Ly49d 4e5

The following antibodies were obtained from Biolegend (San Diego, CA) or eBioscience (San Diego, CA): CD3 (17A2), γδ (GL3), CD8 (53.67), CD4 (GK1.5), NK1.1 (PK136), NKG2D (CX5), CD107a (1D4B), IFN‐γ (XMG1.2), NKp46 (29A1.4), NKG2A (16A11), Ly49D (4E5), Ly49H (3D10), TGF‐β (TW7‐16B4), FasL (MFL3), IL‐10 (JES5‐16E3), IL‐17 (eBio17B7), CD62L (MEL‐14), CD44 (IM7), granzyme B (NG2B), perforin (eBioOMAK‐D), CD25 (PC61.5), Foxp3 (FJK‐16S), GITR (YGITR 765), CTLA‐4 (UC10‐4B9), CD39 (24DMS1), CD69 (LG.3A10), CCR9 (CW‐1.2), CD28 (E18), T‐bet (4B10), GATA‐3 (16E10A23) and ROR‐γt (AFKJS‐9), neutralized TGF‐β antibody (1D11) and RAE‐1ε mAb (205001). C57BL/6 and pCD86‐RAE‐1 transgenic mice 19 were generated and housed in accordance with the rules of Animal Committee of Yangzhou University.

Flow cytometry data were acquired on a FACSCanto (BD Biosciences, San Diego, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR). To determine expression of cell surface proteins, mAb were incubated at 4°C for 20–30 min and cells were fixed using Cytofix/Cytoperm Solution (BD Biosciences) and, in some instances followed by mAb incubation to detect intracellular proteins. The following mAb clones were used: NK1.1 (PK136, eBioscience), CD3 (17A2, eBioscience), Ly49H (3D10, eBioscience), Ly49D (4E5, eBioscience), NKp46 (29A1.4, eBioscience), CD27 (LG.7F9, eBioscience), CD11b (M1/70, eBioscience), IFN-γ (XMG1.2; eBioscience), Granzyme B (MHGB04, Invitrogen), CD107a (1D4B, BD Pharmingen), AKT1 (55/PKBa/AKT, BD Pharmigen), pS473 (M89-61, BD Pharmigen).
Intracellular cytokine staining: For direct ex vivo, staining cells were incubated for 1 additional hour in the presence of Brefeldin A (BFA) before surface and intracellular IFN-γ staining. For cytokine staining following in vitro stimulation BFA was added during the last hour of stimulation. Intracellular signaling staining: For detection of intracellular AKT and pAKT (pS473) cells were methanol fixed and permeabilized according to BD protocol. Apoptosis was evaluated using Vybrant FAM Caspase-3/7 Assay Kit (Invitrogen) according to manufacturer’s protocol.