RESUMEN
Adoptive immunotherapy using genetically engineered patient-derived lymphocytes to express tumor-reactive receptors is a promising treatment for malignancy. However, utilization of autologous T cells in this therapy limits the quality of gene-engineered T cells, thereby inhibiting the timely infusion of the cells into patients. In this study, we evaluated the anti-tumor efficacy and the potential to induce graft-versus-host disease (GVHD) in T cell receptor (TCR) gene-engineered allogeneic T cells that downregulate the endogenous TCR and HLA class I molecules with the aim of developing an "off-the-shelf" cell product with expanded application of genetically engineered T cells. We transduced human lymphocytes with a high-affinity TCR specific to the cancer/testis antigen NY-ESO-1 using a novel retrovirus vector with siRNAs specific to the endogenous TCR (siTCR vector). These T cells showed reduced expression of endogenous TCR and minimized reactivity to allogeneic cells in vitro. In non-obese diabetic/SCID/γcnull mice, TCR gene-transduced T cells induced tumor regression without development of GVHD. A lentivirus-based CRISPR/Cas9 system targeting ß-2 microglobulin in TCR gene-modified T cells silenced the HLA class I expression and prevented allogeneic CD8+ T cell stimulation without disrupting their anti-tumor capacity. This report is the first demonstration that siTCR technology is effective in preventing GVHD. Adoptive cell therapy with allogeneic T cells engineered with siTCR vector may be useful in developing an "off-the-shelf" therapy for patients with malignancy.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Neoplasias , Ratones , Animales , Humanos , ARN Interferente Pequeño/genética , Células Alogénicas/metabolismo , Ratones SCID , Receptores de Antígenos de Linfocitos T , Genes Codificadores de los Receptores de Linfocitos T , Inmunoterapia Adoptiva , Neoplasias/genética , Enfermedad Injerto contra Huésped/prevención & controlRESUMEN
Cell-based immunotherapy has become the new-generation cancer medicine, and "off-the-shelf" cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. Here, by combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we generate human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development.
Asunto(s)
Células Alogénicas/inmunología , Ingeniería Celular , Células Madre Hematopoyéticas/inmunología , Inmunoterapia , Células T Asesinas Naturales/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Células Alogénicas/metabolismo , Animales , Línea Celular Tumoral , Perfilación de la Expresión Génica , Antígenos HLA/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones Endogámicos NOD , Ratones SCID , Células T Asesinas Naturales/metabolismo , Fenotipo , Receptores Quiméricos de Antígenos/metabolismo , Transcriptoma/genéticaRESUMEN
Low haemocompatibility of left ventricular assist devices (LVAD) surfaces necessitates anticoagulative therapy. Endothelial cell (EC) seeding can support haemocompatibility, however, the availability of autologous ECs is limited. In contrast, allogeneic ECs are readily available in sufficient quantity, but HLA disparities induce harmful immune responses causing EC loss. In this study, we investigated the feasibility of using allogeneic low immunogenic ECs to endothelialize LVAD sintered inflow cannulas (SIC). To reduce the immunogenicity of ECs, we applied an inducible lentiviral vector to deliver short-hairpins RNA to silence HLA class I expression. HLA class I expression on ECs was conditionally silenced by up to 70%. Sufficient and comparable endothelialization rates were achieved with HLA-expressing or HLA-silenced ECs. Cell proliferation was not impaired by cell-to-Sintered Inflow Cannulas (SIC) contact or by silencing HLA expression. The levels of endothelial phenotypic and thrombogenic markers or cytokine secretion profiles remained unaffected. HLA-silenced ECs-coated SIC exhibited reduced thrombogenicity. In contrast to native ECs, HLA-silenced ECs showed lower cell lysis rates when exposed to allogeneic T cells or specific anti-HLA antibodies. Allogeneic HLA-silenced ECs could potentially become a valuable source for LVAD endothelialization to reduce immunogenicity and correspondingly the need for anticoagulative therapy which can entail severe side effects.
Asunto(s)
Células Alogénicas/inmunología , Bioprótesis , Células Endoteliales/inmunología , Corazón Auxiliar , Células Alogénicas/citología , Células Alogénicas/metabolismo , Bioprótesis/efectos adversos , Proliferación Celular , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Genes MHC Clase I , Corazón Auxiliar/efectos adversos , Humanos , Ensayo de Materiales , Interferencia de ARN , ARN Interferente Pequeño/genética , Trombosis/etiologíaRESUMEN
Individuals with autism spectrum disorder (ASD) suffer from developmental disabilities that impact communication, behavior, and social interaction. Immune dysregulation and inflammation have been linked to children with ASD, the latter manifesting in serum levels of macrophage-derived chemokine (MDC) and thymus, and activation-regulated chemokine (TARC). Mesenchymal stem cells derived from umbilical cord tissue (UC-MSCs) have immune-modulatory and anti-inflammatory properties, and have been safely used to treat a variety of conditions. This study investigated the safety and efficacy of UC-MSCs administered to children diagnosed with ASD. Efficacy was evaluated with the Autism Treatment Evaluation Checklist (ATEC) and the Childhood Autism Rating Scale (CARS), and with measurements of MDC and TARC serum levels. Twenty subjects received a dose of 36 million intravenous UC-MSCs every 12 weeks (four times over a 9-month period), and were followed up at 3 and 12 months after treatment completion. Adverse events related to treatment were mild or moderate and short in duration. The CARS and ATEC scores of eight subjects decreased over the course of treatment, placing them in a lower ASD symptom category when compared with baseline. MDC and TARC inflammatory cytokine levels also decreased for five of these eight subjects. The mean MDC, TARC, ATEC, and CARS values attained their lowest levels 3 months after the last administration. UC-MSC administration in children with ASD was therefore determined to be safe. Although some signals of efficacy were observed in a small group of children, possible links between inflammation levels and ASD symptoms should be further investigated. Stem Cells Translational Medicine 2019;8:1008-1016.
Asunto(s)
Células Alogénicas/metabolismo , Trastorno del Espectro Autista/terapia , Citocinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/trasplante , Adolescente , Trastorno del Espectro Autista/patología , Niño , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND: Allotransplantation of submandibular salivary glands (SMGs) could be an alternative treatment option for severe keratoconjunctivitis sicca in noncandidates for autologous SMG transplantation. This study was conducted to evaluate the effect of allogeneic mesenchymal stem cell (MSC) therapy on the survival of allotransplanted SMGs. METHODS: Thirty-six SMG allotransplantations (n = 6 per group) were performed in New Zealand white rabbits and randomly divided into the following groups: allograft control (Allo-Ctrl), low-dose FK506 (FK506-L), high-dose FK506 (FK506-H), allogeneic MSCs, MSCs+FK506-L, and MSCs+FK506-H. Rabbits were closely observed for 2 weeks. Gland viability and rejection were assessed by monitoring interleukin-2 levels by ELISA, sialoscintigraphy, M3-muscarinic acetylcholine receptor expression, histological evaluation, and apoptosis assay. RESULTS: Intraoperatively, all glands showed patency and saliva flow except 1 gland. Sialoscintigraphy revealed significantly higher saliva production within the MSC-treated glands. Histologically, MSC-treated glands showed higher glandular tissue preservation and less acini atrophy. The MSCs+FK506-H group revealed significantly lower apoptosis percentage. The highest survival was observed in the MSCs+FK506-H group, followed by the FK506-H and MSCs+FK506-L groups, and lastly less in the FK506-L and MSCs groups. CONCLUSIONS: Concurrent administration of MSCs with FK506-H (0.16 mg/kg) resulted in higher survival rate with greater glandular tissue preservation and salivary secretion. MSCs with FK506-L (0.08 mg/kg) could be an alternative to FK506-H (0.16 mg/kg) in salivary gland allotransplantation.
Asunto(s)
Rechazo de Injerto/prevención & control , Supervivencia de Injerto/efectos de los fármacos , Inmunosupresores/administración & dosificación , Trasplante de Células Madre Mesenquimatosas , Glándula Submandibular/efectos de los fármacos , Glándula Submandibular/trasplante , Tacrolimus/administración & dosificación , Células Alogénicas/inmunología , Células Alogénicas/metabolismo , Células Alogénicas/patología , Animales , Apoptosis/efectos de los fármacos , Atrofia , Femenino , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Interleucina-2/metabolismo , Masculino , Conejos , Receptor Muscarínico M3/metabolismo , Salivación/efectos de los fármacos , Glándula Submandibular/inmunología , Glándula Submandibular/patología , Factores de Tiempo , Supervivencia Tisular/efectos de los fármacos , Trasplante HomólogoRESUMEN
Transplanted stem cells constitute a new therapeutic strategy for the treatment of neurological disorders. Emerging evidence indicates that a negative microenvironment, particularly one characterized by the acute inflammation/immune response caused by physical injuries or transplanted stem cells, severely impacts the survival of transplanted stem cells. In this study, to avoid the influence of the increased inflammation following physical injuries, an intelligent, double-layer, alginate hydrogel system is designed. This system fosters the matrix metalloproeinases (MMP) secreted by transplanted stem cell reactions with MMP peptide grafted on the inner layer and destroys the structure of the inner hydrogel layer during the inflammatory storm. Meanwhile, the optimum concentration of the arginine-glycine-aspartate (RGD) peptide is also immobilized to the inner hydrogels to obtain more stem cells before arriving to the outer hydrogel layer. It is found that blocking Cripto-1, which promotes embryonic stem cell differentiation to dopamine neurons, also accelerates this process in neural stem cells. More interesting is the fact that neural stem cell differentiation can be conducted in astrocyte-differentiation medium without other treatments. In addition, the system can be adjusted according to the different parameters of transplanted stem cells and can expand on the clinical application of stem cells in the treatment of this neurological disorder.