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Although IL-2 has been studied for its immune-stimulating activity against metastatic cancer, its side effects have limited its clinical use; here, an engineered IL-2 ‘superkine’ is shown to have increased activity, particularly in inducing antitumour T cells, but fewer side effects. Engineering an interleukin-2 'superkine' Chris Garcia and colleagues elucidate the molecular mechanism that underlies the sensitization of T cells to the immunostimulatory cytokine interleukin-2 (IL-2). They use this information to engineer a single-chain IL-2 superkine that functions independent of its α-receptor (IL-2Rα or CD25). This new superkine is more efficacious than IL-2 in inducing antitumour T-cell responses and has fewer toxic side effects. The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells 1 , 2 , 3 . Considerable effort has been invested in using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary ‘high affinity’ receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ and IL-2Rγ 4 , 5 , 6 , 7 , 8 . Naive T cells express only a low density of IL-2Rβ and IL-2Rγ, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ and IL-2Rγ. Here, using in vitro evolution, we eliminated the functional requirement of IL-2 for CD25 expression by engineering an IL-2 ‘superkine’ (also called super-2) with increased binding affinity for IL-2Rβ. Crystal structures of the IL-2 superkine in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, reducing the flexibility of a helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in the IL-2 superkine recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation of T cells irrespective of CD25 expression. Compared to IL-2, the IL-2 superkine induced superior expansion of cytotoxic T cells, leading to improved antitumour responses in vivo , and elicited proportionally less expansion of T regulatory cells and reduced pulmonary oedema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαβγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαβγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαβγ to a greater extent than for IL2Rβγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rβγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action.

While IL-2 can potently activate both NK and T cells, its short in vivo half-life, severe toxicity, and propensity to amplify Treg cells are major barriers that prevent IL-2 from being widely used for cancer therapy. In this study, we construct a recombinant IL-2 immunocytokine comprising a tumor-targeting antibody (Ab) and a super mutant IL-2 (sumIL-2) with decreased CD25 binding and increased CD122 binding. The Ab-sumIL2 significantly enhances antitumor activity through tumor targeting and specific binding to cytotoxic T lymphocytes (CTLs). We also observe that pre-existing CTLs within the tumor are sufficient and essential for sumIL-2 therapy. This next-generation IL-2 can also overcome targeted therapy-associated resistance. In addition, preoperative sumIL-2 treatment extends survival much longer than standard adjuvant therapy. Finally, Ab-sumIL2 overcomes resistance to immune checkpoint blockade through concurrent immunotherapies. Therefore, this next-generation IL-2 reduces toxicity while increasing TILs that potentiate combined cancer therapies. Interleukin-2 (IL-2) based cancer therapy is limited by severe toxicity and strong Treg amplification at the therapeutic dosage. Here, the authors develop a recombinant IL-2 immunocytokine which is comprised of a tumor-targeting antibody fused to a super mutant IL-2 and show in mouse models that this next-generation IL2 has reduced toxicity and enhanced antitumor activity.

The differentiation of T follicular helper cells requires the G-protein-coupled receptor Ebi2 as well as the interaction with CD25-producing dendritic cells that quench T-cell-derived interleukin-2. EBI2 required for Tfh cell maturation The T-cell subset known as T follicular helper (Tfh) cells are crucial for mounting antibody responses, but the cues that guide their positioning and development are incompletely understood. Here Jianhua Li et al . demonstrate that the differentiation of Tfh cells requires the G-protein-coupled receptor EBI2 (also called GPR183), as well as the interaction with CD25-producing dendritic cells that quench T-cell-derived interleukin-2. T follicular helper (Tfh) cells are a subset of T cells carrying the CD4 antigen; they are important in supporting plasma cell and germinal centre responses 1 , 2 . The initial induction of Tfh cell properties occurs within the first few days after activation by antigen recognition on dendritic cells, although how dendritic cells promote this cell-fate decision is not fully understood 1 , 2 . Moreover, although Tfh cells are uniquely defined by expression of the follicle-homing receptor CXCR5 (refs 1 , 2 ), the guidance receptor promoting the earlier localization of activated T cells at the interface of the B-cell follicle and T zone has been unclear 3 , 4 , 5 . Here we show that the G-protein-coupled receptor EBI2 (GPR183) and its ligand 7α,25-dihydroxycholesterol mediate positioning of activated CD4 T cells at the interface of the follicle and T zone. In this location they interact with activated dendritic cells and are exposed to Tfh-cell-promoting inducible co-stimulator (ICOS) ligand. Interleukin-2 (IL-2) is a cytokine that has multiple influences on T-cell fate, including negative regulation of Tfh cell differentiation 6 , 7 , 8 , 9 , 10 . We demonstrate that activated dendritic cells in the outer T zone further augment Tfh cell differentiation by producing membrane and soluble forms of CD25, the IL-2 receptor α-chain, and quenching T-cell-derived IL-2. Mice lacking EBI2 in T cells or CD25 in dendritic cells have reduced Tfh cells and mount defective T-cell-dependent plasma cell and germinal centre responses. These findings demonstrate that distinct niches within the lymphoid organ T zone support distinct cell fate decisions, and they establish a function for dendritic-cell-derived CD25 in controlling IL-2 availability and T-cell differentiation.

Humoral immunity is necessary for controlling viral infection. Ballesteros-Tato and colleagues show that development of follicular regulatory T cells is prevented by high concentrations of interleukin 2 at the peak of viral infection, but resumes at later time points to suppress autoantibody production. Interleukin 2 (IL-2) promotes Foxp3 + regulatory T (T reg ) cell responses, but inhibits T follicular helper (T FH ) cell development. However, it is not clear how IL-2 affects T follicular regulatory (T FR ) cells, a cell type with properties of both T reg and T FH cells. Using an influenza infection model, we found that high IL-2 concentrations at the peak of the infection prevented T FR cell development by a Blimp-1-dependent mechanism. However, once the immune response resolved, some T reg cells downregulated CD25, upregulated Bcl-6 and differentiated into T FR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional T reg cells, IL-2 inhibits T FR cell responses.

IL-2 immunotherapy is an attractive treatment option for certain metastatic cancers. However, administration of IL-2 to patients can lead, by ill-defined mechanisms, to toxic adverse effects including severe pulmonary edema. Here, we show that IL-2-induced pulmonary edema is caused by direct interaction of IL-2 with functional IL-2 receptors (IL-2R) on lung endothelial cells in vivo. Treatment of mice with high-dose IL-2 led to efficient expansion of effector immune cells expressing high levels of IL-2Rβγ, including CD8⁺ T cells and natural killer cells, which resulted in a considerable antitumor response against s.c. and pulmonary B16 melanoma nodules. However, high-dose IL-2 treatment also affected immune cell lineage marker-negative CD31⁺ pulmonary endothelial cells via binding to functional αβγ IL-2Rs, expressed at low to intermediate levels on these cells, thus causing pulmonary edema. Notably, IL-2-mediated pulmonary edema was abrogated by a blocking antibody to IL-2Rα (CD25), genetic disruption of CD25, or the use of IL-2Rβγ—directed IL-2/anti-IL-2 antibody complexes, thereby interfering with IL-2 binding to IL-2Rαβγ⁺ pulmonary endothelial cells. Moreover, IL-2/anti-IL-2 antibody complexes led to vigorous activation of IL-2Rβγ⁺ effector immune cells, which generated a dramatic antitumor response. Thus, IL-2/anti-IL-2 antibody complexes might improve current strategies of IL-2-based tumor immunotherapy.

Regulatory T (Treg) cells suppress abnormal/excessive immune responses to self‐ and nonself‐antigens to maintain immune homeostasis. In tumor immunity, Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. There are several Treg cell immune suppressive mechanisms: inhibition of costimulatory signals by CD80 and CD86 expressed by dendritic cells through cytotoxic T‐lymphocyte antigen‐4, interleukin (IL)‐2 consumption by high‐affinity IL‐2 receptors with high CD25 (IL‐2 receptor α‐chain) expression, secretion of inhibitory cytokines, metabolic modulation of tryptophan and adenosine, and direct killing of effector T cells. Infiltration of Treg cells into the tumor microenvironment (TME) occurs in multiple murine and human tumors. Regulatory T cells are chemoattracted to the TME by chemokine gradients such as CCR4‐CCL17/22, CCR8‐CCL1, CCR10‐CCL28, and CXCR3‐CCL9/10/11. Regulatory T cells are then activated and inhibit antitumor immune responses. A high infiltration by Treg cells is associated with poor survival in various types of cancer. Therefore, strategies to deplete Treg cells and control of Treg cell functions to increase antitumor immune responses are urgently required in the cancer immunotherapy field. Various molecules that are highly expressed by Treg cells, such as immune checkpoint molecules, chemokine receptors, and metabolites, have been targeted by Abs or small molecules, but additional strategies are needed to fine‐tune and optimize for augmenting antitumor effects restricted in the TME while avoiding systemic autoimmunity. Here, we provide a brief synopsis of these cells in cancer and how they can be controlled to achieve therapeutic outcomes. Regulatory T cells suppress immune functions through various mechanisms such as cytotoxic T‐lymphocyte antigen‐4‐mediated suppression of antigen‐presenting cell function, consumption of interleukin‐2, production of immunosuppressive cytokines, and production of immune suppressive metabolites.

IL-2 is crucial to T cell homeostasis, especially of CD4⁺ T regulatory cells and memory CD8⁺ cells, as evidenced by vigorous proliferation of these cells in vivo following injections of superagonist IL-2/anti-IL-2 antibody complexes. The mechanism of IL-2/anti-IL-2 antibody complexes is unknown owing to a lack of understanding of IL-2 homeostasis. We show that IL-2 receptor α (CD25) plays a crucial role in IL-2 homeostasis. Thus, prolongation of IL-2 half-life and blocking of CD25 using antibodies or CD25-deficient mice led in combination, but not alone, to vigorous IL-2-mediated T cell proliferation, similar to IL-2/anti-IL-2 antibody complexes. These data suggest an unpredicted role for CD25 in IL-2 homeostasis.

T helper (Th) cell differentiation is fundamental to functional adaptive immunity. Different subsets of dendritic cells (DC) preferentially induce different types of Th cells, but the DC-derived mechanism for Th type 2 (Th2) differentiation is not fully understood. Here, we show that in mice, CD301b + DCs, a major Th2-inducing DC subset, drive Th2 differentiation through cognate interaction by rapidly inducing IL-2 receptor signalling in CD4 + T cells. Mechanistically, CD40 engagement prompts IL-2 production selectively from CD301b + DCs to maximize CD25 expression in CD4 + T cells, which instructs the Th2 fate decision, while simultaneously skewing CD4 + T cells away from the T follicular helper fate. Moreover, CD301b + DCs utilize their own CD25 to facilitate directed action of IL-2 toward cognate CD4 + T cells, as genetic deletion of CD25 in CD301b + DCs results in reduced IL-2-mediated signalling in antigen-specific CD4 + T cells and hence their Th2 differentiation. These results highlight the critical role of DC-intrinsic CD40–IL-2 axis in Th cell fate decision. A subset of migratory type 2 conventional dendritic cells marked by CD301b expression (CD301b + DC), are required specifically for the development of Th2 cells. Here authors show that at the mechanistic level, the CD301b + DCs are triggered by CD40 signalling to produce IL-2, which in turn promotes Th2 cell differentiation while suppressing the Tfh cell fate decision.

The receptors for IL-2 and IL-15 share many characteristics, but mice deficient in either receptor have very different phenotypes. Garcia and colleagues present the quaternary structure of the complex of IL-15 and its receptor, as well as insights into its unique signaling properties. Interleukin 15 (IL-15) and IL-2 have distinct immunological functions even though both signal through the receptor subunit IL-2Rβ and the common γ-chain (γ c ). Here we found that in the structure of the IL-15–IL-15Rα–IL-2Rβ–γ c quaternary complex, IL-15 binds to IL-2Rβ and γ c in a heterodimer nearly indistinguishable from that of the IL-2–IL-2Rα–IL-2Rβ–γ c complex, despite their different receptor-binding chemistries. IL-15Rα substantially increased the affinity of IL-15 for IL-2Rβ, and this allostery was required for IL-15 trans signaling. Consistent with their identical IL-2Rβ–γ c dimer geometries, IL-2 and IL-15 showed similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induced similar signals, and the cytokine specificity of IL-2Rα versus IL-15Rα determined cellular responsiveness. Our results provide new insights for the development of specific immunotherapeutics based on IL-15 or IL-2.