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Haematologica 2020 Volume 105(1):182-192
Chronic Lymphocytic Leukemia
Responsiveness of chronic lymphocytic leukemia cells to B-cell receptor stimulation
is associated with low expression of regulatory molecules of the nuclear factor-kB pathway
Ruud W.J. Meijers,1* Alice F. Muggen,1* Leticia G. Leon, 1Maaike de Bie,1 Jacques J.M. van Dongen,1,2 Rudi W. Hendriks3# and Anton W. Langerak1#
1Laboratory Medical Immunology, Department of Immunology, Erasmus MC, University
2
Medical Center Rotterdam, Rotterdam; Department of Immunohematology and Blood
3
Transfusion, Leiden University Medical Center, Leiden and Department of Pulmonary
Medicine, Erasmus MC, Rotterdam, the Netherlands
*RWJM and AFM share equal responsibility and first authorship #RWH and AWL share equal responsibility and senior authorship
ABSTRACT
Chronic lymphocytic leukemia (CLL) is a disease with heterogeneous clinical and biological characteristics. Differences in Ca2+ levels among cases, both basal and upon B-cell receptor (BCR) stimulation, may reflect heterogeneity in the pathogenesis due to cell-intrinsic factors. Our aim was to elucidate cell-intrinsic differences between BCR-responsive and -unresponsive cases. We therefore determined BCR responsiveness ex vivo based on Ca2+ influx upon α-IgM stimulation of purified CLL cell frac- tions from 52 patients. Phosphorylation levels of various BCR signaling molecules, and expression of activation markers were assessed by flow cytometry. Transcription profiling of responsive (n=6) and unresponsive cases (n=6) was performed by RNA sequencing. Real-time quantitative polymerase chain reaction analysis was used to validate transcript level dif- ferences in a larger cohort. In 24 cases an α-IgM response was visible by Ca2+ influx which was accompanied by higher phosphorylation of PLCγ2 and Akt after α-IgM stimulation in combination with higher surface expres- sion of IgM, IgD, CD19, CD38 and CD43 compared to the unresponsive cases (n=28). Based on RNA sequencing analysis several components of the canonical nuclear factor (NF)-kB pathway, especially those related to NF-kB inhibition, were expressed more highly in unresponsive cases. Moreover, upon α-IgM stimulation, the expression of these NF-kB pathway genes (especially genes coding for NF-kB pathway inhibitors but also NF-kB sub- unit REL) was upregulated in BCR-responsive cases while the level did not change, compared to basal level, in the unresponsive cases. These findings suggest that cells from CLL cases with enhanced NF-kB signaling have a lesser capacity to respond to BCR stimulation.
Introduction
Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy that is charac- terized by a monoclonal expansion of mature B cells with a homogeneous mor- phology and a characteristic immunophenotype.1 CLL is the most common type of leukemia in the Western world and mainly affects the elderly.1 Based on the somatic hypermutation (SHM) status of the immunoglobulin heavy chain (IGHV) gene, CLL can be divided into unmutated CLL (U-CLL) and mutated CLL (M-CLL), with U-CLL generally being a more aggressive form of the disease and M-CLL a more indolent form.2,3 Around 30% of all cases can be grouped into subsets based on so-called stereotypic B-cell receptors (BCR), which are identified by their restricted IGHV/IGHD/IGHJ gene usage plus similarities in length and amino acid sequence of their complementarity-determining region 3 (CDR3). 4
Correspondence:
ANTON W. LANGERAK
a.langerak@erasmusmc.nl
Received: January 1, 2019. Accepted: May 15, 2019. Pre-published: May 16, 2019.
doi:10.3324/haematol.2018.215566
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/1/182
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