Ifn γ xmg1.2

Lymph nodes and spleen were disintegrated into single cells as previously described45 (link) and stained for surface markers with the following antibodies: TCR-β (H57-597), B220 (R43-6B2), CD4 (RM4-5), and CD8 (53-6.7), and all were purchased from BD Biosciences (Franklin Lakes, NJ, USA) or eBioscience (San Diego, CA, USA).
Spleen cells were stimulated as previously described46 , and then stained for intracellular cytokines using a Mouse FOXP3 Buffer Set (BD Biosciences). The following fluorochrome-conjugated antibodies were used: CD4 (GK1.5), CD25 (7D4), CD3 (145-2C11), FOXP3 (FJK-16s), RORγt (AFKJ-9), IFNγ (XMG1.2), and IL-17 (eBio17B7), and all were purchased from BD Biosciences or eBioscience.
Data was collected with a FACSCanto instrument (BD Biosciences).

The following FACS antibodies were purchased from BD Biosciences: CD4 (RM4-5), CD44 (IM7), Ki67 (B56), IFNγ (XMG1.2) and IL-17 (TC11-18H10). The following were purchased from eBioscience: CD73 (eBIOTY/11.8), RORγt (AKFJS9), Foxp3 (FJK-16s) and GM-CSF (MP1-22E9). For cytokine analysis, cells were cultured in complete medium (as described for T cell cultures above) with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of Golgiplug (BD Biosciences) for 3 to 4 hours followed by FACS staining and analysis. For intracellular cytokines, staining was performed using Cytofix-cytoperm kit from BD; RORγt and Foxp3 intracellular stains were performed using eBioscience Foxp3 staining kit according to manufacturer’s instructions. Prior to surface staining, cells were incubated for 20 min on ice with Ghost Dye^™ Violet 510 (TONBO biosciences, CA) to allow exclusion of dead cells from analysis performed in FlowJo.

The isotype-matched control antibodies rat IgG2a (553928), rat IgG1 (553921), and mouse IgG1 (553445), as well as mAbs to CD4 (RM4-5), CD8 (53–6.7), CD11b (M1/70), CD11c (HL3), I-A/I-E (M5/114.15.2), Ly6C (AL-21), Ly6G (1A8), CD62L (MEL-14), CD44 (IM7), CD25 (PC61), Ki67 (B56), and IFN-γ (XMG1.2) were purchased from BD Bioscience. Mabs to CD63 (NVG-2), CD103 (2E7), XCR1 (ZET), Bcl-2 (BCL/10C4), GITR (DTA-1), PD-1 (RMP1-30), Tim3 (RMT3-23), CTLA-4 (UC10-4B9), Foxp3 (150D), CD45.2 (104), and CD40 (3/23) were purchased from Biolegend. Anti-IFN-β(7F-D3) was from Yamasa; control rat IgG (6130-01) was purchased from Southern Biotechnology. Anti-PS antibody (1H6) was purchased from Merck Millipore. The CD300a-specific mAb (EX42) was generated in our laboratory. Anti-CD25 (PC61) was a gift from E. Nakayama (Okayama University). Cells were treated for 10 min with anti-CD16/CD32 mAb (2.4G2; TONBO Bioscience) to prevent binding to FcγR prior to incubation with the indicated combination of antibodies. All samples were evaluated by using a Fortessa flow cytometer (Becton Dickinson) and analyzed by using FlowJo software (Tree Star).

For cell surface staining, cell suspensions were labeled with Abs in PBS, 1% FCS, 2 mM EDTA. For intracellular staining, cells were fixed and permeabilized using Foxp3 staining buffer kit (BD Biosciences) or the BD cytofix/cytoperm kit. For pERK staining, cells were fixed in 2% paraformaldehyde, washed and permeabilized in ice-cold methanol for 30 min, washed twice in PBS 10% FCS, and stained for 1 h. Samples were run on LSR-IIB or Fortessa II (BD Biosciences) and analyzed with FlowJo software (Free Star). An Aria III cell sorter (BD Biosciences) was used to isolate naive CD8 T cells at >97% purity as judged by cell surface marker expression. Coulter CC Size standard beads (Beckman Coulter) were used for calculating cell numbers. Abs were from eBioscience: CD8 (53-6.7), CD44 (IM7), CD122 (5H4), CXCR3 (173), CCR7 (4B12), NKG2D (CX5), CD45.1 (A20), Eomes (Dan11mag), T-bet (4B10), IL-4 (11B11), IFN-γ (XMG1.2), TNF-α (MP6-XT22), and GranzymeB (NGZB); BD Biosciences: Bcl-2 (3F11), CD4 (RM4-5), αβ TCR (β-chain. H57-597), heat stable Ag (HSA; M1/69), and pan ERK; BioLegend: CCL5 (2E9), PLZF (9E12), and IL-4R (I015F8); Santa Cruz Biotechnology: Egr1 (C19); and Cell Signaling Technology: pERK (p42/p44). mCD1d/PBS57 tetramers were generously supplied by the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility.

Anti-mouse CD45 (30-F11), CD8a (53-6.7), and IFN-γ (XMG1.2) antibodies were purchased from BD Biosciences (Franklin Lakes, NJ); CD45.1 (A20), CD4 (RM4-5), and TCRβ (H57-597) antibodies from BioLegend (San Diego, CA); CD8b (H35-17.2) antibody from eBioscience (San Diego, CA); CD4 (RM4-5) and Foxp3 (FJK-16s) antibodies from Invitrogen. Dorsal halves of ears were digested with Liberase TL (Roche) containing 0.05% DNase I (Sigma-Aldrich, St. Louis, MO) for 60 minutes at 37 °C. The digested tissues were meshed through 40 μm of cell strainer to obtain single-cell suspensions. For intracellular staining, 10 μg/ml brefeldin A (Sigma-Aldrich) was put in the digestion buffer, and then collected cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences). For intranuclear staining, cells were fixed and permeabilized using Transcription Factor Buffer Set (BD Biosciences). Flow cytometry was performed using LSRFortessa (BD Biosciences) and data were analyzed with FlowJo software (Tree Star, Ashland, OR).