Anti mouse ifnγ clone xmg1

Anti-IDH2 was from Abcam (# ab131263), anti-IDH1 was from Cell Signaling Technology (#8137S), and anti-HIF1α is from Novus biologicals (#NB100–105). Anti-mouse CD3 (clone 145–2C11, cat#16–0031), anti-mouse CD28 (clone 37.51, cat#16–0281), anti-mouse IFNγ (Clone XMG1.2,Cat#16–7311), anti-mouse IL-4(Clone 11B11, cat#16–7041), anti-mouse FOXP3 (clone FJK-16s, cat#17–5773) are from eBioscience. Anti-mouse CD4 (clone RM4–5, Cat#550954), anti-mouse CD25 (clone 7D4, Cat#558642), anti-mouse CD44 (Clone IM7, Cat#559250), anti-mouse CD62L (clone MEL-14, Cat#560507), anti-mouse IL-17 (clone TC11–18H10, Cat#559502), anti-mouse IFNγ (Clone XMG1.2, Cat#561040, for staining) and anti-CD90.1 (Thy1.1) (clone OX-7, cat#561406) were from BD Bioscience.

T cells were stained with H2-K^b/GP_61–80 tetramers (NIH) and antibodies against mouse CD4 (clone GK1.5, Biolegend), CD11a (clone M17/4, eBioscience), CD25 (clone PC61, Biolegend), CD44 (clone IM7, Tonbo), Ly6C (clone HK1.4, Biolegend), CXCR3 (clone CXCR3-173, Biolegend), and PD-1 (clone RMP1-30, Biolegend). B cells were stained with CD19 (clone 6D5, Tonbo), T and B cell activation antigen (clone GL-7, BD Biosciences), CD95 (clone Jo2, BD Biosciences), IgD (clone 11-26c.2a, BD Biosciences), and IgM (clone RMM-1, eBioscience). Cells were stimulated with either GP_61–80 peptide antigen or PMA and ionomycin in the presence of monensin, permeabilized with cytofix/cytoperm (BD Bioscience), and stained with anti-mouse IFN-γ (clone XMG1.2, eBioscience), anti-IL-2 (clone JES6-5H4, eBioscience), and anti-TNF (clone MP6-XT22, eBioscience). For Tfh analyses, cells were stained with anti-CXCR5 (clone 2G8, BD Biosciences) followed by staining with fluorochrome-conjugated anti-CD4, anti-CD44, anti-Ly6C, anti-CXCR3, and anti-PD-1. Anti-T-bet (clone 4B-10, Biolegend) and anti-Bcl-6 (clone K112-91, BD Biosciences) were applied after fixation and permeabilization of cells using FoxP3/Transcription Factor Staining Buffer Kits (Tonbo). Data were acquired using either Stratedigm S1200Ex or BD LSR II flow cytometers and analyzed using FlowJo software (Tree Star, Inc.).