A CD25-CCR7 complex initiates non-canonical IL-2 signaling

IL-2, a central regulator of immune function, binds to its receptor subunit CD25 (IL-2Rα), promoting IL-2 interaction with β and γ subunits to trigger the canonical IL-2 signaling pathway. An anti-mouse CD25 antibody, PC61, triggers alternative IL-2 signaling, leading to integrin activation. PC61 induces a complex formed by the IL-2-dependent association of CD25 with CCR7, suggesting that the formation of this complex initiates alternative IL-2 signaling. Here, we used structure-based design together with combinatorial screening to identify an IL-2 mutant (denoted IL-2(E52K)) that spares canonical IL-2 signaling but disrupts both PC61-induced complex formation and integrin activation while retaining the full CD25 affinity of the parent molecule. We also report that heparan sulfate (HS), a physiological ligand of IL-2 that triggers alternative signaling, induced IL-2-dependent CD25-CCR7 association, whereas IL-2(E52K) failed to support both HS-induced CD25-CCR7 complex formation and integrin activation. Thus, both anti-CD25 antibody and HS require common features of IL-2 needed for CD25-CCR7 complex assembly and resulting integrin activation. Collectively, these data show that IL-2 promotes CD25 interaction with CCR7, thereby forming the signal initiating complex. Furthermore, canonical and alternative IL-2 signaling can be decoupled by an IL-2 mutation, creating a tool to specify the biological role of alternative IL-2 signaling in immune responses.