7), CD11b (M1/70), CD11c (HL3), CD19 (1D3), CD25 (PC61), CD62L (MEL-14), Ter119 (TER119), and streptavidin (SA)- allophycyanin, SA-allophycyanin Cy7, SA-FITC. Qdot605 anti-CD4 (RM4–5) and SA-Qd605 this website were

obtained from Invitrogen. Alexa Fluor 488 anti-LAG-3 (C9B7W) was obtained from AbD Serotec. PE anti-Egr-2 (erongr2) was obtained from e-Bioscience. Streptavidin-conjugated microbeads were purchased from Miltenyi Biotec. Recombinant murine IL-2, IL-10, IL-12, IL-21, and IL-27 were obtained from R&D Systems. Recombinant human TGF-β1 was purchased from R&D Systems. Recombinant murine IL-23 was obtained from Biolegend. Zymosan was obtained from Sigma. Eα52−68 peptide was purchased from Takara (Otsu, Japan). T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 μg/mL L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol (all purchased from Sigma). Naïve CD4+ T cells (CD4+CD45RBhiCD62LhiCD25−) from C57BL/6 WT, Egr-2 CKO, or Blimp-1 CKO mice, WSX-1 KO mice, and STAT1 KO, or STAT3 CKO mice were isolated from their splenocytes. Briefly, single Stem Cells inhibitor cell suspensions

were first purified by negative selection with MACS (Miltenyi Biotec) using anti-CD8α mAb, anti-CD11b mAb, anti-CD11c mAb, anti-CD19 mAb, anti-CD25mAb, and anti-Ter119 mAb, and were then purified by positive selection with anti-CD62L microbeads. The purity of MACS sorted cells was >90%. Purified cells many were cultured in flat-bottomed 24-well plates coated with anti-CD3ε (2 μg/mL) and anti-CD28 (2 μg/mL). Mouse IL-27 (25 ng/mL) was added at the start of culturing. To assess T-cell proliferation, purified naïve CD4+ T cells were labeled with 1 μM carboxyfluorescein diacetate succinimidyl diester (Invitrogen) by incubation

for 5 min at 37°C in the dark at a density of 2 × 106 cells/mL in RPMI medium. Other cytokines used were as follows: IL-2; 20 ng/mL, IL-6; 10 ng/mL, IL-12; 20 ng/mL, IL-23; 20 ng/mL and IFN-γ; 10 ng/mL. A total of 1 × 106 cells of CD4+ T cells from Eα52−68/I-Ab-specific transgenic mice were purified by positive selection with anti-CD4 microbeads and cultured with 5 × 105 cells of B cells from C57BL/6 WT mice in the presence of Eα52−68 peptide (3 μM) in flat-bottomed 24-well plates. IL-27 (20 ng/mL), TGF-β1 (20 ng/mL), IL-21 (50 ng/mL), IL-10 (50 ng/mL), and zymosan (25 μg/mL) were added, respectively. CD4+ T-cell RNA was prepared using an RNeasy Micro Kit (Qiagen). RNA was reverse-transcribed to cDNA with random primers (Invitrogen) and Superscript III (Invitrogen) in accordance with the manufacturer’s protocol (Invitrogen). The cellular expression level of each gene was determined by quantitative real-time PCR analysis using an iCycler (Bio-Rad).