RESUMO
Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating multisystemic disease characterized by a complex, incompletely understood etiology. Methods: To facilitate future clinical and translational research, a multicenter German ME/CFS registry (MECFS-R) was established to collect comprehensive, longitudinal, clinical, epidemiological, and laboratory data from adults, adolescents, and children in a web-based multilayer-secured database. Results: Here, we present the research protocol and first results of a pilot cohort of 174 ME/CFS patients diagnosed at two specialized tertiary fatigue centers, including 130 (74.7%) adults (mean age 38.4; SD 12.6) and 43 (25.3%) pediatric patients (mean age 15.5; SD 4.2). A viral trigger was identified in 160/174 (92.0%) cases, with SARS-CoV-2 in almost half of them. Patients exhibited severe functional and social impairment, as reflected by a median Bell Score of 30.0 (IQR 30.0 to 40.0) and a poor health-related quality of life assessed with the Short Form-36 health survey, resulting in a mean score of 40.4 (SD 20.6) for physical function and 59.1 (SD 18.8) for mental health. Conclusions: The MECFS-R provides important clinical information on ME/CFS to research and healthcare institutions. Paired with a multicenter biobank, it facilitates research on pathogenesis, diagnostic markers, and treatment options. Trial registration: ClinicalTrials.gov NCT05778006.
RESUMO
Healthy individuals with hybrid immunity, due to a SARS-CoV-2 infection prior to first vaccination, have stronger immune responses compared to those who were exclusively vaccinated. However, little is known about the characteristics of antibody, B- and T-cell responses in kidney disease patients with hybrid immunity. Here, we explored differences between kidney disease patients and controls with hybrid immunity after asymptomatic or mild coronavirus disease-2019 (COVID-19). We studied the kinetics, magnitude, breadth and phenotype of SARS-CoV-2-specific immune responses against primary mRNA-1273 vaccination in patients with chronic kidney disease or on dialysis, kidney transplant recipients, and controls with hybrid immunity. Although vaccination alone is less immunogenic in kidney disease patients, mRNA-1273 induced a robust immune response in patients with prior SARS-CoV-2 infection. In contrast, kidney disease patients with hybrid immunity develop SARS-CoV-2 antibody, B- and T-cell responses that are equally strong or stronger than controls. Phenotypic analysis showed that Spike (S)-specific B-cells varied between groups in lymph node-homing and memory phenotypes, yet S-specific T-cell responses were phenotypically consistent across groups. The heterogeneity amongst immune responses in hybrid immune kidney patients warrants further studies in larger cohorts to unravel markers of long-term protection that can be used for the design of targeted vaccine regimens.
RESUMO
Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.
Assuntos
Vacinas Anticâncer , Neoplasias , Vacinas , Animais , Camundongos , Linfócitos T , Inibidores de Checkpoint Imunológico , Receptor Toll-Like 9 , Camundongos Endogâmicos C57BL , Adjuvantes Imunológicos , Antígenos , PeptídeosRESUMO
A preliminary analysis was conducted on data acquired from RNA sequencing and SomaScan platforms, for the classification of patients with Inflammation of Unknown Origin. To this end, a multimodal data integration approach was designed, by combining the two platforms, in order to assess the potentiality of learning estimators, using the differentially expressed features from the independent profiling experiments of both platforms. The classification framing was the differentiation of Inflammation of Unknown Origin patients against a multitude of Systemic Autoinflammatory disease patients. Separate false discovery rate analyses were performed on each dataset to extract statistically significant features between the two designated sample groups. Genomic analysis managed higher overall classification metrics compared to proteomic analysis, averaging an ~19% increase overall metrics and classifiers, with a ~0.07% increase in standard error. The multimodal data integration approach achieved similar results to the individual platforms' analyses. More specifically, it managed the same classification accuracy, sensitivity, and specificity scores as the best individual analysis, with the simple Logistic Regression estimator.Clinical Relevance- This study highlights the advantage of exploiting RNA sequencing data to identify potential Inflammation of Unknown Origin disease specific biomarkers, even against other Systemic Autoinflammatory diseases. These findings are further emphasized given the non-apparent clinical discrepancy between Inflammation of Unknown Origin and other Systemic Autoinflammatory diseases.
Assuntos
Doenças Hereditárias Autoinflamatórias , Proteômica , Humanos , Proteômica/métodos , RNA-Seq , Genômica/métodos , Análise de Sequência de RNA/métodos , SíndromeRESUMO
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid tumors and is resistant to immunotherapy. B cells play an essential role in PDAC progression and immune responses, both locally and systemically. Moreover, increasing evidence suggests that microbial compositions inside the tumor, as well as in the oral cavity and the gut, are important factors in shaping the PDAC immune landscape. However, the gut-associated lymphoid tissue (GALT) has not previously been explored in PDAC patients. In this study, we analyzed healthy vermiform appendix (VA) from 20 patients with PDAC and 32 patients with colon diseases by gene expression immune profiling, flow cytometry analysis, and microbiome sequencing. We show that the VA GALT of PDAC patients exhibits markers of increased inflammation and cytotoxic cell activity. In contrast, B cell function is decreased in PDAC VA GALT based on gene expression profiling; B cells express significantly fewer MHC class II surface receptors, whereas plasma cells express the immune checkpoint molecule HLA-G. Additionally, the vermiform appendix microbiome of PDAC patients is enriched with Klebsiella pneumoniae, Bifidobacterium animalis, and Adlercreutzia equolifaciens, while certain commensals are depleted. Our findings may suggest impaired B cell function within the GALT of PDAC patients, which could potentially be linked to microbial dysbiosis. Additional investigations are imperative to validate our observations and explore these potential targets of future therapies.
Assuntos
Apêndice , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Apêndice/microbiologia , Apêndice/patologia , Disbiose , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Antígenos HLA-GRESUMO
Introduction: Cytotoxic CD8+ T cell (CTL) exhaustion is a dysfunctional state of T cells triggered by persistent antigen stimulation, with the characteristics of increased inhibitory receptors, impaired cytokine production and a distinct transcriptional profile. Evidence from immune checkpoint blockade therapy supports that reversing T cell exhaustion is a promising strategy in cancer treatment. Ibrutinib, is a potent inhibitor of BTK, which has been approved for the treatment of chronic lymphocytic leukemia. Previous studies have reported improved function of T cells in ibrutinib long-term treated patients but the mechanism remains unclear. We investigated whether ibrutinib directly acts on CD8+ T cells and reinvigorates exhausted CTLs. Methods: We used an established in vitro CTL exhaustion system to examine whether ibrutinib can directly ameliorate T cell exhaustion. Changes in inhibitory receptors, transcription factors, cytokine production and killing capacity of ibrutinib-treated exhausted CTLs were detected by flow cytometry. RNA-seq was performed to study transcriptional changes in these cells. Btk deficient mice were used to confirm that the effect of ibrutinib was independent of BTK expression. Results: We found that ibrutinib reduced exhaustion-related features of CTLs in an in vitro CTL exhaustion system. These changes included decreased inhibitory receptor expression, enhanced cytokine production, and downregulation of the transcription factor TOX with upregulation of TCF1. RNA-seq further confirmed that ibrutinib directly reduced the exhaustion-related transcriptional profile of these cells. Importantly, using btk deficient mice we showed the effect of ibrutinib was independent of BTK expression, and therefore mediated by one of its other targets. Discussion: Our study demonstrates that ibrutinib directly ameliorates CTL exhaustion, and provides evidence for its synergistic use with cancer immunotherapy.
RESUMO
Cytotoxic CD8 + T cell (CTL) exhaustion is driven by chronic antigen stimulation. Reversing CTL exhaustion with immune checkpoint blockade (ICB) has provided clinical benefits in different types of cancer. We, therefore, investigated whether modulating chronic antigen stimulation and T-cell receptor (TCR) signaling with an IL2-inducible T-cell kinase (ITK) inhibitor, could confer ICB responsiveness to ICB resistant solid tumors. In vivo intermittent treatment of 3 ICB-resistant solid tumor (melanoma, mesothelioma or pancreatic cancer) with ITK inhibitor significantly improved ICB therapy. ITK inhibition directly reinvigorate exhausted CTL in vitro as it enhanced cytokine production, decreased inhibitory receptor expression, and downregulated the transcription factor TOX. Our study demonstrates that intermittent ITK inhibition can be used to directly ameliorate CTL exhaustion and enhance immunotherapies even in solid tumors that are ICB resistant.
Assuntos
Mesotelioma , Neoplasias Pancreáticas , Humanos , Inibidores de Checkpoint Imunológico , Proteínas Tirosina QuinasesRESUMO
OBJECTIVE: To combine targeted transcriptomic and proteomic data in an unsupervised hierarchical clustering method to stratify patients with childhood-onset SLE (cSLE) into similar biological phenotypes, and study the immunological cellular landscape that characterises the clusters. METHODS: Targeted whole blood gene expression and serum cytokines were determined in patients with cSLE, preselected on disease activity state (at diagnosis, Low Lupus Disease Activity State (LLDAS), flare). Unsupervised hierarchical clustering, agnostic to disease characteristics, was used to identify clusters with distinct biological phenotypes. Disease activity was scored by clinical SELENA-SLEDAI (Safety of Estrogens in Systemic Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index). High-dimensional 40-colour flow cytometry was used to identify immune cell subsets. RESULTS: Three unique clusters were identified, each characterised by a set of differentially expressed genes and cytokines, and by disease activity state: cluster 1 contained primarily patients in LLDAS, cluster 2 contained mainly treatment-naïve patients at diagnosis and cluster 3 contained a mixed group of patients, namely in LLDAS, at diagnosis and disease flare. The biological phenotypes did not reflect previous organ system involvement and over time, patients could move from one cluster to another. Healthy controls clustered together in cluster 1. Specific immune cell subsets, including CD11c+ B cells, conventional dendritic cells, plasmablasts and early effector CD4+ T cells, differed between the clusters. CONCLUSION: Using a targeted multiomic approach, we clustered patients into distinct biological phenotypes that are related to disease activity state but not to organ system involvement. This supports a new concept where choice of treatment and tapering strategies are not solely based on clinical phenotype but includes measuring novel biological parameters.
Assuntos
Lúpus Eritematoso Sistêmico , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Multiômica , Proteômica , Fenótipo , CitocinasRESUMO
Reactivation of the latent HIV-1 reservoir is a first step toward triggering reservoir decay. Here, we investigated the impact of the BAF complex inhibitor pyrimethamine on the reservoir of people living with HIV-1 (PLWH). Twenty-eight PLWH on suppressive antiretroviral therapy were randomized (1:1:1:1 ratio) to receive pyrimethamine, valproic acid, both, or no intervention for 14 days. The primary end point was change in cell-associated unspliced (CA US) HIV-1 RNA at days 0 and 14. We observed a rapid, modest, and significant increase in (CA US) HIV-1 RNA in response to pyrimethamine exposure, which persisted throughout treatment and follow-up. Valproic acid treatment alone did not increase (CA US) HIV-1 RNA or augment the effect of pyrimethamine. Pyrimethamine treatment did not result in a reduction in the size of the inducible reservoir. These data demonstrate that the licensed drug pyrimethamine can be repurposed as a BAF complex inhibitor to reverse HIV-1 latency in vivo in PLWH, substantiating its potential advancement in clinical studies.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , RNA , Ácido Valproico/farmacologia , Ativação Viral , Latência ViralRESUMO
[This corrects the article DOI: 10.1371/journal.pmed.1003979.].
RESUMO
BACKGROUND: The COVIH study is a prospective coronavirus disease 2019 (COVID-19) vaccination study in 1154 people with HIV (PWH), of whom 14% showed reduced antibody levels after primary vaccination. We evaluated whether an additional vaccination boosts immune responses in these hyporesponders. METHODS: The primary end point was the increase in antibodies 28 days after additional mRNA-1273 vaccination. Secondary end points included neutralizing antibodies, S-specific T-cell and B-cell responses, and reactogenicity. RESULTS: Of the 66 participants, 40 previously received 2 doses ChAdOx1-S, 22 received 2 doses BNT162b2, and 4 received a single dose Ad26.COV2.S. The median age was 63 years (interquartile range [IQR], 60-66), 86% were male, and median CD4+ T-cell count was 650/µL (IQR, 423-941). The mean S1-specific antibody level increased from 35 binding antibody units (BAU)/mL (95% confidence interval [CI], 24-46) to 4317 BAU/mL (95% CI, 3275-5360) (P < .0001). Of all participants, 97% showed an adequate response and the 45 antibody-negative participants all seroconverted. A significant increase in the proportion of PWH with ancestral S-specific CD4+ T cells (P = .04) and S-specific B cells (P = .02) was observed. CONCLUSIONS: An additional mRNA-1273 vaccination induced a robust serological response in 97% of PWH with a hyporesponse after primary vaccination. Clinical Trials Registration. EUCTR2021-001054-57-N.
Assuntos
COVID-19 , Infecções por HIV , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vacina de mRNA-1273 contra 2019-nCoV , Ad26COVS1 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , ChAdOx1 nCoV-19 , Vacinas contra COVID-19 , Estudos Prospectivos , SARS-CoV-2 , Vacinação , IdosoRESUMO
Background: Infectious mononucleosis after primary infection with Epstein-Barr virus (EBV-IM) has been linked to the development of myalgic encephalomyelitis/chronic fatigue-syndrome (ME/CFS) in children, adolescents, and young adults. Here, we present clinical phenotypes and follow-up data from a first German cohort of young people with ME/CFS following EBV-IM. Methods: 12 adolescents and 13 young adults were diagnosed with IM-triggered ME/CFS at our specialized tertiary outpatient service by clinical criteria requiring post-exertional malaise (PEM) and a history of confirmed EBV primary infection as triggering event. Demographic information, laboratory findings, frequency and severity of symptoms, physical functioning, and health-related quality of life (HRQoL) were assessed and re-evaluated 6 and 12 months later. Results: Young adults displayed more severe symptoms as well as worsening of fatigue, physical and mental functioning, and HRQoL throughout the study, compared to adolescents. After one year, 6/12 (54%) adolescents no longer met the diagnostic criteria for ME/CFS while all young adults continued to fulfill the Canadian consensus criteria. Improvement in adolescents was evident in physical functioning, symptom frequency and severity, and HRQoL, while young adults showed little improvement. EBV serology and EBV DNA load did not correlate with distinct clinical features of ME/CFS, and clinical chemistry showed no evidence of inflammation. Remarkably, the median time from symptom onset to ME/CFS diagnosis was 13.8 (IQR: 9.1-34.9) months. Conclusions: ME/CFS following EBV-IM is a severely debilitating disease often diagnosed late and with limited responses to conventional medical care, especially in adults. Although adolescents may have a better prognosis, their condition can fluctuate and significantly impact their HRQoL. Our data emphasize that biomarkers and effective therapeutic options are also urgently needed to improve medical care and pave the way to recovery.
RESUMO
BACKGROUND: Vaccines can be less immunogenic in people living with HIV (PLWH), but for SARS-CoV-2 vaccinations this is unknown. In this study we set out to investigate, for the vaccines currently approved in the Netherlands, the immunogenicity and reactogenicity of SARS-CoV-2 vaccinations in PLWH. METHODS AND FINDINGS: We conducted a prospective cohort study to examine the immunogenicity of BNT162b2, mRNA-1273, ChAdOx1-S, and Ad26.COV2.S vaccines in adult PLWH without prior COVID-19, and compared to HIV-negative controls. The primary endpoint was the anti-spike SARS-CoV-2 IgG response after mRNA vaccination. Secondary endpoints included the serological response after vector vaccination, anti-SARS-CoV-2 T-cell response, and reactogenicity. Between 14 February and 7 September 2021, 1,154 PLWH (median age 53 [IQR 44-60] years, 85.5% male) and 440 controls (median age 43 [IQR 33-53] years, 28.6% male) were included in the final analysis. Of the PLWH, 884 received BNT162b2, 100 received mRNA-1273, 150 received ChAdOx1-S, and 20 received Ad26.COV2.S. In the group of PLWH, 99% were on antiretroviral therapy, 97.7% were virally suppressed, and the median CD4+ T-cell count was 710 cells/µL (IQR 520-913). Of the controls, 247 received mRNA-1273, 94 received BNT162b2, 26 received ChAdOx1-S, and 73 received Ad26.COV2.S. After mRNA vaccination, geometric mean antibody concentration was 1,418 BAU/mL in PLWH (95% CI 1322-1523), and after adjustment for age, sex, and vaccine type, HIV status remained associated with a decreased response (0.607, 95% CI 0.508-0.725, p < 0.001). All controls receiving an mRNA vaccine had an adequate response, defined as >300 BAU/mL, whilst in PLWH this response rate was 93.6%. In PLWH vaccinated with mRNA-based vaccines, higher antibody responses were predicted by CD4+ T-cell count 250-500 cells/µL (2.845, 95% CI 1.876-4.314, p < 0.001) or >500 cells/µL (2.936, 95% CI 1.961-4.394, p < 0.001), whilst a viral load > 50 copies/mL was associated with a reduced response (0.454, 95% CI 0.286-0.720, p = 0.001). Increased IFN-γ, CD4+ T-cell, and CD8+ T-cell responses were observed after stimulation with SARS-CoV-2 spike peptides in ELISpot and activation-induced marker assays, comparable to controls. Reactogenicity was generally mild, without vaccine-related serious adverse events. Due to the control of vaccine provision by the Dutch National Institute for Public Health and the Environment, there were some differences between vaccine groups in the age, sex, and CD4+ T-cell counts of recipients. CONCLUSIONS: After vaccination with BNT162b2 or mRNA-1273, anti-spike SARS-CoV-2 antibody levels were reduced in PLWH compared to HIV-negative controls. To reach and maintain the same serological responses as HIV-negative controls, additional vaccinations are probably required. TRIAL REGISTRATION: The trial was registered in the Netherlands Trial Register (NL9214). https://www.trialregister.nl/trial/9214.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Infecções por HIV , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ad26COVS1 , Anticorpos Antivirais , Vacina BNT162 , COVID-19/epidemiologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Infecções por HIV/imunologia , Imunogenicidade da Vacina , Imunoglobulina G , Países Baixos/epidemiologia , Estudos Prospectivos , RNA Mensageiro , SARS-CoV-2 , Vacinas de mRNARESUMO
Quantitative or qualitative differences in immunity may drive clinical severity in COVID-19. Although longitudinal studies to record the course of immunological changes are ample, they do not necessarily predict clinical progression at the time of hospital admission. Here we show, by a machine learning approach using serum pro-inflammatory, anti-inflammatory and anti-viral cytokine and anti-SARS-CoV-2 antibody measurements as input data, that COVID-19 patients cluster into three distinct immune phenotype groups. These immune-types, determined by unsupervised hierarchical clustering that is agnostic to severity, predict clinical course. The identified immune-types do not associate with disease duration at hospital admittance, but rather reflect variations in the nature and kinetics of individual patient's immune response. Thus, our work provides an immune-type based scheme to stratify COVID-19 patients at hospital admittance into high and low risk clinical categories with distinct cytokine and antibody profiles that may guide personalized therapy.
Assuntos
Anticorpos Antivirais/sangue , COVID-19/patologia , Citocinas/sangue , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Idoso , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Progressão da Doença , Feminino , Hospitalização , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Imunofenotipagem/métodos , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologiaRESUMO
MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139-mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139-/- mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139-/- CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.
Assuntos
Vírus da Influenza A/imunologia , Listeria monocytogenes/imunologia , MicroRNAs/genética , Infecções por Orthomyxoviridae/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Regulação para Baixo/genética , Feminino , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologiaRESUMO
T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or ß CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFßR3 and aberrant TGFß1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFß pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease.
Assuntos
Leucemia Prolinfocítica de Células T , MicroRNAs , Perfilação da Expressão Gênica , Humanos , Leucemia Prolinfocítica de Células T/diagnóstico , Leucemia Prolinfocítica de Células T/genética , Leucemia Prolinfocítica de Células T/terapia , Linfócitos , MicroRNAs/genética , Fator de Crescimento Transformador beta , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaRESUMO
An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNß. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.
Assuntos
Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , RNA Helicases DEAD-box/metabolismo , Infecções por HIV/imunologia , HIV-1/metabolismo , Imidazóis/farmacologia , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antirretrovirais/farmacologia , Apoptose/genética , Azepinas/química , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/química , Inibidores Enzimáticos/farmacologia , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Imidazóis/química , Hibridização in Situ Fluorescente , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Células Jurkat , Simulação de Acoplamento Molecular , RNA Viral/metabolismo , Análise de Célula Única , Survivina/metabolismo , Ativação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
In a randomized clinical trial of 86 hospitalized COVID-19 patients comparing standard care to treatment with 300mL convalescent plasma containing high titers of neutralizing SARS-CoV-2 antibodies, no overall clinical benefit was observed. Using a comprehensive translational approach, we unravel the virological and immunological responses following treatment to disentangle which COVID-19 patients may benefit and should be the focus of future studies. Convalescent plasma is safe, does not improve survival, has no effect on the disease course, nor does plasma enhance viral clearance in the respiratory tract, influence SARS-CoV-2 antibody development or serum proinflammatory cytokines levels. Here, we show that the vast majority of patients already had potent neutralizing SARS-CoV-2 antibodies at hospital admission and with comparable titers to carefully selected plasma donors. This resulted in the decision to terminate the trial prematurely. Treatment with convalescent plasma should be studied early in the disease course or at least preceding autologous humoral response development.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/terapia , Citocinas/sangue , SARS-CoV-2/imunologia , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Doadores de Sangue , COVID-19/sangue , COVID-19/virologia , Progressão da Doença , Feminino , Hospitalização , Humanos , Imunização Passiva , Imunoglobulina G/sangue , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/imunologia , Resultado do Tratamento , Soroterapia para COVID-19RESUMO
PD-1/PD-L1-checkpoint blockade therapy is generally thought to relieve tumor cell-mediated suppression in the tumor microenvironment but PD-L1 is also expressed on non-tumor macrophages and conventional dendritic cells (cDCs). Here we show in mouse tumor models that tumor-draining lymph nodes (TDLNs) are enriched for tumor-specific PD-1+ T cells which closely associate with PD-L1+ cDCs. TDLN-targeted PD-L1-blockade induces enhanced anti-tumor T cell immunity by seeding the tumor site with progenitor-exhausted T cells, resulting in improved tumor control. Moreover, we show that abundant PD-1/PD-L1-interactions in TDLNs of nonmetastatic melanoma patients, but not those in corresponding tumors, associate with early distant disease recurrence. These findings point at a critical role for PD-L1 expression in TDLNs in governing systemic anti-tumor immunity, identifying high-risk patient groups amendable to adjuvant PD-1/PD-L1-blockade therapy.
Assuntos
Antígeno B7-H1/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Linfonodos/imunologia , Melanoma/tratamento farmacológico , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/imunologia , Adulto , Animais , Antígeno B7-H1/antagonistas & inibidores , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Feminino , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Linfonodos/efeitos dos fármacos , Masculino , Melanoma/imunologia , Melanoma/patologia , Camundongos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Linfócitos T/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.