Clone xmg1

Splenocytes of C57BL/6 (1.5 × 10⁵ per well) were incubated with ONX 0914 and stimulated with plate bound anti-CD3 (5 μg/ml, clone 17A2; eBioscience, San Diego, CA, USA) and anti-CD28 (5 μg/ml, clone 37.51; BD Biosciences, Heidelberg, Germany) antibodies for 24 h. GM-CSF in the supernatant was determined by ELISA, according to the manufacturer's protocol (eBioscience). For GM-CSF polarizing conditions, anti-IL-12 (10 μg/ml, clone C15.6; BD Biosciences) and anti-IFN-γ (10 μg/ml, clone XMG1.2; eBioscience) antibodies were added and supernatants were analyzed 96 h post stimulation.
Human PBMC′s from healthy volunteers were incubated with ONX 0914 for 2 h at 37°C. Washed cells (2.5 × 10⁵ per well) were stimulated with plate bound anti-CD3 (1 μg/ml, clone OKT3; eBioscience) and anti-CD28 (5 μg/ml, clone CD28.2; BD Biosciences) antibodies for 24 h. GM-CSF or IL-23 in the supernatant was determined by ELISA, according to the manufacturer's protocol (eBioscience).

Splenic naive T helper cells were purified with the CD4⁺CD62L⁺T Cell Isolation Kit II (Miltenyi Biotec) and polarized in vitro toward differentiated Th2 subtype as described before in [32 (link)]. In brief, naive cells were seeded into anti-CD3e (2 μg/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 μg/ml, clone 37.51, eBioscience) antibody coated 96-well round bottom plates. The medium contained the following cytokines and/or antibodies for Th2 subtype: recombinant murine IL-2 (10 ng/ml, R&D Systems), recombinant murine IL-4 (10 ng/ml, R&D Systems), and neutralizing anti-IFN-g (5 μg/ml, cloneXMG1.2eBioscience). The cells were removed from the activation plate on day 4 (72 h). Th2 cells were cultured for another 2 days in the absence of anti-CD3 and CD28 stimulation. Then, cells were restimulated by anti-CD3e/CD28-coated plate for 6 h. For flow cytometric detection, cells were treated with monensin (2 μM, eBioscience) for the last 3 h.

Surface marker expression was analyzed using the following antibodies: CD3 (clone 17A2) and B220 (clone RA3-6B2) (all from eBiocience according to data sheet). Staining was performed in the presence of Fc-receptor blocking antibody (clone 2.4G8, a kind gift of E. Kremmer, Helmholtz Zentrum München). Intracellular cytokine staining for IFN-ɣ (eBioscience Clone XMG1.2) was performed as follows: 1x10⁶ cells/ well were put in 200μL of stimulation media (RPMI 1640 + 10% FCS supplemented with PMA (20ng/mL), Ionomycin (1μg/mL) and Brefeldin A (10μg/mL)) in a 96 well flat bottom plate and incubated for 4h at 37°C. Unstimulated cells were handled in parallel, without PMA/Iono stimulation but with Brefeldin A. After incubation, cells were washed twice with PBS and used for surface marker staining (B220) followed by intracellular CD3 and IFN-ɣ staining with Fix/Perm-Kit (eBioscience) as described by the manufacturer.
All cells were processed on a LSRII Fortessa Flow Cytometer (BD) and analyzed with FlowJo9.6.2 software. Dead cells were excluded using Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) and doublets by gating on single cells.

CD4⁺ T cells from WT, lmp7^−/− or lmp2^−/− mice were isolated from spleen and lymph nodes by negative magnetic cell sorting (MACS, Miltenyi Biotec). CD4⁺ T cells were cultured containing plate-bound 5 μg/ml anti-CD3 (clone 145-2C11) and 1 μg/ml soluble anti-CD28 (clone 37.51). For Th17 differentiation, cultures were supplemented with 5 μg/ml α-IFN-γ (clone XMG1.2), α-IL-4 (10% culture supernatants of clone 11B11), 50 U/ml rhIL-2 (Novartis), 1ng/ml rhTGF-β1 (PeproTech) and 40 ng/ml IL-6 (PeproTech). 200-400 nM ONX-0914 (Onyx Pharmaceuticals) or 300-400 nM BAY 11-7082 (Sigma-Aldrich) were added to the Th17 differentiation medium for indicated time points. Cells were restimulated with 750 ng/ml of ionomycin, 50 ng/ml of PMA in presence of 10 µg/ml Brefeldin A (all three substances, Sigma-Aldrich) and were analysed for IL-17A (clone eBio17B7, eBioscience) and IFN-γ (clone XMG1-2, eBioscience) production by intracellular staining (ICS). In some experiments, expression of transcription factor IRF4 (clone 3E4, eBioscience) was analysed by ICS.