Identification of cellular and signaling signatures of the Lck^hi subpopulations. (A) BCR-signaling profiles differ between Lck^hi and Lck^−/low subpopulations. Cells from group C CLL patients were left unstimulated or triggered with anti-human IgM/IgG. Graphs show pCD79a, pERK, and pAKT levels at steady state (−) and after BCR triggering (+) among Lck^−/low, Lck^hi, and corresponding T cells, as indicated. The Wilcoxon matched-pair test (dotted line) was used for comparisons between unstimulated samples and the Student t test (solid line) for comparison of responses to BCR crosslinking (n = 8; mean ± SEM; *P < .05; **P < .005; ***P < .0005). Lck^−/low cells from group A and B CLL patients respond to BCR crosslinking in a similar fashion as the Lck^−/low subpopulation from group C, as shown in supplemental Figure 1C. CD79a total protein expression in different subsets is also shown in supplemental Figure 1C. (B) Lck^hi subsets display a hyperactivated phenotype. PBMCs from group C CLL patients were stained with CD3, CD19, pERK and each of Lck, CD38, CD69, and Ki-67. Graphs display the expression levels of CD38, CD69, and Ki-67 in the Lck^hi, Lck^−/low subsets and T cells as indicated. The right graph shows the percentage of Ki-67⁺ cells within the Lck^hi and Lck^−/low subpopulations (n = 11; Student t test; mean ± SEM; **P < .01; ***P < .001). Supplemental Figure 1D-E shows a detailed analysis of a representative experiment. (C) Evaluation of the presence of Lck^hi subsets within proliferative (CXCR4^dim/CD5^bright) and resting (CXCR4^bright/CD5^dim) CLL fractions. Cells from group C patients were stained for CD19, CD5, CXCR4, and Lck. The CD19⁺CD5⁺ populations were analyzed in 2D FACS plots of CXCR4 vs CD5 (a) and of Lck vs CD19 (b). Top rows, The CXCR4^dim/CD5^bright and CXCR4^bright/CD5^dim fractions were defined in (a) as 5% of the cells lying on the 2 extremes of the plots, as previously described.²⁴  Each fraction shown as a 2D FACS plot of Lck vs CD19 was used to calculate the relative proportions of Lck^−/low and Lck^hi subpopulations within the CXCR4^bright/CD5^dim (a1) and CXCR4^dim/CD5^bright (a2) compartments, respectively. The graph on the right shows collective data. Bottom row, The Lck^hi subset from (b) is represented in a 2D FACS plot of CXCR4 vs CD5 (b1). The gates demarcating the CXCR4^bright/CD5^dim and CXCR4^dim/CD5^bright fractions from (a) were applied to (b1) to reveal the distribution of Lck^hi cells within the respective fractions and the dominant intermediate population. The graph on the right shows collective data (n = 8; Wilcoxon matched-pair test; *P < .05; **P < .005).