Abstract 1023: Duration of signaling through IgM and IgD receptors differentially influences cell survival and BCL6-regulated CCL3/4 chemokine production in Chronic Lymphocytic Leukemia (CLL)
Authors: ten Hacken Elisa , Scielzo Cristina , O'Brien Susan , Wierda William G. , Keating Michael J. , Ghia Paolo , Caligaris-Cappio Federico , Burger Jan A. 
Published: August 2015

Abstract
B-cell receptor (BCR) signaling is a central pathogenetic mechanism in CLL. Most CLL cells express both IgM and IgD receptors, and increased responsiveness to IgM stimulation correlates with poor disease outcome and with expression of IGHV-unmutated immunoglobulin receptors (U-CLL). In contrast, the relevance of IgD signaling remains poorly understood.
The aim of the present study is to characterize functional differences between IgM and IgD signaling in CLL in terms of protection from in vitro apoptosis, BCR signaling activation and CCL3/4 chemokine secretion and to explore the mechanism through which CCL3/4 are regulated, with a focus on their transcriptional repressor BCL6.
CLL cells were stimulated with anti-IgM and anti-IgD; 48-hour cell viability and BCR expression were analyzed by flow cytometry; phosphorylation of the BCR-signaling related molecules HS1 and ERK after 2, 5, 15, 30, 60 minutes of stimulation were analyzed by Western Blot and flow cytometry; BCL6 protein expression by Western Blot and CCL3/4 secretion by ELISA were tested after 3, 6, 9, 12, 24 hours of stimulation.
Anti-IgM stimulation protected CLL cells from in vitro apoptosis to a greater extent than anti-IgD and U-CLL were more responsive to anti-IgM as compared to cells carrying IGHV-mutated receptors (M-CLL) (p = 0.0022; n = 13 M-CLL vs. n = 11 U-CLL). In line with this finding, U-CLL expressed higher levels of surface IgM, as compared to M-CLL (p&lt;0.0001; n = 40 M-CLL vs. n = 33 U-CLL). IgM stimulation induced protracted HS1 and ERK phosphorylation up to 30 minutes following receptor engagement, as analyzed in 10 M-CLL and 10 U-CLL cases. In contrast, IgD stimulation induced short-lived phosphorylation of both proteins, which rapidly returned to baseline. U-CLL cells were more responsive than M-CLL to both anti-IgM and anti-IgD stimulation. High CCL3/4 secretion was detected after anti-IgM, in particular in U-CLL. Surprisingly low, if any, induction of both chemokines was detected after stimulation of IgD receptors. Interestingly, increased CCL3/4 production and concomitant BCL6 protein downregulation were observed after 6 and up to 12 hours following anti-IgM but not anti-IgD stimulation.
In conclusion, IgM stimulation induces a long-lived signaling response, increased CLL-cell survival and CCL3/4 secretion, in particular in U-CLL; in contrast, IgD signaling is short-lived and has minimal effects on protection from apoptosis and chemokine secretion. BCL6 protein downregulation induced by anti-IgM parallels CCL3/4 production, suggesting a functional link between BCL6 expression and CCL3/4. Taken together, these evidences suggest that the differential functional outcomes of IgM and IgD signaling might be related to the duration of the signaling response and support the relevance of IgM signaling in CLL pathogenesis.
Citation Format: Elisa ten Hacken, Cristina Scielzo, Susan O'Brien, William G. Wierda, Michael J. Keating, Paolo Ghia, Federico Caligaris-Cappio, Jan A. Burger. Duration of signaling through IgM and IgD receptors differentially influences cell survival and BCL6-regulated CCL3/4 chemokine production in Chronic Lymphocytic Leukemia (CLL). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1023. doi:10.1158/1538-7445.AM2015-1023