Modulation of B Cell Receptor Signalling By IL-4 In Chronic Lymphocytic Leukaemia
Authors: Aguilar-Hernandez Maria , Blunt Matthew D , Strefford Jonathan C , Duncombe Andrew , Forconi Francesco , Stevenson Freda K. , Packham Graham K , Steele Andrew J 
Published: October 2019

Abstract
Chronic Lymphocytic leukaemia (CLL) is currently incurable using conventional therapies, although new drugs aimed as signalling pathways are having some therapeutic success. Features of CLL cells reveal a balance between anergy, apoptosis or increased proliferation, with outcome determined by the B cell receptor (BCR). Engagement of the BCR triggers formation of the signalosome leading to calcium [Ca+2]i mobilization and ERK phosphorylation. However other microenvironmental factors affect CLL cell growth and survival and may modulate the BCR response. We have focused on IL-4, produced mainly by T cells and known to rescue CLL cells from apoptosis when cultured in vitro. IL-4 strongly induces phosphorylated STAT6 (STAT6-P) within CLL cells in vitro, therefore we analysed STAT6-P expression within lymph nodes of SLL/CLL patients. We detected STAT6-P expression in SLL/CLL lymph nodes predominantly within proliferation centres. Induction is likely to be via IL-4, since IL-13 and CD40L, which are also capable of inducing STAT6-P, did not produce a STAT6-P signal in CLL cells in vitro.
To analyze the effect of IL-4 on BCR signalling, CLL samples were exposed to IL-4 for between 1-72h prior to treatment with anti-IgM. Using calcium flux as a marker of BCR signalling we found that IL-4 significantly increased the calcium flux, as compared to the untreated control, in 63.6% (7/11) of the cases. We showed that the increased calcium flux coincided with an upregulation of sIgM expression observed between 24-72h. Furthermore there was no significant up-regulation of sIgD levels even though calcium flux increased in a proportion of cases (6/9) in response to anti-IgD following IL-4 pre-treatment. We further investigated the effect of IL-4 on known positive and negative regulators of BCR signalling. While no significant changes in CD19 expression were found, CD5 expression was significantly downregulated in all cases which showed IL-4-mediated amplification of signal in response to anti-IgM.
Next we evaluated phosphorylated ERK (ERK-P) as a marker of BCR signalling. CLL cells were incubated overnight in the presence or absence of IL-4 and subsequently stimulated with soluble anti-IgM for 15 minutes. We found ERK-P expression was augmented following αIgM activation in the presence of IL-4 compared to the control in a proportion of samples (12/15). When CLL cells were treated with the PI3K inhibitor LY294002 prior to the addition of anti-IgM, ERK-P was reduced due to inhibition of the BCR signalosome upstream of ERK. However pre-treating CLL cells with IL-4 enabled the cells to overcome chemical inhibition of PI3K with LY294002 and restored ERK-P expression. Using densitometry analysis a significantly greater signalling capacity quantified by ERK-P expression (P=0.042) was observed in 15/23 patients. Interestingly, 73.3% of those 15 cases were unmutated CLL (U-CLL), the aggressive form of the disease.
Taken together, these data shows that IL-4 is able to enhance conventional BCR mediated signalling and may partially overcome chemical inhibition of the classical BCR pathway in CLL cells. The augmentation of BCR signalling is partly mediated by an increase in sIgM receptor density but may also be partly regulated by modulation of the positive and negative regulators of BCR signalling. Together these data indicate an important role of the microenvironment in BCR signalling in CLL.

Disclosures:
No relevant conflicts of interest to declare.