PMC7388238_01
PMC7388238_02
PMC7388238_03
LC3B expression in CLL cells. (A) PBMC cells from patients with CLL were treated with 25 nM bafilomycin (Baf) or medium (untreated control) for 24 h. Flow cytometric analysis of LC3B expression in CLL cells is shown. (i) Dot plot of CD19 vs. side scatter (SSC) of B lymphocytes from CLL patients. (ii) Dot plot of CD19 vs. CD5 shows clonal CLL cells and residual T cells. (iii) Histogram showing LC3B expression (MFI) in clonal B cells treated with Baf and untreated cells. (B) LC3B index B/M was calculated by dividing MFI of LC3B of CD19+CD5+ (clonal B cells) treated with bafilomycin by MFI of LC3B of untreated clonal B cells (*p < 0.05, unpaired t-test). (C) Automatic population separator (APS) shows two populations: LC3B+ CLL cells from two patients with unmutated IGHV (Purple) and LC3B+ CLL cells from two patients with mutated IGHV (Green). (D) PBMC cells from all four patients (CLL#1, Patient #1; CLL#2, Patient #2; CLL#3, Patient #3; CLL#4, Patient #4) were treated with bafilomycin 25 nM for 2 h. After that, MFI of LC3B was determined by flow cytometry, as described in (A).
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LC3B. Chronic lymphocytic leukemia B cells were identified and gated using the combination of CD5 plus CD19 staining, for the evaluation of LC3B expression. Lipidated membrane-located LC3B fraction (LC3BII) was accumulated in the presence of bafilomycin (Figure 1A). We observed higher expression of LC3BII in cells from unmutated progressing CLL patients compared to the cells from mutated non-progressing CLL patients (p < 0.05) (Figure 1B). In addition, using InfinicytTM software, we merged files of all the patients into one file and then analyzed immunophenotype-based automatic separation of cell clusters (automatic population separation, APS) (InfinicytTM software, Cytognos S.L., Salamanca, Spain) based on LC3BII expression (Figure 1C). The APS algorithm clearly discriminated two groups of data corresponding to samples from progressing and non-progressing CLL patients, respectively (Figure 1C). In addition, similar results were obtained when LC3BII expression was studied in cells from CLL patients by flow cytometry (Figure 1D) and the classical Western immunoblotting detection of LC3B (Figure 2A). In the presence of bafilomycin, increased LC3BII levels were observed in samples from unmutated progressing CLL patients (Figure 1D). Minimal LC3BII detection was observed in samples from mutated patients (CLL#3 and CLL#4) (Figure 1D). Similar results were obtained by flow cytometry (Figure 1) after Ibrutinib treatment.
(A) PBMC cells from all four patients (CLL#1, Patient #1; CLL#2, Patient #2; CLL#3, Patient #3; CLL#4, Patient #4) were treated as described in Figure 1D, then LC3B and beta-actin protein levels were examined by immunoblotting. The figure shows image and relative band intensity quantification. The results were analyzed by t-test. Error bars represent SEM (**p < 0.01). (B) In addition to LC3B and beta-actin, p62 and Beclin protein levels were examined by immunoblotting in new samples from survivor Patients #3 and #4 (CLL#3 and CLL#4, respectively). The results were analyzed by t-test. No significant differences were observed.
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In addition, similar results were obtained when LC3BII expression was studied in cells from CLL patients by flow cytometry (Figure 1D) and the classical Western immunoblotting detection of LC3B (Figure 2A). Western immunoblotting. We did not find significant differences in the expression levels of autophagy proteins between untreated and bafilomycin-treated samples (Figure 2B). It is important to notice that increased LC3BII expression was detected by Western immunoblotting (Figure 2A) when the patients progressed but before Ibrutinib treatment.