Multimacs

Tumours were harvested at days 8–21 and digested for 40 min at 37 °C according to Tumour Dissociation Kit protocol (Miltenyi Biotec 130–096–730). Tumours were crushed on 100 μm cell strainers and washed twice with PBS 2% FCS. Single-cell suspensions were enriched for CD45⁺ or CD8⁺ cells using the MultiMACS system (Miltenyi Biotec). In brief, cells from tumour tissues were labelled with anti-CD45 or anti-CD8a microbeads (Miltenyi Biotec 130–052–301 or 130–117–044, respectively), and then purified using the POSSEL program on MultiMACS. The positive fraction was recovered for TIL analysis by flow cytometry or ex vivo assays.

Human iPSCs were reprogrammed from fibroblast cultures by nucleofection (Amaxa™ Nucleofector™) of episomal vectors expressing OCT-4, SOX2, KLF4, L-MYC, LIN28 and shRNA against p53 [38 ], in feeder- and serum-free conditions using TeSR™-E7™ medium (STEMCELL Technologies, Canada) and selected by sorting with anti-human TRA-1-60 Microbeads using a MultiMACS (Miltenyi, Germany) as described in [39 (link)] and [40 (link)]. Cells were maintained on vitronectin XF™ (STEMCELL Technologies)-coated plates using TeSR™-E8™ (Stem Cell Technologies). At passage eight, cells were assessed for quality control as described previously [40 (link)].

T cells were isolated from human PBMC from leftover platelet apheresis products or from purchased leukopacks (Miltenyi Biotech, Auburn, CA) using PAN-T kits and the AutoMACS (Miltenyi). CD4 and CD8 cells were purified separately using StraightFrom kits and a MultiMACS (Miltenyi). T cells were cultured as described [15 (link)]. Cells were activated with anti-CD3/CD28 dyna beads (Gibco, Waltham, MA) for 4–6 days or 2 days if guideRNA electroporation was performed.