RESUMO
Elucidating the mechanisms underlying the persistence and location of the HIV reservoir is critical for developing cure interventions. While it has been shown that levels of T-cell activation and the size of the HIV reservoir are greater in rectal tissue and lymph nodes (LN) than in blood, the relative contributions of T-cell subsets to this anatomic difference are unknown. We measured and compared HIV-1 DNA content, expression of the T-cell activation markers CD38 and HLA-DR, and expression of the exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in naive, central memory (CM), transitional memory (TM), and effector memory (EM) CD4+ and CD8+ T-cells in paired blood and LN samples among 14 people with HIV who were receiving antiretroviral therapy. HIV-1 DNA levels, T-cell immune activation, and TIGIT expression were higher in LN than in blood, especially in CM and TM CD4+ T-cell subsets. Immune activation was significantly higher in all CD8+ T-cell subsets, and memory CD8+ T-cell subsets from LN had higher levels of PD-1 expression, compared with blood, while TIGIT expression levels were significantly lower in TM CD8+ T-cells. The differences seen in CM and TM CD4+ T-cell subsets were more pronounced among participants with CD4+ T-cell counts of <500 cells/µL within 2 years after antiretroviral therapy initiation, thus highlighting increased residual dysregulation in LN as a distinguishing feature of and a potential mechanism for individuals with suboptimal CD4+ T-cell recovery during antiretroviral therapy. IMPORTANCE This study provides new insights into the contributions of different CD4+ and CD8+ T-cell subsets to the anatomic differences between LN and blood in individuals with HIV who have optimal versus suboptimal CD4+ T-cell recovery. To our knowledge, this is the first study comparing paired LN and blood CD4+ and CD8+ T-cell differentiation subsets, as well as those subsets in immunological responders versus immunological suboptimal responders.
Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , DNA Viral , Infecções por HIV , Linfonodos , Ativação Linfocitária , Humanos , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/virologia , DNA Viral/análise , HIV-1 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Sangue/imunologia , Sangue/virologia , Ativação Linfocitária/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Masculino , Adulto , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologiaRESUMO
HIV integrates into the host genome, creating a viral reservoir of latently infected cells that persists despite effective antiretroviral treatment. CD4-positive (CD4+) T cells are the main contributors to the HIV reservoir. CD4+ T cells are a heterogeneous population, and the mechanisms of latency establishment in the different subsets, as well as their contribution to the reservoir, are still unclear. In this study, we analyzed HIV latency establishment in different CD4+ T cell subsets stimulated with interleukin 15 (IL-15), a cytokine that increases both susceptibility to infection and reactivation from latency. Using a dual-reporter virus that allows discrimination between latent and productive infection at the single-cell level, we found that IL-15-treated primary human CD4+ T naive and CD4+ T stem cell memory (TSCM) cells are less susceptible to HIV infection than CD4+ central memory (TCM), effector memory (TEM), and transitional memory (TTM) cells but are also more likely to harbor transcriptionally silent provirus. The propensity of these subsets to harbor latent provirus compared to the more differentiated memory subsets was independent of differential expression of pTEFb components. Microscopy analysis of NF-κB suggested that CD4+ T naive cells express smaller amounts of nuclear NF-κB than the other subsets, partially explaining the inefficient long terminal repeat (LTR)-driven transcription. On the other hand, CD4+ TSCM cells display similar levels of nuclear NF-κB to CD4+ TCM, CD4+ TEM, and CD4+ TTM cells, indicating the availability of transcription initiation and elongation factors is not solely responsible for the inefficient HIV gene expression in the CD4+ TSCM subset. IMPORTANCE The formation of a latent reservoir is the main barrier to HIV cure. Here, we investigated how HIV latency is established in different CD4+ T cell subsets in the presence of IL-15, a cytokine that has been shown to efficiently induce latency reversal. We observed that, even in the presence of IL-15, the less differentiated subsets display lower levels of productive HIV infection than the more differentiated subsets. These differences were not related to different expression of pTEFb, and modest differences in NF-κB were observed for CD4+ T naive cells only, implying the involvement of other mechanisms. Understanding the molecular basis of latency establishment in different CD4+ T cell subsets might be important for tailoring specific strategies to reactivate HIV transcription in all the CD4+ T subsets that compose the latent reservoir.
Assuntos
Linfócitos T CD4-Positivos , Infecções por HIV , Interleucina-15 , Latência Viral , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1 , Humanos , Interleucina-15/farmacologia , NF-kappa B/metabolismo , Provírus , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/virologiaRESUMO
The HIV reservoir size in target CD4+ T cells during primary infection remains unknown. Here, we sorted peripheral and intestinal CD4+ T cells and quantified the levels of cell-associated SIV RNA and DNA in rhesus macaques within days of SIVmac251 inoculation. As a major target cell of HIV/SIV, CD4+ T cells in both tissues contained a large amount of SIV RNA and DNA at day 8-13 post-SIV infection, in which productive SIV RNA highly correlated with the levels of cell-associated SIV DNA. Memory CD4+ T cells had much higher viral RNA and DNA than naïve subsets, yet memory CD4+ T cells co-expressing CCR5 had no significant reservoir size compared with those that were CCR5-negative in blood and intestine. Collectively, memory CD4+ T cells appear to be the major targets for primary infection, and viral reservoirs are equally distributed in systemic and lymphoid compartments in acutely SIV-infected macaques.
Assuntos
Linfócitos T CD4-Positivos/virologia , Intestinos/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Intestinos/imunologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Carga ViralRESUMO
Knowing the mechanisms that govern the persistence of infected CD4+ subpopulations could help us to design new therapies to cure HIV-1 infection. We evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ subpopulations from 14 HIV-1-infected individuals on antiretroviral therapy to analyze its relationship with HIV-1 transcription, immune activation, and cell proliferation. A unique large blood donation was used to isolate CD4, CD4 resting (CD4r), CD4 activated (CD4a), T naive (TN), T stem cell memory (TSCM), T central memory (TCM), T transitional memory (TTM), T effector memory (TEM), circulating T follicular helper (cTFH), TCD20, TCD32, and resting memory TCD2high (rmTCD2high) cells. HIV-1 DNA measured by droplet digital PCR ranged from 3,636 copies/106 in TTM to 244 in peripheral blood mononuclear cells (PBMCs), with no subpopulation standing out for provirus enrichment. Importantly, all the subpopulations harbored intact provirus by intact provirus DNA assay (IPDA). TCD32, cTFH, and TTM had the highest levels of HIV-1 transcription measured by fluorescent in situ hybridization with flow cytometry (FISH/flow), but without reaching statistical differences. The subpopulations more enriched in provirus had a memory phenotype, were less activated (measured by CD38+/HLA-DR+), and expressed more programmed cell death 1 (PD-1). Conversely, subpopulations transcribing more HIV-1 RNA were not necessarily enriched in provirus and were more activated (measured by CD38+/HLA-DR+) and more proliferative (measured by Ki-67). In conclusion, the HIV reservoir is composed of a mosaic of subpopulations contributing to the HIV-1 persistence through different mechanisms such as susceptibility to infection, provirus intactness, or transcriptional status. The narrow range of reservoir differences between the different blood cell subsets tested suggests limited efficacy in targeting only specific cell subpopulations during HIV-1 cure strategies. IMPORTANCE The main barrier for HIV-1 cure is the presence of latently infected CD4+ T cells. Although various cell subpopulations have been identified as major HIV-1 reservoir cells, the relative contribution of infected CD4 subpopulations in the HIV-1 reservoir remains largely unknown. Here, we evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ T-cell subpopulations in peripheral blood from HIV-1-infected individuals under suppressive antiretroviral therapy. We found that the HIV-1 reservoir is composed of a mosaic of cell subpopulations, with heterogeneous proviral DNA, HIV-1 transcription, and activation status. Hence, each cell subpopulation contributes to the HIV-1 persistence through different mechanisms such as susceptibility to infection, rates of intact provirus, transcriptional status or half-life. This research provides new insights into the composition of the HIV-1 reservoir, suggesting that, to be effective, eradication strategies must simultaneously target multiple cell subpopulations.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , DNA Viral/genética , DNA Viral/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Memória Imunológica , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Carga Viral/efeitos dos fármacosRESUMO
Clinical outcomes are inferior for individuals with HIV having suboptimal CD4 T-cell recovery during antiretroviral therapy (ART). We investigated if the levels of infection and the response to homeostatic cytokines of CD4 T-cell subsets contributed to divergent CD4 T-cell recovery and HIV reservoir during ART by studying virologically-suppressed immunologic responders (IR, achieving a CD4 cell count >500 cells/µL on or before two years after ART initiation), and virologically-suppressed suboptimal responders (ISR, did not achieve a CD4 cell count >500 cells/µL in the first two years after ART initiation). Compared to IR, ISR demonstrated higher levels of HIV-DNA in naïve, central (CM), transitional (TM), and effector (EM) memory CD4 T-cells in blood, both pre- and on-ART, and specifically in CM CD4 T-cells in LN on-ART. Furthermore, ISR had higher pre-ART plasma levels of IL-7 and IL-15, cytokines regulating T-cell homeostasis. Notably, pre-ART PD-1 and TIGIT expression levels were higher in blood CM and TM CD4 T-cells for ISR; this was associated with a significantly lower fold-changes in HIV-DNA levels between pre- and on-ART time points exclusively on CM and TM T-cell subsets, but not naïve or EM T-cells. Finally, the frequency of CM CD4 T-cells expressing PD-1 or TIGIT pre-ART as well as plasma levels of IL-7 and IL-15 predicted HIV-DNA content on-ART. Our results establish the association between infection, T-cell homeostasis, and expression of PD-1 and TIGIT in long-lived CD4 T-cell subsets prior to ART with CD4 T-cell recovery and HIV persistence on-ART.
Assuntos
Antirretrovirais/farmacologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Infecções por HIV/virologia , Homeostase , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , DNA Viral , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Memória Imunológica/imunologia , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/virologia , Carga ViralRESUMO
In various pathological conditions, cellular immunity plays an important role in immune responses. Amongimmunecells, T lymphocytes pdomotecellular and humoralresponses as well as innate immunity. Therefore, careful investigation of these cells has a significant impact on accurate knowledge in COVID-19diseasepathogenesis. In current research, the frequency and function of various T lymphocytes involved in immune responses examined in SARS-CoV-2 patients with various disease severity compared to normal subjects. In order to make an accurate comparison among patients with various disease severity, this study was performed on asymptomatic recovered cases (n = 20), ICU hospitalized patients (n = 30), non-ICU hospitalized patients (n = 30), and normal subjects (n = 20). To precisely evaluate T cells activity following purification, their cytokine secretion activity was examined. Similarly, immediately after purification of Treg cells, their inhibitory activity on T cells was investigated. The results showed that COVID-19 patients with severe disease (ICU hospitalized patients) not only had a remarkable increase in Th1 and Th17 but also a considerable decrease in Th2 and Treg cells. More importantly, as the IL-17 and IFN-γ secretion was sharply increased in severe disease, the secretion of IL-10 and IL-4 was decreased. Furthermore, the inhibitory activity of Treg cells was reduced in severe disease patients in comparison to other groups. In severe COVID-19 disease, current findings indicate when the inflammatory arm of cellular immunity is significantly increased, a considerable reduction in anti-inflammatory and regulatory arm occurred.
Assuntos
COVID-19/sangue , COVID-19/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Adulto , Idoso , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Imunidade Celular , Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Adulto JovemRESUMO
Severe respiratory viral infectious diseases such as influenza and COVID-19 especially affect the older population. This is partly ascribed to diminished CD8+ T-cell responses a result of aging. The phenotypical diversity of the CD8+ T-cell population has made it difficult to identify the impact of aging on CD8+ T-cell subsets associated with diminished CD8+ T-cell responses. Here we identify a novel human CD8+ T-cell subset characterized by expression of Killer-cell Immunoglobulin-like Receptors (KIR+ ) and CD45RA (RA+ ). These KIR+ RA+ T cells accumulated with age in the blood of healthy individuals (20-82 years of age, n = 50), expressed high levels of aging-related markers of T-cell regulation, and were functionally capable of suppressing proliferation of other CD8+ T cells. Moreover, KIR+ RA+ T cells were a major T-cell subset becoming activated in older adults suffering from an acute respiratory viral infection (n = 36), including coronavirus and influenza virus infection. In addition, older adults with influenza A infection showed that higher activation status of their KIR+ RA+ T cells associated with longer duration of respiratory symptoms. Together, our data indicate that KIR+ RA+ T cells are a unique human T-cell subset with regulatory properties that may explain susceptibility to viral respiratory disease at old age.
Assuntos
Envelhecimento/fisiologia , Linfócitos T CD8-Positivos/virologia , Subpopulações de Linfócitos T/virologia , Idoso , Idoso de 80 Anos ou mais , COVID-19/imunologia , Feminino , Regulação da Expressão Gênica , Humanos , Influenza Humana/imunologia , Masculino , Pessoa de Meia-Idade , Receptores KIR/sangue , Receptores KIR/metabolismo , SARS-CoV-2RESUMO
Chronic active Epstein-Barr virus (CAEBV) typically presents as persistent infectious mononucleosis-like disease and/or hemophagocytic lymphohistocytosis (HLH), reflecting ectopic Epstein-Barr virus (EBV) infection and lymphoproliferation of T and/or NK cells. Clinical behavior ranges from indolent, stable disease through to rapidly progressive, life-threatening disease. Although it is thought the chronicity and/or progression reflect an escape from immune control, very little is known about the phenotype and function of the infected cells vs coresident noninfected population, nor about the mechanisms that could underpin their evasion of host immune surveillance. To investigate these questions, we developed a multicolor flow cytometry technique combining phenotypic and functional marker staining with in situ hybridization for the EBV-encoded RNAs (EBERs) expressed in every infected cell. This allows the identification, phenotyping, and functional comparison of infected (EBERPOS) and noninfected (EBERNEG) lymphocyte subset(s) in patients' blood samples ex vivo. We have characterized CAEBV and HLH cases with monoclonal populations of discrete EBV-activated T-cell subsets, in some cases accompanied by EBV-activated NK-cell subsets, with longitudinal data on the infected cells' progression despite standard steroid-based therapy. Given that cytotoxic CD8+ T cells with relevant EBV antigen specificity were detectable in the blood of the best studied patient, we searched for means whereby host surveillance might be impaired. This revealed a unique feature in almost every patient with CAEBV studied: the presence of large numbers of myeloid-derived suppressor cells that exhibited robust inhibition of T-cell growth. We suggest that their influence is likely to explain the host's failure to contain EBV-positive T/NK-cell proliferation.
Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/virologia , Células Supressoras Mieloides/imunologia , Subpopulações de Linfócitos T/virologia , Adulto , Citometria de Fluxo/métodos , Herpesvirus Humano 4/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor ß (TGF-ß), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-ß signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103+ and CD103- CD4 T cells from the blood and rectum of HIV-negative (HIV-) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV+) individuals. Like gut CD4+ T cells, circulating CD103+ cells harbored more HIV DNA than did CD103- cells but transcribed less HIV RNA per provirus. Circulating CD103+ cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103- cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103+ CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency.IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103+ CD4+ T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103+ T cells share a cellular transcriptome that more closely resembles that of CD4+ T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4+ or circulating CD103+ T cells from circulating CD103- T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV.
Assuntos
Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/virologia , Trato Gastrointestinal/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Cadeias alfa de Integrinas/metabolismo , Transcrição Gênica/genética , Antivirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , DNA Viral/metabolismo , Trato Gastrointestinal/imunologia , Regulação da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Humanos , Linfócitos Intraepiteliais/metabolismo , Linfócitos Intraepiteliais/virologia , Provírus/fisiologia , RNA Viral/metabolismo , Proteínas Ribossômicas/genética , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologia , Latência ViralRESUMO
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 JovemRESUMO
Previous studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further in silico analysis of transcriptional data suggested a critical role for STAT5 transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of STAT5 was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with STAT5 signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/isolamento & purificação , Análise de Sequência de RNA/métodos , Fixação de Tecidos/métodos , Linfócitos T CD8-Positivos/virologia , Infecções por Citomegalovirus/imunologia , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Fator de Transcrição STAT5/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologiaRESUMO
Though recent reports link SARS-CoV-2 infections with hyper-inflammatory states in children, most children experience no/mild symptoms, and hospitalization and mortality rates are low in the age group. As symptoms are usually mild and seroconversion occurs at low frequencies, it remains unclear whether children significantly contribute to community transmission. Several hypotheses try to explain age-related differences in disease presentation and severity. Possible reasons for milder presentations in children as compared to adults include frequent contact to seasonal coronaviruses, presence of cross-reactive antibodies, and/or co-clearance with other viruses. Increased expression of ACE2 in young people may facilitate virus infection, while limiting inflammation and reducing the risk of severe disease. Further potential factors include recent vaccinations and a more diverse memory T cell repertoire. This manuscript reviews age-related host factors that may protect children from COVID-19 and complications associated, and addresses the confusion around seropositivity and immunity.
Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/patogenicidade , Infecções por Coronaviridae/prevenção & controle , Coronaviridae/patogenicidade , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Imunidade Adaptativa/efeitos dos fármacos , Adolescente , Doenças Assintomáticas , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , COVID-19 , Criança , Coronaviridae/efeitos dos fármacos , Coronaviridae/imunologia , Infecções por Coronaviridae/epidemiologia , Infecções por Coronaviridae/imunologia , Infecções por Coronaviridae/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Proteção Cruzada , Feminino , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Imunidade Inata/efeitos dos fármacos , Masculino , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , SARS-CoV-2 , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Reino Unido/epidemiologia , Vacinação , Adulto JovemAssuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Imunidade Coletiva , Pandemias/prevenção & controle , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle , Vacinas Virais/administração & dosagem , Anticorpos Antivirais/biossíntese , Linfócitos B/imunologia , Linfócitos B/virologia , Betacoronavirus/patogenicidade , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Vacinação em Massa , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , SARS-CoV-2 , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Fatores de TempoAssuntos
Linfócitos B/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Citocinas/biossíntese , Imunidade Celular , Pneumonia Viral/imunologia , Subpopulações de Linfócitos T/imunologia , Anticorpos Antivirais/biossíntese , Doenças Assintomáticas , Linfócitos B/virologia , Betacoronavirus/imunologia , COVID-19 , Infecções por Coronavirus/patologia , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Humanos , Imunidade Humoral , Imunização Passiva/métodos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Memória Imunológica , Ativação Linfocitária , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/terapia , Pneumonia Viral/virologia , SARS-CoV-2 , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/virologia , Fatores de Tempo , Soroterapia para COVID-19RESUMO
BACKGROUND: HIV-1 infects a wide range of CD4+ T cells with different phenotypic properties and differing expression levels of entry coreceptors. We sought to determine the viral tropism of subtype C (C-HIV) Envelope (Env) clones for different CD4+ T cell subsets and whether tropism changes during acute to chronic disease progression. HIV-1 envs were amplified from the plasma of five C-HIV infected women from three untreated time points; less than 2 months, 1-year and 3-years post-infection. Pseudoviruses were generated from Env clones, phenotyped for coreceptor usage and CD4+ T cell subset tropism was measured by flow cytometry. RESULTS: A total of 50 C-HIV envs were cloned and screened for functionality in pseudovirus infection assays. Phylogenetic and variable region characteristic analysis demonstrated evolution in envs between time points. We found 45 pseudoviruses were functional and all used CCR5 to mediate entry into NP2/CD4/CCR5 cells. In vitro infection assays showed transitional memory (TM) and effector memory (EM) CD4+ T cells were more frequently infected (median: 46% and 25% of total infected CD4+ T cells respectively) than naïve, stem cell memory, central memory and terminally differentiated cells. This was not due to these subsets contributing a higher proportion of the CD4+ T cell pool, rather these subsets were more susceptible to infection (median: 5.38% EM and 2.15% TM cells infected), consistent with heightened CCR5 expression on EM and TM cells. No inter- or intra-participant changes in CD4+ T cell subset tropism were observed across the three-time points. CONCLUSIONS: CD4+ T cell subsets that express more CCR5 were more susceptible to infection with C-HIV Envs, suggesting that these may be the major cellular targets during the first 3 years of infection. Moreover, we found that viral tropism for different CD4+ T cell subsets in vitro did not change between Envs cloned from acute to chronic disease stages. Finally, central memory, naïve and stem cell memory CD4+ T cell subsets were susceptible to infection, albeit inefficiently by Envs from all time-points, suggesting that direct infection of these cells may help establish the latent reservoir early in infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Subpopulações de Linfócitos T/imunologia , Tropismo Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Adulto , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Feminino , Variação Genética , Infecções por HIV/imunologia , HIV-1/classificação , HIV-1/genética , Humanos , Memória Imunológica , Estudos Longitudinais , Filogenia , Receptores de HIV/metabolismo , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. A disadvantage of this method is the use of a ubiquitous promoter leads not only to genetic modification of the leukocyte subset of interest e.g. T-cells, but also all other subsequent leukocyte progeny of the parent HSCs. To overcome this limitation we tested a bicistronic lentivirus, enabling subset specific modifications. Designed novel lentiviral constructs harbor a global promoter (mPGK) regulating mCherry for HSCs selection and a T-cell specific promoter upstream of eGFP. Two T-cell specific promoters were assessed: the distal Lck-(dLck) and the CD3δ-promoter. Transduced HSCs were FACS sorted by mCherry expression and transferred into sublethally irradiated C57/BL6 mice. Successful transplantation and T-cell specific expression of eGFP was monitored by peripheral blood assessment. Furthermore, recruitment response of lentiviral engineered leukocytes to the site of inflammation was tested in a peritonitis model without functional impairment. Our constructed lentivirus enables fast generation of subset specific leukocyte transgenesis as shown in T-cells in vivo and opens new opportunities to modify other HSCs derived subsets in the future.
Assuntos
Células-Tronco Hematopoéticas/virologia , Infecções por Lentivirus/virologia , Lentivirus/genética , Subpopulações de Linfócitos T/fisiologia , Subpopulações de Linfócitos T/virologia , Animais , Complexo CD3/genética , Linhagem Celular Tumoral , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Inflamação/genética , Inflamação/virologia , Leucócitos/fisiologia , Leucócitos/virologia , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genéticaRESUMO
Immune activation is the hallmark of HIV infection and plays a role in the pathogenesis of the disease. In the context of suppressed HIV RNA replication by combination antiretroviral therapy (cART), there remains immune activation which is associated to the HIV reservoirs. Persistent virus contributes to a sustained inflammatory environment promoting accumulation of "activated/exhausted" T cells with diminished effector function. These T cells show increased expression of immunomodulatory receptors including Programmed cell death protein (PD1), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA4), Lymphocyte activation gene 3 (LAG3), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin domain containing 3 (TIM3) among others. More importantly, recent reports had demonstrated that, HIV infected T cells express checkpoint receptors, contributing to their survival and promoting maintenance of the viral reservoir. Therapeutic strategies are focused on viral reservoir elimination and/or those to achieve sustained cART-free virologic remission. In this review, we will discuss the immunological basis and the latest advances of the use of checkpoint inhibitors to treat HIV infection.
Assuntos
Suscetibilidade a Doenças , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/imunologia , Interações Hospedeiro-Patógeno , Receptores Imunológicos/metabolismo , Animais , Terapia Antirretroviral de Alta Atividade , Biomarcadores , Ensaios Clínicos como Assunto , Gerenciamento Clínico , Suscetibilidade a Doenças/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Proteínas de Checkpoint Imunológico/metabolismo , Imunomodulação , Terapia de Alvo Molecular , Receptores Imunológicos/antagonistas & inibidores , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologia , Resultado do Tratamento , Carga ViralRESUMO
T Follicular Helper (Tfh) cells are an independent CD4+ T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.
Assuntos
Centro Germinativo/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Linfócitos B/imunologia , Linfócitos B/virologia , Centro Germinativo/virologia , Camundongos , Subpopulações de Linfócitos T/virologia , Linfócitos T Auxiliares-Indutores/virologiaRESUMO
Background: Visceral leishmaniasis/HIV-co-infected patients (VL/HIV) accounts for around 8% of VL reported cases in Brazil. Relapses of Leishmania infection after anti-leishmanial treatment constitute a great challenge in the clinical practice because of the disease severity and drug resistance. We have shown that non-relapsing-VL/HIV (NR-) evolved with increase of CD4+ T-cell counts and reduction of activated CD4+ and CD8+ T cells after anti-leishmanial treatment. This immune profile was not observed in relapsing-VL/HIV patients (R-), indicating a more severe immunological compromising degree. Elevated activation status may be related to a deficient immune reconstitution and could help to explain the frequent relapses in VL/HIV co-infection. Our aim was to evaluate if this gain of T cells was related to changes in the peripheral TCRVß repertoire and inflammatory status, as well as the possible thymus involvement in the replenishment of these newly formed T lymphocytes. Methods: VL/HIV patients, grouped into non-relapsing (NR- = 6) and relapsing (R- = 12) were evaluated from the active phase up to 12 months post-treatment (mpt). HIV-infected patients (non-VL) and healthy subjects (HS) were included. The TCRVß repertoire was evaluated ex vivo by flow cytometry, whereas the plasmatic cytokine levels were assessed by Luminex assay. To evaluate the thymic output, DNA was extracted from PBMCs for TCR rearrangement excision circles (TREC) quantification by qPCR. Results: VL/HIV cases presented an altered mobilization profile (expansions or retractions) of the TCRVß families when compared to HS independent of the follow-up phase (p < 0.05). TCRVß repertoire on CD4+ T-cells was more homogeneous in the NR-VL/HIV cases, but heterogeneous on CD8+ T-cells, since different Vß-families were mobilized. NR-VL/HIV had the inflammatory pattern reduced after 6 mpt. Importantly, VL/HIV patients showed number of TREC copies lower than controls during all follow-up. An increase of recent thymic emigrants was observed in NR-VL/HIV individuals at 10 mpt compared to R- patients (p < 0.01), who maintained lower TREC contents than the HIV controls. Conclusions: VL/HIV patients that maintain the thymic function, thus generating new T-cells, seem able to replenish the T lymphocyte compartment with effector cells, then enabling parasite control.
Assuntos
Coinfecção , Infecções por HIV/imunologia , Leishmaniose Visceral/imunologia , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Relação CD4-CD8 , Estudos de Casos e Controles , Proliferação de Células , Citocinas/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Fenótipo , Estudos Prospectivos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Recidiva , Subpopulações de Linfócitos T/parasitologia , Subpopulações de Linfócitos T/virologia , Timo/parasitologia , Timo/virologia , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: We observe changes of the main lymphocyte subsets (CD16+CD56ãCD19ãCD3ãCD4ãand CD8) in COVID-19-infected patients and explore whether the changes are associated with disease severity. METHODS: One-hundred and fifty-four cases of COVID-19-infected patients were selected and divided into 3 groups (moderate group, severe group and critical group). The flow cytometry assay was performed to examine the numbers of lymphocyte subsets. RESULTS: CD3+, CD4+ and CD8 + T lymphocyte subsets were decreased in COVID-19-infected patients. Compared with the moderate group and the sever group, CD3+, CD4+ and CD8+ T cells in the critical group decreased greatly (P < 0.001, P = 0.005 or P = 0.001). CONCLUSIONS: Reduced CD3+, CD4+, CD8+ T lymphocyte counts may reflect the severity of the COVID-19. Monitoring T cell changes has important implications for the diagnosis and treatment of severe patients who may become critically ill.