Ficoll

Sintilimab (Tyvyt) is a monoclonal antibody against programmed cell death protein 1 (PD-1). It could block the interaction between PD-1 and its ligands and help the anti-tumor effect of T-cells to recover. Sintilimab is developed by Innovent Biologics and Eli Lilly and Company (18 (link)). NK cells were expanded by our previously described method (19 (link)). Briefly, blood samples were centrifuged at 1800×g for 10 min, and plasma was transferred to new tubes. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll (Axis-Shield PoC AS, Oslo, Norway) at 800×g for 30 min. PBMCs were resuspended in Aly505 medium (Cell Science & Technology Institute Inc., Yamagata, Japan) medium with 5% auto-plasma, 600 U/mL IL-2, and 10 ng/mL IL-15 (both from Miltenyi Biotec, Germany) and 1 μg/mL OK432 (T&L Biological Technology, Beijing, China) at a concentration of 1×10⁶ cells/mL in a humidified atmosphere of 5% CO₂. The cells were cultured in Aly505 medium supplemented with 5% auto-plasma, 500 U/mL IL-2 and 10 ng/mL IL-15 at 37°C for the following days.

We obtained blood from HLA-A*11:01 positive healthy donors. PBMCs were isolated from peripheral blood with a density gradient technique (Ficoll, Axis-Shield) and cultured in RPMI 1640 supplemented with 10% human serum (Access Biologicals LLC) and 50 U/mL IL-2. NK cells were enriched from peripheral blood with RosetteSep™ Human NK Cell Enrichment Cocktail (Stemcell, 15,025) and cultured in RPMI 1640 supplemented with 10% human serum.

After ethical approval and informed consent, tumours were resected and stored in phosphate‐buffered saline (PBS) or within the cavitron ultrasonic surgical aspirator (CUSA) 20. Samples were washed in PBS, CUSA samples were prepared as shown by Schroeteler et al. 20 and all samples were filtered through a 40‐µm cell strainer, washed twice in PBS, centrifuged at 400 g for 5 min and resuspended in PBS, 0·5% bovine serum albumin (BSA) and 0·05% sodium azide. Matched patient blood was diluted with PBS, layered over Ficoll (Axis‐Shield PoC, Oslo, Norway) and centrifuged at 800 g for 20 min. Tumour and blood‐derived cells were stained with appropriate antibodies and isotype controls (see Supporting information, Table S1), with single stain controls on tumour samples used for compensation during analysis using the cytexpert compensation matrix. All samples were run on a CytoFlex S (Beckman Coulter Life Sciences, Indianapolis, IN, USA) (see Supporting information, Table S1). Gated, isotype control stained, intratumoral or peripheral blood NK cells from each patient (Supporting information, Fig. S1) were assigned a gate of 2% positive, and specific antibody staining is reported within this gate.