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The covalent binding of C3 to antigen-antibody complexes [immune complexes (IC)] plays a pivotal role in the elimination of antigens. C3 prevents the formation of large IC lattices promoting their solubilization. Subsequently, bound C3 fragments determine the efficacy of antigen presentation, and the generation of antibody responses and immunological memory. C3 binding to IgG-IC generates IgG-C3b-C3b complexes which are detected by SDS-PAGE as two major bands: C3alpha65-heavy chain and C3alpha65-C3alpha43 covalent complexes. Using human heat-aggregated IgG1 as a model of IC, a C3b binding site was localized only in the Cgamma1 domain. However, with true IC of ovalbumin and rabbit IgG anti-ovalbumin, C3b binds to both the Fab and Fc regions of IgG. To study the binding of C3b to the different domains of IgG and particularly to evaluate the involvement of the Cgamma1 domain, we have constructed recombinant single-chain antibodies without Cgamma1, which have the structure: V(H)-linker-V(L)-hinge-Cgamma2-Cgamma3 (scAb). The variable domains were from a mouse mAb anti-HSA and the constant region (hinge-C(H)2-C(H)3) from human IgG1 or rabbit IgG. C3 binds very efficiently to IC formed with human (h-scAb) or rabbit (r-scAb) recombinant antibodies (scAb-HSA) and generates also two bands on SDS-PAGE (C3alpha65-scAb and C3alpha65-C3alpha43), which are the counterparts of those of the complete antibody. In addition, IC formed with scAb activate the alternative pathway to a similar extent as IC of the entire IgG. These data indicate that the Cgamma1 domain is a dispensable region for C3b binding and that the remaining constant domains are as efficient as Cgamma1 in C3b binding. Overall these results support the view that C3 does not specifically recognize a unique site in the Cgamma1 domain. Rather it seems to be able to attach along the antibody molecule. Probably this implies an advantage for effective processing of C3b-IC and elimination of antigens in vivo.

The ligand-receptor binding requirements for achieving full B cell activation through the membrane immunoglobulin (mIg) signaling pathway are relatively demanding, and mIg-antigen engagements which fall below these critical thresholds cause, at most, only the partial activation of B cells. In an effort to resolve new means of enhancing the efficacy of mIgM-mediated signal transduction, as well as to further understand the process by which mIgM-mediated signals are initiated, we have explored the mechanism for a previously reported synergy between certain mixtures of murine anti-IgM mAbs in eliciting human B cell DNA synthesis. We here report that striking synergy occurs when any of several relatively high affinity mAbs specific for diverse domains of mIgM are combined in culture with the relatively low affinity C mu 4-specific ligand, mAb IG6. Although B cell activation was dependent upon the bivalency, and hence mIgM cross-linking potential, of the high affinity ligand, low affinity mAb IG6 could enhance the activation process when present as a monovalent Fab' fragment. This did not appear due to F(ab')2 contamination or Fab' aggregation, since IG6 Fab' preparations were notably compromised in several other functions requiring ligand bivalency. Pulsing studies revealed that the C mu 4-specific ligand exhibits its functional effects only when stimulatory mIgM receptor cross-links are being formed by bivalent ligands, and that IG6 Fab' enhancement is most notable during the later interval of the prolonged mIgM signaling process that leads to S phase entry. A unique region of the membrane-proximal IgM domain may be important for Fab'-mediated enhancement, since Fab' fragments that bind with higher affinities to distinct sites on C mu 4 were not as effective at mediating this phenomenon. Several possibilities for the adjuvant effects of this C mu 4-specific Fab' on B cell responses triggered by mIgM crosslinking ligands are discussed, including the possibility that IG6 Fab' influences the potential for mIgM dimer formation or interactions of mIgM with other signal-transducing molecules.

IgD receptor (IgD-R) bearing CD4+ T cells with immunoaugmenting properties in vivo are induced in mice within 24 h after a single injection of dimeric or aggregated IgD. In the present study, we sought to identify the region(s) of IgD responsible for upregulation of IgD-R and for the immunoaugmenting effect of IgD. IgD-R can be upregulated on CD4+ T cells in vitro and in vivo by glutaraldehyde-aggregated mutant IgD or by fragments of enzymatically digested IgD molecules possessing either the C delta 1 domain (Fd delta) or the C delta 3 domain (Fc delta). Neoglycoproteins (D-galactose--BSA and N-acetyl-D-glucosamine--BSA), can competitively block upregulation of IgD-R by IgD in vitro. Furthermore, when injected 1 day before antigen, the aggregated IgD derived molecules, KWD1 (which lacks C delta 1), KWD6 (which lacks C delta 1 plus C delta-hinge), and Fab delta can all cause augmentation of antigen-specific primary and secondary antibody responses comparable to that achieved with intact aggregated IgD. Moreover, the immunoaugmenting effect of intact oligomeric IgD molecules in primary antibody responses is competitively blocked by simultaneous injection of monomeric forms of KWD6 and Fab delta. These results suggest that the binding of IgD to IgD-R, previously shown to be dependent on N-glycans present on Fd delta and Fc delta regions, also contributes to the upregulation of IgD-R and immunoagumentation.