Inferring Cross-Talk Among Interleukin-12, Interferon-γ and Tumor Necrosis Factor Signaling Pathways within T Helper Cells

Interleukin-12 (IL-12) is an important immune regulatory cytokine that exerts potent anti-tumor activity. Cellular response to IL-12 occurs via a member of the JAK/STAT family of signaling networks. The flow of information within the IL-12 signaling pathway is regulated by a series of putative positive and negative feedback loops that remain unclear. Prior work suggests that the IL-12 receptor is controlled via a positive feedback loop mediated directly through STAT4, the central transcription factor in the IL-12 pathway. In contrast, we have recently shown that expression of the IL-12 receptor in naïve T helper cells is controlled by an autonomous program rather than via direct feedback via STAT4 [1]. The objective of this study was to identify the biochemical cues that mediate this autonomous program. The 2D6 cell line, derived from a T helper cell clone, was used as a model system to study how collectively Interferon-γ, Tumor Necrosis Factor, and IL-12 cross-regulate these signaling pathways. In response to IL-12, dynamic changes in cell viability, cytokine production, and intracellular protein expression and activity were measured using flow cytometry and analyzed using R/Bioconductor. These data were used in conjunction with an empirical Bayesian model-based inference approach [2] to infer the relative strength of different regulatory mechanisms that result in signaling cross-talk within the system. Autocrine feedback loops that were inferred from the data were validated experimentally.

[1] Finley, S.D.; Gupta, D.; Cheng, N.; Klinke, D. J.; “Inferring relevant control mechanisms for Interleukin 12 signaling within naïve CD4+ T cells", (2010) Immunol Cell Biol (in press). [2] Klinke, D. J.; “An empirical Bayesian approach for model-based inference of cellular signaling networks", (2009) BMC Bioinformatics 10:371.