fc specificity. Three of eight CB/FL clones expressed the V kappa III subgroup of light chains, and two of these expressed the V kappa III sub-subgroup associated CRI, 17-109. These two clones reacted with IgG Fc, and one also bound to single-stranded DNA. The VHIII-associated idiotope D12 was expressed on IgM from 4 out of 9 FL and 5 out of 12 CB clones. D12 and B6 (also a VHIII-associated CRI) were coexpressed in 4 out of 5 CB clones but not in the four FL clones. Seven out of nine clones expressing these idiotopes were polyreactive, and five had Fc-binding activity. Three of the 12 CB clones expressed the VHI-associated conformational idiotope G8. One of 20 CLL clones expressed both B6 and D12, and another expressed both 17-109 and the VHI-associated G6 and G8 idiotopes. Taken together, these data provide evidence for the frequent usage, in early B cells, of V kappa subgroups and VH-associated idiotopes of RF paraproteins. The expression of these CRI was not a prerequisite for binding to IgG Fc, but there was a frequent association of these idiotopes with it. Differences in expression of CRI between CLL and early B-cell clones may suggest differences in the pattern of VH usage between these subsets of B cells.

B-Lymphocytes↗
PubMed1990

Evaluation of monoclonal antibodies with putative specificity for human IgG allotypes.

Fourteen monoclonal antibodies (MAbs) of putative specificity for human IgG allotypes and isoallotypes were evaluated for reactivity and specificity in 8 different assay systems. The study showed that the MAbs tested could be classified into 1 of 4 groups: those exhibiting allotypic specificity regardless of the assay system, allotypic specificity dependent on the assay system, isoallotypic specificity, and those showing neither allotypic nor isoallotypic specificity. These observations were presumably dependent on antigen presentation, epitope integrity and/or antibody multispecificity. For the G1m(a), G1m(f), G1m(z), G3m(g) and G3m(u) specificities, MAbs have been produced which can be used for routine typing purposes in defined haemagglutination and enzyme-linked immunosorbent assay systems. MAbs are also available that show 'non-g' isoallotypic specificity.

Animals↗
PubMed1990

Qualitative and quantitative expression of VHI associated cross reactive idiotopes within IgM rheumatoid factor from patients with early synovitis.

Monoclonal rheumatoid factors (RFs) of the major Wa cross reactive idiotype group have been shown to express exclusively VKIII subgroup light chains and VHI subgroup heavy chains. A VKIII associated cross reactive idiotope (CRI) (17-109), however, was shown not to be exclusively expressed on IgM paraproteins having rheumatoid factor activity or to be present at increased levels in the sera of patients with rheumatoid arthritis (RA). Three VHI associated CRIs have been defined with monoclonal antibodies and quantitative studies of their representation are reported, together with VKIII, in IgM and IgM RF isolated from the sera of patients with early synovitis, some of whom progressed to classical RA. The results show (a) the probed CRIs were expressed predominantly on IgM RF rather than on non-RF IgM; (b) 5-10% of IgM RFs from patients with classical RA expressed the CRIs, but this represented a lower proportion of IgM RFs than observed for normal individuals or patients with self limiting synovitis; (c) VKIII light chains were highly associated with IgM RFs rather than non-RF IgM (75% and 25% respectively). It is suggested that the CRIs probed are markers for germline gene encoded antibodies or sequences resulting from minimal mutation of germline genes. The lowered proportion of RFs expressing CRIs in RA may therefore be evidence of polyclonal activation or specific antigenic stimulation, or both, resulting in maturation of the RF response with recruitment of further VH genes or extensive mutation of germline genes. These studies show that monoclonal RFs are relevant models of RF produced in RA and that the repertoire of RF autoantibodies may be encompassed within a small number of CRI expressing families.

Cross Reactions↗
PubMed1989

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.

Antibodies, Monoclonal↗
PubMed1989

Interaction of human IgG chimeric antibodies with the human FcRI and FcRII receptors: requirements for antibody-mediated host cell-target cell interaction.

Chimeric monoclonal antibodies (McAb), specific for the hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP), expressing human IgG1, IgG2, IgG3 and IgG4 subclass constant domains, have been examined for their ability to interact with the human FcRII receptor. Human red blood cells (RBC) sensitized by each of these McAbs have been assayed for their ability to form rosettes with the human histiocytic lymphoma U937 cell line, human B cell line Daudi and erythroblastoid K562 cell line. IgG1 and IgG3 sensitized RBC formed significant rosettes with the FcR- and FcRII+ Daudi and K562 cell lines, the percentage of cells forming rosettes being directly proportional to the degree of sensitization of the RBC. Bromelin treating Daudi cells did not alter this pattern of reactivity, whereas bromelin treated FcRI+ and FcRII+ U937 cells formed significant resettes with IgG1, IgG3 and IgG4 sensitized RBC, demonstrating a difference in the IgG subclass specificity between human FcRI and FcRII. Murine IgG2b anti-NIP sensitized RBC did not form rosettes with any cell line tested; however, RBC sensitized by some members of a panel of murine IgG1 McAb, specific for the glycophorin A molecule, were able to form rosettes with Daudi, U937 and K562 cells. This interaction was enhanced by bromelin treating the Daudi or U937 cells and can be correlated to the disposition of the epitopes recognized, relative to the target cell membrane, those McAbs recognizing epitopes furthest from the RBC surface being most effective in interacting with FcRII. The data are interpreted in terms of a simple model for antibody-mediated cell--cell interaction.

Antibodies, Monoclonal↗
PubMed1989

Distinctive role of IgG1 and IgG3 isotypes in Fc gamma R-mediated functions.

Polyclonal and monoclonal anti-Rh (D) antibodies of IgG1 and IgG3 subclass were evaluated for their capacity to sensitize erythrocytes and (i) to trigger monocyte and K-cell mediated antibody-dependent cellular cytotoxicity (ADCC); (ii) to mediate binding to monocyte and lymphocyte Fc gamma R; (iii) to stimulate phagocytosis by monocytes. All antibodies were equally effective in mediating monocyte or activated U937 cell ADCC but IgG1 was more active than IgG3 in K-cell mediated ADCC. IgG3-sensitized erythrocytes inhibited IgG1-induced lysis, suggesting that each subclass engages the same Fc gamma R receptor but that lysis requires a further 'signal' that the IgG3 molecule can not deliver. Two monoclonal IgG3 anti-D antibodies were shown to have higher binding (two times) and phagocytic (three times) indices than IgG1 antibody for monocytes; similar differences were observed for polyclonal IgG1 and IgG3 antibodies. The same pattern was observed in an EA rosette assay when a total lymphocyte population was used; however, this difference was not seen with a B-cell depleted (T+ null cell) lymphocyte population.

Antibodies, Monoclonal↗