ABSTRACT
OBJECTIVES: Bruton's tyrosine kinase (BTK) plays a non-redundant signaling role downstream of the B-cell receptor (BCR) in B cells and the receptors for the Fc region of immunoglobulins (FcR) in myeloid cells. Here, we characterise BIIB091, a novel, potent, selective and reversible small-molecule inhibitor of BTK. METHODS: BIIB091 was evaluated in vitro and in vivo in preclinical models and in phase 1 clinical trial. RESULTS: In vitro, BIIB091 potently inhibited BTK-dependent proximal signaling and distal functional responses in both B cells and myeloid cells with IC50s ranging from 3 to 106 nm, including antigen presentation to T cells, a key mechanism of action thought to be underlying the efficacy of B cell-targeted therapeutics in multiple sclerosis. BIIB091 effectively sequestered tyrosine 551 in the kinase pocket by forming long-lived complexes with BTK with t 1/2 of more than 40 min, thereby preventing its phosphorylation by upstream kinases. As a key differentiating feature of BIIB091, this property explains the very potent whole blood IC50s of 87 and 106 nm observed with stimulated B cells and myeloid cells, respectively. In vivo, BIIB091 blocked B-cell activation, antibody production and germinal center differentiation. In phase 1 healthy volunteer trial, BIIB091 inhibited naïve and unswitched memory B-cell activation, with an in vivo IC50 of 55 nm and without significant impact on lymphoid or myeloid cell survival after 14 days of dosing. CONCLUSION: Pharmacodynamic results obtained in preclinical and early clinical settings support the advancement of BIIB091 in phase 2 clinical trials.
ABSTRACT
Autoreactive B cell-derived antibodies form immune complexes that likely play a pathogenic role in autoimmune diseases. In systemic lupus erythematosus (SLE), these antibodies bind Fc receptors on myeloid cells and induce proinflammatory cytokine production by monocytes and NETosis by neutrophils. Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that signals downstream of Fc receptors and plays a transduction role in antibody expression following B cell activation. Given the roles of BTK in both the production and sensing of autoreactive antibodies, inhibitors of BTK kinase activity may provide therapeutic value to patients suffering from autoantibody-driven immune disorders. Starting from an in-house proprietary screening hit followed by structure-based rational design, we have identified a potent, reversible BTK inhibitor, BIIB068 (1), which demonstrated good kinome selectivity with good overall drug-like properties for oral dosing, was well tolerated across preclinical species at pharmacologically relevant doses with good ADME properties, and achieved >90% inhibition of BTK phosphorylation (pBTK) in humans.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Administration, Oral , Agammaglobulinaemia Tyrosine Kinase/metabolism , Animals , Antigens, T-Independent/chemistry , Antigens, T-Independent/metabolism , Autoimmune Diseases/drug therapy , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Binding Sites , Catalytic Domain , Dogs , Drug Evaluation, Preclinical , Female , Half-Life , Humans , Mice , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/metabolism , Pyrimidines/therapeutic use , Rats , Structure-Activity RelationshipABSTRACT
Aim: Replicate sample testing has long been regarded as a necessity for bioanalytical laboratory testing, especially in the realm of ligand-binding assays (LBAs). In an era in which results were derived from crude test tube-based assays, the replication of results was warranted. Those assays were often imprecise and required multiple replicates to arrive at results that approached accuracy. However, given technological advancements and excellent accuracy and precision of many modern LBAs, the practice of replicate testing should be re-evaluated. Although most regulatory guidelines allow for singlet testing when sufficient robustness and precision are demonstrated during validation, duplicate testing is still common practice. Recently however, several articles have been published that support singlet analysis for LBAs performed on a platform with automated liquid handling. Results: Data from five pharmacokinetic assay validations and five clinical and preclinical studies originally run in duplicate were re-evaluated in singlet and found to be nearly identical to the original duplicate results. Conclusion: We confirm that well-developed LBAs produce comparable data whether evaluated in singlet or duplicate. Additionally, automation is not requisite for singlet testing.
Subject(s)
Biological Assay/methods , Small Molecule Libraries/metabolism , Animals , Ligands , Macaca fascicularis , Management Quality Circles , Small Molecule Libraries/pharmacokinetics , Tissue DistributionABSTRACT
The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of Bioanalysis, issues 22 and 23 (2019), respectively.
Subject(s)
Biological Assay/methods , Biomarkers/metabolism , Flow Cytometry/methods , Genetic Therapy/methods , United States Food and Drug Administration/standards , History, 21st Century , Humans , United StatesABSTRACT
OBJECTIVE: To assess functional changes in lymphocyte repertoire and subsequent clinical implications during delayed-release dimethyl fumarate (DMF) treatment in patients with multiple sclerosis. METHODS: Using peripheral blood from several clinical trials of DMF, immune cell subsets were quantified using flow cytometry. For some patients, lymphocyte counts were assessed after DMF discontinuation. Incidence of adverse events, including serious and opportunistic infections, was assessed. RESULTS: In DMF-treated patients, absolute lymphocyte counts (ALCs) demonstrated a pattern of decline followed by stabilization, which also was reflected in the global reduction in numbers of circulating functional lymphocyte subsets. The relative frequencies of circulating memory T- and B-cell populations declined and naive cells increased. No increased incidence of serious infection or malignancy was observed for patients treated with DMF, even when stratified by ALC or T-cell subset frequencies. For patients who discontinued DMF due to lymphopenia, ALCs increased after DMF discontinuation; recovery time varied by ALC level at discontinuation. T-cell subsets closely correlated with ALCs in both longitudinal and cross-sectional analyses. CONCLUSIONS: DMF shifted the immunophenotype of circulating lymphocyte subsets. ALCs were closely correlated with CD4+ and CD8+ T-cell counts, indicating that lymphocyte subset monitoring is not required for safety vigilance. No increased risk of serious infection was observed in patients with low T-cell subset counts. Monitoring ALC remains the most effective way of identifying patients at risk of subsequently developing prolonged moderate to severe lymphopenia, a risk factor for progressive multifocal leukoencephalopathy in DMF-treated patients. TRIAL REGISTRATION NUMBERS: EUDRA CT 2015-001973-42, NCT00168701, NCT00420212, NCT00451451, and NCT00835770.
Subject(s)
Dimethyl Fumarate/therapeutic use , Immunosuppressive Agents/therapeutic use , Lymphocytes/drug effects , Multiple Sclerosis, Relapsing-Remitting/blood , Adult , B-Lymphocytes/drug effects , CD4-CD8 Ratio , Cross-Sectional Studies , Delayed-Action Preparations , Dimethyl Fumarate/adverse effects , Dimethyl Fumarate/pharmacology , Female , Humans , Immunosuppressive Agents/adverse effects , Immunosuppressive Agents/pharmacology , Longitudinal Studies , Lymphocyte Count , Lymphopenia/blood , Lymphopenia/chemically induced , Male , Middle Aged , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Multiple Sclerosis, Relapsing-Remitting/immunology , Risk Assessment , T-Lymphocytes/drug effectsABSTRACT
The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.
Subject(s)
Antigens/analysis , Biological Assay/standards , Flow Cytometry/standards , Genetic Therapy/standards , Pharmacokinetics , Antigens/immunology , Biological Assay/methods , Biomarkers/analysis , Biotechnology , Flow Cytometry/methods , Government Agencies , Humans , Reference ValuesABSTRACT
OBJECTIVES: To investigate the immune response to vaccinations in patients with relapsing forms of MS treated with delayed-release dimethyl fumarate (DMF) vs nonpegylated interferon (IFN). METHODS: In this open-label, multicenter study, patients received 3 vaccinations: (1) tetanus-diphtheria toxoid (Td) to test T-cell-dependent recall response, (2) pneumococcal vaccine polyvalent to test T-cell-independent humoral response, and (3) meningococcal (groups A, C, W-135, and Y) oligosaccharide CRM197 conjugate to test T-cell-dependent neoantigen response. Eligible patients were aged 18-55 years, diagnosed with relapsing-remitting MS (RRMS), and either treated for ≥6 months with an approved dose of DMF or for ≥3 months with an approved dose of nonpegylated IFN. Primary end point was the proportion of patients with ≥2-fold rise in antitetanus serum IgG levels from prevaccination to 4 weeks after vaccination. RESULTS: Seventy-one patients (DMF treated, 38; IFN treated, 33) were enrolled. The mean age was 45.3 years (range 27-55); 86% were women. Responder rates (≥2-fold rise) to Td vaccination were comparable between DMF- and IFN-treated groups (68% vs 73%). Responder rates (≥2-fold rise) were also similar between DMF- and IFN-treated groups for diphtheria antitoxoid (58% vs 61%), pneumococcal serotype 3 (66% vs 79%), pneumococcal serotype 8 (95% vs 88%), and meningococcal serogroup C (53% vs 53%), all p > 0.05. In a post hoc analysis, no meaningful differences were observed between groups in the proportion of responders when stratified by age category or lymphocyte count. CONCLUSIONS: DMF-treated patients mount an immune response to recall, neoantigens, and T-cell-independent antigens, which was comparable with that of IFN-treated patients and provided adequate seroprotection. CLINICALTRIALSGOV IDENTIFIER: NCT02097849. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that patients with RRMS treated with DMF respond to vaccinations comparably with IFN-treated patients.
ABSTRACT
The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.
Subject(s)
Biomarkers/analysis , Immunity, Active , Chromatography, Liquid , Consensus Development Conferences as Topic , Drug Tolerance , Guidelines as Topic , Ligands , Mass Spectrometry , PharmacokineticsABSTRACT
AIM: Daclizumab high yield process (HYP) is a humanized IgG1 monoclonal antibody that binds to the α-subunit of the interleukin-2 receptor and is being developed for treatment of multiple sclerosis (MS). This manuscript characterized the pharmacokinetic-pharmacodynamic (PK-PD) relationships of daclizumab HYP in subjects with MS. METHODS: Approximately 1400 subjects and 7000 PD measurements for each of three biomarkers [CD25 occupancy, CD56bright natural killer (NK) cell count, regulatory T cell (Treg) count] from four clinical trials were analyzed using non-linear mixed effects modelling. Evaluated regimens included 150 or 300 mg subcutaneous (s.c.) every 4 weeks. RESULTS: CD25 occupancy was characterized using a sigmoidal maximum response (Emax ) model. Upon daclizumab HYP treatment, CD25 saturation was rapid with complete saturation occurring after approximately 7 h and maintained when daclizumab HYP serum concentration was ≥5 mg l-1 . After the last 150 mg s.c. dose, unoccupied CD25 returned to baseline levels in approximately 24 weeks, with daclizumab HYP serum concentration approximately ≤1 mgl-1 1L. CD56bright NK cell expansion was characterized using an indirect response model. Following daclizumab HYP 150 mg s.c. every 4 weeks, expansion plateaus approximately at week 36, at which the average maximum expansion ratio is 5.2. After the last dose, CD56bright NK cells gradually declined to baseline levels within 24 weeks. Treg reduction was characterized by a sigmoidal Emax model. Average maximum reduction of 60% occurred approximately 4 days post 150 mg s.c. dose. After the last dose, Tregs were projected to return to baseline levels in approximately 20 weeks. CONCLUSIONS: Robust PK-PD models of CD25 occupancy, CD56bright NK cell expansion and Treg reduction by daclizumab HYP were developed to characterize its key pharmacodynamic effects in the target patient population.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/pharmacokinetics , CD56 Antigen/drug effects , Interleukin-2 Receptor alpha Subunit/drug effects , Killer Cells, Natural/drug effects , Multiple Sclerosis/blood , T-Lymphocytes, Regulatory/drug effects , Antibodies, Monoclonal, Humanized/blood , Clinical Trials as Topic , Daclizumab , Humans , Killer Cells, Natural/cytology , Lymphocyte Count , Nonlinear Dynamics , T-Lymphocytes, Regulatory/cytologyABSTRACT
Regulatory T cells (Tregs) mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction, and instability are implicated in the pathogenesis of numerous autoimmune diseases. There is considerable interest in therapeutic modulation of the IL-2 pathway to treat autoimmunity, facilitate transplantation tolerance, or potentiate tumor immunotherapy. Daclizumab is a humanized mAb that binds the IL-2 receptor a subunit (IL-2R a or CD25) and prevents IL-2 binding. In this study, we investigated the effect of daclizumab-mediated CD25 blockade on Treg homeostasis in patients with relapsing-remitting multiple sclerosis. We report that daclizumab therapy caused an ~50% decrease in Tregs over a 52-wk period. Remaining FOXP3+ cells retained a demethylated Treg-specific demethylated region in the FOXP3 promoter, maintained active cell cycling, and had minimal production of IL-2, IFN- g, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2R bg signaling induced STAT5 phosphorylation and sustained FOXP3 expression. Treg declines were not associated with daclizumab-related clinical benefit or cutaneous adverse events. These results demonstrate that Treg phenotype and lineage stability can be maintained in the face of CD25 blockade.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Immunoglobulin G/therapeutic use , Immunosuppressive Agents/therapeutic use , Interleukin-2 Receptor alpha Subunit/antagonists & inhibitors , Interleukin-2/immunology , Multiple Sclerosis, Relapsing-Remitting/drug therapy , T-Lymphocytes, Regulatory/drug effects , CD4 Lymphocyte Count , Cell Cycle/drug effects , Cell Cycle/genetics , Daclizumab , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin Receptor Common gamma Subunit/immunology , Interleukin-17/biosynthesis , Interleukin-17/immunology , Interleukin-2/biosynthesis , Interleukin-2/blood , Interleukin-2 Receptor alpha Subunit/immunology , Interleukin-2 Receptor beta Subunit/immunology , Multiple Sclerosis, Relapsing-Remitting/immunology , Phosphorylation , Promoter Regions, Genetic , STAT5 Transcription Factor/metabolism , Self Tolerance/drug effects , Self Tolerance/immunology , T-Lymphocytes, Regulatory/immunologyABSTRACT
Immunogenicity assessments in response to drug treatment are commonly performed using a tiered approach strategy. All samples are initially tested in a screening assay followed by the evaluation of the screened positive samples in a confirmatory assay. Percent inhibition of signal intensity by the competing unlabeled drug in a confirmatory assay is typically used to measure the specificity of antidrug binding activity in samples, and has been successfully applied to most immunogenicity assays. However, the percent inhibition approach may not be suitable in cases where broadly distributed and high percent inhibition values are observed in drug-naïve subjects or when persistent operator-dependent differences in assay performance are encountered. Herein, we present the case studies faced with such challenges and provide appropriate solutions by introducing two novel data analysis methods: (1) Reference Delta, and (2) Reference Percent Inhibition, - in which relative-to-baseline signal inhibition is calculated for each sample. These novel methods significantly improve the confirmatory assay's ability to detect the samples positive for antidrug antibodies (ADA), especially when challenges are encountered using the traditional percent inhibition approach. Furthermore, both methods can be implemented in parallel with the percent inhibition method, enabling not only confirmation of ADA specificity, but also providing additional insights about the relevance of this antidrug binding activity to drug treatment.
Subject(s)
Antibodies/chemistry , Biological Assay/methods , Drug Tolerance/immunology , Antibodies/immunology , Antibody Formation/immunology , Humans , Immunogenetic Phenomena , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Longitudinal StudiesABSTRACT
The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection.
Subject(s)
Fibroblasts/immunology , Immunity, Innate/immunology , Interferon Type I/immunology , Nitric Oxide/metabolism , Signal Transduction , Animals , Antiviral Agents , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibroblasts/virology , Herpesvirus 1, Human/drug effects , Humans , Immunity, Innate/drug effects , Interferon Regulatory Factor-1/metabolism , Interferon Regulatory Factor-3/deficiency , Interferon Regulatory Factor-3/metabolism , Interferon-Stimulated Gene Factor 3, gamma Subunit/deficiency , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Mice , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Poly I-C/pharmacology , RNA, Double-Stranded/metabolism , Signal Transduction/drug effects , Solubility/drug effects , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , Virus Replication/drug effectsABSTRACT
It has been established that a total of 250 microg of monoclonal anti-mouse CD3 F(ab')(2) fragments, administered daily (50 microg per dose), induces remission of diabetes in the non-obese diabetic (NOD) mouse model of autoimmune diabetes by preventing beta cells from undergoing further autoimmune attack. We evaluated lower-dose regimens of monoclonal anti-CD3 F(ab')(2) in diabetic NOD mice for their efficacy and associated pharmacodynamic (PD) effects, including CD3-T-cell receptor (TCR) complex modulation, complete blood counts and proportions of circulating CD4(+), CD8(+) and CD4(+) FoxP3(+) T cells. Four doses of 2 microg (total dose 8 microg) induced 53% remission of diabetes, similarly to the 250 microg dose regimen, whereas four doses of 1 microg induced only 16% remission. While the 250 microg dose regimen produced nearly complete and sustained modulation of the CD3 -TCR complex, lower doses, spaced 3 days apart, which induced similar remission rates, elicited patterns of transient and partial modulation. In treated mice, the proportions of circulating CD4(+) and CD8(+) T cells decreased, whereas the proportions of CD4(+) FoxP3(+) T cells increased; these effects were transient. Mice with greater residual beta-cell function, estimated using blood glucose and C-peptide levels at the initiation of treatment, were more likely to enter remission than mice with more advanced disease. Thus, lower doses of monoclonal anti-CD3 that produced only partial and transient modulation of the CD3-TCR complex induced remission rates comparable to higher doses of monoclonal anti-CD3. Accordingly, in a clinical setting, lower-dose regimens may be efficacious and may also improve the safety profile of therapy with monoclonal anti-CD3, potentially including reductions in cytokine release-related syndromes and maintenance of pathogen-specific immunosurveillance during treatment.
Subject(s)
Antibodies, Monoclonal/administration & dosage , CD3 Complex/drug effects , Diabetes Mellitus, Type 1/drug therapy , Animals , Antibodies, Monoclonal/pharmacokinetics , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cell Separation , Diabetes Mellitus, Type 1/immunology , Dose-Response Relationship, Drug , Female , Flow Cytometry , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Pancreas/drug effects , Pancreas/pathology , Remission Induction , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunologyABSTRACT
Virus infection elicits a robust innate antiviral response dominated by the production of type 1 IFN. In nonprofessional innate immune cells such as fibroblasts, type 1 IFN is rapidly produced following the recognition of viral dsRNA and the subsequent activation of the constitutively expressed transcription factor IFN regulatory factor 3 (IRF3). Although origin, localization, and length are factors in mediating dsRNA recognition and binding by cellular dsRNA-binding proteins, the biological significance of differential dsRNA binding is unclear, since the subsequent signaling pathways converge on IRF3. In this study, we show a dsRNA length-dependent activation of IRFs, IFNs, and IFN-stimulated genes in mouse fibroblasts. The length dependence was exacerbated in fibroblasts deficient in the mitochondria-associated adaptor IFN-beta promoter stimulator 1 and IRF3, suggesting that antiviral gene induction mediated by short and long dsRNA molecules is predominantly IFN-beta promoter stimulator 1 and IRF3 dependent and independent, respectively. Furthermore, we provide evidence of an innate antiviral response in fibroblasts in the absence of both IRF3 and type 1 IFN induction. Even with these key modulators missing, a 60-90% inhibition of virus replication was observed following 24-h treatment with short or long dsRNA molecules, respectively. These data provide evidence of a novel antiviral pathway that is dependent on dsRNA length, but independent of the type 1 IFN system.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Fibroblasts/immunology , Interferon Regulatory Factor-3/immunology , Interferon-beta/immunology , RNA, Double-Stranded/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , DNA Virus Infections/genetics , DNA Virus Infections/immunology , DNA Virus Infections/virology , Fibroblasts/metabolism , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Immunity, Innate/immunology , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-beta/metabolism , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/immunology , RNA Virus Infections/genetics , RNA Virus Infections/immunology , RNA Virus Infections/virology , RNA, Double-Stranded/metabolism , Signal Transduction/genetics , Signal Transduction/immunology , Viruses/immunologyABSTRACT
T helper (Th) 2 cells selectively express IL-21 in addition to the classic Th2 cytokines IL-4, IL-5, and IL-13. In contrast to these clustered Th2 cell cytokine genes, the IL-21 gene resides on a different chromosome and is not coordinately regulated by the same locus control region that directs the expression of other Th2 cytokines. We demonstrate that the proximal promoter of IL-21 controls its Th-cell-subset-specific expression through the action of NFATc2 and T-bet. Whereas NFATc2 directly binds to and activates transcription of the IL-21 promoter in Th2 cells, T-bet represses IL-21 transcription by inhibiting the binding of NFATc2 to the promoter in Th1 cells. These data suggest that there are multiple mechanisms by which Th-cell-subset-specific cytokine genes are regulated.
Subject(s)
DNA-Binding Proteins/immunology , Gene Expression Regulation , Interleukins/immunology , Nuclear Proteins/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/immunology , Transcription Factors/immunology , Animals , Base Sequence , Cell Line , DNA-Binding Proteins/genetics , Humans , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interleukins/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Mutagenesis, Site-Directed , NFATC Transcription Factors , Nuclear Proteins/genetics , Promoter Regions, Genetic , Sequence Alignment , T-Box Domain Proteins , T-Lymphocyte Subsets/cytology , T-Lymphocytes, Helper-Inducer/cytology , Transcription Factors/geneticsABSTRACT
Interleukin-21 (IL-21) is the newest member of the common gamma-chain family of cytokines, which includes IL-2, IL-4, IL-7, IL-9, IL-13, and IL-15. Its private receptor, IL-21R, has been shown to activate the Janus kinase/signal transducers and activators of transcription signaling pathway upon ligand binding. Initial studies have demonstrated that IL-21 has pleiotropic effects on the proliferation, differentiation, and effector functions of B, T, natural killer, and dendritic cells. More recently, the potential therapeutic capacity of IL-21 in the treatment of cancers has been widely investigated. The biological role of IL-21 in the immune system is complex, as IL-21 has been shown to have the ability to both promote and inhibit immune responses. Overall, the current data point to IL-21 being a novel immunomodulatory cytokine, whose regulation of any given immune response is highly dependent on the surrounding environmental context.
Subject(s)
Interleukins/physiology , Receptors, Interleukin/physiology , Animals , B-Lymphocytes/physiology , Dendritic Cells/physiology , Humans , Interleukin-21 Receptor alpha Subunit , Interleukins/genetics , Killer Cells, Natural/physiology , Mice , Receptors, Interleukin/genetics , Receptors, Interleukin-21 , Signal Transduction/physiology , T-Lymphocytes/physiologyABSTRACT
Cytokines play an important role in regulating the development and homeostasis of B cells by controlling their viability. In this study, we show that the recently described T cell-derived cytokine IL-21 induces the apoptosis of resting primary murine B cells. In addition, the activation of primary B cells with IL-4, LPS, or anti-CD40 Ab does not prevent IL-21-mediated apoptosis. The induction of apoptosis by IL-21 correlates with a down-regulation in the expression of Bcl-2 and Bcl-x(L), two antiapoptotic members of the Bcl-2 family. Furthermore, the reconstitution of Bcl-x(L) or Bcl-2 expression protects primary B cells from IL-21-induced apoptosis. In addition, a short-term preactivation of B cells with anti-CD40 Ab confers protection from IL-21-mediated apoptosis through the up-regulation of Bcl-x(L). These studies reveal a novel pathway that mediates B cell apoptosis via the IL-21R and suggest that IL-21 may play a role in regulating B cell homeostasis.