Figure 6. Notch-mediated IL-10 induction in Th1 cells requires Blimp-1. (A) Heat map representation of relative expressions of transcription factors >2× up-regulated in in vitro generated IL-10–producing versus nonproducing Th1 cells stimulated additionally with the Notch ligand Dll4. Cells were FACS-sorted according to their IL-10 secretion after IL-10 cytokine secretion assay 5 d after activation with anti-CD3/anti-CD28 MACSi beads under Th1 skewing conditions. To induce Notch activation, Th1 cells were cultured additionally in the presence of MACSi beads covalently coated with recombinant mouse Dll4. Probes were ranked by the difference (log2-fold) between results obtained from IL-10+Th1 cells and IL-10−Th1 cells (FC > 2). (B) Relative Prdm1 expression measured by qPCR and Blimp-1 protein level determined by immunoblot in Th0 and Th1 cells stimulated either in the presence (+) or absence (−) of rDll4 72 h after priming of Th cells. Indication of protein size is in kD. (C) ChIP analysis of Blimp-1 binding to CNS upstream of Il10 in Th1 cells stimulated in the presence (w/) or absence (w/o) of rDll4 72 h after activation. From left to right: *, P = 0.018; *, P = 0.042. (D) Relative Maf expression measured by qPCR and c-Maf protein level determined by immunoblot (same blot as in B) in Th0 and Th1 cells stimulated either in the presence (+) or absence (−) of rDll4 72 h after priming of Th cells. Indication of protein size is in kD. *, P = 0.036. (E) ChIP analysis of c-Maf binding to CNS upstream of Il10 in Th1 cells stimulated in the presence (w/) or absence (w/o) of rDll4 72 h after activation. From left to right: *, P = 0.027; **, P = 0.007; *, P = 0.01. (F) Wild-type (WT) or Prdm1-deficient (CKO) Th1 cells were transduced with either a control (GFP RV) or with a Notch3IC (Notch RV) retroviral construct. Relative Il10 expression in GFP-sorted cells was measured by qPCR and frequency of IL-10 and IFN-γ producers (gated on CD4+GFP+) was determined by flow cytometry 96 h after Th1 priming after PMA/ionomycin restimulation. From left to right: **, P = 0.002; **, P = 0.003; **, P = 0.002; *, P = 0.038; **, P = 0.009. Data are representative of three independent experiments (mean ± SEM).
Figure 6.

Notch-mediated IL-10 induction in Th1 cells requires Blimp-1. (A) Heat map representation of relative expressions of transcription factors >2× up-regulated in in vitro generated IL-10–producing versus nonproducing Th1 cells stimulated additionally with the Notch ligand Dll4. Cells were FACS-sorted according to their IL-10 secretion after IL-10 cytokine secretion assay 5 d after activation with anti-CD3/anti-CD28 MACSi beads under Th1 skewing conditions. To induce Notch activation, Th1 cells were cultured additionally in the presence of MACSi beads covalently coated with recombinant mouse Dll4. Probes were ranked by the difference (log2-fold) between results obtained from IL-10+Th1 cells and IL-10−Th1 cells (FC > 2). (B) Relative Prdm1 expression measured by qPCR and Blimp-1 protein level determined by immunoblot in Th0 and Th1 cells stimulated either in the presence (+) or absence (−) of rDll4 72 h after priming of Th cells. Indication of protein size is in kD. (C) ChIP analysis of Blimp-1 binding to CNS upstream of Il10 in Th1 cells stimulated in the presence (w/) or absence (w/o) of rDll4 72 h after activation. From left to right: *, P = 0.018; *, P = 0.042. (D) Relative Maf expression measured by qPCR and c-Maf protein level determined by immunoblot (same blot as in B) in Th0 and Th1 cells stimulated either in the presence (+) or absence (−) of rDll4 72 h after priming of Th cells. Indication of protein size is in kD. *, P = 0.036. (E) ChIP analysis of c-Maf binding to CNS upstream of Il10 in Th1 cells stimulated in the presence (w/) or absence (w/o) of rDll4 72 h after activation. From left to right: *, P = 0.027; **, P = 0.007; *, P = 0.01. (F) Wild-type (WT) or Prdm1-deficient (CKO) Th1 cells were transduced with either a control (GFP RV) or with a Notch3IC (Notch RV) retroviral construct. Relative Il10 expression in GFP-sorted cells was measured by qPCR and frequency of IL-10 and IFN-γ producers (gated on CD4+GFP+) was determined by flow cytometry 96 h after Th1 priming after PMA/ionomycin restimulation. From left to right: **, P = 0.002; **, P = 0.003; **, P = 0.002; *, P = 0.038; **, P = 0.009. Data are representative of three independent experiments (mean ± SEM).

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