Cell lines
Human cancer cell lines were obtained from the Peeper lab repository. They were short-tandem-repeat profiled to confirm identity and tested mycoplasma-negative at the start of in vitro experiments. Cell lines were transduced with lentivirus to express HLA-A*02:01-MART1-mPlum plasmid as described previously25. D10, FM6, BLM, A875, M063 and MDA-MB-231 (referred to as MDA-231) cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 41966052, Gibco) with 10% FBS (3101120, Sigma-Aldrich) and 100 U ml−1 penicillin–streptomycin (15140122, Invitrogen). LCLC-103H, EBC-1, DU-145 and SW480 cells were cultured in RPMI (21875034, Thermo Fisher Scientific) with 10% FBS and 100 U ml−1 penicillin–streptomycin. For REP, the suspension cell line EBV-JY was used, which was cultured in IMDM (CA IMDM-A, Capricorn Scientific) supplemented with 10% FBS and 100 U ml−1 penicillin–streptomycin.
Primary human CD8+ T cell isolation, transduction and culture
Primary CD8+ T cells used in in vitro experiments with cell lines were isolated from healthy donor blood (from buffy coats). In brief, PBMCs were isolated by density centrifugation using Ficoll (11743219, Thermo Fisher Scientific) (2,500 rpm, 15 min, no break). CD8+ T cells were positively isolated with Dynabeads (11333D, Invitrogen) and activated for 48 h in a precoated plate with anti-hCD3 and anti-hCD28 (16-0037-85/16-0289-85, eBioscience), 5 mg per well in 24-well plates at 106 cells per ml. CD8+ T cells were then retrovirally transduced in retronectin-coated (T100B, Takara) plates with the MART-1-specific TCR (2,000g, 1.5 h, no break). For the first 2 days after activation, primary CD8+ T cells were cultured in RPMI with 10% human serum (H3667, Sigma-Aldrich) and 100 U ml−1 penicillin–streptomycin, with IL-2, IL-7 and IL-15 (100 IU ml−1, 10 ng ml−1, 10 ng ml−1 respectively) (Proleukin, Novartis; 11340075, Immunotools; 11340155, Immunotools). T cells were then refreshed three times a week with RPMI containing 10% FBS, 100 U ml−1 penicillin–streptomycin and 100 IU ml−1 IL-2.
Patient samples
Resected tumour material was collected from patients with melanoma undergoing surgery at the Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital (NKI-AvL) (Supplementary Table 2). The study was approved by the Medical Ethical Review Board of the NKI-AvL (under studies B16MEL, IRBm23-029) and performed in compliance with the ethical regulations. All of the patients provided prior informed consent to use their anonymized data and tumour material for research, including publication of the results in a manuscript.
Patient tumour digestion
To obtain tumour digests, freshly obtained patient tumours were cut in small pieces and incubated in prewarmed RPMI medium supplemented with pulmozyme (12.6 µg ml−1; Roche), collagenase (1 mg ml−1; 17104-019, Thermo Fisher Scientific) and a pan-caspase inhibitor (Q-VD-Oph, 50 μM; or Z-VAD, 5 μg ml−1; S7311, Selleckchem; sc-3067, Santa Cruz Biotechnology) at 37 °C in a spinning rotor for a maximum of 30 min. The sample was then passed through a 100-μm filter, washed with RPMI containing 10% FBS and frozen in FBS + 10% DMSO until further processing.
In vitro T cell–tumour cell line co-cultures
Before the start of the co-culture, primary CD8+ T cells were labelled with CTV (C34557, Invitrogen) or carboxyfluorescein succinimidyl ester (CFSE; C34554, Invitrogen) according to the manufacturer’s instructions. Tumour cell lines and pre-labelled CD8+ T cells were counted and seeded in a non-tissue-culture-treated 96-well V-bottom plate (781601, Brand) at a 2:1 tumour:T cell ratio for standard flow cytometry and at a 1:1 ratio for image-based flow cytometry assays (50,000 tumour and 25,000 or 50,000 T cells, respectively). Co-culturing was performed in 100 μl per well with 50 μl of tumour cell medium and 50 μl of T cell medium with IL-2. In standard assays, cells were co-cultured for 4 h and subsequently analysed by flow cytometry. For competition assays, non-specific and MART-1-specific T cells were mixed at the indicated ratios before the start of co-culture, based on the measured transduction efficiency. After most co-cultures, the percentage of MART-1-specific T cells in the populations of interest was determined by staining for the mouse TCR β-chain. For the experiment in which the 5:95 and 95:5 ratios (MART-1-specific:non-specific) were studied together (Extended Data Fig. 1k), the T cells were sorted after transduction to obtain a pure MART-1-specific T cell population. Before the co-culture, MART-1-specific T cells were stained with CTV and non-specific T cells with CFSE, after which they were mixed at the ratios described above to perform the co-culture.
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