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1.
J Surg Res ; 296: 742-750, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38368775

RESUMO

INTRODUCTION: Epstein-Barr virus-associated gastric cancer (EBVaGC) may be a meaningful biomarker for potential benefit from immunotherapy. Further investigation is needed to characterize the immune landscape of EBVaGC. We assessed our institutional frequency of surgically treated EBVaGC and analyzed the immunologic biomarker profile and tumor-infiltrating lymphocyte (TIL) phenotypes of a series of EBVaGC compared to non-EBVaGC cases. METHODS: Available tissue samples from all patients with biopsy-confirmed gastric adenocarcinoma who underwent resection with curative intent from 2012 to 2020 at our institution were collected. In situ hybridization was used to assess EBV status; multiplex immunohistochemistry was performed to assess mismatch repair status, Programmed Death-Ligand 1 (PD-L1) expression, and phenotypic characterization of TILs. RESULTS: Sixty-eight samples were included in this study. EBVaGC was present in 3/68 (4%) patients. Among all patients, 27/68 (40%) had positive PD-L1 expression; two of three (67%) EBVaGC patients exhibited positive PD-L1 expression. Compared to non-EBVaGC, EBV-positive tumors showed 5-fold to 10-fold higher density of TILs in both tumor and stroma and substantially elevated CD8+ T cell to Tregulatory cell ratio. The memory subtypes of CD8+ and CD4+ T cells were upregulated in EBVaGC tumors and stromal tissue compared to non-EBVaGC. CONCLUSIONS: The incidence of surgically resected EBVaGC at our center was 4%. EBVaGC tumors harbor elevated levels of TILs, including memory subtypes, within both tumor and tumor-related stroma. Robust TIL presence and upregulated PD-L1 positivity in EBVaGC may portend promising responses to immunotherapy agents. Further investigation into routine EBV testing and TIL phenotype of patients with gastric cancer to predict response to immunotherapy may be warranted.


Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Gástricas , Humanos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Infecções por Vírus Epstein-Barr/complicações , Antígeno B7-H1/metabolismo , Neoplasias Gástricas/patologia , Biomarcadores
2.
PLoS Pathog ; 17(11): e1010019, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34780571

RESUMO

Gammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into detectable lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on properties of latent infection, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::ßla), to enumerate both the cellular distribution and frequency of LANA gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::ßla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::ßla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential LANA expression, and that a primary function of the viral cyclin is to promote LANA expression during latency, a state associated with ex vivo reactivation competence.


Assuntos
Antígenos Virais/metabolismo , Ciclinas/metabolismo , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Virais/metabolismo , Ativação Viral , Replicação Viral , Animais , Antígenos Virais/genética , Ciclinas/genética , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Infecção Persistente , Proteínas Virais/genética , Latência Viral
3.
J Virol ; 95(14): e0007921, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33910955

RESUMO

RNA polymerase III (pol III) transcribes multiple noncoding RNAs (ncRNAs) that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including those of the gammaherpesviruses (γHVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during γHV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 (γHV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via reverse transcription-quantitative PCR (RT-qPCR). We further measured endogenous ncRNA expression at single-cell resolution by flow cytometry. These studies demonstrated that lytic infection with γHV68 increased the transcription from multiple host and viral pol III promoters and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis. IMPORTANCE Gammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small noncoding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III-dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral noncoding RNA expression within the infected cell.


Assuntos
Gammaherpesvirinae/fisiologia , Regulação Viral da Expressão Gênica , Regiões Promotoras Genéticas , RNA Polimerase III/genética , Latência Viral , Gammaherpesvirinae/genética , Células HEK293 , Humanos , Luciferases/genética , Reação em Cadeia da Polimerase , RNA não Traduzido/metabolismo , Transfecção , Proteínas Virais/genética
4.
J Immunol ; 204(8): 2295-2307, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32179637

RESUMO

MHC class II (MHCII) expression is usually restricted to APC but can be expressed by cancer cells. We examined the effect of cancer cell-specific MHCII (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis lung carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, whereas LLC tumors are resistant. RNA sequencing analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, whereas resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII and converted tumors from anti-PD-1 sensitive to anti-PD-1 resistant. This was associated with lower levels of Th1 cytokines, decreased T cell infiltration, increased B cell numbers, and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, which provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.


Assuntos
Adenocarcinoma de Pulmão/terapia , Antígenos de Histocompatibilidade Classe II/genética , Neoplasias Pulmonares/terapia , Proteínas Nucleares/genética , Transativadores/genética , Microambiente Tumoral/genética , Adenocarcinoma de Pulmão/imunologia , Animais , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe II/imunologia , Neoplasias Pulmonares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/imunologia , Receptor de Morte Celular Programada 1/imunologia , Transativadores/imunologia , Microambiente Tumoral/imunologia
5.
Immunology ; 162(1): 68-83, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32931017

RESUMO

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Tolerância Imunológica/imunologia , Memória Imunológica/imunologia , Animais , Antígenos/imunologia , Autoimunidade/imunologia , Ciclo Celular/imunologia , Proliferação de Células/fisiologia , Citocinas/imunologia , Reparo do DNA/imunologia , Feminino , Inflamação/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transcrição Gênica/imunologia
6.
Cancer Immunol Immunother ; 70(4): 989-1000, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33097963

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) has a heterogeneous tumor microenvironment (TME) comprised of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages, neutrophils, regulatory T cells, and myofibroblasts. The precise mechanisms that regulate the composition of the TME and how they contribute to radiotherapy (RT) response remain poorly understood. In this study, we analyze changes in immune cell populations and circulating chemokines in patient samples and animal models of pancreatic cancer to characterize the immune response to radiotherapy. Further, we identify STAT3 as a key mediator of immunosuppression post-RT. We found granulocytic MDSCs (G-MDSCs) and neutrophils to be increased in response to RT in murine and human PDAC samples. We also found that RT-induced STAT3 phosphorylation correlated with increased MDSC infiltration and proliferation. Targeting STAT3 using an anti-sense oligonucleotide in combination with RT circumvented RT-induced MDSC infiltration, enhanced the proportion of effector T cells, and improved response to RT. In addition, STAT3 inhibition contributed to the remodeling of the PDAC extracellular matrix when combined with RT, resulting in decreased collagen deposition and fibrotic tissue formation. Collectively, our data provide evidence that targeting STAT3 in combination with RT can mitigate the pro-tumorigenic effects of RT and improve tumor response.


Assuntos
Carcinoma Ductal Pancreático/radioterapia , Raios gama , Células Supressoras Mieloides/imunologia , Oligonucleotídeos Antissenso/genética , Neoplasias Pancreáticas/radioterapia , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Apoptose , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Feminino , Humanos , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Células Supressoras Mieloides/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Prognóstico , Fator de Transcrição STAT3/genética , Linfócitos T Reguladores/imunologia , Células Tumorais Cultivadas , Microambiente Tumoral
7.
J Transl Med ; 19(1): 43, 2021 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-33485341

RESUMO

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, cetuximab, in the management of this cancer. The degree of response to EGFR inhibitors measured by tumor shrinkage varies widely among HNSCC patients, and the biological mechanisms that underlie therapeutic heterogeneity amongst HNSCC patients remain ill-defined. METHODS: EGFR-dependent human and murine HNSCC cell lines were treated with the EGFR/ERBB inhibitors, gefitinib and AZD8931, and submitted to RNAseq, GSEA, and qRT-PCR. Conditioned media was analyzed by ELISA and Luminex assays. Murine HNSCC tumors were stained for T cell markers by immunofluorescence. Primary HSNCC patient specimens treated with single agent cetuximab were stained with Vectra multispectral immunofluorescence. RESULTS: The transcriptional reprogramming response to EGFR/ERBB-specific TKIs was measured in a panel of EGFR-dependent human HNSCC cell lines and interferon (IFN) α and γ responses identified as top-ranked TKI-induced pathways. Despite similar drug sensitivity, responses among 7 cell lines varied quantitatively and qualitatively, especially regarding the induced chemokine and cytokine profiles. Of note, the anti-tumorigenic chemokine, CXCL10, and the pro-tumorigenic factor, IL6, exhibited wide-ranging and non-overlapping induction. Similarly, AZD8931 exerted potent growth inhibition, IFNα/IFNγ pathway activation, and CXCL10 induction in murine B4B8 HNSCC cells. AZD8931 treatment of immune-competent mice bearing orthotopic B4B8 tumors increased CD8 + T cell content and the therapeutic response was abrogated in nu/nu mice relative to BALB/c mice. Finally, Vectra 3.0 analysis of HNSCC patient tumors prior to and after 3-4 weeks of single agent cetuximab treatment revealed increased CD8 + T cell content in specimens from patients exhibiting a therapeutic response relative to non-responders. CONCLUSIONS: The findings reveal heterogeneous, tumor cell-intrinsic, EGFR/ERBB inhibitor-induced IFN pathway activation in HNSCC and suggest that individual tumor responses to oncogene-targeted agents are a sum of direct growth inhibitory effects and variably-induced participation of host immune cells.


Assuntos
Antineoplásicos , Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Receptores ErbB , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Interferons , Camundongos , Camundongos Endogâmicos BALB C , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico
8.
PLoS Pathog ; 15(6): e1007849, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31166996

RESUMO

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level.


Assuntos
Gammaherpesvirinae/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Infecções por Herpesviridae/metabolismo , RNA Mensageiro/biossíntese , RNA não Traduzido/biossíntese , RNA Viral/biossíntese , Replicação Viral/fisiologia , Animais , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Humanos , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA não Traduzido/genética , RNA Viral/genética
9.
J Immunol ; 202(2): 460-475, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30552164

RESUMO

Aging of established antiviral T cell memory can foster a series of progressive adaptations that paradoxically improve rather than compromise protective CD8+ T cell immunity. We now provide evidence that this gradual evolution, the pace of which is contingent on the precise context of the primary response, also impinges on the molecular mechanisms that regulate CD8+ memory T cell (TM) homeostasis. Over time, CD8+ TM generated in the wake of an acute infection with the natural murine pathogen lymphocytic choriomeningitis virus become more resistant to apoptosis and acquire enhanced cytokine responsiveness without adjusting their homeostatic proliferation rates; concurrent metabolic adaptations promote increased CD8+ TM quiescence and fitness but also impart the reacquisition of a partial effector-like metabolic profile; and a gradual redistribution of aging CD8+ TM from blood and nonlymphoid tissues to lymphatic organs results in CD8+ TM accumulations in bone marrow, splenic white pulp, and, particularly, lymph nodes. Altogether, these data demonstrate how temporal alterations of fundamental homeostatic determinants converge to render aged CD8+ TM poised for greater recall responses.


Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/fisiologia , Memória Imunológica/imunologia , Linfonodos/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Animais , Antígenos Virais/imunologia , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética
10.
J Immunol ; 200(1): 3-22, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29255085

RESUMO

Mass cytometry has revolutionized the study of cellular and phenotypic diversity, significantly expanding the number of phenotypic and functional characteristics that can be measured at the single-cell level. This high-dimensional analysis platform has necessitated the development of new data analysis approaches. Many of these algorithms circumvent traditional approaches used in flow cytometric analysis, fundamentally changing the way these data are analyzed and interpreted. For the beginner, however, the large number of algorithms that have been developed, as well as the lack of consensus on best practices for analyzing these data, raise multiple questions: Which algorithm is the best for analyzing a dataset? How do different algorithms compare? How can one move beyond data visualization to gain new biological insights? In this article, we describe our experiences as recent adopters of mass cytometry. By analyzing a single dataset using five cytometry by time-of-flight analysis platforms (viSNE, SPADE, X-shift, PhenoGraph, and Citrus), we identify important considerations and challenges that users should be aware of when using these different methods and common and unique insights that can be revealed by these different methods. By providing annotated workflow and figures, these analyses present a practical guide for investigators analyzing high-dimensional datasets. In total, these analyses emphasize the benefits of integrating multiple cytometry by time-of-flight analysis algorithms to gain complementary insights into these high-dimensional datasets.


Assuntos
Diagnóstico por Imagem/métodos , Citometria de Fluxo/métodos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Animais , Separação Celular , Biologia Computacional , Citometria de Fluxo/instrumentação , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Imunofenotipagem , Guias de Prática Clínica como Assunto
11.
J Virol ; 92(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29298882

RESUMO

Gammaherpesviruses are common viruses associated with lifelong infection and increased disease risk. Reactivation from latency aids the virus in maintaining infection throughout the life of the host and is responsible for a wide array of disease outcomes. Previously, we demonstrated that the virus-encoded cyclin (v-cyclin) of murine gammaherpesvirus 68 (γHV68) is essential for optimal reactivation from latency in normal mice but not in mice lacking the host tumor suppressor p18INK4c (p18). Whether p18 plays a cell-intrinsic or -extrinsic role in constraining reactivation remains unclear. Here, we generated recombinant viruses in which we replaced the viral cyclin with the cellular p18INK4c gene (p18KI) for targeted expression of p18, specifically within infected cells. We find that the p18KI virus is similar to the cyclin-deficient virus (cycKO) in lytic infection, establishment of latency, and infected cell reservoirs. While the cycKO virus is capable of reactivation in p18-deficient mice, expression of p18 from the p18KI virus results in a profound reactivation defect. These data demonstrate that p18 limits reactivation within latently infected cells, functioning in a cell-intrinsic manner. Further, the p18KI virus showed greater attenuation of virus-induced lethal pneumonia than the cycKO virus, indicating that p18 could further restrict γHV68 pathogenesis even in p18-sufficient mice. These studies demonstrate that host p18 imposes the requirement for the viral cyclin to reactivate from latency by functioning in latently infected cells and that p18 expression is associated with decreased disease, thereby identifying p18 as a compelling host target to limit chronic gammaherpesvirus pathogenesis.IMPORTANCE Gammaherpesviruses are ubiquitous viruses associated with multiple malignancies. The propensity to cycle between latency and reactivation results in an infection that is never cleared and often difficult to treat. Understanding the balance between latency and reactivation is integral to treating gammaherpesvirus infection and associated disease outcomes. This work characterizes the role of a novel inhibitor of reactivation, host p18INK4c, thereby bringing more clarity to a complex process with significant outcomes for infected individuals.


Assuntos
Inibidor de Quinase Dependente de Ciclina p18 , Gammaherpesvirinae , Regulação Viral da Expressão Gênica , Pneumonia Viral , Ativação Viral , Latência Viral , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p18/biossíntese , Inibidor de Quinase Dependente de Ciclina p18/genética , Gammaherpesvirinae/genética , Gammaherpesvirinae/metabolismo , Gammaherpesvirinae/patogenicidade , Técnicas de Silenciamento de Genes , Camundongos , Pneumonia Viral/genética , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia
12.
J Virol ; 92(7)2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29343566

RESUMO

Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.IMPORTANCE SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.


Assuntos
Regulação Viral da Expressão Gênica , Proteínas de Fluorescência Verde , Infecções por Herpesviridae , Doenças dos Macacos , Fases de Leitura Aberta , Varicellovirus , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/veterinária , Macaca mulatta , Doenças dos Macacos/genética , Doenças dos Macacos/metabolismo , Doenças dos Macacos/patologia , Varicellovirus/genética , Varicellovirus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
Kidney Int ; 94(6): 1127-1140, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30249452

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent inherited nephropathy. To date, therapies alleviating the disease have largely focused on targeting abnormalities in renal epithelial cell signaling. ADPKD has many hallmarks of cancer, where targeting T cells has brought novel therapeutic interventions. However, little is known about the role and therapeutic potential of T cells in ADPKD. Here, we used an orthologous ADPKD model, Pkd1 p.R3277C (RC), to begin to define the role of T cells in disease progression. Using flow cytometry, we found progressive increases in renal CD8+ and CD4+ T cells, correlative with disease severity, but with selective activation of CD8+ T cells. By immunofluorescence, T cells specifically localized to cystic lesions and increased levels of T-cell recruiting chemokines (CXCL9/CXCL10) were detected by qPCR/in situ hybridization in the kidneys of mice, patients, and ADPKD epithelial cell lines. Importantly, immunodepletion of CD8+ T cells from one to three months in C57Bl/6 Pkd1RC/RC mice resulted in worsening of ADPKD pathology, decreased apoptosis, and increased proliferation compared to IgG-control, consistent with a reno-protective role of CD8+ T cells. Thus, our studies suggest a functional role for T cells, specifically CD8+ T cells, in ADPKD progression. Hence, targeting this pathway using immune-oncology agents may represent a novel therapeutic approach for ADPKD.


Assuntos
Imunidade Adaptativa , Linfócitos T CD8-Positivos/microbiologia , Rim Policístico Autossômico Dominante/imunologia , Animais , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais , Feminino , Humanos , Imunoterapia/métodos , Rim/citologia , Rim/imunologia , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/terapia , Transdução de Sinais/imunologia , Canais de Cátion TRPP/genética
15.
J Immunol ; 195(10): 4973-85, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26453755

RESUMO

NK cells are innate lymphoid cells important for immune surveillance, identifying and responding to stress, infection, and/or transformation. Whereas conventional NK (cNK) cells circulate systemically, many NK cells reside in tissues where they appear to be poised to locally regulate tissue function. In the present study, we tested the contribution of tissue-resident NK (trNK) cells to tissue homeostasis by studying ischemic injury in the mouse kidney. Parabiosis experiments demonstrate that the kidney contains a significant fraction of trNK cells under homeostatic conditions. Kidney trNK cells developed independent of NFIL3 and T-bet, and they expressed a distinct cell surface phenotype as compared with cNK cells. Among these, trNK cells had reduced asialo-GM1 (AsGM1) expression relative to cNK cells, a phenotype observed in trNK cells across multiple organs and mouse strains. Strikingly, anti-AsGM1 Ab treatment, commonly used as an NK cell-depleting regimen, resulted in a robust and selective depletion of cNKs, leaving trNKs largely intact. Using this differential depletion, we tested the relative contribution of cNK and trNK cells in ischemic kidney injury. Whereas anti-NK1.1 Ab effectively depleted both trNK and cNK cells and protected against ischemic/reperfusion injury, anti-AsGM1 Ab preferentially depleted cNK cells and failed to protect against injury. These data demonstrate unanticipated specificity of anti-AsGM1 Ab depletion on NK cell subsets and reveal a new approach to study the contributions of cNK and trNK cells in vivo. In total, these data demonstrate that trNK cells play a key role in modulating local responses to ischemic tissue injury in the kidney and potentially other organs.


Assuntos
Anticorpos/farmacologia , Gangliosídeo G(M1)/imunologia , Isquemia/imunologia , Nefropatias/imunologia , Rim/irrigação sanguínea , Rim/imunologia , Células Matadoras Naturais/imunologia , Animais , Gangliosídeo G(M1)/antagonistas & inibidores , Isquemia/patologia , Rim/patologia , Nefropatias/patologia , Células Matadoras Naturais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout
16.
J Virol ; 89(21): 10821-31, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26292318

RESUMO

UNLABELLED: Gammaherpesviruses (GHVs) carry homologs of cellular genes, including those encoding a viral cyclin that promotes reactivation from latent infection. The viral cyclin has reduced sensitivity to host cyclin-dependent kinase inhibitors in vitro; however, the in vivo significance of this is unclear. Here, we tested the genetic requirement for the viral cyclin in mice that lack the host inhibitors p27(Kip1) and p18(INK4c), two cyclin-dependent kinase inhibitors known to be important in regulating B cell proliferation and differentiation. While the viral cyclin was essential for reactivation in wild-type mice, strikingly, it was dispensable for reactivation in mice lacking p27(Kip1) and p18(INK4c). Further analysis revealed that genetic ablation of only p18(INK4c) alleviated the requirement for the viral cyclin for reactivation from latency. p18(INK4c) regulated reactivation in a dose-dependent manner so that the viral cyclin was dispensable in p18(INK4c) heterozygous mice. Finally, treatment of wild-type cells with the cytokine BAFF, a known attenuator of p18(INK4c) function in B lymphocytes, was also able to bypass the requirement for the viral cyclin in reactivation. These data show that the gammaherpesvirus viral cyclin functions specifically to bypass the cyclin-dependent kinase inhibitor p18(INK4c), revealing an unanticipated specificity between a GHV cyclin and a single cyclin-dependent kinase inhibitor. IMPORTANCE: The gammaherpesviruses (GHVs) cause lifelong infection and can cause chronic inflammatory diseases and cancer, especially in immunosuppressed individuals. Many GHVs encode a conserved viral cyclin that is required for infection and disease. While a common property of the viral cyclins is that they resist inhibition by normal cellular mechanisms, it remains unclear how important it is that the GHVs resist this inhibition. We used a mouse GHV that either contained or lacked a viral cyclin to test whether the viral cyclin lost importance when these inhibitory pathways were removed. These studies revealed that the viral cyclin was required for optimal function in normal mice but that it was no longer required following removal or reduced function of a single cellular inhibitor. These data define a very specific role for the viral cyclin in bypassing one cellular inhibitor and point to new methods to intervene with viral cyclins.


Assuntos
Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Ciclinas/metabolismo , Gammaherpesvirinae/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Fator Ativador de Células B/farmacologia , Inibidor de Quinase Dependente de Ciclina p18/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p18/deficiência , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Ciclinas/farmacologia , Primers do DNA/genética , Citometria de Fluxo , Gammaherpesvirinae/genética , Immunoblotting , Camundongos , Testes de Neutralização , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Viral/efeitos dos fármacos
17.
Proc Natl Acad Sci U S A ; 109(41): E2784-93, 2012 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-22988108

RESUMO

Recent studies have demonstrated dramatic shifts in metabolic supply-and-demand ratios during inflammation, a process resulting in localized tissue hypoxia within inflammatory lesions ("inflammatory hypoxia"). As part of the adaptive immune response, T cells are recruited to sites of inflammatory hypoxia. Given the profound effects of hypoxia on gene regulation, we hypothesized that T-cell differentiation is controlled by hypoxia. To pursue this hypothesis, we analyzed the transcriptional consequences of ambient hypoxia (1% oxygen) on a broad panel of T-cell differentiation factors. Surprisingly, these studies revealed selective, robust induction of FoxP3, a key transcriptional regulator for regulatory T cells (Tregs). Studies of promoter binding or loss- and gain-of-function implicated hypoxia-inducible factor (HIF)-1α in inducing FoxP3. Similarly, hypoxia enhanced Treg abundance in vitro and in vivo. Finally, Treg-intrinsic HIF-1α was required for optimal Treg function and Hif1a-deficient Tregs failed to control T-cell-mediated colitis. These studies demonstrate that hypoxia is an intrinsic molecular cue that promotes FoxP3 expression, in turn eliciting potent anti-inflammatory mechanisms to limit tissue damage in conditions of reduced oxygen availability.


Assuntos
Fatores de Transcrição Forkhead/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia , Inflamação/genética , Mucosa Intestinal/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Colite/genética , Colite/metabolismo , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Interleucina-1/farmacologia , Mucosa Intestinal/patologia , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia
18.
Eur J Immunol ; 43(2): 521-32, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23172374

RESUMO

Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4(+) CD8(+) thymocyte using an Ikaros null CD4(-) CD8(-) mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR ß gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry nonfunctional rearrangements on both alleles of their TCR α loci. Retroviral reintroduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αßTCR(+) cells. The process is RAG dependent, requires switch/sucrose nonfermentable chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/nucleosome remodeling and deacetylase complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αßTCR(+) CD4(+) CD8(+) thymocyte transition.


Assuntos
Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Linfócitos Nulos/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Timoma/genética , Alelos , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Linfócitos Nulos/metabolismo , Linfócitos Nulos/patologia , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
19.
FASEB J ; 27(6): 2207-19, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23413361

RESUMO

Acute lung injury (ALI) is characterized by alveolar injury and uncontrolled inflammation. Since most cases of ALI resolve spontaneously, understanding the endogenous mechanisms that promote ALI resolution is important to developing effective therapies. Previous studies have implicated extracellular adenosine signaling in tissue adaptation and wound healing. Therefore, we hypothesized a functional contribution for the endogenous production of adenosine during ALI resolution. As a model, we administered intratracheal LPS and observed peak lung injury at 3 d, with resolution by d 14. Treatment with pegylated adenosine-deaminase to enhance extracellular adenosine breakdown revealed impaired ALI resolution. Similarly, genetic deletion of cd73, the pacemaker for extracellular adenosine generation, was associated with increased mortality (0% wild-type and 40% in cd73(-/-) mice; P<0.05) and failure to resolve ALI adequately. Studies of inflammatory cell trafficking into the lungs during ALI resolution revealed that regulatory T cells (Tregs) express the highest levels of CD73. While Treg numbers in cd73(-/-) mice were similar to controls, cd73-deficient Tregs had attenuated immunosuppressive functions. Moreover, adoptive transfer of cd73-deficient Tregs into Rag(-/-) mice emulated the observed phenotype in cd73(-/-) mice, while transfer of wild-type Tregs was associated with normal ALI resolution. Together, these studies implicate CD73-dependent adenosine generation in Tregs in promoting ALI resolution.


Assuntos
5'-Nucleotidase/fisiologia , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/metabolismo , Adenosina/fisiologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , 5'-Nucleotidase/deficiência , Lesão Pulmonar Aguda/patologia , Adenosina/deficiência , Adenosina Desaminase/administração & dosagem , Transferência Adotiva , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Linfócitos T Reguladores/patologia
20.
J Immunol ; 189(9): 4566-73, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23028059

RESUMO

Renal ischemia is among the leading causes of acute kidney injury (AKI). Previous studies have shown that extracellular adenosine is a prominent tissue-protective cue elicited during ischemia, including signaling events through the adenosine receptor 2b (Adora2b). To investigate the functional role of Adora2b signaling in cytokine-mediated inflammatory pathways, we screened wild-type and Adora2b-deficient mice undergoing renal ischemia for expression of a range of inflammatory cytokines. These studies demonstrated a selective and robust increase of TNF-α levels in Adora2b-deficient mice following renal ischemia and reperfusion. Based on these findings, we next sought to understand the contribution of TNF-α on ischemic AKI through a combination of loss- and gain-of-function studies. Loss of TNF-α, through either Ab blockade or study of Tnf-α-deficient animals, resulted in significantly attenuated tissue injury and improved kidney function following renal ischemia. Conversely, transgenic mice with overexpression of TNF-α had significantly pronounced susceptibility to AKI. Furthermore, neutrophil depletion or reconstitution of Adora2b(-/-) mice with Tnf-α-deficient neutrophils rescued their phenotype. In total, these data demonstrate a critical role of adenosine signaling in constraining neutrophil-dependent production of TNF-α and implicate therapies targeting TNF-α in the treatment of ischemic AKI.


Assuntos
Injúria Renal Aguda/imunologia , Injúria Renal Aguda/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptor A2B de Adenosina/fisiologia , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Injúria Renal Aguda/genética , Animais , Modelos Animais de Doenças , Predisposição Genética para Doença , Isquemia/genética , Isquemia/imunologia , Isquemia/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/patologia , Receptor A2B de Adenosina/deficiência , Receptor A2B de Adenosina/genética , Reperfusão/métodos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/deficiência
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