IgM and IgD Receptors Differentially Contribute to CLL Survival and Chemokine Secretion: Implications for CLL Biology and Treatment
Authors: ten Hacken Elisa , Sivina Mariela , Ping Li , O'Brien Susan , Wierda William G. , Ferrajoli Alessandra , Estrov Zeev , Keating Michael J. , Scielzo Cristina , Ghia Paolo , Caligaris-Cappio Federico , Burger Jan A. 
Published: October 2019

Abstract
Background: B-cell receptor (BCR) signaling is a central pathway activated in the tissue microenvironment of patients with Chronic Lymphocytic Leukemia (CLL). CLL cells express IgM and IgD receptors, but the contribution of these isoforms to signaling and functional responses remains incompletely defined. The mutational status of CLL BCRs, on the other hand, has clear correlation with clinical outcome: patients with unmutated IGHV genes (U-CLL) characteristically have more aggressive disease and an inferior prognosis when compared to patients with mutated IGHV (M-CLL).
Aim: The aim of this present study is to characterize functional differences between IgM and IgD signaling, in the context of the CLL microenvironment and of ibrutinib treatment.
Methods: Freshly isolated, purified CLL cells from 80 patients (40 M-CLL and 40 U-CLL) were stimulated with soluble or bead-bound anti-IgM or anti-IgD; IgM, IgD expression, phosphorylation of the BCR-related molecules HS1 and ERK, F-actin polymerization and cell viability were analyzed by flow cytometry; HS1 and ERK phosphorylation, and BCL6 protein expression were also analyzed by Western Blot. CCL3 and CCL4 chemokine secretion was measured by ELISA. BCL6 binding to the CCL3 gene promoter region was assessed by ChIP-PCR. We utilized nurselike cell (NLC) co-cultures to activate BCR signaling and to induce CCL3 and CCL4 production in this model system that resembles the lymph node microenvironment. Effects of ibrutinib treatment on CLL-cell viability and BCR activation were also tested.
Results: Surface IgM and IgD levels were significantly higher in U-CLL (P<0.0001), ZAP70 positive (P<0.01), and/or del17p or del11q CLL cells (P<0.05). Early BCR signaling with activation of the cytoskeletal protein HS1 and F-actin polymerization were similar after anti-IgM and anti-IgD stimulation. Downstream activation of ERK kinases was more prolonged after anti-IgM than after anti-IgD stimulation. Transient activation of IgD signaling was paralleled by rapid IgD receptor endocytosis, as compared to IgM. Furthermore, anti-IgM increased CLL viability to a significantly greater extent than anti-IgD, and exclusively induced secretion of CCL3 and CCL4 chemokines. This effect was consistently observed after stimulation with either soluble or bead-bound anti-IgM and anti-IgD. Expression of BCL6, which functions as a transcriptional repressor for CCL3 gene, was down-regulated following anti-IgM, but not after anti-IgD stimulation. In addition, we observed BCL6 binding to CCL3 promoter regions and noted that BCL6 down-regulation coincided with the onset of CCL3 secretion. In all experimental settings, U-CLL cells were more responsive to BCR stimulation than M-CLL. NLC co-culture induced down-modulation of surface IgM and IgD on CLL cells, increased HS1 and ERK phosphorylation and induced CCL3 chemokine secretion, along with BCL6 down-regulation. When CLL cells were removed from NLCs, recovery of surface IgM and IgD, baseline BCL6 expression, and HS1 and ERK phosphorylation was observed after 72 hours. Ibrutinib treatment preferentially interfered with anti-IgM induced survival signals and BCR signaling activation, in particular of U-CLL cells.
Conclusion: Compared to IgD, IgM stimulation induces more durable signaling responses, CCL3 and CCL4 chemokine secretion and increases CLL-cell survival, in particular in U-CLL cells. BCL6 binding to CCL3 promoter and its down-regulation concomitant to CCL3 production suggests that BCL6 represses CCL3 gene expression in CLL cells. Co-culture of CLL cells with NLCs triggers BCR signaling, with functional outcomes that mainly recapitulate the features of IgM stimulation in vitro. Ibrutinib treatment preferentially interferes with IgM-mediated signals, and reduces CCL3 and CCL4 levels in CLL-cell supernatants and in the plasma of ibrutinib-treated patients. Taken together, these results support the relevance of IgM signaling in the pathogenesis of CLL, and suggest that IgM is the main receptor isotype targeted in vivo during ibrutinib treatment.

Disclosures
Estrov: incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.