Three M-components IgA lambda + IgG kappa n + IgG kappa h in one patient (DA): lack of shared idiotypic determinants between IgA and IgG, and the presence of an unusual kappa h chain of 30,000 M.W

Two apparently homogeneous electrophoretic bands were found in the serum of a patient (DA) with multiple myeloma. These M-components were identified as IgA-lambda and IgG-kappa paraproteins bearing different idiotypic determinants. Further analysis of the L chains showed that the lambda-chain was homogeneous but the kappa-chain could be separated by SDS-polyacrylamide gel electrophoresis into two different bands. Both of them were associated with gamma-chains but one (termed kappa n) had normal m.w. (24,500) whereas the other (termed kappa h) was larger (m.w. 30,000). Sugar content of the two DA IgG, as determined by anthrone reaction, was similar in DA IgG kappa n (0.73%) and in DA IgG kappa h (1.1%), clearly demonstrating that the difference in m.w. was not due to a large sugar chain. Furthermore, the peptide map of the kappa h chain included nine peptides absent in those of four other control kappa-chains. Sequence analysis showed that the first 25 N-terminal amino acids of the kappa n differed from those of the kappa h chain at positions 4, 5, 15, 18, and 21. Thus the two kappa-chains had different framework regions.     

Differential binding of histidine-rich glycoprotein (HRG) to human IgG subclasses and IgG molecules containing kappa and lambda light chains.

In previous studies we showed that the plasma protein histidine-rich glycoprotein (HRG) binds strongly to pooled human IgG. In the present work myeloma proteins consisting of different human IgG subclasses were examined for their ability to interact with human HRG. Using an IAsys optical biosensor we found initially that IgG subclasses differ substantially in their affinity of interaction with HRG. However, the most striking finding was the observation that the kinetics of the HRG interaction was dramatically affected by whether the IgG subclasses contained the kappa or lambda light (L)-chains. Thus, the on-rate for the binding of HRG to the kappa L-chain containing IgG1 and IgG2 (IgG1kappa and IgG2kappa) was approximately 4- and approximately 10-fold faster than that for the binding of HRG to lambda L-chain containing IgG1 and IgG2 (IgG1lambda and IgG2lambda), respectively, with the dissociation constants (K(d)) in the range 3-5 nM and 112-189 nM for the kappa and lambda isoforms, respectively. In contrast, the on-rate for the binding of HRG to IgG3kappa and IgG4kappa was found to be 9- and 20-fold slower than that for the binding of HRG to IgG3lambda and IgG4lambda, respectively, with the K(d) in the range 147-268 nM and 96-109 nM for the kappa and lambda isoforms, respectively. The binding of HRG to immunoglobulins containing the kappa L-chain (particularly IgG1kappa) was generally potentiated in the presence of a physiological concentration (20 microM) of Zn(2+) (K(d) decreased to 0.60 +/- 0.01 for IgG1kappa), but Zn(2+) had no effect or slightly inhibited the binding of HRG to immobilized IgG subclasses possessing the lambda L-chain. Interestingly, HRG also bound differentially to Bence Jones (BJ) proteins containing kappa and lambda L-chains, with HRG having a 14-fold lower K(d) for BJkappa than for BJlambda when 20 microM Zn(2+) was present. HRG also bound to IgM (IgMkappa), but the affinity of this interaction (K(d) approximately 1.99 +/- 0.05 microM) was markedly lower than the interaction with IgG, and the affinity was actually decreased 4-fold in the presence of Zn(2+). The results demonstrate that both the heavy (H)- and L-chain type have a profound effect on the binding of HRG to different IgG subclasses and provide the first evidence of a functional difference between the kappa and lambda L-chains of immunoglobulins.     

T cell hybridomas coexpressing Fc receptors (FcR) for different isotypes. II. IgA-induced formation of suppressive IgA binding factor(s) by a murine T hybridoma bearing Fc gamma R and Fc alpha R

T2D4 murine T hybridoma cells have previously been shown to express Fc receptors (FcR) for IgG (Fc gamma R) and for IgA (Fc alpha R) and to produce an IgG binding factor (IgGBF) that suppresses IgG and IgM responses. In the present work we report on the behavior of IgA bound to T2D4 cells and on the production of IgA binding factor (IgABF) and its ability to suppress IgA antibody production. A dose-dependent binding of MOPC315 IgA with anti-TNP activity by T2D4 cells was demonstrated by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) and fixation of iodinated DNP-BSA. IgA bound to the cells disappeared after a short-term culture of 3 hr at 37 degrees C, but not at 4 degrees C. Because this phenomenon was inhibited by 0.1% sodium azide and 100 microM dansylcadaverine, a transglutaminase inhibitor, Fc alpha R-IgA complexes seemed to be released by an active process involving receptor movement. In the culture supernatant of IgA-treated T2D4 cells, we detected a factor(s) that binds to IgA-Sepharose and competitively inhibits the binding of IgA to T2D4 cells. The factor (IgABF) failed to inhibit the rosette formation of Fc gamma R(+) cells with IgG-sensitized ORBC (EAox gamma), indicating that it binds specifically to IgA. IgABF was undetectable in the culture supernatants of untreated T2D4 cells of Fc alpha R(-) BW5147 T lymphoma cells used as parent cells for the establishment of the hybridoma. To study the effect of IgABF on antibody formation, culture filtrates of IgA-treated or untreated T2D4 cells were fractionated on IgA-Sepharose beads and were added to BALB/c spleen cells cultured with pokeweed mitogen. By use of a reverse plaque assay, it was shown that the IgA plaque-forming cell (PFC) response was suppressed by the acid eluate but not by the effluent of IgA-Sepharose beads incubated with the filtrates of IgA-treated T2D4 cell cultures. The suppression was IgA specific, because neither IgG nor IgM responses were suppressed by the eluate. As expected, there was no significant IgA suppressive activity in the acid eluates of the beads incubated with the culture filtrate of untreated T2D4 cells or IgA-treated BW5147 cells. IgA-specific suppressive activity proved to be due to IgA binding factor(s), because suppressive activity in the eluate was completely adsorbed by IgA-Sepharose but not by IgG- nor BSA-Sepharose.