RESUMO
The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.
Assuntos
Autorrenovação Celular , Células-Tronco Hematopoéticas , Proteínas Nucleares , Animais , Feminino , Humanos , Masculino , Camundongos , Células Cultivadas , Endocitose , Endossomos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Sangue Fetal/citologia , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/embriologia , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Adoptive cell therapy (ACT) using T cells expressing chimeric antigen receptors (CARs) is an area of intense investigation in the treatment of malignancies and chronic viral infections. One of the limitations of ACT-based CAR therapy is the lack of in vivo persistence and maintenance of optimal cell function. Therefore, alternative strategies that increase the function and maintenance of CAR-expressing T cells are needed. In our studies using the humanized bone marrow/liver/thymus (BLT) mouse model and nonhuman primate (NHP) model of HIV infection, we evaluated two CAR-based gene therapy approaches. In the ACT approach, we used cytokine enhancement and preconditioning to generate greater persistence of anti-HIV CAR+ T cells. We observed limited persistence and expansion of anti-HIV CAR T cells, which led to minimal control of the virus. In our stem cell-based approach, we modified hematopoietic stem/progenitor cells (HSPCs) with anti-HIV CAR to generate anti-HIV CAR T cells in vivo. We observed CAR-expressing T cell expansion, which led to better plasma viral load suppression. HSPC-derived CAR cells in infected NHPs showed superior trafficking and persistence in multiple tissues. Our results suggest that a stem cell-based CAR T cell approach may be superior in generating long-term persistence and functional antiviral responses against HIV infection.
Assuntos
Infecções por HIV , HIV-1 , Receptores de Antígenos Quiméricos , Camundongos , Animais , Linfócitos T , Receptores de Antígenos Quiméricos/genética , Células-Tronco Hematopoéticas , Imunoterapia AdotivaRESUMO
Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target.
Assuntos
Apolipoproteína A-I , Infecções por HIV/patologia , Inflamação/patologia , Intestinos/efeitos dos fármacos , Peptídeos/farmacologia , Proteína ADAM17/efeitos dos fármacos , Animais , Fármacos Anti-HIV/farmacologia , Humanos , CamundongosRESUMO
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.
Assuntos
Infecções por HIV/virologia , Células-Tronco Hematopoéticas/citologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Infecções por HIV/imunologia , HIV-1/imunologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/uso terapêuticoRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1009404.].
RESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1006753.].
RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/terapia , Células-Tronco Hematopoéticas/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Animais , Linfócitos T CD4-Positivos/transplante , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Modelos Animais de Doenças , Terapia Genética/métodos , Infecções por HIV/virologia , Transplante de Células-Tronco Hematopoéticas/métodos , Imunoterapia/métodos , Macaca nemestrina , Masculino , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/virologiaRESUMO
The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities.
Assuntos
Infecções por HIV/imunologia , Células-Tronco Hematopoéticas/citologia , Receptores de Antígenos/química , Animais , Antígenos CD34/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Citocinas/metabolismo , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos , Células HEK293 , HIV-1 , Humanos , Células Matadoras Naturais/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Baço/metabolismo , Baço/virologia , Linfócitos T Citotóxicos/imunologiaRESUMO
The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Engenharia Celular , Terapia Genética , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/terapia , HIV-1/fisiologia , Células-Tronco Hematopoéticas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Replicação Viral/fisiologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Feminino , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transplante HeterólogoRESUMO
Neurocognitive deficits are prevalent among people living with HIV, likely due to chronic inflammation and oxidative stress in the brain. To date, no pharmaceutical treatments beyond antiretroviral therapy (ARV) has been shown to reduce risk for, or severity of, HIV-associated neurocognitive disorder. Here we investigate a novel compound, CDDO-Me, with documented neuroprotective effects via activation of the nrf2 and inhibition of the NFkB pathways. Methods: We conducted three studies to assess the efficacy of CDDO-Me alone or in combination with antiretroviral therapy in humanized mice infected with HIV; behavioral, histopathological, and immunohistochemical. Results: CDDO-Me in combination with ARV rescued social interaction deficits; however, only ARV was associated with preserved functioning in other behaviors, and CDDO-Me may have attenuated those benefits. A modest neuroprotective effect was found for CDDO-Me when administered with ARV, via preservation of PSD-95 expression; however, ARV alone had a more consistent protective effect. No significant changes in antioxidant enzyme expression levels were observed in CDDO-Me-treated animals. Only ARV use seemed to affect some antioxidant levels, indicating that it is ARV rather than CDDO-Me that is the major factor providing neuroprotection in this animal model. Finally, immunohistochemical analysis found that several cellular markers in various brain regions varied due to ARV rather than CDDO-Me. Conclusion: Limited benefit of CDDO-Me on behavior and neuroprotection were observed. Instead, ARV was shown to be the more beneficial treatment. These experiments support the future use of this chimeric mouse for behavioral experiments in neuroHIV research.
RESUMO
The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.
Assuntos
Infecções por HIV , HIV-1 , Camundongos , Humanos , Animais , Infecções por HIV/tratamento farmacológico , Antirretrovirais/uso terapêutico , Antirretrovirais/farmacologia , Viremia/tratamento farmacológico , Replicação Viral , Carga Viral , Linfócitos T CD4-PositivosRESUMO
Cannabis (Cannabis sativa) is a widely used drug in the United States and the frequency of cannabis use is particularly high among people living with HIV (PLWH). One key component of cannabis, the non-psychotropic (-)-cannabidiol (CBD) exerts a wide variety of biological actions, including anticonvulsive, analgesic, and anti-inflammatory effects. However, the exact mechanism of action through which CBD affects the immune cell signaling remains poorly understood. Here we report that CBD modulates type I interferon responses in human macrophages. Transcriptomics analysis shows that CBD treatment significantly attenuates cGAS-STING-mediated activation of type I Interferon response genes (ISGs) in monocytic THP-1 cells. We further showed that CBD treatment effectively attenuates 2'3-cGAMP stimulation of ISGs in both THP-1 cells and primary human macrophages. Interestingly, CBD significantly upregulates expression of autophagy receptor p62/SQSTM1. p62 is critical for autophagy-mediated degradation of stimulated STING. We observed that CBD treated THP-1 cells have elevated autophagy activity. Upon 2'3'-cGAMP stimulation, CBD treated cells have rapid downregulation of phosphorylated-STING, leading to attenuated expression of ISGs. The CBD attenuation of ISGs is reduced in autophagy deficient THP-1 cells, suggesting that the effects of CBD on ISGs is partially mediated by autophagy induction. Lastly, CBD decreases ISGs expression upon HIV infection in THP-1 cells and human primary macrophages, leading to increased HIV RNA expression 24 hours after infection. However, long term culture with CBD in infected primary macrophages reduced HIV viral spread, suggesting potential dichotomous roles of CBD in HIV replication. Our study highlights the immune modulatory effects of CBD and the needs for additional studies on its effect on viral infection and inflammation.
Assuntos
Canabidiol , Infecções por HIV , Interferon Tipo I , Anti-Inflamatórios , Canabidiol/farmacologia , Infecções por HIV/tratamento farmacológico , Humanos , Macrófagos , Nucleotidiltransferases , RNA , Proteína Sequestossoma-1RESUMO
A hallmark of HIV-1 infection is chronic inflammation, even in patients treated with antiretroviral therapy (ART). Chronic inflammation drives HIV-1 pathogenesis, leading to loss of CD4+ T cells and exhaustion of antiviral immunity. Therefore, strategies to safely reduce systematic inflammation are needed to halt disease progression and restore defective immune responses. Autophagy is a cellular mechanism for disposal of damaged organelles and elimination of intracellular pathogens. Autophagy is pivotal for energy homeostasis and plays critical roles in regulating immunity. However, how it regulates inflammation and antiviral T cell responses during HIV infection is unclear. Here, we demonstrate that autophagy is directly linked to IFN-I signaling, which is a key driver of immune activation and T cell exhaustion during chronic HIV infection. Impairment of autophagy leads to spontaneous IFN-I signaling, and autophagy induction reduces IFN-I signaling in monocytic cells. Importantly, in HIV-1-infected humanized mice, autophagy inducer rapamycin treatment significantly reduced persistent IFN-I-mediated inflammation and improved antiviral T cell responses. Cotreatment of rapamycin with ART led to significantly reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach to treat persistent inflammation and improve immune control of HIV replication.
Assuntos
Infecções por HIV , HIV-1 , Interferon Tipo I , Camundongos , Animais , Sirolimo/farmacologia , Sirolimo/uso terapêutico , AutofagiaRESUMO
Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.
Assuntos
Síndromes de Imunodeficiência , Purina-Núcleosídeo Fosforilase , Animais , Autoimunidade , Humanos , Camundongos , Nucleosídeos de Purina , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Linfócitos T , Receptor 7 Toll-LikeRESUMO
OBJECTIVES: Despite antiretroviral therapy (ART), there is an unmet need for therapies to mitigate immune activation in HIV infection. The goal of this study is to determine whether the apoA-I mimetics 6F and 4F attenuate macrophage activation in chronic HIV. DESIGN: Preclinical assessment of the in-vivo impact of Tg6F and the ex-vivo impact of apoA-I mimetics on biomarkers of immune activation and gut barrier dysfunction in treated HIV. METHODS: We used two humanized murine models of HIV infection to determine the impact of oral Tg6F with ART (HIV+ART+Tg6F+) on innate immune activation (plasma human sCD14, sCD163) and gut barrier dysfunction [murine I-FABP, endotoxin (LPS), LPS-binding protein (LBP), murine sCD14]. We also used gut explants from 10 uninfected and 10 HIV-infected men on potent ART and no morbidity, to determine the impact of ex-vivo treatment with 4F for 72âh on secretion of sCD14, sCD163, and I-FABP from gut explants. RESULTS: When compared with mice treated with ART alone (HIV+ART+), HIV+ART+Tg6F+ mice attenuated macrophage activation (h-sCD14, h-sCD163), gut barrier dysfunction (m-IFABP, LPS, LBP, and m-sCD14), plasma and gut tissue oxidized lipoproteins. The results were consistent with independent mouse models and ART regimens. Both 4F and 6F attenuated shedding of I-FABP and sCD14 from gut explants from HIV-infected and uninfected participants. CONCLUSION: Given that gut barrier dysfunction and macrophage activation are contributors to comorbidities like cardiovascular disease in HIV, apoA-I mimetics should be tested as therapy for morbidity in chronic treated HIV.
Assuntos
Infecções por HIV , Animais , Apolipoproteína A-I , Biomarcadores , Infecções por HIV/tratamento farmacológico , Receptores de Lipopolissacarídeos , Ativação de Macrófagos , CamundongosRESUMO
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.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Imunoterapia Adotiva/métodos , Células T Matadoras Naturais/fisiologia , Neoplasias/terapia , Animais , Células Cultivadas , Engenharia Genética , Humanos , Camundongos , Camundongos SCID , Células T Matadoras Naturais/transplante , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic immune activation, immunosuppression, and T cell exhaustion are hallmarks of HIV infection, yet the mechanisms driving these processes are unclear. Chronic activation can be a driving force in immune exhaustion, and type I interferons (IFN-I) are emerging as critical components underlying ongoing activation in HIV infection. Here, we have tested the effect of blocking IFN-I signaling on T cell responses and virus replication in a murine model of chronic HIV infection. Using HIV-infected humanized mice, we demonstrated that in vivo blockade of IFN-I signaling during chronic HIV infection diminished HIV-driven immune activation, decreased T cell exhaustion marker expression, restored HIV-specific CD8 T cell function, and led to decreased viral replication. Antiretroviral therapy (ART) in combination with IFN-I blockade accelerated viral suppression, further decreased viral loads, and reduced the persistently infected HIV reservoir compared with ART treatment alone. Our data suggest that blocking IFN-I signaling in conjunction with ART treatment can restore immune function and may reduce viral reservoirs during chronic HIV infection, providing validation for IFN-I blockade as a potential therapy for HIV infection.
Assuntos
Antirretrovirais/farmacologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Interferon Tipo I/antagonistas & inibidores , Animais , Linfócitos T CD8-Positivos/patologia , Doença Crônica , Modelos Animais de Doenças , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/patologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Camundongos , Camundongos KnockoutRESUMO
With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (cγ(-/-))-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.
Assuntos
Infecções por HIV , Animais , Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ-/-) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.