RESUMEN
Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.
Asunto(s)
Traslado Adoptivo/métodos , Ingeniería Celular/métodos , Ingeniería Genética/métodos , Células Asesinas Naturales/inmunología , Neoplasias Ováricas/terapia , Proteína ADAM17/genética , Animales , Sistemas CRISPR-Cas , Citotoxicidad Inmunológica/genética , Dependovirus , Femenino , Técnicas de Inactivación de Genes , Voluntarios Sanos , Humanos , Células K562 , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Ováricas/patología , Parvovirinae/genética , Receptor de Muerte Celular Programada 1/genética , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Social disruption stress (SDR) prior to primary influenza A virus (IAV) infection augments memory to IAV re-challenge in a T cell-specific manner. However, the effect of SDR on the primary anti-viral immune response has not been elucidated. In this study, SDR-infected (INF) mice terminated viral gene expression earlier and mounted an enhanced pulmonary IAV-specific CD8(+)T cell response versus controls. Additionally, SDR-INF mice had a more pro-inflammatory lung profile prior to and during infection and an attenuated corticosterone response. These data demonstrate neuroendocrine modification of the lung microenvironment and increased antigen-specific T cell activation, clonal expansion and viral control in stress-exposed mice.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regulación de la Expresión Génica/inmunología , Infecciones por Orthomyxoviridae/patología , Estrés Psicológico/patología , Análisis de Varianza , Animales , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/virología , Corticosterona/sangre , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/inmunología , Estrés Psicológico/inmunología , Factores de TiempoRESUMEN
Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded ß(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.
Asunto(s)
Corticosterona/fisiología , Reactividad Cruzada/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Células Dendríticas/inmunología , Terapia de Inmunosupresión , Activación de Linfocitos/efectos de los fármacos , Estrés Psicológico/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Células Cultivadas , Técnicas de Cocultivo , Corticosterona/biosíntesis , Reactividad Cruzada/efectos de los fármacos , Citotoxicidad Inmunológica/inmunología , Células Dendríticas/metabolismo , Inmovilización , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Immunological memory (MEM) development is affected by stress-induced neuroendocrine mediators. Current knowledge about how a behavioral interaction, such as social defeat, alters the development of adaptive immunity, and MEM is incomplete. In this study, the experience of social disruption stress (SDR) prior to a primary influenza viral infection enhanced the frequency and function of the T cell memory pool. Socially stressed mice had a significantly enlarged population of CD8(+) T cells specific for the immunodominant NP366-74 epitope of A/PR/8/34 virus in lung and spleen tissues at 6-12 wk after primary infection (resting memory). Moreover, during resting memory, SDR-MEM mice responded with an enhanced footpad delayed-type hypersensitivity response, and more IFN-gamma-producing CD4(+) T cells were detected after ex vivo stimulation. When mice were rechallenged with A/PR/8/34 virus, SDR-MEM mice terminated viral gene expression significantly earlier than MEM mice and generated a greater D(b)NP(366-74)CD8(+) T cell response in the lung parenchyma and airways. This enhancement was specific to the T cell response. SDR-MEM mice had significantly attenuated anti-influenza IgG titers during resting memory. Similar experiments in which mice were primed with X-31 influenza and challenged with A/PR/8/34 virus elicited similar enhancements in the splenic and lung airway D(b)NP(366-74)CD8(+) T cell populations in SDR-MEM mice. This study demonstrates that the experience of repeated social defeat prior to a primary viral infection significantly enhances virus-specific memory via augmentation of memory T cell populations and suggests that social stressors should be carefully considered in the design and analysis of future studies on antiviral immunity.
Asunto(s)
Epítopos de Linfocito T/inmunología , Memoria Inmunológica , Subtipo H3N2 del Virus de la Influenza A/inmunología , Virus de la Influenza A/inmunología , Estrés Psicológico/inmunología , Animales , Antígenos Virales/administración & dosificación , Antígenos Virales/inmunología , Conducta Animal , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/psicología , Pulmón/inmunología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/psicología , Estrés Psicológico/virologíaRESUMEN
The systemic elevation of psychological stress-induced glucocorticoids strongly suppresses CD8(+) T cell immune responses resulting in diminished antiviral immunity. However, the specific cellular targets of stress/glucocorticoids, the timing of exposure, the chronology of immunological events, and the underlying mechanisms of this impairment are incompletely understood. In this study, we address each of these questions in the context of a murine cutaneous HSV infection. We show that exposure to stress or corticosterone in only the earliest stages of an HSV-1 infection is sufficient to suppress, in a glucocorticoid receptor-dependent manner, the subsequent antiviral immune response after stress/corticosterone has been terminated. This suppression resulted in early onset and delayed resolution of herpetic lesions, reduced viral clearance at the site of infection and draining popliteal lymph nodes (PLNs), and impaired functions of HSV-specific CD8(+) T cells in PLNs, including granzyme B and IFN-gamma production and the ability to degranulate. In knockout mice lacking glucocorticoid receptors only in T cells, we show that these impaired CD8(+) T cell functions are not due to direct effects of stress/corticosterone on the T cells, but the ability of PLN-derived dendritic cells to prime HSV-1-specific CD8(+) T cells is functionally impaired. These findings highlight the susceptibility of critical early events in the generation of an antiviral immune response to neuroendocrine modulation and implicate dendritic cells as targets of stress/glucocorticoids in vivo. These findings also provide insight into the mechanisms by which the clinical use of glucocorticoids contributes to altered immune responses in patients with viral infections or tumors.