Anti cd4 rm4 5

Cells isolated from the BAL, HLN, and lung tissue were analyzed via flow cytometry using the following monoclonal antibodies: anti-F4/80 (BM8.1; Tonbo Biosciences, San Diego, CA), anti-CD11b (M1/70; eBioscience, San Diego, CA), anti-CD11c (N418; eBioscience), anti-CD3 (145-2C11; Tonbo Biosciences) and anti-CD4 (RM4-5; Tonbo Biosciences). Samples were stained as previously described [17 ]. Briefly, cells were washed in PBS containing 0.2% bovine serum albumin and 0.1% NaN₃. Aliquots containing 10⁵–10⁶ cells were incubated with anti-mouse CD16/CD32 (FcBlock; eBioscience) for 15 min. Cells were then stained with 100 μl of appropriately diluted Live/Dead Fixable Blue Dead Cell Stain (Invitrogen, Grand Island, NY) and surface antibodies for 30 min at 4°C. Following staining, cells were fixed with 4% paraformaldehyde. For identification of Tregs, cells stained with anti-CD3 and anti-CD4 were treated with Foxp3/Transcription Factor Fixation/Permeabilization buffer (eBioscience) according to the manufacturer’s instructions and stained with anti-Foxp3 (FJK-16s; eBioscience). Samples were run with corresponding isotype controls on a BD LSR II (Becton Dickinson, Franklin Lakes, NJ) and analyzed with FlowJo (Tree Star Software, Ashland, OR).

Whole tumor infiltrating leukocytes population dynamics were determined by enzymatically disassociating primary tumors from Ccr2^GFPFoxp3^DTR-GFP mice as mentioned above. TAM activation markers were determined by disassociating primary tumors of Foxp3^DTR-GFP tumor bearing mice 4 days after intervention as described above. Cell suspensions were stained with anti-CD45 (30F11, 1:1000, BD Bioscience), Ghost Violet Viability (1:2000, Tonbo Bioscience), (anti-CD11B (M1/70, 1:1000, eBioscience), anti-CD4 (RM4–5, 1:1000, Tonbo Bioscience), anti-CD8a (53–6.7, 1:800, eBioscience), anti-CD19 (1D3, 1:1000, Tonbo Bioscience), antiCD11c (HL3, 1:1000, BD Bioscience), anti-NK1.1 (PK136, 1:1000, Tonbo Bioscience), anti-F4/80 (BM8, 1:1000, eBioscience), anti-Ly6C (HK1.4, 1:1000, eBioscience), anti-Ly6G (1A8, 1:1000, eBioscience), anti-MHCII (M5/114.15.2, 1:800, Tonbo Bioscience), anti-CD86 (GL1, 1:500, BD Bioscience), anti-CD80 (16–10A1, 1:500, BD Bioscience), and anti-FoxP3 (FJK-16S, 1:500, Invitrogen). Stained cells were analyzed with a LSRII Fortessa flow cytometer (BD) from the VCU Flow Cytometry Core Facility. Data were analyzed using FlowJo software (Tree Star).