Anti il 10r 1b1.3a

In vivo whole-animal blockade of HMGB-1, PD-L1, and Tim-3 was conducted by intraperitoneal (IP) injection of 300 μg of anti-HMGB-1 (pAb; Shino-Test Corporation, Kanagawa, Japan), anti-PD-L1 (10F.9G2), or anti-Tim-3 (RMT3-23; BioXCell, West Lebanon, NH). For in vitro and in vivo lymph node blockade, CD8⁺ T_reg cells were precoated with 20 μg/mL of anti-PD-L1 and/or anti-Tim-3 for 1 hour at 37°C. Recombinant mouse Gal-9 (rGal-9; 1.0 μg/mL; R&D Systems), 20 μg/mL of anti-Gal-9 (RG9-1), 20 μg/mL of anti-IL-10R (1B1.3A; BioXCell), and 0.5 μg/mL of anti-HMGB-1 (pAb; eBioscience) were added to culture media in relevant experiments.

Mice were infected with 1x10⁶ or 1x10⁷ CFU for Salmonella typhimurium SL7207 strain or 100 CFU for SL1344 strain intravenously (i.v.). For survival and vaccination plus rechallenge experiments mice were first vaccinated with 1x10⁶ CFU of SL7207 given i.v. and 90 days later were rechallenged with 100 CFU of SL1344 or 1x10⁶ or 10⁷ CFU of SL7207. During survival experiments, mice were daily checked and presented as percentage of live animals. The bacterial loads were determined by plating a series of dilutions of homogenized organs on MacConkey agar plates. Heat-killed S. typhimurium (HKST) was prepared by inactivation of the SL1344 strain in water bath at 70°C for 1 hour. Bacteria were suspended in PBS. Salmonella infection was performed in a blinded manner, and identities of the mice were revealed upon termination of the experiment. No randomization was used. Estimation of size groups was based on our previous experience with these disease models, without a priori determination via power calculation. For neutralization of IL-10 signaling, mice were treated i.v. with a combination of anti-IL-10 (JES5-2A5; 500 μg/mouse/injection; BioXCell) and anti-IL-10R (1B1.3A; 500 μg/mouse/injection; BioXCell) on days −3, −1, and +1 after infection.