Nkp44 apc

All participants donated 10 ml of peripheral blood anticoagulated with EDTA, and samples were processed within three hours after extraction. Briefly, 150 μl of peripheral blood was incubated with CD56-Pacific blue (362520), CD16-BrilliantViolet510 (302048), CD19-PercPCy5.5 (302230), NKG2A-APC fyre (375116), NKG2C-PE (375004), NKG2D-FITC (320820) and NKp44-APC (325110) from Biolegend (San Diego, CA, USA) and CD3-ECD (A07748) from Beckman Coulter (Brea, CA, USA), according to manufacturer’s protocols. After monoclonal incubation, red blood cells were lysed with BD FACS lysing solution (349202, Becton Dickinson, Franklin Lakes, NJ, USA). Samples were washed and suspended in 300 μl phosphate saline buffer, and acquired in a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA). For every variable, all files were collected and data was analyzed at the same time with Kaluza v2.1 (Beckman Coulter, Brea, CA) by a blinded operator. NK^bright, NK^dim and NKT populations were gated by expression of CD56, CD16 and CD3. Subpopulations expressing NKG2A, NKG2C and NKG2D were selected, and NKp44 expression was analyzed in every subpopulation with two different gating strategies to confirm the differences. Gating strategies are detailed in Supplementary File.

To assess degranulation, NK cells were co-incubated with opsonized target cell lines in the presence of GolgiPlug (1/1000 dilution, BD Biosciences), monensin (1/1000 dilution, Biolegend) and anti-CD107a PE mAb (clone H4A3, BD biosciences) or an isotype-matched control (PE-IgG1κ; clone MOPC-21, BD) for 4 h at 37°C. After incubation, cells were washed and stained with Zombie NIR viability dye (Biolegend), anti-CD56-AF488 mAb (clone HCD56, Biolegend), anti-CD107a-PE (clone H4A3) and anti-CD16-AF647 (clone; B.731, Biolegend) (42 (link)). To assess expression of other proteins, NK cells were co-incubated with opsonized target cell lines for 4 h, then washed and stained with Zombie NIR viability dye (Biolegend) and LFA-1-PE (Clone M24), NKp46-PercP-Cy5.5 (Clone 9E2) and NKp44-APC (Clone P44-8) or NKp30-AF647 (Clone P30-15) and NKG2D-PE (Clone 1D11, all from Biolegend). Isotype-matched control mAbs were also used accordingly (mouse IgG1 isotype control; clone MOPC-21, Biolegend). Finally, cells were fixed in 2% PFA/PBS and staining was assessed using a BD FACS LSRFortessa™ X-20 flow cytometer and analyzed by FlowJo V10 software (BD Biosciences). The gating strategy used for all flow cytometry assays is shown in Supplementary Figure 5.

Anti-human CD3-FITC/CD16 + 56-PE mixed antibody were purchased from Beckman Coulter (Brea, CA, USA). Anti-human CD16-FITC, NKG2D-PE, NKp44-APC, DNAM-1-FITC, NKp46-Alexa Fluor 647, NKp30-PE, KIR3DL1-FITC, KIR2DL1/DS1-PE, NKp30-APC, Perforin-PE and Granzyme B-FITC antibodis were all purchased from BioLegend (San Diego, CA, USA), as well as Fixation Buffer, Wash Buffer, Annexin V binding buffer, Alexa Fluor 647 Annexin V and 7-AAD Viability Staining Solution. The MPP-9 and IDO inhibitors were 1-Methyl-DL-tryptopan (1-MT; Sigma-Aldrich, St. Louis, MO, USA) and Tissue inhibitor of metalloproteinases 1 (TIMP-1; PeproTech, Rocky Hill, NJ, USA). Human NK Cell Isolation Kit was purchased from Miltenyi Biotec (Auburn, CA, USA) and ELISA kits were purchased from Abcam (Cambridge, MA, USA). Trizol reagent and PrimeScript RT Master Mix (Perfect Real Time) were both obtained from TaKaRa (Shiga, Japan) and Power SYBR Green PCR Master mix was purchased from Applied Biosystems (Carlsbad, CA, USA).