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R.W.J. Meijers et al.
a small series of CLL samples with known high (n=3) or low (n=6) basal Ca2+ levels from our previous study in 2015,15 and cloned their BCR into TKO cells as described by Dühren-von Minden et al.14
Even though we could detect Ca2+ signaling by the BCR in TKO cells for all analyzed CLL-derived BCR expressed as IgM, we did not detect any correlation (R2=0.014, P=0.764) between the Ca2+ signal in CLL and that in TKO cells (Figure 1B) indicating that the high basal Ca2+ levels seen in some CLL samples would result from cell-intrinsic changes rather than from BCR-dependent autonomous signaling.
To determine which cell-intrinsic differences might cause the heterogeneity in Ca2+ signaling in basal condi- tions and upon BCR stimulation, we established a new cohort of patients (n=52, Online Supplementary Table S1). CLL cells were isolated from peripheral blood and imme- diately used for further analysis. First, basal Ca2+ levels were assessed (Figure 1B). Similar to the previous study, basal Ca2+ levels were heterogeneous in both U-CLL and M-CLL cases.15
Next, we examined the responsive capacity of the CLL samples upon BCR stimulation. Figure 1D shows two flow cytometric examples. In line with our previous study,15 we found that U-CLL cells in general responded significantly (P=0.049) better upon α-IgM stimulation compared with M-CLL cells (Figure 1E). Although no dif- ferences were found in the response after α-IgD stimula- tion (Figure 1F), there was a strong correlation between the relative response to α-IgM and α-IgD stimulation (R2=0.508, P<0.0001) (Figure 1G). Based on this, we fur- ther defined CLL subgroups based on BCR responsive- ness upon α-IgM stimulation. Twenty-four cases were classified as responsive (median fluorescence intensity ratio, response/basal signal: 1.1-6.5; n=17 U-CLL and n=7 M-CLL) and 28 cases were unresponsive (median fluores- cence intensity ratio, response/basal signal: <1.1; n=13 U- CLL and n=15 M-CLL).
Higher phosphorylation of PLCγ2 and Akt in chronic lymphocytic leukemia correlated with responsiveness upon B-cell receptor stimulation
expected, CLL cells from responsive cases displayed a sig- nificantly (P=0.0002) higher expression of surface IgM compared to the unresponsive cases; likewise, IgD (P=0.036), CD19 (P=0.029), CD38 (P=0.035), and CD43 (P=0.047) expression levels were also higher in responsive cases than in unresponsive cases (Figure 3A). No differ- ences were found in CD20, CD21, CD27, CD69, CD80, CD86 and CXCR4 expression (Online Supplementary Figure S1).
In order to gain a better understanding of BCR respon- sive capacity, as defined by Ca2+ influx, we examined phosphorylation of Syk, PLCγ2 and Akt upon α-IgM stimulation. First, we evaluated differences in basal phos- phorylation levels of Syk (pSyk), PLCγ2 (pPLCγ2) and Akt (pAkt) (Figure 2A). The responsive cases showed a signif- icantly (P=0.0013) higher basal pPLCγ2 level than unre- sponsive cases but no differences were found in basal pSyk and pAKT levels (Figure 2B). Next we examined the relative response of kinase phosphorylation upon BCR stimulation. Even though no difference in relative response of pSYK after α-IgM stimulation was found, the responsive patients had a higher relative response of pPLCγ2 and pAkt upon α-IgM stimulation (Figure 2C).
IkB-related genes in particular are differentially expressed between B-cell receptor-responsive and -unresponsive cases
Twelve cases from our cohort were selected to evaluate cell-intrinsic differences based on RNA sequencing of total RNA from MACS-purified (>95%) CLL cells. Six patients were classified based on Ca2+ levels as responders upon α-IgM stimulation and were compared to another six patients who were unresponsive. (Online Supplementary Table S3) First, RNA expression profiles of the 12 cases were compared to each other via Spearman correlation (Figure 4A). Based on these results the patients could be divided into three major clusters, which did not correlate with BCR responsiveness or SHM status. In addition, when comparing the variation in total gene expression levels between the samples, as shown by Z- scores in a heat map (Online Supplementary Figure S2), no clear division of responsive and unresponsive cases was found either, probably reflecting the biological hetero- geneity of CLL samples, even when classified as BCR- responsive and -unresponsive.
Next, we therefore focused on genes involved in BCR signaling using Qiagen’s Ingenuity Pathway Analysis (IPA). As illustrated by the volcano plot, responsive cases demonstrated significantly higher expression of EBF1, FCGR2A, SYK and FYN (positive logFC values), whereas the non-responders showed significantly higher expres- sion of NFKBID, NFKB2, CAM2KA, NFKBIE, RAF1, NFK- BIB, NFKB1, RPS6K1, PLCG1 and BCL3 (negative logFC values) (Figure 4B and Online Supplementary Figure S2). Interestingly, the NFKBIB, NFKBID and NFKBIE genes all encode inhibitors of NF-kB (IkB), while NFKB1, NFKB2 and BCL3 are genes coding for NF-kB components that are associated with inhibition.22
B-cell receptor-unresponsive cases have higher expression of genes expressing regulatory molecules of nuclear factor-kB signaling
Additional samples were selected (n=13 unresponsive, n=15 responsive) to validate the differences in transcript levels of NF-kB genes (NFKB1, NFKB2, BCL3, NFKBIB, NFKBID and NFKBIE) using RQ-PCR. RQ-PCR results (displayed as 2-deltaCT values) indeed confirmed that responding cases had significantly lower expression of NFKB1 and NFKB2 (Figure 5A) NFKBIB and NFKBIE (Figure 5B). Furthermore, we found a trend towards lower NFKBID expression, but no difference in BCL3 expres- sion between the subgroups (Figure 5B).
In addition, we investigated whether the transcription-
Taken together, the α-IgM response as determined by Ca2+ influx, is consistent with greater phosphorylation of pPLCy2 and pAkt upon α-IgM stimulation
Chronic lymphocytic leukemia cases showing good B-cell receptor responsiveness have a more activated phenotype
Next we examined whether the expression of activa- tion markers is associated with the response to α-IgM. As
To determine whether the α-IgM responsiveness with- in the responsive cases correlates with the expression level of these markers, we compared surface expression and relative response. The relative response did correlate with surface IgM (R2=0.322, P=0.0038) and CD21 (R2=0.469, P=0.0002) expression levels (Figure 3B).
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