Rat igg2a clone 2a3

Cells were injected intradermally onto the backs of C57BL/6, NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG), or B6. 129S7-Rag1^tm1Mom/J (Rag1 KO) mice. Cell numbers were based on previous publications and past experiments (YUMM1.1 = 1×10⁶ cells; 1014 = 5×10⁵ cells) (Meeth et al., 2016 (link)). Tumors were considered fully formed when they reached ~50mm³. For CD8+ depletion, animals were treated with 300 μg of anti-CD8α (clone 53–6.72) or the corresponding isotype control (Rat IgG2a, clone 2A3) (BioXCell; West Lebanon, NH) by intraperitoneal injection 2 days prior to tumor implantation and 2 times per week for the duration of the experiment. Treatments were determined based on previous publications. Animals were sacrificed when tumors exceeded 650mm³ (Erkes et al., 2020 (link)).

Viruses were administered via the intranasal (i.n.) route to generate respiratory infections. Most studies utilized primary influenza X31 expressing the GP_33–41 epitope from LCMV (X31-GP33, 1.6×10⁵ TCID₅₀) (Laidlaw et al., 2013 (link)). In some studies, primary LCMV Armstrong (10⁴ PFU), vaccinia virus-expressing GP_33–41 from LCMV (VV-GP33, 10⁴ PFU) (Rodriguez et al., 1998 (link)), or influenza strain PR8 expressing the GP_33–41 epitope from LCMV (PR8-GP33, 0.3 LD50) (Mueller et al., 2010 (link)) were used and delivered via the i.n. route. For re-challenge experiments (secondary PR8-GP33 challenge), mice were infected i.n. with PR8-GP33 (10 LD₅₀) at > 35 days after primary infection. Recombinant influenza strains containing the LCMV GP_33–41 epitope were provided by Dr. Richard Webby (St. Jude Children’s Research Hospital, Memphis, TN) (Laidlaw et al., 2013 (link); Mueller et al., 2010 (link)). Viral titers in lungs were determined by quantitative real-time PCR as described (Laidlaw et al., 2013 (link)). For in vivo antibody blockade experiments, WT C57BL/6 mice were given 200 μg per mouse per injection of rat anti-PD-1 (clone 29F.1A12) or isotype control antibody (Rat IgG2a, clone 2A3 from BioXCell) on days −1, 2, and 5 p.i. and analyzed at day 8 p.i., or on days −1, 2, 5, and 8 p.i. and analyzed at day 60⁺ p.i.

Neutrophils were depleted from mice as described previously (20 (link)). Briefly mice were treated with 1.0 mg of anti-mLy-6G antibody (clone 1A8, BioXCell, West Lebanon, NH) or 1.0 mg of isotype control antibody (rat IgG2a, clone 2A3, BioXCell) by intraperitoneal injection (i.p.). After 24 hours the mice were treated with carbon tetrachloride or vehicle. The mice then received a second dose of anti-mLy-6G antibody or isotype control 24 hours after carbon tetrachloride treatment.