Anti cd3 bv421 clone 17a2

For DC activation, 5 × 10⁴ DCs were co-cultured with various concentrations of HBc VLPs for 24 h. The supernatant of the DCs was collected for IL-12-p40 measurement. The cells were collected and stained with anti-CD11c-APC-eF780, anti-CD40-APC, anti-CD86-PE-cy7, anti-I-Ab, and DAPI, and then analyzed on an LSR-II flow cytometer. For B cell activation, 6–12-week-old female C57BL/6J mice were inoculated on the right flanks with 4 × 10⁵ MC-38 cells in 100 μL PBS. When mice displayed established tumors, the mice were sacrificed to collect splenocytes (n = 3). Next, 1 × 10⁶ splenocytes were co-cultured with various concentrations of HBc VLPs for 24 h. The supernatant of the splenocytes was collected to measure the IFN-γ levels using an IFN-γ Mouse ELISA Kit (Invitrogen, Waltham, MA, USA). Thereafter, the cells were washed three times with phosphate-buffered saline (PBS) and stained with anti-CD45.2-APC-ef780 (clone 104, Thermo Fisher, Waltham, MA, USA), anti-CD3-BV421 (clone 17A2, Biolegend, San Diego, CA, USA), anti-CD19-BV655 (clone 6D5, Thermo Fisher, USA), anti-CD40-APC (clone 3/23, Biologend, San Diego, CA, USA), anti-CD86-FTIC (clone GL1, Thermo Fisher, Waltham, MA, USA) before analysis by means of flow cytometry.

Naïve synovial cells were used to analyze their Foxp3 expression profile, while the expression of IL-17A and IFNγ was assessed after in vitro stimulation of 8 × 10⁶ cells with 20 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml Ionomycin (Sigma-Aldrich) in a 6-well flat-bottom plate (Falcon) for 6 h. To prevent cytokine GolgiPlug (BD) (1 μl/ml) and GolgiStop (0,6 μl/ml) (BD) were added after the first hour in culture. Afterwards cells were washed and stained for the expression of CD3 (anti-CD3-BV421, clone 17A2; BioLegend), CD4 (anti-CD4-APC-Fire, clone RM4-5; BioLegend), CD8 (anti-CD8-FITC, clone 53-6.7; BioLegend), CD19 (anti-CD19-PerCP Cy5.5, clone 6D5; BioLegend), CD25 (anti-CD25-PerCP Cy5.5, clone PC61; BioLegend), and living cells using L/D Aqua in BV510 (BioLegend) for 40 min at 4°C. Further, cells were prepared for intracellular staining using the Fixation/Permeabilization solution kit (Thermo Fisher Scientific). Antibodies for Foxp3 (anti-Foxp3 AF647, clone FJK-16s; eBioscience), IFNγ (anti-IFNγ-PE-Cy7, clone XMG1.2; BD), and IL-17A (anti-IL-17A-AF647, clone TC11-18H10.1; BioLegend) were diluted in permbuffer (Thermo Fisher Scientific) and applied to the cells for 30 min at 4°C. The flow cytometric analyses were performed using a cytofluorometer (FACS Canto II, BD) and data were evaluated using FCS-express 5 (De Novo Software).