Uncoupling of BCR Internalization and Signaling in Treatment Naive and Btki-Treated Patients with Chronic Lymphocytic Leukemia
Authors: Coulter Eve M , Buggins Andrea , Mele Silvia , Folarin Najeem , Townsend William , Cuthill Kirsty , Phillips Elizabeth , Patten Piers , Devereux Stephen 
Published: October 2019

Abstract
Ligation of the B-cell receptor (BCR) results in activation of intracellular signaling as well as internalization and processing of ligand/receptor complexes. BCR responsiveness has been shown to vary markedly between patients with chronic lymphocytic leukaemia (CLL) and is linked to prognosis. Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, relatively little is known about the capacity of CLL B-cells to internalize ligands that bind to the BCR.
In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR following stimulation. First, we assessed to what extent this varies between various prognostic subgroups. Second, given that BCR signaling is thought to be more pronounced within lymphoid tissue, we investigated whether internalization varies between different anatomic sites of the same individual. Finally, we examined the effect of agents that inhibit BCR function by comparing BCR expression and internalization in a cohort of patient before and during therapy with Bruton's tyrosine kinase inhibitors (BTKi).
BCR internalization was assessed in two ways. First, we used a pH sensitive dye linked to agonistic anti-IgM (pHrodo-αIgM) to detect the uptake and retention of ligand/receptor complexes in acidified endosomes. Second, BCR internalization was assessed directly by measuring the rate of disappearance of surface IgM following ligation by agonistic anti-IgM. An increase in the percentage of cells showing pHrodo fluorescence above control was detected in all CLL cases studied (mean percentage pHrodo-αIgM uptake = 30.2±2.5%, p<0.0001, n=40) although there was considerable inter-patient variation. We observed a significant correlation between pHrodo-αIgM uptake and high surface IgM (sIgM) expression (r2=0.529, p=0.0001, n=40), and an association with markers of adverse prognosis including, CD38 positivity (p=0.001, n=40), and the capacity to mobilize calcium ions following BCR ligation (p=0.001, n=26). Cases of CLL with unmutated IGHV genes showed higher uptake of pHrodo-αIgM than mutated cases however this did not reach statistical significance (p=0.053, n=40).
Similarly, when BCR function within individual CLL patients was examined, we also found that pHrodo-αIgM uptake varied substantially and was maximal in lymph node (LN) derived CLL cells (p=0.03, n=6) and those in the peripheral blood (PB) that express the highest levels of CD5 (p=0.0001, n=26), a marker that is upregulated following BCR activation. In addition, we found that LN CLL cells expressed higher levels of sIgM than those derived from the PB (p=0.03, n=6). This was a surprising finding, as BCR stimulation is thought to occur within LNs, which might be expected to result in down regulated BCR expression.
When the level of BCR internalization and accumulation in the endosomes was adjusted for the number of sIgM molecules, we found that BCR internalization and retention was actually more efficient in anergic cases of CLL (defined by lack of ability to mobilize Ca2+ following stimulation with anti-IgM; p=0.0002, n=26). This observation was supported by direct measurement of the rate of BCR endocytosis, which showed a more rapid internalization in anergic B-cells compared to signaling competent and normal B-cells. Similar findings have previously been reported in a murine model of B-cell anergy.
Finally, data from BTKi treated CLL patients showed that, 12 months after commencing treatment, CLL B-cells exhibit lower levels of sIgM expression (p=0.02, n=7) and have more efficient BCR internalization than at the outset (p=0.05, n=7). Since low sIgM expression and more efficient BCR internalization is a feature of B-cell anergy, these results suggest that therapy with BTK inhibitors selectively depletes B-cells that are capable of signaling through the BCR and enriches for those displaying features of anergy.
Overall our data show that BCR internalization is uncoupled from intracellular signaling in CLL and is most efficient in cells that demonstrate poor downstream BCR signaling or show features of recent BCR stimulation. These observations are similar to those previously reported in anergic B-cells and provide further evidence for ongoing BCR activation and anergy in CLL.

Disclosures
Patten: Gilead: Research Funding. Devereux:Gilead: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Consultancy, Other: Travel, Accommodations, Expenses ; GSK: Consultancy.