Dissecting IL-2 Signaling Dynamics with an Irreversible JAK3 Inhibitor
The common γ-chain cytokines (IL-2, 4, 7, 9, 15 and 21) play an essential role in lymphocyte development and function. Their signals are transduced through receptor complexes comprised of the IL-2Rγ (γc) and one or two private subunits. These subunits lack intrinsic kinase activity and thus require the cytoplasmic kinases JAK1 and JAK3 for signal transduction. Cytokine binding induces a conformational change that activates JAK1 and JAK3, which in turn phosphorylate key tyrosines within one of the receptor subunits. These phosphorylated tyrosines recruit downstream signaling molecules, principally the STAT transcription factor family members. It is unknown what the precise non-redundant roles are for JAK1 and JAK3 in the signaling complex. Furthermore, although it is known that proliferation or differentiation requires hours to days of continuous cytokine exposure, how the signaling dynamics change over time has not been studied.
To address both the specific role of JAK3 and the requirements for JAK3 catalytic activity over time, we identified a potent and highly selective JAK3 inhibitor with in vivo activity. We took advantage of a cysteine found in JAK3 (Cys909 human/Cys905 mouse) but not in other JAK kinases to design selective inhibitors based on three different scaffolds, ultimately identifying JAK3i. By monitoring STAT5 phosphorylation over 20 hours in IL-2-stimulated CD4+ T cells, we document a previously unappreciated second wave of signaling that is much more sensitive to JAK3i than the first wave. Selective inhibition of this second wave is sufficient to block cyclin expression and S-phase entry.
Finally, we extended this study of IL-2 signaling dynamics to the closely related CD8+ T cell, which has an earlier and greater proliferative response to IL-2. When STAT5 phosphorylation was followed over time, CD8 T cells sustained this signaling throughout a 6-hour time course, in contrast to the biphasic CD4 response. A 50% knockdown of the IL-2Rβ receptor chain converted CD8 T-cells to a CD4-like signaling pattern and reduced early S-phase entry. This cell type variability in IL-2Rβ expression appears to tune responses, potentially preventing extensive, autoimmune expansion of CD4 T-cells while still enabling sufficient CD8 expansion to control viral infections.