Figure 1. Tumour interactions induce CD25^high NK cells with heightened metabolism and effector function.

(a–d) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with or without B16 melanoma cells at an E:T ratio of 2:1 (a), with B16 melanoma cells at an E:T ratio of 4:1, 2:1 or 1:2 (b), with or without YAC-1, CT26 and LLC tumour cells at an E:T ratio of 1:4 (c), or with or without RMA/RMA-S cells at an E:T ratio of 1:4 (d) for 18 h before analysis of CD25 expression by flow cytometry. (e–k) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells for 18 h at an E:T ratio of 2:1, washed and put back into culture with IL15 (7.5 ng/mL) with or without ± IL2 (20 ng/mL) for 18 h before analysis by flow cytometry. (e) IFNγ production by CD25^high and CD25^neg NK cells cultured in IL2 alone, with B16 cells or with B16 cells plus IL2. (f) IFNγ production in CD25^high NK cells cultured with B16 ± IL2. (g,h) Analysis of NK cells cultured with B16 cells + IL2 comparing CD25^high and CD25^neg NK cells (g, left panel) for effector functions, IFNγ production and granzyme (Gnzb) B expression. (i,j) Analysis of cell size (FSC), cMyc and CD71 expression, and levels of phosphorylated S6 ribosomal protein (pS6) in CD25^high and CD25^neg NK cells from B16 cells + IL2 co-cultures. (k,l) Rates of fluorescent transferrin uptake were measured in CD25^high and CD25^neg NK cells from B16 cells + IL2 co-cultures. Data is representative (a,c,d,e,g,i,k) or mean ± SEM (b,f,h,j,l) of 3–5 independent experiments. Data was analyzed using a paired students t-test. (* p < 0.05, ** p < 0.01, *** p < 0.001).