Anti cd28 clone 37

Naïve CD4+ cells were purified from spleens of B6 mice using positive selection with anti-CD4 MACS beads (Miltenyi Biotec). Purified cells were activated with anti-CD3 (clone 145–2C11, 2.5μg/mL) and anti-CD28 (clone 37.51, 2.5μg/mL) antibodies in the presence of the indicated concentration of anti-IL-2 neutralizing antibodies (JES6–1A12 and S4B6–1, BioXcell) with or without rIL6 (R&D) at indicated concentrations. Cells were cultured for 48 h at 37 °C in 125 μl in round- bottomed 96-well plates in RPMI-1640 supplemented with sodium pyruvate, HEPES (pH 7.2–7.6 range), nonessential amino acids, penicillin, streptomycin, 2-mercaptoethanol and 10% heat-inactivated FCS (all from Gibco).

FACS-sorted or magnetic bead-selected naïve CD4 T cells were cultured in Iscove’s Modified Dulbecco’s medium (IMDM) (Mediatech; Manassas, VA) with 10% FBS (Gemini Bio Products; West Sacramento, CA), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine. Cells were activated with 10 μg/ml plate-bound anti-CD3 (clone X145-2C11) and anti-CD28 (clone 37.51) (Bio X Cell; West Lebanon, NH). Where indicated cultures included: 50 ng/ml recombinant mouse IL-23, 20 ng/ml recombinant mouse IL-6, 20 ng/ml recombinant mouse IL-1β, 3.5 ng/ml recombinant mouse IL-12 (all from eBioscience), 2 ng/ml recombinant human TGF-β (R&D Systems; Minneapolis, MN), 200 nM 6-formylindolo(3,2-b)carbazole (FICZ) (Enzo Life Sciences; Farmingdale, NY), 10 μg/ml anti-IFNγ mAb (clone XMG1.2), 10 μg/ml anti-IL-4 mAb (clone 11B11) (Bio X Cell). Our Th cultures conditions were: Th0: anti-IL-4 mAb, anti-IFNγ mAb; Th1: anti-IL-4 Ab, IL-12; Th17 inflammatory: anti-IL-4 mAb, anti-IFNγ mAb, IL-6, TGF-β, IL-1β, IL-23; Th17 classic: anti-IL-4 mAb, anti-IFNγ mAb, IL-6, TGF-β; and Th22: anti-IL-4 mAb, anti-IFNγ mAb, IL-23, IL-6, IL-1β, FICZ. For proliferation assays, sorted naïve CD4 T cells were labeled with 5 μM CFSE (eBioscience) prior to culture. Cells were cultured in a standard CO₂ incubator, with 5% CO₂, approximately 70% humidity and approximately 17% O₂.

Total CD4⁺ T cells were isolated from single-cell spleen suspensions using magnetic negative selection (eBioscience). Cells were stimulated at 37 °C, 5% CO₂ for 16 h in RPMI culture media supplemented with 10% fetal calf serum (Sigma), 1% penicillin and streptomycin (pen/strep; Gibco), and 50μM β-mercaptoethanol, non-essential amino acids (Gibco) and Glutamax (Gibco). Cells were plated at density of 1×10⁵ cells/well on 96-well plates in the presence or absence of 10ng/mL recombinant human IL-2 (Peprotech) with or without 300nM Tofacitinib (Sigma) or 10μM BI 605906 (Tocris). Where indicated, plates were coated overnight with 5 μg/mL anti-CD3 (clone 145.2C11; BioXcell Cat #BE0001-1) and 5 μg/mL anti-CD28 (clone 37.51 BioXcell Cat #BE0015-1) monoclonal antibodies in PBS before washing and plating of cells for stimulation. Cells were harvested and analysed by flow cytometry. To detect LPS-induced GARP expression on B cells, cells from erythrocyte-lysed blood were stimulated in RPMI complete media (RPMI, 10% FCS, 1% pen/strep, and 50μM β-mercaptoethanol) containing 0.5 mg/ml lipopolysaccharide (Sigma) at 37 °C, 5% CO₂ for 48 hr. At the end of the stimulation period, cells were collected and analysed using flow cytometry.