RESUMEN
BACKGROUND: Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity. METHODOLOGY/PRINCIPAL FINDINGS: We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NKâ¶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses. CONCLUSIONS AND SIGNIFICANCE: These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch.
Asunto(s)
Citomegalovirus/genética , Ingeniería Genética , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Proteínas Virales/genética , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Regulación hacia Abajo/inmunología , Expresión Génica , Antígenos HLA/metabolismo , Humanos , Kanamicina Quinasa/inmunología , Células Asesinas Naturales/inmunología , Hígado/citología , Hígado/cirugía , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Ovinos , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
In our previous phase 1/2 study aimed at controlling graft-versus-host disease, 12 patients received Herpes simplex virus thymidine kinase (HSV-tk(+))/neomycin phosphotransferase (NeoR(+))-expressing donor gene-modified T cells (GMCs) and underwent an HLA-identical sibling T-cell-depleted bone marrow transplantation (BMT). This study's objective was to follow up, to quantify, and to characterize persistently circulating GMCs more than 10 years after BMT. Circulating GMCs remain detectable in all 4 evaluable patients. However, NeoR- and HSV-tk-polymerase chain reaction (PCR) differently quantified in vivo counts, suggesting deletions within the HSV-tk gene. Further experiments, including a novel "transgene walking" PCR method, confirmed the presence of deletions. The deletions were unique, patient-specific, present in most circulating GMCs expressing NeoR, and shown to occur at time of GMC production. Unique patient-specific retroviral insertion sites (ISs) were found in all GMCs capable of in vitro expansion/cloning as well. These findings suggest a rare initial gene deletion event and an in vivo survival advantage of rare GMC clones resulting from an anti-HSV-tk immune response and/or ganciclovir treatment. In conclusion, we show that donor mature T cells infused with a T-cell-depleted graft persist in vivo for more than a decade. These cells, containing transgene deletions and subjected to significant in vivo selection, represent a small fraction of T cells infused at transplantation.
Asunto(s)
Secuencia de Bases/genética , Trasplante de Médula Ósea , Transfusión de Linfocitos , Eliminación de Secuencia , Simplexvirus , Timidina Quinasa/genética , Transgenes , Proteínas Virales/genética , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Femenino , Estudios de Seguimiento , Terapia Genética , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Neoplasias Hematológicas/enzimología , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Kanamicina Quinasa/genética , Kanamicina Quinasa/inmunología , Depleción Linfocítica , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia/inmunología , Simplexvirus/enzimología , Simplexvirus/genética , Simplexvirus/inmunología , Timidina Quinasa/inmunología , Trasplante Homólogo , Proteínas Virales/inmunologíaRESUMEN
Biochemical and functional studies have demonstrated major histocompatibility complex (MHC) class II-restricted presentation of peptides derived from cytosolic proteins, but the underlying processing and presentation pathways have remained elusive. Here we show that endogenous presentation of an epitope derived from the cytosolic protein neomycin phosphotransferase II (NeoR) on MHC class II is mediated by autophagy. This presentation pathway involves the sequestration of NeoR into autophagosomes, and subsequent delivery into the lytic compartment. These results identify endosomes/lysosomes as the processing compartment for cytosolic antigens and furthermore link endogenous antigen presentation on MHC class II with the process of cellular protein turnover by autophagy.
Asunto(s)
Presentación de Antígeno , Autofagia , Citosol/inmunología , Antígenos de Histocompatibilidad Clase II/fisiología , Kanamicina Quinasa/inmunología , Línea Celular , Antígenos HLA-DP/fisiología , Cadenas beta de HLA-DP , Humanos , Kanamicina Quinasa/análisis , Lisosomas/química , Lisosomas/metabolismo , Vacuolas/enzimologíaRESUMEN
Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.