Evidence for an upper affinity threshold for anti-IgM-induced apoptosis in a human B-cell lymphoma.

The influence of ligand:receptor affinity on B-cell antigen receptor (BCR)-induced apoptosis in the IgM+ Burkitt lymphoma line, Ramos, was evaluated with a group of affinity-diverse murine monoclonal antibodies (MoAbs) specific for human B-cell IgM. The studies showed not only a minimal affinity threshold for the induction of apoptosis, but, interestingly, also a maximal affinity threshold above which increases in affinity were associated with diminished apoptosis. The lesser capacity of high-affinity MoAb to induce apoptosis was paralleled by a lesser capacity to induce receptor cross-linking. At high ligand concentration, high MoAb affinity was also associated with a diminished capacity to induce early protein tyrosine phosphorylation. The compromised capacity of two high-affinity MoAbs to trigger apoptosis may be, at least in part, explained by two separate phenomena that can impair the formation of mIgM cross-links: (1) more stable univalent binding and (2) a tendency for monogamous binding of both MoAb Fab to two Fab epitopes on mIgM. These in vitro studies suggest that the use of the highest affinity MoAbs for antireceptor immunotherapies that depend on receptor cross-linking might, on occasion, be contraindicated.

Membrane IgM-stimulated human B lymphocytes succumb to activation-related apoptosis at a G1-->S transition: influence of ligand affinity and valency.

Culture of human B lymphocytes with polyclonally activating surrogates for type II T-cell-independent antigen, i.e., anti-IgM mAb and anti-IgM:dextran, resulted in both membrane IgM (mIgM)-triggered S/G2/M entry and apoptosis. Although high ligand valency could compensate for low affinity, and high affinity could compensate for low valency, in achieving mIgM-triggered apoptosis, the phenomenon was most pronounced when the soluble "antigen" had both high binding site affinity and valency. Most of the mIgM-triggered apoptosis may represent B cells which progress into G1 but fail to receive a sufficient level of continuous mIgM-mediated signaling during G1 for passage through a G1 --> S phase restriction point(s). This was supported by the findings that (a) a lesser proportion of mIg-triggered cells enter S phase than G1; (b) maximal mIgM-triggered apoptosis was noted at 48-72 h of culture and surrounding activated cell clusters; (c) mIgM-triggered apoptosis was not inhibited by pharmacologic blockers of S phase; and (d) a high proportion of viable mIgM-triggered B cell blasts in G1 succumb to apoptosis rather than enter S phase, if high-affinity multivalent ligand is washed from the cultures. In addition to quantitative aspects of initial receptor engagement, the potential for a protracted period of recurrent mIgM signaling events may influence whether apoptosis or cell cycle progression is the functional outcome of B cell encounter with a multivalent antigen.

The affinity threshold for human B cell activation via the antigen receptor complex is reduced upon co-ligation of the antigen receptor with CD21 (CR2).

The present studies have examined whether the potential of an Ag to co-ligate the complement (C3d)-binding CD21 receptor complex with the membrane IgM (mIgM) receptor complex can reduce the mIgM:Ag affinity threshold for triggering human B cell S phase entry. A series of Ab:dextran conjugates consisting of affinity-diverse anti-IgM mAb, with and without anti-CD21 mAb, were synthesized as polyclonally reactive, moderately multivalent ligands that mimic C3d-bearing and non-C3d-bearing Ag. Co-ligation of mIgM and CD21 significantly diminished both the ligand concentration threshold and the IgM:ligand affinity threshold for eliciting S phase entry in the presence of IL-4. Furthermore, such co-engagement ablated the triggering bonus associated with high mlgM:ligand affinity, suggesting that B cells with a high affinity for Ag are not preferentially activated over B cells of intermediate affinity upon encountering a multivalent Ag with bound C3d. The enhancing effects of mIgM:CD21 co-ligation were restricted to low concentrations of ligand; at high concentrations, a decrease in B cell DNA synthesis was often observed. The findings suggest that the ability a moderately multivalent Ag substrate to engage B cells through both mIgM and CD21 is critical for B cell activation at limiting Ag concentrations, and furthermore, that mIgM:CD21 co-engagement may be particularly important in eliciting an immune response to such Ags in unprimed individuals in whom the majority of specific B cells are of low affinity.