Skip to main content
. 2018 Dec 20;9:2985. doi: 10.3389/fimmu.2018.02985

Figure 3.

Figure 3

NKP cells in the BM of Cd47−/− mice are reduced with a concomitant increase of iNK and mNK cells. Bone marrow (BM) single cell suspensions from WT and littermate Cd47−/− mice were stained for Aqua live/dead, Lin (CD3, CD4, CD8, B220, CD19, CD11c, Gr1, and Ter119), CD127, CD122, CD49b, NK1.1, and NKp46. Cells were then fixed, permeabilized and intracellularly stained for Eomes. (A,B) Representative contour plots (values indicate percentage of parent population), frequency and count of NK cell precursors (NKP, gated on Lin−NK1.1−CD49b−CD122+ cells) (C,D) immature NK cells (iNK, gated on Lin−CD127−NK1.1+CD49b−CD122+ cells) and (E,F) mature NK cells (mNK, gated on Lin−NK1.1+NKp46+Eomes+ cells) are shown, n = 5. (G) Splenocytes from WT and Cd47−/− littermate mice were enriched for Lin (B220, CD19, CD11b, CD11c, CD49b, CD105, MHC-II, and Ter119)-depleted cells, pulsed with CFSE and cultured on uncoated or anti-CD3 (2 μg/ml) plus anti-CD28 (1 μg/ml) coated plates for 48 h in complete RPMI (10% FCS), CFSE dilutions were measured for cultured CD4−CD8−NK1.1+ cells. (H) CTV dilutions are depicted from Lin (B220, CD19, CD11b, CD11c, CD49b, CD105, MHC-II and Ter119)−CD4−CD8− cells which were sorted from the spleens of WT and Cd47−/− mice and cultured for 1 week in complete RPMI + 60 IU/ml IL-2. (I,J) Representative contour plots (values indicate percentage of live cells) and frequency of NK1.1+ cells within a week in culture were shown, n=3. Data derived are representative of two experiments involving three mice per experiment (Mean ± SEM). (K) Incorporation of BrdU in BM iNK and mNK cells of WT and Cd47−/− mice, 3 h after BrdU i.p. injection. (L) Unlabeled NK cells were isolated from spleens of WT and littermate Cd47−/− mice and cultured with/without IL15. Cell proliferation was estimated for 24 and 48 hours using a tetrazolium [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) proliferation assay. (M,N) levels of the secreted cytokine IFNγ and granzyme B (GzmB) in the NK cell culture media were quantified using ELISA kits.