TILs were washed once, resuspended at 200,000 cells/50 μl in MACS-buffer and stained with the following fluorochrome-conjungated antibodies: Alexa Fluor 488 anti-human CD3 (clone: UCHT1), APC anti-human CD279 (PD1) (clone: EH12.2H7), PE/Cy7 anti-human Tim-3 (clone F38-2E2) (BioLegend). Cells were then washed and analyzed on an Accuri C6 flow cytometer equipped with the BD Accuri C6 software.
For detailed immune phenotypning, cells were analyzed by flow cytometry at the department of Clinical Immunology and Transfusion Medicine, Sahlgrenska University Hospital (accredited to EN ISO 15189). For ex vivo characterization of TILs in ACT treated mice, TILs were injected in hIL2-NOG mice bearing established autologous tumors. Tumors were harvested 40 days after injection of TILs, homogenized using a tissue chopper (McIlwain) and filtered repeatedly through 70 and 40 μm cell strainers before analysis. For PBMCs, blood was harvested from anesthesized mice through cardiac puncture 48 days after injection of TILs. PBMCs were then isolated using Ficoll-Paque before analysis.
For detailed immune phenotypning, cells were analyzed by flow cytometry at the department of Clinical Immunology and Transfusion Medicine, Sahlgrenska University Hospital (accredited to EN ISO 15189). For ex vivo characterization of TILs in ACT treated mice, TILs were injected in hIL2-NOG mice bearing established autologous tumors. Tumors were harvested 40 days after injection of TILs, homogenized using a tissue chopper (McIlwain) and filtered repeatedly through 70 and 40 μm cell strainers before analysis. For PBMCs, blood was harvested from anesthesized mice through cardiac puncture 48 days after injection of TILs. PBMCs were then isolated using Ficoll-Paque before analysis.