CD161 and CD56 expression on CD3⁻ NK cells from cultures of progenitor cells with feeder cells and IL-2 or IL-15, or Flt3-L and IL-2, to determine the conditions that support accumulation of immature CD56⁻ NK cells.

(A, B) 2-color and (C) 3-color immunofluorescence was performed, with the indicated FITC- or PE-, and biotin-labeled mAb detected with streptavidin-PE or CyC, on cells generated from Lin⁻ (A, B) or CD34⁺ cells (C) after 30-day culture with IL-15 (A) or IL-2 (B) and the Sl/Sl⁴hSCF²²⁰ feeder cells, or with Flt3-L and IL-2 (C), as described in “Materials and methods.” Correlate measurements of red and green fluorescence (x and y axis, respectively, log₁₀ scale) are displayed as 2-dimensional contour plots. In C, analysis was performed on gated CD3⁻ cells (CD3-FITC). The contours were divided into quadrants in which less than 0.5% control cells (treated with irrelevant isotype-matched mAbs) were included: top left, cells with green fluorescence (binding FITC-labeled Ab only); top right, double positive cells; bottom right, cells with red fluorescence (binding PE-labeled Ab only); bottom left, double-negative cells. Histograms in (C) are from samples treated with biotin-labeled anti-CD56 +/− anti-CD161 mAb detected with streptavidin-CyC on the same CD3⁻ cells (dotted line, negative control; solid line, mAb⁺ cells; x axis, fluorescence intensity, y axis, relative cell number). The experiment in A and B is representative of 10, and that in C is representative of 3 performed with similar results.