The effect of microenvironmental interactions on CD20 expression in CLL cells. (A) Relative CD20 mRNA (MS4A1) expression in samples obtained before and during ibrutinib treatment of CLL patients (N = 8 patients; for characterization of CLL samples, see supplemental Table 1, sample no. CLL102-109). Samples were acquired the day before the first administration of ibrutinib (Pre), and on day 15, and/or week 5 and/or week 12 after the first ibrutinib administration. (B) A representative example of the gating of CXCR4^dimCD5^bright and CXCR4^brightCD5^dim CLL cell populations analyzed using flow cytometry (top). The histogram of the surface CD20 expression on CLL cells gated in the top panel (bottom). (C) The mean fluorescence intensity (MFI) for the surface CD20 on CXCR4^dimCD5^bright and CXCR4^brightCD5^dim CLL cells (N = 21 pairs; for characterization of CLL samples, see supplemental Table 2). The statistical difference was tested by paired Student t test. (D) Normalized CD20 mRNA (MS4A1) expression in sorted CXCR4^dimCD5^bright and CXCR4^brightCD5^dim CLL cells (N = 7 pairs; purity >99% CD5⁺CD19⁺ cells; for characterization of CLL samples, see supplemental Table 2). The statistical difference was tested by paired Student t test. (E) Freshly obtained CLL cells (N = 6, purity >99% CD5⁺CD19⁺ cells; for characterization of CLL samples, see supplemental Table 2) were seeded (2.5 × 10⁶ cells per mL) on plastic (control) or an HS-5 monolayer. CLL cells were pretreated with vehicle (labeled HS-5) or ibrutinib (HS-5 + ibrutinib) for 2 hours prior to being seeded on the HS-5 stromal cells. The CLL cells were treated with ibrutinib (1 μM) prior to seeding on stromal cells to ensure full BTK inhibition before the contact of B cells with HS-5 cells. The control stands for CLL cells treated with vehicle and cultured on plastic with no contact with stromal cells. After 48 hours in cultivation, all cells in the wells were harvested and labeled with anti-CD20 antibody and Annexin-V, and CD20 MFI was analyzed on viable CD105-negative cells (ie, CLL cells) by flow cytometry. CD105 was used as a stromal cell marker. (F) A representative plot showing gating strategy of 5 subpopulations based on CXCR4 and CD5 expression (i). The relative surface CD20 expression in CLL subpopulations gated according to CXCR4/CD5 levels (ii; N = 9 CLL samples). The statistical difference was tested by paired Student t test. (G) Freshly obtained CLL cells (N = 4, purity >99% CD5⁺CD19⁺ cells; for characterization of CLL samples, see supplemental Table 2) were seeded on a plastic surface at a concentration of 2.5 × 10⁶ cells per mL and treated with recombinant human SDF-1α (100 or 500 ng/mL), or vehicle (control) and cultured for 24/48/72 hours. After the indicated cultivation period, the CLL cells were harvested and labeled with anti-CD20 antibody and Annexin-V, and CD20 MFI was analyzed on viable cells using flow cytometry. MFI on the control cells was set as 1. *P ≤ .05. (H) Freshly obtained primary CLL cells (N = 5, purity >99% CD5⁺CD19⁺ cells; for characterization of CLL samples, see supplemental Table 2) were seeded on plastic (2.5 × 10⁶ cells per mL) and treated with SDF-1α (labeled SDF-1α) or plerixafor and SDF-1α (SDF-1α + plerixafor) or ibrutinib and SDF-1α (SDF-1α + ibrutinib). Ibrutinib (1 μM) or plerixafor (5 μg/mL) was added to the cell culture 2 hours prior to SDF-1α treatment (500 ng/mL) to ensure full BTK/CXCR4 inhibition before the contact of B cells with the SDF-1α chemokine. The control stands for cells that were treated with vehicle and cultured on plastic with no inhibitor or SDF-1α treatment. After 48 hours in cultivation, all cells in the wells were harvested and labeled with anti-CD20 antibody and Annexin-V, and CD20 MFI was analyzed on viable cells using flow cytometry.