Since the initial discovery of IL-15 almost 20 years ago, numerous mechanisms have been offered to explain how IL-2 and IL-15 can produce divergent functional effects despite sharing common signaling receptors. In this work, we report the x-ray crystal structure of the quaternary complex of IL-15 bound to the ectodomains of IL-15Rα, IL-2Rβ, and γc and find the complex to have an approximately identical heterodimeric IL-2Rβ—γc architecture to that of the IL-2—IL-2R quaternary complex. The lack of significant deviations in dimer topology between the signaling complexes of IL-2 and IL-15 suggest that any functional differences between the two cytokines are unlikely to arise from instructive extracellular structural influences. However, differences in the cytokine interaction affinities and kinetics of the respective cytokine associations with the IL-2Rβ and γc extracellular domains could result in overall complex stability differences that would be manifested as distinct signaling outcomes. Thus, using phospho-flow cytometry, we compared IL-2 and IL-15 mediated signaling over a broad range of cytokine concentrations and kinetic intervals, finding that many of the apparent signaling differences between IL-2 and IL-15 may be explained by differences in receptor affinity between the two cytokines. Similarly, we found the gene expression profiles of cells stimulated with IL-2 and IL-15 to be highly correlated and that differences in gene expression were generally diminished at saturating concentrations of the cytokines. When differences persisted at saturation, they remained modest, bringing into question their true biological relevance. While our results do not rule out the possibility of additional mechanisms of IL-15 action, they indicate these mechanisms are not necessary to explain the complex and diverse functional behaviors of IL-15 and IL-2 observed in vivo. Rather, we find that alpha-receptor expression and cytokine concentration drastically affect the signaling behavior of IL-2 and IL-15, producing differences in gene expression when the cytokines lie at different points on their respective concentration-response curves. Presumably, the disparate spatial and temporal expression of the alpha receptors, as well as their absolute expression level, dynamically regulates the sensitivity of cells for each respective cytokine and their ensuing response to stimulation.
Underscoring the importance of their respective alpha receptors in their functions, a striking difference between IL-2 and IL-15 can be seen in the manner in which the cytokines are presented to effector cells. Since IL-15 binds to IL-15Rα with an extremely high affinity and IL-15Rα is widely expressed in tissues, IL-15 is believed to mainly exist in the body as a complex with IL-15Rα, and is therefore primed for trans-presentation to cells expressing IL-2Rβ and γc6
. As previously mentioned, soluble complexes of IL-15—IL-15Rα mimicking trans-presentation show enhanced potency compared to free IL-1520–22
. Through our studies, we elucidated the mechanism underlying this phenomenon, finding that binding of IL-15Rα increases the affinity of IL-15 for IL-2Rβ approximately 150-fold. This affinity increase for IL-2Rβ subsequently manifests as a left-shift in the concentration-response relationship of IL-15 signaling in cells lacking IL-15Rα. The structural basis for the affinity enhancement of IL-15 for IL-2Rβ appears to be a consequence of a global stabilization of IL-15 upon binding IL-15Rα to a much greater degree than seen for IL-2 upon binding IL-2Rα23,24
. From a teleological perspective, the affinity enhancement endowed by IL-15Rα onto the IL-15—IL-2Rβ interaction may serve to compensate for the lack of surface-capture in the setting of trans-presentation.
IL-2 is administered clinically as immunotherapy in the treatment of renal cell carcinoma and metastatic melanoma. However, IL-2 therapy is hampered by dose-limiting toxicity from vascular leakage and the counter-productive activation of regulatory T cells (Treg) that abrogate anti-tumor responses. Both of these undesirable side-effects are attributable to the activation of cells that express IL-2Rα: pulmonary vascular endothelial cells and IL-2Rα+
. Recently, we demonstrated that IL-2 variants that bind to IL-2Rβ with high affinity independently of IL-2Rα (“Super-2” or “H9”) produce greater anti-tumor efficacy and decreased pulmonary edema compared to wild-type IL-224
. Compared to wild-type IL-2, Super-2 more efficiently activates anti-tumor responses from IL-2Rα−
cells such as naive T cells and NK cells, with proportionally less activation IL-2Rα+
cells such as Treg and pulmonary endothelial cells. In recent years, the potential use of IL-15 for the treatment of cancer has garnished considerable enthusiasm and it is presently undergoing evaluation in phase I clinical trials (ClinicalTrials.gov identifier NCT01021059). Notably, IL-15 does not produce vascular leak syndrome or stimulate Treg, but preferentially activates cytotoxic T lymphocytes and NK cells thought to mediate anti-tumor effects3
, in many ways similar to Super-2. Similarly, a single-chain fusion protein of IL-15 and IL-15Rα (RLI) has been proposed as a potential anti-tumor agent, with enhanced potency and bioavailability compared to free IL-1535
In comparing the therapeutic anti-tumor potential of IL-2, IL-15, and their respective variants, Super-2 and IL-15—IL-15α complexes, it is important to consider the degree of alpha-receptor dependence inherent to each molecule. While IL-2 and IL-15 represent the extreme ends of the spectrum, showing great dependence on their alpha-receptors for potency, Super-2 and the single-chain IL-15—IL-15Rα fusion RLI appear to lie in-between the two wild-type cytokines, showing little to no preference for cells expressing IL-2Rα or IL-15Rα. Super-2 and RLI can be further distinguished by their interactions with IL-2Rα and IL-15Rα. Since the IL-15Rα binding site is sterically-obscured in RLI, it represents the exact midpoint between IL-2 and IL-15 on the spectrum, unaffected by the presence or absence of either alpha-receptor. By contrast, Super-2 is capable of binding IL-2Rα and subsequently shows some preference for IL-2Rα+ cells over IL-2Rα− cells, albeit at a much-decreased degree than wild-type IL-2. This subtle distinction may yield important differences in efficacy and toxicity. For example, though IL-2Rα is responsible for many of IL-2’s undesirable side effects, some IL-2Rα+ cells may be beneficial to target, such as activated T cells. Similarly, IL-15Rα+ cells such as NK and cytotoxic CD8 cells are critical determinants of anti-tumor efficacy in vivo.
In light of these considerations, it may be possible to enhance IL-2 and/or IL-15 immunotherapy by modulating their dependencies on IL-2Rα and IL-15Rα, respectively, thus “tuning” the distribution of immune cells activated for therapeutic effect. To this end, Super-2 and RLI represent good starting points for such immunological manipulation. Just as the structure of the IL-2 quaternary complex enabled the engineering of Super-2, we hope to leverage the information from the IL-15 quaternary complex presented here for the design of improved IL-15 therapies.