IL-15 complex enhances agonistic anti-CD40 + anti-PDL1 by correcting the T-bet to Tox ratio in CD8+ T cells infiltrating pancreatic ductal adenocarcinoma

Single cell RNA sequencing paired with single cell V(D)J sequenction of immune cells infiltrating orthotopic KPC2a tumors, spleen cells, and CD8 T cells enriched from spleen cells on day 14 posttumor. Cohorts received no treatment, agonistic anti-CD40, anti-PD-L1 or the combination of agonistic anti-CD40 + anti-PDL1. Treatments were initated on day 7 posttumor. Mice with orthotopic KPC2a tumors were left untreated (control) or treated with anti-PDL1 and/or agonistic anti-CD40 according to detailed methods in manuscript. On day 14 post tumor implantation, immune cells were were isolated and samples were pooled by treatment group. Spleen and tumor single cell suspensions were prepared. Immune cells were enriched from the tumor single cell suspensions using a 44/67% percoll density gradient. Briefly, samples were resuspended in 3 mL of 44% percoll in a 15 mL conical tube and then underlaid with 3 mL of 67% percoll using a glass Pasteur pipette. Samples were centrifuged for 20 minutes at 600 x g at 22°C with an acceleration of 1 and brake of 0. After spinning, cells at the density interface were isolated with a P100 pipette and transferred to 20 mL of complete media for washing. CD8 T cells were isolated from half of the spleen mononuclear cell suspension using a CD8a negative selection magnetic cell separation kit (Miltenyi). For each tissue (intratumoral cells, enriched CD8 T cells from spleen, and bulk unenriched mononuclear cells from spleen), samples from individual treatment groups (control, aPDL1, aCD40, aPDL1+aCD40) were stained with unique hashtags (TotalSeq C, Biolegend) at a concentration of 0.2 mg per 1 x 106 cells. Next, samples were pooled by tissue containing the 4 treatment groups for proceeding with 3 independent captures. Cells were submitted to University of Minnesota Genomics Core for single cell 10X Chromium 5’ GEX and TCR capture targeting 40,000 cells per sample and NovaSeq 2 x 150 S4 sequencing targeting 35,000 reads per cell for GEX and 25,000 reads per cell for V(D)J sequencing.