Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma

Egil S Blix,Anne Husebekk,June H Myklebust,Arne Kolstad,Jan Delabie,Jonathan M Irish,Anne M Tierens,Lise Forfang

doi:10.1186/1471-2407-12-478

Abstract

BackgroundKnowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. B-cell receptor (BCR) and co-receptor CD40 signaling is essential for normal B cells, and there is increasing evidence that signaling via BCR and CD40 plays an important role in the pathogenesis of B-cell lymphoma. The aim of this study was to investigate basal and induced signaling in lymphoma B cells and infiltrating T cells in single-cell suspensions of biopsies from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and marginal zone lymphoma (MZL) patients.MethodsSamples from untreated SLL/CLL and MZL patients were examined for basal and activation induced signaling by phospho-specific flow cytometry. A panel of 9 stimulation conditions targeting B and T cells, including crosslinking of the B cell receptor (BCR), CD40 ligand and interleukins in combination with 12 matching phospho-protein readouts was used to study signaling.ResultsMalignant B cells from SLL/CLL patients had higher basal levels of phosphorylated (p)-SFKs, p-PLCγ, p-ERK, p-p38, p-p65 (NF-κB), p-STAT5 and p-STAT6, compared to healthy donor B cells. In contrast, anti-BCR induced signaling was highly impaired in SLL/CLL and MZL B cells as determined by low p-SFK, p-SYK and p-PLCγ levels. Impaired anti-BCR-induced p-PLCγ was associated with reduced surface expression of IgM and CD79b. Similarly, CD40L-induced p-ERK and p-p38 were also significantly reduced in lymphoma B cells, whereas p-p65 (NF-κB) was equal to that of normal B cells. In contrast, IL-2, IL-7 and IL-15 induced p-STAT5 in tumor-infiltrating T cells were not different from normal T cells.ConclusionsBCR signaling and CD40L-induced p-p38 was suppressed in malignant B cells from SLL/CLL and MZL patients. Single-cell phospho-specific flow cytometry for detection of basal as well as activation-induced phosphorylation of signaling proteins in distinct cell populations can be used to identify aberrant signaling pathways.

Highlights

Knowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy
Low CD79b expression correlates with impaired BCRinduced p-PLCγ We investigated if impaired B-cell receptor (BCR) signaling in malignant B cells from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) or marginal zone lymphoma (MZL) patients could be explained by loss or reduced cell surface levels of IgM, CD79a or CD79b
We found that BCR-induced p-Src family kinases (SFKs), p-SYK/p-Zap70, p-PLCγ and p-ERK were highly impaired in SLL/CLL and MZL lymphoma B cells, compared to normal B cells

Summary

Introduction

Knowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and marginal zone lymphoma (MZL) are indolent lymphoid malignancies that arise from mature B cells. PLCγ activates PKC via DAG, and this further phosphorylates downstream signaling proteins like ERK, p38 and leads to activation of the pleiotropic transcription factor NF-κB (p65) [5]. The balance of these signals determines the B-cell fate [3]. There is increasing evidence that signaling via BCR plays an important role in the pathogenesis of CLL [1]. There are conflicting results whether exposure to anti-IgM in vitro promotes or suppresses apoptosis in CLL cells [6] and other signals provided by the tumor microenvironment likely determines the outcome [7]

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