![Figure 2. Extrinsic microenvironmental agonists generate resistance to IBR+VEN. (A) Diagram showing the patient PBMCs cocultured with stromal cells or preincubated with soluble agonists for 12 hours. Drugs as well as a second dose of agonists were added at 12 hours and cultured for an additional 12 hours, and cleaved PARP or dead cell staining in CD5+/CD19+ cells in CLL or MCL was analyzed by flow cytometry. (B) PBMCs from CLL patients (Pts 01, 03, and 12) were cocultured with HS-5 or HK cell line at a 10:1 ratio or preincubated with sCD40L (2 μg/mL), IL-10 (0.015 μg/mL), CpG-ODN (1.5 μg/mL), CXCL13 (0.5 μg/mL), IL-2 (1.54 ng/mL), BAFF (0.25 μg/mL), or IgM (25 μg/mL) and treated with IBR (0.1 μM) + VEN (25 nM) or DMSO as well as second doses of agonists for 12 hours as shown in panel A. Cleaved PARP in CD5+/CD19+ cells was analyzed by flow cytometry. (C-D) PBMCs from 22 CLL (Pts 1-3, 6-7, 12, 14, 21, 25-28, 33, 35, 39, 43, 46, 48, 51-52, 56, and 59) and 8 MCL (Pts 22-23, 38, 45, 49, 55, 60, and 66) patients and the Mino MCL cell line were preincubated with agonist mix (sCD40L [2 μg/mL] + IL-10 [0.015 μg/mL] + CpG-ODN [1.5 μg/mL]) for 12 hours. Samples were then incubated with IBR (0.1 μM) + VEN (25 nM) or DMSO for an additional 12 hours as well as a second dose of agonist mix. Cleaved PARP in CD5+/CD19+ cells in CLL samples (C) and MCL samples/Mino cell line (D) were analyzed by flow cytometry. (E) Ki67+/CD5+/CD19+ cells in CLL and MCL patient PBMCs (Pts 06, 35, 39, 43, and 45) preincubated with or without 2 doses of agonist mix at 12-hour intervals were analyzed by flow cytometry. (F) PBMCs from CLL patients (Pt 01 and 25) were preincubated with agonist mix for 12 hours and treated with increasing concentrations of IBR (0.1, 1, and 10 μM) and VEN (6.25, 12, 25, 100, 400 nM) for 12 hours along with a second dose of agonist mix. The cleaved PARP in CD5+/CD19+ cells was analyzed by flow cytometry. Data are expressed as means ± SD. The statistical significance was determined by ANOVA. *P < .05, ***P < .001, ****P < .0001. FACS, fluorescence-activated cell sorter.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/1/14/10.1182_bloodadvances.2016004176/3/advances004176f2.jpeg?Expires=1724940873&Signature=PPIjhbir6gYX4ekOYnWRtGM56NnjW9waR7Oti3x4BBJK8yirZqVfoW544batk~HnKM-kMPuT7HH4i5mY7XAuhHDYxkz7HOHK4kLhDarj6jcGIKwctsejure7g8JC3w8Sp8Xaui4HXm5EEq-iiRCY86mM~lN-wqVd7oP1S5N4n0OS0LXoIEs8tIiMwsKRhLyVeYk0ljT6RRgPQ3Zk51vfamO7gH3IGkdMCyqYG9~VVYkoVffVujJl9cSTLHDCiF3fDu6f5VyjaM2259XJ8Lm8zhv2gPQVqtw4hkR50~jfiMwvkGZVnarCoCk5cPdhls2cxqD-ltgCUxA7RATG53Xj-g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Extrinsic microenvironmental agonists generate resistance to IBR+VEN. (A) Diagram showing the patient PBMCs cocultured with stromal cells or preincubated with soluble agonists for 12 hours. Drugs as well as a second dose of agonists were added at 12 hours and cultured for an additional 12 hours, and cleaved PARP or dead cell staining in CD5+/CD19+ cells in CLL or MCL was analyzed by flow cytometry. (B) PBMCs from CLL patients (Pts 01, 03, and 12) were cocultured with HS-5 or HK cell line at a 10:1 ratio or preincubated with sCD40L (2 μg/mL), IL-10 (0.015 μg/mL), CpG-ODN (1.5 μg/mL), CXCL13 (0.5 μg/mL), IL-2 (1.54 ng/mL), BAFF (0.25 μg/mL), or IgM (25 μg/mL) and treated with IBR (0.1 μM) + VEN (25 nM) or DMSO as well as second doses of agonists for 12 hours as shown in panel A. Cleaved PARP in CD5+/CD19+ cells was analyzed by flow cytometry. (C-D) PBMCs from 22 CLL (Pts 1-3, 6-7, 12, 14, 21, 25-28, 33, 35, 39, 43, 46, 48, 51-52, 56, and 59) and 8 MCL (Pts 22-23, 38, 45, 49, 55, 60, and 66) patients and the Mino MCL cell line were preincubated with agonist mix (sCD40L [2 μg/mL] + IL-10 [0.015 μg/mL] + CpG-ODN [1.5 μg/mL]) for 12 hours. Samples were then incubated with IBR (0.1 μM) + VEN (25 nM) or DMSO for an additional 12 hours as well as a second dose of agonist mix. Cleaved PARP in CD5+/CD19+ cells in CLL samples (C) and MCL samples/Mino cell line (D) were analyzed by flow cytometry. (E) Ki67+/CD5+/CD19+ cells in CLL and MCL patient PBMCs (Pts 06, 35, 39, 43, and 45) preincubated with or without 2 doses of agonist mix at 12-hour intervals were analyzed by flow cytometry. (F) PBMCs from CLL patients (Pt 01 and 25) were preincubated with agonist mix for 12 hours and treated with increasing concentrations of IBR (0.1, 1, and 10 μM) and VEN (6.25, 12, 25, 100, 400 nM) for 12 hours along with a second dose of agonist mix. The cleaved PARP in CD5+/CD19+ cells was analyzed by flow cytometry. Data are expressed as means ± SD. The statistical significance was determined by ANOVA. *P < .05, ***P < .001, ****P < .0001. FACS, fluorescence-activated cell sorter.
