Realease cd3 microbead kit

Pierce streptavidin-coated high-capacity flat-bottom 96-well plates (Thermo Fisher Scientific) were coated with 50 μl biotinylated MR1/5-OP-RU monomer (NIH Tetramer Core Facility) at 10 μg per ml in PBS (Sigma-Aldrich) overnight at 4 °C. Cryopreserved PBMCs were thawed in complete medium. CD8⁺ T cells were isolated using CD8 MicroBeads (Exp 3; Miltenyi Biotec) and CD3⁺ T cells using the REAlease CD3 MicroBead Kit (Exp 4 and validation experiments; Miltenyi Biotec) following the manufacturer’s instructions. Isolated CD8⁺/CD3⁺ T cells were washed in complete medium and resuspended at 1 × 10⁷ cells per ml. One million (20 h stimulation) or 500,000 (68 h stimulation) cells were added per well to the appropriate 96-well plates (MR1/5-OP-RU-coated plate for TCR and TCR+cytokine stimulation, round-bottom plate for unstimulated and cytokine stimulation). IL-12 (50 ng ml⁻¹; R&D Systems) and IL-18 (50 ng ml⁻¹; R&D Systems) were added for cytokine stimulation; αCD28 (1 μg ml⁻¹; clone: CD28.2; BioLegend) for TCR stimulation; IL-12, IL-18 and αCD28 for TCR+cytokine stimulation; and complete medium for unstimulated cells (final volume 200 μl per well). Cells were incubated for 20 h or 68 h at 37 °C, 5% CO₂. For intracellular cytokine staining, brefeldin A (BioLegend) and monensin (BioLegend) were added for the final 4 h.

Peripheral blood mononuclear cells (PBMCs) were derived from healthy human donors. Primary human CD3⁺ T cells were isolated from PBMCs by positive selection using REAlease CD3 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated CD3⁺ T cells were stimulated for 24 h with recombinant human IL-2 (100 U/mL; PeproTech, Rocky Hill, NJ, USA) plus anti-CD3 (10 ng/mL; eBioscience, Pittsburgh, PA, USA) antibodies in accordance with the manufacturer’s instructions. CD3⁺ T cells were then transduced with the lentiviral vector at a multiplicity of infection of 16 U/cell. The transfected T cells were cultured at 8 × 10⁵ cells/mL in the presence of recombinant human IL-2 (300 IU/mL) every other day. Genetically modified T cells were isolated using a flow sorter (FACSAria III; BD Biosciences, San Jose, CA, USA) prior to being used for functional assays.

Samples of tumor and peripheral blood mononuclear cells (PBMCs) from 29 patients with stage IV melanoma were obtained under the internal review board–approved protocols UPCI 05–140 and UPCI 96-099, including 22 men and 7 women, ranging from age 31 to 82, recruited from UPMC Hillman Cancer Center. Metastatic sites included skin or soft tissue (20%), lymph nodes (40%), lung (20%), and other visceral locations (20%). The tumor and blood samples were collected before therapy for stage IV melanoma or more than 3 years after the end of IFN adjuvant therapy. Tumor samples were dissociated and enzymatically digested with a tumor dissociation kit (Miltenyi Biotec). Cell suspensions were then aspirated through an 18G needle 10 times and strained through a 70 μm mesh (Miltenyi Biotec) before RBC lysis using ACK lysing buffer (Thermo Fisher Scientific). Human CD45⁺CD3⁺ T lymphocytes and CD45⁺CD3^– cells were purified from tumor single-cell suspensions with magnetic separation using REAlease CD3 MicroBead Kit (Miltenyi Biotec). Cells were cultured in complete Iscove’s DMEM (10% human serum, 1% penicillin-streptomycin, 1% l-glutamine, 1% nonessential amino acids [NEAA], and 25 mM HEPES) (Gibco, Thermo Fisher Scientific) at 37°C in a 5% CO₂ incubator.