Monocytes and NLCs from CLL patients express CD1d. (A-B) CD1d expression by NLCs in vivo. Frozen lymph node sections from 2 patients with progressive CLL co-stained with anti-CD1d mAb plus goat anti-mouse fluorescein isothiocyanate (FITC) (green), anti-CD68 mAb plus goat anti-rabbit phycoerythrin (PE) (red) Abs, and DAPI (blue). Macrophages co-expressing CD1d and CD68 (yellow arrows) are detectable scattered within (A) dense macrophage infiltrates (original magnification ×400) or in (B) macrophage clusters (original magnification ×200), surrounded by lymphoid elements compatible with CLL cell morphology. Sections were analyzed with a Zeiss AXIO Scope.A1 optical microscope (Zeiss, Oberkochen, Germany). Images were collected with Zeiss Axiocam 503 Color. (C) Representative CD1d expression detected by flow cytometry in circulating monocytes from 1 of 10 CD1d^neg CLL patients. (D) CD1d expression by NLCs differentiated in vitro from patients’ PBMCs. Adherent cells were stained with anti-CD1d mAb plus goat anti-mouse PE (green) and DAPI (blue). (Left panel) CD1d expression by immunofluorescence analysis in 1 representative field (original magnification ×20) of 20 to 40 fields per patient performed on 6 patients. (Middle panel) Bright field (gray) merged with CD1d immunofluorescence and DAPI nuclei staining (original magnification ×20) showing NLCs with typical stellate morphology. Immunofluorescence was performed by In Cell Analyzer 1000 GE Healthcare and In Cell Analyzer 1000 Workstation or Arrayscan XTI (Thermo Fisher) with HCS studio software. (Right panel) The same NLCs were stained with anti-CD1d PE mAb and DAPI for confocal imaging. CD1d is localized on the cell surface and in punctate structures (original magnification ×63). Confocal imaging was performed with Confocal Microscope Leica TCS SP2.