(670g) Dynamics and Regulation of Il-12 Receptor Signaling

Engineering individualized immunotherapy is dependent on understanding the mechanistic basis for variations in disease susceptibility in outbred human populations. Inbred mouse strains provide model systems for quantifying functional differences among genetic variants of a species. The first objective of this study was to quantify dynamic differences in the Interleukin-12 receptor signaling (IL-12) pathway in two inbred mouse strains: C57Bl/6 and Balb/C. IL-12 is a key cytokine known to be involved in shaping cell-mediated immunity to infectious pathogens [1]. IL-12 initiates a cellular response via the IL-12 receptor, which is composed of two subunits: IL-12Rβ1 and IL-12Rβ2. The IL-12 signaling pathway is a member of the JAK/STAT family of signaling networks. Signaling via the JAK/STAT pathway culminates in the activation of STATs, a family of transcription factors that, upon activation, translocate to the nucleus and initiate protein synthesis. Suppressors of cytokine signaling (SOCS) proteins act as negative regulators of the JAK/STAT pathway. However, it has been shown that cellular context plays an important role in influencing the strength of signaling via particular reaction pathways [2]. The second objective of this study was to quantify how well the generalized JAK/STAT signaling mechanism represents the dynamics of STAT4 activation via the IL-12 pathway in naïve T helper cells, a cell critical for coordinating the adaptive immune response.

To quantify differences in IL-12 signaling, naïve CD4⁺ CD62L⁺ T-cells were isolated from murine Balb/C and C57Bl/6 splenocytes. The cells were activated for 44 hours using plate-bound anti-CD3 and anti-CD28 to provide nonspecific activation of the T cell receptor and co-stimulation, respectively. Activated naïve CD4⁺ T cells were subdivided into three treatment groups: those receiving no stimulation, cells treated with anti-IL-4 only, and cells stimulated with anti-IL-4 and IL-12. Untreated cells and cells treated with anti-IL-4 only served as controls. Frequent samples of the cell populations were taken and stained with markers for IL-12Rβ1, IL-12Rβ2, SOCS3, and pSTAT4. Dynamic changes in protein expression were measured using flow cytometry and analyzed using R/Bioconductor. A mathematical model of the JAK/STAT pathway was created to quantify the differences in IL-12 signaling between mouse strains. Separate models incorporating various regulation mechanisms were created to test the strength of proposed negative regulators of the IL-12 signaling pathway.

IL-12R expression is dynamically regulated in naïve CD4⁺ T-cells following activation by anti-CD3 and anti-CD28. Stimulation by IL-12 increased active STAT4 during the first 30 minutes and was followed by a steady decrease. Active STAT4 expression decreased to a greater extent in Balb/C cells compared to C57BI/6. The decrease in active STAT4 was correlated to a decrease in receptor number. Preliminary modeling results suggested that regulation of this pathway occurs via modulation of receptor expression rather than changes in activity of receptor. In summary, we have found dynamic differences between the two inbred mouse strains. These dynamic differences in IL-12 signaling appear to be regulated through different mechanisms than other members of the JAK/STAT signaling family. Reaction pathway analysis was helpful for discriminating between varying hypotheses regarding regulation of this particular pathway.

This work was supported in part by NIH RR016440 and NIH RR020866.

References

1. O'Garra, A., Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity, 1998. 8: p. 275-283.

2. Klinke, D.J., et al., Modulating temporal control of NF-kappaB activation: implications for therapeutic and assay selection. Biophysical Journal, 2008. In press.