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1.
J Virol ; 94(4)2020 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-31776272

RESUMEN

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and, to a lesser extent, cycling T cells. Virion-packaged Vpr is released in target cells shortly after entry, suggesting it is required in the early phase of infection. Previously, we described REAF (RNA-associated early-stage antiviral factor; RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without an intact vpr gene is more highly restricted by REAF and, using delivery by virus-like particles (VLPs), that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells, and we detected, by coimmunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts quickly during the early phase of replication and induces the degradation of REAF within 30 min of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localization and interaction with cullin 4A-DBB1 (DCAF1) E3 ubiquitin ligase are required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, that impedes HIV infection in macrophages.IMPORTANCE For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages or to explain why Vpr is carried in the virus particle. Here, we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one that inhibits virus replication during reverse transcription. REAF is degraded by Vpr within 30 min of virus entry in a manner dependent on the nuclear localization of Vpr and its interaction with the cell's protein degradation machinery.


Asunto(s)
Antivirales/metabolismo , VIH-1/metabolismo , Replicación Viral/fisiología , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/fisiología , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Productos del Gen vpr/metabolismo , Productos del Gen vpr/fisiología , Células HEK293 , Infecciones por VIH/virología , VIH-1/fisiología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Macrófagos/metabolismo , Cultivo Primario de Células , Ubiquitina-Proteína Ligasas/metabolismo , Virión/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo
2.
Haematologica ; 101(4): 482-90, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26802053

RESUMEN

Due to the lack of specificity for tumor antigens, allogeneic T-cell therapy is associated with graft-versus-host disease. Enhancing the anti-tumor specificity while reducing the graft-versus-host disease risk of allogeneic T cells has remained a research focus. In this study, we demonstrate that the introduction of 'dominant' T-cell receptors into primary murine T cells can suppress the expression of endogenous T-cell receptors in a large proportion of the gene-modified T cells. Adoptive transfer of allogeneic T cells expressing a 'dominant' T-cell receptor significantly reduced the graft-versus-host toxicity in recipient mice. Using two bone marrow transplant models, enhanced anti-tumor activity was observed in the presence of reduced graft-versus-host disease. However, although transfer of T-cell receptor gene-modified allogeneic T cells resulted in the elimination of antigen-positive tumor cells and improved the survival of treated mice, it was associated with accumulation of T cells expressing endogenous T-cell receptors and the development of delayed graft-versus-host disease. The in-vivo deletion of the engineered T cells, mediated by endogenous mouse mammary tumor virus MTV8 and MTV9, abolished graft-versus-host disease while retaining significant anti-tumor activity of adoptively transferred T cells. Together, this study shows that the in-vitro selection of allogeneic T cells expressing high levels of a 'dominant' T-cell receptor can lower acute graft-versus-host disease and enhance anti-tumor activity of adoptive cell therapy, while the in-vivo outgrowth of T cells expressing endogenous T-cell receptors remains a risk factor for the delayed onset of graft-versus-host disease.


Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Inmunoterapia Adoptiva/métodos , Virus del Tumor Mamario del Ratón/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Trasplante de Médula Ósea/métodos , Línea Celular Tumoral , Femenino , Expresión Génica , Genes Dominantes , Vectores Genéticos/inmunología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Humanos , Depleción Linfocítica/métodos , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Receptores de Antígenos de Linfocitos T/genética , Análisis de Supervivencia , Linfocitos T/citología , Linfocitos T/inmunología , Transgenes , Trasplante Homólogo , Irradiación Corporal Total
3.
Respir Res ; 17(1): 140, 2016 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-27793198

RESUMEN

BACKGROUND: Some COPD patients are more susceptible to exacerbations than others. Mechanisms underlying these differences in susceptibility are not well understood. We hypothesized that altered cell mediated immune responses may underlie a propensity to suffer from frequent exacerbations in COPD. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from 24 stable COPD patients, eight frequent exacerbators (≥3 diary-card exacerbations/year) and 16 infrequent exacerbators (< 3 diary-card exacerbations/year). Detailed multi-parameter flow cytometry was used to study differences in innate and adaptive systemic immune function between frequent and infrequently exacerbating COPD patients. RESULTS: The 24 COPD patients had a mean (SD) age of 76.3 (9.4) years and FEV1 1.43 (0.60)L, 53.3 (18.3)% predicted. PBMCs of frequent exacerbators (FE) contained lower frequencies of CD4+ T central memory cells (CD4+ Tcm) compared to infrequent exacerbators (IE) (FE = 18.7 %; IE = 23.9 %; p = 0.035). This observation was also apparent in absolute numbers of CD4+ Tcm cells (FE = 0.17 × 10^6/mL; IE = 0.25 × 10^6/mL; p = 0.035). PBMCs of FE contained a lower frequency of CD8+ T effector memory cells expressing HLA-DR (Human Leukocyte Antigen - D Related) compared to IE COPD patients (FE = 22.7 %; IE = 31.5 %; p = 0.007). CONCLUSION: Differences in the adaptive systemic immune system might associate with exacerbation susceptibility in the 'frequent exacerbator' COPD phenotype. These differences include fewer CD4+ T central memory cells and CD8+ T effector memory cells. TRIAL REGISTRATION: Not applicable.


Asunto(s)
Inmunidad Celular , Inmunidad Innata , Leucocitos Mononucleares/inmunología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Anciano , Anciano de 80 o más Años , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Estudios Transversales , Progresión de la Enfermedad , Femenino , Humanos , Memoria Inmunológica , Modelos Lineales , Pulmón/inmunología , Pulmón/fisiopatología , Masculino , Análisis Multivariante , Fenotipo , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Índice de Severidad de la Enfermedad
4.
J Immunol ; 193(4): 1567-77, 2014 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-25000983

RESUMEN

The mechanisms whereby different vaccines may expand distinct Ag-specific T cell clonotypes or induce disparate degrees of protection are incompletely understood. We found that several delivery modes of a model retroviral Ag, including natural infection, preferentially expanded initially rare high-avidity CD4(+) T cell clonotypes, known to mediate protection. In contrast, the same Ag vectored by human adenovirus serotype 5 induced clonotypic expansion irrespective of avidity, eliciting a predominantly low-avidity response. Nonselective clonotypic expansion was caused by relatively weak adenovirus serotype 5-vectored Ag presentation and was reproduced by replication-attenuated retroviral vaccines. Mechanistically, the potency of Ag presentation determined the speed and, consequently, completion of the CD4(+) T cell response. Whereas faster completion retained the initial advantage of high-avidity clonotypes, slower completion permitted uninhibited accumulation of low-avidity clonotypes. These results highlighted the importance of Ag presentation patterns in determining the clonotypic composition of vaccine-induced T cell responses and ultimately the efficacy of vaccination.


Asunto(s)
Afinidad de Anticuerpos/inmunología , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Selección Clonal Mediada por Antígenos/inmunología , Virus de la Leucemia Murina de Friend/inmunología , Productos del Gen env/inmunología , Animales , Presentación de Antígeno/inmunología , Proliferación Celular , Perfilación de la Expresión Génica , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunología , Receptores OX40/genética , Vacunas Virales/inmunología
6.
Int J Syst Evol Microbiol ; 63(Pt 11): 4294-4302, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23832969

RESUMEN

Two Gram-negative, facultatively anaerobic, rod-shaped bacteria, strains EF212(T) and PS125(T), were isolated from the octocorals Eunicea fusca and Plexaura sp., respectively. EF212(T) was isolated from a specimen of E. fusca collected off the coast of Florida, USA, and PS125(T) was isolated from a specimen of Plexaura sp. collected off the coast of Bimini, Bahamas. Analysis of the nearly full-length 16S rRNA gene sequences showed that these novel strains were most closely related to Endozoicomonas montiporae CL-33(T), E. elysicola MKT110(T) and E. numazuensis HC50(T) (EF212(T), 95.6-97.2 % identity; PS125(T), 95.1-96.4 % identity). DNA-DNA hybridization values among EF212(T), PS125(T), E. montiporae LMG 24815(T) and E. elysicola KCTC 12372(T) were far below the 70 % cut-off, with all values for duplicate measurements being less than 35 %. Both EF212(T) and PS125(T) required NaCl for growth and showed optimal growth at 2-3 % NaCl, 22-30 °C and pH 8.0. The predominant cellular fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c), C16 : 0 and C14 : 0. The DNA G+C content of EF212(T) was 48.6 mol% and that of PS125(T) was 47.5 mol%. In addition to the genotypic differences observed between the two novel strains and related type strains, phenotypic and chemotaxonomic experiments also revealed differences between strains. Thus, strains EF212(T) and PS125(T) represent novel species of the genus Endozoicomonas, for which the names Endozoicomonas euniceicola sp. nov. and Endozoicomonas gorgoniicola sp. nov., respectively, are proposed. The type strains are EF212(T) ( = NCCB 100458(T) = DSM 26535(T)) for Endozoicomonas euniceicola sp. nov. and PS125(T) ( = NCCB 100438(T) = CECT 8353(T)) for Endozoicomonas gorgoniicola sp. nov. An emended description of the genus Endozoicomonas is also provided to encompass differences observed in the results of genotypic, chemotaxonomic and phenotypic tests compared from the original and amended genus descriptions.


Asunto(s)
Antozoos/microbiología , Gammaproteobacteria/clasificación , Filogenia , Animales , Técnicas de Tipificación Bacteriana , Bahamas , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Florida , Gammaproteobacteria/genética , Gammaproteobacteria/aislamiento & purificación , Genotipo , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ubiquinona/química
7.
J Virol ; 83(21): 11211-22, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19692462

RESUMEN

Retroviruses can establish persistent infection despite induction of a multipartite antiviral immune response. Whether collective failure of all parts of the immune response or selective deficiency in one crucial part underlies the inability of the host to clear retroviral infections is currently uncertain. We examine here the contribution of virus-specific CD4(+) T cells in resistance against Friend virus (FV) infection in the murine host. We show that the magnitude and duration of the FV-specific CD4(+) T-cell response is directly proportional to resistance against acute FV infection and subsequent disease. Notably, significant protection against FV-induced disease is afforded by FV-specific CD4(+) T cells in the absence of a virus-specific CD8(+) T-cell or B-cell response. Enhanced spread of FV infection in hosts with increased genetic susceptibility or coinfection with Lactate dehydrogenase-elevating virus (LDV) causes a proportional increase in the number of FV-specific CD4(+) T cells required to control FV-induced disease. Furthermore, ultimate failure of FV/LDV coinfected hosts to control FV-induced disease is accompanied by accelerated contraction of the FV-specific CD4(+) T-cell response. Conversely, an increased frequency or continuous supply of FV-specific CD4(+) T cells is both necessary and sufficient to effectively contain acute infection and prevent disease, even in the presence of coinfection. Thus, these results suggest that FV-specific CD4(+) T cells provide significant direct protection against acute FV infection, the extent of which critically depends on the ratio of FV-infected cells to FV-specific CD4(+) T cells.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Virus de la Leucemia Murina de Friend/inmunología , Leucemia Experimental/inmunología , Activación de Linfocitos/inmunología , Infecciones por Retroviridae/inmunología , Infecciones Tumorales por Virus/inmunología , Animales , Virus Elevador de Lactato Deshidrogenasa/inmunología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Receptor de Interferón gamma
8.
Front Immunol ; 7: 126, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27148254

RESUMEN

We tested whether multi-parameter immune phenotyping before or after renal -transplantation can predict the risk of rejection episodes. Blood samples collected before and weekly for 3 months after transplantation were analyzed by multi-parameter flow cytometry to define 52 T cell and 13 innate lymphocyte subsets in each sample, producing more than 11,000 data points that defined the immune status of the 28 patients included in this study. Principle component analysis suggested that the patients with histologically confirmed rejection episodes segregated from those without rejection. Protein death 1 (PD-1)-expressing subpopulations of regulatory and conventional T cells had the greatest influence on the principal component segregation. We constructed a statistical tool to predict rejection using a support vector machine algorithm. The algorithm correctly identified 7 out of 9 patients with rejection, and 14 out of 17 patients without rejection. The immune profile before transplantation was most accurate in determining the risk of rejection, while changes of immune parameters after transplantation were less accurate in discriminating rejection from non-rejection. The data indicate that pretransplant immune subset analysis has the potential to identify patients at risk of developing rejection episodes, and suggests that the proportion of PD1-expressing T cell subsets may be a key indicator of rejection risk.

9.
Cancer Res ; 75(13): 2641-52, 2015 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-25904681

RESUMEN

A key challenge in the field of T-cell immunotherapy for cancer is creating a suitable platform for promoting differentiation of effector cells while at the same time enabling self-renewal needed for long-term memory. Although transfer of less differentiated memory T cells increases efficacy through greater expansion and persistence in vivo, the capacity of such cells to sustain effector functions within immunosuppressive tumor microenvironments may still be limiting. We have therefore directly compared the impact of effector versus memory differentiation of therapeutic T cells in tumor-bearing mice by introducing molecular switches that regulate cell fate decisions via mTOR. Ectopic expression of RAS homolog enriched in brain (RHEB) increased mTORC1 signaling, promoted a switch to aerobic glycolysis, and increased expansion of effector T cells. By rapidly infiltrating tumors, RHEB-transduced T cells significantly reduced the emergence of immunoedited escape variants. In contrast, expression of proline-rich Akt substrate of 40 kDa (PRAS40) inhibited mTORC1, promoted quiescence, and blocked tumor infiltration. Fate mapping studies following transient expression of PRAS40 demonstrated that mTORC1(low) T cells made no contribution to initial tumor control but instead survived to become memory cells proficient in generating recall immunity. Our data support the design of translational strategies for generating heterogeneous T-cell immunity against cancer, with the appropriate balance between promoting effector differentiation and self-renewal. Unlike pharmacologic inhibitors, the genetic approach described here allows for upregulation as well as inhibition of the mTORC1 pathway and is highly selective for the therapeutic T cells without affecting systemic mTORC1 functions.


Asunto(s)
Linfocitos T CD8-positivos/fisiología , Memoria Inmunológica/genética , Inmunoterapia Adoptiva/métodos , Neoplasias Experimentales/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Línea Celular Tumoral , Humanos , Memoria Inmunológica/inmunología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Proteínas de Unión al GTP Monoméricas/biosíntesis , Proteínas de Unión al GTP Monoméricas/genética , Complejos Multiproteicos/inmunología , Neoplasias Experimentales/prevención & control , Neuropéptidos/biosíntesis , Neuropéptidos/genética , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro , Serina-Treonina Quinasas TOR/inmunología , Transducción Genética
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