Sema3a fc

Purified CD45⁺ TIL were co-cultured for 4 h with B16F10 tumour cells, pulsed with Trp2 (1 µg) and gp100 (1 µg) peptides, in the presence of Brefeldin A (Invitrogen 00-4506-51, 1/1000), monensin (Merck M5273, 2 µm) and anti-CD107a mAb (clone 1D4B, Biolegend 121619, 1/50). TIL were stained with mAb specific for surface proteins prior to fixation and permeabilization. Permeabilized cells were then stained with anti-IFNγ (clone XMG1.2, Biolegend 505808, 1/200), anti-TNF (clone MP6-XT22, Biolegend 506321, 1/100) and anti-perforin (clone eBioOMAK-D, eBioscience 17-9392-80, 1/50) mAb.
For cytotoxicity experiments, freshly isolated CD8⁺ TIL were either kept in medium or pretreated with neutralising anti-Nrp-1 (clone 761704, R&D systems MAB59941, 10 µg/ml), anti-PD-1 (clone RMP1-14, Bio-X-Cell BE0146, 10 µg/ml) or isotype controls (clone 2A3, Bio-X-Cell BE0089, 10 µg/ml). Cytotoxic activity toward the B16F10 cell line, pulsed with Trp2 and gp100 peptides, or the MC-38 cell line was evaluated using a conventional 4 h and overnight chromium (⁵¹Cr) release assay.
For experiments with human freshly isolated lung TIL and CTL clone P62, activated T cells were incubated for 30 min to 1 h either with bovine serum albumin (BSA, Merck) or Sema-3A-Fc (R&D systems 1250-S3-025, 100 ng/ml), and their cytotoxic activity toward the autologous tumour cell was evaluated.

Five microliters of biotinylated mouse NRP1 (R&D Systems, biotinylated in-house, final concentration 1.6 nM) were incubated with 5 µL of a dilution series of Nb1 for 1 h at room temperature. After 1 h, 5 µL of human Sema3A-Fc (R&D Systems, final concentration 6 nM) were added and incubated for an additional hour. Next, a mixture of 5 µL of 1/50 diluted AlphaScreen streptavidin donor beads and 5 µL of 1/50 Anti-human IgG (Fc specific) AlphaLISA Acceptor Beads was added, After 1 h, the AlphaCounts were measured according to the manufacturer’s instructions on an Envision multimode microplate reader (PerkinElmer).

HUVECs where seeded at 4000 cells/well of a human fibronectin-coated 96-well plate in endothelial cell growth medium (ECGM) medium (C-22010 (Bioconnect, TE Huissen, The Netherlands). After 6 h, the HUVEC medium was replaced by DMEM supplemented with 10 % FBS. After 24 h, a dilution series of the Nbs was added (0.04–1000 nM) and incubated for 30 min, followed by a 20 min incubation with 8.8 nM Sema3A-Fc (R&D systems). Cells were fixed in 4% paraformaldehyde (PFA) and stained with phalloidin-488 and Hoechst (Thermo Fisher Scientific, Waltham, MA, USA). Imaging and analysis were performed with the Opera Phenix High Content Screening System (10× air magnification) (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s protocol.