RESUMO
Chronological aging correlates with epigenetic modifications at specific loci, calibrated to species lifespan. Such 'epigenetic clocks' appear conserved among mammals, but whether they are cell autonomous and restricted by maximal organismal lifespan remains unknown. We used a multilifetime murine model of repeat vaccination and memory T cell transplantation to test whether epigenetic aging tracks with cellular replication and if such clocks continue 'counting' beyond species lifespan. Here we found that memory T cell epigenetic clocks tick independently of host age and continue through four lifetimes. Instead of recording chronological time, T cells recorded proliferative experience through modification of cell cycle regulatory genes. Applying this epigenetic profile across a range of human T cell contexts, we found that naive T cells appeared 'young' regardless of organism age, while in pediatric patients, T cell acute lymphoblastic leukemia appeared to have epigenetically aged for up to 200 years. Thus, T cell epigenetic clocks measure replicative history and can continue to accumulate well-beyond organismal lifespan.
Assuntos
Senescência Celular , Epigênese Genética , Animais , Humanos , Camundongos , Senescência Celular/genética , Senescência Celular/imunologia , Envelhecimento/imunologia , Envelhecimento/genética , Linfócitos T/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Células T de Memória/imunologia , Células T de Memória/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Senescência de Células TRESUMO
PURPOSE OF REVIEW: Successful sustained remission of HIV infection has been achieved after CCR5Δ32/Δ32 allogeneic hematopoietic stem cell transplantation for treatment of leukemia in a small cohort of people living with HIV (PLWH). This breakthrough demonstrated that the goal of curing HIV was achievable. However, the high morbidity and mortality associated with bone marrow transplantation limits the routine application of this approach and provides a strong rationale for pursuing alternative strategies for sustained long-term antiretroviral therapy (ART)-free HIV remission. Notably, long-term immune-mediated control of HIV replication observed in elite controllers and posttreatment controllers suggests that potent HIV-specific immune responses could provide sustained ART-free remission in PLWH. The capacity of chimeric antigen receptor (CAR)-T cells engineered to target malignant cells to induce remission and cure in cancer patients made this an attractive approach to provide PLWH with a potent HIV-specific immune response. Here, we review the recent advances in the design and application of anti-HIV CAR-T-cell therapy to provide a functional HIV cure. RECENT FINDINGS: HIV reservoirs are established days after infection and persist through clonal expansion of infected cells. The continuous interaction between latently infected cells and the immune system shapes the landscape of HIV latency and likely contributes to ART-free viral control in elite controllers. CAR-T cells can exhibit superior antiviral activity as compared with native HIV-specific T cells, particularly because they can be engineered to have multiple HIV specificities, resistance to HIV infection, dual costimulatory signaling, immune checkpoint inhibitors, stem cell derivation, CMV TCR coexpression, and tissue homing ligands. These modifications can significantly improve the capacities of anti-HIV CAR-T cells to prevent viral escape, resist HIV infection, and enhance cytotoxicity, persistence, and tissue penetration. Collectively, these novel modifications of anti-HIV CAR-T cell design have increased their capacity to control HIV infection. SUMMARY: Anti-HIV CAR-T cells can be engineered to provide potent and sustained in-vitro and in-vivo antiviral function. The combination of anti-HIV CAR-T cells with other immunotherapeutics may contribute to long-term HIV remission in PLWH.
Assuntos
Infecções por HIV , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Infecções por HIV/imunologia , Infecções por HIV/terapia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Imunoterapia Adotiva/métodos , HIV-1/imunologia , Linfócitos T/imunologiaRESUMO
The inability of people living with HIV (PLWH) to eradicate human immunodeficiency virus (HIV) infection is due in part to the inadequate HIV-specific cellular immune response. The antiviral function of cytotoxic CD8+ T cells, which are crucial for HIV control, is impaired during chronic viral infection because of viral escape mutations, immune exhaustion, HIV antigen downregulation, inflammation, and apoptosis. In addition, some HIV-infected cells either localize to tissue sanctuaries inaccessible to CD8+ T cells or are intrinsically resistant to CD8+ T cell killing. The novel design of synthetic chimeric antigen receptors (CARs) that enable T cells to target specific antigens has led to the development of potent and effective CAR-T cell therapies. While initial clinical trials using anti-HIV CAR-T cells performed over 20 years ago showed limited anti-HIV effects, the improved CAR-T cell design, which enabled its success in treating cancer, has reinstated CAR-T cell therapy as a strategy for HIV cure with notable progress being made in the recent decade.Effective CAR-T cell therapy against HIV infection requires the generation of anti-HIV CAR-T cells with potent in vivo activity against HIV-infected cells. Preclinical evaluation of anti-HIV efficacy of CAR-T cells and their safety is fundamental for supporting the initiation of subsequent clinical trials in PLWH. For these preclinical studies, we developed a novel humanized mouse model supporting in vivo HIV infection, the development of viremia, and the evaluation of novel HIV therapeutics. Preclinical assessment of anti-HIV CAR-T cells using this mouse model involves a multistep process including peripheral blood mononuclear cells (PBMCs) harvested from human donors, T cell purification, ex vivo T cell activation, transduction with lentiviral vectors encoding an anti-HIV CAR, CAR-T cell expansion and infusion in mice intrasplenically injected with autologous PBMCs followed by the determination of CAR-T cell capacity for HIV suppression. Each of the steps described in the following protocol were optimized in the lab to maximize the quantity and quality of the final anti-HIV CAR-T cell products.
Assuntos
Infecções por HIV , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Animais , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Camundongos , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T CD8-Positivos/imunologia , HIV-1/imunologia , Linfócitos T/imunologia , Transdução GenéticaRESUMO
To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8+ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8+ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8+ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8+ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8+ T cells, enabling optimization of individualized therapies, including HIV cure strategies.
Assuntos
Infecções por Citomegalovirus/metabolismo , Infecções por HIV/metabolismo , Imunoterapia/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/virologia , Animais , Células Apresentadoras de Antígenos/imunologia , Produtos Biológicos , Linfócitos T CD8-Positivos/citologia , Citomegalovirus , Células HEK293 , Antígeno HLA-A2/metabolismo , Humanos , Técnicas In Vitro , Células Jurkat , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Ligantes , Camundongos , Camundongos SCID , Peptídeos , Baço/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Adoptive immunotherapy using chimeric antigen receptor-modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1-based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4+ T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.
Assuntos
Antivirais/uso terapêutico , Infecções por HIV/imunologia , Infecções por HIV/terapia , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Células Cultivadas , Citocinas/biossíntese , Citotoxicidade Imunológica , Modelos Animais de Doenças , HIV-1/imunologia , Humanos , Lentivirus/metabolismo , Ativação Linfocitária/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Linfócitos T/imunologia , Células Th1/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
We previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.
Assuntos
Vacinas contra a AIDS/farmacocinética , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/farmacologia , Vacinas contra a AIDS/química , Animais , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Amplamente Neutralizantes , Antígenos CD4 , HIV-1/imunologia , Meia-Vida , Humanos , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/farmacologia , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/farmacologia , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Engenharia de Proteínas/métodos , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
UNLABELLED: Natural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the capacity of NK cells to suppress acute in vivo HIV-1 infection would be augmented by activating them via treatment with an interleukin-15 (IL-15) superagonist, IL-15 bound to soluble IL-15Rα, an approach that potentiates human NK cell-mediated killing of tumor cells. In vitro stimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of the cytotoxic effector molecules granzyme B and perforin; their degranulation upon exposure to K562 cells, as indicated by cell surface expression of CD107a; and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acute in vivo HIV-1 infection was investigated by using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMCs) which develop productive in vivo infection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until 3 days after intrasplenic HIV-1 inoculation. Removal of NK cells from human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression of in vivo HIV-1 infection. Thus, the in vivo activation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acute in vivo HIV-1 infection. These results indicate that in vivo activation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection. IMPORTANCE: Epidemiological studies have indicated that NK cells contribute to the control of HIV-1 infection, and in vitro studies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated that in vivo activation of NK cells by treatment with an IL-15 superagonist that potently stimulates the antitumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell depletion from PBMCs prior to their intrasplenic injection abrogated the suppression of in vivo HIV-1 infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus, in vivo immunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals, particularly in the earliest stages of infection.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Interleucina-15/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos SCIDRESUMO
Insulin-like growth factor 2 receptor (IGF2R), also known as cation-independent mannose 6-phosphate (M6P) receptor, is a transmembrane glycoprotein localized in the trans-Golgi region and is involved in targeting both M6P-bearing enzymes and IGF2 to the lysosomal compartment. During development, IGF2R plays a crucial role in removing excess growth factors from both tissue and blood. Due to the perinatal lethality of the global Igf2r knockout, the function of IGF2R in adults, particularly in the CNS, is not known. We made a novel observation that IGF2R is highly expressed in microglial nodules in human brains with HIV encephalitis. In vitro, microglial IGF2R expression was uniquely enhanced by IFNγ among the several cytokines and TLR ligands examined. Furthermore, in several in vitro models of HIV infection, including human and murine microglia, macrophages, and nonmacrophage cells, IGF2R is repeatedly shown to be a positive regulator of HIV infection. IGF2R RNAi also down-regulated the production of the IP-10 chemokine in HIV-infected human microglia. Injection of VSVg env HIV into mouse brain induced HIV p24 expression in neurons, the only cell type normally expressing IGF2R in the adult brain. Our results demonstrate a novel role for IGF2R as an inducible microglial protein involved in regulation of HIV and chemokine expression. Mice with the Csf1r- driven Igf2r knockout should be useful for the investigation of macrophage-specific IGF2R function.
Assuntos
Complexo AIDS Demência/fisiopatologia , HIV/fisiologia , Interferon gama/metabolismo , Microglia/metabolismo , Receptor IGF Tipo 2/metabolismo , Replicação Viral , Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Animais , Astrócitos/citologia , Astrócitos/virologia , Encéfalo/citologia , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , Células Cultivadas , HIV/genética , HIV/ultraestrutura , Infecções por HIV/patologia , Infecções por HIV/fisiopatologia , Humanos , Macrófagos/citologia , Macrófagos/virologia , Camundongos , Camundongos Knockout , Microglia/citologia , Microglia/virologia , Interferência de RNA , Receptor IGF Tipo 2/genética , Vírion/ultraestruturaRESUMO
Humanized Rag2(-/-)gamma(c)(-/-) mice (Hu-DKO mice) become populated with functional human T cells, B cells, and dendritic cells following transplantation with human hematopoietic stem cells (HSC) and represent an improved model for studying HIV infection in vivo. In the current study we demonstrated that intrasplenic inoculation of hu-DKO mice with HIV-1 initiated a higher level of HIV infection than intravenous or intraperitoneal inoculation, associated with a reciprocal decrease in peripheral CD4(+) T cells and increase in peripheral CD8(+) T cells. HIV infection by intrasplenic injection increased serum levels of human IgG and IgM including human IgM and IgG specific for HIV-1 gp120. There was a significant inverse correlation between the level of HIV-1 infection and the extent of CD4(+) T cell depletion. Highly active antiretroviral therapy (HAART) initiated 1 week after HIV-1 inoculation markedly suppressed HIV-1 infection and prevented CD4(+) T cell depletion. Taken together, these findings demonstrate that intrasplenic injection of hu-DKO mice with HIV is a more efficient route of HIV infection than intravenous or intraperitoneal injection and generates increased infection associated with an increased anti-HIV humoral response. This animal model can serve as a valuable in vivo model to study the efficacy of anti-HIV therapies.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade/métodos , Proteínas de Ligação a DNA/deficiência , Modelos Animais de Doenças , Infecções por HIV/tratamento farmacológico , Receptores de Interleucina-2/deficiência , Animais , Animais Recém-Nascidos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Anticorpos Anti-HIV/sangue , HIV-1/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Camundongos , Camundongos KnockoutRESUMO
Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/gamma(c)(null) mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/gamma(c)(null) mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1(JR-CSF), mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/gamma(c)(null) mice inoculated with equivalent high-titer HIV-1(JR-CSF). These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1/imunologia , Células-Tronco Hematopoéticas/imunologia , Imunoterapia/métodos , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Vetores Genéticos , Humanos , Lentivirus/genética , Infecções por Lentivirus , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Baço/virologia , Carga ViralRESUMO
There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR). Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.
Assuntos
Antígenos Virais/imunologia , Epitopos/imunologia , Engenharia Genética , Células-Tronco Hematopoéticas/imunologia , Linfócitos T/imunologia , Animais , Antivirais/metabolismo , Diferenciação Celular/imunologia , Clonagem Molecular , HIV/imunologia , Humanos , Camundongos , Camundongos SCID , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/isolamento & purificação , Especificidade da Espécie , Linfócitos T/virologiaRESUMO
To delineate the mechanistic basis for the epidemiological association between methamphetamine use and accelerated progression to AIDS, we evaluated the direct in vitro and in vivo effects of methamphetamine on HIV-1 replication. Methamphetamine administration significantly increased HIV-1 production by both HIV-infected monocytes and CD4 T lymphocytes in vitro. In addition, in vivo methamphetamine treatment increased HIV production and viremia in mice transgenic for a replication-competent HIV provirus and human cyclin T1. Methamphetamine activated transcription of the HIV long terminal repeat (LTR) regulatory region, was associated with nuclear translocation of NF-kappaB. Our results provide further insights into the mechanisms by which methamphetamine accelerates disease course in HIV-infected individuals.
Assuntos
Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , HIV-1 , Metanfetamina , Replicação Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Ciclina T/genética , Ciclina T/metabolismo , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , Repetição Terminal Longa de HIV/genética , Repetição Terminal Longa de HIV/fisiologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Metanfetamina/administração & dosagem , Metanfetamina/farmacologia , Camundongos , Camundongos Transgênicos , Viremia/tratamento farmacológico , Viremia/virologiaRESUMO
Inflammatory mediators and viral products produced by human immunodeficiency virus (HIV)-infected microglia and astrocytes perturb the function and viability of adjacent uninfected neuronal and glial cells and contribute to the pathogenesis of HIV-associated neurocognitive disorders (HAND). In vivo exposure to lipopolysaccharide (LPS) activates parenchymal microglia and astrocytes and induces cytokine and chemokine production in the brain. HIV-infected individuals display increased circulating LPS levels due to microbial translocation across a compromised mucosa barrier. We hypothesized that HIV-infected microglia and astrocytes display increased sensitivity to the proinflammatory effects of LPS, and this combines with the increased levels of systemic LPS in HIV-infected individuals to contribute to the development of HAND. To examine this possibility, we determined the in vivo responsiveness of HIV-infected microglia and astrocytes to LPS using our mouse model, JR-CSF/human cyclin T1 (JR-CSF/hu-cycT1) mice, which are transgenic for both an integrated full-length infectious HIV type 1 (HIV-1) provirus derived from the primary R5-tropic clinical isolate HIV-1(JR-CSF) regulated by the endogenous HIV-1 long terminal repeat and the hu-cycT1 gene under the control of a CD4 promoter. In the current report, we demonstrated that in vivo-administered LPS more potently activated JR-CSF/hu-cycT1 mouse microglia and astrocytes and induced a significantly higher degree of monocyte chemoattractant protein production by JR-CSF/hu-cycT1 astrocytes compared to that of the in vivo LPS response of control littermate mouse microglia and astrocytes. These results indicate that HIV infection increases the sensitivity of microglia and astrocytes to inflammatory stimulation and support the use of these mice as a model to investigate various aspects of the in vivo mechanism of HIV-induced neuronal dysfunction.
Assuntos
Astrócitos/imunologia , Encéfalo/imunologia , Antígenos CD4/imunologia , Ciclinas/metabolismo , HIV-1/imunologia , Lipopolissacarídeos/farmacologia , Microglia/imunologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Antígenos CD4/metabolismo , Quimiocina CCL2/biossíntese , Ciclina T , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Provírus/imunologiaRESUMO
Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal-lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/toxicidade , Células Dendríticas/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Metanfetamina/toxicidade , Fagocitose/efeitos dos fármacos , Animais , Apresentação de Antígeno/imunologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Candida albicans/imunologia , Fracionamento Celular , Células Cultivadas , Cloroquina/farmacologia , Cryptococcus neoformans/imunologia , Células Dendríticas/microbiologia , Células Dendríticas/fisiologia , Feminino , Terapia de Imunossupressão , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/imunologiaRESUMO
The human immunodeficiency virus type 1 (HIV-1)-specific CD8 cytotoxic T-lymphocyte (CTL) response plays a critical role in controlling HIV-1 replication. Augmenting this response should enhance control of HIV-1 replication and stabilize or improve the clinical course of the disease. Although cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection in immunocompromised patients can be treated by adoptive transfer of ex vivo-expanded CMV- or EBV-specific CTLs, adoptive transfer of ex vivo-expanded, autologous HIV-1-specific CTLs had minimal effects on HIV-1 replication, likely a consequence of the inherently compromised qualitative function of HIV-1-specific CTLs derived from HIV-1-infected individuals. We hypothesized that this limitation could be circumvented by using as an alternative source of HIV-1-specific CTLs, autologous peripheral CD8(+) T lymphocytes whose antigen specificity is redirected by transduction with lentiviral vectors encoding HIV-1-specific T-cell receptor (TCR) alpha and beta chains, an approach used successfully in cancer therapy. To efficiently convert peripheral CD8 lymphocytes into HIV-1-specific CTLs that potently suppress in vivo HIV-1 replication, we constructed lentiviral vectors encoding the HIV-1-specific TCR alpha and TCR beta chains cloned from a CTL clone specific for an HIV Gag epitope, SL9, as a single transcript linked with a self-cleaving peptide. We demonstrated that transduction with this lentiviral vector efficiently converted primary human CD8 lymphocytes into HIV-1-specific CTLs with potent in vitro and in vivo HIV-1-specific activity. Using lentiviral vectors encoding an HIV-1-specific TCR to transform peripheral CD8 lymphocytes into HIV-1-specific CTLs with defined specificities represents a new immunotherapeutic approach to augment the HIV-1-specific immunity of infected patients.
Assuntos
Vetores Genéticos , HIV-1/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Engenharia Genética , HumanosRESUMO
HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effective in the control of viral replication in vivo. In this study, we studied human CD8+ T cell responses to the subdominant HLA-A2-restricted epitope TV9 (Gag p24(19-27), TLNAWVKVV) to explore the possibility of increasing its immune recognition. We confirmed in a cohort of 313 patients, infected by clade B or clade C viruses, that TV9 is rarely recognized. Of interest, the functional sensitivity of the TV9 response can be relatively high. The potential T cell repertoires for TV9 and the characteristics of constituent clonotypes were assessed by ex vivo priming of circulating CD8+ T cells from healthy seronegative donors. TV9-specific CTLs capable of suppressing viral replication in vitro were readily generated, suggesting that the cognate T cell repertoire is not limiting. However, these cultures contained multiple discrete populations with a range of binding avidities for the TV9 tetramer and correspondingly distinct functional dependencies on the CD8 coreceptor. The lack of dominant clonotypes was not affected by the stage of maturation of the priming dendritic cells. Cultures primed by dendritic cells transduced to present endogenous TV9 were also incapable of clonal maturation. Thus, a diffuse TCR repertoire appeared to be an intrinsic characteristic of TV9-specific responses. These data indicate that subdominance is not a function of poor immunogenicity, cognate TCR repertoire availability, or the potential avidity properties thereof, but rather suggest that useful responses to this epitope are suppressed by competing CD8+ T cell populations during HIV-1 infection.
Assuntos
Produtos do Gene gag/imunologia , HIV-1/imunologia , Antígeno HLA-A2/imunologia , Epitopos Imunodominantes/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Reações Cruzadas , Células Dendríticas/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Existing antiviral therapies produce a therapeutic effect by suppressing viral replication and reducing viral burden and the associated inflammatory reaction. However, infection with many viruses results in chronic infections that cannot be eradicated by the immune response or available antiviral drugs. As viruses are obligate intracellular pathogens, it should be possible to eradicate chronic infections by targeting and eliminating the infected host cells. Radioisotope-conjugated antibodies that specifically bind viral antigens can deliver cytotoxic radiation to virally infected cells. This approach was recently shown to target and eliminate HIV-1-infected cells in vitro and in mouse models, and provides a new approach for eliminating virally infected cells.
Assuntos
Anticorpos Antivirais/uso terapêutico , Antígenos Virais/imunologia , Radioimunoterapia/métodos , Viroses/radioterapia , Animais , Humanos , Radioisótopos/uso terapêutico , Viroses/virologiaRESUMO
BACKGROUND: The HIV epidemic is a major threat to health in the developing and western worlds. A modality that targets and kills HIV-1-infected cells could have a major impact on the treatment of acute exposure and the elimination of persistent reservoirs of infected cells. The aim of this proof-of-principle study was to demonstrate the efficacy of a therapeutic strategy of targeting and eliminating HIV-1-infected cells with radiolabeled antibodies specific to viral proteins in vitro and in vivo. METHODS AND FINDINGS: Antibodies to HIV-1 envelope glycoproteins gp120 and gp41 labeled with radioisotopes bismuth 213 ((213)Bi) and rhenium 188 ((188)Re) selectively killed chronically HIV-1-infected human T cells and acutely HIV-1-infected human peripheral blood mononuclear cells (hPBMCs) in vitro. Treatment of severe combined immunodeficiency (SCID) mice harboring HIV-1-infected hPBMCs in their spleens with a (213)Bi- or (188)Re-labeled monoclonal antibody (mAb) to gp41 resulted in a 57% injected dose per gram uptake of radiolabeled mAb in the infected spleens and in a greater than 99% elimination of HIV-1-infected cells in a dose-dependent manner. The number of HIV-1-infected thymocytes decreased 2.5-fold in the human thymic implant grafts of SCID mice treated with the (188)Re-labeled antibody to gp41 compared with those treated with the (188)Re-control mAb. The treatment did not cause acute hematologic toxicity in the treated mice. CONCLUSIONS: The current study demonstrates the effectiveness of HIV-targeted radioimmunotherapy and may provide a novel treatment option in combination with highly active antiretroviral therapy for the eradication of HIV.
Assuntos
Anticorpos Antivirais/farmacologia , Infecções por HIV/fisiopatologia , HIV-1 , Imunoterapia , Monócitos/virologia , Linfócitos T/virologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/administração & dosagem , Bismuto , Morte Celular , Células Cultivadas , Citotoxinas/administração & dosagem , Citotoxinas/farmacologia , Relação Dose-Resposta a Droga , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Humanos , Camundongos , Camundongos SCID , Radioisótopos , Rênio , Baço/virologia , Timo/transplante , Timo/virologia , Transplante HeterólogoRESUMO
Human immunodeficiency virus type 1 (HIV-1)-encoded Tat provides transcriptional activation critical for efficient HIV-1 replication by interacting with cyclin T1 and recruiting P-TEFb to efficiently elongate the nascent HIV transcript. Tat-mediated transcriptional activation in mice is precluded by species-specific structural differences that prevent Tat interaction with mouse cyclin T1 and severely compromise HIV-1 replication in mouse cells. We investigated whether transgenic mice expressing human cyclin T1 under the control of a murine CD4 promoter/enhancer cassette that directs gene expression to CD4(+) T lymphocytes and monocytes/macrophages (hu-cycT1 mice) would display Tat responsiveness in their CD4-expressing mouse cells and selectively increase HIV-1 production in this cellular population, which is infected primarily in HIV-1-positive individuals. To this end, we crossed hu-cycT1 mice with JR-CSF transgenic mice carrying the full-length HIV-1(JR-CSF) provirus under the control of the endogenous HIV-1 long terminal repeat and demonstrated that human cyclin T1 expression is sufficient to support Tat-mediated transactivation in primary mouse CD4 T lymphocytes and monocytes/macrophages and increases in vitro and in vivo HIV-1 production by these stimulated cells. Increased HIV-1 production by CD4(+) T lymphocytes was paralleled with their specific depletion in the peripheral blood of the JR-CSF/hu-cycT1 mice, which increased over time. In addition, increased HIV-1 transgene expression due to human cyclin T1 expression was associated with increased lipopolysaccharide-stimulated monocyte chemoattractant protein 1 production by JR-CSF mouse monocytes/macrophages in vitro. Therefore, the JR-CSF/hu-cycT1 mice should provide an improved mouse system for investigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacies of therapeutic interventions.
Assuntos
Linfócitos T CD4-Positivos/virologia , Ciclinas/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Células Mieloides/virologia , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/metabolismo , Ciclina T , Ciclinas/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Proteína do Núcleo p24 do HIV/análise , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Imuno-Histoquímica , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Células Mieloides/metabolismo , Provírus/genética , Baço/virologiaRESUMO
HIV-1-infected monocyte/macrophages located in lymph nodes and tissues are highly productive sources of HIV-1 and may function as a persistent reservoir contributing to the rebound viremia observed after highly active antiretroviral therapy is stopped. Mechanisms activating latently infected, primary monocyte/macrophages to produce HIV-1 were investigated using monocytes isolated from a transgenic mouse line carrying a full-length proviral clone of a monocyte-tropic HIV-1 isolate, HIV-1(JR-CSF), regulated by the endogenous long terminal repeat (LTR) (JR-CSF mice). Granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with lipopolysaccharide (LPS) induced infectious HIV-1 production by JR-CSF mouse monocytes over 10-fold and 100-fold higher than that stimulated by GM-CSF or LPS alone, respectively. We examined mechanisms of GM-CSF synergy with LPS and demonstrated that GM-CSF up-regulated the LPS receptor, TLR-4, and also synergized with LPS to activate mitogen-activated protein (MAP) kinase/ERK kinase and the Sp1 transcription factor. Inhibitors of either MAP kinase/ERK kinase or p38 kinase but not PI 3-kinase potently suppressed GM-CSF and LPS-induced HIV-1 production by JR-CSF mouse monocytes. Because Sp1 is activated by both the MAP kinase/ERK kinase and p38 kinase pathways, we postulate that synergistic activation of these pathways by GM-CSF and LPS induced sufficient levels of Sp1 to activate the HIV-1 LTR in a Tat-independent manner and induced HIV-1 production by JR-CSF mouse monocytes. Thus, our study delineated the pathway of HIV-1 LTR activation by GM-CSF and LPS and indicated that JR-CSF transgenic mice may provide a new in vitro and in vivo system for investigating the mechanism by which inflammatory and infectious stimuli activate HIV-1 production from latently infected monocytes.