RESUMEN
Activation of a conditional safety switch has the potential to reverse serious toxicities arising from the administration of engineered cellular therapies, including chimeric antigen receptor (CAR) T cells. The functionally inert, non-immunogenic cell surface marker derived from human epidermal growth factor receptor (EGFRt) is a promising safety switch that has been used in multiple clinical constructs and can be targeted by cetuximab, a clinically available monoclonal antibody. However, this approach requires high and persistent cell surface expression of EGFRt to ensure that antibody-mediated depletion of engineered cells is rapid and complete. Here we show that incorporating a short juxtamembrane sequence into the EGFRt polypeptide enhances its expression on the surface of T cells and their susceptibility to antibody-dependent cellular cytotoxicity (ADCC). Incorporating this optimized variant (EGFRopt) into bicistronic and tricistronic CAR designs results in more rapid in vivo elimination of CAR T cells and robust termination of their effector activity compared to EGFRt. These studies establish EGFRopt as a superior safety switch for the development of next-generation cell-based therapeutics.
RESUMEN
Effective vaccines inducing lifelong protection against many important infections such as respiratory syncytial virus (RSV), HIV, influenza virus, and Epstein-Barr virus (EBV) are not yet available despite decades of research. As an alternative to a protective vaccine, we developed a genetic engineering strategy in which CRISPR-Cas9 was used to replace endogenously encoded antibodies with antibodies targeting RSV, HIV, influenza virus, or EBV in primary human B cells. The engineered antibodies were expressed efficiently in primary B cells under the control of endogenous regulatory elements, which maintained normal antibody expression and secretion. Using engineered mouse B cells, we demonstrated that a single transfer of B cells engineered to express an antibody against RSV resulted in potent and durable protection against RSV infection in RAG1-deficient mice. This approach offers the opportunity to achieve sterilizing immunity against pathogens for which traditional vaccination has failed to induce or maintain protective antibody responses.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Ingeniería Metabólica/métodos , Infecciones por Virus Sincitial Respiratorio/terapia , Virus Sincitial Respiratorio Humano/inmunología , Células 3T3 , Traslado Adoptivo/métodos , Animales , Sistemas CRISPR-Cas , Femenino , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Infecciones por Virus Sincitial Respiratorio/virologíaRESUMEN
During infection, antigen-specific T cells undergo tightly regulated developmental transitions controlled by transcriptional and post-transcriptional regulation of gene expression. We found that the microRNA miR-31 was strongly induced by activation of the T cell antigen receptor (TCR) in a pathway involving calcium and activation of the transcription factor NFAT. During chronic infection with lymphocytic choriomeningitis virus (LCMV) clone 13, miR-31-deficent mice recovered from clinical disease, while wild-type mice continued to show signs of disease. This disease phenotype was explained by the presence of larger numbers of cytokine-secreting LCMV-specific CD8+ T cells in miR-31-deficent mice than in wild-type mice. Mechanistically, miR-31 increased the sensitivity of T cells to type I interferons, which interfered with effector T cell function and increased the expression of several proteins related to T cell dysfunction during chronic infection. These studies identify miR-31 as an important regulator of T cell exhaustion in chronic infection.
Asunto(s)
Infecciones por Arenaviridae/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , MicroARNs/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Anticuerpos Antivirales/inmunología , Infecciones por Arenaviridae/genética , Linfocitos T CD8-positivos/efectos de los fármacos , Calcio/metabolismo , Inmunoprecipitación de Cromatina , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , Immunoblotting , Interferón Tipo I/farmacología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Noqueados , MicroARNs/genética , Factores de Transcripción NFATC/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Therapies using T cells that are programmed to express chimeric antigen receptors (CAR T cells) consistently produce positive results in patients with hematologic malignancies. However, CAR T cell treatments are less effective in solid tumors for several reasons. First, lymphocytes do not efficiently target CAR T cells; second, solid tumors create an immunosuppressive microenvironment that inactivates T cell responses; and third, solid cancers are typified by phenotypic diversity and thus include cells that do not express proteins targeted by the engineered receptors, enabling the formation of escape variants that elude CAR T cell targeting. Here, we have tested implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors, thereby exposing them to high concentrations of immune cells for a substantial time period. In immunocompetent orthotopic mouse models of pancreatic cancer and melanoma, we found that CAR T cells can migrate from biopolymer scaffolds and eradicate tumors more effectively than does systemic delivery of the same cells. We have also demonstrated that codelivery of stimulator of IFN genes (STING) agonists stimulates immune responses to eliminate tumor cells that are not recognized by the adoptively transferred lymphocytes. Thus, these devices may improve the effectiveness of CAR T cell therapy in solid tumors and help protect against the emergence of escape variants.
Asunto(s)
Biopolímeros/administración & dosificación , Carcinoma Ductal Pancreático/terapia , Melanoma Experimental/terapia , Neoplasias Pancreáticas/terapia , Traslado Adoptivo , Animales , Células Presentadoras de Antígenos/fisiología , Antineoplásicos/administración & dosificación , Carcinoma Ductal Pancreático/inmunología , Línea Celular Tumoral , GMP Cíclico/administración & dosificación , GMP Cíclico/análogos & derivados , Portadores de Fármacos/administración & dosificación , Femenino , Implantes Experimentales , Melanoma Experimental/inmunología , Proteínas de la Membrana/agonistas , Ratones Endogámicos C57BL , Ratones Transgénicos , Trasplante de Neoplasias , Neoplasias Pancreáticas/inmunología , Linfocitos T/fisiologíaRESUMEN
An emerging approach for treating cancer involves programming patient-derived T cells with genes encoding disease-specific chimeric antigen receptors (CARs), so that they can combat tumour cells once they are reinfused. Although trials of this therapy have produced impressive results, the in vitro methods they require to generate large numbers of tumour-specific T cells are too elaborate for widespread application to treat cancer patients. Here, we describe a method to quickly program circulating T cells with tumour-recognizing capabilities, thus avoiding these complications. Specifically, we demonstrate that DNA-carrying nanoparticles can efficiently introduce leukaemia-targeting CAR genes into T-cell nuclei, thereby bringing about long-term disease remission. These polymer nanoparticles are easy to manufacture in a stable form, which simplifies storage and reduces cost. Our technology may therefore provide a practical, broadly applicable treatment that can generate anti-tumour immunity 'on demand' for oncologists in a variety of settings.
Asunto(s)
ADN/química , Portadores de Fármacos , Técnicas de Transferencia de Gen , Inmunidad Celular/efectos de los fármacos , Leucemia/terapia , Nanopartículas/química , Receptores Quiméricos de Antígenos , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Inmunidad Celular/genética , Leucemia/genética , Leucemia/inmunología , Leucemia/patología , Ratones , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunologíaRESUMEN
The maintenance of immune homeostasis requires regulatory T cells (T(regs)). Given their intrinsic self-reactivity, T(regs) must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3(+) CD4 and Qa-1-restricted CD8 T(regs) results in defective regulatory activity and autoimmunity in mice. Helios-deficient T(regs) develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 T(regs) also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes T(regs) in the face of inflammatory responses provides a genetic explanation for a core property of T(regs).
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de Unión al ADN/biosíntesis , Linfocitos T Reguladores/inmunología , Factores de Transcripción/biosíntesis , Animales , Autoinmunidad/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/inmunología , Expresión Génica , Riñón/inmunología , Hígado/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Páncreas/inmunología , Factor de Transcripción STAT5/metabolismo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: With its unique ability to produce high-voltage electric discharges in excess of 600 volts, the South American strong voltage electric eel (Electrophorus electricus) has played an important role in the history of science. Remarkably little is understood about the molecular nature of its electric organs. RESULTS: We present an in-depth analysis of the genome of E. electricus, including the transcriptomes of eight mature tissues: brain, spinal cord, kidney, heart, skeletal muscle, Sachs' electric organ, main electric organ, and Hunter's electric organ. A gene set enrichment analysis based on gene ontology reveals enriched functions in all three electric organs related to transmembrane transport, androgen binding, and signaling. This study also represents the first analysis of miRNA in electric fish. It identified a number of miRNAs displaying electric organ-specific expression patterns, including one novel miRNA highly over-expressed in all three electric organs of E. electricus. All three electric organ tissues also express three conserved miRNAs that have been reported to inhibit muscle development in mammals, suggesting that miRNA-dependent regulation of gene expression might play an important role in specifying an electric organ identity from its muscle precursor. These miRNA data were supported using another complete miRNA profile from muscle and electric organ tissues of a second gymnotiform species. CONCLUSIONS: Our work on the E. electricus genome and eight tissue-specific gene expression profiles will greatly facilitate future research on determining the coding and regulatory sequences that specify the function, development, and evolution of electric organs. Moreover, these data and future studies will be informed by the first comprehensive analysis of miRNA expression in an electric fish presented here.
Asunto(s)
Órgano Eléctrico/metabolismo , Electrophorus/metabolismo , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Transcriptoma , Animales , Electrophorus/genética , MicroARNs/genética , América del SurRESUMEN
Little is known about the genetic basis of convergent traits that originate repeatedly over broad taxonomic scales. The myogenic electric organ has evolved six times in fishes to produce electric fields used in communication, navigation, predation, or defense. We have examined the genomic basis of the convergent anatomical and physiological origins of these organs by assembling the genome of the electric eel (Electrophorus electricus) and sequencing electric organ and skeletal muscle transcriptomes from three lineages that have independently evolved electric organs. Our results indicate that, despite millions of years of evolution and large differences in the morphology of electric organ cells, independent lineages have leveraged similar transcription factors and developmental and cellular pathways in the evolution of electric organs.
Asunto(s)
Evolución Biológica , Pez Eléctrico/genética , Órgano Eléctrico/citología , Órgano Eléctrico/fisiología , Electrophorus/anatomía & histología , Electrophorus/genética , Animales , Bagres/anatomía & histología , Bagres/genética , Bagres/fisiología , Diferenciación Celular , Pez Eléctrico/anatomía & histología , Pez Eléctrico/fisiología , Órgano Eléctrico/anatomía & histología , Electrophorus/fisiología , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Filogenia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+) and IgV(H) unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.
Asunto(s)
Linfocitos B/inmunología , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Activación de Linfocitos/genética , MicroARNs/genética , Linfocitos B/patología , Humanos , Leucemia Linfocítica Crónica de Células B/terapia , MicroARNs/metabolismo , Fenotipo , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Epstein-Barr Virus (EBV) establishes a long-term latent infection and is associated with a number of human malignancies that are thought to arise from deregulation of different stages of the viral life cycle. Recently, a large number of microRNAs (miRNAs) have been described for EBV, and it has been suggested that their expression may vary between the different latency states found in normal and malignant tissue. To date, however, no technique has been utilized to comprehensively and quantitatively test this idea by profiling expression of the EBV miRNAs in primary infected tissues. We describe here a multiplex reverse transcription-PCR assay that allows the profiling of 39 of the 40 known mature EBV miRNAs from as little as 250 ng of RNA. With this approach, we present a comprehensive profile of EBV miRNAs in primary nasopharyngeal carcinoma (NPC) tumors including estimates of miRNA copy number per tumor cell. This is the first comprehensive profiling of EBV miRNAs in any EBV-associated tumor. In contrast to previous suggestions, we show that the BART-derived miRNAs are present in a wide range of copy numbers from < or =10(3) per cell in both primary tumors and the widely used NPC-derived C666-1 cell line. However, we confirm the hypothesis that the BHRF1 miRNAs are not expressed in NPC. Lastly, we demonstrate that EBV miRNA expression in the widely used NPC line C666-1 is, with some caveats, broadly representative of primary NPC tumors.
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Carcinoma/virología , Perfilación de la Expresión Génica , Herpesvirus Humano 4/genética , MicroARNs/genética , Neoplasias Nasofaríngeas/virología , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/genética , Humanos , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
RNA interference (RNAi) describes a set of natural processes in which genes are silenced by small RNAs. RNAi has been widely used as an experimental tool that has recently become the focus of drug development efforts to treat a variety of diseases and disorders. Like all molecular therapies, in vivo delivery is the major hurdle to realizing therapeutic RNAi. Several strategies have been developed that increase small RNA half-life in the blood, facilitate transduction across biological membranes, and mediate cell-specific delivery. Importantly, these strategies permit targeting of mRNAs as well as microRNAs (miRNAs), a class of small RNAs encoded in the genome. miRNAs are required for multiple developmental and cellular processes. Dysfunction of miRNAs can result in a host of pathologies, suggesting that miRNAs are potential targets of therapy. Recent studies of miRNA function in immune-specific pathways indicate that specific miRNAs might be exploited as therapeutic targets to treat immune disorders, including autoimmunity, allergy, and hematopoietic cancers.
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MicroARNs/uso terapéutico , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Hipersensibilidad/tratamiento farmacológico , Enfermedades del Sistema Inmune/tratamiento farmacológico , MicroARNs/administración & dosificación , MicroARNs/efectos adversosRESUMEN
The first highly specific knockouts of a microRNA, miR155, in mice result in multiple defects in adaptive immunity, and also show the feasibility of investigating at least some microRNAs by gene knockout.
Asunto(s)
Regulación de la Expresión Génica , Inmunidad/genética , MicroARNs/genética , Animales , Ratones , Ratones NoqueadosRESUMEN
Antagonism of T cell responses by variants of the cognate peptide is a potential mechanism of viral escape from immune responses and may play a role in the ability of HIV to evade immune control. We show here a rarely described mechanism of antagonism by a peptide shorter than the minimum length epitope for an HIV p24-specific CD4+ T cell clone. The shorter antagonist peptide-MHC complex bound the T cell receptor (TCR), albeit with lower affinity than the full-length agonist peptide. Prior work showing the crystal structure of the peptide-MHC complex revealed a unique glycine hinge near the C-terminus of the agonist peptide, allowing the generation of full-length antagonist peptide lacking the hinge. These results confirm the dependence of productive TCR engagement on residues spilling out from the C-terminus of the MHC binding groove and show that partial engagement of the TCR with a truncated, low-affinity ligand can result in T cell antagonism.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Proteína p24 del Núcleo del VIH/inmunología , Secuencia de Aminoácidos , Epítopos/química , Epítopos/inmunología , Proteína p24 del Núcleo del VIH/química , Proteína p24 del Núcleo del VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Antígeno HLA-DR1/química , Antígeno HLA-DR1/metabolismo , Humanos , Técnicas In Vitro , Ligandos , Activación de Linfocitos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Activating receptors in cells of hematopoetic origin include members of two unrelated protein families, the immunoglobulin (Ig) and C type lectins, which differ even in the orientation of the transmembrane (TM) domains. We examined assembly of four receptors with diverse function: the NK receptors KIR2DS and NKG2C/CD94, the Fc receptor for IgA, and the GPVI collagen receptor. For each of the four different receptors studied here, assembly results in the formation of a three-helix interface in the membrane involving two acidic TM residues from the signaling dimer and a basic TM residue from the ligand recognition module, an arrangement remarkably similar to the T cell receptor (TCR)-CD3 complex. The fact that the TM domains of Ig family and C type lectins adopt opposite orientations proves that these receptor families independently evolved toward the same structural arrangement of the interacting TM helices. This assembly mechanism is thus widely utilized by receptors in cells of hematopoetic origin.
Asunto(s)
Receptores Inmunológicos/química , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células Sanguíneas/química , Células Sanguíneas/inmunología , Dimerización , Inmunoglobulina A/química , Inmunoglobulina A/genética , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Subfamília C de Receptores Similares a Lectina de Células NK , Receptores de Colágeno/química , Receptores de Colágeno/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores KIR , Receptores de Células Asesinas Naturales , Alineación de Secuencia , Regulación hacia ArribaRESUMEN
Strong CD4(+) and CD8(+) T cell responses are considered important immune components for controlling HIV infection, and their priming may be central to an effective HIV vaccine. We describe in this study an approach by which multiple CD4(+) and CD8(+) T cell epitopes are processed and presented from an exogenously added HIV-1 Gag-p24 peptide of 32 aa complexed to heat shock protein (HSP) gp96. CD8(+) T cell recognition of the HSP/peptide complex, but not the peptide alone, was inhibited by brefeldin A, suggesting an endoplasmic reticulum-dependent pathway. This is the first report to describe efficient processing and simultaneous presentation of overlapping class I- and class II-restricted epitopes from the same extracellularly added precursor peptide complexed to HSP. Given previous reports of the strong immunogenicity of HSP/peptide complexes, the present data suggest that HSP-complexed peptides containing multiple MHC class I- and class II-restricted epitopes represent potential vaccine candidates for HIV and other viral infections suitable to induce effective CTL memory by simultaneously providing CD4 T cell help.
Asunto(s)
Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Antígenos VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , VIH-1/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Fragmentos de Péptidos/inmunología , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Antígenos CD/análisis , Linfocitos B/inmunología , Brefeldino A/farmacología , Linfocitos T CD8-positivos/inmunología , Línea Celular Transformada , Células Dendríticas/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Proteína p24 del Núcleo del VIH/química , Antígenos HLA-B/inmunología , Antígenos HLA-DQ/inmunología , Herpesvirus Humano 4 , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Datos de Secuencia Molecular , Fragmentos de Péptidos/químicaRESUMEN
The immune correlates of protection in human immunodeficiency virus type 1 (HIV-1) infection remain poorly defined, particularly the contribution of CD4(+) T cells. Here we explore the effector functions of HIV-1-specific CD4(+) T cells. We demonstrate HIV-1 p24-specific CD4(+)-T-cell cytolytic activity in peripheral blood mononuclear cells directly ex vivo and after enrichment by antigen-specific stimulation. We further show that in a rare long-term nonprogressor, both an HIV-1-specific CD4(+)-T-cell clone and CD4(+) T cells directly ex vivo exert potent suppression of HIV-1 replication. Suppression of viral replication was dependent on cell-cell contact between the effector CD4(+) T cells and the target cells. While the antiviral effector activity of CD8(+) T cells has been well documented, these results strongly suggest that HIV-1-specific CD4(+) T cells are capable of directly contributing to antiviral immunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , VIH-1/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T CD4-Positivos/virología , Citotoxicidad Inmunológica , Proteína p24 del Núcleo del VIH/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Replicación Viral/inmunologíaRESUMEN
Despite growing evidence that HIV-1-specific CD4(+) T helper (Th) cells may play a role in the control of viremia, discrete Th cell epitopes remain poorly defined. Furthermore, it is not known whether Th cell responses generated using vaccines based on clade B virus sequences will elicit immune responses that are effective in regions of the world where non-clade B viruses predominate. To address these issues we isolated CD4(+) T cell clones from individuals with vigorous HIV-1-specific Th cell responses and identified the minimum epitopes recognized. The minimum peptide length required for induction of CD4(+) T cell proliferation, IFN-gamma secretion, and cytolytic activity ranged from 9 to 16 amino acids in the five epitopes studied. Cross-clade recognition of the defined epitopes was examined for variant peptides from clades A, B, C, D, and AE. Over half the variant epitopes (17 of 32) exhibited impaired recognition, defined as less than 50% of the IFN-gamma secretion elicited by B clade consensus sequence. There was no evidence for antagonistic activity mediated by the variant peptides, and despite strong responses there was no escape of autologous virus from Th responses in the epitopes we studied. Abrogated recognition of variant CD4(+) T cell epitopes presents a potential obstacle to vaccine development.