RESUMO
Recombinant adeno-associated virus (AAV) vectors have emerged as the preferred platform for gene therapy of rare human diseases. Despite the clinical promise, host immune responses to AAV vectors and transgene remain a major barrier to the development of successful AAV-based human gene therapies. Here, we assessed the human innate immune response to AAV9, the preferred serotype for AAV-mediated gene therapy of the CNS. We showed that AAV9 induced type I interferon (IFN) and IL-6 responses in human blood from healthy donors. This innate response was replicated with AAV6, required full viral particles, but was not observed in every donor. Depleting CpG motifs from the AAV transgene or inhibiting TLR9 signaling reduced type I IFN response to AAV9 in responding donors, highlighting the importance of TLR9-mediated DNA sensing for the innate response to AAV9. Remarkably, we further demonstrated that only seropositive donors with preexisting antibodies to AAV9 capsid mounted an innate immune response to AAV9 in human whole blood and that anti-AAV9 antibodies were necessary and sufficient to promote type I IFN release and plasmacytoid dendritic (pDC) cell activation in response to AAV9. Thus, our study reveals a previously unidentified requirement for AAV preexisting antibodies for TLR9-mediated type I IFN response to AAV9 in human blood.
Assuntos
Dependovirus , Vetores Genéticos , Imunidade Humoral , Interferon Tipo I , Receptor Toll-Like 9 , Humanos , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/genética , Dependovirus/genética , Dependovirus/imunologia , Interferon Tipo I/imunologia , Vetores Genéticos/genética , Imunidade Inata , Células Dendríticas/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Terapia Genética , Interleucina-6/sangue , Interleucina-6/imunologiaRESUMO
T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.
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Linfócitos T CD4-Positivos , Células Dendríticas , Humanos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Medição de Risco , Técnicas de Cocultura , Ativação Linfocitária/imunologia , Leucócitos Mononucleares/imunologia , Proliferação de Células , Linfócitos T CD8-Positivos/imunologia , Avaliação Pré-Clínica de Medicamentos , Produtos Biológicos/imunologia , Produtos Biológicos/efeitos adversos , Anticorpos/imunologia , Células CultivadasRESUMO
Immunotoxicology/immunosafety science is rapidly evolving, with novel modalities and immuno-oncology among the primary drivers of new tools and technologies. The Immunosafety Working Group of IQ/DruSafe sought to better understand some of the key challenges in immunosafety evaluation, gaps in the science, and current limitations in methods and data interpretation. A survey was developed to provide a baseline understanding of the needs and challenges faced in immunosafety assessments, the tools currently being applied across the industry, and the impact of feedback received from regulatory agencies. This survey also focused on current practices and challenges in conducting the T-cell-dependent antibody response (TDAR) and the cytokine release assay (CRA). Respondents indicated that ICH S8 guidance was insufficient for the current needs of the industry portfolio of immunomodulators and novel modalities and should be updated. Other challenges/gaps identified included translation of nonclinical immunosafety assessments to the clinic, and lack of relevant nonclinical species and models in some cases. Key areas of emerging science that will add future value to immunotoxicity assessments include development of additional in vitro and microphysiological system models, as well as application of humanized mouse models. Efforts are ongoing in individual companies and consortia to address some of these gaps and emerging science.
Assuntos
Fatores Imunológicos , Humanos , Animais , Inquéritos e Questionários , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/toxicidade , Citocinas/imunologia , Medição de Risco , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Toxicidade/métodosRESUMO
Biologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals and its one-day training course gives experts and newcomers across academia, industry, and regulatory agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we report the discussions that took place at the EIP's 14th Symposium, held in April 2023. The topics covered included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assessment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated with new modalities, which were discussed in a dedicated session.
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Produtos Biológicos , Humanos , Anticorpos , Desenvolvimento de Medicamentos , Medição de RiscoRESUMO
Introduction: Cellular immune responses against AAV vector capsid represent an obstacle for successful gene therapy. Previous studies have used overlapping peptides spanning the entire capsid sequence to identify T cell epitopes recognized by AAV-specific CD8+ T cells. However, the repertoire of peptides naturally displayed by HLA class I molecules for CD8 T cell recognition is unknown. Methods: Using mRNA transfected monocyte-derived dendritic cells (MDDCs) and MHC-associated peptide proteomics (MAPPs), we identified the HLA class I immunopeptidomes of AAV2, AAV6 and AAV9 capsids. MDDCs were isolated from a panel of healthy donors that have diverse alleles across the US population. mRNA-transfected MDDCs were lysed, the peptide:HLA complexes immunoprecipitated, and peptides eluted and analyzed by mass spectrometry. Results: We identified 65 AAV capsid-derived peptides loaded on HLA class I molecules of mRNA transfected monocyte derived dendritic cells. The HLA class I peptides are distributed along the entire capsid and more than 60% are contained within HLA class II clusters. Most of the peptides are organized as single species, however we identified twelve clusters containing at least 2 peptides of different lengths. Only 9% of the identified peptides have been previously identified as T cell epitopes, demonstrating that the immunogenicity potential for the vast majority of the AAV HLA class I immunopeptidome remains uncharacterized. In contrast, 12 immunogenic epitopes identified before were not found to be naturally processed in our study. Remarkably, 11 naturally presented AAV peptides were highly conserved among the three serotypes analyzed suggesting the possibility of cross-reactive AAV-specific CD8 T cells. Discussion: This work is the first comprehensive study identifying the naturally displayed HLA class I peptides derived from the capsid of AAVs. The results from this study can be used to generate strategies to assess immunogenicity risk and cross-reactivity among serotypes during gene therapies.
Assuntos
Proteínas do Capsídeo , Epitopos de Linfócito T , Capsídeo , Alelos , RNA MensageiroRESUMO
A survey conducted by the Therapeutic Product Immunogenicity (TPI) community within the American Association of Pharmaceutical Scientists (AAPS) posed questions to the participants on their immunogenicity risk assessment strategies prior to clinical development. The survey was conducted in 2 phases spanning 5 years, and queried information about in silico algorithms and in vitro assay formats for immunogenicity risk assessments and how the data were used to inform early developability effort in discovery, chemistry, manufacturing and control (CMC), and non-clinical stages of development. The key findings representing the trends from a majority of the participants included the use of high throughput in silico algorithms, human immune cell-based assays, and proteomics based outputs, as well as specialized assays when therapeutic mechanism of action could impact risk assessment. Additional insights into the CMC-related risks could also be gathered with the same tools to inform future process development and de-risk critical quality attributes with uncertain and unknown risks. The use of the outputs beyond supporting early development activities was also noted with participants utilizing the risk assessments to drive their clinical strategy and streamline bioanalysis.
Assuntos
Desenvolvimento de Medicamentos , Humanos , Consenso , Medição de Risco/métodosRESUMO
BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.
Assuntos
Cardiopatias , Insuficiência Cardíaca , Relaxina , Animais , Feminino , Humanos , Camundongos , Gravidez , Ratos , Cardiomegalia/tratamento farmacológico , Cardiopatias/tratamento farmacológico , Insuficiência Cardíaca/tratamento farmacológico , Relaxina/farmacologia , Relaxina/uso terapêutico , Relaxina/metabolismo , Volume SistólicoRESUMO
Introduction: Gene therapies are using Adeno-associated viruses (AAVs) as vectors, but immune responses against the capsids pose challenges to their efficiency and safety. Helper T cell recognition of capsid-derived peptides bound to human leukocyte antigen (HLA) class II molecules is an essential step in the AAV-specific adaptive immunity. Methods: Using MHC-associated peptide proteomics, we identified the HLA-DR and HLA-DQ immunopeptidomes of the capsid proteins of three different AAV serotypes (AAV2, AAV6, and AAV9) from a panel of healthy donors selected to represent a majority of allele usage. Results: The identified sequences span the capsids of all serotypes, with AAV2 having the highest peptide count. For all the serotypes, multiple promiscuous peptides were identified and displayed by both HLA-DR and -DQ. However, despite high sequence homology, there were few identical peptides among AAV2, AAV6, and AAV9 immunopeptidomes, and none were promiscuous. Discussion: Results from this work represent a comprehensive immunopeptidomics research of potential CD4+ T cell epitopes and provide the basis for immunosurveillance efforts for safer and more efficient AAV-based gene therapies.
Assuntos
Proteínas do Capsídeo , Capsídeo , Humanos , Proteínas do Capsídeo/genética , Dependovirus , Peptídeos/metabolismo , Antígenos HLA/metabolismoRESUMO
Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.
Assuntos
Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacosRESUMO
Treatment-emergent antidrug antibodies (TE-ADA) pose a major challenge to the development of biotherapeutics. The antidrug antibody responses are highly orchestrated and involve many types of immune cells and biological processes. Biological drug internalization and processing by antigen-presenting cells (APCs) are two initial and critical steps in the cascade of events leading to T cell-dependent ADA production. The assays thus far described in literature to evaluate immunogenicity potential/risk as a function of APC activity mainly focus on internalization of labeled drug candidates in vitro. Herein, we describe a high-throughput Förster Resonance Energy Transfer (FRET)-based assay for assessing both internalization and processing using CD14+ monocyte-derived dendritic cells (DCs) as APCs. Antigen-binding fragment F(ab')2 against IgG fragment crystallizable gamma (Fcγ) was labeled with the activatable FRET pair TAMRA-QSY7 as a universal probe for antibodies and proteins with a fragment crystallizable (Fc) domain. The assay was qualified using six mAbs of known clinical immunogenicity and one IgG1 isotype antibody using Design of Experiment (DoE). Correlation analysis of internalization and clinical immunogenicity data showed that this FRET-based internalization assay was able to detect clinically immunogenic antibodies. This method provides a tool for analyzing/screening the immunogenicity risk of biological candidates by assessing one of the critical components of the ADA formation process within the broader context of an immunogenicity risk assessment strategy.
Assuntos
Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Células Dendríticas/fisiologia , Fenômenos Imunogenéticos , Sondas Moleculares , Transferência Ressonante de Energia de Fluorescência , Lisossomos/metabolismo , Medição de RiscoRESUMO
Many autoimmune diseases are characterized by the production of autoantibodies. The current view is that CD4+ T follicular helper (Tfh) cells are the main subset regulating autoreactive B cells. Here we report a CXCR5+PD1+ Tfh subset of CD8+ T cells whose development and function are negatively modulated by Stat5. These CD8+ Tfh cells regulate the germinal center B cell response and control autoantibody production, as deficiency of Stat5 in CD8 T cells leads to an increase of CD8+ Tfh cells, resulting in the breakdown of B cell tolerance and concomitant autoantibody production. CD8+ Tfh cells share similar gene signatures with CD4+ Tfh, and require CD40L/CD40 and TCR/MHCI interactions to deliver help to B cells. Our study thus highlights the diversity of follicular T cell subsets that contribute to the breakdown of B-cell tolerance.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica/imunologia , Receptor de Morte Celular Programada 1/imunologia , Receptores CXCR5/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos B/metabolismo , Antígenos CD40/genética , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Tolerância Imunológica/genética , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Fator de Transcrição STAT5/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Immunomodulatory monoclonal IgG1 antibodies developed for cancer and autoimmune disease have an inherent risk of systemic release of pro-inflammatory cytokines. In vitro cytokine release assays are currently used to predict cytokine release syndrome (CRS) risk, but the validation of these preclinical tools suffers from the limited number of characterized CRS-inducing IgG1 antibodies and the poor understanding of the mechanisms regulating cytokine release. Here, we incubated human whole blood from naïve healthy volunteers with four monoclonal IgG1 antibodies with different proven or predicted capacity to elicit CRS in clinic and measured cytokine release using a multiplex assay. We found that, in contrast to anti-CD52 antibodies (Campath-1H homolog) that elicited high level of multiple inflammatory cytokines from human blood cells in vitro, other IgG1 antibodies with CRS-inducing potential consistently induced release of a single tested cytokine, interferon (IFN)-γ, with a smaller magnitude than Campath. IFN-γ expression was observed as early as 2-4 h after incubation, mediated by natural killer cells, and dependent upon tumor necrosis factor and FcγRIII. Importantly, the magnitude of the IFN-γ response elicited by IgG1 antibodies with CRS-inducing potential was determined by donor FcγRIIIa-V158F polymorphism. Overall, our results highlight the importance of FcγRIIIa-dependent IFN-γ release in preclinical cytokine release assay for the prediction of CRS risk associated with therapeutic IgG1 antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Interferon gama/imunologia , Receptores de IgG/imunologia , Alemtuzumab/imunologia , Alemtuzumab/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Citocinas/sangue , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Imunoensaio/métodos , Imunoglobulina G/uso terapêutico , Interferon gama/sangue , Interferon gama/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Polimorfismo Genético/imunologia , Prognóstico , Receptores de IgG/genética , SíndromeRESUMO
Unfortunately, after publication of this article [1], it was noticed that corrections to the legends of Figs. 1 and 2 were not correctly incorporated. The correct legends can be seen below.
RESUMO
BACKGROUND: Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. RESULTS: Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. CONCLUSIONS: LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Imunoglobulina G/imunologia , Neoplasias/imunologia , Animais , Linhagem Celular , Cricetulus , Feminino , Humanos , Macaca fascicularis , Camundongos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Manipulation of host cellular pathways is a strategy employed by gammaherpesviruses, including mouse gammaherpesvirus 68 (MHV68), in order to negotiate a chronic infection. Ataxia-telangiectasia mutated (ATM) plays a unique yet incompletely understood role in gammaherpesvirus infection, as it has both proviral and antiviral effects. Chronic gammaherpesvirus infection is poorly controlled in a host with global ATM insufficiency, whether the host is a mouse or a human. In contrast, ATM facilitates replication, reactivation, and latency establishment of several gammaherpesviruses in vitro, suggesting that ATM is proviral in the context of infected cell cultures. The proviral role of ATM is also evident in vivo, as myeloid-specific ATM expression facilitates MHV68 reactivation during the establishment of viral latency. In order to better understand the complex relationship between host ATM and gammaherpesvirus infection, we depleted ATM specifically in B cells, a cell type critical for chronic gammaherpesvirus infection. B cell-specific ATM deficiency attenuated the establishment of viral latency due to compromised differentiation of ATM-deficient B cells. Further, we found that during long-term infection, peritoneal B-1b, but not related B-1a, B cells display the highest frequency of gammaherpesvirus infection. While ATM expression did not affect gammaherpesvirus tropism for B-1 B cells, B cell-specific ATM expression was necessary to support viral reactivation from peritoneal cells during long-term infection. Thus, our study reveals a role of ATM as a host factor that promotes chronic gammaherpesvirus infection of B cells.IMPORTANCE Gammaherpesviruses infect a majority of the human population and are associated with cancer, including B cell lymphomas. ATM is a unique host kinase that has both proviral and antiviral roles in the context of gammaherpesvirus infection. Further, there is insufficient understanding of the interplay of these roles in vivo during chronic infection. In this study, we show that ATM expression by splenic B cells is required for efficient establishment of gammaherpesvirus latency. We also show that ATM expression by peritoneal B cells is required to facilitate viral reactivation during long-term infection. Thus, our study defines a proviral role of B cell-specific ATM expression during chronic gammaherpesvirus infection.
Assuntos
Linfócitos B/metabolismo , Infecções por Herpesviridae/virologia , Rhadinovirus/crescimento & desenvolvimento , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/biossíntese , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Infecções por Herpesviridae/imunologia , Interações Hospedeiro-Patógeno/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peritônio/citologia , Peritônio/imunologia , Rhadinovirus/imunologia , Baço/citologia , Baço/imunologia , Ativação Viral/genéticaRESUMO
Gammaherpesviruses are cancer-associated pathogens that establish life-long infection in most adults. Insufficiency of Ataxia-Telangiectasia mutated (ATM) kinase leads to a poor control of chronic gammaherpesvirus infection via an unknown mechanism that likely involves a suboptimal antiviral response. In contrast to the phenotype in the intact host, ATM facilitates gammaherpesvirus reactivation and replication in vitro. We hypothesized that ATM mediates both pro- and antiviral activities to regulate chronic gammaherpesvirus infection in an immunocompetent host. To test the proposed proviral activity of ATM in vivo, we generated mice with ATM deficiency limited to myeloid cells. Myeloid-specific ATM deficiency attenuated gammaherpesvirus infection during the establishment of viral latency. The results of our study uncover a proviral role of ATM in the context of gammaherpesvirus infection in vivo and support a model where ATM combines pro- and antiviral functions to facilitate both gammaherpesvirus-specific T cell immune response and viral reactivation in vivo.
Assuntos
Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Células Mieloides/virologia , Ativação Viral , Adulto , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Doença Crônica , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
TCR signal strength during priming is a key determinant of CD4 T cell activation, but its impact on effector CD4 T functions in vivo remains unclear. In this study, we compare the functionality of CD4 T cell responses induced by peptides displaying varying binding half-lives with MHC class II before and after influenza virus infection. Although significant quantitative and qualitative differences in CD4 T cell responses were observed before infection between mice vaccinated with low- or high-stability peptides, both mice mounted robust early Th1 effector cytokine responses upon influenza challenge. However, only effector CD4 T cells induced by low-stability peptides proliferated and produced IL-17A after influenza challenge. In contrast, effector T cells elicited by higher-stability peptides displayed a terminally differentiated phenotype and divided poorly. This defective proliferation was T cell intrinsic but could not be attributed to a reduced expression of lymph node homing receptors. Instead, we found that CD4 T cells stimulated with higher-stability peptides exhibited decreased responsiveness to low levels of Ag presentation. Our study reveals the critical role of TCR signal strength during priming for the function and Ag sensitivity of effector CD4 T cells during viral challenge.
Assuntos
Ativação Linfocitária/imunologia , Infecções por Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Células Th1/imunologia , Animais , Formação de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Proliferação de Células , Células Cultivadas , Interleucina-17/biossíntese , Camundongos , Camundongos Transgênicos , Orthomyxoviridae/imunologia , Transdução de Sinais/imunologiaRESUMO
Gammaherpesviruses, such as Epstein-Barr virus (EBV), are ubiquitous cancer-associated pathogens that interact with DNA damage response, a tumor suppressor network. Chronic gammaherpesvirus infection and pathogenesis in a DNA damage response-insufficient host are poorly understood. Ataxia-telangiectasia (A-T) is associated with insufficiency of ataxia-telangiectasia mutated (ATM), a critical DNA damage response kinase. A-T patients display a pattern of anti-EBV antibodies suggestive of poorly controlled EBV replication; however, parameters of chronic EBV infection and pathogenesis in the A-T population remain unclear. Here we demonstrate that chronic gammaherpesvirus infection is poorly controlled in an animal model of A-T. Intriguingly, in spite of a global increase in T cell activation and numbers in wild-type (wt) and ATM-deficient mice in response to mouse gammaherpesvirus 68 (MHV68) infection, the generation of an MHV68-specific immune response was altered in the absence of ATM. Our finding that ATM expression is necessary for an optimal adaptive immune response against gammaherpesvirus unveils an important connection between DNA damage response and immune control of chronic gammaherpesvirus infection, a connection that is likely to impact viral pathogenesis in an ATM-insufficient host.
Assuntos
Ataxia Telangiectasia/imunologia , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Gammaherpesvirinae , Infecções por Herpesviridae/imunologia , Ativação Linfocitária/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/deficiência , Linhagem Celular , Proteínas de Ligação a DNA/deficiência , Citometria de Fluxo , Infecções por Herpesviridae/enzimologia , Camundongos , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/deficiência , Linfócitos T/imunologia , Proteínas Supressoras de Tumor/deficiênciaRESUMO
Chemokines regulate T cell trafficking into secondary lymphoid organs and migration across endothelial cells in response to inflammatory signals. The small guanosine triphosphatase Rap1 is a critical regulator of chemokine signaling in T cells, but how chemokines activate Rap1 has been unclear. A study showed that Abl family tyrosine kinases were essential for chemokine-induced Rap1 activation, T cell polarization, and migration. Abl family kinases promoted Rap1 activation by phosphorylating the adaptor protein human enhancer of filamentation 1 (HEF1), thus establishing a critical Abl-HEF1-Rap1 signaling axis for chemokine-induced T cell migration.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/imunologia , Quimiocinas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Proteínas rap1 de Ligação ao GTP/imunologia , Humanos , Modelos Imunológicos , FosforilaçãoRESUMO
The maintenance of B-cell anergy is essential to prevent the production of autoantibodies and autoimmunity. However, B-cell extrinsic mechanisms that regulate B-cell anergy remain poorly understood. We previously demonstrated that regulatory T (Treg) cells are necessary for the maintenance of B-cell anergy. We now show that in Treg-cell-deficient mice, helper T cells are necessary and sufficient for loss of B-cell tolerance/anergy. In addition, we show that the absence of Treg cells is associated with an increase in the proportion of CD4(+) cells that express GL7 and correlated with an increase in germinal center follicular helper T (GC-T(FH) ) cells. These GC-T(FH) cells, but not those from Treg-cell-sufficient hosts, were sufficient to drive antibody production by anergic B cells. We propose that a function of Treg cells is to prevent the expansion of T(FH) cells, especially GC-T(FH) cells, which support autoantibody production.