R. A. Mageed

Auto‐ and Polyreactivity of IgM from CD5+ and CD5− Cord Blood B Cells

The presence of the CD5 (67 kDa) molecule on the surface of B cells has been considered a marker for cells producing auto‐and polyreactive antibodies. Cord blood B lymphocytes (rich in CD5+ B cells) have been sorted into CD5 positive and negative populations by flow cytometry using monoclonal antibodies to CD20 and CD5. Clones of these populations were obtained by immortalization mill Epstein Barr virus. Clones derived from both CD5+ and CD5− B cells produced IGM which was auto‐ and polyreactive with a higher frequency of these specificities in the CD5+ population. These data indicate Ihm expression of surface CD5 on cord blood B cells Is not a definitive marker of an auto/polyreactive population.

Human Antibodies Elicited by a Pneumococcal Vaccine Express Idiotypic Determinants Indicative of Vh3 Gene Segment Usage

Human immunodeficiency virus (HIV)-infected persons manifest decreased antibody responses to pneumococcal polysaccharide vaccines. Since human antibody responses to polysaccharides are often restricted, the molecular structure of antibodies elicited by a 23-valent pneumococcal vaccine was analyzed. Anti-idiotypic reagents were used to detect V(H)1, V(H)3, and V(H)4 gene usage by antibodies to pneumococcal capsular polysaccharides in HIV-uninfected and HIV-infected subjects by ELISA. HIV-uninfected persons generated beta-mercaptoethanol-sensitive and -resistant antibodies to pneumococcal capsular polysaccharides expressing V(H)3 determinants recognized by the D12, 16.84, and B6 monoclonal antibodies; antibodies expressing V(H)1 determinants were not detected, and V(H)4 determinants were expressed by beta-mercaptoethanol-sensitive antibodies only; and HIV-infected subjects had significantly lower capsular polysaccharide-specific and V(H)3-positive antibody responses. These findings confirm decreased antibody responses to pneumococcal vaccination in HIV-infected persons and suggest that their poor responses may result from HIV-associated depletion of restricted B cell subsets.

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.

Immunogenic and antigenic epitopes of immunoglobulins. XXIII. Idiotypy and molecular specificity of human rheumatoid factors: analysis of cross-reactive idiotype of rheumatoid factor paraproteins from the Wa idiotype group in relation to their IgG subclass specificity.

The expression of idiotypic and variable region‐associated isotypic determinants on a panel of human monoclonal rheumatoid factors (RF) was studied by means of murine hybridoma antibodies produced to two IgM‐RF paraproteins. Fourteen RF paraproteins from patients with cryoglobulinaemia and one (RF‐AN) from an Epstein‐Barr virus (EBV)‐established B‐cell line from a patient with rheumatoid arthritis (RA) were studied. Nine RF paraproteins expressed a VkIIIb light chain sub‐subgroup‐associated cross‐reactive idiotope and seven of these nine also expressed a heavy chain‐associated cross‐reactive idiotope. The reactivity of the monoclonal RF with human IgG subclass paraproteins revealed four patterns of molecular specificities: (1) RF reactive with an epitope common to all IgG subclasses; (2) RF reactive with an epitope expressed on IgG 1,2,4 and G3m(s,t) which has histidine al 435, but not G3m(b) or G3m(g) which have arginine at 435; (3) RF reactive with an epitope expressed on IgG 1,2, and 4, but not IgG3 irrespective of allotypic markers; (4) RF reactive with epitopes expressed on some, but not all paraproteins within the subclasses. Four of five RF paraproteins that expressed both the heavy and light chain‐associated idiotopes showed a similar pattern of reactivity with IgG subclass proteins.

Anti-idiotypic antibody D12 and superantigen SPA both interact with human VH3-encoded antibodies on the external face of the heavy chain involving FR1, CDR2 and FR3.

The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V(H)3 encoded antibodies. We used mutational analysis to determine the subregions of a V(H)3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FRI, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V(H)3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.

Restricted immunoglobulin variable region gene usage by hybridoma rheumatoid factors from patients with systemic lupus erythematosus and rheumatoid arthritis

Rheumatoid factors (RFs) are autoantibodies that are produced by approximately 75% of patients with rheumatoid arthritis (RA). Their role in pathogenesis is not well understood. In this study of 81 human hybridoma IgM antibodies derived from unstimulated peripheral blood B-cells of patients with RA and systemic lupus erythematosus (SLE), we have demonstrated that idiotypes associated with RFs derived from patients with mixed cryoglobulinemia were expressed by approximately 60% of RFs and 6% of IgM antibodies lacking RF activity. The specificity of the RFs for the Fc portion of IgG only (monospecificity) or for Fc and additional self antigens (polyreactivity) was found to correlate with the expression of specific heavy chain associated idiotypes. The VH3 associated RF idiotypes, D12 and B6, were expressed by 0/16 (0%) of monospecific RFs compared with 6/22 (27%) of polyreactive RFs. The predominant use of VH3 was verified by analysis of the expressed Ig with VH family specific anti-peptide antibodies. The light chains expressed by both populations of IgM RFs were found to be predominantly VKIII, both by detection of specific epitopes/idiotypes and V family analysis. This non-random gene usage of both the heavy and light chains suggests that there is a selective expression of V regions in the RF producing B-cells in patients with RA and SLE. We suggest that different antigen-driven, clonal selection events may occur which result in either monospecific RFs or polyreactive RFs.

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 Sjögrens 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 Sjögrens syndrome were similar. Lymphoid infiltrates of labial glands from Sjögrens syndrome patients contained a higher proportion of kappa III+ cells reactive for the kappa IIIb‐associated 17–109 idiotope (P<0.01) and larger G6 (P<0.02) and HI (P<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 < 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 Sjögrens syndrome and support the idea that this condition is associated with proliferation of immature B cell clones retaining germ‐line V genes.

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 VRIII 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.

The Genetic Basis of Human Vh4 Gene Family-Associated Cross-Reactive Idiotype Expression in CD5+ and CD5- Cord Blood B-Lymphocyte Clones

In a recent study we have observed a high frequency expression of cross‐reactive idiotypes encoded by genes from the relatively small VH4 family of immunoglobulin heavy chain genes in cord blood B‐lymphocyte lines. Furthermore, we have demonstrated a selective pattern of expression of two VH4‐associated cross‐reactive idiotype (CRI) in B‐lytnphocyte lines established from CD5+ and CD5‐ cord blood B‐lymphocytes. There was a restricted expression of one CRI marker recognized by the 9G4 monoclonal antibody in lines established from CD5+ B‐lymphocytes but not in those established from the CD5‐ population. In the current study we examine the molecular basis for the selective pattern of CRI expression. Nucleotide‐sequence analysis of functional immunoglobulin heavy chain (IgH) gene rearrangements in three CD5 + lines expressing the CRI recognised by 9G4 reveal that all use a single gene from the Vh4 family, the V4.21 gene. However, all three lines have distinct third complementarity determining regions (CDR3) implying different clonal origins. In contrast, four cord blood cell lines (two established from CD5+ B‐lymphocytes) expressing the CRI recognized by MoAb Lcl have functional IgH gene rearrangements involving two ditferent genes from the Vh4 family, the V71–4, and V2–1 genes. Antigen specificity analysis reveals that all three 9G4‐reactive lines produce antibodies that react with the I and/or i red blood cell carbohydrate antigens. These data suggest that the distinction in VH4 gene use in CD5+ B‐lymphocytes in cord blood results from a selection process in vivo that shapes the repertoire of CD5+ B‐lymphocytes. This study extends recent observations that the monoclonal anti‐CRI antibodies 9G4and Lc1 are markers of two distinct subgroups of proteins encoded by two subsets of genes within the VH4 family. Furthermore, it appears that amino acid residues in framework region one and complementarity determining region two are critical for the expression of the cross reactive idiotypes and the serological distinction between the two subgroups of proteins.

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 IgGl 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%.