RESUMEN
Background: While megakaryocytes are known for making platelets, recent single-cell RNA sequencing data have revealed subpopulations of megakaryocytes with predicted immunoregulatory and bone marrow niche-supporting roles. Although these studies uncovered interesting information regarding the transcriptional variation of megakaryocytes, the generation, localization, and regulation of these subsets have not yet been studied and therefore remain incompletely understood. Considering the complex organization of the bone marrow, we reasoned that the application of spatial transcriptomic approaches could help dissect megakaryocyte heterogeneity within a spatiotemporal context. Objectives: The aim of this study was to combine spatial context and transcriptomics to assess the heterogeneity of murine bone marrow megakaryocytes in situ at a single-cell level. Methods: Bone marrow sections were obtained from femurs of C57BL/6J mice. Using the murine whole transcriptome array on the Nanostring GeoMx digital spatial profiling platform, we profiled 44 individual megakaryocytes (CD41+ by immunofluorescence) in situ throughout the bone marrow, both adjacent and nonadjacent to the endothelium (directly in contact with vascular endothelial-cadherin-positive cells). Results: Principal component analysis revealed no association between transcriptomic profile and adjacency to the vasculature. However, there was a significant effect of proximal vs distal regions of the bone. Two and 3 genes were found overexpressed in the proximal and distal sides, respectively. Of note, proplatelet basic protein and platelet factor 4, 2 genes associated with platelet production, had higher expression in proximal megakaryocytes. Conclusion: This study indicates a possible effect of spatial location on megakaryocyte heterogeneity and substantiate further interest in investigating megakaryocyte subpopulations in the context of their spatial orientation.
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The persistence of virally infected cells as reservoirs despite effective antiretroviral therapy is a major barrier to an HIV/SIV cure. These reservoirs are predominately contained within cells present in the B cell follicles (BCFs) of secondary lymphoid tissues, a site that is characteristically difficult for most cytolytic antiviral effector cells to penetrate. Here, we identified a population of NK cells in macaque lymph nodes that expressed BCF-homing receptor CXCR5 and accumulated within BCFs during chronic SHIV infection. These CXCR5+ follicular NK cells exhibited an activated phenotype coupled with heightened effector functions and a unique transcriptome characterized by elevated expression of cytolytic mediators (e.g., perforin and granzymes, LAMP-1). CXCR5+ NK cells exhibited high expression of FcγRIIa and FcγRIIIa, suggesting a potential for elevated antibody-dependent effector functionality. Consistently, accumulation of CXCR5+ NK cells showed a strong inverse association with plasma viral load and the frequency of germinal center follicular Th cells that comprise a significant fraction of the viral reservoir. Moreover, CXCR5+ NK cells showed increased expression of transcripts associated with IL-12 and IL-15 signaling compared with the CXCR5- subset. Indeed, in vitro treatment with IL-12 and IL-15 enhanced the proliferation of CXCR5+ granzyme B+ NK cells. Our findings suggest that follicular homing NK cells might be important in immune control of chronic SHIV infection, and this may have important implications for HIV cure strategies.
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Infecciones por VIH , Interleucina-15 , Humanos , Interleucina-12/metabolismo , Interleucina-15/metabolismo , Células Asesinas Naturales , Ganglios Linfáticos , Receptores CXCR5/metabolismoRESUMEN
Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.
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Receptores CXCR6/metabolismo , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral , Animales , Antígeno B7-H1/metabolismo , Comunicación Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Quimiocina CXCL16 , Células Dendríticas/metabolismo , Interleucina-12/metabolismo , Interleucina-15/metabolismo , Ligandos , Ganglios Linfáticos/metabolismo , Melanoma/inmunología , Melanoma/patología , Ratones Endogámicos C57BLRESUMEN
NAFLD is a leading comorbidity in HIV with an exaggerated course compared to the general population. Tesamorelin has been demonstrated to reduce liver fat and prevent fibrosis progression in HIV-associated NAFLD. We further showed that tesamorelin downregulated hepatic gene sets involved in inflammation, tissue repair, and cell division. Nonetheless, effects of tesamorelin on individual plasma proteins pertaining to these pathways are not known. Leveraging our prior randomized-controlled trial and transcriptomic approach, we performed a focused assessment of 9 plasma proteins corresponding to top leading edge genes within differentially modulated gene sets. Tesamorelin led to significant reductions in vascular endothelial growth factor A (VEGFA, log2-fold change - 0.20 ± 0.35 vs. 0.05 ± 0.34, P = 0.02), transforming growth factor beta 1 (TGFB1, - 0.35 ± 0.56 vs. - 0.05 ± 0.43, P = 0.05), and macrophage colony stimulating factor 1 (CSF1, - 0.17 ± 0.21 vs. 0.02 ± 0.20, P = 0.004) versus placebo. Among tesamorelin-treated participants, reductions in plasma VEGFA (r = 0.62, P = 0.006) and CSF1 (r = 0.50, P = 0.04) correlated with a decline in NAFLD activity score. Decreases in TGFB1 (r = 0.61, P = 0.009) and CSF1 (r = 0.64, P = 0.006) were associated with reduced gene-level fibrosis score. Tesamorelin suppressed key angiogenic, fibrogenic, and pro-inflammatory mediators. CSF1, a regulator of monocyte recruitment and activation, may serve as an innovative therapeutic target for NAFLD in HIV. Clinical Trials Registry Number: NCT02196831.
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Proteínas Sanguíneas/metabolismo , Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Sustancias de Crecimiento/farmacología , Infecciones por VIH/complicaciones , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Proteómica/métodos , Transcriptoma , Adolescente , Adulto , Anciano , Método Doble Ciego , Femenino , Hormona Liberadora de Hormona del Crecimiento/farmacología , Infecciones por VIH/sangre , Humanos , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/genética , Placebos , Ensayos Clínicos Controlados Aleatorios como Asunto , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto JovenRESUMEN
BACKGROUND: The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis modulates critical metabolic pathways; however, little is known regarding effects of augmenting pulsatile GH secretion on immune function in humans. This study used proteomics and gene set enrichment analysis to assess effects of a GH releasing hormone (GHRH) analog, tesamorelin, on circulating immune markers and liver tissue in people with human immunodeficiency virus (HIV) (PWH) and nonalcoholic fatty liver disease (NAFLD). METHODS: 92 biomarkers associated with immunity, chemotaxis, and metabolism were measured in plasma samples from 61 PWH with NAFLD who participated in a double-blind, randomized trial of tesamorelin versus placebo for 12 months. Gene set enrichment analysis was performed on serial liver biopsies targeted to immune pathways. RESULTS: Tesamorelin, compared to placebo, decreased interconnected proteins related to cytotoxic T-cell and monocyte activation. Circulating concentrations of 13 proteins were significantly decreased, and no proteins increased, by tesamorelin. These included 4 chemokines (CCL3, CCL4, CCL13 [MCP4], IL8 [CXCL8]), 2 cytokines (IL-10 and CSF-1), and 4 T-cell associated molecules (CD8A, CRTAM, GZMA, ADGRG1), as well as ARG1, Gal-9, and HGF. Network analysis indicated close interaction among the gene pathways responsible for these proteins, with imputational analyses suggesting down-regulation of a closely related cluster of immune pathways. Targeted transcriptomics using liver tissue confirmed a significant end-organ signal of down-regulated immune activation pathways. CONCLUSIONS: Long-term treatment with a GHRH analog reduced markers of T-cell and monocyte/macrophage activity, suggesting that augmentation of the GH axis may ameliorate immune activation in an HIV population with metabolic dysregulation, systemic and end organ inflammation. Clinical Trials Registration. NCT02196831.
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Infecciones por VIH , Enfermedad del Hígado Graso no Alcohólico , Biomarcadores , Método Doble Ciego , Hormona Liberadora de Hormona del Crecimiento , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Humanos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológicoRESUMEN
Among transgender women (TGW), the effects of feminizing hormone therapy use on rectal mucosal (RM) HIV transmission are largely unknown. In this small pilot study, we compared the RM transcriptome in TGW utilizing feminizing hormone therapy with a group of cisgender men who have sex with men (MSM) engaging in condomless receptive anal intercourse (AI) and to a group of cisgender men who had never engaged in AI. There were 498 differentially expressed genes (DEGs) in TGW compared with men who had never engaged in AI, and 154 DEGs compared with the MSM. Among TGW, a unique RM transcriptome was identified that implicated pathways critical for mucosal immune responses, including upregulation of genes that mediate immune cell activation and the production of cytokines and other immune signaling molecules. Furthermore, gene set enrichment analyses identified immune signatures that implicated enrichment of proinflammatory immunological pathways in TGW, specifically involving interferon-α, interleukin-6, and tumor necrosis factor-α signaling, whereas metabolic pathways were shown to be enriched among the cisgender male groups. These findings suggest that TGW have a distinct RM immune environment influenced by the use of feminizing hormones, and consequently, there is an urgent need for further investigation into the immunological effects of gender-affirming hormone therapy and its potential impact on HIV mucosal transmission risk for transgender individuals.
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Infecciones por VIH , Minorías Sexuales y de Género , Personas Transgénero , Femenino , Infecciones por VIH/tratamiento farmacológico , Homosexualidad Masculina , Hormonas , Humanos , Masculino , Proyectos Piloto , TranscriptomaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a common comorbidity among people living with HIV that has a more aggressive course than NAFLD among the general population. In a recent randomized placebo-controlled trial, we demonstrated that the growth hormone-releasing hormone analog tesamorelin reduced liver fat and prevented fibrosis progression in HIV-associated NAFLD over 1 year. As such, tesamorelin is the first strategy that has shown to be effective against NAFLD among the population with HIV. The current study leveraged paired liver biopsy specimens from this trial to identify hepatic gene pathways that are differentially modulated by tesamorelin versus placebo. Using gene set enrichment analysis, we found that tesamorelin increased hepatic expression of hallmark gene sets involved in oxidative phosphorylation and decreased hepatic expression of gene sets contributing to inflammation, tissue repair, and cell division. Tesamorelin also reciprocally up- and downregulated curated gene sets associated with favorable and poor hepatocellular carcinoma prognosis, respectively. Notably, among tesamorelin-treated participants, these changes in hepatic expression correlated with improved fibrosis-related gene score. Our findings inform our knowledge of the biology of pulsatile growth hormone action and provide a mechanistic basis for the observed clinical effects of tesamorelin on the liver.
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Expresión Génica/efectos de los fármacos , Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Infecciones por VIH/complicaciones , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/virología , Carcinoma Hepatocelular/genética , Femenino , Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/farmacología , Infecciones por VIH/genética , Hepatitis/tratamiento farmacológico , Hepatitis/genética , Hepatitis/virología , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Hígado/efectos de los fármacos , Hígado/fisiopatología , Hígado/virología , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Fosforilación Oxidativa/efectos de los fármacos , Placebos , PronósticoRESUMEN
HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac-infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML NBs are hyper-SUMOylated and that PML protein is degraded via the ubiquitin-proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV-1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.
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Redes Reguladoras de Genes , Infecciones por VIH/virología , VIH-1/fisiología , Hierro/metabolismo , Proteína de la Leucemia Promielocítica/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Humanos , Macaca , Oxidación-Reducción , Proteolisis , Análisis de Secuencia de ARN , Sumoilación , Regulación hacia Arriba , Latencia del VirusRESUMEN
BACKGROUND: Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion, resulting in a mutant huntingtin protein. While it is now clear that astrocytes are affected by HD and significantly contribute to neuronal dysfunction and pathogenesis, the alterations in the transcriptional and epigenetic profiles in HD astrocytes have yet to be characterized. Here, we examine global transcription and chromatin accessibility dynamics during in vitro astrocyte differentiation in a transgenic non-human primate model of HD. RESULTS: We found global changes in accessibility and transcription across different stages of HD pluripotent stem cell differentiation, with distinct trends first observed in neural progenitor cells (NPCs), once cells have committed to a neural lineage. Transcription of p53 signaling and cell cycle pathway genes was highly impacted during differentiation, with depletion in HD NPCs and upregulation in HD astrocytes. E2F target genes also displayed this inverse expression pattern, and strong associations between E2F target gene expression and accessibility at nearby putative enhancers were observed. CONCLUSIONS: The results suggest that chromatin accessibility and transcription are altered throughout in vitro HD astrocyte differentiation and provide evidence that E2F dysregulation contributes to aberrant cell-cycle re-entry and apoptosis throughout the progression from NPCs to astrocytes.
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Astrocitos/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Enfermedad de Huntington/patología , Células Madre Pluripotentes/metabolismo , Animales , Astrocitos/citología , Ensamble y Desensamble de Cromatina , Modelos Animales de Enfermedad , Factores de Transcripción E2F/metabolismo , Ontología de Genes , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Macaca mulatta , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células Madre Pluripotentes/citología , Transducción de Señal , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: An intravaginal ring that releases the tenofovir prodrug, tenofovir disoproxil fumarate, provided 100% protection in macaques against simian HIV and was safe in a 14-day clinical trial in sexually abstinent women. We aimed to assess the safety and pharmacokinetics of this intravaginal ring over 90 days in sexually active women. METHODS: We did a phase 1, single-blind, randomised, placebo-controlled trial to assess safety, pharmacokinetics, and acceptability of a tenofovir disoproxil fumarate intravaginal ring used continuously with monthly ring changes for 3 months. Sexually active women who were HIV negative were randomly assigned (3:1) to a tenofovir disoproxil fumarate ring or placebo ring. Primary safety endpoint was the proportion of women who had grade 2 or higher genitourinary adverse events judged related to study product and any grade 2 or higher adverse event as defined by the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events. We quantified tenofovir disoproxil fumarate and tenofovir concentrations in cervicovaginal fluid, tenofovir in plasma, and tenofovir diphosphate, the active metabolite, in cervical tissue and dried blood spots 1 month after each ring insertion. We compared changes over time in cervicovaginal fluid cytokine and chemokine concentrations and vaginal microbiota. The study was electively stopped early and is registered with ClinicalTrials.gov, number NCT02762617. FINDINGS: Between Feb 24 and July 20, 2017, 17 women were enrolled before study termination. 12 were assigned to receive the tenofovir disoproxil fumarate ring and five were assigned to receive the placebo ring. Two participants in the tenofovir disoproxil fumarate ring group completed 3 months of continuous ring use; eight were asked to discontinue ring use early because of ulcerations (grade 1) near the ring; in the remaining two women, rings were electively removed by study staff on day 20 and day 23. Ulcers were detected a mean of 32 days after ring use (range 23-56). Four of eight participants with ulcers were symptomatic with vaginal discharge; four had ulcers identified when examined; three had two ulcers; all ulcers resolved after ring removal. No participants in the placebo group developed ulcers. No grade 2 product-related adverse events were reported in either group and four non-product-related grade 2 adverse events were reported in the tenofovir disoproxil fumarate ring group. Cervicovaginal fluid tenofovir concentrations did not differ at day 14 (p=0·14) comparing the eight patients who did (median 1·0â×â105 ng/mL [IQR 9·1â×â104-1·1â×â105]) with the four who did not (6·0â×â104 ng/mL [5·6â×â104-1·1â×â105]) develop ulcers. No significant changes in vaginal microbiota were detected in either group. Concentrations of multiple inflammatory cytokines and chemokines were significantly higher at days 14 and 28 compared with baseline in the tenofovir disoproxil fumarate ring group but not the placebo group. INTERPRETATION: Future studies are needed to establish whether the unanticipated finding of ulcerations is specific to this tenofovir disoproxil fumarate ring or generalisable to other sustained topical release formulations of tenofovir or its prodrugs. FUNDING: National Institutes of Health.
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Infecciones por VIH/prevención & control , Profilaxis Pre-Exposición , Tenofovir/administración & dosificación , Administración Intravaginal , Adulto , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Método Simple Ciego , Tenofovir/efectos adversos , Tenofovir/farmacocinética , Adulto JovenRESUMEN
Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.
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Fármacos Anti-VIH/uso terapéutico , Expresión Génica/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Células TH1/patología , Células Th17/patología , Perfilación de la Expresión Génica , Infecciones por VIH/virología , Humanos , Receptores CXCR5/metabolismo , Células TH1/citología , Células TH1/inmunología , Células Th17/citología , Células Th17/inmunología , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Chronic activation of the immune system in HIV infection is one of the strongest predictors of morbidity and mortality. As such, approaches that reduce immune activation have received considerable interest. Previously, we demonstrated that administration of a type I interferon receptor antagonist (IFN-1ant) during acute SIV infection of rhesus macaques results in increased virus replication and accelerated disease progression. Here, we administered a long half-life PASylated IFN-1ant to ART-treated and ART-naïve macaques during chronic SIV infection and measured expression of interferon stimulated genes (ISG) by RNA sequencing, plasma viremia, plasma cytokines, T cell activation and exhaustion as well as cell-associated virus in CD4 T cell subsets sorted from peripheral blood and lymph nodes. Our study shows that IFN-1ant administration in both ART-suppressed and ART-untreated chronically SIV-infected animals successfully results in reduction of IFN-I-mediated inflammation as defined by reduced expression of ISGs but had no effect on plasma levels of IL-1ß, IL-1ra, IL-6 and IL-8. Unlike in acute SIV infection, we observed no significant increase in plasma viremia up to 25 weeks after IFN-1ant administration or up to 15 weeks after ART interruption. Likewise, cell-associated virus measured by SIV gag DNA copies was similar between IFN-1ant and placebo groups. In addition, evaluation of T cell activation and exhaustion by surface expression of CD38, HLA-DR, Ki67, LAG-3, PD-1 and TIGIT, as well as transcriptome analysis showed no effect of IFN-I blockade. Thus, our data show that blocking IFN-I signaling during chronic SIV infection suppresses IFN-I-related inflammatory pathways without increasing virus replication, and thus may constitute a safe therapeutic intervention in chronic HIV infection.
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Antirretrovirales/farmacología , Inflamación/prevención & control , Interferón Tipo I/antagonistas & inhibidores , Síndrome de Inmunodeficiencia Adquirida del Simio , Linfocitos T/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Antirretrovirales/uso terapéutico , Enfermedad Crónica , Inflamación/inmunología , Inflamación/virología , Interferón Tipo I/metabolismo , Activación de Linfocitos/efectos de los fármacos , Macaca mulatta , Receptores de Interferón/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/inmunologíaRESUMEN
The major obstacle to human immunodeficiency type 1 (HIV-1) eradication is a reservoir of latently infected cells that persists despite long-term antiretroviral therapy (ART) and causes rapid viral rebound if treatment is interrupted. Type I interferons are immunomodulatory cytokines that induce antiviral factors and have been evaluated for the treatment of HIV-infected individuals, resulting in moderate reduction of viremia and inconclusive data about their effect on reservoir size. Here, we assessed the potential of pegylated IFN-α2a (pIFN-α2a) to reduce the viral reservoir in simian immunodeficiency virus (SIV)-infected, ART-treated rhesus macaques (RMs). We found that pIFN-α2a treatment of animals in which virus replication is effectively suppressed with ART is safe and well tolerated, as no major clinical side effects were observed. By monitoring the cellular immune response during this intervention, we established that pIFN-α2a administration is not associated with either CD4+ T cell depletion or increased immune activation. Importantly, we found that interferon-stimulated genes (ISGs) were significantly upregulated in IFN-treated RMs compared to control animals, confirming that pIFN-α2a is bioactive in vivo To evaluate the effect of pIFN-α2a administration on the viral reservoir in CD4+ T cells, we performed cell-associated proviral SIV DNA measurements in multiple tissues and assessed levels of replication-competent virus by a quantitative viral outgrowth assay (QVOA). These analyses failed to reveal any significant difference in reservoir size between IFN-treated and control animals. In summary, our data suggest that short-term type I interferon treatment in combination with suppressive ART is not sufficient to induce a significant reduction of the viral reservoir in SIV-infected RMs.IMPORTANCE The potential of type I interferons to reduce the viral reservoir has been recently studied in clinical trials in HIV-infected humans. However, given the lack of mechanistic data and the potential for safety concerns, a more comprehensive testing of IFN treatment in vivo in SIV-infected RMs is critical to provide rationale for further development of this intervention in humans. Utilizing the SIV/RM model in which virus replication is suppressed with ART, we addressed experimental limitations of previous human studies, in particular the lack of a control group and specimen sampling limited to blood. Here, we show by rigorous testing of blood and lymphoid tissues that virus replication and reservoir size were not significantly affected by pIFN-α2a treatment in SIV-infected, ART-treated RMs. This suggests that intensified and/or prolonged IFN treatment regimens, possibly in combination with other antilatency agents, are necessary to effectively purge the HIV/SIV reservoir under ART.
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Antivirales/farmacología , Interferón-alfa/farmacología , Polietilenglicoles/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Linfocitos T/virología , Viremia/virología , Replicación Viral/efectos de los fármacos , Animales , Células Cultivadas , Macaca mulatta , Masculino , Proteínas Recombinantes/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Carga Viral/efectos de los fármacos , Viremia/tratamiento farmacológicoRESUMEN
We report on prime-boost vaccine regimens with two simian adenovirus (Ad) vectors (SAdV) or two human serotype Ad vectors (HAdV) expressing Gag and gp160 of simian immunodeficiency virus (SIV)mac239 tested in HAdV-seropositive rhesus macaques (RMs) repeatedly challenged rectally with low doses of SIVmac251. Both vaccine regimens reduced set point and peak viral loads (PVL) and accelerated viral clearance. In SAdV-vaccinated controller genotype RMs resistance against infection correlated with levels of envelope (Env)-specific antibody (Ab) titers. In both vaccine groups CD8+T cells controlled viral loads (VL) upon infection. Circulating CD4+ and CD8+ T cells showed significant changes in their transcriptome over time following vaccination, which differed between the vaccine groups. T cells from SIV-resistant RMs had unique transcriptional profiles indicating that both follicular T helper (TFH) cell responses and highly activated CD8+ T cells may play a role in protection.
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Adenoviridae/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Productos del Gen env/inmunología , Vectores Genéticos/inmunología , Inmunidad Celular , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Adenoviridae/genética , Animales , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Productos del Gen env/genética , Vectores Genéticos/genética , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/genética , VacunaciónRESUMEN
HIV-1 causes chronic inflammation and AIDS in humans, whereas related simian immunodeficiency viruses (SIVs) replicate efficiently in their natural hosts without causing disease. It is currently unknown to what extent virus-specific properties are responsible for these different clinical outcomes. Here, we incorporate two putative HIV-1 virulence determinants, i.e., a Vpu protein that antagonizes tetherin and blocks NF-κB activation and a Nef protein that fails to suppress T cell activation via downmodulation of CD3, into a non-pathogenic SIVagm strain and test their impact on viral replication and pathogenicity in African green monkeys. Despite sustained high-level viremia over more than 4 years, moderately increased immune activation and transcriptional signatures of inflammation, the HIV-1-like SIVagm does not cause immunodeficiency or any other disease. These data indicate that species-specific host factors rather than intrinsic viral virulence factors determine the pathogenicity of primate lentiviruses.
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VIH-1/patogenicidad , Especificidad del Huésped , Proteínas del Virus de la Inmunodeficiencia Humana/inmunología , Lentivirus de los Primates/crecimiento & desarrollo , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Proteínas Reguladoras y Accesorias Virales/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Animales , Antígeno 2 del Estroma de la Médula Ósea/genética , Antígeno 2 del Estroma de la Médula Ósea/inmunología , Complejo CD3/genética , Complejo CD3/inmunología , Chlorocebus aethiops , Femenino , Regulación de la Expresión Génica , VIH-1/crecimiento & desarrollo , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Lentivirus de los Primates/patogenicidad , Activación de Linfocitos , FN-kappa B/genética , FN-kappa B/inmunología , Alineación de Secuencia , Transducción de Señal , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Transcripción Genética , Carga Viral , Proteínas Reguladoras y Accesorias Virales/genética , Virulencia , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Pathogenic human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection of humans and rhesus macaques (RMs) induces persistently high production of type I interferon (IFN-I), which is thought to contribute to disease progression. To elucidate the specific role of interferon alpha (IFN-α) in SIV pathogenesis, 12 RMs were treated prior to intravenous (i.v.) SIVmac239 infection with a high or a low dose of an antibody (AGS-009) that neutralizes most IFN-α subtypes and were compared with six mock-infused, SIV-infected controls. Plasma viremia was measured postinfection to assess the effect of IFN-α blockade on virus replication, and peripheral blood and lymphoid tissue samples were analyzed by immunophenotypic staining. Consistent with the known antiviral effect of IFN-I, high-dose AGS-009 treatment induced a modest increase in acute-phase viral loads versus controls. Four out of 6 RMs receiving a high dose of AGS-009 also experienced an early decline in CD4+ T cell counts that was associated with progression to AIDS. Interestingly, 50% of the animals treated with AGS-009 (6/12) developed AIDS within 1 year of infection compared with 17% (1/6) of untreated controls. Finally, blockade of IFN-α decreased the levels of activated CD4+ and CD8+ T cells, as well as B cells, as measured by PD-1 and/or Ki67 expression. The lower levels of activated lymphocytes in IFN-α-blockaded animals supports the hypothesis that IFN-α signaling contributes to lymphocyte activation during SIV infection and suggests that this signaling pathway is involved in controlling virus replication during acute infection. The potential anti-inflammatory effect of IFN-α blockade should be explored as a strategy to reduce immune activation in HIV-infected individuals.IMPORTANCE Interferon alpha (IFN-α) is a member of a family of molecules (type I interferons) that prevent or limit virus infections in mammals. However, IFN-α production may contribute to the chronic immune activation that is thought to be the primary cause of immune decline and AIDS in HIV-infected patients. The study presented here attempts to understand the contribution of IFN-α to the natural history and progression of SIV infection of rhesus macaques, the primary nonhuman primate model system for testing hypotheses about HIV infection in humans. Here, we show that blockade of IFN-α action promotes lower chronic immune activation but higher early viral loads, with a trend toward faster disease progression. This study has significant implications for new treatments designed to impact the type I interferon system.
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Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón-alfa/antagonistas & inhibidores , Activación de Linfocitos/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Recuento de Linfocito CD4 , Interferón-alfa/inmunología , Antígeno Ki-67/biosíntesis , Células Asesinas Naturales/inmunología , Macaca mulatta , Receptor de Muerte Celular Programada 1/biosíntesis , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Carga Viral/efectos de los fármacos , Replicación Viral/inmunologíaRESUMEN
Although multiple restriction factors have been shown to inhibit HIV/SIV replication, little is known about their expression in vivo Expression of 45 confirmed and putative HIV/SIV restriction factors was analyzed in CD4+ T cells from peripheral blood and the jejunum in rhesus macaques, revealing distinct expression patterns in naive and memory subsets. In both peripheral blood and the jejunum, memory CD4+ T cells expressed higher levels of multiple restriction factors compared to naive cells. However, relative to their expression in peripheral blood CD4+ T cells, jejunal CCR5+ CD4+ T cells exhibited significantly lower expression of multiple restriction factors, including APOBEC3G, MX2, and TRIM25, which may contribute to the exquisite susceptibility of these cells to SIV infection. In vitro stimulation with anti-CD3/CD28 antibodies or type I interferon resulted in upregulation of distinct subsets of multiple restriction factors. After infection of rhesus macaques with SIVmac239, the expression of most confirmed and putative restriction factors substantially increased in all CD4+ T cell memory subsets at the peak of acute infection. Jejunal CCR5+ CD4+ T cells exhibited the highest levels of SIV RNA, corresponding to the lower restriction factor expression in this subset relative to peripheral blood prior to infection. These results illustrate the dynamic modulation of confirmed and putative restriction factor expression by memory differentiation, stimulation, tissue microenvironment and SIV infection and suggest that differential expression of restriction factors may play a key role in modulating the susceptibility of different populations of CD4+ T cells to lentiviral infection.IMPORTANCE Restriction factors are genes that have evolved to provide intrinsic defense against viruses. HIV and simian immunodeficiency virus (SIV) target CD4+ T cells. The baseline level of expression in vivo and degree to which expression of restriction factors is modulated by conditions such as CD4+ T cell differentiation, stimulation, tissue location, or SIV infection are currently poorly understood. We measured the expression of 45 confirmed and putative restriction factors in primary CD4+ T cells from rhesus macaques under various conditions, finding dynamic changes in each state. Most dramatically, in acute SIV infection, the expression of almost all target genes analyzed increased. These are the first measurements of many of these confirmed and putative restriction factors in primary cells or during the early events after SIV infection and suggest that the level of expression of restriction factors may contribute to the differential susceptibility of CD4+ T cells to SIV infection.
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Linfocitos T CD4-Positivos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Inmunidad Innata , Yeyuno/inmunología , Yeyuno/virología , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Transcriptoma/inmunologíaRESUMEN
We recently completed a phase I/IIa trial of RNActive® CV9201, a novel mRNA-based therapeutic vaccine targeting five tumor-associated antigens in non-small cell lung cancer (NSCLC) patients. The aim of the study presented here was to comprehensively analyze changes in peripheral blood during the vaccination period and to generate hypotheses facilitating the identification of potential biomarkers correlating with differential clinical outcomes post RNActive® immunotherapy. We performed whole-genome expression profiling in a subgroup of 22 stage IV NSCLC patients before and after initiation of treatment with CV9201. Utilizing an analytic approach based on blood transcriptional modules (BTMs), a previously described, sensitive tool for blood transcriptome data analysis, patients segregated into two major clusters based on transcriptional changes post RNActive® treatment. The first group of patients was characterized by the upregulation of an expression signature associated with myeloid cells and inflammation, whereas the other group exhibited an expression signature associated with T and NK cells. Patients with an enrichment of T and NK cell modules after treatment compared to baseline exhibited significantly longer progression-free and overall survival compared to patients with an upregulation of myeloid cell and inflammatory modules. Notably, these gene expression signatures were mutually exclusive and inversely correlated. Furthermore, our findings correlated with phenotypic data derived by flow cytometry as well as the neutrophil-to-lymphocyte ratio. Our study thus demonstrates non-overlapping, distinct transcriptional profiles correlating with survival warranting further validation for the development of biomarker candidates for mRNA-based immunotherapy.
RESUMEN
The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVΔnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from naïve animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVΔnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data support the hypothesis that ongoing stimulation by SIVΔnef promotes a distinct protective balance of CD8+ T cell differentiation and activation states.
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Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Factores de Transcripción/inmunología , Proteínas Reguladoras y Accesorias Virales , Animales , Linfocitos T CD8-positivos/patología , Diferenciación Celular/inmunología , Femenino , Perfilación de la Expresión Génica , Macaca mulatta , Vacunas contra el SIDAS/genética , Virus de la Inmunodeficiencia de los Simios/genética , Factores de Transcripción/genéticaRESUMEN
Immunization of macaques with attenuated simian immunodeficiency virus (SIV) with deletions in nef (SIVΔnef) is shown to elicit protective immunity to infection by pathogenic SIV, yet the mechanisms that orchestrate protection and prevent pathogenesis remains unknown. We utilized whole-genome transcriptional profiling to reveal molecular signatures of protective immunity in circulating CD8+ T cells of rhesus macaques vaccinated with SIVmac239Δnef and challenged with pathogenic SIVmac251. Our findings suggest that protective immunity to pathogenic SIV infection induced by SIVmac239∆nef is associated with balanced induction of T cell activation and immunoregulatory mechanisms and dampened activation of interferon-induced signaling pathways and cytolytic enzyme production as compared with pathogenic SIVmac251 infection of unvaccinated controls. We provide evidence that protective immunity to SIVmac251 correlates with induction of biomarkers of T cell activation, differentiation, signaling, and adhesion that were down regulated in unvaccinated controls. The study highlights potential immunomodulatory networks associated with protective immunity against the virus.