Figure 1.
Figure 1. Profiling of ectopic Lck expression patterns in CLL. (A) Interpatient and intratumoral heterogeneity of CLL according to ectopic Lck expression. PBMCs from HDs (n = 15) and from CLL patients (n = 43) were stained with anti-Lck, anti-CD19, and anti-CD3. The gating strategy and additional CD19/CD5 double staining is shown in supplemental Figure 1A. The lymphocyte populations gated to exclusively include CD3+ (representing the T-cell population) and CD19+ (normal B cells or CLL cells) cells are presented as 2-dimensional (2D) fluorescence-activated cell sorting (FACS) plots of Lck vs CD3 staining, to enable a visual comparison of Lck expression between the B/CLL cells (CD19+/CD3−) and T cells (CD3+). CLL cells were gated as Lck−, Lcklow, Lck−/low, and Lckhi relative to the Lck expression in corresponding T cells and used to classify samples in 3 groups A, B, and C (Gp.A, Gp.B, and Gp.C), as indicated. B cells and T cells from HDs were used as negative and positive controls for anti-Lck staining, respectively. Representative FACS plots are shown. (B) Lckhi subsets contain elevated SFK activity and constitutive pERK. PBMCs from HDs and CLL patients were stained with CD3, CD19, Lck, and either pY416 (which measures global SFK activity) or pERK. Graphs display the geometrical mean fluorescence intensity values for Lck, pY416, and pERK staining within gated cell populations as defined in panel A. Data points from individual samples and medians (horizontal lines) are shown. The Mann-Whitney U test was used for comparisons between samples (solid lines) and the Wilcoxon matched-pair test between populations within the same sample (dotted lines). Relevant comparisons are shown. *P < .05; ***P < .0005. CD19 and CD5 expression levels in the different CLL subsets are shown in supplemental Figure 1B. (C) Lck inhibition diminishes constitutive ERK phosphorylation in Lckhi subpopulations. Cells from group C CLL patients were either left untreated or treated with the specific Lck inhibitors Lck-i and A770041. Graphs show the parallel reduction in SFK activity (anti-pY416 staining) and pERK levels (anti-pERK staining) in the Lckhi subset and corresponding T cells after treatment, as indicated (n = 6; Student t test; mean ± standard error of the mean [SEM]; *P < .05; **P < .005). ns, not significant.

Profiling of ectopic Lck expression patterns in CLL. (A) Interpatient and intratumoral heterogeneity of CLL according to ectopic Lck expression. PBMCs from HDs (n = 15) and from CLL patients (n = 43) were stained with anti-Lck, anti-CD19, and anti-CD3. The gating strategy and additional CD19/CD5 double staining is shown in supplemental Figure 1A. The lymphocyte populations gated to exclusively include CD3+ (representing the T-cell population) and CD19+ (normal B cells or CLL cells) cells are presented as 2-dimensional (2D) fluorescence-activated cell sorting (FACS) plots of Lck vs CD3 staining, to enable a visual comparison of Lck expression between the B/CLL cells (CD19+/CD3−) and T cells (CD3+). CLL cells were gated as Lck−, Lcklow, Lck−/low, and Lckhi relative to the Lck expression in corresponding T cells and used to classify samples in 3 groups A, B, and C (Gp.A, Gp.B, and Gp.C), as indicated. B cells and T cells from HDs were used as negative and positive controls for anti-Lck staining, respectively. Representative FACS plots are shown. (B) Lckhi subsets contain elevated SFK activity and constitutive pERK. PBMCs from HDs and CLL patients were stained with CD3, CD19, Lck, and either pY416 (which measures global SFK activity) or pERK. Graphs display the geometrical mean fluorescence intensity values for Lck, pY416, and pERK staining within gated cell populations as defined in panel A. Data points from individual samples and medians (horizontal lines) are shown. The Mann-Whitney U test was used for comparisons between samples (solid lines) and the Wilcoxon matched-pair test between populations within the same sample (dotted lines). Relevant comparisons are shown. *P < .05; ***P < .0005. CD19 and CD5 expression levels in the different CLL subsets are shown in supplemental Figure 1B. (C) Lck inhibition diminishes constitutive ERK phosphorylation in Lckhi subpopulations. Cells from group C CLL patients were either left untreated or treated with the specific Lck inhibitors Lck-i and A770041. Graphs show the parallel reduction in SFK activity (anti-pY416 staining) and pERK levels (anti-pERK staining) in the Lckhi subset and corresponding T cells after treatment, as indicated (n = 6; Student t test; mean ± standard error of the mean [SEM]; *P < .05; **P < .005). ns, not significant.

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