Anti ctla 4 antibody

Inflammation was induced in the retina and choroid, as previously described^{11 (link)}. Briefly, animals were intraperitoneally injected with 300 μg/100 μL of anti-CTLA4 antibody (Bio X Cell, Lebanon, NH, USA; InvivoMab 9D9; Cat. #BE0164), which was adjusted with phosphate-buffered saline (PBS), three times weekly (days 0, 1, and 3) for 5 weeks. To enhance the immune response, 100 μL of complete Freund’s adjuvant (Rocklands Immunochemicals Inc., Pottstown, PA, USA; D614-0050) was injected on the first day of the weekly protocol, except during the fourth week (Fig. 1).Figure 1

Schedule of anti-CTLA4 and complete Freund adjuvant injections in mice. IP: intraperitoneal.

YSK12-C4 was synthesized as previously described [14 (link)]. The commercially available reagents used are as follows: Cholesterol (Sigma-Aldrich, St. Louis, MO, USA); 1,2-Dimirystoyl-sn-glycerol methoxyethyleneglycol 2000 ether (DMG-PEG2k) (NOF Corporation, Tokyo, Japan); Cyclic di GMP (c-di-GMP) (Cayman chemical, Ann Arbor, MI, USA); anti-programmed cell death ligand 1 (anti-PD-L1) antibody (clone: 10F.9G2), anti-CTLA-4 antibody (clone: 9H10), and anti-lymphocyte activation gene 3 (anti-LAG-3) antibody (clone: C9B7W) (Bio X Cell, West Lebanon, NH, USA).
Renca cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured with RPMI1640 medium (high glucose) containing 10% fetal bovine serum (FBS), 100 units/mL of penicillin/streptomycin (P/S), and 1 mM of sodium pyruvate.
Female BALB/c mice (6–8 weeks old) (Japan SLC Inc., Shizuoka, Japan) were housed under specific pathogen-free (SPF) conditions. The animal experiments described herein were approved by the Animal Committee of Hokkaido University (approval number: 20-0176). All methods were conducted based on the guidelines set by Hokkaido University and the guidelines established for Animal Research: Reporting of In Vivo Experiments (ARRIVE).

HLJD decoction was prepared as above and stored at − 20 °C. The following flow cytometric antibodies were obtained from Biolegend (San Diego, CA, USA): anti-mouse CD45-APC/Cy7 (Cat#157,617), anti-mouse F4/80-PE (Cat#123,110), anti-mouse CD11b-FITC (Cat#101,206), anti-mouse CD11c-PE/Cy7 (Cat#117,318), anti-mouse CD8a-FITC (Cat#155,004), anti-mouse CD4 PerCP/Cy5.5 (Cat#100,434) and anti-mouse CD3ε-PE/Cy7 (Cat#155,706). Fixable Viability Stain (FVS) 700 was purchased from BD Bioscience (San Jose, CA, USA, Cat#564,997). Primary antibody against mouse TLR7 was purchased from Proteintech (Wuhan, China), and secondary antibodies and DAPI were obtained from Servicebio (Wuhan, China). ROS staining solution (Cat#D7008) was purchased from SIGMA (St. Louis, MO, USA). Mouse anti-PD-1 antibody and anti-CTLA-4 antibody were purchased from Bioxcell (Lebanon, NH, USA).