Ctla 4

For surface staining, cells were labeled with mAbs against various targets including CD8, CD62L, CCR7, CD45RA, CD45RO, CD25, CD44, OX40, PD-1, CTLA-4, and KLRG-1 (Ebiosciences), incubated at 4 °C for 15 minutes, then washed twice with PBS 1% FBS (FACS buffer), and finally fixed in PBS containing 1% paraformaldehyde (Fix buffer). For T-bet (4B10) and Eomes (Dan11mag), and Foxp1 intracellular staining, cells were first labeled with surface markers CD8, CD62L, OX40 (Ebiosciences) for T-bet and Eomes, or CD25 (BD Biosciences), CD8, CD27, and CD44 (Ebiosciences) for Foxp1 detection, respectively, and then fixed and permeabilized with Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent (Ebiosciences) in a 96-round-well plate. Intracellular labeling for T-bet and Eomes (Ebiosciences), and Foxp1 (LifeSpan Technologies) was performed according to manufacturer’s instruction. For evaluation of Perforin, Granzyme B, and IFN-ɤ cytokine secretion, effector cells were incubated with brefeldin A for 4 hours at 37 °C to disrupt Golgi-mediated transport and accumulate cytokines. Cells were then surface stained, permeabilized with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Biosciences), and intracellularly stained as recommended. Flow cytometry data was analyzed using FlowJo Version 10.0.7 software.

Mononuclear cells from central draining lymph nodes of mice were isolated and labeled using specific antibodies for murine CD4 (eBioscience), PD-1 (Novus), FoxP3 (eBioscience) and CTLA-4 (eBioscience). Background nonspecific fluorescence was measured using control isotype antibodies. Labeled cells were analyzed by flow cytometry using a FACS Canto II flow cytometer (BD Bioscience) and data analyzed with Kaluza analysis software (Beckman Coulter). All determination were performed in at least n = 5 mice per experimental group and three separate experiments.

For cell surface staining of mouse PD-1H, MH5A (hamster anti-mouse PD-1H) was used^{23 (link)}, followed by anti-hamster-IgG-PE (eBioscience); hamster IgG (eBioscience) was used as the isotype control. PD-1H agonist mAb clone mam82 (mouse anti-mouse IgG1) were described previously^{26 (link)}. All other fluorescently labelled antibodies including CD4, CD25, CD44, CD69, Foxp3, TCR Vβ5.1/5.2, p-STAT3, p-STAT5, CTLA-4, Lag-3, GITR, IOCS, CD45.1, and CD45.2 were purchased from eBioscience and BD Pharmingen. For neutralizing assays, the anti-IFN-γ (clone XMG1.2), anti-IL-4 (clone 11B11), anti-IL-6 (clone MP5–20F3) neutralizing Abs were purchased from R&D System. The intracellular staining for Foxp3 and other intracellular cytokines were performed according to BD’s Cytofix/Cytoperm kit manual. Cytokine analysis was performed using the mouse Th1/Th2/Th17 CBA kits (BD Bioscience). Mouse pan-T isolation kit, CD8⁺ T cell isolation kit, CD4⁺ T cell isolation kit, CD25 micro beads kit, and naïve CD4⁺ T cell isolation kits were purchased from Miltenyi Biotec (Cambridge, MA). Flow cytometry analysis was performed using a BD FACSVerse (BD Biosciences) and the data was analysed using FlowJo software (Tree Star).

Anti-mouse CD3 (clone 145-2C11) and anti-CD28 (clone PV-1) antibodies were prepared in our laboratory. DMEM, penicillin and streptomycin from GIBCO Inc. (Grand Island, NY, USA); fetal bovine serum (FBS) from HyClone Inc. (Logan, UT, USA); anti-mouse antibodies including Alexa 488 anti-Foxp3, -IFNγ and -perforin, PE anti-CD25, -CD39, -CD101, -CTLA-4, -granzyme B, ICOS and -IL-2, PerCP-Cy5.5 anti-CD73 and APC anti-CD45.2, FITC anti-human CD8, PE anti-human CD28, APC anti-human CD39 and PE-Cy7 anti-human CD73 from eBioscience (San Diego, CA, USA); PE anti-TNFα, -CCR6, -CD103, -Galectin-9 and -Helios antibodies from BioLegend (San Diego, CA, USA); PE anti-CD122, -CTLA-4, -LAG3 and -PD-1, APC anti-FR4, and PE-Cy7 anti-GITR antibodies from BD Bioscience (San Jose, CA, USA); Anti-CD73 antibody from Abcam (Cambridge, MA, USA); Mitomycin C, LPS, MTT, α, β-methylene adenosine 5’-diphosphate (AMPCP) from Sigma (St. Louis, MO, USA); KC7F2 from Calbiochem (Burlington, MA, USA); ZM241385 and MRS1754 from Tocris (Minneapolis, MN, USA); CFSE from Molecular Probes (Eugene, OR, USA); M2 peptides (LCMV gp33-41, KAVYNFATM) from AnaSpec, Inc. (San Jose, CA, USA); LY294002 from Cell signaling Technology (Danvers, MA, USA) were purchased.