PD-1H Expression and Signaling Analysis

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).