Rhil 2

Primary NK cells were isolated from at least 5 x 10⁶ cells of cryopreserved PBMCs using the EasySep Human NK cell Enrichment Kit according to the manufacturer’s instructions (STEMCELL Technologies, Canada). The purity of NK cells was > 95% of CD3^-CD56⁺ cells as determined by flow cytometry. Primary NK cells obtained from healthy donors (n =4) were cultured and expanded in NK MACs medium (Miltenyi Biotec, Bergisch-Gladbach, Germany) supplemented with 10% human AB serum (Sigma,), 1 mM Penicillin/Streptomycin (Sigma), 5 ng/ml of rhIL-2 and rhIL-15 (Biolegend, CA, USA) at 37°C with 5% CO₂ for 7-10 days. Expanded NK cells were used in the experimental validation of the NK cell memory assay.

The MC38 and CT26 cell lines were obtained from the Einstein Cytogenetics Facility cell line repository. The SKBR-3, MDA MB-468, OVCAR-3, OVCAR-4, and NCI HC322M cell lines were derived from the National Cancer Institute Developmental Therapeutics Program cell line repository. The Hepa1-6 cell line was obtained from the Marion Bessin Liver Center at the Albert Einstein College of Medicine. MC38-derived cell lines were cultured in RPMI (Corning) supplemented with 10% FBS. Hepa1-6 and CT26-derived cell lines were cultured in DMEM (Corning) supplemented with 10% FBS. Primary T cells were cultured in T cell medium: RPMI supplemented with 10% FBS, 100 U/mL penicillin-streptomycin, 10 mM HEPES, L-glutamine, 1 mM sodium pyruvate, MEM non-essential amino acids, and 55 µM β-mercaptoethanol. T cell medium was also supplemented with cytokines (Biolegend) as needed; 10 ng/mL rhIL-2, and variable concentration of rhTGFβ1 (Biolegend) as noted per experiment.

Ficoll (GE Healthcare) was applied to separate human PBMCs. CD8⁺ T cells were isolated from purified PBMCs or tumour tissue‐digested single‐cell suspensions by human CD8 nanobeads (MojoSort, BioLegend). During a 4‐day incubation, bead‐purified peripheral CD8⁺ T cells (1 × 10⁵ cells/well in 24‐well plates) were co‐cultured with IgG_2B‐treated TAMs or α‐VISTA‐treated TAMs isolated from tumour tissues in 1 mL Roswell Park Memorial Institute (RPMI) 1640 (Gibco) with 10% FBS (Gibco), rhIL‐2 (80 ng/mL, BioLegend), anti‐CD3 (2 μg/mL, BioLegend) and anti‐CD28 (1 μg/mL, BioLegend) antibodies (Figure 6G‐H). After 4‐day incubation, CD8⁺ T cells were harvested for flow cytometry (FC) or intracellular flow cytometry (ICFC).

The NK cell line NK-92 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and the human HCC SK-Hep1-Luc cell line was provided by Professor Kuroda (Osaka University). NK-92 cells were cultured in alpha-MEM (Gibco, Grand Island, NY, USA) with 20% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA), and rhIL-2 (200 U/mL, BioLegend, San Diego, CA, USA). SK-Hep1-Luc cells were cultured in Minimum Essential Media (GenDEPOT, Katy, TX, USA) supplied with 10% FBS (Gibco) and 1% penicillin and streptomycin (Gibco). Both were maintained at 37 °C in a humidified atmosphere with 5% CO₂.

MAIT cell cloning was performed as described by Cansler et al. (22 ). Briefly, MR1 tetramer-positive T cells were sorted from cryopreserved bronchoalveolar lavage cells and rested overnight. Limiting dilution assay was performed to seed single cells in 96-well plates in RPMI media containing 10% human serum (HuS), 2% L-glutamine, 0.1% gentamycin and irradiated feeder cells (lymphoblastoid cell lines and peripheral blood mononuclear cells). Media was supplemented with recombinant human (rh)IL-2 (5 ng/mL), rhIL-7 (0.5 ng/mL), rhIL-12 (0.5 ng/mL), rhIL-15 (0.5 ng/mL) (BioLegend) and anti-CD3 (0.03 µg/mL) (eBiosciences). Plates were assessed weekly for growth with clones taking 2 – 3 weeks to grow. Clones were taken forward for subsequent analyses only from plates, which had growth in approximately less than 30% of the wells. Resulting clones were subjected to surface MR1 5-OP-RU tetramer and monoclonal antibody staining with 1:200 of Live/dead viability dye (Aqua, Life Technologies), 1:25 of anti-CD3 PerCP/Cy5.5 (UCHT1, BioLegend), 1:25 of anti-CD4 BV785 (OKT4, BioLegend), 1:50 of anti-CD8 (FITC, RPA-T8) and 1:25 of anti-CD26 PE-Cy7 (BA5b, BioLegend), as well as IFN-γ ELISPOT assay to confirm MAIT cell identify and clonal purity.

T cells were maintained in T cells Serum‐free Medium (RC‐003‐500; STEMERY) after separation, supplemented with 20 ng/mL rh‐IL‐2 (589102; Biolegend), 100 U/mL penicillin and 100 μg/mL streptomycin and activated with 5 μg/mL plate‐coated CD3 antibody (16‐0037‐85; eBioscience) and 2 μg/mL free CD28 antibody (16‐0037‐28; eBioscience). For chemotaxis assays, PBMCs were cultured in the same condition as T cells and activated for 3 days. Jurkat cells were incubated in RPMI 1640 supplemented with 10% exosome‐free foetal calf serum and activated with CD3 and CD28 for 48 hours.

Peripheral blood mononuclear cells (PBMCs) of asymptomatic HTLV-1 carriers (proviral load: 6.3–13.1%) and HTLV-1^- individuals were used in this study. CD4⁺ T cells in the live cell gate were sorted from the PBMCs by using a FACSAria II (BD Biosciences) and cultured in RPMI 1640 medium containing 10% FCS and 10 ng/mL rhIL-2 (BioLegend). The antibodies used for staining were as follows: PE-anti-CD3 (OKT3), Pacific Blue-anti-CD3 (OKT3), APC-anti-CD4 (RPA-T4), Pacific Blue-anti-CD14 (M5E2; all from BioLegend), and PE-anti-CADM1 (3E1; MBL, Nagoya, Japan). Total RNA and genomic DNA were isolated using the RNeasy micro kit and QIAamp DNA micro kit (both from Qiagen), respectively. The RNA and DNA samples were used for qRT-PCR (M-Sec mRNA) and qPCR (provirus), respectively. The cells were also used for immunofluorescence (Gag).