Isotype matched igg controls

The single‐cell suspension was obtained from the spleen, lymphocyte node (LN), blood and tumour of HNSCC mouse model as previously described 21. The following antimouse antibodies were used for staining: FITC‐conjugated CD4, CD8 and CD11b, PE‐conjugated B7‐H3 and Gr‐1 (all from Becton Dickinson, Mountain View, CA, USA); PerCP‐Cy5.5‐conjugated F4/80, PE‐conjugated IFN‐γ, mouse regulatory T cell staining kit #3 (all from eBioscience, San Diego, CA, USA); and isotype‐matched IgG controls (eBioscience). The cells were analysed using FlowJo (Tree Star, Ashland, OR, USA) and gated by the side scatter and forward scatter filters. Dead cells were excluded by staining 7AAD (Invitrogen, Carlsbad, USA).

FACS was performed on single cell suspensions from Spleen and blood in Tgfbr1/Pten 2cKO mice with or without STAT3 blockade. Single cell suspension from tumor tissues were acquired according to a protocol previously reported^{30 (link)}. Briefly, tumor tissue was processed with a gentleMACS™ Dissociator and a murine tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) to create a single cell suspension. Wild type controls with same dose tamoxifen were set for flow cytometry analysis. Single cell suspensions from spleen, blood and tumor were processed according to a standardized protocol. These cells were labeled with FITC-conjugated anti-mouse CD4, CD8, and CD11b (all from BD Biosciences, 554046, 553030, 561689); Percp-Cy5.5-conjugated anti-mouse F4/80 (from eBioscience,45–4801), PE-conjugated anti-mouse Gr-1 (from BD Bioscience, 562060); isotype-matched IgG controls (eBioscience, 12–4732). These cells were analyzed on a FACS caliber flow cytometer with CellQuest software and gated by the side scatter and forward scatter filters (Becton Dickinson, Mountain View, CA). Live cells were gated by 7AAD (Invitrogen) and populations phenotyped as described above.