Figure 3
Figure 3. The phenotype of CD19− BMPC indicates terminal PC differentiation and prosurvival capacity. (A) CD19+ and CD19− BMPC were isolated from 4 donors using a combined magnetic-activated cell-sorting/FACS protocol (supplemental Figure 4) and were subjected to global gene expression profiling. A heat map resulting from unsupervised clustering of data of 859 significantly differentially expressed probe sets reflecting 689 genes (supplemental Table IIIA) according to high-performance chip data analysis (HPCDA) after Bonferroni correction is shown. (B) CD19+ and CD19− BMPC (CD38highCD3−CD14−DAPI−) were analyzed by flow cytometry for the expression of CD28, CD56, HLA-DR, or CD95. Histograms representative of 8 (CD28), 14 (CD56), 18 (HLA-DR), or 19 (CD95) donors are shown. Numbers represent median values of the geometric mean fluorescence intensity values of all donors analyzed, frequencies represent median values of frequencies of PC expressing a marker gated as illustrated by the black bars in the histogram plots, where applicable (gray, CD19− BMPC; black CD19+ BMPC). Both median fluorescence intensity and frequency data were compared using the Wilcoxon test (**P < .01; ***P < .001). (C) Affymetrix signal intensity data for selected molecules involved in apoptosis regulation not contained in the list of differentially expressed genes as delivered by HPCDA, that is, BCL2 (B-cell lymphoma 2, probeset 203685_at), TNFRSF6 (CD95), TNFRSF10B (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), BID (BH3 interacting-domain death agonist), and for caspases 8 and 3 (supplemental Table IIIB), were extracted from global gene expression analysis of CD19+ and CD19− BMPC. A distinct probeset for BCL2 (203685_at) was identified as differentially expressed by HPCDA. Student t test P values (BCL2, 0.05; TNFRSF6, 0.01; TNFRSF10B, 0.10; BID, 0.002; CASP8, 5.3 × 10−5; and CASP3, 1.1 × 10−5, were not considered significant according to the significance level of 0.05 corrected for multiple comparisons in the overall analysis (P = 2.29 × 10−8).

The phenotype of CD19− BMPC indicates terminal PC differentiation and prosurvival capacity. (A) CD19+ and CD19− BMPC were isolated from 4 donors using a combined magnetic-activated cell-sorting/FACS protocol (supplemental Figure 4) and were subjected to global gene expression profiling. A heat map resulting from unsupervised clustering of data of 859 significantly differentially expressed probe sets reflecting 689 genes (supplemental Table IIIA) according to high-performance chip data analysis (HPCDA) after Bonferroni correction is shown. (B) CD19+ and CD19− BMPC (CD38highCD3−CD14−DAPI−) were analyzed by flow cytometry for the expression of CD28, CD56, HLA-DR, or CD95. Histograms representative of 8 (CD28), 14 (CD56), 18 (HLA-DR), or 19 (CD95) donors are shown. Numbers represent median values of the geometric mean fluorescence intensity values of all donors analyzed, frequencies represent median values of frequencies of PC expressing a marker gated as illustrated by the black bars in the histogram plots, where applicable (gray, CD19− BMPC; black CD19+ BMPC). Both median fluorescence intensity and frequency data were compared using the Wilcoxon test (**P < .01; ***P < .001). (C) Affymetrix signal intensity data for selected molecules involved in apoptosis regulation not contained in the list of differentially expressed genes as delivered by HPCDA, that is, BCL2 (B-cell lymphoma 2, probeset 203685_at), TNFRSF6 (CD95), TNFRSF10B (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), BID (BH3 interacting-domain death agonist), and for caspases 8 and 3 (supplemental Table IIIB), were extracted from global gene expression analysis of CD19+ and CD19− BMPC. A distinct probeset for BCL2 (203685_at) was identified as differentially expressed by HPCDA. Student t test P values (BCL2, 0.05; TNFRSF6, 0.01; TNFRSF10B, 0.10; BID, 0.002; CASP8, 5.3 × 10−5; and CASP3, 1.1 × 10−5, were not considered significant according to the significance level of 0.05 corrected for multiple comparisons in the overall analysis (P = 2.29 × 10−8).

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