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Combination treatments of s.c. 4T1 tumors with Clec9A-mAFN do not cause toxicity.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I IFN has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1,000-fold more potent on target cells, allowing specific signaling in selected cell types only. Type I IFN-derived AcTaferon (AFN)-targeting Clec9A+ dendritic cells (DC) displayed strong antitumor activity in murine melanoma, breast carcinoma, and lymphoma models and against human lymphoma in humanized mice without any detectable toxic side effects. Combined with immune checkpoint blockade, chemotherapy, or low-dose TNF, complete tumor regression and long-lasting tumor immunity were observed, still without adverse effects. Our findings indicate that DC-targeted AFNs provide a novel class of highly efficient, safe, and broad-spectrum off-the-shelf cancer immunotherapeutics with no need for a tumor marker.Significance: Targeted type I interferon elicits powerful antitumor efficacy, similar to wild-type IFN, but without any toxic side effects. Cancer Res; 78(2); 463–74. ©2017 AACR.

Type I Interferon (IFN) was the very first drug approved for the treatment of Multiple Sclerosis (MS), and is still frequently used as a first line therapy. However, systemic IFN also causes considerable side effects, affecting therapy adherence and dose escalation. In addition, the mechanism of action of IFN in MS is multifactorial and still not completely understood. Using AcTaferons (Activity-on-Target IFNs, AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting, we have previously demonstrated that specific targeting of IFN activity to dendritic cells (DCs) can protect against experimental autoimmune encephalitis (EAE), inducing in vivo tolerogenic protective effects, evidenced by increased indoleamine-2,3-dioxygenase (IDO) and transforming growth factor β (TGFβ) release by plasmacytoid (p) DCs and improved immunosuppressive capacity of regulatory T and B cells. We here report that targeting type I IFN activity specifically towards B cells also provides strong protection against EAE, and that targeting pDCs using SiglecH-AFN can significantly add to this protective effect. The superior protection achieved by simultaneous targeting of both B lymphocytes and pDCs correlated with improved IL-10 responses in B cells and conventional cDCs, and with a previously unseen very robust IDO response in several cells, including all B and T lymphocytes, cDC1 and cDC2.

International audience; Globally, autosomal recessive IFNAR1 deficiency is a rare inborn error of immunity underlying susceptibility to live attenuated vaccine and wild-type viruses. We report seven children from five unrelated kindreds of western Polynesian ancestry who suffered from severe viral diseases. All the patients are homozygous for the same nonsense IFNAR1 variant (p.Glu386*). This allele encodes a truncated protein that is absent from the cell surface and is loss-of-function. The fibroblasts of the patients do not respond to type I IFNs (IFN-α2, IFN-ω, or IFN-β). Remarkably, this IFNAR1 variant has a minor allele frequency >1% in Samoa and is also observed in the Cook, Society, Marquesas, and Austral islands, as well as Fiji, whereas it is extremely rare or absent in the other populations tested, including those of the Pacific region. Inherited IFNAR1 deficiency should be considered in individuals of Polynesian ancestry with severe viral illnesses.

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An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I IFN has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1,000-fold more potent on target cells, allowing specific signaling in selected cell types only. Type I IFN-derived AcTaferon (AFN)-targeting Clec9A+ dendritic cells (DC) displayed strong antitumor activity in murine melanoma, breast carcinoma, and lymphoma models and against human lymphoma in humanized mice without any detectable toxic side effects. Combined with immune checkpoint blockade, chemotherapy, or low-dose TNF, complete tumor regression and long-lasting tumor immunity were observed, still without adverse effects. Our findings indicate that DC-targeted AFNs provide a novel class of highly efficient, safe, and broad-spectrum off-the-shelf cancer immunotherapeutics with no need for a tumor marker. Significance: Targeted type I interferon elicits powerful antitumor efficacy, similar to wild-type IFN, but without any toxic side effects. Cancer Res; 78(2); 463–74. ©2017 AACR.

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity‐on‐Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF‐based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T‐cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8‐targeted type I interferon AcTakine. Co‐treatment with a CD13‐targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible., TNF and IFN‐γ are cytokines with great anticancer potential but with limited clinical application due to side‐effects. In the present study, the tumor endothelium is identified as target cell for their antitumor activity and new biologics that allow their safe targeting are developed.

International audience; Type I Interferon (IFN) is widely used for multiple sclerosis (MS) treatment, but its side effects are limiting and its mechanism of action still unknown. Furthermore, 30-50% of MS patients are unresponsive, and IFN can even induce relapses. Fundamental understanding of the cellular target(s) of IFN will help to optimize treatments by reducing side effects and separating beneficial from detrimental effects. To improve clinical systemic IFN usage, we are developing AcTaferons (Activity-on-Target IFNs = AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting. In experimental autoimmune encephalitis (EAE) in mice, high dose WT mIFNα could delay disease, but caused mortality and severe hematological deficits. In contrast, AFN targeted to dendritic cells (DC, via Clec9A) protected without mortality or hematological consequences. Conversely, CD8-targeted AFN did not protect and exacerbated weight loss, indicating the presence of both protective and unfavorable IFN effects in EAE. Comparing Clec9A-, XCR1-and SiglecH-targeting, we found that targeting AFN to plasmacytoid (p) and conventional (c) DC is superior and non-toxic compared to WT mIFN. DC-targeted AFN increased pDC numbers and their tolerogenic potential, evidenced by increased TGFβ and IDO synthesis and regulatory T cell induction. In addition, both regulatory T and B cells produced significantly more immunosuppressive TGFβ and IL-10. In conclusion, specific DC-targeting of IFN activity induces a robust in vivo tolerization, efficiently protecting against EAE, without noticeable side effects. Thus, dissecting positive and negative IFN effects via cell-specific targeting may result in better and safer MS therapy and response rates.

Sensing and reciprocating cellular systems (SARs) are important for the operation of many biological systems. Production in interferon (IFN) SARs is achieved through activation of the Jak-Stat pathway, and downstream upregulation of IFN regulatory factor (IRF)-3 and IFN transcription, but the role that high and low affinity IFNs play in this process remains unclear. We present a comparative between a minimal spatio-temporal partial differential equation (PDE) model and a novel spatio-structural-temporal (SST) model for the consideration of receptor, binding, and metabolic aspects of SAR behaviour. Using the SST framework, we simulate single- and multi-cluster paradigms of IFN communication. Simulations reveal a cyclic process between the binding of IFN to the receptor, and the consequent increase in metabolism, decreasing the propensity for binding due to the internal feed-back mechanism. One observes the effect of heterogeneity between cellular clusters, allowing them to individualise and increase local production, and within clusters, where we observe `sub popular quiescence'; a process whereby intra-cluster subpopulations reduce their binding and metabolism such that other such subpopulations may augment their production. Finally, we observe the ability for low affinity IFN to communicate a long range signal, where high affinity cannot, and the breakdown of this relationship through the introduction of cell motility. Biological systems may utilise cell motility where environments are unrestrictive and may use fixed system, with low affinity communication, where a localised response is desirable., Comment: 34 pages

Type I IFNs (interferons) are pathogen-induced immunoregulatory cytokines that exert anti-viral and anti-proliferative activities through binding to a common cell-surface receptor. Among the 17 human IFN subtypes, IFNβ binds the IFNAR (IFNα receptor) 1/IFNAR2 receptor chains with particularly high affinity and is especially potent in select bioactivities (e.g. anti-proliferative and pro-apoptotic) when compared with IFNα2. However, no molecular basis has been ascribed to this differential action, since the two ligands are equipotent in immediate early signalling events. In the present study we report that IFNβ induces Stat (signal transducer and activator of transcription) phosphorylation and transcriptional activation of ISGs (interferon-stimulated genes), including two genes with pro-apoptotic functions, for a considerably longer time frame than does IFNα2. We show that the diversification of α2/β responses progressively builds up at the receptor level as a result of accumulating USP18 (ubiquitin specific protease 18), itself an ISG, which exerts its negative feedback action by taking advantage of the weakness of IFNα2 binding to the receptor. This represents a novel type of signalling regulation that diversifies the biological potential of IFNs α and β.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I interferon (IFN), for instance, has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its optimal clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1000-fold more potent on target cells, allowing specific signaling in selected cell types only. As conventional Dendritic Cells (cDC) are essential for IFN antitumor efficacy, we targeted type I IFN-derived “AcTaferon (AFN)” to Clec9A+ cDC. Clec9A-AFN therapy displayed strong antitumor activity in murine melanoma (B16), breast carcinoma (4T1) and lymphoma models (A20), as well as against human RL lymphoma in immunodeficient NSG mice reconstituted with a human immune system. In sharp contrast to wild-type IFN therapy, the antitumor efficacy of Clec9A-AFN was not accompanied by any detectable toxicity, assessed by body weight and several hematological parameters. Clec9A-AFN effects were lost in CD8-depleted or Batf3-/- mice, and depended on IFN signaling in cDCs but not in T lymphocytes. Combined with α-PDL1 immune checkpoint blockade, Treg-depleting anti-CTLA4 + anti-OX40 therapy, immunogenic chemotherapy, or low-dose TNF, complete tumor regressions and long-lasting tumor immunity (memory) were obtained, still without any adverse effects. Our findings thus indicate that DC-targeted AFN provides a highly efficient, off-the-shelf and safe cancer immunotherapy, with possible application in a broad range of malignancies. Citation Format: Jan H. Tavernier, Anje Cauwels, Sandra Van Lint, Franciane Paul, Geneviève Garcin, Alexander Van Parys, Nikolai Kley, Gilles Uzé. Delivering type I interferon to dendritic cells empowers tumor eradication and immune combination treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 629.

Tracking the motion of individual proteins on the surface of live cells has contributed considerably towards unveiling the functional organization of proteins in the plasma membrane. Individual proteins labeled with quantum dots (QDs) can be imaged over long time periods with ultrahigh spatial and temporal resolution, yielding powerful information on the spatiotemporal dynamics of proteins at the plasma membrane in live cells. A key challenge for the application of QDs is to site-specifically attach proteins to the surface of these nanoparticles in a stoichiometric manner without affecting protein function. Several procedures for rendering surfaces of QDs biocompatible have been described, thus reducing non-specific binding and protein denaturation on the QD surface. However, functionalized biocompatible QDs used to target cell surface proteins generally result in multipoint attachment to the target proteins because the number of functional groups on the QDs is very difficult to control. Such multiply functionalized QDs induce clustering of target proteins on the cell surface, biasing not only lateral diffusion but also the functional properties of these proteins. As a consequence, increased endocytosis has been observed upon binding of QDs functionalized with multiple epidermal grow factor (EGF) molecules to cell-surface EGF receptors. Stochastic functionalization of multiple reactive sites on the QD offers the choice of obtaining only a minor fraction of the QDs with a single functional group, or a significant fraction of QDs with multiple functional groups. Preparation of homogeneous, monofunctional QDs currently relies on electrophoretic purification, which has been achieved only for very small QDs. These compact QDs are designed with very thin surface coatings, which have the disadvantage of showing relatively strong non-specific interactions. Most approaches for targeting proteins using QDs in live cells are based on biotin–streptavidin interactions, which form quasi-irreversible complexes. For multiplexed, generic labeling of proteins on the cell surface, further targeting strategies are required. We have recently described tris(hydroxymethyl)methylamine–nitrilotriacetic acid (Tris-NTA) moieties for highly specific and stable attachment of fluorophores and other functional units to histidine-tagged proteins in vitro and on the surface of live cells. The lifetime of Tris-NTA complexes with His-tagged proteins is in the order of several hours, which is well-suited to medium-term single molecule tracking applications. Herein, we have attempted to control the functionalization degree of QDs with Tris-NTA by means of electrostatic repulsion. We devised a bottom-up coupling chemistry based on a novel Tris-NTA derivative (1; Figure 1a), which comprises a thiol-terminated hepta(ethylene glycol) linker. This compound was generated in situ by reduction of the disulfide-linked dimer (1a ; see the Supporting Information, Scheme S1) and coupled to commercially available polymer-coated and amine-functionalized QDs by means of a hetero-bifunctional cross-linker (Figure 1b). Covalently attachment of 1 to surfaces modified with maleimide-functionalized polyethylene glycol (PEG) polymer brush and specific immobilization of His-tagged proteins was confirmed by label-free detection (Supporting Information, Figure S1). To control the degree of functionalization with Tris-NTA on the QD surface, the reaction of 1 with surface maleimide groups was performed at low ionic strength. Under these conditions, all QDs were reacted with Tris-NTA, as confirmed by an increase in negative charges detected by anion exchange chromatography and agarose gel electrophoreses (Figure 1c,d). These assays indicated relatively monodisperse electrostatic properties after coupling of 1, despite the fact that it was reacted at a large excess (660 mm of compound 1 to 1 mm QD). Coupling of 1 at higher ionic strength yielded QDs with a substantially higher degree of functionalization, as confirmed by a further shift of the signals both in anion exchange chromatography and agarose gel electrophoresis (Figure 1c,d). To characterize the functional properties of Tris-NTAcoupled QDs, binding to immobilized hexahistidine (H6) [*] Dr. C. You, S. Wilmes, O. Beutel, S. L chte, Y. Podoplelowa, F. Roder, C. Richter, T. Seine, D. Schaible, Prof. Dr. J. Piehler Division of Biophysics, Universit t Osnabr ck Barbarstrasse 11, 49076 Osnabr ck (Germany) Fax: (+49)541-969-2262 E-mail: piehler@uos.de Homepage: http://www.biologie.uni-osnabrueck.de/Biophysik/ Piehler/

IL-28 and IL-29 were recently described as members of a new cytokine family that shares with type I interferon (IFN) the same Jak/Stat signalling pathway driving expression of a common set of genes. Accordingly, they have been named IFN lambda. IFNs lambda exhibit several common features with type I IFNs: antiviral activity, antiproliferative activity and in vivo antitumour activity. Importantly, however, IFNs lambda bind to a distinct membrane receptor, composed of IFNLR1 and IL10R2. This specific receptor usage suggests that this cytokine family does not merely replicate the type I IFN system and justifies its designation as type III IFN by the nomenclature committee of the International Society of Interferon and Cytokine Research.

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunmodulatory responses by binding to a shared cell surface receptor comprising the transmembrane proteins ifnar1 and ifnar2. Activation of differential response patterns by IFNs has been observed, suggesting that members of the family play different roles in innate immunity. The molecular basis for differential signaling has not been identified yet. Here, we have investigated the recognition of various IFNs including several human IFNalpha species, human IFNomega and human IFNbeta as well as ovine IFNtau2 by the receptor subunits in detail. Binding to the extracellular domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC) was monitored in real time by reflectance interference and total internal reflection fluorescence spectroscopy. For all IFNs investigated, competitive 1:1 interaction not only with ifnar2-EC but also with ifnar1-EC was shown. Furthermore, ternary complex formation was studied with ifnar1-EC and ifnar2-EC tethered onto solid-supported membranes. These analyses confirmed that the signaling complexes recruited by IFNs have very similar architectures. However, differences in rate and affinity constants over several orders of magnitude were observed for both the interactions with ifnar1-EC and ifnar2-EC. These data were correlated with the potencies of ISGF3 activation, antiviral and anti-proliferative activity on 2fTGH cells. The ISGF3 formation and antiviral activity correlated very well with the binding affinity towards ifnar2. In contrast, the affinity towards ifnar1 played a key role for antiproliferative activity. A striking correlation was observed for relative binding affinities towards ifnar1 and ifnar2 with the differential antiproliferative potency. This correlation was confirmed by systematically engineering IFNalpha2 mutants with very high differential antiproliferative potency.

International audience; The human interferon α2 (IFNα2) was the first highly active IFN subtype to be cloned in the early eighties. It was also the first IFN and the first cytokine to be produced and commercialized by the pharmaceutical industry. Ipso facto it became the favorite IFNα subtype for academic researchers. For this fortunate reason IFNα2 has been at the origin of most discoveries related to the mechanism of action of type I interferons.

Quantitative single-molecule receptor dimerization assays show dimerization of IFNAR1 and IFNAR2 upon IFN treatment, and reveal the limiting role of IFNAR1 binding affinity in complex assembly and the regulatory role of USP18., Type I interferons (IFNs) activate differential cellular responses through a shared cell surface receptor composed of the two subunits, IFNAR1 and IFNAR2. We propose here a mechanistic model for how IFN receptor plasticity is regulated on the level of receptor dimerization. Quantitative single-molecule imaging of receptor assembly in the plasma membrane of living cells clearly identified IFN-induced dimerization of IFNAR1 and IFNAR2. The negative feedback regulator ubiquitin-specific protease 18 (USP18) potently interferes with the recruitment of IFNAR1 into the ternary complex, probably by impeding complex stabilization related to the associated Janus kinases. Thus, the responsiveness to IFNα2 is potently down-regulated after the first wave of gene induction, while IFNβ, due to its ∼100-fold higher binding affinity, is still able to efficiently recruit IFNAR1. Consistent with functional data, this novel regulatory mechanism at the level of receptor assembly explains how signaling by IFNβ is maintained over longer times compared with IFNα2 as a temporally encoded cause of functional receptor plasticity.

The λ interferons (IFN-λs), also known as IL-28 and IL-29, are coexpressed with IFN-β after Toll-like–receptor (TLR) stimulation in human monocyte–derived dendritic cells (DCs). IFN-λ shares with type I IFNs an intracellular signaling pathway that drives the expression of a common set of genes. However, IFN-λ signaling is initiated through a membrane receptor system distinct from that of type I IFNs. Because IFNs produced by DCs in response to TLR stimulation are critical in the differentiation and maturation of DCs, we sought to investigate whether IFN-λ exhibits specific effects on DC differentiation. In this work, we show that DCs acquire IFN-λ responsiveness through the expression of the specific IFN-λ receptor chain during their differentiation from monocytes. IFN-λ–treated DCs express high levels of major histocompatibility complex class I (MHC class I) and MHC class II but low levels of costimulatory molecules. However, they express CCR7 and acquire the ability to migrate to lymph nodes when intravenously injected into SCID/Bg mice. In mixed lymphocyte reaction (MLR) cultures, IFN-λ–treated DCs specifically induced IL-2–dependent proliferation of a CD4+CD25+Foxp3+ T-cell subset with contact-dependent suppressive activity on T-cell proliferation initiated by fully mature DCs. IFN-λs are thus able to generate tolerogenic DCs, an activity that could thwart IFN-β functions.

In humans, the type I interferon (IFN) family consists of 13 IFN-alpha subtypes, IFN-beta and IFN-omicron the newly discovered IFN-like family consists of IFN-lambda1, -lambda2 and -lambda3. We have investigated the expression of type I and lambda IFN genes following virus infections or Toll-like receptor (TLR) triggering in monocyte-derived DC (MDDC) and plasmacytoid DC (pDC). We found that all IFN-alpha, -beta, -omicron and -lambda subtypes are expressed in influenza-virus-infected MDDC or pDC. Conversely, differential type I IFN gene transcription was induced in MDDC and pDC stimulated by specific TLR agonists. TLR-9 stimulation by CpG DNA induced the expression of all IFN-alpha, -beta, -omicron and -lambda subtypes in pDC, whereas TLR-4 stimulation by LPS, or TLR-3 stimulation by poly I:C, induced only IFN-beta and IFN-lambda gene expression in MDDC. The expression pattern of IFN regulatory factor (IRF)-5 and IRF-7 in MDDC and pDC was also determined. IRF-5 was constitutively expressed in the two DC subsets whereas IRF-7 was constitutive in pDC but its expression was induced along MDDC maturation. Overall, our data indicate that the coordinated expression of IFN-lambda with IFN-beta would be of crucial importance for the maturation of DC.

International audience; Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-l-positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-l-positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.

International audience; Type-I interferon (IFN)-induced activation of the mammalian target of rapamycin (mTOR) signaling pathway has been implicated in translational control of mRNAs encoding inter-feron-stimulated genes (ISGs). However, mTOR-sensitive translatomes commonly include mRNAs with a 5' terminal oligopyrimidine tract (TOP), such as those encoding ribosomal proteins, but not ISGs. Because these translatomes were obtained under conditions when ISG expression is not induced, we examined the mTOR-sensitive translatome in human WISH cells stimulated with IFN β. The mTOR inhibitor Torin1 resulted in a repression of global protein synthesis, including that of ISG products, and translation of all but 3 ISG mRNAs (TLR3, NT5C3A, and RNF19B) was not selectively more sensitive to mTOR inhibition. Detailed studies of NT5C3A revealed an IFN-induced change in transcription start site resulting in a switch from a non-TOP to a TOP-like transcript variant and mTOR sensitive translation. Thus, we show that, in the cell model used, translation of the vast majority of ISG mRNAs is not selectively sensitive to mTOR activity and describe an uncharacterized mechanism wherein the 5'-UTR of an mRNA is altered in response to a cytokine, resulting in a shift from mTOR-insensitive to mTOR-sensitive translation.

Intracellular ISG15 is an interferon (IFN)-alpha/beta-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-alpha/beta-dependent antiviral immunity in mice(1-4). We previously published a study describing humans with inherited ISG15deficiency but without unusually severe viral diseases(5). We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-gamma-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-alpha/beta immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi-Goutieres syndrome and spondyloenchondrodysplasia(6-9). We further show that an absence of intracellular ISG15 in the patients' cells prevents the accumulation of USP18(10,11), a potent negative regulator of IFN-alpha/beta signalling, resulting in the enhancement and amplification of IFN-alpha/beta responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-alpha/beta immunity. In humans, intracellular ISG15 is IFN-alpha/beta-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-alpha/beta and prevention of IFN-alpha/beta-dependent autoinflammation.

International audience; We have previously shown that the development of a major histocompatibility complex class I (MHC-I)-deficient tumor was favored in protein kinase C-θ knockout (PKC-θ-/-) mice compared to that occurring in wild-type mice. This phenomenon was associated with scarce recruitment of natural killer (NK) cells to the tumor site, as well as impaired NK cell activation and reduced cytotoxicity ex vivo. Poly-inosinic:cytidylic acid (poly I:C) treatment activated PKC-θ in NK cells depending on the presence of a soluble factor produced by a different splenocyte subset. In the present work, we sought to analyze whether interleukin-15 (IL-15) and/or interferon-α (IFNα) mediate PKC-θ-dependent antitumor NK cell function. We found that IL-15 improves NK cell viability, granzyme B expression, degranulation capacity and interferon-γ (IFNγ) secretion independently of PKC-θ. In contrast, we found that IFNα improves the degranulation capability of NK cells against target cancer cells in a PKC-θ-dependent fashion both ex vivo and in vivo. Furthermore, IFNα induces PKC-θ auto-phosphorylation in NK cells, in a signal transduction pathway involving both phosphatidylinositol-3-kinase (PI3K) and phospholipase-C (PLC) activation. PKC-θ dependence was further implicated in IFNα-induced transcriptional upregulation of chemokine (C-X-C motif) ligand 10 (CXCL10), a signal transducer and activator of transcription-1 (STAT-1)-dependent target of IFNα. The absence of PKC-θ did not affect IFNα-induced STAT-1 Tyr701 phosphorylation but affected the increase in STAT-1 phosphorylation on Ser727, attenuating CXCL10 secretion. This connection between IFNα and PKC-θ in NK cells may be exploited in NK cell-based tumor immunotherapy.

International audience; The immune system is routinely confronted with cell death resulting from the physiological turnover of renewable tissues, as well as from pathological insults of several types. We hypothesize the existence of a mechanism that allows the immune system to discriminate between physiological and pathological instances of cell death, but the factors that determine whether cellular demise is perceived as a neutral, tolerogenic or immunogenic event remain unclear 1. Infectious insults are accompanied by so-called microbe-associated molecular patterns (MAMPs), i.e., viral or bacterial products that activate immune cells through a panel of pattern-recognition receptors (PRRs) 2. Moreover, intracellular pathogens generally trigger adaptive mechanisms aimed toward the re-establishment of homeosta-sis, including the unfolded protein response (UPR) and autophagy 3,4. In mammals, MAMPs coupled to the activation of stress responses Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell-mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN- and IFN- receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN-related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy. npg

In this short review, we summarize how insights into the structure and dynamics of interferon-receptor complex assembly and activation guided the design of a novel class of engineered type I interferons that combine a largely lost potency on non-targeted cells with high activity on targeted cells. These novel interferons are expected to exhibit lower systemic toxicities compared to other interferon therapy modalities and could open avenues to revive these cytokines for the treatment of patients suffering of cancer and viral infections.

In contrast to the large number of class I and II cytokine receptors, only four Janus kinase (Jak) proteins are expressed in mammalian cells, implying the shared use of these kinases by many different receptor complexes. Consequently, if receptor numbers exceed the amount of available Jak, cross-interference patterns can be expected. We have engineered two model cellular systems expressing two different exogenous Tyk2-interacting receptors. A receptor chimera was generated wherein the extracellular part of the interferon type 1 receptor (Ifnar1) component of the interferon-alpha/beta receptor is replaced by the equivalent domain of the erythropoietin receptor. Despite Tyk2 activation, erythropoietin treatment of cells expressing this erythropoietin receptor/Ifnar1 chimera did not evoke any detectable IFN-type response. However, a dose-dependent interference with signal transduction via the endogenous Ifnar complex was found for STAT1, STAT2, STAT3, Tyk2, and Jak1 activation, for gene induction, and for antiviral activity. In a similar approach, cells expressing the beta1 chain of the interleukin-12 receptor showed a reduced transcriptional response to IFN-alpha as well as reduced STAT and kinase activation. In both model systems, titration of the Tyk2 kinase away from the Ifnar1 receptor chain accounts for the observed cross-interference.

Janus kinases have so far been viewed as enzymatic intermediates that couple a variety of cell surface receptors to downstream substrates with diverse effector functions. Tyk2 is a member of this family that is involved in the interferon-alpha/beta and interleukin-12 signaling pathways via its specific interaction with the IFNAR1 and the beta1 receptor subunits. Here, we have analyzed the subcellular distribution of the wild-type Tyk2 protein and of several mutants expressed in Tyk2-deficient human cells. Direct GFP-associated fluorescence and immunostaining showed a diffuse localization of Tyk2 throughout the cell, including the nuclear compartment. The nuclear localization of Tyk2 requires a nuclear localization signal-like motif rich in arginine residues that maps within the region mediating interaction with cytokine receptors. To address the question of the role of the Tyk2 nuclear pool in interferon-alpha/beta-induced biological effects, cells expressing a membrane-targeted form of Tyk2-green fluorescent protein were analyzed for their interferon-alpha responses. Our studies demonstrate that Tyk2 can reside in the nucleus independently of receptor binding and that the nuclear pool is dispensable for the transcriptional and anti-vesicular stomatitis virus responses induced by interferon-alpha.

International audience; Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the interferon alpha/beta receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein. Using an in vitro assay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22-221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1-591) of Tyk2. Moreover, substitution of the JR7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor interferon Lu signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-3) contribute in a specific manner to the in vivo assembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in interferon alpha/beta receptor signaling.

The type I interferon family includes 13 alpha, one omega and one beta subtypes recognized by a complex containing the receptor subunits ifnar1 and ifnar2 and their associated Janus tyrosine kinases, Tyk2 and Jak1. To investigate the reported differences in the way that alpha and beta interferons signal through the receptor, we introduced alanine-substitutions in the ifnar2 extracellular domain, and expressed the mutants in U5A cells, lacking endogenous ifnar2. A selection, designed to recover mutants that responded preferentially to alpha or beta interferon yielded three groups: I, neutral; II, sensitive to alpha interferon, partially resistant to beta interferon; III, resistant to alpha interferon, partially sensitive to beta interferon. A mutant clone, TMK, fully resistant to alpha interferon with good sensitivity to beta interferon, was characterized in detail and compared with U5A cells complemented with wild-type ifnar2 and also with Tyk2-deficient 11.1 cells, which exhibit a similar alpha-unresponsive phenotype with a partial beta interferon response. Using anti-receptor antibodies and mutant forms of beta interferon, three distinct modes of ligand interaction could be discerned: (i) alpha interferon with ifnar1 and ifnar2; (ii) beta interferon with ifnar1 and ifnar2; (iii) beta interferon with ifnar2 alone. We conclude that alpha and beta interferons signal differently through their receptors because the two ligand subtypes interact with the receptor subunits ifnar 1 and ifnar2 in entirely different ways.

International audience; Type I interferons (IFN-I) were identified over 50 years ago as cytokines critical for host defense against viral infections. IFN-I promote anti-viral defense through two main mechanisms. First, IFN-I directly reinforce or induce de novo in potentially all cells the expression of effector molecules of intrinsic anti-viral immunity. Second, IFN-I orchestrate innate and adaptive anti-viral immunity. However, IFN-I responses can be deleterious for the host in a number of circumstances, including secondary bacterial or fungal infections, several autoimmune diseases, and, paradoxically, certain chronic viral infections. We will review the proposed nature of protective versus deleterious IFN-I responses in selected diseases. Emphasis will be put on the potentially deleterious functions of IFN-I in human immunod-eficiency virus type 1 (HIV-1) infection, and on the respective roles of IFN-I and IFN-III in promoting resolution of hepatitis C virus (HCV) infection. We will then discuss how the balance between beneficial versus deleterious IFN-I responses is modulated by several key parameters including (i) the subtypes and dose of IFN-I produced, (ii) the cell types affected by IFN-I, and (iii) the source and timing of IFN-I production. Finally, we will speculate how integration of this knowledge combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments in patients. Specifically, we will discuss how induction or blockade of specific IFN-I responses in targeted cell types could promote the beneficial functions of IFN-I and/or dampen their deleterious effects, in a manner adapted to each disease.

International audience; Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-bytargeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

We describe how constraints on the binding of human interferons (IFNs), alpha1 and alpha2 and alpha8 on mouse cells are partially relieved by the expression of the bovine (Bo) or human (Hu) IFN alpha/beta receptor (IFNAR) component in these cells. We show that, while the binding of all three is substantially increased by the transfection of Bo IFNAR, it is accompanied by an increase in activity only in the case of alpha2 and alpha8 (IFNs that otherwise have little activity on mouse cells). IFN alpha1, which shows some partial activity on mouse cells, responds to the presence of Bo IFNAR by acting, at low concentrations, as a competitive antagonist to IFNs alpha2 and alpha8. A review of published results on IFN hybrid scanning and on the effects of expressing Bo IFNAR in human cells led us to propose that an N-terminal segment of the IFN molecule interacts directly with IFNAR. Applying site-directed mutagenesis to an IFN hybrid; alpha8[60]alpha1[92]alpha8, we show that the point mutations K84 to E84 and Y90 to D90 act synergistically to cause the hybrid to behave as the parental IFN alpha8, switching the preference from Mu to Hu IFNAR in transfected mouse cells. The published structural models for IFN reveal that positions 84 and 90 span the exposed residues of the alpha-helix C of the IFN molecule. We derive a model of IFN-receptor interaction in which the A helix and the C helix of IFN interact with IFNAR and in which a binding phase can be distinguished from a binding/activity phase. We propose that the so-called "hot" domains of the IFN molecule (the AB loop and the D helix) are presented by IFNAR to interact with an additional component of the functional receptor.

Differences in activity among the family of human IFNs alpha are much reduced if these ligands are assayed on bovine cells. In particular, the activity of IFN alpha D is much higher on bovine than on human cells. To examine these differences, the bovine counterpart of the human IFNAR has been cloned and expressed in a human cell line. The transfected cell line now recognizes the human IFN alpha D as a high-specific-activity IFN subtype, indicating that the differences in sensitivity between the bovine and human cells to the human IFN alpha lie in the structure of the IFNAR chain rather than in the other components of the functional receptor.

International audience; Multiple type I interferons (IFN-alpha/beta) 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-alpha/beta 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-beta was more potent than IFN-alpha2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-alpha2 matched or even surpassed IFN-beta 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.

A cDNA coding for the human interferon alpha receptor has been cloned using a gene transfer approach. This consists of transferring human DNA to mouse cells and selecting for cells sensitive to human interferon alpha. The transfected cells expressed the human interferon alpha receptor, and a 5 kb human DNA was isolated from a secondary transfectant. This DNA defects an mRNA present in human cells and was used to clone a 2.7 kb cDNA from a library constructed from human Daudi cells. The sequence of the cDNA is presented. It codes for a glycoprotein of 557 amino acids with an N-terminal hydrophobic region and a single transmembrane-spanning segment. Mouse cells expressing the cDNA become sensitive to the antiviral activity of and express binding sites for human interferon alpha, demonstrating that the cloned cDNA encodes a functional human interferon alpha receptor.

Key Points On the 50th anniversary of the discovery of interferon (IFN), we offer a perspective from more than 100,000 published papers, highlighting initial pivotal discoveries and more recent findings of conceptual importance. This covers the mechanisms of IFN induction, the cellular actions of IFN and IFN-stimulated genes (ISGs), and human therapeutic applications.The synthesis of IFNs requires stimulation by viruses or microbial products binding to Toll-like receptors, or chemical inducers. The development of small-molecule modulators is still in its infancy, but the delineation of the responsible signalling pathways has identified many target proteins.IFNs constitute a large protein family that can be subdivided into three types, binding to different receptors. These receptors initiate signalling by activating a complex signalling cascade regulated at many levels, resulting in a diverse pattern of ISG induction.ISGs are a diverse group of more than 300 genes, which can have direct antiviral and antitumour functions. These are attractive targets for high-throughput screening for the identification of new modulators of the IFN system.IFNs were initially investigated for their potential as antivirals, and are now commonly used in anti-HBV (hepatitis B virus) and anti-HCV (hepatitis C virus) therapy. They might also have prophylactic or therapeutic effectiveness in SARS (severe acute respiratory syndrome), influenza or another virus pandemic.The first FDA approval of an IFN was, however, not for virus infection but for cancer. The mechanisms of antitumour action are incompletely understood. Aberrations of the IFN system are also emerging as important contributors to cancer development.IFNs also proved effectiveness in relapsing, remitting multiple sclerosis. It is now common practice to initiate IFN-β treatment at the time of diagnosis.Because of the effectiveness of IFNs in limiting virus replication, reducing tumour cell mass, controlling disease symptoms and prolonging survival, market sales of IFNs approach US$4 billion. As all the effects of IFNs are mediated through ISGs, understanding of the function of these genes might lead to more efficacious antiviral and anti-cancer drugs., Interferons (IFNs) provide fundamental cellular defence mechanisms against viral infections and cancer. On the 50th anniversary of the discovery of IFNs, the authors provide a comprehensive overview of IFN biology, human therapeutic applications and potential drug targets within the IFN system., The family of interferon (IFN) proteins has now more than reached the potential envisioned by early discovering virologists: IFNs are not only antivirals with a spectrum of clinical effectiveness against both RNA and DNA viruses, but are also the prototypic biological response modifiers for oncology, and show effectiveness in suppressing manifestations of multiple sclerosis. Studies of IFNs have resulted in fundamental insights into cellular signalling mechanisms, gene transcription and innate and acquired immunity. Further elucidation of the multitude of IFN-induced genes, as well as drug development strategies targeting IFN production via the activation of the Toll-like receptors (TLRs), will almost certainly lead to newer and more efficacious therapeutics. Our goal is to offer a molecular and clinical perspective that will enable IFNs or their TLR agonist inducers to reach their full clinical potential.

International audience; All type I IFNs act through a single cell surface receptor composed of the IFNAR1 and IFNAR2 subunits and two associated cytoplasmic tyrosine kinases of the Janus family, Tyk2 and Jak1. A central issue in type I IFN biology is to understand how a multitude of subtypes can generate similar signaling outputs but also govern specific cellular responses. This review summarizes results from the last decade that contributed to our current state of knowledge of IFN-receptor complex structure and assembly.

In human monocyte-derived dendritic cells (DC), infection with Mycobacterium tuberculosis and viruses or stimulation with Toll-like receptor type 3 and 4 agonists causes the release of type I interferon (IFN). Here, we describe that the IFN-β released upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) is responsible for a rapid and sustained signal transducer and activator of transcription 1 and 2 activation and expression of IFN-stimulated genes, such as the transcription factor IFN regulatory factor 7 and the chemokine CXC chemokine ligand 10. The autocrine production of IFN-β from LPS and poly I:C-matured DC (mDC) induced a temporary saturation of the response to type I IFN and a marked decline in the level of the two IFN receptor (IFNAR) subunits. It is interesting that we found that upon clearing of the released cytokines, LPS-stimulated DC reacquired full responsiveness to IFN-β but only partial responsiveness to IFN-α, and their maturation process was unaffected. Monitoring of surface and total levels of the receptor subunits showed that maximal expression of IFNAR2 resumed within 24 h of clearing, and IFNAR1 expression remained low. Thus, mDC can modulate their sensitivity to two IFN subtypes through a differential regulation of the IFNAR subunits.

Initially described for their antiviral activities, type I Interferons are now recognized as central regulatory elements of the immune response, primarily for their effect on the differentiation of monocytes into dendritic cells and osteoclasts. They are routinely used in clinic for the treatment of several diseases, including viral hepatitis, multiple sclerosis and several forms of cancer. Interferons are however not devoid of toxic effects when high doses are administered to patients, indicating that interferon action must be timely and spatially down regulated. We review here the molecular mechanisms which have been described to shut off the interferon initiated signals.

Epithelial cells of the lung are the primary targets for respiratory viruses. Virus-carried single-stranded RNA (ssRNA) can activate Toll-like receptors (TLRs) 7 and 8, whereas dsRNA is bound by TLR3 and a cytoplasmic RNA helicase, retinoic acid-inducible protein I (RIG-I). This recognition leads to the activation of host cell cytokine gene expression. Here we have studied the regulation of influenza A and Sendai virus-induced alpha interferon (IFN-alpha), IFN-beta, interleukin-28 (IL-28), and IL-29 gene expression in human lung A549 epithelial cells. Sendai virus infection readily activated the expression of the IFN-alpha, IFN-beta, IL-28, and IL-29 genes, whereas influenza A virus-induced activation of these genes was mainly dependent on pretreatment of A549 cells with IFN-alpha or tumor necrosis factor alpha (TNF-alpha). IFN-alpha and TNF-alpha induced the expression of the RIG-I, TLR3, MyD88, TRIF, and IRF7 genes, whereas no detectable TLR7 and TLR8 was seen in A549 cells. TNF-alpha also strongly enhanced IKK epsilon mRNA and protein expression. Ectopic expression of a constitutively active form of RIG-I (deltaRIG-I) or IKK epsilon, but not that of TLR3, enhanced the expression of the IFN-beta, IL-28, and IL-29 genes. Furthermore, a dominant-negative form of RIG-I inhibited influenza A virus-induced IFN-beta promoter activity in TNF-alpha-pretreated cells. In conclusion, IFN-alpha and TNF-alpha enhanced the expression of the components of TLR and RIG-I signaling pathways, but RIG-I was identified as the central regulator of influenza A virus-induced expression of antiviral cytokines in human lung epithelial cells.

Alpha and beta interferons (IFN-alpha and IFN-beta) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-alpha2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-beta. Compared to wild-type (wt) IFN-alpha2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-beta, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-beta, promotes a differentially higher antiproliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-beta and higher levels of gene induction or repression than those for wt IFN-alpha2. Thus, we show that the differential activities of IFN-beta are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-alpha subtypes suggests that IFN-alpha has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-beta.

Quantitative polymerase chain reaction (PCR) is as old as PCR, but it has had to wait for the introduction of real-time PCR instruments to become widely used. These instruments allow monitoring of the PCR reaction on line; they involve the use of a fluorescent probe that allows quantification of the amplified DNA. Different fluorescent formats and different applications have been developed for quantitative PCR, but this chapter focuses on the use of the SYBR Green label for the quantification of specific cDNAs in reverse transcription mixes: RT-PCR. We propose optimal reaction conditions for the reactions to be performed on the different available instruments and discuss the important parameters for setting up experiments: specificity, efficiency, and reproducibility. We also introduce the reader to the problems of relative quantification.

In humans, type I interferon (IFN) is a family of 17 cytokines, among which the α subtypes and the β subtype are differentially expressed. It has been suggested that IFN-β activates a specific signaling cascade in addition to those activated by all type I IFNs. Nevertheless, no true biological relevance for a differential activity of α and β IFN subtypes has been identified so far. Because type I IFNs are critical for the regulation of osteoclastogenesis in mice, we have compared the effect of IFN-α2 and IFN-β on the differentiation of human monocytes into osteoclasts. Primary monocytes undergoing osteoclastic differentiation are highly and equally sensitive to both α2 and β IFNs as determined by measuring the induction levels of several IFN-stimulated genes. However, IFN-β was 100-fold more potent than the α2 subtype at inhibiting osteoclastogenesis. Expression profiling of the genes differentially regulated by IFN-α2 and IFN-β in this cellular system revealed the chemokine CXCL11 as the only IFN-induced gene differentially up-regulated by IFN-β. We show that recombinant CXCL11 by itself inhibits osteoclastic differentiation. These results indicate that autocrine-acting CXCL11 mediates, at least in part, the regulations of osteoclastogenesis by type I IFNs.