Flow cytometry

PANC-1 tumour cells were incubated with 1 µM of CFSE reagent overnight. Cells were subsequently washed in PBS and incubated with indicated concentration of chemotherapeutic agents for 24 h. The cells were washed twice and incubated with 1 × 10⁵ MDDC for 4 h. The non-adherent MDDC were stained with an PE-CY5 anti-human HLA-class II antibody and assessed for uptake of CFSE labelled PANC-1 cells by flow cytometry (Biolegend, CA).

Tumor cells were washed with PBS and stained with phycoerythrin (PE)‐conjuncted PD‐L1 antibody (29E.2A3, Biolegend) and then determined with flow cytometry (Beckman Coulter). Data were analyzed using Flow Jo software (Tree Star).

We tested neutrophils chemotaxis using a Transwell system (Corning) with 5 mm polycarbonate membranes. PBNs suspended in RPMI-1640 containing 2% foetal bovine serum (1 × 10⁵ cells/100 μL) were added to the upper wells and incubated for 24 h at 37 °C, and 5% CO₂. Supernatants from EOC with or without control Ab (1 μg/mL), anti-CXCL1/2/3 (1 μg/mL), anti-CXCL5 (1 μg/mL), anti-IL-8(1 μg/mL), and SB225002 (CXCR2 inhibitor) were added to the lower chamber. Cells in the lower chamber were collected and analysed for CD66b using flow cytometry (Biolegend).

The A-375 (malignant human melanoma) cells that express the NY-ESO-1 antigen were labeled with 1 µM of Incucyte Cytolight Rapid Dyes (4706) and plated in a 96-well plate at the seeding density of 50,000 cells. 2 h after seeding, a caspase-3/7 green apoptosis reagent (2272582; Invitrogen) and IG4 TCR KI or KO controls (50,000 cells per well) were added to A-375 cells. Cell killing was measured by evaluating the number of A375 cells present in each well expressing caspase-3/7 reagent. The co-culture was monitored for growth and apoptosis using the IncuCyte imaging system for 18 h. After co-culturing, CD137 expression on CD8⁺ T cells was measured by Flow Cytometry (clone 4B4-1; BioLegend).

The Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology Ltd., Shanghai, China) was used to determine cell apoptosis. After a 24-h incubation period, the cells were washed with phosphate buffer solution (PBS), digested with trypsin, and centrifuged at 1000 × g for 5 min. Prior to resuspension into 195 μl of annexin V-FITC binding buffer, the transfected cells were rinsed with PBS, centrifuged, and the supernatant was discarded. The cell suspension was supplemented with 5 µl of annexin V-FITC and 10 µl of propidium iodide (PI). Then, the cells were placed in the dark at 20–25 °C for 15 min. The early and late apoptotic cells were detected via flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
To determine the cell cycle status, the transfected cells were collected using the experimental steps described above and fixed in 1 ml of 70% ice-cold ethanol at 4 °C for 2 h. After washing the cells with ice-cold PBS, they were centrifuged at 1000 × g for 5 min and resuspended in 425 µl of cell staining buffer, which was supplemented with 50 µl of RNase and 25 µl of PI (all from BioLegend, San Diego, CA, USA). The cell cycle status was analyzed via flow cytometry.

WT DCs were treated with 50 µM of CoPP, SnPP; 60 µM of CORM-2, iCORM-2, or an equivalent volume of vehicle (NaOH) for 6 h; or transgenic DCs were treated with 1.5 µg/mL of Dox or PBS for 16 h, then infected at an MOI of 3 with HSV-1 KOS or HSV-2 G for 1 h at 37 °C. Then, the supernatants were removed, washed with culture media, and incubated with the corresponding treatments. DCs were collected 6 h later and co-cultured with 1 × 10⁵ HSV-specific T cells/well. T cells, either CD8⁺ gBT-I or CD4⁺ gDT-II, were purified from the spleens of transgenic mice using negative-selection kits for T cells (MiltenyiBiotec, Bergisch Gladbach, Germany). Uninfected DCs and DCs pulsed with gD_290–302 or gB_498–505 peptides were used as controls, as previously described [13 (link)]. T cell activation and differentiation were determined by measuring IL-2 and IFN-γ using ELISA. Additionally, for CD4⁺ T cells, IL-4 and IL-17 were measured in the supernatants [34 (link)]. Cell viability, CD4, CD8, CD25, and CD71 surface markers were assessed by flow cytometry (BioLegend, San Diego, CA, USA). To further determine CD4⁺ T cell differentiation, antibodies against the surface expression markers OX40 and PD-1 were used, while antibodies against FoxP3 and RORγt were used for intracellular staining (Biolegend, San Diego, CA, USA), followed by FACS analysis.