Ignacio Moraga

Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'

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. 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. 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.

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

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 (γc). Here we found that in the structure of the IL-15–IL-15Rα–IL-2Rβ–γc quaternary complex, IL-15 binds to IL-2Rβ and γc in a heterodimer nearly indistinguishable from that of the IL-2–IL-2Rα–IL-2Rβ–γc 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–γc 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.

Receptor density is key to the alpha2/beta interferon differential activities.

ABSTRACT Multiple type I interferons (IFN-α/β) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-α/β differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-β was more potent than IFN-α2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-α2 matched or even surpassed IFN-β in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.

Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines

Cytokines dimerize their receptors, with binding of the “second chain” triggering signaling. In the interleukin (IL)-4/13 system, different cell types express varying levels of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct Type-I or Type-II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A Type-I receptor-selective IL-4 ‘superkine’ with 3700-fold higher affinity for γc was 3-10 fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the Type-II receptor, and induced differentiation of dendritic cells from monocytes, implicating the Type-II receptor in this process. Superkines exhibited signaling advantages on cells with lower second chain levels. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain levels can be exploited to redirect cytokines towards distinct cell subsets and elicit novel actions, potentially improving the selectivity of cytokine therapy.

Insights into cytokine-receptor interactions from cytokine engineering.

Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. However, the desired immunotherapeutic effects of native cytokines are often mitigated by toxicity or lack of efficacy, either of which results from cytokine receptor pleiotropy and/or undesired activation of off-target cells. As our understanding of the structural principles of cytokine-receptor interactions has advanced, mechanism-based manipulation of cytokine signaling through protein engineering has become an increasingly feasible and powerful approach. Modified cytokines, both agonists and antagonists, have been engineered with narrowed target cell specificities, and they have also yielded important mechanistic insights into cytokine biology and signaling. Here we review the theory and practice of cytokine engineering and rationalize the mechanisms of several engineered cytokines in the context of structure. We discuss specific examples of how structure-based cytokine engineering has opened new opportunities for cytokines as drugs, with a focus on the immunotherapeutic cytokines interferon, interleukin-2, and interleukin-4.

Role of the Diacylglycerol Kinase α-Conserved Domains in Membrane Targeting in Intact T Cells

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKα is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKα domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr335. This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

The role of RPGR in cilia formation and actin stability

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) protein cause one of the most common and severe forms of inherited retinal dystrophy. In spite of numerous studies, the precise function of RPGR remains unclear, as is the mechanism by which RPGR mutations cause retinal degeneration. We have analysed the function of RPGR by RNA interference-mediated translational suppression [knockdown (KD)] using a model cellular system for studying the formation, maintenance and function of primary cilia (human telomerase-immortalized retinal pigmented epithelium 1 cells). We observed that RPGR-deficient cells exhibited reduced numbers of cilia, slower cell cycle progression and impaired attachment to fibronectin, but showed no migration defects in a wound-healing assay. RPGR KD cells showed stronger actin filaments, associated with basal dysregulation of the Akt, Erk1/2, focal adhesion kinase and Src signalling pathways, as well as a 20% reduction in β1-integrin receptors at the cell surface and impaired fibronectin-induced signalling. Stronger actin filaments and impairment of the above signalling pathways suggest a common underlying mechanism for all of the cellular phenotypes observed in RPGR KD cells. Our data underline a novel function for RPGR in cilia formation and in the regulation of actin stress filaments, suggesting that, in the retina, it may regulate nascent photoreceptor disc formation by regulating actin-mediated membrane extension.

Lck-dependent tyrosine phosphorylation of diacylglycerol kinase alpha regulates its membrane association in T cells.

TCR engagement triggers phospholipase Cγ1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca2+, both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase α (DGKα) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGKα to the membrane have not been fully elucidated, although Ca2+ influx and tyrosine kinase activation were proposed to be required. We show that DGKα is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGKα during TCR activation and shows that pY335DGKα is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGKα function and suggest a mechanism by which Lck-dependent phosphorylation and Ca2+ elevation regulate DGKα membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGKα relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.

Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency.

Mathematical modeling of the cellular responses to cytokine variants of differing binding affinities may help design better therapies. Modeling cytokine behavior The use of cytokines, such as interleukin-2 (IL-2) or IL-13, as therapies has been hampered by the fact that many cytokines share receptor subunits on different cell types. Moraga et al. generated recombinant variants of IL-13 with a wide range of binding affinities for the IL-13 receptor. Mathematical modeling of the correlation between the receptor binding affinities of the variants and the extents to which they differentially stimulated early and late cellular responses highlighted aspects of receptor-ligand binding properties that should aid in the development of more effective cytokine therapies. Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.

Multifarious Determinants of Cytokine Receptor Signaling Specificity

Cytokines play crucial roles in regulating immune homeostasis. Two important characteristics of most cytokines are pleiotropy, defined as the ability of one cytokine to exhibit diverse functionalities, and redundancy, defined as the ability of multiple cytokines to exert overlapping activities. Identifying the determinants for unique cellular responses to cytokines in the face of shared receptor usage, pleiotropy, and redundancy will be essential in order to harness the potential of cytokines as therapeutics. Here, we discuss the biophysical (ligand-receptor geometry and affinity) and cellular (receptor trafficking and intracellular abundance of signaling molecules) parameters that contribute to the specificity of cytokine bioactivities. Whereas the role of extracellular ternary complex geometry in cytokine-induced signaling is still not completely elucidated, cytokine-receptor affinity is known to impact signaling through modulation of the stability and kinetics of ternary complex formation. Receptor trafficking also plays an important and likely underappreciated role in the diversification of cytokine bioactivities but it has been challenging to experimentally probe trafficking effects. We also review recent efforts to quantify levels of intracellular signaling components, as second messenger abundance can affect cytokine-induced bioactivities both quantitatively and qualitatively. We conclude by discussing the application of protein engineering to develop therapeutically relevant cytokines with reduced pleiotropy and redirected biological functionalities.