iNKT cells control CLL survival by restraining proleukemia functions of NLCs. (A) iNKT cells impair NLC generation in vitro. Shown is the number of adherent NLCs differentiated from CD14⁺ monocytes of healthy donors upon culturing in vitro for 7 days with or without purified CD1d^neg CLL cells, healthy iNKT cells, anti-CD1d blocking mAb. (Left panel) Quantitative image analyses of 60 different fields were taken from each well. (Right panel) CLL cells cultured with or without iNKT cells or monocytes were collected and counted by flow cytometry in duplicates. Results are expressed as mean ± SD and are represent 1 of 3 consistent experiments. **P ≤ .005; ***P ≤ .0005 1-way ANOVA. (B-F) iNKT cells impair CLL and NLC survival in vitro. T-cell–depleted PBMCs from a CD1d^neg CLL patient were cultured in vitro with or without purified healthy iNKT cells. After 14 days of culture, CLL cells were collected and adherent cells were assessed for morphology in (B) bright field (original magnification ×20) and (C) stained with CD68-FITC (green) and DAPI (blue) for immunofluorescence (original magnification ×20). Quantitative image analyses of 20 different fields were taken directly from the same culture wells to determine (D) total adherent cell counts and (E) CD68⁺ adherent cell counts. (F) The CLL cells that were harvested from the same wells were counted by flow cytometry in quadruplicate. Bars show mean ± SD. Data refer to 1 of 2 independent experiments performed with different patients that gave comparable results.*P < .05; ****P ≤ .00005 Wilcoxon test. Immunofluorescence and counts were performed by In Cell Analyzer 1000 GE Healthcare and In Cell Analyzer 1000 Workstation or Arrayscan XTI (Thermo Fisher) with HCS studio software. Bright field image acquisitions were performed with Zeiss Axio Observer.z1 with QImaging EXI-Blue equipped with Velocity acquisition software.