Patients and sample preparation

Surgical specimens (one piece of rectal tumor and one piece of unaffected recto-sigmoidal mucosa per patient) and heparin-anticoagulated peripheral blood at surgery and one follow-up time point were obtained from five treatment-naïve rectal cancer patients. All patients gave written informed consent and the study was approved by the local ethics committee (protocol EA1/007/16 to L.H.). TILs and T_UM were isolated from fresh specimens immediately after surgery as previously described.²² In short, tissue was cut into small pieces (2–4 mm³) and incubated in PBS containing 10 mM Ethylendiaminetetraacetic acid (EDTA, Invitrogen) for 30 min. Cells in suspension were passed through a 100 μm cell strainer (Corning), tissue was incubated in RPMI1640 containing 5% fetal bovine serum (FBS) and 0.5 mg/ml collagenase (Serva, Collagenase NB 4) for 30 min. Finally, cells were enriched through Percoll (GE Healthcare) gradient centrifugation and cryopreserved. Distances between the tumor and unaffected mucosa specimens varied between patients but unaffected mucosa samples were taken at least 4 cm apart from the macroscopic tumor margin (^{Figure 1}). PBMCs were isolated with Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation. All cell preparations were cryopreserved in RPMI1640 containing 50% FBS, 10% DMSO before further processing.