Clone 4s b3

To investigate T cell activation, PBMCs (1 × 10⁶ cells/ml) were preincubated with DMSO or ML604440 (300 nM) or ONX-0914 (30 nM) for 2h and were seeded in 96-well microplates coated with anti-CD3/CD28 antibodies. After treated for 10h, the cells were harvested and incubated with CD69-PE(Biolegend), CD4-APC (Biolegend). In parallel, the cells were harvested after 72h to measure the expression of CD25 by incubating with CD25-APC (Biolegend)and CD4-FITC (Biolegend) in the dark at Room temperature(RT) for 30 min, washed with cold PBS, and analyzed by flow cytometry within 1 h.
Magnetically purified CD4⁺ T cells (MACS; Miltenyi Biotec) were exposed (continuous treatment) to DMSO, ML604440 (300 nm) or ONX-0914 (30 nM) for 2 h before stimulation with plate-bound antibodies against CD3 and CD28 (Biolegend) for 3 days. The intracellular expression of IL-17A and IFN-γ in CD3⁺CD8^- cells (CD3-PE/cy7, clone SK3; CD8-APC-eFluor780, clone RPA-78, eBioscience) was measured by using the appropriate antibodies (clone eBio64Cap17 and clone 4SB3; eBioscience) 4 h after exposure to 50 ng/ml PMA (Sigma) and 500 ng/mL ionomycin (Sigma) in the presence of 10 µg/mL brefeldin A (Sigma) by flow cytometry (Accuri C6; BD Biosciences).

PBMCs or T cell assays were prepared as described above. Brefeldin A (5μg/ml, Sigma) was added to test wells for the final 16 h of culture, while positive control wells (without antigen) were stimulated with PMA (50ng/ml), ionomycin (500ng/ml) and Brefeldin A (5μg/ml) for the final 4 h. Cells were stained for viability with Aqua amine-reactive dye (Invitrogen) before surface staining with fluorochrome-conjugated antibodies against CD3 (eBioscience, clone OKT3), CD4 (eBioscience, clone RPA-T4), CD8 (eBioscience, clone RPA-T8), CD45RO (eBioscience, clone UCHL1), CCR7 (eBioscience, clone 3D12). Cells were fixed and permeabilised using the Dako IntraStain Fixation and Permeabilization Kit, followed by intracellular staining with fluorochrome-conjugated antibodies against IL-17A (eBioscience, clone eBio64DEC17) and IFNγ (eBioscience, clone 4S.B3). Flow cytometric data was acquired with a BD LSRFortessa and analysed using FlowJo software (Tree Star, Inc.). Gates are set on respective Fluorescence Minus One (FMOs). For patient responses, antigen-specific proliferation was calculated by subtracting any background CFSE_lo proportions in the negative control wells (i.e. media only) from test CFSE_lo populations. Antigen-specific cytokine production in patient samples was similarly calculated by subtracting any background cytokine production seen in negative control wells.