Clone uchl1

Total human lymphocytes were isolated using the Elutra Cell Separation System (Gambro, Lakewood, CO, USA) from fresh leukapheresis material and frozen until further use. Naïve human CD4+ T cells were isolated from buffy coats. First, the PBMC fraction was isolated by density gradient centrifugation (Lymphoprep, Axis-shield, Alere Technologies AS, Oslo, Norway). From PBMC fraction, CD4+ cells were isolated by negative selection using magnetic separation followed by negative selection of CD45RA population (both Miltenyi Biotech, Leiden, Netherlands). Purity of isolated naïve CD4+ T cells ≥95% as determined by staining with FITC-labeled anti-CD45RA (Clone HI100, eBioscience) and BV421-labeled anti-CD45RO (Clone UCHL1, BD Bioscience).

An support vector machine (SVM) based tumor microenvironment signature integrating seven features, including CD3 _{invasive margin (IM)}, CD8 _IM, CD45RO _{center of tumor (CT)}, CD66b _IM, CD34, periostin, and cyclooxygenase-2, was previously developed and validated (7 (link)). Formalin-fixed paraffin-embedded (FFPE) samples were processed for IHC staining as previously described (7 (link), 28 (link)–30 (link)). Following incubation with an antibody against human CD3 (pan T lymphocytes; NeoMarker, clone SP7), CD34 (Abcam, ab81289), CD8 (cytotoxic T lymphocytes; NeoMarker, clone SP16), CD45RO (memory T lymphocytes; Invitrogen, clone UCHL1) and CD66b (neutrophils; BD Pharmingen), periostin (Abcam,ab92460), and cyclooxygenase-2 (Abcam, Cambridge, MA), the sections were stained in an EnVision System (Dako) (Table S1). Two pathologists who were blinded to clinical outcomes independently scored all samples. If there was a difference opinion between the two primary pathologists, the third pathologist was consulted to give the final decision. As the previously described (7 (link)) and the result of IHC, every patient was classified into a high-SVM group and a low-SVM group. Detailed information is provided in the Supplementary Materials.

An aliquot of the T cell-enriched samples from each donor was stained with anti-human CD3 (cat No. 300426, clone UCHT1, BioLegend, San Diego, CA, USA) for 30 minutes at room temperature to validate the isolation of T cells. The aliquots were also stained with anti-human CD3 (cat No. 300426, clone UCHT1, BioLegend), anti-human CD8 (cat No. 560662, clone RPA-T8, BD Biosciences, Franklin Lakes, NJ, USA), and anti-human CD45RO (cat No. 562299, clone UCHL1, BD Biosciences). The cells were then treated with fixation/permeabilization concentrate (cat No. 00-5123-43, Invitrogen, Waltham, MA, USA) to fix and permeabilize cells for 1 h at 4°C, and intracellular staining was conducted according to the manufacturer’s recommendations. The cells were stained with anti-human interferon γ (IFN-γ) (cat No. 56-7319-41, clone 4S.B3, Invitrogen), anti-human tumor necrosis factor α (TNF-α) (cat No. 25-7349-41, clone MAb11, Invitrogen), and anti-human IL-2 (cat No. 500307, clone MQ1-17H12, BioLegend). Sample data were acquired using the LSR Fortessa X20 and were analyzed using FlowJo software.