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Regulating Interleukin-2 activity with engineered receptor signaling clamps

ABSTRACT: Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates lymphocyte function by signaling through heterodimerization of the IL-2Rβ and γc receptor subunits. Previously, we engineered an IL-2 “superkine” (H9) with enhanced affinity for IL-2Rβ. Here, we describe next-generation IL-2 variants that function as “receptor signaling clamps.” They retain high-affinity for IL-2Rβ, thereby inhibiting binding of endogenous IL-2, but their engagement of γc is weakened, thereby attenuating IL-2Rβ-γc heterodimerization. These IL-2 analogues act as partial agonists and can differentially affect lymphocytes poised at distinct activation thresholds. Moreover, one of these variants potently antagonized IL-2 and IL-15 signaling and function better than blocking antibodies against IL-2Rα or IL-2Rβ. Furthermore, this mutein prolonged survival in a model of graft versus host disease and blocked spontaneous proliferation of smoldering adult T-cell leukemia (ATL) T cells ex vivo. This receptor-clamping approach may be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE64713 | GEO | 2015/06/16

SECONDARY ACCESSION(S): PRJNA271704

REPOSITORIES: GEO

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Regulating Interleukin-2 activity with engineered receptor signaling clamps

Project description:Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates lymphocyte function by signaling through heterodimerization of the IL-2Rβ and γc receptor subunits. Previously, we engineered an IL-2 “superkine” (H9) with enhanced affinity for IL-2Rβ. Here, we describe next-generation IL-2 variants that function as “receptor signaling clamps.” They retain high-affinity for IL-2Rβ, thereby inhibiting binding of endogenous IL-2, but their engagement of γc is weakened, thereby attenuating IL-2Rβ-γc heterodimerization. These IL-2 analogues act as partial agonists and can differentially affect lymphocytes poised at distinct activation thresholds. Moreover, one of these variants potently antagonized IL-2 and IL-15 signaling and function better than blocking antibodies against IL-2Rα or IL-2Rβ. Furthermore, this mutein prolonged survival in a model of graft versus host disease and blocked spontaneous proliferation of smoldering adult T-cell leukemia (ATL) T cells ex vivo. This receptor-clamping approach may be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation. Genome-wide transcription factors binding of STAT5 and mRNA-Sequencing of gene expression profiles in human pre-activated CD8+ T cells.

2015-06-16 | E-GEOD-64713 | biostudies-arrayexpress

Mechanistic and structural insight into the functional dichotomy between interleukin-2 and interleukin-15

Project description:Interleukin-15 (IL-15) and IL-2 possess distinct immunological functions despite both signaling through IL-2Rβ and the common cytokine receptor γ-chain, γc, We find that in the IL-15/IL-15Rα/IL-2Rβ/γc quaternary complex structure, IL-15 heterodimerizes IL-2Rβ and γc identically to the IL-2/IL-2Rα/IL-2Rβ/γc complex, despite differing receptor-binding chemistries. IL-15Rα dramatically increases the affinity of IL-15 for IL-2Rβ, and this allostery is required for IL-15 trans-signaling versus IL-2 cis-signaling. Consistent with the identical IL-2Rβ/γc dimer geometry, IL-2 and IL-15 exhibited similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induce similar signals, and the cytokine-specificity of IL-2Rα versus IL-15Rα determines cellular responsiveness. These results provide important new insights for specific development of IL-15- versus IL-2-based immunotherapeutics. RNA-Seq is conducted in mouse CD8+ T cells, not treated or treated with IL2 or IL15 for indicated concentrations (1nM or 500nM) and times (4hr or 24hr).

2012-11-01 | E-GEOD-40350 | biostudies-arrayexpress

Mechanistic and structural insight into the functional dichotomy between interleukin-2 and interleukin-15

2012-11-01 | GSE40350 | GEO

Facile Diversification of Interleukin-2 Pharmacology with Surrogate Agonists

Project description:Cytokines, such as interleukin-2, initiate signaling by dimerizing their receptors into orientations and proximities that induce intracellular signaling. We wished to explore a new pharmacological strategy for cytokine discovery based on variation of receptor dimer geometries using surrogate ligands, since natural cytokines are structurally limited as engineering scaffolds. Here, we report a structurally agonistic approach based on a modular, ‘plug and play’ class of ligands, that is amenable to high-throughout screening. We isolated nanobodies (Nb) and scFvs against human IL-2Rβ and γc, and generated a 40-member combinatorial matrix of tandem single-chain bispecific (IL-2Rβ:γc) molecules. We identified 28 surrogate IL-2 ligands exhibiting a wide range of agonist strengths and biased signaling properties, including cell-type bias for NK expansion and cytotoxity relative to T cells. Crystal structures of select Nb:receptor complexes validated that alternative receptor dimer geometries underly the functional diversification. This “cytokine med-chem” approach is generalizable to many dimeric cell surface receptor systems.

2022-04-27 | GSE183436 | GEO

Selective activation and expansion of regulatory T cells using lipid encapsulated mRNA encoding a long-acting IL-2 mutein

Project description:Interleukin-2 (IL-2) is critical for regulatory T cell (Treg) function and homeostasis. At low doses, IL-2 can suppress immune pathologies by expanding Tregs that constitutively express the high affinity IL-2Rα subunit. However, even low doses IL-2, signaling through the IL2-Rβ/γ complex, may lead to the activation of proinflammatory, non-Treg T cells, so improving specificity toward Tregs may be desirable. Here we used messenger RNAs (mRNA) to encode a half-life-extended human IL-2 mutein (HSA-IL2m) with mutations promoting reliance on IL-2Rα. Our data show that IL-2 mutein subcutaneous delivery in lipid-encapsulated mRNA nanoparticles selectively activates and expands Tregs in mice and non-human primates, as well as reduces disease severity in mouse models of acute graft versus host disease and experimental autoimmune encephalomyelitis. Single cell RNA sequencing of murine splenic CD4+ T cells identifies multiple Treg states with distinct response dynamics following IL-2 mutein treatment. Our results thus demonstrate the potential of mRNA-encoded HSA-IL2m immunotherapy to treat autoimmune diseases.

2022-04-07 | GSE200142 | GEO

Characterization of receptor-associated protein complex assembly in Interleukin (IL)-2- and IL-15-activated T-lymphocytes

Project description:Despite extensive investigation, it remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. In our previous work, we dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells unveiling subtle differences that may contribute to their functional dichotomy. In the present study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is fine tune orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents such as FAM59A and SOCS2. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and –independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2Rβ. We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Whereas IL-2Rβ pS431 binds to clathrins, which are involved in the receptor internalization and subsequent signal attenuation, IL-2Rγ pY325 and pY357 attract phosphatases, adaptor proteins, kinases as well as the newly identified member of the interleukin receptor complex SOCS2. Overall our study sheds new light into the initial molecular mechanisms triggered by IL-2 and IL-15 and constitutes a further step towards a better understanding of the early signaling aspects of the two closely-related cytokines in T-lymphocytes.

2016-08-08 | PXD002386 | Pride

IL-2Rβ- and CD103-dependent genes in regulatory T cells in the gut mucosa

Project description:This study determined the genes that are differentially expressed when regulatory T cells (Tregs) were isolated from the lamina propria of the small and large intestine from mice with impaired IL-2Rβ signaling (designated Y3) or impaired IL-2Rβ signaling and lack of CD103 expression (designated Y3/CD103-/-) when compared to Tregs from WT mice. 204 unique annotated mRNAs were differentially expressed by ≥1.5 fold between these 3 groups (Fig. 6B). Very few mRNAs were uniquely up or down regulated in relationship to impaired IL-2Rβ signaling or the combination of impaired IL-2Rβ signaling and lack of CD103 expression. Thus, lack of CD103 does not obviously regulated signaling in Tregs in the gut mucosa and most differentially expressed genes were due to impaired IL_2Rβ signaling. Gene enrichment analysis of these differentially expressed genes identified 4 major enrichment groups (EG) are: EG1, Cytokine-cytokine receptor interaction and the JAK-STAT signaling pathway; EG2, regulation of lymphocyte activation and proliferation; EG3, regulation of cell death and the caspase pathway in apoptosis; and EG4, transcription.

2015-01-06 | GSE56438 | GEO

Blocking common gamma chain cytokine signaling ameliorates T-cell-mediated pathogenesis in disease models

Project description:The common γ chain (γc; IL2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL2, IL4, IL7, IL9, IL15, and IL21. Because of the lack of appropriate neutralizing antibodies recognizing IL2RG, it has been difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. To determine whether γc cytokines might be targeted for T-cell-mediated disease prevention and treatment, we generated a new γc cytokine receptor antibody, REGN7257. Biochemical, structural and in vitro analysis showed that REGN7257 binds with high affinity to IL2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T-cells without impacting granulocytes, platelets or red blood cells. Using REGN7257, we showed that γc cytokines drive T-cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis, by impacting multiple aspects of the pathogenic response. Importantly, we discovered that our xenogeneic GVHD mouse model recapitulates hallmarks of both acute and chronic GVHD, with T-cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. And we showed that γc cytokines contribute to disease pathology by driving all of these features. Overall, by demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T-cell responses, offering a potentially novel strategy for the management of T-cell-mediated diseases.

2022-10-04 | GSE214625 | GEO

IL-2Rβ- and CD103-dependent genes in regulatory T cells in the gut mucosa

2015-01-06 | E-GEOD-56438 | biostudies-arrayexpress

An IL-2 mutein increases IL-10 and CTLA-4-dependent suppression of dendritic cells by regulatory T cells [10x scRNA-seq]

Project description:Interleukin-2 (IL-2) variants with increased CD25 dependence that selectively expand Foxp3+ regulatory T (TR) cells are under clinical investigation for treating autoimmune and inflammatory diseases. We previously described an Fc-fused CD25-biased IL-2 mutein (Fc.IL-2 mutein) that promotes TR expansion and prevents the onset of autoimmune diabetes in non-obese diabetic (NOD) mice; however, the effects of this treatment on TR function in vivo are unclear. Here we show that Fc.IL-2 mutein treatment induces an activated Foxp3+ TR population characterized by elevated proliferation, a unique transcriptional program associated with Stat5- and TCR-dependent gene modules, and high levels of the immunosuppressive molecules IL-10 and CTLA-4. Increased IL-10 receptor signaling limits the upregulation of surface MHC class II expression during conventional dendritic cell (cDC) maturation, while increased CTLA-4-dependent transendocytosis leads to the transfer of CD80 and CD86 costimulatory ligands from maturing cDCs to Tregs. In NOD mice, Fc.IL-2 mutein treatment resulted in TR-mediated regulation of cDCs in the inflamed pancreas and draining lymph nodes accompanied by an increase in anergic T cells. Thus, we demonstrate that IL-2 mutein-expanded TR cells have enhanced functional properties and potently act to restrict cDC function in the target tissue and at the site of autoimmune induction, offering promising prospects for targeted immunotherapy.

2023-12-08 | GSE249481 | GEO

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