Flow cytometry was performed using CytoFLEX (Beckman Coulter, Brea, CA, USA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). For immunolabeling TILs, fluorophore-conjugated monoclonal antibodies against the following proteins were used: CD8 (RPA-T8, Cat# 301048, Biolegend, San Diego, CA, USA, 1:50), CD3 (SK7, Cat# 344808, Biolegend, San Diego, CA, USA, 1:100), PD-1 (EH12.2H7, Cat# 329933, Biolegend, San Diego, CA, USA, 1:20), CD103 (Ber-ACT8, Cat# 350230, Biolegend, San Diego, CA, USA, 1:20), CD39 (A1, Cat# 328210, Biolegend, San Diego, CA, USA, 1:20), GZMB (QA16A02, Cat# 372214, Biolegend, San Diego, CA, USA, 1:50), and CD4 (RPA-T4, Cat# 560837, BD Biosciences, San Diego, CA, USA, 1:50). The LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit was from Invitrogen (L34973, Waltham, Massachusetts, USA, 1:100). The sequential gating strategies used for analysis for data obtained from flow cytometry are described in Supplementary Fig. S15 .
For t-distributed stochastic neighbor embedding (tSNE) visualization of flow cytometry data, we used three samples for each group (EGFR-WT and EGFR-MT). Each sample was down-sampled to 7000 randomly selected CD3+ live and singlet-gated cells, yielding 42,000 cells. A tSNE plot for the merged 42,000 cells was constructed using FlowJo (v10.6.2) with default settings (3000 iterations and perplexity = 100).
For t-distributed stochastic neighbor embedding (tSNE) visualization of flow cytometry data, we used three samples for each group (EGFR-WT and EGFR-MT). Each sample was down-sampled to 7000 randomly selected CD3+ live and singlet-gated cells, yielding 42,000 cells. A tSNE plot for the merged 42,000 cells was constructed using FlowJo (v10.6.2) with default settings (3000 iterations and perplexity = 100).