Ova peptide ova 323 339

20,000 sorted APCs/iCAFs/myCAFs/normal mesothelial cells/apCAFs or PanMeso cells were seeded in U-bottom 96-well plates and incubated with or without 25 μg/ml OVA peptide (OVA 323–339) (GenScript) in DMEM with 10% FBS for 4 hrs at 37°C. Spleens from 6-to-8-week-old OT II mice were harvested and CD4⁺ T cells were isolated using a MojoSort mouse CD4⁺ T cell isolation kit (Biolegend). APCs/mesothelial cells/CAFs were washed 3 times and co-cultured with 50,000 CD4⁺ T cells in DMEM with 10% FBS for 18 hrs. T cells were then washed twice with PBS and stained with Fixable Viability Stain (BD Biosciences) for 1 hour. The cell suspensions were then washed and stained with antibodies against CD4 (BD Biosciences, 1:100), CD25 (BD Biosciences, 1:100) and CD69 (BD Biosciences, 1:100) for 1 hr at 4°C. Surface-stained cells were fixed, permeabilized (FoxP3 Buffer Set, BD Biosciences), and stained for intracellular markers FoxP3 (BD Biosciences, 1:100) and Ki67 (Biolegend, 1:100). Cells were analyzed using FACS LSRFortessa SORP, and the FlowJo software. For CAF proportion analysis, cell suspension from digested KPfC tumors were stained with antibodies against podoplanin (Biolegend, 1:200), I-A/I-E (Biolegend, MHC class II, 1:200), αSMA (Thermo Fisher Scientific, 1:200) and IL-6 (Biolegend, 1:200).

20,000 sorted apCAFs from KPfC tumors were seeded in U-bottom 96-well plates and incubated with or without 25 μg/ml OVA peptide (OVA 323–339) (GenScript) in DMEM with 10% FBS for 4 hrs at 37°C. Spleens from 6-to-8-week-old OT II mice were harvested and CD4⁺ T cells were isolated using a MojoSort mouse CD4⁺ T cell isolation kit (Biolegend). apCAFs were washed 3 times and co-cultured with 50,000 CD4⁺ T cells in DMEM with 10% FBS for 18 hrs. CD4⁺ T cells were then collected and co-cultured with CD8⁺ T cells (1:1 ratio) from splenocytes of C57BL/6 mice that were isolated using MojoSort mouse CD8⁺ T cell isolation kit (Biolegend) and labeled with CellTrace CFSE dye (Thermo Fisher Scientific, 1 μM). After 72 hours, cells were stained with CD8 antibody (BD Biosciences, 1:100) and the percentage of proliferating CD8⁺ T cells was analyzed by CFSE signal with flow cytometry.