Ack lysis

Tumors or livers were finely chopped and processed on a gentleMACS^™ Octo Dissociator using the pre-set 37C_m_TDK_2 or 37C_m_LDK_1 program. Following dissociation, tumors were quenched with 5ml of RPMI media supplemented with 10% FBS, 1% penicillin/streptomycin and 0.5% L-glutamine, and passed through 100mm cell strainers (Falcon, cat. #08-771-19) to further separate cells. An additional 5ml wash was performed on the C-tube and passed through the cell strainer, followed by a 5ml wash over the cell strainer to flush any remaining cells. The flow through was spun and ACK lysis (Quality Biological, cat. #118-156-101) was performed with 4mls of ACK lysis buffer followed by a 4-minute incubation on ice. Cells were quenched with RPMI complete media and spun down. For the formation of a Percoll gradient, a stock Percoll solution was prepared by diluting the supplied Percoll (GE Healthcare Life Sciences, cat. #17-0891-01) with 10X PBS. This was further diluted with 1X PBS to form 80% and 40% Percoll solutions. Cell pellets were resuspended in 40% Percoll and underlayed with 80% Percoll and then spun at 3200 rpm (no brake) for 25 minutes at room temperature. The immune cell layer was then removed and 1×10⁶ cells were plated per well in a 96-well plate for flow cytometry analysis.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood by Ficoll gradient density centrifugation. In short, blood was diluted 1:1 in phosphate buffered saline (PBS, Quality Biological). One volume of diluted blood was added to one volume of Ficoll-Paque PLUS density gradient media (GE Healthcare) and centrifuged at 1200 rpm for 20 min with brakes off. PBMCs were isolated from the interface of Ficoll and plasma and underwent ACK lysis (Quality Biological) to remove erythrocytes. PBMCs were aliquoted and cryopreserved in liquid nitrogen in fetal bovine serum (FBS) containing 10 % dimethyl sulfoxide (DMSO).

Dissected orthotopic pancreatic tumors and murine livers with diffuse metastases were collected on Day 16 following tumor inoculation by the orthotopic and hemispleen procedure respectively, for analysis of tumor infiltrating lymphocytes (TILs). Each was mechanically processed sequentially through 40-μm and 100-μm nylon filters and brought to a volume of 20 mL of CTL medium. Suspensions were centrifuged at 1500 rpm for 5 minutes. Cell pellets were suspended in 4 mL of ACK lysis (Quality Biological) and subsequently spun at 1500 rpm for 5 minutes. Liver cell pellets were then resuspended in 6 mL 80% Percoll (GE Healthcare LifeSciences), overlaid with 6 mL 40% Percoll and centrifuged at room temperature for 25 minutes at 3200 rpm without brake. The lymphocyte layer was removed and quenched with 30mL of CTL media.
Direct Technique isolation for fibroblast cells was performed using the Cellection Biotin Binder Kit (LifeTechnologies) and the sheep anti-human FAP biotinylated affinity purified antibody (R&D Systems Inc.) from processed tumor. Isolated TILs were enriched for CD8⁺ cells using negative isolation kits (LifeTechnologies) and CD11b⁺ cells using CD11b positive beads (LifeTechnologies) according to manufacturers’ protocols.