Anti tigit

Freshly obtained peripheral blood mononuclear cell (PBMC) were stained with anti-CD4 (-FITC from Biolegend, OKT4, USA), anti-CD25 (-PE from Biolegend, BC96, USA), anti-CD127 (-PECY7 from Biolegend, A019D5, USA), anti-CD226 (-APC from Biolegend, 11A8, USA), anti-TIGIT (-APC from eBioscience, MBSA43, USA). Intracellular detection of Foxp3 with anti-Foxp3 (-Percp-Cy5.5 from eBioscience, 236A/E7, USA) and Helios with anti-Helios (-APC from Biolegend, 22F6, USA) was performed on fixed and permeabilized cells via Foxp3 Staining Buffer Set (eBioscience, USA). Cell fluorescence was acquired on BD LSR Fortessa (BD Biosciences, USA) and analyzed with FlowJo software (version 7.6.5; Tree Star). We usually acquired 10, 000 events in FSC. CD4-FITC positive and SSC gates were used to delineate CD4⁺ cells, then gated with CD25-PE and CD127-PECY7 in these cells, and the acquisition gate was designed on the CD4⁺CD25^highCD127^low/− cells.

FACS staining was performed using standard procedures. Staining was performed with the following conjugated antibodies: anti CD56, anti CD3, anti CD16 (all from Biolegend), anti 2B4, anti NKp46, anti CD69 (BD), anti NKp30, anti NKp44, anti NKG2D, anti DNAM1, anti NKG2C (R&D), anti CEACAM1 (R&D), anti TIGIT (eBioscience), anti NKG2A (R&D). Antibodies against chemokine receptors that were used included anti CCR1, anti CCR2, anti CCR3, anti CCR5, anti CCR7, anti CCR10, anti CXCR1, anti CXCR2, anti CXCR3, anti CXCR4, and anti CX3CR1. Other antibodies used were: anti CD57 and antibodies against the killer cell immunoglobulin-like receptors-KIR2DL1/DS1, anti KIR2DL2/DL3, and anti KIR3DL1/KIR3DS1. Unless otherwise noted, all antibodies in this study were purchased from Biolegend. In all FACS results shown in this paper, dead cells, neutrophils, and monocytes were excluded from analysis. We used the FCS Express version 4 program for FACS analysis. We performed correction for the MFI of different unrelated FACS staining, by dividing the individual staining MFI (of blood and SFs NK cells) by the background isotype control of the same staining (normalized MFI).