Diphtheria toxin

For Treg depletion, Foxp3^DTR, KC;Foxp3^DTR mice were treated with diphtheria toxin (DT) (50 ng/g) (Enzo Life Science) by intraperitoneal injection (i.p.). DT injections were repeated according to the specific experimental design shown in Figures. Control mice lacking Foxp3^DTR alleles received the same DT treatments. In KC, KC;Foxp3^DTR and KC;CD4^−/− mice, mild acute pancreatitis was induced in 4–5-week-old mice by a series of 8 hourly intraperitoneal injections of caerulein (Sigma-Aldrich, 75 μg/kg) over 1-day period. For CCR1 inhibition, mice were subcutaneously dosed with CCR1 antagonist BX471 (Sigma-Aldrich, 50 mg/kg) for 7 days at 12-hour intervals. DMSO was used as vehicle to dissolve BX471 at a concentration of 50 mg/ml.
To establish the orthotopic pancreatic cancer model, 1×10⁵ of 7940B cells (C57BL/6J strain)(46 (link)) derived from KPC tumor (Ptf1a-Cre; LSL-Kras^G12D; p53^flox/+) were injected into Foxp3^DTR mice of compatible genetic background. Cells were tested for mycoplasma free by MycoAlertTM PLUS Mycoplasma Detection Kit (Lonza) and passage 15–20 were used for all experiments. For CD8⁺ T cell depletion, anti-CD8 mAb (BioXcell clone 2.43; 200 μg/mouse) was injected i.p. twice per week. For CD4⁺ T cell depletion, anti-CD4 mAb (BioXcell clone GK1.5; 200 μg/mouse) was injected i.p. at least every three days.

1-methyl-D-tryptophan (1-MT; Sigma-Aldrich) was formulated and administered to mice as previously described (30 (link)). Briefly, mice were treated with 2mg/ml 1-MT in their drinking water; starting 2 days post infection and continued for the duration of the experiment.
Depletion of Foxp3+ cells in DEREG mice was performed as previously described (31 (link)). Briefly three weeks post infection, mice were administered 0.5μg diphtheria toxin (DT; Enzo Life Sciences), intraperitoneally on 2 consecutive days per week for 2 weeks. PBMCs were isolated from mice one day following the last DT injection; flow cytometry was employed to confirm T regulatory cell depletion.