Be0249

T cell activation and migration assays were performed following the protocol by Albert et al. (23 (link)). Briefly, splenocytes from OT-1 transgenic mice were stimulated with 1 nM TCR-specific peptide ovalbumin 257–264 ( Sigma-Aldrich, S7951) for 1 hour. Seven days after culture in complete RPMI 1640 medium with 50 U/mL of recombinant IL-2 (R&D, 402-ML-020/CF), CD8⁺ T cells with high CXCR3 expression were used for T cell migration assay. To inhibit receptor binding of CXCR3, anti-mouse CXCR3 mAbs (10 μg/mL, BioXcell, BE0249) were applied to treat activated OT-1 cells for 1 hour at 37°C; IgG2b-pretreated (10 μg/mL, BioXcell, BP0090) cells were used as isotype control. Then, 0.1 × 10⁶ activated T cells were placed into 5 μm pore size polystyrene Transwell inserts (Corning, 3421) in serum-free RPMI 1640 and allowed to migrate for 1.5 hours at 37°C toward EO771-GFP/Bic cell conditioned medium. Cells that had migrated to the receipt chamber were collected and counted using Precision Counting Beads (BioLegend, 424902) by flow cytometry.

Ptf1a/p48^cre/+ and LSL-Kras^G12D/+ mouse strains and genotyping of mice have been described previously (Liou et al., 2015 (link)). Seven to 8 week old Ptf1a/p48^cre;LSL-Kras^G12D (KC) or non-transgenic (ntg) mice with the same background were injected intraperitoneally (IP) with a CXCR3 neutralizing antibody (CXCR3 NAB; BE0249) (Bio X Cell, West Lebanon, NH) or an isotype control IgG; BE0091 (Bio X Cell) at 200 µg/mouse for 9 weeks. Males and females were randomly allocated to different groups since there are no sex-based differences observed in this model. All animal experiments were conducted under IACUC-approved protocols (A50214-14-R17, A30615-15-R18) and were run in accordance with institutional guidance and regulation.
For T-cell depletion, 6 week old Ptf1a/p48^cre;LSL-Kras^G12D mice were intraperitoneally injected with both anti-mouse CD4 (BP0003, Bio X Cell) and anti-mouse CD8α (BP0061, Bio X Cell) antibodies, or their IgG2b isotype control (BP0090, Bio X Cell). Two hundred micrograms of each antibody was injected per mouse for five consecutive days. After T-cell depletion, mice were segregated into different groups and injected with CXCR3 NAB (BE0249, Bio X Cell) or IgG isotype control antibodies (BE0091, Bio X Cell).

For the in vivo CXCR3- and CXCL9-neutralizing experiments, heart transplant recipient mice received different treatments after surgery. In the anti-CXCL9 group, recipient mice were treated by intraperitoneal injection (i.p.) of 200 µg anti-CXCL9 monoclonal antibody (mAb) (BioXCell BE0309, clone: MIG-2F5.5, USA) every 2 days. In the anti-CXCR3 group, recipient mice were treated by i.p. injection of 200 µg anti-CXCR3 mAb (BioXCell BE0249, clone: CXCR3-173, USA) every 2 days. In the anti-CXCL9 + anti-CXCR3 group, recipient mice were treated by i.p. injection of 200 µg anti-CXCL9 mAb and 200 μg anti-CXCR3 mAb every 2 days until complete cessation of graft beating. In the isotype control group, recipient mice were treated by i.p. injection of 200 μg Armenian hamster IgG (BioXCell BP0091, USA).