Live dead nir

J774M cells treated with entinostat or DMSO vehicle for 16 hours were co-cultured with stimulated T cells, and T-cell proliferation was measured via CFSE dilution. T cells were isolated from spleens of BALB/c mice as described above and subsequently labeled with CFSE (ThermoFisher, cat. C34554) per the manufacturer’s instructions. 2.5 x10⁵ CFSE-labeled CD8⁺ T cells were co-cultured with J774M cells at varying ratios (1:1, 1:2, 1:4, and 1:8 J774M:T cells) and anti-CD3/CD28 beads (ThermoFisher, cat. 11453D) at a bead-to-T cell ratio of 1:1, per the manufacturer’s instructions, for T-cell activation. T cells were allowed to proliferate for 52 hours. Subsequently, the cultures were harvested, stained with Live/Dead NIR (ThermoFisher, cat. L10119), as well as anti-CD8, and analyzed via flow cytometric analysis as described above. Dilutions of initial CFSE were indications of T-cell division, where fewer divisions indicated greater suppressive activity. All antibodies used are listed in Supplemental File S1.

Redirected cytotoxicity assays were performed as described(4 (link)). Briefly, P815 cells were incubated with 5 μg/ml of the indicated combinations of mAbs to CD28H (R&D MAB83162), 2B4 (BioLegend 329502), NKp46 (BD 557847), NKG2D (R&D MAB139), CD2 (BD 555323), DNAM-1 (BD Biosciences 559787), CD16 (BD 555404) and CD56 (BD 555513) for 15 minutes at room temperature. Resting NK cells were added at an E:T ratio of 1:2, mixed and gently centrifuged at 300 rpm for 1 minute. After 2 hours at 37°C, cells were stained with Live/Dead-NIR (Thermo Fisher), anti–CD56-Bv421 (BD 562751) and anti–CD107a-PE (BD 555801) and analyzed by flow cytometry. Target cell lysis assays were either performed using the ToxiLight Non-Destructive Cytotoxicity BioAssay Kit (Lonza) following the manufacturer’s instruction, or through a flow-based assay. Briefly, NK cells were incubated with PKH67-labeled target cells for 6 hours in IMDM medium with 10% FCS at the indicated E:T ratios. Cells were stained with Live/Dead NIR, and the lysis of target cells were determined by flow cytometry. For CD28H blocking, NK cells were pre-incubated with 10 μg/ml CD28H antibody (R&D Systems) for 15 min before mixing with 221.B7H7 cells. KHYG-1 and NKL cells were rested in complete IMDM medium without IL-2 for 1 day, prior to use in cytotoxicity assays.

Clodronate (CCL) liposomes (5mg/mL) (n=3) and PBS liposomes (n=3) were injected intraperitoneally in animals at 2-day intervals beginning 3 days pre-implantation through endpoint (post-operative days 4 and 14). Systemic macrophage depletion was confirmed using flow cytometry. In brief, bone marrow was extracted from mice femur and long bones. Extracted cells were treated with RBC lysis buffer (Thermo Fisher, MA, USA). Cells were blocked with Fc-block and stained with Live/Dead-NIR (Thermo Fisher), CD45-BV510 (Biolegend, San Diego, CA, USA), Ly6C-PE (Biolegend), and F4/80-BV421 (Biolegend), fixed with 4% paraformaldehyde, then sorted using BD LSRFortessa. Data analysis was performed using the FloJo software (FlowJo LLC, Ashland, Oregon).