Phenotype and NKR expression of CD34⁺-derived NK cells. (A) Forward and side scatter appearance of cells at days 14, 21, 28, and 35 of culture (top panels). Early in culture, large cells with high side and forward scatter are seen. Over time these cells decrease and there is an increase in cells within the lymphocyte gate. Concurrent with the increasing percentages of lymphocytes, both CD56⁺ and CD16⁺ cells can be detected (bottom panels, gated on viable cells using forward and side scatter). By day 35 of culture, the vast majority of cells are CD56⁺ with a significant proportion coexpressing CD16. Results were representative of more than 20 healthy UCB donors. (B) Expression of NK-cell receptors on in vitro–generated CD56⁺ cells at day 28. Shown are data from cells falling within the lymphoid gate. Results representative of more than 20 donors. (C) The fraction of CD56⁺ cells expressing an individual NKR as a function of the percentage of CD56⁺ cells in culture. Shown are the percentage of NK cells in culture (x-axis) and the percentage of receptor-expressing cells (y-axis). At all time points studied (days 14, 17, 19, 21, 25, 28), the majority of CD56⁺ cells (> 75%) expressed NKp44 and CD161. There was progressive acquisition of NKp30, NKp46, NKG2A, CD94, and NKG2D as the percentage of NK cells increased in the culture. A third group of receptors (CD16 and KIR) was acquired more gradually. Results were representative of more than 20 donors.