Antibodies used were anti-CD8-PeCy7, -CD3-PERCP Cy5.5, -CD44-PeCy5, -CD25-PeCy7, -CD335-APC (NKP46) (BioLegend), -CD8-eFluor450, CD3-Pecy7, -CD122-FITC, -IL15-receptorα-PE, -CD279-eFluor780 (PD-1), -CD314-PE (NKG2D) (eBioscience), -CD4-PE, -CD3-FITC (Beckman Coulter), and -CD69-PE (Pharmingen). Samples were collected on a LSRII (BD Biosciences) or a Cytomics FC 500 (Beckman Coulter) and analyzed with FlowJo software (Tree Star, Ashland, OR).
For analysis of phosphorylated proteins, splenocytes were incubated with IL-2 (200 U/ml) or IL-15 (100 ng/ml) (20 min), fixed in 1% paraformaldehyde (10 min, room temperature (RT)) and permeabilized using Phosflow Perm Buffer III (BD Bioesciences; 30 min, 4 °C). Cells were stained for surface markers (15 min, RT), washed, incubated with anti-pS6 (Ser235/236, D57.2.2E) or -pSTAT5 (Tyr694, D47E7) (both from Cell Signaling; 1 h, RT), washed and stained with secondary antibody (goat F(ab′)2 anti-rabbit IgG (H + L)-PE; Beckman Coulter).
For proliferation experiments, cells were stained with CellTrace Violet (Molecular Probes) and cultured with IL-2 (200 U/ml) or IL-15 (100 ng/ml). After 72 h, cells were harvested, stained for surface markers and analyzed.
For analysis of phosphorylated proteins, splenocytes were incubated with IL-2 (200 U/ml) or IL-15 (100 ng/ml) (20 min), fixed in 1% paraformaldehyde (10 min, room temperature (RT)) and permeabilized using Phosflow Perm Buffer III (BD Bioesciences; 30 min, 4 °C). Cells were stained for surface markers (15 min, RT), washed, incubated with anti-pS6 (Ser235/236, D57.2.2E) or -pSTAT5 (Tyr694, D47E7) (both from Cell Signaling; 1 h, RT), washed and stained with secondary antibody (goat F(ab′)2 anti-rabbit IgG (H + L)-PE; Beckman Coulter).
For proliferation experiments, cells were stained with CellTrace Violet (Molecular Probes) and cultured with IL-2 (200 U/ml) or IL-15 (100 ng/ml). After 72 h, cells were harvested, stained for surface markers and analyzed.