Low Affinity Interaction with CLL B-Cell Receptors Induces Apoptosis by Down-Regulating Mcl-1 and Up-Regulating BimEL.
Authors: Paul Santanu , Abu-Helu Rasmi , Mongini Patricia , Metz Christine N. , Chu Charles C. , Steinberg Bettie , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
The B-cell receptor (BCR) engages antigens and triggers divergent signaling pathways, inducing survival, proliferation, and differentiation or anergy and apoptosis. The decision as to which pathway is activated and which functional outcome ensues is regulated by a series of receptor and cellular interactions. In particular, signal transduction via the BCR is influenced by the affinity of receptor-ligand binding and by the number of antigen-binding sites engaged. To address the role of BCR-antigen-binding affinity and valency in CLL, we used a series of anti-μ chain mAbs that differ in their affinities for the ligand as surrogates of antigen binding to the BCR (HB57: Ka = 5x108, “high”; Mu53: Ka = 2x107, “intermediate”‘ and P24: Ka = ∼2x106 M−1, “low”). We also mimicked the valency of the surrogate antigens (and hence the number of binding sites occupied) by preparing these mAbs in either their native dimeric state or in multimeric forms by conjugating them to dextran and to DYNA beads M-450. The native mAbs and the mAbs bound to dextran are soluble; the mAbs bound to beads are insoluble. The functional effects on CLL B cells of each mAb, in all three forms (i.e., soluble dimeric, soluble multimeric on dextran, and insoluble multimeric on beads), were then assessed in vitro. We noted striking and consistent differences in CLL cell responses when the mAb of different affinities for the BCR were used in the soluble, native dimeric state. Whereas the higher affinity anti-μ mAbs (HB57 and Mu53) induced minimal to modest proliferation, the low affinity mAb P24 drastically reduced proliferation. Similarly the soluble, dimeric forms of the same three mAbs had striking and consistently different effects on CLL cell survival. In particular, the low affinity mAb induced significant apoptosis as measured by Annexin V/propidium iodide immunofluorescent analyses (mean 61.2%; range 32–99.2%) as well as by PARP cleavage. Western blotting studies of cell lysates from CLL cells stimulated with the low affinity mAb vs. the higher affinity mAbs or isotype matched control mAbs revealed downregulation of the anti-apoptotic protein Mcl-1 and upregulation of the pro-apoptotic protein BimEL. The multimeric forms of the three anti-μ mAbs, both soluble and insoluble, increased CLL cell survival and proliferation, irrespective of affinities, by maintaining Mcl-1 levels and failing to upregulate Bim. This study illustrates the importance of the “quality” of BCR-antigen interaction for CLL cell survival and growth. Soluble low affinity interactions with antigens of reduced valency lead to CLL cell death, whereas soluble low affinity interactions with multivalent antigens lead to cell survival and growth. The use of soluble, monomeric, low affinity ligands reactive with shared stereotypic BCRs, therefore, should be considered as an immunotherapeutic approach in CLL.