96 u bottom well plates

After either MACS or FACS isolation, naive cells were plated under Th17 differentiation conditions at 5x10⁵ cells/well in 96 U-bottom well plates (Falcon). For cell stimulation, plates were coated at least 5 hours prior to use with 3 ug/ml plate-bound anti-CD3 antibody (clone OKT3; BioLegend, LEAF grade), 1 ug/ml soluble anti-CD28 antibody (BioLegend, LEAF grade) and 2 ng/ml IL-2 (carrier-free; Tocris R&D Systems). Cells treated with only these functioned as the mock control. For Th17 induction, TGF- β (30 ng/ml; carrier-free; Tocris R&D Systems), IL-6 (30 ng/ml; carrier-free; Tocris R&D Systems), IL-1 β (10 ng/ml; carrier-free; Tocris R&D Systems), IL-23 (50 ng/ml; carrier-free; Tocris R&D Systems), anti-IFN γ antibody (2 ug/ml; clone MD-1, Biolegend), anti-IL-4 antibody (2 ug/ml; clone MD-1, Biolegend), bexarotene (1 µM, Abcam), or atRA (100 nM; Sigma-Aldrich) were added additionally. The DMSO control (for bexarotene, atRA and AGN190) had no effect on IL-17A cytokine expression. Cells were incubated for 7 days unless otherwise stated.

This assay was performed based on a previous work by our group (31). The peptides corresponding to the unspecific or specific groups (P1 or P2) were associated in one single tube in a stock concentration of 1 mg/mL. 2 × 10⁵ PBMCs from all evaluated groups (AD, PT, and CT) were deposited per well in 96-U-bottom well plates (BD Falcon, USA) and stimulated individually with 15 μg/mL of the peptide pools P1 or P2. Each pool was tested in triplicates per group. The plates were incubated at 37 °C with 5% CO₂ for 96 h. After that period, the plates were centrifuged for 10 min at 400× g, the supernatants were collected and stored at −80 °C for cytokine evaluation and cells were retrieved for the subsequent tests.

Naive CD4+ T cells were plated under iTreg differentiation conditions at 1.0x10⁵ cells/well in 96 U-bottom well plates (Falcon). For cell stimulation, plates were coated at least 5 hours prior to use with 5 ug/ml plate-bound anti-CD3 antibody (clone OKT3; BioLegend, LEAF grade), which is then washed off with PBS, with 1 ug/ml soluble anti-CD28 antibody (BioLegend, LEAF grade) and 5 ng/ml IL-2 (carrier-free; Tocris R&D Systems) added with cells. Cells treated with only these functioned as the mock control. For iTreg induction, TGF- β (5ng/ml; carrier-free; Tocris R&D Systems), anti-IFN γ antibody (2 ug/ml; clone MD-1, Biolegend), bexarotene (1 µM, Abcam) or atRA (100 nM; Sigma-Aldrich) were added additionally. The DMSO control (for bexarotene, atRA and AGN190) had no effect on FOXP3 expression. Cells were incubated for 7 days unless otherwise stated.