Human cd8 isolation kit

PBMCs were depleted of CD8⁺ T cells using a human CD8⁺ isolation kit (Miltenyi Biotec). CD3⁺ T cells were then selected via the human CD3 Microbead kit (Miltenyi Biotec). To isolate the different subsets of memory CD4 T cells (central memory (TCM), transitional memory (TTM) and effector memory (TEM)), the CD3⁺ CD8^- T cells were stained with Fixable Near-Infra Red (LDNIR, Invitrogen, Massachusetts, USA) for 30 minutes at 4°C followed by a wash with MACS buffer [PBS, 1% (v/v) human AB serum and 2 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich)]. Cells were then resuspended in Brilliant Stain Buffer (Becton Dickinson (BD), New Jersey, USA) and stained for 30 minutes at 4°C with the following antibodies: CD3 (AF700, clone UCHT1), CD4 (BUV496, clone SK3), HLA-DR (BB515, clone G46-6), CD25 (BV711, clone MA251), CD38 (BV421, clone HIT2), CD69 (PECy7, clone FN50), CD45RO (BV605, clone UCHL1), CCR7 (BB700, clone 3D12) and CD27 (BUV395, clone L128). All antibodies were purchased from BD Biosciences or BioLegend. This was followed by a wash in MACS buffer and resuspension at 10⁸ cells/mL for cell sorting on the BD Influx Cell Sorter. Sort purity was assessed post cell sorting and was consistently >95%. Resting memory cells were sorted as CD3⁺CD4⁺CD69^–CD25^–CD38^–HLA-DR^–CD45RO⁺ (S7A Fig) and were further subdivided into TCM (CCR7⁺CD27⁺), TTM (CCR7^-CD27⁺) and TEM (CCR7^-CD27^-).

CD8⁺ T cells were enriched from propagated TIL by depletion of CD4⁺ cells with a human CD8⁺ isolation kit (Miltenyi Biotech, Germany) according to the manufacturer’s protocol. CD8⁺ TIL were expanded for 8 days using a mini-REP in T25 flasks.