Anti ly6g 1a8

Extracted tumors were fixed with 10% formalin, embedded in paraffin and sliced in 4 μm thickness. The multiplex IHC was performed with an Opal 7-Color Automation IHC Kit (Akoya Biosciences, Hopkinton, MA, USA) and BOND RXm automated stainer (Leica Biosystems, Wetzlar, Germany) using the following antibodies: anti-pan-cytokeratin (CK) (rabbit poly; Bioss, Woburn, MA, USA), anti-CD8α (EPR20305; Abcam, Cambridge, United Kingdom), anti-CD44 (IM7; Bio X Cell, Lebanon, NH, USA), anti-F4/80 (D2S9R; Cell Signaling Technology, Danvers, MA, USA), anti-Ly6G (1A8; Bio X Cell, Lebanon, NH, USA), anti-CD11b (EPR1344; Abcam, Cambridge, United Kingdom) and anti-Gzmb (rabbit polyclonal; Abcam, Cambridge, United Kingdom). The slides were imaged in the Mantra Quantitative Pathology Workstation (Akoya Biosciences, Menlo Park, CA, USA). The multiplex IHC images were analyzed using inForm Tissue Finder software (Akoya Biosciences, Menlo Park, CA, USA). Cell phenotype was defined based on the antigen expressions as the following: CD8+ = CD8+ T cell, F4/80+ = macrophage, Ly6G+CD11b+ = neutrophil, Gzmb+CD3− = NK cell. Tissue phenotype was determined as the following: pan-cytokeratin positive = tumor. CD8+ T cells in the tumor area were counted as tumor-infiltrating CD8+ T cells.

Mice were depleted of CD4⁺ and/or CD8⁺ T cell subsets via intraperitoneal (ip) administration of 200 μg anti-CD4 (GK1.5, rat IgG2b) and 200 μg anti-CD8α (2.43, rat IgG2b) antibodies (National Cell Culture Center) in 200 μl PBS as previously described beginning at 48 hours prior to challenge and administered weekly [11 (link)]. For neutrophil depletions each mouse received ip injections of 200 ug anti-Ly6G (1A8)(BioXcell) in 100 μl every other day. For NK cell depletion, each mouse received ip injections of 0.5 ug anti-asialo-GM (Wako USA) every 3 days. Neutrophil and NK cell depletions were started 24 hours prior to challenge. Control IgG2a and IgG2b isotype control antibodies were used (eBioscience Inc., San Diego, CA). All depletions were maintained through the entirety of the study at concentrations and schedules chosen following studies testing different dosages and schedules to determine the optimum dosage and schedule for each antibody. The efficiency of all cell depletions in the lungs and spleens was assessed by flow cytometric analysis using antibodies that adhere to epitopes distinct from those adhered to by the depletion antibodies (S1 Fig). Antibodies depleted approximately 95% of neutrophils, 87% of NK cells, 98% of CD4⁺ T cells, and 98% of CD8⁺ T cells.

B16F10, MC38, A20, and 4T1 cells were purchased from ATCC. TUBO was cloned from a spontaneous mammary tumor in a BALB/c Neu-transgenic mouse^{39 (link)}. B16-EGFR5 and MC38-EGFR5 were selected from single-cell clones after being transduced by lentivirus-expressing the mutant mouse EGFR which can bind to cetuximab. TUBO, MC38, B16F10, and the derivatives were cultured in 5% CO₂ and maintained in vitro in Dulbecco’s modified Eagle’s medium, supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/l l-glutamine, 0.1 mmol/l Minimum Essential Medium nonessential amino acids, 100 U/ml penicillin, and 100 mg/ml streptomycin. 4T1 and A20 cells were maintained in vitro in RPMI 1640 medium. All cell lines were routinely tested using mycoplasma contamination kit (R&D). Anti-CD8 (TIB210), FcγRII/III blocking Ab (2.4G2), anti-CD4 (GK1.5), and anti-NK1.1 (PK136) Abs were produced in-house. Anti-PD-L1 (10 F.9G2) and anti-Ly-6G (1A8) Abs were purchased from Bio X Cell (USA). The TKI-Afatinib was purchased from Shanghai Bojing Chemical Co., Ltd FTY720 was purchased from Sigma.