Contribution of IL-12R mediated feedback loop to Th1 cell differentiation

Abstract

T helper 1 (Th1) cell fate is induced by overlapping signaling pathways, whose kinetic principles and regulatory motifs are largely unknown. We identified a simple positive feedback loop in the STAT4 signaling pathway, whereby activation by IL-12 leads to the increased expression in IL-12 receptor. A computational analysis shows that this feedback loop synergizes with the one mediated by the IFN-gamma secreted by differentiating cells, when the induction of Th1 cell fate is weak. Positive feedback loops are often utilized to enhance phenotypic differentiation. This effect was confirmed by experiments showing that stochastic fluctuations in the expression of IL-12 receptor gene were amplified, leading to two discrete levels of expression in a cell population.

Fig. 1

IFN-γ production by wild-type lymphocytes in response to different cytokines. Cells were labeled with CFSE to detect the number of cell divisions and were stained with APC labeled anti-IFN-γ following activation. All concentration units are given in ng/ml. Under neutral conditions (in the absence of IL-12 and IL-27), 1% of the cells are IFN-γ positive, which corresponds to a background production.

Fig. 2

Interaction of IL-27 and IL-12 on IFN-γ and IL-12Rβ2 mRNA production in wild-type and Stat1^−/− cells (A) Proportion of IFN-γ positive wild-type cells as a function of IL-27 in the absence (black empty squares) and presence of 10 ng/ml IL-12 (black filled squares). The proportion of IFN-γ positive cells corresponds to the sum of relative cell counts in the upper two quadrants as shown in Fig. 1. (B) IL-12Rβ2 mRNA levels in wild-type cells as a function of IL-27 in the absence (black empty squares) and presence of 10 ng/ml IL-12 (black filled squares). (C) Proportion of IFN-γ positive Stat1^−/− cells as a function of IL-27 concentration 5 days after induction with IL-27 in the absence (red empty triangles) or presence (red filled triangles) of 10 ng/ml IL-12. (D) IL-12Rβ2 mRNA levels in Stat1^−/− cells as a function of IL-27 concentration 5 days after induction in the absence (red empty triangles) or presence (red filled triangles) of 10 ng/ml IL-12

Fig. 3

Modeling the Th1 cell fate induction pathways (A) Schematic representation of the signaling network. The dashed line stands for the predicted interaction to close the IL-12R feedback loop. (B, C) Simulation of IL-12Rβ2 mRNA levels in Stat1^−/− (B, red lines) and wild-type (C, black lines) background using fitted parameters (see Methods). Empty and filled lines denote were obtained using zero and saturating IL-12 concentrations, respectively. (D, E) The parameter v_x4 was set to zero to eliminate the activation of IL-12Rβ2 by STAT4 in and thereby the positive feedback loop. IL-12Rβ2 mRNA levels were simulated in the presence (continuous line) and absence of the feedback loop (dashed line) in Ifngr1^−/− (D, green lines) and wild-type networks (E, black lines).

Fig. 4

Self-activation of Th1 cells through IFN-γ (A) IL-12Rβ2 mRNA levels in wild-type (black empty squares) and Ifngr1^−/− (green empty circles) cells as a function of IL-27 concentrations. (B) IL-12Rβ2 mRNA levels in wild-type (black filled squares) and Ifngr1^−/− (green filled circles) cells as a function of IL-27 concentrations when IL-12 is present at 10 ng/ml concentration. (C) Simulation of IL-12Rβ2 mRNA levels in Ifngr1^−/− cells using fitted parameters (see Methods). Empty and filled green lines denote were obtained using zero and saturating IL-12 concentrations, respectively. (D) Proportion of IFN-γ positive wild-type cells as a function of IL-27 in wild-type (BALB background, (black empty squares) and Stat4^−/− cells (BALB background, blue empty diamonds). (E) T-bet mRNA levels in Stat1^−/− cells, 5 days after induction with IL-27 in the presence (red filled triangles) or absence (red empty triangles) of IL-12 (10 ng/ml). (F) T-bet mRNA levels in wild-type (black empty squares), and Ifngr1^−/− cells in the absence (green empty cicles) or presence of 10 ng/ml IL-12 (green filled circle) after induction with different concentrations of IL-27.

Fig. 5

Stochastic differentiation of Th1 cells. Lymphocytes from 129S6 mice were activated in the presence of indicated cytokines and 2 days later they were stained using the IFN-γ capture assay. Cells expressing low (off-cells) and high (on-cells) levels of IFN-γ were separated, and RNA was isolated from the sorted cells and the relative IL-12Rβ2 mRNA levels were measured. (A) 2 ng/ml IL-27 was used for induction of Th1 cell fate. The IL-12Rβ2 mRNA content of off and on-cells (77.5% and 13.2% of total population) was 121 and 393, respectively (B) 0.4 ng/ml IL-27 and 10 ng/ml IL-12 were used for induction of Th1 cell fate. The IL-12Rβ2 mRNA content of on and off-cells (84.5% and 11.7% of total population) was 49 and 455, respectively.