RESUMO
Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulate the immune response against many RNA viruses. We previously identified that a small molecule compound, KIN1148, led to the activation of IFN regulatory factor 3 (IRF3) and served to enhance protection against influenza A virus (IAV) A/California/04/2009 infection. We have now determined direct binding of KIN1148 to RIG-I to drive expression of IFN regulatory factor 3 and NF-κB target genes, including specific immunomodulatory cytokines and chemokines. Intriguingly, KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I IFN treatment. When administered in combination with a vaccine against IAV, KIN1148 induces both neutralizing Ab and IAV-specific T cell responses compared with vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal A/California/04/2009 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing noncanonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination.
Assuntos
Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Vacinas contra Influenza , Influenza Humana , Humanos , Animais , Camundongos , Proteína DEAD-box 58/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Adjuvantes Imunológicos , Antivirais/farmacologia , Imunidade InataRESUMO
OBJECTIVE: People with chronic HIV exhibit lower regional brain volumes compared to people without HIV (PWOH). Whether imaging alterations observed in chronic infection occur in acute HIV infection (AHI) remains unknown. DESIGN: Cross-sectional study of Thai participants with AHI. METHODS: One hundred and twelve Thai males with AHI (age 20-46) and 18 male Thai PWOH (age 18-40) were included. Individuals with AHI were stratified into early (Fiebig I-II; n â=â32) and late (Fiebig III-V; n â=â80) stages of acute infection using validated assays. T1-weighted scans were acquired using a 3 T MRI performed within five days of antiretroviral therapy (ART) initiation. Volumes for the amygdala, caudate nucleus, hippocampus, nucleus accumbens, pallidum, putamen, and thalamus were compared across groups. RESULTS: Participants in late Fiebig stages exhibited larger volumes in the nucleus accumbens (8% larger; P â=â0.049) and putamen (19%; P â<â0.001) when compared to participants in the early Fiebig. Compared to PWOH, participants in late Fiebig exhibited larger volumes of the amygdala (9% larger; P â=â0.002), caudate nucleus (11%; P â=â0.005), nucleus accumbens (15%; P â=â0.004), pallidum (19%; P â=â0.001), and putamen (31%; P â<â0.001). Brain volumes in the nucleus accumbens, pallidum, and putamen correlated modestly with stimulant use over the past four months among late Fiebig individuals ( P sâ<â0.05). CONCLUSIONS: Findings indicate that brain volume alterations occur in acute infection, with the most prominent differences evident in the later stages of AHI. Additional studies are needed to evaluate mechanisms for possible brain disruption following ART, including viral factors and markers of neuroinflammation.
Assuntos
Infecções por HIV , Humanos , Masculino , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Adolescente , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Estudos Transversais , Encéfalo/diagnóstico por imagem , HIV , Imageamento por Ressonância Magnética/métodosRESUMO
Activation of the NF-κB signaling pathway by Protein Kinase C (PKC) agonists is a potent mechanism for human immunodeficiency virus (HIV) latency disruption in vitro. However, significant toxicity risks and the lack of evidence supporting their activity in vivo have limited further evaluation of PKC agonists as HIV latency-reversing agents (LRA) in cure strategies. Here we evaluated whether GSK445A, a stabilized ingenol-B derivative, can induce HIV/simian immunodeficiency virus (SIV) transcription and virus production in vitro and demonstrate pharmacological activity in nonhuman primates (NHP). CD4+ T cells from people living with HIV and from SIV+ rhesus macaques (RM) on antiretroviral therapy (ART) exposed in vitro to 25 nM of GSK445A produced cell-associated viral transcripts as well as viral particles at levels similar to those induced by PMA/Ionomycin, indicating that GSK445A can potently reverse HIV/SIV latency. Importantly, these concentrations of GSK445A did not impair the proliferation or survival of HIV-specific CD8+ T cells, but instead, increased their numbers and enhanced IFN-γ production in response to HIV peptides. In vivo, GSK445A tolerability was established in SIV-naïve RM at 15 µg/kg although tolerability was reduced in SIV-infected RM on ART. Increases in plasma viremia following GSK445A administration were suggestive of increased SIV transcription in vivo. Collectively, these results indicate that GSK445A is a potent HIV/SIV LRA in vitro and has a tolerable safety profile amenable for further evaluation in vivo in NHP models of HIV cure/remission.
Assuntos
Diterpenos/farmacologia , HIV , Proteína Quinase C/efeitos dos fármacos , Vírus da Imunodeficiência Símia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Animais , Humanos , Macaca mulatta , Proteína Quinase C/metabolismo , RNA Viral/efeitos dos fármacos , Transcrição GênicaRESUMO
The mitochondrial antiviral signaling protein (MAVS) is part of the cell's innate immune mechanism of defense. MAVS mRNA is bicistronic and can give rise to a full length-MAVS and a shorter isoform termed miniMAVS. In response to viral infections, viral RNA can be sensed by the cytosolic RNA sensors retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation-associated protein 5 (MDA5) and activate NF-κB through interaction with MAVS. MAVS can also sense cellular stress and activate an anti-oxidative stress (AOS) response through the activation of NF-κB. Because NF-κB is a main cellular transcription factor for HIV-1, we wanted to address what role MAVS plays in HIV-1 reactivation from latency in CD4 T cells. Our results indicate that RIG-I agonists required full length-MAVS whereas the AOS response induced by Dynasore through its catechol group can reactivate latent HIV-1 in a MAVS dependent manner through miniMAVS isoform. Furthermore, we uncover that PKC agonists, a class of latency-reversing agents, induce an AOS response in CD4 T cells and require miniMAVS to fully reactivate latent HIV-1. Our results indicate that the AOS response, through miniMAVS, can induce HIV-1 transcription in response to cellular stress and targeting this pathway adds to the repertoire of approaches to reactivate latent HIV-1 in 'shock-and-kill' strategies.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Proteínas Mitocondriais/metabolismo , Ativação Viral , Latência Viral , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Humanos , Modelos Biológicos , NF-kappa B/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio , Transdução de Sinais/efeitos dos fármacos , Ativação Viral/imunologia , Latência Viral/imunologiaRESUMO
People living with HIV (PWH) often exhibit poor responses to influenza vaccination despite effective combination anti-retroviral (ART) mediated viral suppression. There exists a paucity of data in identifying immune correlates of influenza vaccine response in context of HIV infection that would be useful in improving its efficacy in PWH, especially in younger individuals. Transcriptomic data were obtained by microarray from whole blood isolated from aviremic pediatric and adolescent HIV-infected individuals (4-25 yrs) given two doses of Novartis/H1N1 09 vaccine during the pandemic H1N1 influenza outbreak. Supervised clustering and gene set enrichment identified contrasts between individuals exhibiting high and low antibody responses to vaccination. High responders exhibited hemagglutination inhibition antibody titers >1:40 post-first dose and 4-fold increase over baseline. Baseline molecular profiles indicated increased gene expression in metabolic stress pathways in low responders compared to high responders. Inflammation-related and interferon-inducible gene expression pathways were higher in low responders 3 wks post-vaccination. The broad age range and developmental stage of participants in this study prompted additional analysis by age group (e.g. <13yrs and ≥13yrs). This analysis revealed differential enrichment of gene pathways before and after vaccination in the two age groups. Notably, CXCR5, a homing marker expressed on T follicular helper (Tfh) cells, was enriched in high responders (>13yrs) following vaccination which was accompanied by peripheral Tfh expansion. Our results comprise a valuable resource of immune correlates of vaccine response to pandemic influenza in HIV infected children that may be used to identify favorable targets for improved vaccine design in different age groups.
Assuntos
Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Vacinas contra Influenza/imunologia , Pandemias/prevenção & controle , Transcrição Gênica/genética , Adolescente , Adulto , Anticorpos Antivirais/imunologia , Criança , Pré-Escolar , Feminino , Testes de Inibição da Hemaglutinação/métodos , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/genética , Influenza Humana/imunologia , Masculino , Receptores CXCR5/imunologia , Células T Auxiliares Foliculares/imunologia , Vacinação/métodos , Adulto JovemRESUMO
BACKGROUND: Early antiretroviral therapy (ART) restricts the size of the human immunodeficiency virus (HIV) reservoir in infants. However, whether antiretroviral (ARV) prophylaxis given to exposed vertically infected children exerts similar effects remains unknown. METHODS: We measured total and integrated HIV DNA, as well as the frequency of CD4 T cells producing multiply spliced RNA (msRNA) after stimulation (inducible reservoir) in vertically infected Thai infants. Eighty-five infants were followed longitudinally for up to 3 years. We compared the size of the reservoir in children who received continuous ARV prophylaxis since birth vs those who never received or discontinued prophylaxis before initiating ART. We used samples from a cross-sectional cohort of 37 Thai children who had initiated ART within 6 months of life to validate our findings. RESULTS: Before ART, levels of HIV DNA and the frequencies of cells producing msRNA were significantly lower in infants who received continuous ARV prophylaxis since birth compared to those in whom ARV prophylaxis was discontinued or never initiated (P < .020 and P < .001, respectively). Upon ART initiation, total and integrated HIV DNA levels decayed significantly in both groups (P < .01 in all cases). Interestingly, the initial differences in the frequencies of infected cells persisted during 3 years on ART. The beneficial effect of prophylaxis on the size of the HIV reservoir was confirmed in the cross-sectional study. Importantly, no differences were observed between children who discontinued prophylactic ARVs before starting ART and those who delayed ART initiation without receiving prior prophylaxis. CONCLUSIONS: Neonatal ARV prophylaxis with direct transition to ART durably limits the size of the HIV reservoir.
Assuntos
Antirretrovirais , Infecções por HIV , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos , Criança , Estudos Transversais , HIV , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Humanos , Lactente , Recém-NascidoRESUMO
The innate immune response to cytosolic DNA involves transcriptional activation of type I interferons (IFN-I) and proinflammatory cytokines. This represents the culmination of intracellular signaling pathways that are initiated by pattern recognition receptors that engage DNA and require the adaptor protein Stimulator of Interferon Genes (STING). These responses lead to the generation of cellular and tissue states that impair microbial replication and facilitate the establishment of long-lived, antigen-specific adaptive immunity. Ultimately this can lead to immune-mediated protection from infection but also to the cytotoxic T cell-mediated clearance of tumor cells. Intriguingly, pharmacologic activation of STING-dependent phenotypes is known to enhance both vaccine-associated immunogenicity and immune-based anti-tumor therapies. Unfortunately, the STING protein exists as multiple variant forms in the human population that exhibit differences in their reactivity to chemical stimuli and in the intensity of molecular signaling they induce. In light of this, STING-targeting drug discovery efforts require an accounting of protein variant-specific activity. Herein we describe a small molecule termed M04 that behaves as a novel agonist of human STING. Importantly, we find that the molecule exhibits a differential ability to activate STING based on the allelic variant examined. Furthermore, while M04 is inactive in mice, expression of human STING in mouse cells rescues reactivity to the compound. Using primary human cells in ex vivo assays we were also able to show that M04 is capable of simulating innate responses important for adaptive immune activation such as cytokine secretion, dendritic cell maturation, and T cell cross-priming. Collectively, this work demonstrates the conceivable utility of a novel agonist of human STING both as a research tool for exploring STING biology and as an immune potentiating molecule.
Assuntos
Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Proteínas de Membrana/agonistas , Alelos , Animais , Descoberta de Drogas , Humanos , Imunidade Inata/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , CamundongosAssuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV/crescimento & desenvolvimento , HIV/imunologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Simulação por Computador , Humanos , Resultado do TratamentoRESUMO
PURPOSE OF REVIEW: Increasing evidence suggests that purging the latent HIV reservoir in virally suppressed individuals will require both the induction of viral replication from its latent state and the elimination of these reactivated HIV-infected cells ('Shock and Kill' strategy). Boosting potent HIV-specific CD8 T cells is a promising way to achieve an HIV cure. RECENT FINDINGS: Recent studies provided the rationale for developing immune interventions to increase the numbers, function and location of HIV-specific CD8 T cells to purge HIV reservoirs. Multiple approaches are being evaluated including very early suppression of HIV replication in acute infection, adoptive cell transfer, therapeutic vaccination or use of immunomodulatory molecules. New assays to measure the killing and antiviral function of induced HIV-specific CD8 T cells have been developed to assess the efficacy of these new approaches. The strategies combining HIV reactivation and immunobased therapies to boost HIV-specific CD8 T cells can be tested in in-vivo and in-silico models to accelerate the design of new clinical trials. SUMMARY: New immunobased strategies are explored to boost HIV-specific CD8 T cells able to purge the HIV-infected cells with the ultimate goal of achieving spontaneous control of viral replication without antiretroviral treatment.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV/imunologia , HIV/fisiologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Pesquisa Biomédica/tendências , Terapia Combinada/métodos , Descoberta de Drogas/tendências , Tratamento Farmacológico/métodos , Humanos , Imunoterapia/métodosRESUMO
Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.
Assuntos
Linfócitos B/imunologia , Infecções por HIV/imunologia , HIV , Interleucina-2/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Linfócitos B/virologia , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Doença Crônica , Humanos , Memória Imunológica , Pessoa de Meia-Idade , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/virologia , Adulto JovemRESUMO
UNLABELLED: The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. IMPORTANCE: The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antivirais/biossíntese , RNA Helicases DEAD-box/imunologia , Vacinas contra Influenza/imunologia , Oligorribonucleotídeos/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Adjuvantes Imunológicos/genética , Animais , Proteína DEAD-box 58 , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Células HEK293 , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunização , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/imunologia , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Cultura Primária de Células , Receptores Imunológicos , Análise de Sobrevida , Equilíbrio Th1-Th2/efeitos dos fármacos , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
UNLABELLED: The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5'-triphosphate (5'ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5'ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5'pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5'pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5'pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses. IMPORTANCE: The development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral response in vitro and in vivo and was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5'pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.
Assuntos
Vírus Chikungunya/imunologia , RNA Helicases DEAD-box/imunologia , Vírus da Dengue/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , RNA Viral/agonistas , RNA Viral/imunologia , Viroses/imunologia , Animais , Linhagem Celular , Vírus Chikungunya/química , Vírus Chikungunya/genética , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Vírus da Dengue/química , Vírus da Dengue/genética , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/genética , Camundongos , Camundongos Endogâmicos BALB C , Conformação de Ácido Nucleico , RNA Viral/genética , Receptores Imunológicos , Viroses/genética , Viroses/virologiaRESUMO
Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/10(6) cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Citometria de Fluxo/métodos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Comunicação Celular/imunologia , Citotoxicidade Imunológica , Humanos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Immediate-early host-virus interactions that occur during the first weeks after HIV infection have a major impact on disease progression. The mechanisms underlying the failure of HIV-specific CD8 T-cell response to persist and control viral replication early in infection are yet to be characterized. In this study, we performed a thorough phenotypic, gene expression and functional analysis to compare HIV-specific CD8 T cells in acutely and chronically infected subjects. We showed that HIV-specific CD8 T cells in primary infection can be distinguished by their metabolic state, rate of proliferation, and susceptibility to apoptosis. HIV-specific CD8 T cells in acute/early HIV infection secreted less IFN-γ but were more cytotoxic than their counterparts in chronic infection. Importantly, we showed that the levels of IL-7R expression and the capacity of HIV-specific CD8 T cells to secrete IL-2 on antigenic restimulation during primary infection were inversely correlated with the viral set-point. Altogether, these data suggest an altered metabolic state of HIV-specific CD8 T cells in primary infection resulting from hyperproliferation and stress induced signals, demonstrate the discordant function of HIV-specific CD8 T cells during early/acute infection, and highlight the importance of T-cell maintenance for viral control.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Regulação da Expressão Gênica/imunologia , Infecções por HIV/metabolismo , Doença Aguda , Citotoxicidade Celular Dependente de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Apoptose , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Doença Crônica , HIV/fisiologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/patologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Fatores de Tempo , Carga ViralRESUMO
Chronic HIV infection, which is primarily characterized by the progressive depletion of total CD4(+) T cells, also causes persistent inflammation and immune activation. This is followed by profound changes in cellular and tissue microenvironments that often lead to prolonged immune dysfunction. The global nature of this immune dysfunction suggests that factors that are involved in immune cell survival, proliferation, differentiation and maturation are all affected. Of particular interest is the transcriptional factor Foxo3a that regulates a number of genes that are critical in the development and the maintenance of T and B cells, dendritic cells (DCs) and macrophages. Alterations in the microenvironment mediated by HIV infection cause significant increase in the transcriptional activity of Foxo3a; this has major impact on T cell and B cell immunity. In fact, recent findings from HIV infected individuals highlight three important points: (1) the alteration of Foxo3a signaling during HIV infection deregulates innate and adaptive immune responses; (2) Foxo3a-mediated effects are reversible and could be restored by interfering with the Foxo3a pathway; and (3) down-regulation of Foxo3a transcriptional activity in elite controllers (ECs) represents a molecular signature, or a correlate of immunity, associated with natural protection and lack of disease progression. In this review, we will discuss how HIV-infection altered microenvironments could result in impaired immune responses via the Foxo3a signaling pathway. Defining precisely the molecular mechanisms of how persistent inflammation and immune activation are able to influence the Foxo3a pathway could ultimately help in the development of novel approaches to improve immune responses in HIV infected subjects.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/imunologia , Infecções por HIV/imunologia , Hematopoese/imunologia , Transdução de Sinais/imunologia , Imunidade Adaptativa/imunologia , Linfócitos T CD4-Positivos/metabolismo , Sobrevivência Celular/imunologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Infecções por HIV/metabolismo , Humanos , Imunidade Inata/imunologia , Modelos ImunológicosRESUMO
Persistent exposure to cognate Ag leads to the functional impairment and exhaustion of HIV-specific CD8 T cells. Ag withdrawal, attributable either to antiretroviral treatment or the emergence of epitope escape mutations, causes HIV-specific CD8 T cell responses to wane over time. However, this process does not continue to extinction, and residual CD8 T cells likely play an important role in the control of HIV replication. In this study, we conducted a longitudinal analysis of clonality, phenotype, and function to define the characteristics of HIV-specific CD8 T cell populations that persist under conditions of limited antigenic stimulation. Ag decay was associated with dynamic changes in the TCR repertoire, increased expression of CD45RA and CD127, decreased expression of programmed death-1, and the emergence of polyfunctional HIV-specific CD8 T cells. High-definition analysis of individual clonotypes revealed that the Ag loss-induced gain of function within HIV-specific CD8 T cell populations could be attributed to two nonexclusive mechanisms: 1) functional improvement of persisting clonotypes; and 2) recruitment of particular clonotypes endowed with superior functional capabilities.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/virologia , HIV/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Clonais , Regulação da Expressão Gênica/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-7 , Antígenos Comuns de Leucócito , Estudos Longitudinais , Receptor de Morte Celular Programada 1RESUMO
The major function of CD8 T cells is to kill specifically target cells. Moreover in certain incurable diseases, antigen-specific human CD8 T cells are impaired, and assessment of their cytolytic activity could bring insights into their physiopathological role and ways to restore immune dysfunctions for immunotherapeutic purposes. Despite this, T cell cytolytic function has been seldom analyzed thoroughly in humans, due to the lack of approaches well suited for ex vivo assessment of T cell cytotoxicity. Current techniques require prior in vitro expansion of antigen-specific CD8 T cell populations and the use of immortalized cells as targets to measure the cell-mediated killing. Furthermore, bulk cytotoxic activity is frequently measured using percentage of specific lysis calculations that do not quantify actual target cell death and effector numbers at the single cell level. Here we established a new flow cytometry-based assay that allows accurate single-cell analysis of cytotoxic capacity of primary antigen-specific CD8 T cells generated in vivo in humans after antigenic exposure without in vitro amplification that can be used for specificities restricted by different HLAs as target cells are autologous cells. We show that this assay is robust, highly sensitive irrespective of the frequency of antigen-specific CD8 T cells, and allows accurate calculation of the index of cytotoxic capacity in lytic units. This new assay provides a sensitive method to measure the intrinsic cytotoxic activity of antigen-specific CD8 T cells directly ex vivo on human primary cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos HLA/imunologia , Linfócitos T Citotóxicos/imunologia , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Célula Única/métodosRESUMO
HIV persists in a reservoir of latently infected CD4(+) T cells in individuals treated with highly active antiretroviral therapy (HAART). Here we identify central memory (T(CM)) and transitional memory (T(TM)) CD4(+) T cells as the major cellular reservoirs for HIV and find that viral persistence is ensured by two different mechanisms. HIV primarily persists in T(CM) cells in subjects showing reconstitution of the CD4(+) compartment upon HAART. This reservoir is maintained through T cell survival and low-level antigen-driven proliferation and is slowly depleted with time. In contrast, proviral DNA is preferentially detected in T(TM) cells from aviremic individuals with low CD4(+) counts and higher amounts of interleukin-7-mediated homeostatic proliferation, a mechanism that ensures the persistence of these cells. Our results suggest that viral eradication might be achieved through the combined use of strategic interventions targeting viral replication and, as in cancer, drugs that interfere with the self renewal and persistence of proliferating memory T cells.
Assuntos
Proliferação de Células , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Linfócitos T/fisiologia , Carga Viral , Latência Viral/imunologia , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Sobrevivência Celular , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Homeostase/imunologia , Humanos , Memória Imunológica , Dados de Sequência Molecular , Linfócitos T/imunologia , Replicação Viral/fisiologiaRESUMO
The majority of acute hepatitis C virus (HCV) infections progress to chronicity and progressive liver damage. Alpha interferon (IFN-alpha) antiviral therapy achieves the highest rate of success when IFN-alpha is administered early during the acute phase, but the underlying mechanisms are unknown. We used a panel of major histocompatibility complex class I tetramers to monitor the phenotypic and functional signatures of HCV-specific T cells during acute HCV infection with different infection outcomes and during early IFN therapy. We demonstrate that spontaneous resolution correlates with the early development of polyfunctional (IFN-gamma- and IL-2-producing and CD107a(+)) virus-specific CD8(+) T cells. These polyfunctional T cells are distinguished by the expression of CD127 and Bcl-2 and represent a transitional memory T-cell subset that exhibits the phenotypic and functional signatures of both central and effector memory T cells. In contrast, HCV-specific CD8(+) T cells in acute infections evolving to chronicity expressed low levels of CD127 and Bcl-2, exhibited diminished proliferation and cytokine production, and eventually disappeared from the periphery. Early therapeutic intervention with pegylated IFN-alpha rescued polyfunctional memory T cells expressing high levels of CD127 and Bcl-2. These cells were detectable for up to 1 year following discontinuation of therapy. Our results suggest that the polyfunctionality of HCV-specific T cells can be predictive of the outcome of acute HCV infection and that early therapeutic intervention can reconstitute the pool of long-lived polyfunctional memory T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C , Interferon-alfa , Subpopulações de Linfócitos T/imunologia , Adulto , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/citologia , Epitopos , Feminino , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatite C/imunologia , Humanos , Memória Imunológica/imunologia , Interferon-alfa/imunologia , Interferon-alfa/uso terapêutico , Subunidade alfa de Receptor de Interleucina-7/imunologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Subpopulações de Linfócitos T/citologiaRESUMO
Generation of memory T cells, which mediate immunity against microbes and cancers, relies, for optimal activity, on the interactions of multiple cell types that are highly regulated through the expression of soluble factors and negative and positive receptors. Their disruption will lead to aberrant immune responses, which can result in the invasion of the host by foreign pathogens. In chronic viral infections including HIV and hepatitis C virus, persistence of antigen and lack of CD4 help (HIV) disrupt memory T-cell function and induce defects in memory T-cell responses, which have been defined as T-cell exhaustion. In this review, we examine the molecular mechanisms involved in such T-cell dysfunction. Better understanding of these mechanisms will assist in the development of novel therapies to prevent the immune damage mediated by HIV infection.