Profiling Tumor-Infiltrating CD8+ T Cells

Tumor samples were disposed with Tumor Dissociation Kit (Miltenyi Biotec). Then single-cell suspensions from tumors were lysed to remove red blood cells using RBC Lysis/Fixtion Solution (BioLegend) and filtered through a 70 μm MACS SmartStrainer (Miltenyi Biotec). After that, the single-cell suspensions were incubated with fluorescently labeled antibodies (BioLegend) against CD45, CD3, CD4, CD8 and PD-1 at 4℃ for 45 min. For intracellular staining, cells were fixed and permeabilized, and then incubated with TIM-3 (BioLegend), TOX (eBioscience), TCF-1 (BD) at 4℃ for 6 h. Multicolor flow cytometry analysis was conducted with BD Fortessa flow cytometer. Expression level of PD-1 on CD8⁺ T cells was gated by its low controls of CD8⁺ T cells from spleen of untreated mice (PD-1^low), high controls of cells stained by both anti-TOX and anti-PD-1(PD-1^hi) and between them (PD-1^int) [15 (link)] (Details are provided in the Supplementary Methods section). The proportion of early Tex (CD8⁺PD-1^int) and terminal Tex (CD8⁺PD-1^hi) in CD8⁺ T cells, the ratio of early to terminal Tex (E/T), and mean fluorescence intensity (MFI) of PD-1 expression on CD8⁺ T cells were further analyzed using FlowJo software (Treestar).