Anti ifn γ xmg1

Total CD4⁺ T cells were isolated from peripheral lymph nodes and spleens of age- and sex-matched mice and purified with CD4 microbeads (103-104-454, Miltenyi Biotec). Naïve CD4⁺ T cells were purified by naïve CD4⁺ T cell isolation kit (130-104-503, Miltenyi Biotec). Purified CD4⁺ T cells were activated with plate coated with 10 µg/ml anti-CD3 (145-2C11, BioXCell) and 10 µg/ml anti-CD28 (37.51, BioXCell) antibodies and cultured in serum-free X-VIVO 20 medium (Lonza). For pathogenic Th17 cell differentiation, 20 ng/ml IL-1β (Biolegend), 40 ng/ml IL-6 (Biolegend), 50 ng/ml IL-23 (Biolegend) and 20 µg/ml anti-IFN-γ (XMG1.2, BioXcell) were added to the culture. For TGFβ1 induced non-pathogenic Th17 cell differentiation, 2 ng/ml TGFβ1 (Biolegend), 40 ng/ml IL-6 (Biolegend) and 20 µg/ml anti-IFN-γ (XMG1.2, BioXcell) were added to the culture. For Th0 condition, 40 U/ml of IL-2 was added to the culture. TGFβ receptor I (Alk5) inhibitor SB431542 (S1067, Selleckchem) was used to block TGFβ signaling.
For the in vitro maintenance of pathogenic Th17 cells, cells from 4 days of pathogenic Th17 cell differentiation were harvested and transferred into 24-well plated coated with 2 µg/ml anti-CD3 and 2 µg/ml anti-CD28 antibodies and cultured in serum-free X-VIVO 20 medium for another 4 days. Samples for FACS, immunoblotting and realtime PCR were harvested at indicated time points.

Agonistic mouse anti-CD40 (clone FGK4.5/FGK45, BioXCell, Lebanon, NH, USA), anti-CD154/CD40L (MR-1, BioXCell), anti-NK1.1 (PK136, BioXCell), anti-CD19 (1D3, BioXCell), anti-IL12 (R1-5D9, BioXCell), and anti-IFNγ (XMG1.2, BioXCell) antibodies were diluted to 200 µg in PBS and administered 24 h prior to viral challenge. Anti-CD4 (YTS 177, BioXCell) and anti-CD8 (2.43, BioXCell) antibodies were diluted to 200 µg/100 µL in PBS, pooled to a total of 400 µg (200 µL), and administered 24 h prior to downstream use. Isotype IgG controls (BioXCell) were used at equivalent doses in all experiments. All antibodies were administered in a final volume of 100–200 µL via intraperitoneal (i.p.) injection. Cytokines used in this study include IL-12 (StemCell Technologies, Vancouver, CA, USA, catalog #78028.1) and IFN-γ (StemCell Technologies, catalog #78020.1), which were given at 5 µg in 100 µL of PBS via i.p. injection 24 h prior to viral challenge. For the disruption of IL-12 production, the inhibitor apilimod (MedChemExpress, Monmouth Junction, NJ, USA, catalog #HY-14644) was administered via i.p. injection at a concentration of 2.5 mg/kg. The drug was administered on day −1, day 0, and day 1 post-infection with rVSV-EBOV GP.

Naive CD4⁺ T cells were sorted from spleens as CD4⁺TCRβ⁺CD44^–CD25^–Il4^gfp–PI⁻(Il4^gfp reporter) or CD4⁺TCRβ⁺CD44^–CD25^–Il4^gfp-Ifng^yfp–Il17a^FP635–PI⁻(triple reporter). Th2 cells were cultured for 2 weeks from splenic CD4⁺ cells in vitro with 10 ng/mL IL-4 (R&D), 5 ng/mL IL-2 (R&D), 10 μg/mL anti-IFNγ (XMG1.2, BioXcell), and Mouse T-Activator CD3/CD28 Dynabeads (Life Technologies) in IMDM with 10% FCS. Th2 cells were sorted as CD4⁺TCRβ⁺Il4^gfp+PI⁻(Il4^gfp reporter) or CD4⁺TCRβ⁺Il4^gfp+Ifng^yfp–Il17a^FP635–PI⁻(triple reporter). For each experiment, 0.2x10⁶ to 1x10⁶ cells were adoptively transferred i.v. into recipient C57BL/6 Rag1^–/- mice. Blocking antibodies diluted in PBS (anti-IFNγ, XMG1.2, anti-IL12p40 C17.8, BioXcell) were used at 0.4 or 0.5 mg/ dose.