Tumor and Spleen Single-cell Isolation and Analysis

Single-cell suspensions isolated from tumors and spleen were acquired as previously described^{26 (link)}. In brief, tumors were dissociated by sufficient mincing in complete RPMI 1640 medium followed by collagenase Type IV (Sigma-Aldrich) and DNase I (Sigma-Aldrich) digestion for 1 hour at 37°C and filtering through 70-μm nylon cell strainers in phosphate buffer saline. The cells were immediately analyzed by flow cytometry. Human-specific antibodies were purchased from ENZO Life Science (LAG-3, 17B4) and BD Pharmingen (CD3, CD4, CD8). Isotype controls were purchased from BD Pharmingen. Mouse-specific antibodies were purchased from eBioscience (Cd3e, Cd4, and Cd8a). Intracellular staining for IFN-γ was performed using the Fixation and Permeabilization Buffer Kit (BD Bioscience). Cells were sorted using the Beckman flow cytometer, and the data were analyzed using FlowJo software.