Phenotype of NLC derived from the blood of patients with CLL.

CLL B cells from representative patients were cultured for 14 to 21 days, and nonadherent cells were vigorously (A-D) or carefully (E) removed before immunohistochemical examination. Cells were stained with antibody–biotin conjugates that subsequently were developed with an avidin–biotin–peroxidase complex and DAB, resulting in a brown-red stain for positive cells. Specimens were lightly counterstained with hematoxylin (Vector) and were photographed at 400× magnification. Displayed are pictures of NLC derived from the blood of patients with CLL (A-C, E) or adherent cells derived from the blood of a normal adult donor (D). Adherent NLC from the blood of patients with CLL expressed stromal cell markers vimentin (A, E) and STRO-1 (C). CLL B cells, on the other hand, did not express vimentin (E). Control staining with the MOPC-21 mAb of irrelevant specificity was negative (B). In contrast, most adherent cells derived from the blood of normal adult donors (D) were smaller and strongly positive for CD14. The close physical interaction of NLC and CLL B cells is demonstrated in (E), where vimentin-negative CLL cells are attached to vimentin-positive NLC. This is typically seen in long-term cultures of CLL B cells, suggesting a close, symbiotic relationship of these 2 cell types.