Figure 1.
Circulating leukemic B cells recently emigrated from the LN in patients with CLL are enriched for cells simultaneously overexpressing multiple antiapoptotic proteins. (A-B) Patient PBMCs (N = 20) were examined using FCM for expression of apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2, Bim, Puma, Bak, and Bax) and markers of microenvironmental interactions (CD69 and CXCR4) in CLL cells (viability dye−/CD5+/CD19+). (A) Flow cytometry images present the gating strategy for identification of CLL cells, CD69/CXCR4-expressing CLL cells, and expression of apoptotic proteins in CD69−/CXCR4High and CD69+/CXCR4Low CLL cells. (B) The expression of antiapoptotic proteins Mcl-1, Bcl-xL, and Bcl-2 in CD69+/CXCR4Low CLL cells as compared with CD69−/CXCR4High CLL cells. Data are presented as fold difference in geometric mean fluorescence intensity (GMFLI). (C) FCS files were generated from pregated CD69+/CXCR4Low- or CD69−/CXCR4High-expressing CLL cells in every patient (N = 19) using FlowJo software, and cell clusters expressing different levels of antiapoptotic proteins (Mcl-1, Bcl-xL, and Bcl-2) were identified through unsupervised clustering analysis, as described in "Materials and methods." A heatmap was generated based on GMFLI values to show the expression of various antiapoptotic proteins in different clusters (left). The clusters were visualized using a bubble graph in which every bubble represents 1 cluster and the area of the bubble (size) is proportional to the mean percentage of cells in that cluster (middle). A table presenting the mean percentage of cells in each cluster identified in CD69+/CXCR4Low- or CD69−/CXCR4High-expressing CLL cells (right). The mean percentage of cells in a cluster was determined by calculating the average for that cluster across patients analyzed (N = 19). (D) PBMCs of patients with CLL (patients 25, 27, 52, 81, and 95), either fresh or after culturing ex vivo for 24 hours in RPMI containing 10% fetal calf serum without added agonists, were analyzed for expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and Bcl-2 using FCM. Data are presented as fold difference in GMFLI in CD69+ or CD69− CLL cells as compared with CD69− CLL cells from fresh PBMCs (ie, processed without ex vivo culture). Statistical significance was determined by ANOVA with Sidak’s post-hoc test for multiple comparisons. *P < .05; **P < .01; ****P < .0001; ns, not significant. Data are presented as mean ± SD. Neg, negative; Pos, positive.

Circulating leukemic B cells recently emigrated from the LN in patients with CLL are enriched for cells simultaneously overexpressing multiple antiapoptotic proteins. (A-B) Patient PBMCs (N = 20) were examined using FCM for expression of apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2, Bim, Puma, Bak, and Bax) and markers of microenvironmental interactions (CD69 and CXCR4) in CLL cells (viability dye−/CD5+/CD19+). (A) Flow cytometry images present the gating strategy for identification of CLL cells, CD69/CXCR4-expressing CLL cells, and expression of apoptotic proteins in CD69−/CXCR4High and CD69+/CXCR4Low CLL cells. (B) The expression of antiapoptotic proteins Mcl-1, Bcl-xL, and Bcl-2 in CD69+/CXCR4Low CLL cells as compared with CD69−/CXCR4High CLL cells. Data are presented as fold difference in geometric mean fluorescence intensity (GMFLI). (C) FCS files were generated from pregated CD69+/CXCR4Low- or CD69−/CXCR4High-expressing CLL cells in every patient (N = 19) using FlowJo software, and cell clusters expressing different levels of antiapoptotic proteins (Mcl-1, Bcl-xL, and Bcl-2) were identified through unsupervised clustering analysis, as described in "Materials and methods." A heatmap was generated based on GMFLI values to show the expression of various antiapoptotic proteins in different clusters (left). The clusters were visualized using a bubble graph in which every bubble represents 1 cluster and the area of the bubble (size) is proportional to the mean percentage of cells in that cluster (middle). A table presenting the mean percentage of cells in each cluster identified in CD69+/CXCR4Low- or CD69−/CXCR4High-expressing CLL cells (right). The mean percentage of cells in a cluster was determined by calculating the average for that cluster across patients analyzed (N = 19). (D) PBMCs of patients with CLL (patients 25, 27, 52, 81, and 95), either fresh or after culturing ex vivo for 24 hours in RPMI containing 10% fetal calf serum without added agonists, were analyzed for expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and Bcl-2 using FCM. Data are presented as fold difference in GMFLI in CD69+ or CD69− CLL cells as compared with CD69− CLL cells from fresh PBMCs (ie, processed without ex vivo culture). Statistical significance was determined by ANOVA with Sidak’s post-hoc test for multiple comparisons. *P < .05; **P < .01; ****P < .0001; ns, not significant. Data are presented as mean ± SD. Neg, negative; Pos, positive.

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