Characterization of the Intraclonal Complexity of Chronic Lymphocytic Leukemia B Cells: Potential Influences of B-Cell Receptor Crosstalk with Other Stimuli

SIMPLE SUMMARY: Chronic lymphocytic leukemia (CLL) clones contain cells differing in age: recently born, proliferative (PF), intermediate (IF), and resting (RF) fractions. We used deuterium incorporation into newly synthesized DNA in leukemic cells from patients with CLL to refine the “aging” kinetics, characterizing additional fractions differing in surface membrane (sm) CXCR4/CD5 levels, i.e., CXCR4(Dim)CD5(Dim) double dim fraction (DDF) and CXCR4(Bright)CD5(Bright) double bright fraction (DBF); and fractions differing in (sm)IgM and IgD densities. Although DDF was enriched in younger and DBF in older cells, PF and RF remained the youngest and oldest cells, respectively. Similarly, when using smIG to define subsets, cells with high smIgM and smIgD were the youngest, while cells with low smIgM and smIgD were the oldest. The youngest cells bore high levels of smIG and stimulating them via TLR9 and smIG yielded a phenotype that is more consistent with this in vivo observation. Finally, older cells were less sensitive to in vivo inhibition by ibrutinib. These data define additional CLL subpopulations; suggest that smIGs stimulation alone might not be responsible for the observed smIgM phenotype; and suggest that differential sensitivities of distinct fractions to the actions of ibrutinib might account, in part, for therapeutic relapse. ABSTRACT: Chronic lymphocytic leukemia (CLL) clones contain subpopulations differing in time since the last cell division (“age”): recently born, proliferative (PF; CXCR4(Dim)CD5(Bright)), intermediate (IF; CXCR4(Int)CD5(Int)), and resting (RF; CXCR4(Bright)CD5(Dim)) fractions. Herein, we used deuterium ((2)H) incorporation into newly synthesized DNA in patients to refine the kinetics of CLL subpopulations by characterizing two additional CXCR4/CD5 fractions, i.e., double dim (DDF; CXCR4(Dim)CD5(Dim)) and double bright (DBF; CXCR4(Bright)CD5(Bright)); and intraclonal fractions differing in surface membrane (sm) IgM and IgD densities. Although DDF was enriched in recently divided cells and DBF in older cells, PF and RF remained the most enriched in youngest and oldest cells, respectively. Similarly, smIgM(High) and smIgD(High) cells were the youngest, and smIgM(Low) and smIgD(Low) were the oldest, when using smIG levels as discriminator. Surprisingly, the cells closest to the last stimulatory event bore high levels of smIG, and stimulating via TLR9 and smIG yielded a phenotype more consistent with the in vivo setting. Finally, older cells were less sensitive to in vivo inhibition by ibrutinib. Collectively, these data define additional intraclonal subpopulations with divergent ages and phenotypes and suggest that BCR engagement alone is not responsible for the smIG levels found in vivo, and the differential sensitivity of distinct fractions to ibrutinib might account, in part, for therapeutic relapse.