Anti cd28 37.51

Naïve CD4⁺ T cells were isolated from spleen and lymph nodes of C57BL/6 mice using naïve CD4 negative selection kit (StemCell) and activated under Th17-polarizing conditions in the presence of irradiated wild-type splenocytes at a 5:1 ratio, with anti-CD3 (2.5 μg/ml) (145-2C11, BioXCell), anti-IL-4 (10 μg/ml) (11B11, BioXCell), anti-IFNγ (10 μg/ml) (XMG1.2, BioXCell), mIL-6 (30 ng/ml) (Miltenyi Biotec) and hTGF-β1 (3 ng/ml) (StemCell). The following day, cells were treated with either DMSO or of FGIN-1-27 (10 μM). For phospho-ZAP70 analyses, cells were stained after 24 hours of treatment using 10 μg/ml of biotinylated anti-CD3 (145-2C11, BD Biosciences) and anti-CD28 (37.51, BD Biosciences) antibodies on ice for 15 minutes, washed, and stimulated in presence of 20 μg/ml of Streptavidin for 10 minutes at 37 degrees. Cells were then fixed in 2% PFA, permeabilized in Methanol/Acetone and stained with anti-phospho ZAP70 antibody (n3kobu5, eBioscience) and anti-CD4 (RM4–5, eBioscience). For phospho-STAT3 analyses, cells were cultured for additional 24 hours in IL-6 free media, before stimulation with 100 ng/ml of mIL-6 for 30 minutes. Cells were then fixed in 2% PFA, permeabilized in Methanol/Acetone and stained with anti-phospho-STAT3 antibody (4/P-STAT3, BD Biosciences) and anti-CD4. Dead cells were excluded using Live/Dead Amcyan (Thermo Fisher) staining prior to stimulation.

Induced Tregs (iTregs) were generated as described previously.⁷ (link) Briefly, CD4⁺ cells were isolated from B6 spleen and lymph nodes by magnetic bead sorting (L3T4 Microbeads, Miltenyi Biotec), and cultivated in plates coated with 10 µg/ml anti-CD3 (145-2C11), 1 µg/ml anti-CD28 (37.51) (BD Pharmingen) in the presence of 100 U/ml interleukin-2 (IL-2; Sigma) and 5 ng/ml recombinant human transforming growth factor-beta (rhTGF-β; R&D Systems) for 5 days. At the end of culture, the Treg-enriched cell population was used for therapeutic administration without additional sorting steps. FoxP3 expression was usually >80%. Then 3 × 10⁶ cells were injected intravenously per mouse.

Lymph node cells were stimulated with 2 µg/ml H56 together with anti-CD28 (37.51; BD Biosciences) and anti-CD49d (9C10; BD Biosciences) for 1 h. Subsequently, 10 µg/well brefeldin A (Sigma-Aldrich, St. Louis, MO) and 0.7 µl/well monensin/GolgiStop (BD Biosciences Franklin Lakes, NJ) were added and cells were incubated for 5 h at 37 °C. After overnight storage at 4 °C, cells were washed in FACS buffer (1% FCS (VWR-Bie & Berntsen, Herlev, Denmark), 0.1% sodium azide (VWR, Radnor, PA) in PBS (Life Technologies, Carlsbad, CA), and stained for surface markers with 1 µg/ml anti-CD4-APC-eFlour780 (clone GK1.5) and 1 µg/ml anti-CD44-FITC (clone IM7) (both eBiosciences, San Diego, CA) for 30 min at 4 °C. Cells were washed with FACS buffer, before fixation and permeabilization using Cytofix/Cytoperm kit (BD Biosciences Franklin Lakes, NJ). Subsequently, cells were stained for intracellular cytokines with 1 µg/ml anti-IFN-γ PE-Cy7 (XMG1.2; eBiosciences, San Diego, CA), 1 µg/ml anti-TNF-PE (MP6-XT22; eBiosciences, San Diego, CA) and 1 µg/ml IL-17A for 20 min. Finally, cells were washed, re-suspended in FACS buffer, and analyzed using a FACSCanto flow cytometer (BD Biosciences Franklin Lakes, NJ) and FlowJo software version 10.

Naïve (CD4⁺CD25⁻CD44^loCD62L^hi) CD4⁺ T cells were isolated by negative selection using EasySep (StemCell), then activated with plate-bound anti-CD3 (145-2C11, BD) and anti-CD28 (37.51, BD) under neutral (anti-IFNγ, anti-IL-4, anti-TGFβ) or T_FH-like (anti-IFNγ, anti-IL-4, anti-TGFβ, rmIL-6, rmIL-21) condition. The cells were transduced with retrovirus (MigR-GFP or MigR-ETV5-GFP) at 24 h and at 36 h after in vitro stimulation. Two days after culture, the cells were rested for 24 h in the presence of rmIL-7, then re-stimulated with pre-coated anti-CD3 for 2 h for RNA samples and for 12 h for protein samples.
For STAT3 inhibitor treatment experiments, naïve CD4⁺ T cells were polarized under T_FH-like condition as described above. The cells were transduced with retrovirus (MigR1-GFP or MigR1-ETV5-GFP) at 24 h after in vitro stimulation, then treated with AG490 (Tokyo Chemical, 50 μM) for 16 h or Stattic (Santa Cruz Biotechnology, 20 μM) for 12 h. Two days after culture, the cells were rested for 24 h. The cells were collected for RNA samples.