RESUMEN
Background: Latency reversing agents (LRAs) such as protein kinase C (PKC) modulators can reduce rebound-competent HIV reservoirs in small animal models. Furthermore, administration of natural killer (NK) cells following LRA treatment improves this reservoir reduction. It is currently unknown why the combination of a PKC modulator and NK cells is so potent and whether exposure to PKC modulators may augment NK cell function in some way. Methods: Primary human NK cells were treated with PKC modulators (bryostatin-1, prostratin, or the designed, synthetic bryostatin-1 analog SUW133), and evaluated by examining expression of activation markers by flow cytometry, analyzing transcriptomic profiles by RNA sequencing, measuring cytotoxicity by co-culturing with K562 cells, assessing cytokine production by Luminex assay, and examining the ability of cytokines and secreted factors to independently reverse HIV latency by co-culturing with Jurkat-Latency (J-Lat) cells. Results: PKC modulators increased expression of proteins involved in NK cell activation. Transcriptomic profiles from PKC-treated NK cells displayed signatures of cellular activation and enrichment of genes associated with the NFκB pathway. NK cell cytotoxicity was unaffected by prostratin but significantly decreased by bryostatin-1 and SUW133. Cytokines from PKC-stimulated NK cells did not induce latency reversal in J-Lat cell lines. Conclusions: Although PKC modulators have some significant effects on NK cells, their contribution in "kick and kill" strategies is likely due to upregulating HIV expression in CD4+ T cells, not directly enhancing the effector functions of NK cells. This suggests that PKC modulators are primarily augmenting the "kick" rather than the "kill" arm of this HIV cure approach.
RESUMEN
Human immunodeficiency virus (HIV) has infected millions of people worldwide and continues to be a major global health problem. Scientists required a small animal model to study HIV pathogenesis and immune responses. To this end, humanized mice were created by transplanting human cells and/or tissues into immunodeficient mice to reconstitute a human immune system. Thus, humanized mice have become a critical animal model for HIV researchers, but with some limitations. Current conventional humanized mice are prone to death by graft versus host disease induced by the mouse signal regulatory protein α and CD47 signaling pathway. In addition, commonly used humanized mice generate low levels of human cytokines required for robust myeloid and natural killer cell development and function. Here, we describe recent advances in humanization procedures and transgenic and knock-in immunodeficient mice to address these limitations.
RESUMEN
HIV can establish a long-lived latent infection in cells harboring integrated non-expressing proviruses. Latency reversing agents (LRAs), including protein kinase C (PKC) modulators, can induce expression of latent HIV, thereby reducing the latent reservoir in animal models. However, PKC modulators such as bryostatin-1 also cause cytokine upregulation in peripheral blood mononuclear cells (PBMCs), including cytokines that might independently reverse HIV latency. To determine whether cytokines induced by PKC modulators contribute to latency reversal, primary human PBMCs were treated with bryostatin-1 or the bryostatin analog SUW133, a superior LRA, and supernatant was collected. As anticipated, LRA-treated cell supernatant contained increased levels of cytokines compared to untreated cell supernatant. However, exposure of latently-infected cells with this supernatant did not result in latency reactivation. These results indicate that PKC modulators do not have significant indirect effects on HIV latency reversal in vitro and thus are targeted in their latency reversing ability.
Asunto(s)
Infecciones por VIH , VIH-1 , Animales , Humanos , Latencia del Virus , Brioestatinas/farmacología , Leucocitos Mononucleares , Linfocitos T CD4-Positivos , VIH-1/fisiología , Citocinas/metabolismo , Activación ViralRESUMEN
NK cells are an important subset of innate immune effectors with antiviral activity. However, NK cell development and immune responses in different tissues during acute and chronic HIV infection in vivo have been difficult to study due to the impaired development and function of NK cells in conventional humanized mouse models. In this issue of the JCI, Sangur et al. report on a transgenic MISTRG-6-15 mouse model with human IL-6 and IL-15 knocked into the previously constructed MISTRG mice. The predecessor model was deficient in Rag2 and γ chain (γc) with knock-in expression of human M-CSF, IL-3, GM-CSF, and TPO, and transgenic expression of human SIRPα. The researchers studied tissue-specific NK cell immune responses during HIV infection and clearly show that the endogenous human NK cells in the humanized mouse model suppressed HIV-1 replication in vivo. These findings provide insight into harnessing the innate immune response for clinical antiviral therapies.
Asunto(s)
Infecciones por VIH , VIH-1 , Ratones , Humanos , Animales , Infecciones por VIH/genética , Células Asesinas Naturales , Modelos Animales de Enfermedad , Ratones Transgénicos , AntiviralesRESUMEN
HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir.
Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/terapia , VIH-1/efectos de los fármacos , Células Asesinas Naturales/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Viremia/terapia , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Médula Ósea/virología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Técnicas de Cocultivo , Femenino , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Células Asesinas Naturales/trasplante , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa C/genética , Proteína Quinasa C/inmunología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/virología , Carga Viral/efectos de los fármacos , Viremia/genética , Viremia/inmunología , Viremia/virología , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009404.].
RESUMEN
Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study.
Asunto(s)
Infecciones por VIH/virología , Células Madre Hematopoyéticas/citología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Animales , Infecciones por VIH/inmunología , VIH-1/inmunología , Células Madre Hematopoyéticas/inmunología , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/uso terapéuticoRESUMEN
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.
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Infecciones por VIH/inmunología , Infecciones por VIH/terapia , Trasplante de Células Madre Hematopoyéticas , Inmunoterapia Adoptiva , Animales , Linaje de la Célula/inmunología , Modelos Animales de Enfermedad , Reservorios de Enfermedades/virología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/virología , Centro Germinal/inmunología , Centro Germinal/patología , Centro Germinal/virología , Infecciones por VIH/virología , VIH-1 , Humanos , Inmunohistoquímica , Macaca nemestrina , Masculino , Receptores Quiméricos de Antígenos/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Trasplante HomólogoRESUMEN
HIV latency prevents cure of infection with antiretroviral therapy (ART) alone. One strategy for eliminating latently infected cells involves the induction of viral protein expression via latency-reversing agents (LRAs), allowing killing of host cells by viral cytopathic effects or immune effector mechanisms. Here, we combine a barcoded HIV approach and a humanized mouse model to study the effects of a designed, synthetic protein kinase C modulating LRA on HIV rebound. We show that administration of this compound during ART results in a delay in rebound once ART is stopped. Furthermore, the rebounding virus appears composed of a smaller number of unique barcoded viruses than occurs in control-treated animals, suggesting that some reservoir cells that would have contributed virus to the rebound process are eliminated by LRA administration. These data support the use of barcoded virus to study rebound and suggest that LRAs may be useful in HIV cure efforts.
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Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Humanos , Ratones , Proteína Quinasa C/farmacología , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
"Shock and kill" strategies focus on purging the latent HIV-1 reservoir by treating infected individuals with therapeutics that activate the latent virus and subsequently eliminating infected cells. We have previously reported that induction of non-canonical nuclear factor κB (NF-κB) signaling through a class of small-molecule antagonists known as Smac mimetics can reverse HIV-1 latency. Here, we describe the development of Ciapavir (SBI-0953294), a molecule specifically optimized for HIV-1 latency reversal that was found to be more efficacious as a latency-reversing agent than other Smac mimetics under clinical development for cancer. Critically, this molecule induced activation of HIV-1 reservoirs in vivo in a bone marrow, liver, thymus (BLT) humanized mouse model without mediating systemic T cell activation. This study provides proof of concept for the in vivo efficacy and safety of Ciapavir and indicates that Smac mimetics can constitute a critical component of a safe and efficacious treatment strategy to eliminate the latent HIV-1 reservoir.
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Antirretrovirales/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Animales , Médula Ósea/efectos de los fármacos , Células Cultivadas , Infecciones por VIH/metabolismo , Seropositividad para VIH/tratamiento farmacológico , Humanos , Hígado/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T/efectos de los fármacos , Timo/efectos de los fármacos , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
AIDS is a pandemic disease caused by HIV that affects 37 million people worldwide. Current antiretroviral therapy slows disease progression but does not eliminate latently infected cells, which resupply active virus, thus necessitating lifelong treatment with associated compliance, cost, and chemoexposure issues. Latency-reversing agents (LRAs) activate these cells, allowing for their potential clearance, thus presenting a strategy to eradicate the infection. Protein kinase C (PKC) modulators-including prostratin, ingenol esters, bryostatin, and their analogs-are potent LRAs in various stages of development for several clinical indications. While LRAs are promising, a major challenge associated with their clinical use is sustaining therapeutically meaningful levels of the active agent while minimizing side effects. Here we describe a strategy to address this problem based on LRA prodrugs, designed for controllable release of the active LRA after a single injection. As intended, these prodrugs exhibit comparable or superior in vitro activity relative to the parent compounds. Selected compounds induced higher in vivo expression of CD69, an activation biomarker, and, by releasing free agent over time, significantly improved tolerability when compared to the parent LRAs. More generally, selected prodrugs of PKC modulators avoid the bolus toxicities of the parent drug and exhibit greater efficacy and expanded tolerability, thereby addressing a longstanding objective for many clinical applications.
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Fármacos Anti-VIH/farmacología , Brioestatinas/farmacología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Profármacos/farmacología , Proteína Quinasa C/metabolismo , Latencia del Virus/efectos de los fármacos , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/uso terapéutico , Brioestatinas/síntesis química , Brioestatinas/uso terapéutico , Línea Celular Tumoral , Células Cultivadas , Diterpenos/química , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ésteres del Forbol/química , Profármacos/síntesis química , Profármacos/uso terapéutico , Proteína Quinasa C/efectos de los fármacosRESUMEN
Bryostatin 1 is a marine natural product under investigation for HIV/AIDS eradication, the treatment of neurological disorders, and enhanced CAR T/NK cell immunotherapy. Despite its promising activity, bryostatin 1 is neither evolved nor optimized for the treatment of human disease. Here we report the design, synthesis, and biological evaluation of several close-in analogs of bryostatin 1. Using a function-oriented synthesis approach, we synthesize a series of bryostatin analogs designed to maintain affinity for bryostatin's target protein kinase C (PKC) while enabling exploration of their divergent biological functions. Our late-stage diversification strategy provides efficient access to a library of bryostatin analogs, which per our design retain affinity for PKC but exhibit variable PKC translocation kinetics. We further demonstrate that select analogs potently increase cell surface expression of CD22, a promising CAR T cell target for the treatment of leukemias, highlighting the clinical potential of bryostatin analogs for enhancing targeted immunotherapies.
Asunto(s)
Brioestatinas/biosíntesis , Brioestatinas/farmacología , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Proteína Quinasa C/metabolismo , Brioestatinas/química , Línea Celular Tumoral , Humanos , Leucemia/tratamiento farmacológico , Modelos Moleculares , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Linfocitos TRESUMEN
Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development.
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Células Madre Hematopoyéticas/fisiología , Inmunoterapia Adoptiva/métodos , Células T Asesinas Naturales/fisiología , Neoplasias/terapia , Animales , Células Cultivadas , Ingeniería Genética , Humanos , Ratones , Ratones SCID , Células T Asesinas Naturales/trasplante , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human T cell leukemia virus type 1 (HTLV-1) is the ethological agent of adult T cell leukemia/lymphoma (ATLL) and a number of lymphocyte-mediated inflammatory conditions, including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 orf-I encodes two proteins, p8 and p12, whose functions in humans are to counteract innate and adaptive responses and to support viral transmission. However, the in vivo requirements for orf-I expression vary in different animal models. In macaques, the ablation of orf-I expression by mutation of its ATG initiation codon abolishes the infectivity of the molecular clone HTLV-1p12KO In rabbits, HTLV-1p12KO is infective and persists efficiently. We used humanized mouse models to assess the infectivity of both wild-type HTLV-1 (HTLV-1WT) and HTLV-1p12KO We found that NOD/SCID/γC-/- c-kit+ mice engrafted with human tissues 1 day after birth (designated NSG-1d mice) were highly susceptible to infection by HTLV-1WT, with a syndrome characterized by the rapid polyclonal proliferation and infiltration of CD4+ CD25+ T cells into vital organs, weight loss, and death. HTLV-1 clonality studies revealed the presence of multiple clones of low abundance, confirming the polyclonal expansion of HTLV-1-infected cells in vivo HTLV-1p12KO infection in a bone marrow-liver-thymus (BLT) mouse model prone to graft-versus-host disease occurred only following reversion of the orf-I initiation codon mutation within weeks after exposure and was associated with high levels of HTLV-1 DNA in blood and the expansion of CD4+ CD25+ T cells. Thus, the incomplete reconstitution of the human immune system in BLT mice may provide a window of opportunity for HTLV-1 replication and the selection of viral variants with greater fitness.IMPORTANCE Humanized mice constitute a useful model for studying the HTLV-1-associated polyclonal proliferation of CD4+ T cells and viral integration sites in the human genome. The rapid death of infected animals, however, appears to preclude the clonal selection typically observed in human ATLL, which normally develops in 2 to 5% of individuals infected with HTLV-1. Nevertheless, the expansion of multiple clones of low abundance in these humanized mice mirrors the early phase of HTLV-1 infection in humans, providing a useful model to investigate approaches to inhibit virus-induced CD4+ T cell proliferation.
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Linfocitos T CD4-Positivos/virología , Proliferación Celular , Infecciones por HTLV-I/patología , Infecciones por HTLV-I/virología , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Modelos Animales de Enfermedad , Transmisión de Enfermedad Infecciosa , Ratones , Ratones Noqueados , Ratones SCID , Proteínas Reguladoras y Accesorias Virales/deficienciaRESUMEN
Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.
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VIH-1/inmunología , Latencia del Virus/inmunología , Latencia del Virus/fisiología , Animales , Antirretrovirales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Modelos Animales de Enfermedad , Infecciones por VIH/virología , Seropositividad para VIH/tratamiento farmacológico , VIH-1/patogenicidad , Humanos , Ratones , Receptor de Muerte Celular Programada 1/inmunología , Receptores Inmunológicos/inmunología , Nucleasas de los Efectores Tipo Activadores de la Transcripción/inmunología , Activación Viral , Replicación ViralRESUMEN
The original article [1] contains an error in the legend of Fig 5 whereby the descriptions for panels 5d and 5e are incorrect; as such, the corrected legend can be viewed below with its respective figure images.
RESUMEN
HIV latency in resting CD4+ T cell represents a key barrier preventing cure of the infection with antiretroviral drugs alone. Latency reversing agents (LRAs) can activate HIV expression in latently infected cells, potentially leading to their elimination through virus-mediated cytopathic effects, host immune responses, and/or therapeutic strategies targeting cells actively expressing virus. We have recently described several structurally simplified analogs of the PKC modulator LRA bryostatin (termed bryologs) designed to improve synthetic accessibility, tolerability in vivo, and efficacy in inducing HIV latency reversal. Here we report the comparative performance of lead bryologs, including their effects in reducing cell surface expression of HIV entry receptors, inducing proinflammatory cytokines, inhibiting short-term HIV replication, and synergizing with histone deacetylase inhibitors to reverse HIV latency. These data provide unique insights into structure-function relationships between A- and B-ring bryolog modifications and activities in primary cells, and suggest that bryologs represent promising leads for preclinical advancement.
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Brioestatinas/química , Brioestatinas/farmacología , Diseño de Fármacos , VIH-1/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Inhibidores de Histona Desacetilasas , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006753.].
RESUMEN
Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/terapia , Células Madre Hematopoyéticas/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Animales , Linfocitos T CD4-Positivos/trasplante , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Modelos Animales de Enfermedad , Terapia Genética/métodos , Infecciones por VIH/virología , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunoterapia/métodos , Macaca nemestrina , Masculino , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virologíaRESUMEN
BACKGROUND: Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. METHODS: We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. RESULTS: Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. CONCLUSIONS: This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.