Anti il 4

Naïve (CD25-) CD4+ T cells were isolated using the CD4+ T Cell Isolation Kit, mouse and the CD25 MicroBead Kit, mouse by Miltenyi Biotec. Instead of the recommended buffer in the kit, R10 medium (RPMI-1640 with 10% FCS (Gibco) and 1% Penicillin/Streptomycin (Sigma-Aldrich)) was used and the amount of reagents suggested per 10⁷ cells was instead used per 1.5 · 10⁷ cells.
For the polarization of the CD4+ CD25- T cells, a 24-well-plate was coated with 10 μg/ml anti-CD3 and 10 μg/ml anti-CD28 in PBS for 1 h at 37 °C and washed one time with PBS before the cells were seeded at a concentration of 10⁶ cells in R10 medium for Th0 and Th1 polarization. Lastly, 5 μg/ml anti-IL-4 (BioXcell) and 10 ng/ml IL-12 (R&D systems) diluted in PBS were added for Th1 polarization. For IL-6 treatment during polarization, 10 ng/ml or 100 ng/ml IL-6 (Miltenyi Biotec) were supplemented. The cells were in total cultured for five days at 37 °C and 5% CO₂, with a medium change on day four. On day five, the cells were stimulated by addition of eBioscience™ Cell Stimulation Cocktail (plus protein transport inhibitors) (500X) (Invitrogen) for 3.5 h before harvest of supernatant for ELISA and cells for flow cytometry. The shown data after in vitro polarization are representative of at least four (Figs. 2, 3) or two (Fig. 4) independent experiments.

For Th0 conditions, sorted naïve CD4⁺ T cells were stimulated (day 0) with plate-bound anti-CD3ε (1 μg/ml; BD Biosciences) and anti-CD28 (3 μg/ml; BD Biosciences) on 48-well-plates (0.3 × 10⁶ cells/well) in 1 ml T cell medium/well (RPMI1640 supplemented with 10% FBS [Sigma/biowest], antibiotics, 50 mM β-mercaptoethanol) supplemented with 20 U/ml recombinant human IL-2 (rhIL-2) (Peprotech) for 3 days, unless otherwise stated. For the assessment of cell proliferation, 1–10 × 10⁶ naïve CD4⁺ T cells were labeled using Cell Proliferation Dye eFluor™ 450 (Thermo Scientific) according to the manufacturer's protocol prior to activation. Th1 and Th17 cells were generated from sorted naïve CD4⁺ T cells activated with anti-CD3ε/anti-CD28 for 3 days in the presence of 20 U/ml rhIL-2 (Peprotech), 5 ng/ml IL-12 (R&D Systems) and 3 μg/ml anti-IL-4 (BioXcell) for Th1 cells, and in the presence of 2 ng/ml TGFβ, 10 μg/ml IL-6, 10 μg/ml IL-1α, and 10 μg/ml IL-1β for Th17 cells. For cytokine detection, activated cells were restimulated for 4 h with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) and ionomycin (Iono, 750 ng/ml) (both from Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences).

To differentiate in vitro Tfh-like cells, naïve CD4 T cells were isolated from spleen and lymph nodes of WT or Hif1a-KO mice, and plated in IMDM 10% FCS with 10 ug/ml each of anti-IFN-g, anti-IL-4, anti-IL-12, anti-TGF-b (all from Bioxcell), 100 ng/ml IL-6, and 50 ng/ml IL-21 (both cytokines from Peprotech), on plates coated with 3 ug/ml anti-CD3 and 5 ug/ml anti-CD28. For inhibitor studies, CAL-101 (Santa Cruz Bio) was added at 4 nM from the beginning of culture, rapamycin (Calbiochem) was added at 10 nM for the last 24 h of culture. For hypoxia studies, cultures were incubated in a hypoxic cabinet (Coy Labs) supplied with a 94/5/1 N₂/CO₂/O₂ mixture.