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Basal NF-κB inhibition impacts BCR responsiveness in CLL
BCR stereotypy would be indicative of the involve- ment of similar specific antigens and underlines the importance of antigenic stimulation and BCR specificity in the pathogenesis of CLL.4 In general, most U-CLL express a BCR that is polyreactive and recognizes self- and non-self-antigens with low-affinity binding.5-8 In addition, for some stereotypic CLL subsets the antigens recognized by their BCR have been identified.9-13
However, it was previously shown that the BCR from CLL cells could also be stimulated independently of exter- nal antigens, as the CDR3 regions are able to recognize an internal epitope in framework 2 (FR2) of the IGHV domain.14 This induces a higher level of antigen-indepen- dent autonomous BCR signaling, since these cells exhibit a higher Ca2+ level in their cytoplasm, as demonstrated in vitro using a triple knockout (TKO) cell system.14
We previously demonstrated that primary CLL cells generally have higher basal Ca2+ levels compared with peripheral B cells from healthy individuals.15 Basal Ca2+ levels correlated with IGHV mutational status, as we found on average higher basal Ca2+ levels in M-CLL than in U-CLL.14,15 However, our data also showed large varia- tion within the subgroups, as cases with high and low basal Ca2+ levels could be found in both M-CLL and U- CLL groups.15 Since there was no correlation with BCR characteristics (e.g., Ig expression level, HCDR3 length, charge and composition) or with cytogenetic aberrations, it is conceivable that high basal Ca2+ levels are partly directed by the SHM status and that cell-intrinsic differ- ences caused by cell anergy could explain the variation.15
Anergy is an immune state in which the cell is silenced upon low-affinity recognition of self-antigens.16 Anergic cells remain capable of antigen binding, but have a reduced ability to respond to BCR-dependent antigenic stimulation.16 Anergy has been linked to CLL based on low surface BCR expression, reduced responsive capabil- ity,17,18 and increased basal Ca2+ levels.15 M-CLL in particu- lar shows these increased basal Ca2+ levels in combination with a poorer response to BCR stimulation15 which is in line with other studies showing that the α-IgM response is associated with IGHV mutational status and with the surface expression of markers of prognosis, such as CD38.18,19 Moreover, a high level of surface IgM is associ- ated with a clinically aggressive form of the disease, which has potential implications as a diagnostic parame- ter for disease progression.20
However, Ca2+ levels, both basal and upon BCR stimu- lation, vary within the U-CLL and M-CLL groups. We hypothesized that this heterogeneity in BCR responsive- ness could reflect a diverse disease pathogenesis involving cell-intrinsic differences. In this study we aimed to eluci- date potential cell-intrinsic differences underlying the observed differences in Ca2+ levels between CLL cases.
Methods
Study population
Fifty-two patients were included of whom 30 (58%) had U-CLL and 22 (42%) had M-CLL as determined by the IGHV SHM status (Online Supplementary Methods). The patients’ characteristics are shown in Online Supplementary Table S1. The majority of the included patients (n=41, 79%) were treatment-naïve. Purified CLL cells were isolated (Online Supplementary Methods) upon informed consent and anonymized for further use, following the guidelines
of the institutional review board (METC-2015-741) and in accor- dance with the Declarations of Helsinki.
Flow cytometry
Flow cytometry was used to assess the responsive capacity upon α-IgM stimulation by measuring Ca2+ levels (Online Supplementary Methods) and to determine the expression of activa- tion markers by using antibodies listed in Online Supplementary Table S2. Phospho-flow analysis was done to study the phospho- rylation of Spleen tyrosine kinase (Syk), Phopholipase Cγ2 (PLCγ2) and Protein kinase B (Akt) upon α-IgM stimulation. (Online Supplementary Methods).
Cell culture and retroviral transduction of triple knockout cells
TKO cells, derived from a signaling-competent mouse pre-B-cell line lacking the expression of endogenous pre-BCR due to inacti- vation of RAG2 and λ5 genes,21 and Phoenix cells (ATCC CRL- 3214) were both cultured as described by Meixlsperger et al.21 The protocol used for the transduction of TKO cells was also docu- mented before by Meixlsperger et al.21
RNA sequencing
Twelve cases from our cohort were selected based on their responsiveness to α-IgM stimulation (6 responsive, 6 unrespon- sive) and their RNA was sequenced. The RNA was extracted using Allprep DNA/RNA/miRNA Universal (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA sequencing was performed on a TruSeq platform (Illumina, San Diego, CA, USA) at the Human Genome Facility (Erasmus Medical Center, Rotterdam, the Netherlands). Reads were extracted from the raw sequencing data using CASAVA 1.8.2 (Illumina) and aligned to the human reference genome (UCSC’s hg19) using the STAR (2.5.0c) splice aware aligner with gencode v19 transcriptome annotations as an additional template. The BAM files were processed using various tools from the picard software suite (v1.90), as well as tools from the Genome Analysis ToolKit (GATK, v3.5). Quality control metrics were collected at various steps using picard and evaluated, along with coverage metrics using GATK. Read counts per exon/gene were then determined by the featureCounts func- tion of the subread package (v1.4.6-p1) using the gencode v19 annotation as markers. The raw read counts were normalized through the fragments per kilobase of exon model per million reads mapped (FPKM) methodology, normalizing for library yield and gene size.
For classification analysis, the calculated Spearman correlation as a distance (1/similarity) measurement and Ward.D2 for the unsupervised clustering were applied to the samples used. R pack- ages (version 3.4.4) weres used for differential expression analysis and to create plots for visualization. We analyzed the sample fit- ting with edgeR, the gene-wise negative binomial generalized lin- ear models for contrast.
To validate transcript level differences in a larger cohort, RNA was synthesized to cDNA and real-time quantitative polymerase chain reaction (RQ-PCR) was performed (Online Supplementary Methods and Online Supplementary Table S2).
Results
Unmutated cases of chronic lymphocytic leukemia are generally more responsive than mutated cases to α-IgM stimulation
To determine whether high basal Ca2+ levels are BCR- dependent or caused by cell-intrinsic factors, we selected
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