Rmt3 23

For phenotypic analysis of DCs, splenic lymphocyte suspensions were stained with fluorochrome conjugated antibodies specific for the following surface markers: CD11c (N418), CD80 (16–10 A1), CD86 (GL1), CD40 (39164), MHCII (10.2.16), and PDL1 (10F.9G2), all of which were purchased from eBioscience (eBioscience, San Diego, CA, USA). Cell suspensions of thymus, spleen, and pancreatic lymph nodes (PLNs) were stained and analyzed with antibodies against CD4 (RM4-5), CD8α (53-6.7), CD25 (PC61.5), Foxp3 (FJK-16s), IFN-γ (XMG1.2), IL-4 (11B11), IL-2 (JES6-5H4), programmed cell death protein-1 (PD-1) (J43), T-bet (4B10), Eomesodermin (Eomes) (Dan11mag), CD160 (CNX46-3), CD244 (C9.1), CD272 (8F4), KLRG1, or killer cell lectin-like receptor G1 (2F1), all purchased from eBioscience. Antibodies against TNF-α (MP6-XT22), LAG3 or lymphocyte-activation gene 3 (C9B7W), and TIM3 or T cell immunoglobulin and mucin domain 3 (RMT3-23) were purchased from BioLegend (San Diego, CA, USA). For intracellular cytokine staining, splenocytes were stimulated with phorbol 12-myristate 13-acetate/PMA, ionomycin, and monensin (Sigma-Aldrich, St. Louis, MO, USA) for 6 h, followed by staining with antibodies. All analyses were performed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using CellQuest software (BD Biosciences).

Tumors were harvested, fixed with 4% paraformaldehyde, embedded in CryoplastR (Biopack), and cut into 5 µm thick sections using a Cryostat 0620E (Thermo Scientific Shandon, USA). Sections were rehydrated in Tris buffer 50 mM, heated to 90 °C in antigen retrieval solution (citrate buffer, pH 6) for 20 min, and then blocked for 90 min with 10% rat serum-Tris buffer. After blocking, slides were incubated overnight at 4 °C with PE-labeled anti-mouse F4/80 (1:250 dilution, #123110 BioLegend, clone BM8) or PE-labeled anti-mouse TIM3 (1:50 dilution, #119704, BioLegend, clone RMT3-23) in blocking buffer. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Slices were mounted with FluorSave (Merck Millipore, Burlington, MA, USA). Images were collected with a Leica microscope (DMi8).

For PD-1 blockade, 0.2 mg of anti-PD-1 mAb (RMP1-14, BioLegend) was administered i.p. at the time of infection and a second dose, 7 days post-infection. Control mice were administered with the same amount of rat IgG2a isotype control (RTK2758, BioLegend). For blockade of PD-1 and TIM-3, 0.2 mg of anti-PD-1 mAb and 0.2 mg of anti-TIM-3 (RMT3-23, BioLegend) were administered following the same scheme as applied for the PD-1 blockade. The control group received 0.2 mg of rat IgG2a isotype control.