Ficoll paque plus

For the functional assay of peripheral blood mononuclear cells (PBMCs), 10 mL of blood, containing 1 mL of 1% EDTA (Merck, Darmstadt, Germany) at pH 7.5–8.0, was collected and centrifuged at 1811× g, 4 °C, for 30 min. The buffy coat was harvested and diluted in 6 mL of RPMI-1640 medium (Gibco) for subsequent density gradient centrifugation. The diluted buffy coat was gently applied to an equal volume of Ficoll-Paque^TM PLUS (GE Healthcare), and centrifuged at 1811× g, 20 °C, for 30 min. The isolated PBMCs, located at the interface of RPMI-1640 and Ficoll-Paque^TM PLUS (GE Healthcare), were collected and mixed with three volumes of sterile ammonium chloride potassium (ACK) lysis buffer, containing 0.15 M NH₄Cl, 1.0 M KHCO₃, and 0.01 M EDTA at pH 7.2–7.4. After incubation at 4 °C for 5 min, the cells were centrifuged at 201× g, 20 °C, for 10 min to collect the erythrocyte-free pellets. The pellet was resuspended in RPMI-1640 medium and centrifuged at 129× g, 20 °C, for 10 min to get rid of the platelets. The platelet-free pellets were then diluted to a final concentration of 3 × 10⁶ PBMCs/mL in CTL-Test^TM medium (Cellular Technology, LLC, Cleveland, OH, USA) for subsequent use.

PBMC from healthy donors and patients were isolated by density gradient centrifugation (Ficoll Paque PLUS, density 1.077 g/mL, GE Healthcare, Chicago, IL, USA). Then, we negatively enriched NK cells with an NK Cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany) using the AutoMACS Pro (Magnetic-Activated Cell Sorting, Miltenyi, Bergisch Gladbach, Germany, version 2.3.0.2), following the manufacturer’s protocol.
Bone marrow mononuclear cells (BMMC) were isolated from bone marrow aspirates by density centrifugation (Ficoll Paque PLUS, density 1.077 g/mL, GE Healthcare, Chicago, IL, USA), and abnormal plasmatic cell (aPC) enrichment was performed with CD138 MicroBeads (Miltenyi, Bergisch Gladbach, Germany) using the AutoMACS Pro, following the manufacturer’s protocol. The phenotype of separated aPC was verified by FACSAria III (BD Biosciences-US, San Jose, CA, USA, version 8.0.1) using the following fluorescent antibodies: CD19-PECy7 (Beckman Coulter, Brea, CA, USA), CD56-PE (Agilent Dako, Santa Clara, CA, USA), CD38-FITC (Cytognos, Salamanca, Spain), CD138-APC (Sony, San Jose, CA, USA), and CD45-PB (Agilent Dako, Santa Clara, CA, USA).

Peripheral blood, metastatic lymph nodes, and tumor samples were obtained from individuals with HNSCC or MSS colon cancers. At the time of sample collection, patients were not undergoing therapy. Previously, they had undergone a range of therapies, including chemotherapy, radiotherapy, surgery and immunotherapy, or none of the above.
PBMCs were purified from whole blood over a Ficoll-Paque PLUS (GE Healthcare) gradient and cryopreserved prior to analysis. Tumor specimens were prepared as follows: under sterile conditions, specimens were cut into small pieces and digested in RPMI-1640 medium in the presence of hyaluronidase at 0.5 mg/mL (MilliporeSigma, H6254), collagenase at 1 mg/mL (MilliporeSigma, C5138), and DNase at 30 U/mL (Roche, 04536282001) as well as human serum albumin (MP Biomedicals, catalog IC08823051) at a 1.5% final concentration. Cells were digested for 1 hour at room temperature (RT) under agitation with a magnetic stir bar. Cell suspensions were filtered through a 70 μm filter. In some cases, TILs were enriched as described above by Ficoll-Paque PLUS (GE Healthcare, catalog 17144003) density centrifugation. Tumor single-cell suspensions were cryopreserved until further analysis.