RESUMO
A subset of people living with HIV (PLWH) can produce broadly neutralizing antibodies (bNAbs) against HIV, but the lymph node (LN) dynamics that promote the generation of these antibodies are poorly understood. Here, we explored LN-associated histological, immunological, and virological mechanisms of bNAb generation in a cohort of anti-retroviral therapy (ART)-naïve PLWH. We found that participants who produce bNAbs, termed neutralizers, have a superior LN-associated B cell follicle architecture compared with PLWH who do not. The latter was associated with a significantly higher in situ prevalence of Bcl-6hi follicular helper CD4 T cells (TFH), expressing a molecular program that favors their differentiation and stemness, and significantly reduced IL-10 follicular suppressor CD4 T cells. Furthermore, our data reveal possible molecular targets mediating TFH- B cell interactions in neutralizers. Together, we identify cellular and molecular mechanisms that contribute to the development of bNAbs in PLWH.
RESUMO
Human immunodeficiency virus (HIV) cure efforts are increasingly focused on harnessing CD8+ T cell functions, which requires a deeper understanding of CD8+ T cells promoting HIV control. Here we identifiy an antigen-responsive TOXhiTCF1+CD39+CD8+ T cell population with high expression of inhibitory receptors and low expression of canonical cytolytic molecules. Transcriptional analysis of simian immunodeficiency virus (SIV)-specific CD8+ T cells and proteomic analysis of purified CD8+ T cell subsets identified TOXhiTCF1+CD39+CD8+ T cells as intermediate effectors that retained stem-like features with a lineage relationship with terminal effector T cells. TOXhiTCF1+CD39+CD8+ T cells were found at higher frequency than TCF1-CD39+CD8+ T cells in follicular microenvironments and were preferentially located in proximity of SIV-RNA+ cells. Their frequency was associated with reduced plasma viremia and lower SIV reservoir size. Highly similar TOXhiTCF1+CD39+CD8+ T cells were detected in lymph nodes from antiretroviral therapy-naive and antiretroviral therapy-suppressed people living with HIV, suggesting this population of CD8+ T cells contributes to limiting SIV and HIV persistence.
Assuntos
Linfócitos T CD8-Positivos , Linfonodos , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T CD8-Positivos/imunologia , Animais , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Linfonodos/imunologia , Humanos , Macaca mulatta , Infecções por HIV/imunologia , Infecções por HIV/virologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Patients with COVID-19 may develop abnormal inflammatory response, followed in some cases by severe disease and long-lasting syndromes. We show here that in vitro exposure to SARS-CoV-2 activates the expression of the human endogenous retrovirus (HERV) HERV-W proinflammatory envelope protein (ENV) in peripheral blood mononuclear cells from a subset of healthy donors, in ACE2 receptor and infection-independent manner. Plasma and/or sera of 221 COVID-19 patients from different cohorts, infected with successive SARS-CoV-2 variants including the Omicron, had detectable HERV-W ENV, which correlated with ENV expression in T lymphocytes and peaked with the disease severity. HERV-W ENV was also found in postmortem tissues of lungs, heart, gastrointestinal tract, brain olfactory bulb, and nasal mucosa from COVID-19 patients. Altogether, these results demonstrate that SARS-CoV-2 could induce HERV-W envelope protein expression and suggest its involvement in the immunopathogenesis of certain COVID-19-associated syndromes and thereby its relevance in the development of personalized treatment of patients.
RESUMO
Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.
Assuntos
Infecções por HIV , HIV-1 , Humanos , HIV-1/fisiologia , Linfócitos T CD4-Positivos , Latência Viral/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Epigênese Genética , Carga Viral , Antirretrovirais/uso terapêuticoRESUMO
The main barrier to a cure for HIV is the persistence of long-lived and proliferating latently infected CD4+ T-cells despite antiretroviral therapy (ART). Latency is well characterized in multiple CD4+ T-cell subsets, however, the contribution of regulatory T-cells (Tregs) expressing FoxP3 as well as immune checkpoints (ICs) PD-1 and CTLA-4 as targets for productive and latent HIV infection in people living with HIV on suppressive ART is less well defined. We used multiplex detection of HIV DNA and RNA with immunohistochemistry (mIHC) on formalin-fixed paraffin embedded (FFPE) cells to simultaneously detect HIV RNA and DNA and cellular markers. HIV DNA and RNA were detected by in situ hybridization (ISH) (RNA/DNAscope) and IHC was used to detect cellular markers (CD4, PD-1, FoxP3, and CTLA-4) by incorporating the tyramide system amplification (TSA) system. We evaluated latently infected cell lines, a primary cell model of HIV latency and excisional lymph node (LN) biopsies collected from people living with HIV (PLWH) on and off ART. We clearly detected infected cells that coexpressed HIV RNA and DNA (active replication) and DNA only (latently infected cells) in combination with IHC markers in the in vitro infection model as well as LN tissue from PLWH both on and off ART. Combining ISH targeting HIV RNA and DNA with IHC provides a platform to detect and quantify HIV persistence within cells identified by multiple markers in tissue samples from PLWH on ART or to study HIV latency.
Assuntos
DNA Viral/análise , Infecções por HIV/diagnóstico , HIV/genética , Inibidores de Checkpoint Imunológico/análise , Imuno-Histoquímica , Hibridização In Situ , Infecção Latente/diagnóstico , Linfonodos/imunologia , Linfonodos/virologia , RNA Viral/análise , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Células Jurkat , Infecção Latente/imunologia , Infecção Latente/virologia , Valor Preditivo dos Testes , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/virologiaRESUMO
CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination.
Assuntos
Reservatórios de Doenças/virologia , Infecções por HIV/virologia , Processamento de Imagem Assistida por Computador , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Microscopia Confocal , Imagem Molecular , Biomarcadores , Imunofluorescência , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Centro Germinativo/virologia , Infecções por HIV/diagnóstico , Infecções por HIV/imunologia , Interações Hospedeiro-Patógeno , Humanos , Processamento de Imagem Assistida por Computador/métodos , Linfonodos/imunologia , Linfonodos/virologia , Tecido Linfoide/virologia , Microscopia Confocal/métodos , Imagem Molecular/métodosRESUMO
The integration of HIV DNA into the host genome contributes to lifelong infection in most individuals. Few studies have examined integration in lymphoid tissue, where HIV predominantly persists before and after antiretroviral treatment (ART). Of particular interest is whether integration site distributions differ between infection stages with paired blood and tissue comparisons. Here, we profiled HIV integration site distributions in sorted memory, tissue-resident, and/or follicular helper CD4+ T cell subsets from paired blood and lymphoid tissue samples from acute, chronic, and ART-treated individuals. We observed minor differences in the frequency of nonintronic and nondistal intergenic sites, varying with tissue and residency phenotypes during ART. Genomic and epigenetic annotations were generally similar. Clonal expansion of cells marked by identical integration sites was detected, with increased detection in chronic and ART-treated individuals. However, overlap between or within CD4+ T cell subsets or tissue compartments was only observed in 8 unique sites of the 3540 sites studied. Together, these findings suggest that shared integration sites between blood and tissue may, depending on the tissue site, be the exception rather than the rule and indicate that additional studies are necessary to fully understand the heterogeneity of tissue-sequestered HIV reservoirs.
Assuntos
DNA Viral/genética , Infecções por HIV/genética , Interações Hospedeiro-Patógeno/genética , Integração Viral/genética , Adulto , Antirretrovirais/administração & dosagem , Linfócitos T CD4-Positivos/virologia , Genoma Humano/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Tecido Linfoide/virologia , Masculino , Subpopulações de Linfócitos T/virologia , Carga Viral/genética , Adulto JovemAssuntos
Endossonografia , Gastrostomia/métodos , Abscesso Hepático/cirurgia , Fígado/cirurgia , Cirurgia Assistida por Computador/métodos , Adenocarcinoma/complicações , Procedimentos Cirúrgicos do Sistema Biliar , Colangiopancreatografia Retrógrada Endoscópica , Colestase/etiologia , Drenagem , Feminino , Humanos , Abscesso Hepático/diagnóstico por imagem , Abscesso Hepático/etiologia , Pessoa de Meia-Idade , Neoplasias Pancreáticas/complicações , Stents , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: The incidence of bile duct injuries (BDI) after cholecystectomy, which is a life-threatening condition that has several medical and legal implications, currently stands at about 0.6%. The aim of this study is to describe our experience as the first center to use a laparoscopic approach for BDI repair. METHODS: A prospective study between June 2012 and September 2014 was developed. Twenty-nine consecutive patients with BDI secondary to cholecystectomy were included. Demographics, comorbidities, presenting symptoms, details of index surgery, type of lesion, preoperative and postoperative diagnostic work-up, and therapeutic interventions were registered. Videos and details of laparoscopic hepaticojejunostomy (LHJ) were recorded. Injuries were staged using Strasberg classification. A side-to-side anastomosis with Roux-en-Y reconstruction was always used. In patients with E4 and some E3 injuries, a segment 4b or 5 section was done to build a wide anastomosis. In E4 injuries, a neo-confluence was performed. Complications, mortality, and long-term evolution were recorded. RESULTS: Twenty-nine patients with BDI were operated. Women represented 82.7% of the cases. The median age was 42 years (range 21-74). Injuries at or above the confluence occurred in 62%, and primary repair at our institution was performed at 93.1% of the cases. Eight neo-confluences were performed in all E4 injuries (27.5%). The median operative time was 240 min (range 120-585) and bleeding 200 mL (range 50-1100). Oral intake was started in the first 48 h. Bile leak occurred in 5 cases (17.2%). Two patients required re-intervention (6.8%). No mortality was recorded. The maximum follow-up was 36 months (range 2-36). One patient with E4 injury developed a hepaticojejunostomy (HJ) stenosis after 15 months. This was solved with endoscopic dilatation. CONCLUSIONS: The benefits of minimally invasive approaches in BDI seem to be feasible and safe, even when this is a complex and catastrophic scenario.