Cd122 pe

NK cell markers expression was verified using mouse anti-human antibodies to CD45-APC-CY7, CD25-FITC, CD16-PE, CD3-PE-CY7, CD56-PAC BLUE and CD122-PE, were from BD Biosciences. Anti-human antibodies to NKG2D-APC, MHC-1 HLA-A2-APC, NKP30-PE, NKP44-PE-CY7, NKP46-FITC, Granzyme B-FITC, Perforin1-PE, Interferon-γ-APC, TNF-α1- APC-CY7, were from BioLegend, DAPI from Invitrogen, mouse anti-cMyc: sureLight APC was from Columbia Biosciences. Cells were sorted at MGH Flow Cytometry Core facility using a BD 5 laser SORP FACS Vantage SE Diva system (BD Biosciences). FACS data and ∑Median statistics were analyzed using FlowJo Software (Tree Star, Inc.). Human primary NK (hNK) cells were extracted from peripheral blood of healthy donors using the Rosettesep™ human enrichment kit (StemCell technologies).

Spleens and lymph nodes were collected in Eppendorf tubes containing RPMI media. We mechanically dissociated spleens or lymph nodes through a 70 μm strainer to obtain single cell suspensions. Red blood cell (RBC) lysing buffer were used to lyse RBCs. After washing with PBS, cells were counted using hemocytometer. Cells (2 × 10⁶) were pipetted for a flow panel. Conjugated antibodies for CD3-PercpCy5 (Cat. #55116, 145-2C11), CD4-APCH7 (Cat. #560181, GK1.5), CD8-V450 (Cat. #560469, 53-6.7), CD44-FITC (Cat. #553133, IM7), CD122-PE (Cat. #553362, TM-Beta1) were purchased from BD Biosciences. TCRβ-PECy (Cat. #25-5961, H56-597) was purchased from eBioscience. For Treg flow cytometry we used the Mouse Regulatory T-cell Staining Kit from eBioscience (Cat. #88-8111-40) containing CD4-FITC (RM4-5), CD25-APC (PC61.5), and Foxp3-PE (FJK-16s). All flow cytometry was performed using a BD LSRII. The results were analyzed with Flowjo.