Resected B78 tumors were mechanically dissociated for 45 min using a gentleMACS dissociator (Miltenyi Biotec) in HBSS supplemented with 1 mg/mL collagenase type D and 100 µg/mL DNAse I (Sigma-Aldrich) to obtain single cell suspensions.22 (link) Ghost Dye Red 780 (Tonbo Biosciences) was used for viability staining. For cell surface staining, cells were preincubated with mouse FC Block anti-mouse CD16/32 (clone 2.4G2, BD Biosciences). After blocking, the cells were labeled with CD3-PE-Cy5 (clone 145–2 C11, Biolegend), CD4-PE-Dazzle594 (clone GK1.5, Biolegend), CD8a-APC-R700 (clone 53–6.7, Biolegend), CD25-BB515 (clone PC61, BD Biosciences), CD45-BV510 (clone 30-F11, Biolegend), GD2-APC (clone 14G2a, Biolegend), and NK1.1-BV421 (clone PK136, Biolegend). Cells were then fixed and permeabilized overnight using Foxp3/Transcription Factor Staining Buffer Set (eBioscience). FoxP3 intracellular staining was then performed prior to flow cytometry. Flow cytometry data were acquired using an Attune NxT Flow Cytometer and analyzed using FlowJo V.10.7.1. The flow cytometry gating strategy is shown in online supplemental figure 1.