RESUMEN
The mouse preimplantation embryo is an excellent system for studying how mammalian cells organize dynamically into increasingly complex structures. Accessible to experimental and genetic manipulations, its normal or perturbed development can be scrutinized ex vivo by real-time imaging from fertilization to late blastocyst stage. High-resolution imaging of multiple embryos at the same time can be compromised by embryos displacement during imaging. We have developed an inexpensive and easy-to-produce imaging device that facilitates greatly the imaging of preimplantation embryo. In this chapter, we describe the different steps of production and storage of the imaging device as well as its use for live imaging of mouse preimplantation embryos expressing fluorescent reporters from genetically modified alleles or after in vitro transcribed mRNA transfer by microinjection or electroporation.
Asunto(s)
Blastocisto/ultraestructura , Microscopía Confocal/métodos , Animales , Electroporación/métodos , Técnicas de Cultivo de Embriones/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Microinyecciones/métodosRESUMEN
Aim: Peritoneal metastasis is often present in end-stage neoplastic diseases, including recurrent colorectal cancer and is associated with decreased overall survival. Novel methods are needed. Materials & methods: We constructed first-, second- and third-generation chimeric antigen receptors (CARs) specific for NKG2D ligands and modified human T cells with mRNA electroporation. Results: NKG2D CAR expression was detectable for at least 6 days postelectroporation and mediated efficient cytotoxicity against NKG2DL+ tumor cells, but not NKG2DL-cells. Multiple infusions of the first-generation CAR-T cells into immunodeficient mice bearing established peritoneal colorectal xenografts led to significantly reduced tumor burden. Conclusion: mRNA CAR is an economical way to test new CARs and potentiates controlling on-target/off-tumor toxicity and cytokine storms. The use of NKG2D RNA CARs to treat colorectal peritoneal metastasis warrants further investigation.
Asunto(s)
Neoplasias del Colon/terapia , Neoplasias Colorrectales/terapia , Inmunoterapia Adoptiva/métodos , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , ARN/genética , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Neoplasias Colorrectales/inmunología , Humanos , Ratones , Linfocitos T/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. METHODS: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. RESULTS: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. CONCLUSIONS: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.
Asunto(s)
Células Dendríticas/metabolismo , Electroporación/métodos , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/terapia , Glicoproteína Mielina-Oligodendrócito/metabolismo , ARN Mensajero/metabolismo , Animales , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Tolerancia Inmunológica/fisiología , Células K562 , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Glicoproteína Mielina-Oligodendrócito/inmunología , ARN Mensajero/administración & dosificación , ARN Mensajero/inmunologíaRESUMEN
Chimeric antigen receptor (CAR)-T cells have been used successfully for cancer immunotherapy. While substantial tumor regression was observed in leukaemia and lymphoma, CAR therapy of solid tumors needs further improvement. A major obstacle to the efficiency of engineered T cells is posed by triggering of inhibitory receptors, for example programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), leading to an impaired antitumor activity. To boost CAR-T-cell function, we co-electroporated T cells with both, mRNA encoding a CAR specific for chondroitin sulphate proteoglycan 4 (CSPG4) and small-interfering RNAs (siRNAs) to downregulate PD-1 (siPD-1) and CTLA-4 (siCTLA-4). Flow cytometry revealed that activation-induced upregulation of both PD-1 and CTLA-4 was suppressed when compared to CAR-T cells electroporated with negative control siRNA. The siRNA transfection showed no influence on CAR expression of engineered T cells. Functionality assays were performed using PD-L1- and CD80-transfected melanoma cells endogenously expressing CSPG4. CAR-T cells transfected with siPD-1 alone showed improvement in cytokine secretion. Additionally, CAR-T cells transfected with either siPD-1 alone or together with siCTLA-4 exhibited a significantly increased cytotoxicity. No or only little effects were observed when CAR-T cells were co-transfected with siCTLA-4 only. Taken together, it is feasible to optimize CAR-T cells by co-transfection of CAR-encoding mRNA and siRNAs to downregulate inhibitory receptors. Our in vitro data indicate an improvement of the functionality of these CAR-T cells, suggesting that this strategy could represent a novel method to enhance CAR-T-cell immunotherapy of cancer.
Asunto(s)
Antígeno CTLA-4/antagonistas & inhibidores , Inmunoterapia Adoptiva/métodos , Melanoma/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias Cutáneas/terapia , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/genética , Citocinas/metabolismo , Citotoxicidad Inmunológica , Regulación hacia Abajo , Electroporación , Humanos , Melanoma/genética , Melanoma/inmunología , Receptor de Muerte Celular Programada 1/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , TransfecciónRESUMEN
The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.
Asunto(s)
Antígenos CD28 , Electroporación , Inmunidad Celular , Neoplasias Experimentales/inmunología , ARN Mensajero , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Animales , Antígenos CD28/genética , Antígenos CD28/inmunología , Humanos , Interleucina-2/inmunología , Células K562 , Ratones , Muromonab-CD3/inmunología , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , ARN Mensajero/genética , ARN Mensajero/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) infects about 20 million people world-wide. Around 5% of the infected individuals develop adult T-cell leukemia (ATL) or a neurological disease termed tropical spastic paraparesis (TSP) after a clinical latency of years to decades. Through the use of two promoters and alternative splicing HTLV-1 expresses at least 12 different proteins. HTLV-1 establishes a life-long persistent infection by inducing the clonal expansion of infected cells, a property largely ascribed to the viral genes Tax and HBZ. However, the fact that ATL arises in a minority of infected individuals after a long clinical latency suggests the existence of factors counterbalancing the oncogenic potential of HTLV-1 in the context of natural infection.To study the role of the different HTLV-1 gene products in the HTLV-1 life cycle, we optimized a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA. Results showed that the RNA transfection technique combines a high transfection efficiency with low toxicity, not only in Jurkat T-cells but also in primary T-cells. These findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Electroporación , Expresión Génica , Productos del Gen tax , Genes Virales , Virus Linfotrópico T Tipo 1 Humano , ARN Viral , Proteínas de los Retroviridae , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Productos del Gen tax/biosíntesis , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de los Retroviridae/biosíntesis , Proteínas de los Retroviridae/genéticaRESUMEN
The use of tolerance-inducing dendritic cells (tolDCs) has been proven to be safe and well tolerated in the treatment of autoimmune diseases. Nevertheless, several challenges remain, including finding ways to facilitate the migration of cell therapeutic products to lymph nodes, and the site of inflammation. In the treatment of neuroinflammatory diseases, such as multiple sclerosis (MS), the blood-brain barrier (BBB) represents a major obstacle to the delivery of therapeutic agents to the inflamed central nervous system (CNS). As it was previously demonstrated that C-C chemokine receptor 5 (CCR5) may be involved in inflammatory migration of DCs, the aim of this study was to investigate CCR5-driven migration of tolDCs. Only a minority of in vitro generated vitamin D3 (vitD3)-treated tolDCs expressed the inflammatory chemokine receptor CCR5. Thus, messenger RNA (mRNA) encoding CCR5 was introduced by means of electroporation (EP). After mRNA EP, tolDCs transiently displayed increased levels of CCR5 protein expression. Accordingly, the capacity of mRNA electroporated tolDCs to transmigrate toward a chemokine gradient in an in vitro model of the BBB improved significantly. Neither the tolerogenic phenotype nor the T cell-stimulatory function of tolDCs was affected by mRNA EP. EP of tolDCs with mRNA encoding CCR5 enabled these cells to migrate to inflammatory sites. The approach used herein has important implications for the treatment of MS. Using this approach, tolDCs actively shuttle across the BBB, allowing in situ down-modulation of autoimmune responses in the CNS.
RESUMEN
The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.
Asunto(s)
Animales , Humanos , Ratones , Antígenos CD28 , Genética , Alergia e Inmunología , Electroporación , Inmunidad Celular , Interleucina-2 , Alergia e Inmunología , Células K562 , Muromonab-CD3 , Alergia e Inmunología , Neoplasias Experimentales , Genética , Alergia e Inmunología , Patología , ARN Mensajero , Genética , Alergia e Inmunología , Linfocitos T , Alergia e Inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Genética , Alergia e InmunologíaRESUMEN
Monocyte-derived dendritic cells (DCs) are used as immunoadjuvant cells in cancer vaccines and have made great progress. However, an optimal DCs subset is vital for this treatment effect, the current 'gold standard' cytokine cocktail DCs have a shortcoming in their cytokines secretion, especially IL-12p70, mainly because of the existence of PGE2. Therefore, it is necessary to find an appropriate DCs-based immunotherapeutic protocol. In this study, we compared a novel 'improved' maturation cytokine cocktail with the current 'gold standard' maturation cytokine cocktail used for generating standard DCs. The 'improved' maturation cytokine cocktail DCs showed a higher levels surface markers expression (CD80, CD83, CD86 and HLA-DR), the chemokine receptors CXCR4 and CCR7 and chemokine CCL19, CCL21 and CXCL21, whereas CCR5 expression was reduced. Most importantly, in contrast to 'gold standard' DCs, which secrete little IL-12p70 and as a result induce mainly Th2 immunity, 'improved' cytokine cocktail DCs secreted higher levels IL-12p70 and also secreted similar concentration IL-10. To removal of PGE2 from the 'improved' DCs did increase the IL-12p70 production. In conclusion, we here present the 'improved' DCs, as an optimal maturation cocktail protocol, can induce high migratory potential, generate immunostimulatory DCs, produce higher levels IL-12p70 with superior capacity to induce Th1 immunity, when compared with the 'gold standard' DCs.