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Biomedical subjects

R Jefferis

Publications and source records attributed to R Jefferis.

At least 73 records · Page 4Linked to original sources
PubMed1993

Modulation and high frequency expression of autoantibody-associated cross-reactive idiotypes linked to the VHI subgroup in CD5-expressing B lymphocytes from patients with chronic lymphocytic leukaemia (B-CLL).

Leukaemic B cells from patients with chronic lymphocytic leukaemia (B-CLL) are known to express the pan T-cell marker CD5 and a restricted set of immunoglobulin (Ig) variable region heavy (VH) and light (VL) chains encoded by germline or minimally mutated germline genes. We have studied surface expression of certain VH and VK gene products on peripheral blood B lymphocytes from 23 patients with B-CLL, using a panel of monoclonal antibodies (MoAbs) recognizing germline encoded cross-reactive idiotypes (CRI) associated with VHI (G6, G8), VHIII (B6, D12), VKIIIb (17-109) and an epitope linked to the VKIII light chain subgroup (C7). While only 1.7-3.2% of peripheral blood B lymphocytes from normal individuals expressed the VHI-associated CRI (VHI-CRI), these CRI were expressed on virtually all the leukaemic B cells from 17-22% of the CLL patients. The VHIII-associated CRI (VHIII-CRI), however, were found in 8.5-13% of the CLL B cells. Fifty per cent of the IgMK-expressing CLL cells (7/14) expressed the VKIII light chain subgroup of which only one expressed the VKIIIb-associated CRI (VKIIIb-CRI), 17-109. The anti-VHI-associated CRI antibodies were used to study their regulatory effect on in vitro Ig synthesis by the leukaemic cells. A significant suppression of spontaneous and mitogen-driven Ig production was observed in all cases studied. These results demonstrate an over-expression of VHI and VKIII gene products in B-CLL and suggest that B cells expressing these CRI are particularly susceptible to lymphoproliferative stimuli. The anti-CRI antibodies can be used to modulate Ig production by the leukaemic cells and may be of potential value for selective immunotherapy.

Aged↗
PubMed1993

Glycosylation of human IgG subclass and mouse IgG2b heavy chains secreted by mouse J558L transfectoma cell lines as chimeric antibodies.

Oligosaccharide profiles for monoclonal chimeric mouse-human and mouse IgG2b antibodies produced in the mouse J558L cell line have been determined. These chimeric antibodies share the same mouse light chain and VH region and have a single, complex, oligosaccharide moiety attached to residue 297 of the heavy chain. Since the panel of chimeric antibodies included each of the human IgG subclasses and a set of IgG3 proteins having single residue differences, it was possible to evaluate the contributions of the protein template, the cell line, and the culture conditions to glycosylation. The study shows that the J558L cell line glycosylates human heavy chains with oligosaccharides characteristic of mouse immunoglobulins. This includes a galactose alpha 1-->3 galactose structure not found in human IgG and for which most humans have naturally occurring "anti-Gal" antibodies. The extent of galactosylation was very dependent on the culture conditions and was maximal for still culture. The findings have significance for the production of antibodies for in vivo administration.

Animals↗
PubMed1992

Immunoaffinity chromatography in biorecovery: an application of recombinant DNA technology to generic adsorption processes.

The constant region of human kappa light chain (Ck) was linked to Escherichia coli beta-galactosidase, using standard molecular cloning techniques. The binding of Ck-beta-galactosidase fusions to a number of different murine monoclonal antibodies, specific for Ck, was improved by the insertion of spacers between Ck and beta-galactosidase: a cleavable linker was then introduced. Over-expressed Ck-beta-galactosidase fusion protein was purified using monoclonal antibodies immobilised on Sepharose 4B. Elution conditions were found that maintained beta-galactosidase activity so purified enzyme could be released on breaking the cleavable linker. A number of practical problems associated with maintaining stable fusion proteins and immunoaffinity column performance were identified.

Adsorption↗
PubMed1992

Specificity and idiotope expression of IgM produced by CD5+ and CD5- cord blood B-cell clones.

Epstein-Barr virus (EBV)-immortalized monoclonal B-cell lines were established from CD5+ and CD5- cord-blood B cells. IgM from many of both CD5+ and CD5- clones reacted with IgG-Fc, ssDNA, and a variety of other autoantigens. More CD5+ B cells that used light chains of the kappa isotype reacted with IgG-Fc and ssDNA than kappa-bearing CD5- B cells. Because many of the clones reacted with IgG-Fc, they were analyzed for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor and cold agglutinin paraproteins using murine antibodies (mAb) recognizing V kappa and VH subgroup-associated determinants. Expression of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17.109 mAb was expressed at significantly higher frequency (32%; p less than 0.05) and IgM antibodies derived from the CD5+ compared with the CD5- clones (5%). Both CD5+ and CD5- clones expressed the RF paraprotein-associated idiotope recognized by G8 mAb to the same extent. Similar results were obtained using binding to SpA as a marker of VH III family usage. Furthermore, no differences in frequency of expression of RF paraprotein-associated idiotopes recognized by B6 and/or D12, and characteristic of some antibodies using VH III family genes, were found between the CD5+ and CD5- populations. Although a higher than expected frequency of VH IV-gene expression was demonstrated (around 30%) in both CD5+ and CD5- cells, there were differences in expression of CRI recognized by mAb Lc1 and R2.1A2 with specificities for two VH IV subfamilies. While some CD5+ and CD5- clones were identified in which their IgM reacted with mAb Lc1, only CD5+ clones were recognized by another mAb R2.1A2. Analysis of the relationships between antigen specificities and V kappa- and VH-family gene usage indicated that auto- or polyreactivity was not associated with V kappa III nor any particular VH family. The higher frequency of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17-109 in the CD5+ clones and the association of CD5+ B cells with the VH IV subfamily recognized by mAb R2.1A2 and 9G4 may suggest that CD5+ B cells in cord blood are expanded as a result of recruitment within the fetal environment.

Animals↗
PubMed1992

A human monoclonal IgA rheumatoid factor using the VkIV light chain gene.

A human hybridoma stably secreting IgA rheumatoid factor (RF) was produced by cell hybridization with peripheral blood lymphocytes of a patient with rheumatoid arthritis. The RF was of the IgA1 isotype with kappa-light chains and was useful for standardization or specificity controls in class-specific RF assays. RF activity was detected only when the IgA molecular were in a polymeric state, and could be measured by enzyme linked immunosorbent assay as well as in conventional agglutination based tests. The RF had the modified Ga fine specificity described previously for several RFs and for protein A. The immunoglobulin V genes used were isolated and sequenced. The light chain was encoded by the VkIV gene rearranged to Jk2; compared to the published VkIV germ line gene there was 90% nucleotide homology. The heavy chain gene used belonged to the VHI family and was rearranged to JH4. Comparisons with published sequences revealed 90% homology with the recently characterized VH gene expressed by RF-TS3, a rheumatoid synovia RF hybridoma.

Aged↗
PubMed1992

Multiple binding sites on the CH2 domain of IgG for mouse Fc gamma R11.

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.

Animals↗
PubMed1992

Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc gamma receptor.

In the present study 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific antibodies were compared for induction of antibody dependent lysis of NIP-derivatised red blood cells effected by pre-stimulated U937 or HL-60 cells and by K cells. The chimaeric antibodies have heavy chains corresponding to human IgG subclasses 1-4, and include site-directed mutants of IgG3 as well as the aglycosylated form of IgG3; a mouse IgG2b antibody and a site-directed mutant IgG2b were also examined. rIFN stimulated U937 or HL-60 cells express increased levels of Fc gamma R1 compared to unstimulated cells; PMA stimulated HL-60 and U937 cells express an increased level of Fc gamma R11 compared to unstimulated cells; K cells express Fc gamma R111. Using these effector cell populations and the target cells mentioned above, we have compared anti-NIP antibodies with different heavy chain constant domains for their ability to induce ADCC through human Fc gamma R1, Fc gamma R11 and Fc gamma R111. The results suggest that all three human Fc gamma receptors appear to recognise a binding site on IgG within the lower hinge (residues 234-237) and trigger ADCC via this site, but that each receptor sees this common site in a different way. The possibility that other amino acid residues also participate in the binding/triggering site(s) cannot be excluded.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1992

Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of the 2nd IUIS/WHO collaborative study.

Following the 1st IUIS/WHO Collaborative Study of monoclonal anti-IgG subclass antibodies, a panel of WHO Specificity Reference Reagents (SRR) was established [Jefferis, R., et al. (1985) Immunol. Lett., 10, 223]. At the time, the hope was expressed that further reagents particularly for IgG2, and other allotypic specificities would become available which could be applied in a wide range of assay protocols. The 2nd study reports the evaluation of nineteen anti-subclass and seven anti-allotype monoclonal antibodies. The anti-IgG1 antibody HP6187 was equivalent in performance to the SRR. Others, that were not of the mouse IgG1 isotype, may be useful for particular applications. The anti-IgG2 antibody HP6200 could be a valuable addition to the WHO SRR; it is specific for an epitope in the Fab region but does not have the light chain bias of HP6014. Antibodies of putative allotype specificity exhibited the claimed specificity when used within protocols similar to those employed by the originating laboratory. It appears to be inherent in the nature of the epitopes (allotopes) recognized that it will take several years before reagents applicable to a wide range of techniques will become available.

Antibodies, Anti-Idiotypic↗
PubMed1992

Repertoire of CD5+ and CD5- cord blood B cells: specificity and expression of VH I and VH III associated idiotopes.

Epstein-Barr (EBV)-immortalized B cell clones were established from CD5+ and CD5- cord blood B cells separated by flow cytometry. We have previously shown that IgM from many of the clones was polyreactive, exhibiting reactivity with a number of autoantigens. In this study, IgM produced by the clones was analysed by MoAb for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor paraproteins and from defined VH and V kappa subgroups of immunoglobulin heavy and light chains. IgM produced by clones established from CD5+ and CD5- B cells expressed the VH I associated idiotope G8. Furthermore, IgM produced by both sets of clones exhibited a similar frequency of VH III heavy chain subgroup expression, as determined by reactivity with staphylococcal protein A (SpA) and VH III-associated CRI expression (B6 and/or D12). In contrast, expression of the V kappa III-associated 17.109 CRI was significantly higher in IgM antibodies produced by clones established from CD5+ compared with the CD5- clones (32 versus 5%: P less than 0.05). Analysis of the VH and VL subgroup expression by IgM produced by the CD5+ and CD5- cord blood clones, and their autoantigen reactivity profile did not reveal restriction or selection within CD5+ and CD5- populations. However, our data suggest that differences may exist in the expression of certain germ-line genes between CD5+ and CD5- cord blood B cells and might indicate an expansion of CD5+ B cells within the fetal environment.

Animals↗
PubMed1992

Analysis of monoclonal rheumatoid factors obtained from the B-cell repertoire in rheumatoid arthritis.

We have sought to determine whether rheumatoid factors (RF) produced in rheumatoid arthritis (RA) were different from physiological RF produced in normal, healthy adults. RF-secreting clones were established following Epstein-Barr virus (EBV) stimulation of peripheral blood lymphocytes. Ten RF-secreting clones were established from seven RA patients and 16 from six healthy controls. All monoclonal RF (MRF), except two in each group, were monoreactive and ten of these were shown to have low to medium affinity for IgG,Fc, irrespective of their origin. A majority (74%) of the MRF bound to protein A, indicating that genes of the VHIII family were preferentially used for synthesizing these autoantibodies. The expression of cross-reactive idiotypes (CRI) by the MRF did not allow distinction between those derived from RA patients and controls. The VHI-associated CRI G8 and VHIII-associated CRID12 were expressed at low frequency in both panels of RF. These CRI have been shown to be expressed at high frequency in RF paraproteins. However, the idRQ idiotype was expressed within both panels of RF. A possible distinction between polyreactive and monoreactive MRF appeared to be light chain usage since all (four) polyreactive RF used lambda chains while the normal kappa/lambda ratio was observed for monoreactive RF. The frequency of EBV-activated cells secreting IgM bearing CRI or secreting RF was determined and showed that CRI expression occurred with a higher frequency than did RF, suggesting a dissociation between CRI expression and RF activity.

Animals↗
PubMed1991

Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG.

Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII.

Antigens, Differentiation↗
PubMed1991

Selective expression of a VHIV subfamily of immunoglobulin genes in human CD5+ B lymphocytes from cord blood.

Human B lymphocytes expressing the CD5 surface antigen (CD5+ B cells) constitute a subset capable of producing polyspecific antibodies recognizing a variety of self antigens. The repertoire of antibodies produced by CD5+ and CD5- B cells is different. However, it is not yet established whether this distribution is reflected in different immunoglobulin variable region gene (IgV) use. Rearrangement of heavy chain IgV (IgVH) genes represents one of the first identifiable stages in the maturation of B cells, and occurs in a developmentally ordered fashion. The repertoire of IgVH gene expression is highly restricted during fetal life but diversifies progressively after birth. A high frequency of VH gene use from the relatively small VHIV gene family has previously been demonstrated in human fetal liver B cells. In the present study, 102 B cell lines established by Epstein-Barr Virus-transformation of separated CD5+ and CD5- cord blood B cells, were examined for the frequency of IgV expression using monoclonal antibodies to cross-reactive idiotypes (CRI). The results demonstrate a relatively high frequency of VHIV gene use (30%) in B cells from cord blood. Furthermore, two mutually exclusive CRI associated with distinct subgroups of the VHIV family are segregated in their association with either subset of B cells. One CRI is exclusively expressed in lines established from CD5+ B cells while the other is associated with lines established from CD5- B cells.

Antigens, CD↗
PubMed1991

Assay restriction profiles of three monoclonal antibodies recognizing the G3m(u) allotype. Development of an allotype specific assay.

Three monoclonal antibodies raised against a purified human IgG3 paraprotein were found to exhibit a restriction profile for IgG3/G3m(u) and pan-IgG specificity which was dependent on the assay system. When adapted to an IgG3 subclass capture ELISA, all three McAbs discriminated between paraproteins expressing G3m(u) and antithetical markers G3m(st). One of the antibodies (PNF69C) was selected and conditions were optimised for Gm typing purposes. Using this system G3m(u) could be detected on captured IgG3 derived from human sera. This system may prove useful in the elucidation of Gm allotype profiles.

Animals↗
PubMed1991

Expression of VHIII-associated cross-reactive idiotype on human B lymphocytes. Association with staphylococcal protein A binding and Staphylococcus aureus Cowan I stimulation.

It has been demonstrated that staphylococcal protein A (SPA) has an "alternative" binding site with specificity for human Ig H chain V region of the VHIII subgroup. Because the major mitogenic component of Staphylococcus aureus Cowan I (SAC) is SPA, it is possible that SAC stimulates a subpopulation of B cells expressing Ig of the VHIII H chain subgroup. In the present study, we have investigated further the relationship between SPA binding and the expression of VHI- or VHIII-associated cross-reactive idiotype (CRI) on the surface of tonsillar B lymphocytes enriched for the expression or nonexpression of the CRI, and we examined the Ig secreted by cell lines established from these populations of B cells by EBV transformation. The VHIII CRI (D12)-enriched population yielded 21 cell lines, with 67% of them secreting SPA-reactive Ig; in contrast, only 6% (1 of 16) of VHI CRI-expressing lines secreted SPA-reactive Ig. The CRI-negative B cell population yielded 54 cell lines, of which 20% secreted SPA-reactive Ig, as might be anticipated because a majority of VHIII Ig+ B cells will be CRI-. SAC stimulation of CRI+ and CRI- populations showed preferential stimulation of the D12 population. These data support the proposal that SAC stimulation of human B cells is mediated through binding of SPA by its alternative binding site to IgV regions of the VHIII subgroup.

Animals↗
PubMed1991

Expression of rheumatoid factor associated cross-reactive idiotopes by glandular B cells in Sjögren's syndrome.

B cell expression of the germline gene-encoded, kappa IIIb-associated, rheumatoid factor (RF) cross-reactive idiotope (CRI) 17-109 and three VHI associated RF CRIs (G6, G8, H1) was investigated immunocytochemically in labial salivary glands from nine patients with primary and six with secondary Sjögren's syndrome, and in inflamed submandibular salivary glands from 10 patients with no history of connective tissue disease. Expression of CRIs by B cell infiltrates in labial glands from patients with primary and secondary Sjögren's syndrome were similar. Lymphoid infiltrates of labial glands from Sjögren's syndrome patients contained a higher proportion of kappa III+ cells reactive for the kappa IIIb-associated 17-109 idiotope (P less than 0.01) and larger G6 (P less than 0.02) and H1 (P less than 0.01) positive B cell populations than those within inflamed submandibular salivary glands. Furthermore, in labial glands there was a significant correlation between numbers of 17-109 and G6 idiotope reactive cells (r = 0.61; P less than 0.02), reflecting the known association between these H and L chain CRIs in RF IgM paraproteins. These results indicate that B cells bearing both VKIII and VHI-associated CRI are increased in the glandular infiltrates in Sjögren's syndrome and support the idea that this condition is associated with proliferation of immature B cell clones retaining germ-line V genes.

B-Lymphocytes↗