Translating the biology of ß common receptor-engaging cytokines into clinical medicine.

The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called ß-common or ßc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.

Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies.

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.

Targeting the human ßc receptor inhibits inflammatory myeloid cells and lung injury caused by acute cigarette smoke exposure.

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a devastating disease commonly caused by cigarette smoke (CS) exposure that drives tissue injury by persistently recruiting myeloid cells into the lungs. A significant portion of COPD patients also present with overlapping asthma pathology including eosinophilic inflammation. The ßc cytokine family includes granulocyte monocyte-colony-stimulating factor, IL-5 and IL-3 that signal through their common receptor subunit ßc to promote the expansion and survival of multiple myeloid cells including monocytes/macrophages, neutrophils and eosinophils. METHODS: We have used our unique human ßc receptor transgenic (hßc Tg) mouse strain that expresses human ßc instead of mouse ßc and ßIL3 in an acute CS exposure model. Lung tissue injury was assessed by histology and measurement of albumin and lactate dehydrogenase levels in the bronchoalveolar lavage (BAL) fluid. Transgenic mice were treated with an antibody (CSL311) that inhibits human ßc signalling. RESULTS: hßc Tg mice responded to acute CS exposure by expanding blood myeloid cell numbers and recruiting monocyte-derived macrophages (cluster of differentiation 11b+ [CD11b+ ] interstitial and exudative macrophages [IM and ExM]), neutrophils and eosinophils into the lungs. This inflammatory response was associated with lung tissue injury and oedema. Importantly, CSL311 treatment in CS-exposed mice markedly reduced myeloid cell numbers in the blood and BAL compartment. Furthermore, CSL311 significantly reduced lung CD11b+ IM and ExM, neutrophils and eosinophils, and this decline was associated with a significant reduction in matrix metalloproteinase-12 (MMP-12) and IL-17A expression, tissue injury and oedema. CONCLUSION: This study identifies CSL311 as a therapeutic antibody that potently inhibits immunopathology and lung injury caused by acute CS exposure.

Sialylation-dependent pharmacokinetics and differential complement pathway inhibition are hallmarks of CR1 activity in vivo.

Human Complement Receptor 1 (HuCR1) is a potent membrane-bound regulator of complement both in vitro and in vivo, acting via interaction with its ligands C3b and C4b. Soluble versions of HuCR1 have been described such as TP10, the recombinant full-length extracellular domain, and more recently CSL040, a truncated version lacking the C-terminal long homologous repeat domain D (LHR-D). However, the role of N-linked glycosylation in determining its pharmacokinetic (PK) and pharmacodynamic (PD) properties is only partly understood. We demonstrated a relationship between the asialo-N-glycan levels of CSL040 and its PK/PD properties in rats and non-human primates (NHPs), using recombinant CSL040 preparations with varying asialo-N-glycan levels. The clearance mechanism likely involves the asialoglycoprotein receptor (ASGR), as clearance of CSL040 with a high proportion of asialo-N-glycans was attenuated in vivo by co-administration of rats with asialofetuin, which saturates the ASGR. Biodistribution studies also showed CSL040 localization to the liver following systemic administration. Our studies uncovered differential PD effects by CSL040 on complement pathways, with extended inhibition in both rats and NHPs of the alternative pathway compared with the classical and lectin pathways that were not correlated with its PK profile. Further studies showed that this effect was dose dependent and observed with both CSL040 and the full-length extracellular domain of HuCR1. Taken together, our data suggests that sialylation optimization is an important consideration for developing HuCR1-based therapeutic candidates such as CSL040 with improved PK properties and shows that CSL040 has superior PK/PD responses compared with full-length soluble HuCR1.

Blocking the human common beta subunit of the GM-CSF, IL-5 and IL-3 receptors markedly reduces hyperinflammation in ARDS models.

Acute respiratory distress syndrome (ARDS) is triggered by various aetiological factors such as trauma, sepsis and respiratory viruses including SARS-CoV-2 and influenza A virus. Immune profiling of severe COVID-19 patients has identified a complex pattern of cytokines including granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-5, which are significant mediators of viral-induced hyperinflammation. This strong response has prompted the development of therapies that block GM-CSF and other cytokines individually to limit inflammation related pathology. The common cytokine binding site of the human common beta (ßc) receptor signals for three inflammatory cytokines: GM-CSF, IL-5 and IL-3. In this study, ßc was targeted with the monoclonal antibody (mAb) CSL311 in engineered mice devoid of mouse ßc and ßIL-3 and expressing human ßc (hßcTg mice). Direct pulmonary administration of lipopolysaccharide (LPS) caused ARDS-like lung injury, and CSL311 markedly reduced lung inflammation and oedema, resulting in improved oxygen saturation levels in hßcTg mice. In a separate model, influenza (HKx31) lung infection caused viral pneumonia associated with a large influx of myeloid cells into the lungs of hßcTg mice. The therapeutic application of CSL311 potently decreased accumulation of monocytes/macrophages, neutrophils, and eosinophils without altering lung viral loads. Furthermore, CSL311 treatment did not limit the viral-induced expansion of NK and NKT cells, or the tissue expression of type I/II/III interferons needed for efficient viral clearance. Simultaneously blocking GM-CSF, IL-5 and IL-3 signalling with CSL311 may represent an improved and clinically applicable strategy to reducing hyperinflammation in the ARDS setting.

Targeting the Human ßc Receptor Inhibits Contact Dermatitis in a Transgenic Mouse Model.

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (ßc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit ßc, a property that has made ßc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of ßc has precluded testing of inhibitors of human ßc in mouse models. To overcome this problem, we developed a human ßc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human ßc instead of mouse ßc. Human ßc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human ßc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human ßc receptor transgenic mouse as a unique platform to test the inhibitors of ßc in vivo.

Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics.

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation. Preclinical proof-of-concept studies suggest that human plasma-derived Hp is a promising therapeutic candidate for several Hb-driven diseases. Optimizing the efficacy and safety of Hb-targeting biotherapeutics may require structural and functional modifications for specific indications. Improved Hp variants could be designed to achieve the desired tissue distribution, metabolism, and elimination to target hemolytic disease states effectively. However, it is critical to ensure that these modifications maintain the function of Hp. Using transient mammalian gene expression of Hp combined with co-transfection of the pro-haptoglobin processing protease C1r-LP, we established a platform for generating recombinant Hp-variants. We designed an Hpß-scaffold, which was expressed in this system at high levels as a monomeric unit (mini-Hp) while maintaining the key protective functions of Hp. We then used this Hpß-scaffold as the basis to develop an initial proof-of-concept Hp fusion protein using human serum albumin as the fusion partner. Next, a hemopexin-Hp fusion protein with bispecific heme and Hb detoxification capacity was generated. Further, we developed a Hb scavenger devoid of CD163 scavenger receptor binding. The functions of these proteins were then characterized for Hb and heme-binding, binding of the Hp-Hb complexes with the clearance receptor CD163, antioxidant properties, and vascular nitric oxide sparing capacity. Our platform is designed to support the generation of innovative Hb scavenger biotherapeutics with novel modes of action and potentially improved formulation characteristics, function, and pharmacokinetics.

Glycoproteomic measurement of site-specific polysialylation.

Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) due to its high negative charge and size. Most analytical approaches for analysis of PSA measure its degree of polymerization and monosaccharide composition, but do not address the key questions of site specificity and occupancy. Here, we developed a high-throughput LC-ESI-MS/MS glycoproteomics method to measure site-specific polysialylation of glycoproteins. This method measures site-specific PSA modification by using mild acid hydrolysis to eliminate PSA and sialic acids while leaving the glycan backbone intact, together with protease digestion followed by LC-ESI-MS/MS glycopeptide detection. PSA-modified glycopeptides are not detectable by LC-ESI-MS/MS, but become detectable after desialylation, allowing measurement of site-specific PSA occupancy. This method is an efficient analytical workflow for the study of glycoprotein polysialylation in biological and therapeutic settings.

Mining the Plasma Cell Transcriptome for Novel Cell Surface Proteins.

Antibody Secreting Cells (ASCs) are a fundamental component of humoral immunity, however, deregulated or excessive antibody production contributes to the pathology of autoimmune diseases, while transformation of ASCs results in the malignancy Multiple Myeloma (MM). Despite substantial recent improvements in treating these conditions, there is as yet no widely used ASC-specific therapeutic approach, highlighting a critical need to identify novel methods of targeting normal and malignant ASCs. Surface molecules specifically expressed by the target cell population represent ideal candidates for a monoclonal antibody-based therapy. By interrogating the ASC gene signature that we previously defined we identified three surface proteins, Plpp5, Clptm1l and Itm2c, which represent potential targets for novel MM treatments. Plpp5, Clptm1l and Itm2c are highly and selectively expressed by mouse and human ASCs as well as MM cells. To investigate the function of these proteins within the humoral immune system we have generated three novel mouse strains, each carrying a loss-of-function mutation in either Plpp5, Clptm1l or Itm2c. Through analysis of these novel strains, we have shown that Plpp5, Clptm1l and Itm2c are dispensable for the development, maturation and differentiation of B-lymphocytes, and for the production of antibodies by ASCs. As adult mice lacking either protein showed no apparent disease phenotypes, it is likely that targeting these molecules on ASCs will have minimal on-target adverse effects.

Role of the ß Common (ßc) Family of Cytokines in Health and Disease.

The ß common ([ßc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use ßc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the ßc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the ßc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease.

An Optimized Hepatitis C Virus E2 Glycoprotein Core Adopts a Functional Homodimer That Efficiently Blocks Virus Entry.

The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects ∼2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81.

CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common ß chain of the IL-3, GM-CSF and IL-5 receptors.

The ß common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared ß common (ßc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human ßc receptor. The binding epitope of CSL311 on the ßc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human ßc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 ß common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human ßc receptor is central to pathogenesis. The coordinates for the ßc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).

Dual mechanism of interleukin-3 receptor blockade by an anti-cancer antibody.

Interleukin-3 (IL-3) is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα) in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD), a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-13 receptors, adopting unique "open" and classical "closed" conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas "open-like" IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a "double hit" cytokine receptor blockade.

Identification of potent antagonist antibodies against mouse IL-13Rα1 using novel bioassays.

Interleukin-13 (IL-13) is a cytokine implicated in airway diseases such as asthma and idiopathic pulmonary fibrosis. IL-13 signals through a heterodimeric receptor complex consisting of IL-13Rα1 and IL-4Rα, known as the type II IL-4R. IL-4 also signals through this receptor and as such many of the biological effects of IL-13 and IL-4 are similar. Here we describe the development of two sensitive bioassays to determine the potency of antagonists of the mouse type II IL-4R. Both IL-13 and IL-4 dose-dependently induce CCL17 production from J774 mouse monocytic cells and CCL11 production from NIH3T3 mouse fibroblasts in the presence of TNFα. The assays were optimized to minimize TNFα concentration, cell number and incubation time whilst retaining a suitable signal-to-background ratio. Anti-cytokine antibodies or recombinant soluble receptors completely neutralized IL-13 or IL-4 activity in these bioassays. The J774 assay was used to screen a panel of anti-mIL-13Rα1 antibodies for neutralizing activity against this receptor. We report the identification of the first monoclonal antibodies that bind mouse IL-13Rα1 and neutralize both IL-13-induced and IL-4-induced cellular function. These antibodies should prove useful for determining the effects of neutralizing IL-13Rα1 in mouse models of disease. In addition, these bioassays may be used for measuring the bioactivity of mouse IL-13 and IL-4 and for the discovery of additional antagonists of the mouse IL-13Rα1/IL-4Rα complex.

Soluble IFN receptor potentiates in vivo type I IFN signaling and exacerbates TLR4-mediated septic shock.

Circulating levels of a soluble type I IFNR are elevated in diseases, such as chronic inflammation, infections, and cancer, but whether it functions as an antagonist, agonist, or transporter is unknown. In this study, we elucidate the in vivo importance of the soluble type I IFNAR, soluble (s)IFNAR2a, which is generated by alternative splicing of the Ifnar2 gene. A transgenic mouse model was established to mimic the 10-15-fold elevated expression of sIFNAR2a observed in some human diseases. We generated transgenic mouse lines, designated SolOX, in which the transgene mRNA and protein-expression patterns mirrored the expression patterns of the endogenous gene. SolOX were demonstrated to be more susceptible to LPS-mediated septic shock, a disease model in which type I IFN plays a crucial role. This effect was independent of "classical" proinflammatory cytokines, such as TNF-α and IL-6, whose levels were unchanged. Because the increased levels of sIFNAR2a did not affect the kinetics of the increased interferonemia, this soluble receptor does not potentiate its ligand signaling by improving IFN pharmacokinetics. Mechanistically, increased levels of sIFNAR2a are likely to facilitate IFN signaling, as demonstrated in spleen cells overexpressing sIFNAR2a, which displayed quicker, higher, and more sustained activation of STAT1 and STAT3. Thus, the soluble IFNR is an important agonist of endogenous IFN actions in pathophysiological processes and also is likely to modulate the therapeutic efficacy of clinically administered IFNs.

Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined.

EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE).

The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc) as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

Characterization of the type I interferon locus and identification of novel genes.

The human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. We have identified two novel mouse Ifna genes (Ifna12, Ifna13) and Ifnl2 (IFN-like 2, a homologue of Limitin/IFN-like 1). Another type I IFN gene was designated Ifne1. Mouse Ifne1 was expressed in ovaries and uterus but not in tissues of hematopoietic origin. IFN-epsilon1 has general structural characteristics of a type I IFN. These studies represent the first detailed annotation of the mouse type I IFN locus, and the products of these novel genes may have important functions in reproduction and host defense.

Characterization and transcriptional analysis of the mouse Chromosome 16 cytokine receptor gene cluster.

The class II cytokine receptor (CIICR) genes Il10r2 and Ifnar1 are localized on mouse Chr 16 in a cluster that also contains the CIICR genes Ifnar2 and Ifngr2. The structure of the Il10r2 gene was deduced and consisted of 7 exons and 6 introns arrayed in an organization similar to its human ortholog. We also present a revised Il10r2 cDNA sequence with a total of 100 bp of additional nucleotide sequence in the 5' and 3' untranslated regions, and report the first extensive profiles of Il10r2 and Ifnar1 mRNA developmental stage and adult tissue expression. Promoter-luciferase reporter constructs were used to define the major region (-108 to +67) that conferred basal expression of the Il10r2 gene. Long-range comparative genomic sequence analysis between the mouse and the orthologous human CIICR genomic loci revealed several conserved non-coding regions. The most proximal conserved non-coding sequence was a 204-bp element located 1.6 kb upstream of the transcriptional start site of Ifnar2 that had repressor-like activity in transient transfection assays with an SV40 promoter-luciferase reporter construct. The identification of multiple conserved non-coding sequences will provide the basis for further investigations to elucidate CIICR gene regulation.