Following culture with CSA, there are fewer KIR-expressing cells due to a reduction in CD56⁺CD16⁺ cell proliferation. (A) Results from cells from a representative donor after 7 days of culture with IL-2 (100 U/mL) and IL-15 (10 ng/mL), with and without CSA, showing a reduction in CD16 or KIR (CD158a, CD158b, CD158e1) in the CSA-treated cultures (bottom), relative to controls (EtOH) (top). Results are representative of more than 3 individual donors. Numbers in upper right are the percentage of positive cells. (B) Cultured CD56⁺CD16⁻ cells differ in KIR expression compared with CD56⁺CD16⁺ cells regardless of whether they are cultured in CSA or vehicle (EtOH). Purified NK cells were cultured with IL-2 (100 U/mL) and IL-15 (10 ng/mL) with and without CSA for 7 days and then analyzed by FACS. Results are from cells from a single donor and are representative of more than 3 donors. Numbers in upper right are the percentage of positive cells. (C) Reduction in CD16 and KIR on NK-cell exposure to CSA. NK cells were cultured in IL-2 (100 U/mL) and IL-15 (10 ng/mL) with and without CSA for 7 days and analyzed by FACS. Shown is the percentage relative change in CD16 or KIR between CSA- and vehicle control–treated NK cultures (percent relative change = 100 × (% receptor-expressing cells in CSA − % receptor-expressing cells in control)/% receptor-expressing cells in control). Results are the average of cells from 13 healthy donors for CD16, CD158a, and CD158b and from 11 donors for CD158e1. Cells that expressed less than 5% CD158e1 were considered to not express this gene and samples from those donors were excluded (n = 2). Wilcoxon signed rank test was used to calculate statistics. (D) CSA differentially affects the proliferation of CD56⁺CD16⁻ and CD56⁺CD16⁺ NK-cell subpopulations. NK cells were freshly isolated and stained with the membrane dye CFSE. Cells were analyzed at days 3, 5, 7, and 10 of culture after staining with CD56-APC and CD16-PE. CFSE content was determined after gating on the CD56⁺CD16⁻ and the CD56⁺CD16⁺ subpopulations and the differences between CSA- and vehicle control–treated cultures are shown as overlaid histograms. Results are representative of samples from 3 individual donors. (E,F) CD56^brightCD16⁻ and CD56^dimCD16⁺ NK-cell subpopulations were FACS purified from healthy donor buffy coats. Purity after sorting was more than 97%. After culture in IL-2 (100 U/mL) and IL-15 (10 ng/mL) with and without CSA for 7 days, cell phenotype was determined using FACS. As shown, some CD56^brightCD16⁻ cells acquired CD16 and some CD56^dimCD16⁺ NK cells lost CD16. (E) Cells from a representative donor are shown. (F) The average percentage of CD56⁺CD16⁺ cells found after CD56^brightCD16⁻ cells were cultured with (▧) or without (▒) CSA (left). The average percentage of CD56⁺CD16⁻ cells generated after culture of the CD56^dimCD16⁺ purified fraction either with (▧) or without (▒) CSA (right). Results are the average plus and minus the SD for cells from 5 separate donors.