The WA cross-idiotype (XId) is the major XId among human monoclonal rheumatoid factors (mRF) and is almost always associated with the light (L) chain XId, 17 .109, and the heavy (H) chain XId, G6. A cell line, 35G6, was cloned that bears the WA XId, but shows no reactivity with immunoglobulin G (IgG) and is negative for the 17 .109 and G6 XIds. The 35G6 L chain appears to be derived from the same V,,III JKI genes as most WA mRFs L chains. In contrast to the WA mRFs H chains in which V 1 genes are used, the 35G6 IgM expresses a V.3 gene. Sequence comparisons with other WA XId-positive mRF suggested several common structural features that may be related to the WA XId and differences that may relate to lack ofIgG reactivity. Cells similar to 35G6 have previously been described in pokeweed mitogen-stimulated cell lines ofperipheral blood lymphocytes from normal individuals and patients with rheumatoid arthritis and type II mixed cryoglobulinemia. These observations were confirmed, and in addition, it was shown that the majority of WA XId-positive cells in these cultures were negative for the 17 .109 and G6 XIds. The presence of the WA XId in the absence of IgG reactivity suggests that the WA XId is more directly associated with an antigen specificity other than IgG, and its association with RF activity may be incidental. It is postulated that these WA XId-positive RF-negative antibodies may serve a physiologic role as natural antibodies to a pervasive pathogen, and that IgG reactivity is a consequence of somatic diversification accompanying proliferation of the WA XId-positive RF-negative cell.T he WA cross-idiotype (XId)', the major cross-idiotype among human monoclonal rheumatoid factors (mRF), is a conformational antigenic determinant involving both H and L chains and appears to be located in the antigen binding site (1, 2). The L chain-associated XId identified by the mAb 17 .109 and the H chain-associated XId detected by the G6 mAb have been reported to occur in almost all WA XIdpositive mRFs (3) and has led to the notion that there is restricted expression of these germline genes with little or no 1 Abbreviations used in this paper: mRF, monoclonal RF; XId, WA crossidiotype.The sequences reported in this paper have been deposited in the GenBank data base under accession numbers M97268 and M87269. 1903 somatic mutation in the WA mUs . Earlier, it had been postulated that the VKIIIb L chain detected by 17 .109 mAb and encoded by the Humkv325 gene was the structural basis for the mRF WA XId and was unique to IgM with RF activity (4) ; however, this hypothesis was disproved (2), and it has been shown that the 17 .109 and G6 XIds occur separately (5) and together (6) in IgM without RF activity.Although the WA XId occurs in high frequency among mRF in serum cryoglobulins from patients with type II cryoglobulinemia, it appears to occur in only small amounts among polyclonal RFs in patients with rheumatoid arthritis (7,8
Three human monoclonal rheumatoid factor (mRF) crossidiotype (XId) groups, WA, PO, and BLA, were defined using polyclonal antisera (1, 2) . Both the WA and the BLA XIds appeared to be associated with the antigen-combining site (2), and serologic studies (3, 4) indicated that the antisera defining the WA XId appeared to recognize a determinant requiring both H and L chains . Structural characterization of the Ids was not done. Extensive studies (5-7) have recently been performed, however, to determine the structural basis of the WA XId. Investigators using antisera to primary structures on the H and L chains postulated that the WA XId resides on the L chain . Our study, in which we used the original antisera that defined the WA and BLA XIds and additional polyclonal antisera, presents evidence that the WA and BLA XIds are conformational antigens requiring both L and H chains, and that with denaturation, the antigens that define the XId and the antigen-binding activity are lost in parallel . Volume 164 November 1986 1809-1814 Materials and Methods BriefDefinitive ReportPreparation of Reagents and Antisera . IgM mRFs were obtained and isolated as previously described (2). Antisera used to type the WA XId (anti-McD) and BLA XId (antiBla) were previously described (2). Anti-PSL2, a synthetic peptide corresponding to the second complementarity-determining region (CDR) and adjacent residues on the L chain of the IgM RF Sie (8), was provided by Drs. P. P. Chen and D. A. Carson (Scripps Clinic, La Jolla, CA).Preparation of H and L Chains. H and L chains of mRFs were prepared by routine partial reduction with 10 mM DTT, alkylated and isolated in 1 M propionic acid on a *100 Sephadex column . Purity of H and L chains was established, and recombination of H and L chains was performed by mixing H and L chain pools in a 1 :2 M ratio, respectively, and by dialyzing them for 72 h at 4°C against a 0 .5 M Tris, 0 .15 M NaCl buffer (pH 7 .2) . Recombined H and L chains were isolated with G200 Sephadex column chromatography . The recombinant pools were tested for free K or w chains by immunodiffusion in agarose gel using nonspecific antisera to x and ju chains .Immunodotting and Blotting. 100 A1 of each mRF diluted to 10 ug/ml in PBS was "dotted" onto 0 .45-gm nitrocellulose paper using a hybridot filtration manifold (Schleicher & Schuell, Inc ., Keene, NH) . Parallel sample sets were stained with Coomassie blue or blocked with 1 % BSA/PBS and probed with WA or BLA XId-typing reagents . The filter This work was supported by National Institutes of Health (Bethesda, MD) grant ROI AM-35487 and Veterans Administration merit review grant. J. Exp. MED.
The amino acid sequence of the L-CDR2 (complementarity-determining region) of Bla mRF (monoclonal rheumatoid factor) is identical to that of the Wa mRFs. The PSL2-CRI (crossreactive idiotype), as determined by anti-PSL2, which has been shown to be present on all Wa mRFs, is also present on the Bla mRF and other monoclonal autoantibodies. PSL2-CRI is, therefore, not unique to Wa mRFs and may be present on most IgM kappa monoclonal autoantibodies. Whether PSL2-CRI is a crossidiotype (XId) that is selectively present on autoantibodies or represents an allotypic marker for a V kappa III gene is undetermined.
The BLA cross-idiotype (XId) is present on a unique subset of rheumatoid factors (RF) that crossreact with DNA-histone. In this study, prototype Bla monoclonal RF was shown from serologic investigations and N-terminal amino acid sequence analysis to have distinct K chains related to the VK 111 subgroup and v H 4 heavy chains. The amino terminus of the heavy chain was cyclized, rendering the protein resistant to Edman degradation and providing a possible investigator bias to the published Ig sequence data to date. This appears to be the first definitive report of a serum IgM that expresses the v H 4 gene. RF with DNA cross-reactivity have been reported to be produced by human and mouse cloned cells that have the V,4 or homologous mouse Vh36-60 gene.The BLA cross-idiotype (XId) antigen is present on a unique subset of rheumatoid factors (RF) that also have antibody activity to DNA-histone (I).
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