Nk1.1 pe

Heparinized whole blood was used for flow cytometry using the following antibody cocktail: CD45-PerCP (Clone 30-F11; Biolegend 103130), CD3-eFLUO450 (Clone 17A2; eBioscience 48-0032), NK1.1-PE (Clone PK136; BD557391), Ly6G-APC-CY7 (Clone 1A8; BD560600), CD11b PE-CY7 (Clone M1/70; BD552850), Ly6C-APC (Clone 1G7.G10; Miltenyi 130-093-136), CD19-APC-H7 (Clone 1D3, eBioscience 47-0193) and Siglec-F-PE (Clone E50-2440; BD552126). Further, cells from the whole brain were isolated for FACS. Anesthetized mice were perfused with PBS to remove the peripheral blood. Subsequently, brains were dissected, mechanically dissociated and digested with a collagenase mix (including collagenase IX, collagenase I, DNAse and RPMI-HEPES). Immune cells were separated using a Percoll-gradient and stained with CD45 PerCP (Clone 30-F11; Biolegend 103130), CD11c PE-Cy7 (Clone N418, eBioscience 25-0114)), F4/80 (Clone BM8; Biolegend 123116), CD11b BV510 (Clone M1/70; Biolegend 101245), CD3 BV421 (Clone 17A2; eBioscience 48-0032-82), CD19 BV421 (Clone 6D5; Biolegend 115538), Ly6G BV421 (Clone eBio927; eBioscience 48-3172), MHC II-APC-eFluor780 (Clone M5/114.15.2; eBioscience 47-5321-82). All samples were measured with a FACS-Canto II (BD Biosciences). Results were analysed with FACSdiva version 8 (BD Biosciences).

Mononuclear cells from the spleens and brains were surface-labeled with anti-mouse CD4 FITC (eBioscience, Cat #: 11-0042), CD25 PE (eBioscience, Cat #: 12-0251), NK1.1 PE (eBioscience, Cat #: 12-5941-82), CD8 FITC (eBioscience, Cat #: 11-0081-81), and B220 APC (eBioscience, Cat #: 17-0452-81). For Tregs labeling, the cells were further fixed and permeabilized using a Foxp3/transcription Factor Staining Buffer kit (eBioscience, Cat #: 00-5523) and then stained with anti-mouse Foxp3 APC (eBioscience, Cat #: 17-5773). Cells were washed and suspended in flow cytometry staining buffer (eBioscience, Cat #: 00-4222-57). FACS analysis was performed using Accuri C6 software (BD Biosciences, San Jose, CA, USA).

Isolated liver mononuclear cells (LMNCs) were re-suspended at a concentration of approximately 1 × 10⁶ cells per 50 μL in FACS staining buffer containing anti-mouse CD16/CD32 mAb (2.4G2) from BD Pharmingen (San Jose, CA, USA) to block nonspecific binding to Fcγ receptors and incubated for 10 min at 4°C. Cells were stained with live/dead Fixable Blue Dead Cell Stain Kit from Life Technologies (Grand Island, NY, USA) to gate out dead cells. Further, we added the antibodies: CD11b APC, NK1.1 PE, CD3 Pacific Blue, Ly6C FITC (eBioscience, San Diego, CA, USA) to the cells and incubated for 30 min in the dark at 4°C. For the negative control, the cells were stained with isotype-matched control antibodies (eBioscience, San Diego, CA, USA). The cells were washed with FACS staining buffer by centrifugation at 1,500 rpm for 5 min at 4°C and fixed with 1% paraformaldehyde. The cells were acquired on a BD LSR II instrument (BD Biosciences, San Jose, CA, USA) and the data was analyzed by FlowJo software.

Single cell suspensions were prepared from harvested melanoma tissues. Tissues were dissected and digested with collagenase 1U/ml, passed through 70-μm cell strainers and red blood cells (RBC) were lysed. Cell samples were pre-incubated with anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA) to block non-specific Fc-mediated interactions. Antibodies against CD11c-FITC, CD11b-PeCy5.5, Gr1-PE or Gr1-allophycocyanin, CD3-PeCy5.5; CD8-allophycocyanin or CD8-PE; CD4-allophycocyanin; NK1.1-PE were obtained from eBioscience and BioLegend. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences).

PD-1 FITC (1:300; J43; eBioscience), NK1.1 PE (1:300; PK136; eBioscience), CD3e PE-Dazzle594 (1:300; 145-2C11; BioLegend), CD3e PE (1:200; 145-2C11; eBioscience), FoxP3 PerCP-Cy5.5 (1:200; FJK-16s; eBioscience), CD8a PE-Cy7 (1:2000; 53-6.7; eBioscience), CD8a APC (1:600; 53-6.7; eBioscience), CD8a PerCP-Cy5.5 (1:400; 53-6.7; eBioscience), TCRb AF700 (1:100; H57-597; BioLegend), CD45 BV785 (1:1000; 30-F11; BioLegend), CD4 BV711 (1:400; GK1.5; BioLegend), CD19 BV650 (1:400; 6D5; BioLegend), CD19 PE (1:500; 1D3; eBioscience), CD11b BV605 (1:1000; M1/70; BioLegend), CD11b BV510 (1:400; M1/70; BioLegend), CD11c BV605 (1:400; N418; BioLegend), Siglec-F PE (1:300; E50-2440; BD Biosciences), F4/80 APC (1:200; BM8; BioLegend), Ly-6G AF 700 (1:300; 1A8; BioLegend), MHC II BV650 (1:4000; M5/114.15.2; BioLegend), CD64 BV421 (1:200; X54-5/7.1; BioLegend), TNF FITC (1:300; MP6-XT22; BioLegend), IFNg PE-Cy7 (1: 1000; XMG1.2; BD Biosciences), Eomes PE (1:200; W17001A; BioLegend), Ki-67 BV 650 (1:200, 11F6; Biolegend), GzmB FITC (1:100; GB11; BioLegend). Gating strategy is depicted in Supplementary Fig. 6d.

PBMCs and periodontal-infiltrating leukocytes from enrolled subjects were surface-stained with anti-human CD3-PerCP (BD Biosciences, San Jose, CA), CD19-APC-H7 (BD Pharmingen), NKp46-PE-Cy7 (BD Pharmingen), and CD14-FITC (eBioscience, San Diego, CA), and were intracellularly stained with anti-human IL-18 (Abcam), which was conjugated by PE/R-Phycoerythrin Conjugation Kit (Abcam). PBMCs, splenocytes, and periodontal-infiltrating leukocytes from mice were surface-stained with anti-mouse CD3-FITC, CD4-APC, CD8-PerCP Cy5.5, NK1.1-PE, CD11b-FITC, CD11c-PE, CD19-APC, and/or Ly-6G (Gr-1)-PE Cy7 (eBioscience) for detection of leukocyte subsets. Periodontal ligament cells were stained with Annexin V-FITC and propidium iodide. Data were acquired using FACS Aria II flow cytometer (BD Biosciences) and were analyzed using FlowJo Version 10 (Tree Star, Ashland, OR).