αcd40 fgk4

All antibody dosing was performed via intraperitoneal injection in PBS. αCD4 (GK1.5, BioXCell) and αCD8 (2.43, BioXCell) depleting antibodies were administered at 200 ug every 4 days. αPD-1 (29F.1A12, BioXCell) was administered at 200 μg three times a week. αCTLA (9H10, BioXCell) was administered at an initial dose of 200 μg, with all subsequent doses at 100 μg, three times a week. αCD40 (FGK4.5, BioXCell) was administered once at the beginning of treatment at 100 μg.
The adjuvant amphiphile-CpG (amph-CpG) and antigen amphiphile (amph-peptide) were produced as previously described^{53 (link)}. Briefly, class B CpG 1826 oligonucleotide with a G₂ spacer (5’-diacyl lipid-GGTCCATGACGTTCCTGACGTT- 3’) was conjugated via the 5’ end to an 18 carbon diacyl tail. Antigen peptide OVA_250–270 (CGLEQLESIINFEKLTEWTSS) and non-specific mutant gp100_20–39 (optimized S27P, EGP long^{52 (link)}, CAVGALEGPRNQDWLGVPRQL) were conjugated via N’ cysteine residue to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol-2000] (Avanti Polar Lipids). Mice were vaccinated subcutaneously at the base of the tail with 1.24 nmol amph-CpG and 25 μg of amph-peptide, with half dose given to each side. Vaccination was performed once weekly starting 14 days post-transplant of lo^SIIN organoids.

On day 7 post orthotopic tumor implantation and following tumor establishment within the pancreas, 100 μg of agonistic αCD40 (FGK45, BioXcell) was diluted in sterile saline and injected intraperitoneally (i.p.). A separate cohort of tumor bearing mice received 200 μg of αPD-L1 (10F.9G2, BioXcell) i.p on days 7, 10 and 12 post orthotopic tumor implantation. Cohorts in Figure 1 received 200 μg αLag3 (clone C9B7W, BioXcell), αTim3 (RMT3–23, BioXcell) or αTIGIT (clone 1G9, BioXcell) alone, or in combination with αPD-L1 (10F.9G2) i.p. on days 7,10 and 12. Combination treated animals received both antibodies at the same timepoints. For in vivo antibody blockade of IL-27 or IL-10, 200 μg of αIL-27p28 (clone MM27.7B1, BioXcell) or αIL-10R (clone 1B1.3A, BioXcell) was diluted in sterile saline and injected i.p. on days 6, 10, 14, and 18 post orthotopic tumor implantation.

Tumor bearing mice were vaccinated i.p. with 100 μg αCD40 (FGK45; BioXcell), 75 μg polyI:C (Invivogen), and 500 μg ovalbumin (Sigma) or 200 μg OVA_257–264 (Genscript) at day ten after tumor injection. FTY720 (Sigma) was provided in the drinking water (2 μg/mL) starting at day nine after tumor implantation and supplemented with daily i.p. injection (25 μg) on days nine through twelve. Depletion antibodies (250 μg) for CD8 (2.43; BioXCell) and/or CD4 (GK1.5; BioXCell) were administered i.p. at day eight after tumor injection and again every four days for the remainder of the experiment. FTY720 and depletion efficiency were confirmed by flow cytometric analysis of blood in each experiment. LFA blocking antibody (M17/4; BioXCell) was administered i.p. (200 μg) every two days. Brefeldin A (Thermo Fisher) was injected i.v. (250 μg) five hours prior to harvest to block cytokine secretion. For OT1 transfer, spleens were harvested from OT1 mice originally from Taconic and maintained at the University of Virginia.