Cellular and Intracellular Network Characterization of Cytokine JAK/STAT Signaling In Whole Blood Across Multiple Healthy Individuals: Defining “Normal”

As aberrant JAK/STAT signaling in hematopoietic cells is associated with certain hematologic and immune diseases, understanding the regulation of JAK/STAT signaling is critical. Signaling pathway- and cell type-specific responses to various cytokines in immune system signaling networks can elicit a wide range of biological outcomes due to the combinatorial use of a limited set of kinases and STAT proteins. Although advances have been made in uncovering the intracellular mechanisms relating to cytokine signaling, the biological outcome may vary depending on composition and activation state of the cellular network. Single Cell Network Profiling (SCNP) by flow cytometry allows the interrogation of intracellular signaling networks within a heterogeneous cellular network, such as in unfractionated whole blood. We applied SCNP to investigate cytokine induced JAK/STAT signaling in whole blood across healthy human donors (n=11) to 1) measure the relative contribution of distinct cytokines to induce signaling across multiple cell subsets; 2) measure the kinetics of signaling activation across cytokines and cell subsets; 3) measure the variation among donors in their overall signaling characteristics. Our aim was to better characterize “normal” cytokine responses across healthy individuals as a basis to eventually describe abnormal states.

Whole blood from 11 healthy donors (20-65 yrs, 7 males, 4 females, 8 Caucasians, 2 Hispanics, 1 East Asian) was stimulated at 37°C in 96-well plates with a low, medium, and high dose of GM-CSF, IFN-α, IL-27 and IL-6, each added separately. For each dose, a stimulation time course was run with 6 time points between 3 and 45 minutes. Each well had a final concentration of 90% whole blood. SCNP assay was performed using a fluorophore-labeled antibody cocktail to simultaneously measure signaling in six distinct cell populations, including: neutrophils, CD20+ B cells, CD3+CD4+T cells, CD3+CD4- T cells (CD8 enriched), CD3-CD20- lymphocytes (NK cell enriched), and CD14+ monocytes. The median fluorescent intensity of phospho (p)-STAT1(Y701), p-STAT3(Y705), and p-STAT5(Y694) were measured in each defined cell population for each experimental condition.

This SCNP assay was relatively high-throughput and provided high-content data, that equates to 19,000 gel lanes if attempted by Western analysis (11 donors × 4 cytokines × 4 concentrations × 6 time points × 6 cell subsets × 3 p-readouts). In general, each cytokine demonstrated unique dose-dependent signaling characteristics (e.g. activation/termination kinetics, magnitude of response) for each cell type analyzed, and in some cases, the kinetics differed between p-STAT readouts within the same cell subset for the same cytokine. For instance, IL-6 induced signaling was only observed in CD4+ T cells and monocytes with peak p-STAT3 levels at 3 minutes followed by p-STAT1 and p-STAT5 at 10–15 minutes. In addition, signal resolution fell to baseline levels at 45 minutes in monocytes, while the CD4+ T cells showed sustained elevated signaling, suggesting a cell-type specific regulation. In contrast to IL-6, IFN-α stimulation activated all 3 STAT proteins, peaking at 10 minutes with similar kinetics in all cell subsets. However, termination of IFN-α signaling was faster and almost complete at 45 minutes in monocytes, while in the all other subsets the signal was sustained. This efficient signal termination in monocytes was also observed with GM-CSF→p-STAT5, while neutrophils maintained persistent p-STAT5 levels. IL-27 induced p-STAT1 and p-STAT3 in T cell subsets, B cells, and monocytes with peak activation at 30 minutes. In general, signaling characteristics were remarkably uniform across the healthy donors. IL-6→p-STAT3 was particularly consistent across time points and ligand concentrations, while p-STAT1 and p-STAT5 showed more variation.

Approaching cell signaling from the perspective of the cellular network under physiological conditions (whole blood) allows for a more comprehensive and clinically relevant view of the signaling state of complex tissues. As many small molecule compounds targeting JAK/STAT pathways enter the clinic, this study provides an important reference point for comparison of healthy signaling networks with networks altered either by pathological disease state or therapy.

Covey:Nodality Inc.: Employment, Equity Ownership. Gulrajani:Nodality Inc.: Employment, Equity Ownership. Marimpietri:Nodality Inc.: Employment, Equity Ownership. Chiang:Nodality Inc.: Employment, Equity Ownership. Panganiban-Lustan:Nodality Inc.: Employment, Equity Ownership. Cordeiro:Nodality Inc.: Employment, Equity Ownership. Longo:Nodality Inc.: Employment, Equity Ownership. Rosen:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership.