Anti cd56

TLR activation of NK cells was analysed concerning induction IFN-gamma and TNF-alpha as well as CD107a degranulation in response to K562 target cells. Purified NK cells were pre-activated overnight with IL-2 and then cultured with and without Pam3CSK4, LPS-B5 and CpG-ODN-2216, respectively. After 16 h, NK cells were divided into either co-culture with K562 effector cells (effector to target ratio of 1:2) or kept in culture with medium alone. Next, FITC-conjugated CD107a (BD Biosciences, Heidelberg, Germany) was added. After 1 h, Golgi Stop (BD Biosciences) and BFA (Enzo Life Sciences GmbH) were added for additional 3 h. Then, cells were stained with Zombie AquaTM (BioLegend, London, UK) followed by staining with anti-CD3 (APC-Cy7-labelled), anti-CD56 (Brilliant Violet 421-labelled) and anti-CD16 (PerCP-labelled). After fixation and permeabilization, cells were stained intracellularly with anti-IFN-gamma (PE-labelled) and anti-TNF-alpha (PE-Cy7-labelled) (all BioLegend). Percent IFN-gamma-, TNF-alpha- and CD107a-positive CD56^dimCD16^pos, CD56^dimCD16^neg and CD56^highCD16^neg NK cells were measured before and after TLR stimulation as well as after co-culture of NK cells with K562 target cells in the presence and absence of TLR stimulation (Supplementary Figure 2).

Human single cells were stained with fluorochrome-conjugated antibodies after pre-blocking with human TruStain FcX™ (BioLegend) in FACS buffer (PBS with 1% BSA and 0.1% sodium azide) for 20 min at 4 °C. For intracellular staining, the cells were fixed and permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences).The following fluorochrome-conjugated antibodies were purchased from BioLegend: anti-CD3 (HIT3a), anti-CD8 (SK1), anti-CD16 (3G8), anti-CD19 (HIB19), anti-CD20 (rituximab), anti-CD25 (BC96), anti-CD56 (5.1H11), anti-CD69 (FN50), anti-CD107a (H4A3), anti-CD226(11A8), anti-PD-1 (EH12.2.H7), anti-TIGIT (A15153G), anti-SLAMF7 (162.1), anti-Granzyme B(GB11), anti-IFN-γ (4 S.B3), anti-TNF-α (W19063E) and anti-PD-L1 (29E.2A3).

All lymphocytes, pre- and post-selection, were analyzed using flow cytometry and the following antibodies: anti-CD45 (AF700; clone HI300), anti-CD3 (PE, FITC or PE-Cy7; clone OKT3), anti-CD8 (PE or BV510; clone HIT8a), anti-CD4 (BV421 or BV785; clone OKT4), anti-CD27 (BV785; clone 323), anti-CD14 (PC7; clone HCD14), anti-CD56 (APC; clone MEM-188), anti-EGFR (PE; clone AY13), TCRαβ (APC-Fire750; clone IP26) and anti-CD19 (BV421; clone HIB19) (all from Biolegend). To determine T cell activation state, T cells were stained with CD69 antibody (APC-Fire750; clone FN50) and CD25 antibody (BV650; clone BC96) (Biolegend) at indicated time points after initial stimulation. For live/dead discrimination propidium iodide (Sigma) was used. CAR expression has been monitored using an anti-idiotype antibody specific for the CD19-directed CAR, an in-house developed antibody. Cell associated fluorescence was analyzed by flow cytometry using either CytoFLEX or CytoFLEX LX flow cytometers (Beckman Coulter), unless indicated otherwise.
Live nucleated cells were enumerated pre- and post-selection (including fractions and downstream culture) using either NuceloCounter NC-3000 (Chemotec) or XN-350 Cell Counter (Sysmex) according to manufacturers’ instructions.

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).

Harvested and processed PBMCs were block and stained as follows. For the ILC panel, cells were stained with lineage markers (anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD14, anti-CD19 [eBioscience], anti-CD56, anti-FcεR1, anti-CD11b and anti-CD11c [Biolegend, San Diego, CA]), all labeled with Pacific Blue, anti-CD45-Pacific Orange (Invitrogen), anti-cKit-PerCP-eF710 (eBioscience) and anti-IL-7Rα-APC-Cy7 (eBioscience). Cells were then permeabilized and stained with anti-human IL-13-FITC (eBioscience) and data were collected on a BD FACS Canto II (BD Biosciences).
For the T cell panel, PBMCs were stained with anti-CD3-V500, anti-CD4-Qdot 605 and anti-CD8-PE-Texas Red. Cells were then fixed, permeabilized and stained with anti-IL-4-FITC, anti-IL-2-PerCP, anti-IL-10-Pacific Blue, anti-IL-22-APC, anti-TNF-α, anti-IL-17A-APC-Cy7, anti-IL-5-PE and anti-IFN-γ-PE-Cy7 for 30 minutes. Data were collected on a BD LSRFortessa (BD Biosciences). All flow data were analyzed using FlowJo version 9.4.10.

Reagents: Roswell Park Memorial Institute (RPMI-1640) medium was purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was obtained from Clark Bioscience (Richmond, VA, USA). NADΙ was purchased from Sigma (St. Louis, MO, USA), and nitrotetrazolium blue chloride (NBT) was purchased from Biosharp (Hefei, China). Anti-CD3, anti-CD16, and anti-CD56 were purchased from Biolegend (San Diego, CA, USA). The Human CORT ELISA KIT (XL-Eh0551), Human GNLY ELISA KIT (XL-Eh1850), Human Gzms-A ELISA KIT (XL-Eh1375), Human Gzms-B ELISA KIT (XL-Eh1374), and Human PF T ELISA KIT (XL-Eh0770) were purchased from Abcam (Cambridge, MA, USA).