Fig. 1

Expansion and phenotype of expanded natural killer (NK) cells from breast cancer patients and healthy donors. a A schematic showing the expansion protocol of NK cells. b Frozen peripheral blood mononuclear cells (PBMCs) from breast cancer patients (red) or healthy donors (blue) were cultured with irradiated feeder cells K562mbIL21 and 100 U/μL of IL-2. Co-culture was supplemented with IL-2 and new media every 2–3 days, and with irradiated feeder cells on a weekly basis. Cells were counted every week using trypan blue. Fold expansion was calculated assuming that NK cells comprise 15% of PBMCs (n = 3, mean ± SEM). Unpaired Student’s t test was used to compare the difference between groups. c Freshly isolated PBMCs from healthy donors (HD), expanded HD NK cells, or expanded breast cancer patient (BCP) NK cells were stained for the expression of CD45, CD56, CD3, CD16, NKG2D, NKp44, NKp46, NKp30, CD69, CD160, CD11b, CD27, CD25, NKG2A, CD158a, CD158b, and CD158e1. Cells were gated on a CD45⁺CD56⁺CD3^– population and then analysed using FlowJo Software for the expression of other markers. Pecent-positive expression of the markers was then tabulated and graphed (n = 3–8, mean ± SEM). Welch’s t test was used for statistical comparison between HD NK from freshly isolated PBMC and HD expanded NK cell groups and separately between expanded HD and BCP NK cells. *P < 0.05, **P < 0.005, ***P < 0.0005, only significant differences are indicated on graphs