Soluble anti cd3

Fresh non-small cell lung cancer (NSCLC) tumor tissues were processed into single cells suspensions by fine cutting with a scalpel, followed by a 30 min incubation at 37°C in digestion media composed of 10 mL DMEM with 0.5 mg/mL collagenase type I, and 400 U/mL DNase I. Single cells were separated from undigested material with a 70 µM strainer and washed. If cell viability was less than 30%, a Ficoll-density gradient separation was performed to enrich live cells. A total of 0.1×10⁶ tumor digested cells per well were cultured in 96-well round-bottom plates and stimulated with 10 ng/mL soluble anti-CD3 (BioLegend, clone OKT3) in the presence of indicated concentration of MK-5890 or Fc mutant hCD27.131A mIgG1 D265A, 10 µg/mL pembrolizumab, or 20 µg/mL IgG1 isotype control mAb. The supernatants were collected on day 6 and IFNγ was measured using a human IFNγ tissue culture kit (Meso Scale Discovery).

The cervical draining lymph nodes, located beneath the superficial layer of skin on the neck, near the jugular vein, were identified as two or three yellowish, round nodes and collected. Single-cell suspensions were created from cervical draining lymph nodes to isolate the CD4⁺CD25^– T effector cell fraction by magnetic activated cell sorting (MACS) sorting (130-091-041; Miltenyi Biotec, Bergisch-Gladbach, Germany), a protocol that removes non-CD4⁺ cells and positively selects CD4⁺CD25⁺ cells, allowing collection of CD4⁺CD25^– T effectors. T effector cells from allogeneic transplant recipient (week 2 post-transplantation) or naïve mice were co-cultured with MK/T-1 cells, a mouse corneal fibroblast cell line at 70% confluence in a 24-well plate and activated with soluble anti-CD3 (10 µg/mL, 100359; BioLegend) and anti-CD28 (4 µg/mL, 102116; BioLegend).²¹ (link) For IFN-γ neutralization, the co-culture system was incubated with IFN-γ neutralizing antibody or isotype control for 24 hours at a concentration of 1 mg/mL (BE0055; Bio X Cell, Lebanon, NH, USA).