Collagenase 4

Livers were collected at the indicated times after transcardial perfusion to clear organs of red blood cells. Liver samples were digested in collagenase IV (MP Biomedicals, LLC) at 37 °C for one hour and then processed on a 70 µm nylon cell strainer (Falcon) into a single cell suspension. Liver samples were resuspended in 30% Percoll solution and centrifuged at 750g for 20 min without brake. Supernatants were discarded and pellets were resuspended in RBC Lysis Buffer (BioLegend) for 5 min. Cells were then washed and ready for magnetic separation. The CD11c MicroBead kit UltraPure (Cat#130-125-835, Miltenyi Biotec) was then used according to the manufacturer’s conditions in order to isolate CD11c⁺ cells. Total RNA was isolated using MicroRNAeasy (Qiagen, Valencia, California) from freshly isolated hepatic CD11c⁺ cells. 10 ng of input RNA was used to produce cDNA for downstream library preparation. Samples were sequenced on a NextSeq 500 (Illumina) system as previously described^{63 (link)–65} . Raw reads were aligned, normalized, and further analyzed using Partek Genomics Suite software, version 7.0; Partek Inc., St. Louis, MO, USA. Pathway analysis was performed using the Qiagen IPA software.

All human studies were approved by USC Institutional review board and conducted in accordance to the principles of the Declaration of Helsinki. Informed consent was obtained from all human participants. For human ILC2s from blood, peripheral blood mononuclear cells (PBMCs) were first isolated from human fresh blood by diluting the blood 1:1 in PBS and adding to SepMate^TM-50 separation tubes (STEMCELL Technologies Inc, Vancouver, Canada) prefilled with 15 mL Lymphoprep^TM each (Axis-Shield, Oslo, Norway) and centrifugation at 1200 × g for 15 min. Human ILC2s were then isolated by cell sorting based on the lack of expression of classical lineage markers (CD3, CD14, CD16, CD19, CD20, CD56, CD235a, CD1a, CD123) and expression of CD45, CRTH2 and CD161. Purified human ILC2s were stimulated (5 × 10⁴/mL) with recombinant human (rh)-IL-2 (20 ng/mL), rh-IL-7 (20 ng/mL) and rh-IL-33 (20 ng/mL) for the indicated times at 37 °C in presence or absence of plate-bound GITR-L-Fc obtained from Dr. Alan Epstein at USC as described previously^{41 (link)}. For human ILC2s from adipose tissue, adipose tissue samples were digested in collagenase IV (MP Biomedicals, LLC) at 37 °C for one hour and then processed on a 70 µm nylon cell strainer (Falcon®) into a single cell suspension.

Pancreatic acinar cells (PACs) were isolated from the pancreas of C57BL6/J mice as previously described [31 (link)]. Briefly, the pancreas was injected with 2 mg/mL collagenase IV (MP Biomedicals, CA, USA) and digested at 37°C for 20 minutes. Acinar cells were carefully isolated with blunted 1-mL pipette by repeated pipetting, and then filtered through a 70 μm cell strainer (Corning, Corning, NY). After being washed twice in wash buffer, PACs were resuspended in culture DME/F-12 medium containing 5% FBS and recovered for 2 h. To investigate the effect of quinpirole and CA-074Me on cell damage, PACs were pretreated with quinpirole (5 μM) or CA-074Me (10 μM) for 0.5 h and then stimulated with 200 nM CCK with or without LLOMe (0.5 mM) for 6 h.

Surplus tumor tissue obtained at diagnostic biopsy or tumor resection was processed immediately after receipt in the histopathology laboratory (<1 hour after interventional radiology/surgical procedure). Tissue was minced using a scalpel and then incubated in RPMI 1640, supplemented with 10% fetal calf serum, 1% l-glutamine, and 1% penicillin/streptomycin, with collagenase IV (1.6 mg/ml; catalog no. 11410982; MP Biomedicals), for 30 min at 37°C, inverting the tube every 10 min. The digested tissue was passed through a 70-μm filter and incubated in 1× RBC lysis buffer (catalog no. 420301; BioLegend) for 10 min at room temperature. The obtained single-cell suspension was used for downstream processing. Part of the single-cell suspension was depleted of CD45⁺ cells to enrich for tumor cells using a CD45 MicroBeads kit (catalog no. 130-045-801; Miltenyi Biotec), following the manufacturer’s protocol. Both CD45 nondepleted and CD45-depleted single-cell suspensions were depleted of dead cells using a Dead Cell Removal kit (catalog no. 130-090-101; Miltenyi Biotec), following the manufacturer’s protocol. Obtained viable single-cell suspensions (CD45 depleted: two channels and CD45 nondepleted: one channel) were processed on the 10x Chromium platform.

Lymphocytes were isolated from the female reproductive tract of mice 16 days post infection. The FRT was harvested into complete RPMI, diced, then incubated while stirring with collagenase IV (386mg/L MP Biomedicals) for 1 hour at 37°C. Cells were filtered (70μm cell strainer, Corning), then lymphocytes were isolated out of the supernatant using a Percoll gradient (GE Healthcare). Cells were stimulated with PMA (0.2 mM, Millipore Sigma) and Ionomycin (1ug/mL, Millipore Sigma) with Brefeldin A (71.4uM Millipore) for 3.5 hours at 37°C 5% CO₂. Cells were stained for viability with Zombie Yellow (BioLegend) and surface markers B220-APC-eF780 (RA3-6B2, eBioscience), CD11b-APC-eF780 (M1/70, eBioscience), CD11c-APC-eF780 (N418, eBioscience), F4/80-APC-eF780 (BMB, eBioscience), CD4-PE (RM4-4, eBioscience), CD4-eF450 (RM4-5, eBioscience), CD8-PerCPCy5.5 (2.43, Tonbo), CD44-APC (IM7, eBioscience) CD62L-PETexasRed (MEL-14, Invitrogen), followed by intracellular stains IFN-γ-BV785 (XMG1.2, BioLegend), IL-4-PE (11B11, eBioscience), T-bet-PECy7 (4B10, eBioscience), RORγt-BV421 (Q31-378, BD Biosciences), and IL-17A-FITC (17B7, eBioscience) using the Foxp3 Transcription Factor Staining Kit (eBioscience). Data was acquired on an LSRFortessa (BD) and analyzed using FlowJo (Tree Star, San Carlos, CA). Contour plots are shown with 5% outliers.