Cd40l

The human ovarian cancer cell lines SKOV3 and A2780 (5 × 10⁵) were co-cultured with CAR THP-1 cells or vehicle control (5 × 10⁵) and incubated for 4 h (at 37 °C and 5% CO₂). Whole blood from healthy blood donors was diluted 1:1 in PBS and layered in human lymphocyte isolation medium. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation in a 50 ml tube. CD3⁺ T cells in PBMC were separated using CD3 magnetic beads (130-097-043, Miltenyi Biotec). Purified CD3 ⁺ T cells (5 × 10⁵) were immediately added to the co-culture system of tumor cells and CAR THP-1 or vehicle control and cultured for 48 h at 37 °C and 5% carbon dioxide incubator. T cells were harvested, resuspended in PBS, and labeled with anti-human CD8 (344,747, BioLengend), anti-human IFN-γ (2,331,102, Invitrogen), Fasl (306,421, BioLegend) and CD40L (310,824, BioLegend) antibodies at 4 ℃ for 30 min. After centrifugation at 370 g for 5 min, 200 µl PBS was used to resuspended cells and detected by flow cytometry. CAR-M cells were harvested, resuspended in PBS, and labeled with anti-humanCD40 (40,334,308, BioLegend) antibodies at 4 ℃ for 30 min. After centrifugation at 370 g for 5 min, 200 µl PBS was used to resuspended cells and detected by flow cytometry.

CD19+ B cells were seeded at 200,000 cells/mL in RPMI 1640 medium (1% penicillin/streptomycin and 10% FBS depleted of extracellular vesicles) and co-cultured for 96 h as follows: only culture medium; TEX (5, 10, or 20 µg/mL); ATP (20 µM or 2000 µM; #R0441, Thermo Fisher Scientific, Waltham, MA, USA); TEX and 20 µM ATP, or TEX and 2000 µM ATP. During these 96 h, B cells were either kept in a resting state (resting B cells, Brest) or activated (activated B cells, Bact) with human IL-4 (200 IU/mL, #574004, BioLegend, San Diego, CA, USA) and CD40L (2 µg/mL, #591706, BioLegend, San Diego, CA, USA).

Peripheral blood mononuclear cells (PBMCs) were purified with Ficoll density gradient centrifugation. For flow cytometry or cell sorting, PBMCs were stained in 1X PBS 0.5% BSA with appropriate antibodies at 4 °C for 30 min. Flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed with FlowJo version 10 (Tree Star). For assessing in vitro immunoglobulin CSR, CD19⁺CD27⁻IgM⁺ naïve B cells were sorted on a FACSAria III (BD Biosciences) and stimulated with CD40L (5 µg/mL; Biolegend) and IL-21 (100 ng/mL; Preprotech). After 5 days, expression of IgG and IgA was assessed on CD19⁺CD27⁺B cells using flow cytometry.