Figure 3: IL-27-mediated Tim-3 expression is dependent on the functional cooperation between T-bet and NFIL3.

(a) Naïve CD4⁺ T cells from wild-type (WT) C56BL/6 mice and NFIL3^−/− mice were activated with anti-CD3 and anti-CD28 under Th0 or Th1 (IL-12 treatment) condition with or without IL-27. Five days after TcR activation, cells were restimulated with anti-CD3 and anti-CD28 for 24 h and subjected to detection of the expression of Tim-3 and IL-10 by flow cytometry. (b) Naïve CD4⁺ T cells were transduced with retrovirus carrying NFIL3 cDNA (NFIL3), T-bet cDNA (T-bet), or both NFIL3 and T-bet. Retrovirus empty vector-transduced T cells were used as controls (GFP for NFIL3, Thy1.1 for T-bet). Four days after TcR activation, the expression of Tim-3, IL-10 and PD-1 was examined by flow cytometry. The percentage represents the expression of Tim-3, IL-10 or PD-1 in NFIL3- or T-bet-transduced, or NFIL3/T-bet-co-transduced cells relative to GFP- or Thy1.1-expressing empty vector-transduced, or GFP/Thy1.1 empty vector-co-transduced cells, respectively. (c) Naïve CD4⁺ T cells were activated by anti-CD3 and anti-CD28 under Th0 and Th1 condition (IL-12 treatment) with or without IL-27. Three days after TcR activation, the expression of NFIL3 expression was detected by western blot analysis (left) and its relative expression to β-actin was quantified by LI-COR Odyssey Imaging System (right). Data are representative of at least three independent experiments with similar results.