Fig 1. NK cells acquire intrinsic memory features 21 days after in vivo infection.

(A) Experimental scheme. C57BL/6 mice were intranasally injected with either PBS (black symbols) or sub-lethal dose of S. pneumoniae (SPN, red symbols, 5x10⁵ CFU) for two consecutive days. After 21 days, NK cells were highly purified from spleens of D21PBS or D21SPN mice (98% of purity) and stimulated in vitro with cytokines (IL-15 at 2 ng/ml, IL-18 at 1,5 ng/ml, IL-12 at 1,25 ng/ml) and formaldehyde inactivated S. pneumoniae (SPN, MOI 20) for 24 hours. (B) Intensity of IFNγ expression in NK cells (MFI, left panel), percentage of IFNγ⁺ NK cells (middle panel), representative overlay histogram upon IL-15+IL-18+SPN stimulation (right panel, gray represents isotype control). D21PBS NK cells and D21SPN NK cells are purified from spleen, pooled from n ≥ 4 mice/group and incubated in n ≥ 3 experimental replicates/group. Box plots where each dot represents an experimental replicate (black dots for D21PBS NK cells, red dots for D21SPN NKs cells), lines are median, error bars show min to max. Data are representative of at least three experiments. ns, not significant. **** p < 0.0001. 2way ANOVA test comparing D21PBS NKs and D21SPN NKs values in each condition.