Clone fgk4

Prior to injection, Panc02 and KPC cells were harvested using TryplE (Life Technologies), washed thrice with sterile PBS and put through a 70‐µm cell strainer to assure single‐cell suspension without any contaminants. Next, mice were injected subcutaneously with either 0.5 × 10⁶ Panc02 or KPC cells suspended in 100 µL sterile PBS at the left abdominal flank. When tumors reached an average size of 25–35 mm², mice were randomised based on tumor size and divided over four different treatment groups (day 0): (1) Isotype control; (2) IL‐15; (3) CD40 agonist; and (4) IL‐15 + CD40 agonist. Mice were given i.p. 2.5 µg IL‐15 (NCI) at days 0–3, 6–10 and 13–14. A mouse agonistic CD40 monoclonal antibody (Clone FGK‐45, BioXCell, obtained via Bio‐connect, Huissen, The Netherlands) or corresponding isotype control (Clone 2A3, BioXCell) was administered i.p. at days 0, 3, 7, 10 and 14 at a dosage of 12.5 µg per mouse for Panc02 or 200 µg (day 0) and 100 µg (days 3, 7, 10 and 14) for KPC tumors.
Tumor size was measured twice a week using a digital calliper (Chicago Brand, Medford, OR, USA). Tumor area was calculated using the formula length × width. Mice were sacrificed when a tumor size of 150 mm² was reached or were stated as long‐term survivor when they survived 100 days post‐treatment without reaching the 150 mm² threshold.

Activated OT1 or Pmel T cells were generated by transferring 10,000 OT1 or Pmel T cells i.v. into naïve mice and priming with CD40 agonistic antibody (100 ug, clone FGK45, BioXcell), poly I:C (75 ug, Invivogen), and ovalbumin (500 μg, Sigma) or gp100 25-33 peptide (200 ug, GenScript). Seven days later CD8 T cells were magnetically enriched (eBioscience 8804-6822-74) from spleens. Approximately 1 million CD8 T cells were transferred i.v. following FUS + MB BTB/BBB opening. Activated wild-type T cells were generated by culturing splenocytes for three days in vitro with agonistic antibodies for CD3 (5 ug/mL, eBioscience) and CD28 (2 ug/mL, eBioscience) plus IL-2 (10 IU/mL). Approximately 3 million CD8 T cells were transferred i.v. following FUS + MB BTB/BBB opening. Tumors, spleens and meninges were harvested 5 or 24 hours after cell transfer. CD45 antibody was injected i.v. 3 minutes prior to harvest to label circulating cells. This allowed us to separate the extravascular cells (no labeling with CD45 antibody) of interest from those in blood vessels (labeled with CD45 antibody). Transferred OT1 cells were identified by Thy mismatch (i.e. OT1 cells from Thy1.1 donor into Thy1.2 host). Pmel cells were identified by Thy or CD45 mismatch (i.e. Pmel cells from Thy1.1 CD45.2 donor into Thy1.2 CD45.1 hosts).