Flojo software

Freshly thawed PHHs, PHMs detached using Accutase, fully differentiated iPSC-Heps detached using 0.25% trypsin-EDTA and M1/M0 iPSC-Macs detached using Accutase were incubated with the respective antibodies in PBS including 0.1% BSA and 2 mM EDTA for 20 min at 4 °C and washed once before flow cytometry analysis. Antibodies are listed in Supplementary Table 2. Viable cells were distinguished using SYTOX Green/Blue Dead Cell Stain (Invitrogen). Unstained cells were used as control. Cells were analyzed using a LSRFortessa flow cytometer (BD Biosciences) and FloJo software (v10.6.1; BD Biosciences). A representative gating strategy example is shown in Supplementary Fig. 8.

3 × 10⁵ PBMCs were stained for cell surface markers for 30 min at 4°C. The following anti-human antibodies were used: FITC-labeled CD19 (clone HIB19), APC-labeled CD24 (clone eBioSN3), PE-Cy7-labeled CD38 (clone HB7), PE-labeled CD5 (clone UCHT2), and APC-labeled CD1d (clone 51.1). To analyze the intracellular expression of IL-10, cells were fixed for 30 min with Fix and Perm and stained with PerCP-Cy5.5-labeled IL-10 (clone JES3-9D7; all reagents ebioscience, San Diego, USA).
T cells were analyzed as follows: FITC-labeled CD4 (clone RPA-T4) and PerCP-Cy5.5-labeled CD25 (clone BC96) were stained at the cell surface. Following a fixation for 30 min with Fix and Perm, the intracellular staining of APC-labeled Foxp3 (clone 236A/E7) was performed for 30 min at 4°C. To ensure correct gating of rare cell populations, we used Fluorescence Minus One (FMO) controls for each antibody (18 (link)). Measurements were performed with LSR Fortessa (BD Biosciences, Heidelberg, Germany) and analyzed with FloJo software (Ashland, Oregon, USA).

Tumor cells were treated with 25 nM LBH589 (Selleckchem, München, Germany), 2.5 mM VPA (Sigma-Aldrich, Darmstadt, Germany) or solvent DMSO (Sigma-Aldrich, Darmstadt, Germany) for 16 to 72 h before they were collected. The cells were centrifuged at 300 g for 5 min, the supernatant was discarded and the cells were resuspended in 200 µL PBS. Samples were then split in two and one half was incubated with 10 ng/100 µL sample anti-ULBP-2/5/6 PE-conjugated antibody (R&D Systems, Wiesbaden, Germany) and the other half with the corresponding IgG2a control (Biolegend, Koblenz, Germany) for 30 min on ice in the dark. The cells were washed with PBS and then analyzed by flow cytometry. Alternatively, cells were incubated with 0.15 µg/100 µL sample recombinant human NKG2D Fc chimera protein (R&D systems) or corresponding recombinant IgG1 Fc protein (R&D Systems, Wiesbaden, Germany) for 30 min on ice. Cells were washed with PBS and then incubated with AF647-coupled anti-human IgG Fc antibody for 25 min. Cells were washed with PBS and then analyzed by flow cytometry. Data were analyzed using FloJo software (BD Bioscience, Heidelberg, Germany).

An AnnexinV-FITC apoptosis detection kit (Abcam; Cambridge, UK) was used to confirm the effect of Mubritinib and other compounds of interest on cell viability. BC1 (KSHV⁺, EBV⁺), BCBL1 (KSHV⁺, EBV^-), and LCL352 (KSHV^-, EBV⁺) cells were seeded in 6-well plates and exposed to camptothecin (4 μM), rapamycin (40 nM), vargatef (40 nM), AST-1306 (10 nM), cytarabine (1 μM), or Mubritinib (15 nM and 7.5 nM) in biological triplicates per each condition. After 72 h, the percentages of live and apoptotic cells were analyzed after double staining the cells with FITC conjugated Annexin V and propidium iodide (PI). Flow cytometry was performed on a BD-LSR II (BD Biosciences; Bedford, MA, USA) and data were analyzed using FloJo software (Ashland, OR, USA).