Ml rr s2 cda adu s100

Tumor-infiltrating lymphocytes (TIL) were analyzed by 16-color multi-parametric flow cytometry using the following antibodies: BUV737 anti-CD3 (17A2), Alexa Fluor 700 anti-Granzyme B (GB11), BV605 anti-CD11c (HL3), APC-Cy7 anti-CD11b (M1/70), anti-mouse CD16/32 (2.4G2, mouse Fc-block) from BD Bioscience (San Jose, CA), BV650 anti-CD8 (53–6.7), APC anti-CTLA-4 (UC10-4B9), PerCP-ef710 anti-Lag3 (C9B7W), PE-Cy7 anti-PD-1 (RMP1–30), BV711 anti-PD-L1 (10F.9G2), PE-Cy5 anti-F4/80 (BM8) from Biolegend (San Diego, CA), Alexa Fluor 488 anti-FoxP3 (150D/E4) and e450 anti-Gr-1 (RB6-8C5) from eBioscience (Waltham, MA). The following antibodies for in vivo administration were purchased from BioXcell (West Lebanon, NH) and used at the concentrations shown: α-PD-1 (RMP1–14 at 250 μg per dose), α-CTLA-4 (9H10 at 100 μg per dose) and α-Lag-3 (C9B7W at 200 μg per dose). The STING agonist ML-RR-S2 CDA (ADU-S100) was procured from MedChemExpress (Monmouth Junction, NJ). For fluorescence immunohistochemistry, rabbit monoclonal anti-mouse PD-L1 antibody was purchased from Abcam (Cambridge, MA) and chicken anti-rabbit IgG cross-absorbed antibody Alexa Fluor 594 conjugate from Invitrogen (Carlsbad, CA).

STING agonist (ML-RR-S2 CDA, ADU-S100, Med Chem Express) was resuspended in DMSO at a concentration of 6.9 mg/ml and diluted in 1× phosphate buffer saline (PBS, Gibco) prior to injection.