Cd3 sk7

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).

The WB MDSC 9-color, 11-parameter flow cytometric assay included the following fluorescently-labeled antibodies: CD45-V500-C (2D1), CD19-FITC (HIB19), CD20-FITC (L27), CD56-PECY7 (NCAM 16.2), HLA-DR-APC-H7 (L243), CD33-PE (WM53), CD11b-APC (ICRF44), and CD14-BV421 (MΦP9) (all, BD Biosciences); CD3-FITC (SK7), CD16-BV785 (3G8), and CD15-BV650 (W6D3) (all Biolegend). Samples were treated with BD FACS™ Lysing Solution (BD Biosciences) to lyse RBC. Total MDSC were defined as CD45 + CD3⁻CD19⁻CD20⁻CD56⁻CD16⁻HLA-DR⁻CD33 + CD11b + cells, while the monocytic (M-MDSC) and granulocytic or polymorphonuclear (PMN-MDSC) subsets were defined as CD14+ and CD15+, respectively. Absolute cell numbers were obtained using Trucount tubes (BD Biosciences). Presence of intracellular Ki67 was analyzed using antibodies to CD14 (MΦP9, exclusion marker), CD56 (NCAM16.2), CD4 (SK3), CD8 (SK1) (all, BD Biosciences); CD3 (SK7) and Ki67 (both, Biolegend) as well as Fixable Viability Dye eFluor 780 and FoxP3/Transcription Factor Staining Buffer Set (both eBioscience, San Diego, CA). All samples were fixed with 2% paraformaldehyde and data acquired on the same day. Flow cytometric data analysis was performed using a BD LSRFortessa and FlowJo software v9.9.5 (Treestar, Ashland, OR). Statistical analyses utilized paired or unpaired t tests, as appropriate.