RESUMEN
BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are two highly aggressive and generally incurable gliomas with little therapeutic advancements made in the past several decades. Despite immense initial success of chimeric antigen receptor (CAR) T cells for the treatment of leukemia and lymphoma, significant headway into the application of CAR-T cells against solid tumors, including gliomas, is still forthcoming. The integrin complex alphav beta3 (αvß3) is present on multiple and diverse solid tumor types and tumor vasculature with limited expression throughout most normal tissues, qualifying it as an appealing target for CAR-T cell-mediated immunotherapy. METHODS: Patient-derived DIPG and GBM cell lines were evaluated by flow cytometry for surface expression of αvß3. Second-generation CAR-T cells expressing an anti-αvß3 single-chain variable fragment were generated by retroviral transduction containing either a CD28 or 4-1BB costimulatory domain and CD3zeta. CAR-T cells were evaluated by flow cytometry for CAR expression, memory phenotype distribution, and inhibitory receptor profile. DIPG and GBM cell lines were orthotopically implanted into NSG mice via stereotactic injection and monitored with bioluminescent imaging to evaluate αvß3 CAR-T cell-mediated antitumor responses. RESULTS: We found that patient-derived DIPG cells and GBM cell lines express high levels of surface αvß3 by flow cytometry, while αvß3 is minimally expressed on normal tissues by RNA sequencing and protein microarray. The manufactured CAR-T cells consisted of a substantial frequency of favorable early memory cells and a low inhibitory receptor profile. αvß3 CAR-T cells demonstrated efficient, antigen-specific tumor cell killing in both cytotoxicity assays and in in vivo models of orthotopically and stereotactically implanted DIPG and GBM tumors into relevant locations in the brain of NSG mice. Tumor responses were rapid and robust with systemic CAR-T cell proliferation and long-lived persistence associated with long-term survival. Following tumor clearance, TCF-1+αvß3 CAR-T cells were detectable, underscoring their ability to persist and undergo self-renewal. CONCLUSIONS: These results highlight the potential of αvß3 CAR-T cells for immunotherapeutic treatment of aggressive brain tumors with reduced risk of on-target, off-tumor mediated toxicity due to the restricted nature of αvß3 expression in normal tissues.
Asunto(s)
Neoplasias del Tronco Encefálico/inmunología , Glioma Pontino Intrínseco Difuso/inmunología , Glioblastoma/inmunología , Integrina beta3/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Animales , Modelos Animales de Enfermedad , Glioblastoma/patología , Humanos , RatonesRESUMEN
ABSTRACT: The successful application of chimeric antigen receptor (CAR) T cells for the treatment of relapsed and refractory B-cell malignancies has ushered in a new frontier for the immunotherapy of cancer. Despite its successes, CAR T-cell therapy presents several challenges. Cytokine release syndrome (CRS) triggered by robust and exponential CAR T-cell expansion is the most common adverse effect and may be severe or life-threatening. Although modulation of the interleukin 6 axis was appreciated early on as a means to manage CRS, the exact underlying mechanisms leading to severe CRS remain to be elucidated. What is clear is that severe CRS involves recruitment of the broader immune system into a hyperinflammatory and unregulated state. Myeloid-derived cells appear to play a critical role in this regard and are at the center of active investigation. In this article, we will focus on important elements of CRS, the clinical manifestations, underlying biology, and management strategies including grading, supportive care, and treatment via immunosuppression.
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COVID-19 , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/terapia , Inmunoterapia Adoptiva/efectos adversos , Animales , Modelos Animales de Enfermedad , Humanos , Inmunoterapia Adoptiva/métodos , Ratones , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células Mieloides/patologíaRESUMEN
Hepatitis C virus-mediated immune suppression is an underlying feature leading to the establishment of viral persistence and chronic infection. In particular, HCV core protein has been shown to exhibit significant immunosuppressive activity of T cells and antigen presenting cells. Using an HCV core transgenic mouse system, in which liver hepatocytes express core protein, it is possible to study the effects of core-mediated immune suppression in vivo during viral infection. In this protocol, we describe the procedures for evaluating antigen-specific CD8+ T cell responses in response to recombinant adenovirus infection in HCV core transgenic mice.
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Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Adenoviridae/genética , Animales , Separación Celular/instrumentación , Separación Celular/métodos , Centrifugación por Gradiente de Densidad/instrumentación , Centrifugación por Gradiente de Densidad/métodos , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Vectores Genéticos/genética , Hepatitis C/virología , Hepatocitos , Leucocitos Mononucleares , Hígado/citología , Hígado/inmunología , Hígado/virología , Ratones , Ratones Transgénicos , Cultivo Primario de Células/instrumentación , Cultivo Primario de Células/métodos , Transducción Genética/instrumentación , Transducción Genética/métodos , Proteínas del Núcleo Viral/inmunologíaRESUMEN
Hepatitis C virus (HCV) is a global health concern that can cause severe liver disease, such as cirrhosis and hepatocellular carcinoma. Control of HCV requires vigorous T-cell responses, yet CD4+ T cells in chronic HCV patients are dysfunctional. T follicular regulatory (Tfr) cells are a subset of regulatory T cells that suppress T follicular helper (Tfh) cells and the generation of high affinity antibody-producing B cells. In this study, we examined the accumulation of Tfr cells in the liver compartment during chronic HCV infection and defined the cellular and molecular mechanisms underlying their expansion. Our analysis revealed a substantial population of Tfr cells in livers of chronic HCV patients that is absent in liver tissues from nonviral hepatitis or healthy subjects. Coculture of PBMCs from healthy subjects with HCV-infected hepatoma cells resulted in preferential expansion of circulating Tfr cells, leading to suppression of Tfh cells. Additionally, coculture of tonsillar cells with infected hepatoma cells lead to an expansion of germinal center Tfr. Notably, expansion was mediated by transforming growth factor beta (TGF-ß)-containing exosomes released from HCV-infected hepatocytes given that blockade of exosome-associated TGF-ß or inhibition of exosome release abrogated Tfr expansion. CONCLUSION: These results show that liver-derived exosomes play a pivotal role in the accumulation of Tfr cells, likely leading to suppression of Tfh responses in HCV-infected patients. Our study identifies a novel pathway in which HCV infection in hepatocytes exacerbates Tfr cell responses to subvert antiviral immunity. (Hepatology 2018;67:71-85).
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Proliferación Celular/fisiología , Exosomas/inmunología , Hepatitis C/inmunología , Hepatocitos/inmunología , Linfocitos T Reguladores/inmunología , Biopsia con Aguja , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Exosomas/metabolismo , Citometría de Flujo , Hepacivirus/inmunología , Hepatitis C/patología , Hepatocitos/metabolismo , Humanos , Inmunohistoquímica , Técnicas In Vitro , Estadísticas no Paramétricas , Linfocitos T Reguladores/metabolismoRESUMEN
CD8, but not CD4, T cells are considered critical for control of chronic toxoplasmosis. Although CD8 exhaustion has been previously reported in Toxoplasma encephalitis (TE)-susceptible model, our current work demonstrates that CD4 not only become exhausted during chronic toxoplasmosis but this dysfunction is more pronounced than CD8 T cells. Exhausted CD4 population expressed elevated levels of multiple inhibitory receptors concomitant with the reduced functionality and up-regulation of Blimp-1, a transcription factor. Our data demonstrates for the first time that Blimp-1 is a critical regulator for CD4 T cell exhaustion especially in the CD4 central memory cell subset. Using a tamoxifen-dependent conditional Blimp-1 knockout mixed bone marrow chimera as well as an adoptive transfer approach, we show that CD4 T cell-intrinsic deletion of Blimp-1 reversed CD8 T cell dysfunction and resulted in improved pathogen control. To the best of our knowledge, this is a novel finding, which demonstrates the role of Blimp-1 as a critical regulator of CD4 dysfunction and links it to the CD8 T cell dysfunctionality observed in infected mice. The critical role of CD4-intrinsic Blimp-1 expression in mediating CD4 and CD8 T cell exhaustion may provide a rational basis for designing novel therapeutic approaches.
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Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Toxoplasmosis/inmunología , Factores de Transcripción/fisiología , Animales , Enfermedad Crónica , Femenino , Ratones , Ratones Endogámicos C57BL , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Receptor de Muerte Celular Programada 1/fisiologíaRESUMEN
Chronic infectious diseases such as HIV, HBV, and HCV, among others, cause severe morbidity and mortality globally. Progressive decline in CD8 functionality, survival, and proliferative potential-a phenomenon referred to as CD8 exhaustion-is believed to be responsible for poor pathogen control in chronic infectious diseases. While the role of negative inhibitory receptors such as PD-1 in augmenting CD8 exhaustion has been extensively studied, the role of positive costimulatory receptors remains poorly understood. In this review, we discuss how one such costimulatory pathway, CD40-CD40L, regulates CD8 dysfunction and rescue. While the significance of this pathway has been extensively investigated in models of autoimmunity, acute infectious diseases, and tumor models, the role played by CD40-CD40L in regulating CD8 exhaustion in chronic infectious diseases is just beginning to be understood. Considering that monotherapy with blocking antibodies targeting inhibitory PD-1-PD-L1 pathway is only partially effective at ameliorating CD8 exhaustion and that humanized CD40 agonist antibodies are currently available, a better understanding of the role of the CD40-CD40L pathway in chronic infectious diseases will pave the way for the development of more robust immunotherapeutic and prophylactic vaccination strategies.
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Antígenos CD40/inmunología , Linfocitos T CD8-positivos/metabolismo , Infecciones/inmunología , Transducción de Señal , Animales , Antígenos CD8/inmunología , Enfermedad Crónica , HumanosRESUMEN
Functional exhaustion of CD8(+) T cells due to increased expression of inhibitory molecule PD-1 (Programmed Death-1) causes reactivation of latent disease during later phases of chronic toxoplasmosis. Onset of disease recrudescence results in decreased parasite cyst burden concomitant with parasites undergoing stage conversion from a primarily encysted, quiescent bradyzoite to a fast-replicating, highly motile tachyzoite. Thus, reduced cyst burden is one of the early hallmarks of disease recrudescence. This was further validated by depleting gamma interferon (IFN-γ), a cytokine known to control latent toxoplasmosis, in chronically infected prerecrudescent mice. Since CD8(+) T cells (an important source of IFN-γ) lose their functionality during the later phases of chronic toxoplasmosis, we next examined if adoptive transfer of functional CD8(+) T cells from acutely infected donors to the chronically infected prerecrudescent hosts could impede parasite de-encystation and rescue exhausted CD8(+) T cells. While the transfer of immune CD8(+) T cells temporarily restricted the breakdown of cysts, the exhausted endogenous CD8(+) T cell population was not rescued. Over time, the donor population got deleted, resulting in parasite de-encystation and host mortality. Considering that donor CD8(+) T cells fail to become long-lived, one of the cardinal features of memory CD8(+) T cells, it bears the implication that memory CD8 differentiation is impaired during chronic toxoplasmosis. Moreover, our data strongly suggest that while adoptive immunotherapy can prevent parasite de-encystation transiently, reduced antigen burden in the chronic phase by itself is insufficient for rescue of exhausted CD8(+) T cells. The conclusions of this study have profound ramifications in designing immunotherapeutics against chronic toxoplasmosis.
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Linfocitos T CD8-positivos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Traslado Adoptivo/métodos , Animales , Femenino , Inmunoterapia Adoptiva/métodos , Interferón gamma/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Although the highest numbers of studies linking an infectious agent with schizophrenia has involved the parasite Toxoplasma gondii, the mechanistic underpinnings of this correlation has remained unaddressed. Incidentally, CD8 T cells, which play a pivotal role in mediating long-term immunity to Toxoplasma, are downregulated in schizophrenia patients. Recent studies have demonstrated that CD8 response is also impaired during chronic toxoplasmosis in murine models. In light of these new findings, in this article, we discuss the potential role of CD8 T cells in causing altered mental status in Toxoplasma seropositive schizophrenia patients.