RESUMEN
Dendritic cells (DCs), macrophages (MPs), and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous Ags preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum translocation, trimming, and MHC-I presentation. In this study, we compared the capacity of DCs, MPs, and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848, and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs, and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs, and monocytes. Differences in Ag-processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load.
Asunto(s)
Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Epítopos/inmunología , Infecciones por VIH/inmunología , VIH/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/metabolismo , Línea Celular Transformada , Citosol/inmunología , Citosol/metabolismo , Células Dendríticas/metabolismo , Humanos , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Péptido Hidrolasas/metabolismo , Péptidos/química , Péptidos/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , Linfocitos T Citotóxicos/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
UNLABELLED: Antigen persistence in chronic infections and cancer upregulates inhibitory networks, such as the PD-1 and interleukin-10 (IL-10) pathways, that impair immunity and lead to disease progression. These pathways are attractive targets for immunotherapy, as demonstrated by recent clinical trials of PD-1/PD-L1 blockade in cancer patients. However, in HIV-1 infection not all subjects respond to inhibition of either pathway and the mechanistic interactions between these two networks remain to be better defined. Here we demonstrate that in vitro blockade of PD-L1 and/or IL-10Rα results in markedly different profiles of HIV-1-specific CD4 T cell restoration. Whereas PD-L1 blockade leads to balanced increase in gamma interferon (IFN-γ), IL-2, and IL-13 secretion, IL-10Rα blockade preferentially restores IFN-γ production. In viremic subjects, combined PD-L1/IL-10Rα blockade results in a striking 10-fold increase in IFN-γ secretion by HIV-1-specific CD4 T cells that is not observed in subjects with spontaneous (elite controllers) or therapy-induced control of viral replication. In contrast to the dramatic increase in IFN-γ production, concurrent blockade has a marginal additive effect on IL-2 production, IL-13 secretion, and HIV-1-specific CD4 T cell proliferation. IFN-γ produced by Thelper cells upregulates PD-L1, HLA I/II, and IL-12 expression by monocytes. The effect of combined blockade on IFN-γ was dependent on reciprocal reinforcement through IL-12. These studies provide crucial information on the different immunoregulatory qualities of PD-1 and IL-10 in progressive disease and link exhausted virus-specific CD4 T cells and monocytes in the regulation of IFN-γ and IL-12 secretion. IMPORTANCE: Infection with HIV results in most people in uncontrolled viral replication and progressive weakening of the body defenses. In the absence of antiviral therapy, this process results in clinical disease, or AIDS. An important reason why HIV continues to multiply is that a population of white blood cells called CD4 T cells that targets the virus fails to work properly. At least part of this impairment is under the control of inhibitory mechanisms that can be blocked to improve the function of these CD4 T cells. In this report, we show that blocking one or two of the molecules involved, called PD-1 and IL-10, has different effects on the individual functions of these cells and that one is strongly improved. We investigate how these effects are caused by interactions between CD4 T cells and antigen-presenting cells. These observations can have implications for new therapeutic approaches in HIV infection.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , VIH-1/inmunología , Interleucina-10/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Anticuerpos Monoclonales/farmacología , Células Presentadoras de Antígenos/virología , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Citocinas/biosíntesis , Epítopos de Linfocito T/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Humanos , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-10/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Various cosignaling molecules on T cells can contribute to activation, inhibition, or exhaustion, depending on context. The surface receptor signaling lymphocytic activation molecule (SLAM) family receptor CD244 (2B4/SLAMf4) has been shown to be capable of either inhibitory or enhancing effects upon engagement of its ligand CD48 (SLAMf2). We examined phenotypes of CD8 T cells from HIV(+) and HIV(neg) human donors, specific for HIV and/or respiratory syncytial virus. Cultured and ex vivo CD8 T cells expressed PD-1, CD244, and TIM-3. We found that ex vivo CD8 T cells downregulated CD244 in response to superantigen. Furthermore, cognate peptide induced rapid downregulation of both CD244 and TIM-3, but not PD-1, on CD8 T cell clones. CD244 downmodulation required simultaneous signaling via both TCR and CD244 itself. Using a pH-sensitive fluorophore conjugated to avidin-Ab tetramers, we found that CD244 crosslinking in the presence of TCR signaling resulted in rapid transport of CD244 to an acidic intracellular compartment. Downregulation was not induced by PMA-ionomycin, or prevented by PI3K inhibition, implicating a TCR-proximal signaling mechanism. CD244 internalization occurred within hours of TCR stimulation and required less peptide than was required to induce IFN-γ production. The degree of CD244 internalization varied among cultured CD8 T cell lines of different specificities, and correlated with the enhancement of IFN-γ production in response to CD48 blockade in HIV(+), but not HIV(neg), subjects. Our results indicate that rapid CD244 internalization is induced by a two-signal mechanism and plays a role in modulation of antiviral CD8 T cell responses by CD48-CD244 signaling.
Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Inmunológicos/inmunología , Transducción de Señal , Antígenos CD/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Infecciones por VIH/inmunología , Humanos , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Inmunológicos/metabolismo , Infecciones por Virus Sincitial Respiratorio/inmunología , Transducción de Señal/inmunología , Familia de Moléculas Señalizadoras de la Activación LinfocitariaRESUMEN
Viruses evade immune detection partly through immune-associated mutations. Analyses of HIV sequences derived from infected individuals have identified numerous examples of HLA-associated mutations within or adjacent to T cell epitopes, but the potential impact of most mutations on epitope production and presentation remains unclear. The multistep breakdown of proteins into epitopes includes trimming of N-extended peptides into epitopes by aminopeptidases before loading onto MHC class I molecules. Definition of sequence signatures that modulate epitope production would lead to a better understanding of factors driving viral evolution and immune escape at the population level. In this study, we identified cytosolic aminopeptidases cleavage preferences in primary cells and its impact on HIV Ag degradation into epitopes in primary human cell extracts by mass spectrometry and on epitope presentation to CTL. We observed a hierarchy of preferred amino acid cleavage by cytosolic aminopeptidases. We demonstrated that flanking mutations producing more or less cleavable motifs can increase or decrease epitope production and presentation by up to 14-fold. We found that the efficiency of epitope production correlates with cleavability of flanking residues. These in vitro findings were supported by in vivo population-level analyses of clinically derived viral sequences from 1134 antiretroviral-naive HIV-infected individuals: HLA-associated mutations immune pressures drove the selection of residues that are less cleavable by aminopeptidases predominantly at N-flanking sites, leading to reduced epitope production and immune recognition. These results underscore an important and widespread role of Ag processing mutations in HIV immune escape and identify molecular mechanisms underlying impaired epitope presentation.
Asunto(s)
Aminopeptidasas/inmunología , Presentación de Antígeno/inmunología , Epítopos de Linfocito T/inmunología , Infecciones por VIH/inmunología , Proteínas del Virus de la Inmunodeficiencia Humana/inmunología , Evasión Inmune/inmunología , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Presentación de Antígeno/genética , Separación Celular , Epítopos de Linfocito T/metabolismo , Citometría de Flujo , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Evasión Inmune/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , MutaciónRESUMEN
T cell dysfunction in the presence of ongoing antigen exposure is a cardinal feature of chronic viral infections with persistent high viremia, including HIV-1. Although interleukin-10 (IL-10) has been implicated as an important mediator of this T cell dysfunction, the regulation of IL-10 production in chronic HIV-1 infection remains poorly understood. We demonstrated that IL-10 is elevated in the plasma of individuals with chronic HIV-1 infection and that blockade of IL-10 signaling results in a restoration of HIV-1-specific CD4 T cell proliferation, gamma interferon (IFN-γ) secretion, and, to a lesser extent, IL-2 production. Whereas IL-10 blockade leads to restoration of IFN-γ secretion by HIV-1-specific CD4 T cells in all categories of subjects investigated, significant enhancement of IL-2 production and improved proliferation of CD4 T helper cells are restricted to viremic individuals. In peripheral blood mononuclear cells (PBMCs), this IL-10 is produced primarily by CD14(+) monocytes, but its production is tightly controlled by regulatory T cells (Tregs), which produce little IL-10 directly. When Tregs are depleted from PBMCs of viremic individuals, the effect of the IL-10 signaling blockade is abolished and IL-10 production by monocytes decreases, while the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), increases. The regulation of IL-10 by Tregs appears to be mediated primarily by contact or paracrine-dependent mechanisms which involve IL-27. This work describes a novel mechanism by which regulatory T cells control IL-10 production and contribute to dysfunctional HIV-1-specific CD4 T cell help in chronic HIV-1 infection and provides a unique mechanistic insight into the role of regulatory T cells in immune exhaustion.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Interleucina-10/inmunología , Monocitos/inmunología , Linfocitos T Reguladores/inmunología , Regulación hacia Arriba , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Interferón gamma/inmunología , Interleucina-10/sangre , Subunidad alfa del Receptor de Interleucina-2/inmunología , Leucocitos Mononucleares/inmunologíaRESUMEN
Defining the T helper functions impaired by programmed death-1 (PD-1) is crucial for understanding its role in defective HIV control and determining the therapeutic potential of targeting this inhibitory pathway. We describe here the relationships among disease stage, levels of PD-1 expression, and reversibility of CD4 T-cell impairment. PD-L1 blockade in vitro enhanced HIV-specific production of Th0 (IL-2), Th1 (IFN-γ), Th2 (IL-13), and TFH (IL-21) cytokines by CD4 T cells. PD-L1 blockade caused an early increase in cytokine transcription and translation that preceded cell proliferation. Although the impact of PD-L1 blockade on cytokine expression and, to a lesser extent, cell proliferation was associated with markers of disease progression, restoration of cytokine secretion was also observed in most subjects with undetectable viremia. PD-L1 blockade restored cytokine secretion in both PD-1intermediate and PD-1high sorted CD4 T-cell subsets. Compared with PD-1high HIV-specific CD8 T cells, PD-1high HIV-specific CD4 T cells showed lower expression of the inhibitory molecules CD160 and 2B4, demonstrating marked differences in expression of inhibitory receptors between T-cell subsets. These data show that PD-1 impairs HIV-specific T helper responses both by limiting expansion of these cells and by inhibiting effector functions of multiple differentiated CD4 T-cell subsets.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , Subgrupos de Linfocitos T/inmunología , Antígenos CD/metabolismo , Antígeno B7-H1 , Linfocitos T CD4-Positivos/metabolismo , Separación Celular , Citocinas/biosíntesis , Citocinas/inmunología , Citometría de Flujo , Infecciones por VIH/metabolismo , Humanos , Inmunofenotipificación , Receptor de Muerte Celular Programada 1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/metabolismoRESUMEN
Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape. The associations most frequently observed are between expression of an HLA class I molecule and variation from the consensus sequence. However, a substantial number of sites have been identified in which particular HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind this is so far unexplained. The current studies, focusing on two examples of "negatively associated" or apparently preserved epitopes, suggest an explanation for this phenomenon: negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. Such negative associations may only be in evidence transiently, because the statistical power to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation in the population, the epitope will be lost as a potential target to the immune system. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development.
Asunto(s)
Epítopos de Linfocito T/inmunología , Infecciones por VIH/transmisión , VIH-1/inmunología , Antígenos HLA/inmunología , Polimorfismo Genético/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas contra el SIDA , Adulto , Alelos , Secuencia de Aminoácidos , Niño , Preescolar , Secuencia de Consenso/genética , Secuencia de Consenso/inmunología , Epítopos de Linfocito T/genética , Evolución Molecular , Femenino , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/genética , Antígenos HLA/genética , Humanos , Masculino , Datos de Secuencia Molecular , Mutación/genética , Mutación/inmunología , Polimorfismo Genético/genéticaRESUMEN
Murine models indicate that interleukin-10 (IL-10) can suppress viral clearance, and interventional blockade of IL-10 activity has been proposed to enhance immunity in chronic viral infections. Increased IL-10 levels have been observed during HIV infection and IL-10 blockade has been shown to enhance T-cell function in some HIV-infected subjects. However, the categories of individuals in whom the IL-10 pathway is up-regulated are poorly defined, and the cellular sources of IL-10 in these subjects remain to be determined. Here we report that blockade of the IL-10 pathway augmented in vitro proliferative capacity of HIV-specific CD4 and CD8 T cells in individuals with ongoing viral replication. IL-10 blockade also increased cytokine secretion by HIV-specific CD4 T cells. Spontaneous IL-10 expression, measured as either plasma IL-10 protein or IL-10 mRNA in peripheral blood mononuclear cells (PBMCs), correlated positively with viral load and diminished after successful antiretroviral therapy. IL-10 mRNA levels were up-regulated in multiple PBMC subsets in HIV-infected subjects compared with HIV-negative controls, particularly in T, B, and natural killer (NK) cells, whereas monocytes were a major source of IL-10 mRNA in HIV-infected and -uninfected individuals. These data indicate that multiple cell types contribute to IL-10-mediated immune suppression in the presence of uncontrolled HIV viremia.
Asunto(s)
Infecciones por VIH/inmunología , Interleucina-10/inmunología , Linfocitos T/inmunología , Regulación hacia Arriba/inmunología , Viremia/inmunología , Proliferación Celular , Células Cultivadas , Enfermedad , Regulación de la Expresión Génica , Infecciones por VIH/complicaciones , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , ARN Mensajero/genética , Receptores de Interleucina-10/inmunología , Transducción de Señal/inmunología , Linfocitos T/citología , Linfocitos T/metabolismo , Viremia/complicaciones , Viremia/genética , Viremia/metabolismoRESUMEN
Biodegradable core--shell structured nanoparticles with a poly(ß-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell were developed for in vivo mRNA delivery with a view toward delivery of mRNA-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Messenger RNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of ~30%. Particles loaded with mRNA administered intranasally (i.n.) in mice led to the expression of the reporter protein luciferase in vivo as soon as 6 h after administration, a time point when naked mRNA given i.n. showed no expression. At later time points, luciferase expression was detected in naked mRNA-treated mice, but this group showed a wide variation in levels of transfection, compared to particle-treated mice. This system may thus be promising for noninvasive delivery of mRNA-based vaccines.
Asunto(s)
Portadores de Fármacos/química , Lípidos/química , Nanopartículas/química , Polímeros/química , ARN Mensajero/administración & dosificación , ARN Mensajero/química , Animales , Línea Celular , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificaciónRESUMEN
In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Antígeno HLA-B8/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Proteínas no Estructurales Virales/inmunología , Enfermedad Aguda , Secuencia de Aminoácidos/genética , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Enfermedad Crónica , Evolución Molecular , Femenino , Variación Genética/genética , Variación Genética/inmunología , Hepacivirus/genética , Hepatitis C/genética , Hepatitis C/patología , Humanos , Activación de Linfocitos/inmunología , Masculino , Datos de Secuencia Molecular , Mutación/genética , Mutación/inmunología , Proteínas no Estructurales Virales/genética , Viremia/inmunología , Viremia/patologíaRESUMEN
Hepatitis C virus (HCV) infection frequently persists despite eliciting substantial virus-specific immune responses. Thus, HCV infection provides a setting in which to investigate mechanisms of immune escape that allow for viral persistence. Viral amino acid substitutions resulting in decreased MHC binding or impaired Ag processing of T cell epitopes reduce Ag density on the cell surface, permitting evasion of T cell responses in chronic viral infection. Substitutions in viral epitopes that alter TCR contact residues frequently result in escape, but via unclear mechanisms because such substitutions do not reduce surface presentation of peptide-MHC complexes and would be expected to prime T cells with new specificities. We demonstrate that a known in vivo HCV mutation involving a TCR contact residue significantly diminishes T cell recognition and, in contrast to the original sequence, fails to effectively prime naive T cells. This mutant epitope thus escapes de novo immune recognition because there are few highly specific cognate TCR among the primary human T cell repertoire. This example is the first on viral immune escape via exploitation of a "hole" in the T cell repertoire, and may represent an important general mechanism of viral persistence.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatitis C/virología , Latencia del Virus/inmunología , Enfermedad Aguda , Adolescente , Adulto , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Presentación de Antígeno/genética , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Línea Celular Transformada , Células Cultivadas , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Hepatitis C/genética , Hepatitis C/patología , Humanos , Mutación , Estudios Prospectivos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Adulto JovenRESUMEN
HIV-1 directly activates human plasmacytoid DCs (pDCs) by upregulating the expression of costimulatory and MHC molecules and maturation markers, increasing T cell stimulatory activity, and inducing the production of type I interferons and TNF-alpha. A consequence of this activation is the bystander maturation of myeloid DCs and overall enhancement of antigen-presenting function. However, little is known about the mechanism(s) of pDC activation by HIV-1. Here we demonstrate by in vitro studies that IFN-alpha production by pDC in response to HIV-1 requires at least 2 interactions between the cell and virus. Initially, envelope-CD4 interactions mediate endocytosis of HIV-1, as demonstrated through the use of inhibitors of binding, fusion, endocytosis, and endosomal acidification. Subsequently, endosomally delivered viral nucleic acids, particularly RNA, stimulate pDCs through TLRs, as activation is reproduced with purified genomic RNA but not viral RNA packaging-deficient HIV-1 and blocked with different inhibitory TLR ligands. Finally, by using genetic complementation, we show that TLR7 is the likely primary target. Viral RNA rather than DNA in early retrotranscripts appears to be the active factor in HIV-1 that induces IFN-alpha secretion by pDCs. Since the decline in pDCs in chronic HIV-1 infection is associated with high viral loads and opportunistic infections, exploiting this natural adjuvant activity of HIV-1 RNA might be useful in the development of vaccines for the prevention of AIDS.
Asunto(s)
Células Dendríticas/inmunología , Endocitosis/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , ARN Viral/metabolismo , Receptores Toll-Like/metabolismo , Antígenos CD4/metabolismo , Células Cultivadas , Células Dendríticas/virología , Proteínas gp160 de Envoltorio del VIH/metabolismo , HumanosRESUMEN
BACKGROUND: HIV-1 eradication may require reactivation of latent virus along with stimulation of HIV-1-specific immune responses to clear infected cells. Immunization with autologous dendritic cells (DCs) transfected with viral mRNA is a promising strategy for eliciting HIV-1-specific immune responses. We performed a randomized controlled clinical trial to evaluate the immunogenicity of this approach in HIV-1-infected persons on antiretroviral therapy. METHODS: Fifteen participants were randomized 2:1 to receive intradermal immunization with HIV-1 Gag- and Nef-transfected DCs (vaccine) or mock-transfected DCs (placebo) at weeks 0, 2, 6, and 10. All participants also received DCs pulsed with keyhole limpet hemocyanin (KLH) to assess whether responses to a neo-antigen could be induced. RESULTS: After immunization, there were no differences in interferon-gamma enzyme-linked immunospot responses to HIV-1 Gag or Nef in the vaccine or placebo group. CD4 proliferative responses to KLH increased 2.4-fold (P = 0.026) and CD8 proliferative responses to KLH increased 2.5-fold (P = 0.053) after vaccination. There were increases in CD4 proliferative responses to HIV-1 Gag (2.5-fold vs. baseline, 3.4-fold vs. placebo, P = 0.054) and HIV-1 Nef (2.3-fold vs. baseline, 6.3-fold vs. placebo, P = 0.009) among vaccine recipients, but these responses were short-lived. CONCLUSION: Immunization with DCs transfected with mRNA encoding HIV-1 Gag and Nef did not induce significant interferon-gamma enzyme-linked immunospot responses. There were increases in proliferative responses to HIV-1 antigens and to a neo-antigen, KLH, but the effects were transient. Dendritic cell vaccination should be optimized to elicit stronger and long-lasting immune responses for this strategy to be effective as an HIV-1 therapeutic vaccine.
Asunto(s)
Vacunas contra el SIDA/inmunología , Células Dendríticas/inmunología , Infecciones por VIH/terapia , ARN Mensajero/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/uso terapéutico , Adulto , Ensayo de Immunospot Ligado a Enzimas , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Humanos , Inmunización , Inyecciones Intradérmicas , Masculino , Persona de Mediana Edad , ARN Mensajero/inmunología , Transfección , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single-cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to address a variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T-cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by ImageStream technology.
Asunto(s)
Citometría de Flujo/métodos , Hibridación Fluorescente in Situ/métodos , MicroARNs/análisis , ARN Mensajero/análisis , Linfocitos T/metabolismo , Citomegalovirus/inmunología , VIH/inmunología , Infecciones por VIH/inmunología , Humanos , Interferón gamma/genética , Interleucina-2/genética , MicroARNs/genética , Linfocitos T/citología , Linfocitos T/inmunología , Regulación hacia ArribaRESUMEN
PD-1 plays an important role in T cell exhaustion during HIV infection. PD-1 has two ligands: PD-L1, expressed on hematopoietic and nonhematopoietic cells, and PD-L2, limited to DCs and macrophages. Little is known about PD-L1 expression and regulation in human macrophages. Previous reports have found few immediate effects of macrophage exposure to HIV, suggesting that macrophages lack PRRs for this virus. Using quantitative confocal microscopy and a multiplexed cytokine bead array, we measured induction of PD-L1, PD-L2, and innate response cytokines in human MDMs in response to chemically inactivated HIV virions. Consistent with previous reports, no cytokines were induced by HIV virion exposure. Whereas PD-L1 and PD-L2 had low baseline expression, TLR ligands (LPS and CL097) up-regulated PD-L1 but not PD-L2. Unlike what we found for cytokine expression, PD-L1 and PD-L2 were up-regulated in response to exposure with inactivated HIV virions or with replication-competent HIV. Expression of PD-L1 was differentially modulated by IL-10, which induced up-regulation of PD-L1 but not of PD-L2, and IL-10 blockade enhanced only PD-L2 expression. We discuss implications for innate recognition of HIV by macrophages and potential, different roles for PD-L1 and PD-L2 in immunity and pathogenesis.
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Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/inmunología , Interleucina-10/farmacología , Macrófagos/metabolismo , Antígeno B7-H1 , Citocinas/metabolismo , Citometría de Flujo , Humanos , Lipopolisacáridos/farmacología , Activación de Linfocitos , Proteína 2 Ligando de Muerte Celular Programada 1 , Linfocitos T/efectos de los fármacos , Virión , Replicación ViralRESUMEN
Persistent levels of IL-10 play a central role in progressive immune dysfunction associated with chronic viral infections such as HIV, but the underlying mechanisms are poorly understood. Because IL-10 affects the phenotypic and functional properties of DCs, which are responsible for initiating adaptive immune responses, we investigated whether IL-10 induces changes in DC phenotype and function in the context of HIV infection. Here, we show that IL-10 treatment of immature and mature human DCs in culture induced contrasting phenotypic changes in these populations: immature DCs exhibited aberrant resistance to NK cell-mediated elimination, whereas mature DCs exhibited increased susceptibility to NKG2D-dependent NK elimination. Treatment of immature and mature DCs with HIV resulted in potent IL-10 secretion and the same phenotypic and functional changes observed in the IL-10-treated cells. Consistent with these in vitro data, LNs isolated from individuals infected with HIV exhibited aberrant accumulation of a partially "immature" DC population. Together, these data suggest that the progressive immune dysfunction observed in chronic viral infections might be caused in part by IL-10-induced reversal of DC susceptibility to NK cell-mediated elimination, resulting in the accumulation of poorly immunogenic DCs in LNs, the sites of adaptive immune response induction.
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Células Dendríticas/citología , Células Dendríticas/virología , Infecciones por VIH/inmunología , Interleucina-10/inmunología , Células Asesinas Naturales/inmunología , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , VIH/inmunología , Infecciones por VIH/virología , Humanos , Células Asesinas Naturales/virologíaRESUMEN
BACKGROUND: The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission. METHODOLOGY: The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited. FINDINGS: Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naïve T cells. Responses included several CD4(+) and CD8(+) T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4(+) T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4(+) T cell depletion which renders the newly activated HIV-specific CD4(+) T cells prime targets for elimination. CONCLUSION: Our studies highlight the ability of DCs to efficiently prime naïve T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo.
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Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/virología , Infecciones por VIH/sangre , Infecciones por VIH/virología , VIH-1/metabolismo , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/metabolismo , Antígenos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Comunicación Celular , Células Dendríticas/citología , Disulfuros/metabolismo , Epítopos , Humanos , Selectina L/biosíntesis , Antígenos Comunes de Leucocito/biosíntesis , Leucocitos Mononucleares/virología , Activación de Linfocitos , FenotipoRESUMEN
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the brain caused by JC virus (JCV) for which there is no cure. PML patients who have JCV-specific CD8(+) cytotoxic T lymphocytes (CTL) in their blood have a better clinical outcome. We compared JCV-specific CTL responses in vitro elicited either by JCV peptide-loaded dendritic cells (DC) or by direct peptide stimulation of lymphocytes from 20 HLA-A0201(+) healthy controls, HIV(+) and PML patients. JCV peptide-loaded DC elicited a stronger CTL expansion in 13/15 responders. DC can induce a potent JCV-specific CTL response in vitro, and may constitute a promising approach for PML immunotherapy.
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Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Virus JC/inmunología , Leucoencefalopatía Multifocal Progresiva/inmunología , Adulto , Anciano , Proliferación Celular , Infecciones por VIH/complicaciones , Humanos , Técnicas In Vitro , Persona de Mediana EdadRESUMEN
BACKGROUND: The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1) are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. METHODOLOGY AND PRINCIPAL FINDINGS: To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per microg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. CONCLUSIONS: We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA.
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Codón , Productos del Gen gag/genética , VIH-1/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Linfocitos T/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Células JurkatRESUMEN
Increased PD-L1 expression has been reported in HIV-1-infected individuals, but the mechanisms leading to PD-L1 upregulation remain to be elucidated. Here we demonstrate that HIV-1-derived Toll-like receptor (TLR)7/8 ligands can induce MyD88-dependent upregulation of PD-L1 on plasmacytoid dendritic cells, myeloidic dendritic cells and monocytes. These data suggest a mechanism through which HIV-1-derived TLR ligands might contribute to the functional impairment of virus-specific PD-1-positive T cells by inducing the upregulation of PD-L1 on antigen-presenting cells.