Isotype control mab

T cells were isolated from the spleen and inguinal lymph nodes of the iTreg-treated, iTreg_mtDC-treated, and CIA mice on day 49 after the primary immunization. These cells were incubated with an anti-mouse CD4-FITC mAb, fixed, permeabilized, stained with anti-mouse FoxP3-PE, IL-17-PE, or isotype control mAbs (BD Biosciences Pharmingen), and then assessed by flow cytometry.

AnnexinV-FITC/PI Kit (KeyGen Biotechnology, Nanjing, China) was used to evaluate the effect of FICZ and ITE on the apoptosis of PBMCs. For analysis of the frequency of Th1 and Th17, the cells were stimulated by adding PMA (50 ng/mL, Sigma-Aldrich, St. Louis, MO) and ionomycin (1 ug/mL, Sigma) for 1 h at 37°C. Then, brefeldin A (10 ug/mL, Sigma) was added for another 4 h; the cells were fixed and permeabilized using the eBioscience Cytofix/Cytoperm kit according to the manufacturer's instructions and then incubated with CD3-(PerCP)-Cy5.5, anti-human CD8-APC, anti-human IL-17A-PE, and anti-human IFN-γ-FITC (BD Biosciences). For phosphorylated STAT staining, stimulated CD4⁺T cells were fixed with Fix buffer (BD Biosciences) for 10 min at 37°C, permeabilized with Perm buffer (BD Biosciences) for 30 min on ice, and stained with anti-human pSTAT3-PE, anti-human pSTAT4-Per-cy5.5, anti-human pSTAT4-Per-cy5.5, anti-human pSTAT5-PE, or isotype control mAbs (BD Biosciences). Flow cytometric analysis was performed on a FACScan flow cytometer (BD Biosciences) to measure mean fluorescence intensity (MFI). Results were expressed as the percentage difference compared with isotypic control (IC) using the formula [mean fluorescence intensity (MFI) of sample − MFI of IC]/MFI of IC.

FACS sorted FCRL6⁺ and FCRL6⁻ pro B (CD43⁺CD19⁺B220^hiIgM⁻) cells from adult BM were treated with the phosphatase inhibitor pervanadate (NaVO₄) for 10 min. Stimulated cells were fixed with prewarmed Phosflow Lyse/Fix buffer (BD) at 37°C for 10 min and permeablized with Phosflow Perm Buffer III (BD). After Fc blockade (CD16/32), cells were stained with anti-phospho ERK pT202/pY204, STAT5 Y694, or isotype control mAbs (BD) for 30 min at room temperature. Phosphorylation was analyzed using a FACSCalibur flow cytometer (BD) and plotted with FlowJo software. The fold induction change in phosphorylation for FCRL6⁻ and FCRL6⁺ pro B cells was calculated by comparing the MFI ratios of FCRL6⁻/FCRL6⁺ pro B cells with and without stimulation.