Figure 2.

Breg-like CLL cells produce IL-10 and are highly sensitive to the cytotoxic effects of anti-CD38 agents. (A-C) PBMCs from patients with CLL (n = 10) and healthy donors (n = 5), were CD19⁺CD5⁺ flow sorted and further gated based on CD24⁺CD38⁺ surface expression (A) (representative density plot shown to illustrate gating strategy: Q1, CD24^loCD38^hi; Q2, CD24^hiCD38^hi; Q3, CD24^loCD38^lo; and Q4, CD24^hiCD38^lo) followed by measurement of intracellular IL-10 within the PBMC fraction from healthy donors (B) and patients with CLL (C). (D) PBMCs from patients with CLL (n = 5) were treated with vehicle, daratumumab (1 µg/mL), kuromanin (30 µM), or 78c (0.5 µM), followed by quantification of Breg-like CLL cells. (E) PBMCs from patients with CLL (n = 5) were sorted to enrich for Breg-like CLL cells (CD19⁺CD5⁺CD24⁺CD38⁺) and non-Breg CLL cells (CD19⁺CD5⁺CD24⁺CD38⁻) and treated with vehicle, daratumumab (1 µg/mL), or kuromanin (30 µM). After 24 hours, cells were stained with annexin V and propidium iodide (PI) and percentage apoptotic cells measured on a flow cytometer. Contour or density plots are representative and compiled data are presented as mean ± SEM, with individual data points overlaid. Each experiment was performed at least twice in duplicate. *P < .05; **P < .001. NS, not significant.