RESUMO
Natural killer (NK) cells are potent immune modulators that can quickly lyse tumor cells and elicit inflammatory responses. These characteristics make them ideal candidates for immunotherapy. However, unlike T cells, NK cells do not possess clonotypic receptors capable of specific antigen recognition and cannot expand via activating receptor signals alone. To enable NK cells with these capabilities, we created and have previously described a tri-specific killer engager (TriKE) platform capable of inducing antigen specificity and cytokine-mediated NK-cell expansion. TriKE molecules have three arms: (i) a single-chain variable fragment (scFv) against the activating receptor CD16 on NK cells to trigger NK-cell activation, (ii) an scFv against a tumor-associated antigen (CD33 here) to induce specific tumor target recognition, and (iii) an IL15 moiety to trigger NK-cell expansion and priming. Here, we demonstrate that by modifying the anti-CD16 scFv with a humanized single-domain antibody against CD16, we improved TriKE functionality. A CD33-targeting second-generation TriKE induced stronger and more specific NK-cell proliferation without T-cell stimulation, enhanced in vitro NK-cell activation and killing of CD33-expressing targets, and improved tumor control in preclinical mouse models. Given these improved functional characteristics, we propose rapid translation of second-generation TriKEs into the clinic.
Assuntos
Imunoterapia Adotiva/métodos , Interleucina-15/administração & dosagem , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Animais , Modelos Animais de Doenças , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/imunologia , Leucemia Promielocítica Aguda/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The adult mammalian heart has a limited regenerative capacity. Therefore, identification of endogenous cells and mechanisms that contribute to cardiac regeneration is essential for the development of targeted therapies. The side population (SP) phenotype has been used to enrich for stem cells throughout the body; however, SP cells isolated from the heart have been studied exclusively in cell culture or after transplantation, limiting our understanding of their function in vivo. We generated a new Abcg2-driven lineage-tracing mouse model with efficient labeling of SP cells. Labeled SP cells give rise to terminally differentiated cells in bone marrow and intestines. In the heart, labeled SP cells give rise to lineage-traced cardiomyocytes under homeostatic conditions with an increase in this contribution following cardiac injury. Instead of differentiating into cardiomyocytes like proposed cardiac progenitor cells, cardiac SP cells fuse with preexisting cardiomyocytes to stimulate cardiomyocyte cell cycle reentry. Our study is the first to show that fusion between cardiomyocytes and non-cardiomyocytes, identified by the SP phenotype, contribute to endogenous cardiac regeneration by triggering cardiomyocyte cell cycle reentry in the adult mammalian heart.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/fisiologia , Diferenciação Celular , Isquemia Miocárdica/patologia , Miócitos Cardíacos/citologia , Regeneração , Células da Side Population/citologia , Animais , Transplante de Medula Óssea , Linhagem da Célula , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Knockout , Isquemia Miocárdica/terapia , Miócitos Cardíacos/metabolismo , Células da Side Population/metabolismoRESUMO
Age-related thymic involution is characterized by a decrease in thymic epithelial cell (TEC) number and function parallel to a disruption in their spatial organization, resulting in defective thymocyte development and proliferation as well as peripheral T cell dysfunction. Deficiency of Klotho, an antiaging gene and modifier of fibroblast growth factor signaling, causes premature aging. To investigate the role of Klotho in accelerated age-dependent thymic involution, we conducted a comprehensive analysis of thymopoiesis and peripheral T cell homeostasis using Klotho-deficient (Kl/Kl) mice. At 8 wk of age, Kl/Kl mice displayed a severe reduction in the number of thymocytes (10-100-fold reduction), especially CD4 and CD8 double-positive cells, and a reduction of both cortical and medullary TECs. To address a cell-autonomous role for Klotho in TEC biology, we implanted neonatal thymi from Klotho-deficient and -sufficient mice into athymic hosts. Kl/Kl thymus grafts supported thymopoiesis equivalently to Klotho-sufficient thymus transplants, indicating that Klotho is not intrinsically essential for TEC support of thymopoiesis. Moreover, lethally irradiated hosts given Kl/Kl or wild-type bone marrow had normal thymocyte development and comparably reconstituted T cells, indicating that Klotho is not inherently essential for peripheral T cell reconstitution. Because Kl/Kl mice have higher levels of serum phosphorus, calcium, and vitamin D, we evaluated thymus function in Kl/Kl mice fed with a vitamin D-deprived diet. We observed that a vitamin D-deprived diet abrogated thymic involution and T cell lymphopenia in 8-wk-old Kl/Kl mice. Taken together, our data suggest that Klotho deficiency causes thymic involution via systemic effects that include high active vitamin D levels.
Assuntos
Senilidade Prematura/genética , Envelhecimento/fisiologia , Células Epiteliais/fisiologia , Glucuronidase/metabolismo , Linfócitos T/fisiologia , Timócitos/fisiologia , Timo/fisiologia , Transferência Adotiva , Animais , Células Cultivadas , Dietoterapia , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/genética , Proteínas Klotho , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Timo/transplante , Transplante , Vitamina D/metabolismoRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating and ultimately lethal blistering disease caused by mutations to the Col7a1 gene. Development of novel cell therapies for the treatment of RDEB would be fostered by having immunodeficient mouse models able to accept human cell grafts; however, immunodeficient models of many genodermatoses such as RDEB are lacking. To overcome this limitation, we combined the clustered regularly interspaced short palindromic repeats and associated nuclease (CRISPR/Cas9) system with microinjection into NOD/SCID IL2rγcnull (NSG) embryos to rapidly develop an immunodeficient Col7a1-/- mouse model of RDEB. Through dose optimization, we achieve F0 biallelic knockout efficiencies exceeding 80%, allowing us to quickly generate large numbers of RDEB NSG mice for experimental use. Using this strategy, we clearly demonstrate important strain-specific differences in RDEB pathology that could underlie discordant results observed between independent studies and establish the utility of this system in proof-of-concept human cellular transplantation experiments. Importantly, we uncover the ability of a recently identified skin resident immunomodulatory dermal mesenchymal stem cell marked by ABCB5 to reduce RDEB pathology and markedly extend the lifespan of RDEB NSG mice via reduced skin infiltration of inflammatory myeloid derivatives.
Assuntos
Colágeno Tipo VII/genética , Modelos Animais de Doenças , Epidermólise Bolhosa Distrófica , Transplante de Células-Tronco Mesenquimais , Pele/citologia , Animais , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/terapia , Feminino , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Knockout , Pele/patologiaRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing COL7A1 mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in vitro in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB.
RESUMO
The potential of mesenchymal stromal cells (MSCs) to inhibit anti-tumor immunity is becoming increasingly well recognized, but the precise steps affected by these cells during the development of an anti-tumor immune response remain incompletely understood. Here, we examined how MSCs affect the steps required to mount an effective anti-tumor immune response following administration of adenovirus Fas ligand (Ad-FasL) in the Lewis lung carcinoma (LL3) model. Administration of bone marrow-derived MSCs with LL3 cells accelerated tumor growth significantly. MSCs inhibited the inflammation induced by Ad-FasL in the primary tumors, precluding their rejection; MSCs also reduced the consequent expansion of tumor-specific T cells in the treated hosts. When immune T cells were transferred to adoptive recipients, MSCs impaired, but did not completely abrogate the ability of these T cells to promote elimination of secondary tumors. This impairment was associated with a modest reduction in tumor-infiltrating T cells, with a significant reduction in tumor-infiltrating macrophages, and with a reorganization of the stromal environment. Our data indicate that MSCs in the tumor environment reduce the efficacy of immunotherapy by creating a functional and anatomic barrier that impairs inflammation, T cell priming and expansion, and T cell function-including recruitment of effector cells.
Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Inflamação/prevenção & controle , Células-Tronco Mesenquimais/fisiologia , Linfócitos T/imunologia , Microambiente Tumoral , Adenoviridae/genética , Animais , Citotoxicidade Imunológica , Proteína Ligante Fas/genética , Proteína Ligante Fas/fisiologia , Camundongos , Linfócitos T/fisiologiaRESUMO
Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a congenital deficiency of α-L-iduronidase, leading to lysosomal storage of glycosaminoglycans that is ultimately fatal following an insidious onset after birth. Hematopoietic cell transplantation (HCT) is a life-saving measure in MPS IH. However, because a suitable hematopoietic donor is not found for everyone, because HCT is associated with significant morbidity and mortality, and because there is no known benefit of immune reaction between the host and the donor cells in MPS IH, gene-corrected autologous stem cells may be the ideal graft for HCT. Thus, we generated induced pluripotent stem cells from 2 patients with MPS IH (MPS-iPS cells). We found that α-L-iduronidase was not required for stem cell renewal, and that MPS-iPS cells showed lysosomal storage characteristic of MPS IH and could be differentiated to both hematopoietic and nonhematopoietic cells. The specific epigenetic profile associated with de-differentiation of MPS IH fibroblasts into MPS-iPS cells was maintained when MPS-iPS cells are gene-corrected with virally delivered α-L-iduronidase. These data underscore the potential of MPS-iPS cells to generate autologous hematopoietic grafts devoid of immunologic complications of allogeneic transplantation, as well as generating nonhematopoietic cells with the potential to treat anatomical sites not fully corrected with HCT.
Assuntos
Diferenciação Celular , Sistema Hematopoético/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Pré-Escolar , Metilação de DNA , Células HEK293 , Sistema Hematopoético/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Iduronidase/genética , Iduronidase/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Queratinócitos/citologia , Queratinócitos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Mucopolissacaridose I/genética , Mucopolissacaridose I/metabolismo , Mucopolissacaridose I/patologia , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , TransfecçãoRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.
Assuntos
Epidermólise Bolhosa Distrófica , Genes Recessivos , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/terapia , Epigênese Genética/fisiologia , Fibroblastos/citologia , Humanos , Técnicas In Vitro , Queratinócitos/citologia , Medicina de PrecisãoRESUMO
The recessive dystrophic form of epidermolysis bullosa (RDEB) is a disorder of incurable skin fragility and blistering caused by mutations in the type VII collagen gene (Col7a1). The absence of type VII collagen production leads to the loss of adhesion at the basement membrane zone due to the absence of anchoring fibrils, which are composed of type VII collagen. We report that wild-type, congenic bone marrow cells homed to damaged skin, produced type VII collagen protein and anchoring fibrils, ameliorated skin fragility, and reduced lethality in the murine model of RDEB generated by targeted Col7a1 disruption. These data provide the first evidence that a population of marrow cells can correct the basement membrane zone defect found in mice with RDEB and offer a potentially valuable approach for treatment of human RDEB and other extracellular matrix disorders.
Assuntos
Membrana Basal/metabolismo , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Epidermólise Bolhosa Distrófica/terapia , Animais , Membrana Basal/patologia , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Modelos Animais de Doenças , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/metabolismo , Epidermólise Bolhosa Distrófica/patologia , Humanos , Camundongos , Camundongos Knockout , MutaçãoRESUMO
OBJECTIVE: Multipotent adult progenitor cells (MAPCs) are adult stem cells derived from bone marrow. We investigated the capacity of MAPCs to aid in tissue healing after myocardial ischemia in mice with different levels of immune competence. METHODS: Adult murine C57BL/6 MAPCs were labeled with firefly luciferase and DsRed2 fluorescent protein and injected into the myocardium of immunocompetent C57BL/6 or T-, B- and natural killer-cell severe combined immunodeficient C57BL/6 Rag2/IL-2Rgammac(-/-) mice at the time of myocardial infarction (MI). Mice were sequentially analyzed using in vivo whole body bioluminescent imaging for MAPC persistence and high-resolution ultrasound biomicroscopy to assess cardiac function. RESULTS: Luciferase signals emitted from donor MAPCs were significantly higher in Rag2/IL-2Rgammac(-/-) mice compared with C57BL/6 recipients of labeled MAPCs. At 100, 200, and 365 days after MI, left ventricular contractile function was significantly improved (and normalized) in C57BL/6 MAPC recipients. In contrast, despite a greater degree of MAPC persistence compared with C57BL/6 recipients, no cardiac improvement occurred in Rag2/IL-2Rgammac(-/-) recipients of MAPCs. The improved cardiac contractile performance in response to syngeneic MAPC infusion correlated with a prominent increase of vascular density in infarcted and peri-infarcted myocardium, which was dependent upon host immune competency. CONCLUSION: These data indicate that immune competence of the recipient modulates the therapeutic impact of the adult nonhematopoietic stem cells infused after acute MI injury and that a more vigorous immune response is advantageous for therapeutic myocardial repair after MI.
Assuntos
Células-Tronco Adultas , Transplante de Células , Isquemia Miocárdica/terapia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/imunologiaRESUMO
To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon-related genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.
Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/patologia , Sarcoma/patologia , Animais , Diferenciação Celular , Transformação Celular Neoplásica , Células Cultivadas , Células Clonais , Extremidades/patologia , Cariotipagem , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Pulmão/fisiopatologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Sarcoma/genética , Imagem Corporal TotalRESUMO
Multipotent adult progenitor cells (MAPCs) are marrow-derived pluripotent stem cells with a broad differentiation potential. We sought to identify factors that affect adoptively transferred MAPCs. In vitro, MAPCs expressed low levels of major histocompatibility complex (MHC) antigens, failed to stimulate CD4(+) and CD8(+) T-cell alloresponses, and were targets of NK cytolysis. To study in vivo biodistribution, we labeled MAPCs with luciferase for sequential quantification of bioluminescence and DsRed2 for immunohistochemical analysis. C57BL /6 MAPCs were infused intravenously into C57BL /6, Rag-2(-/-) (T- and B-cell-deficient), and Rag-2(-/-)/IL-2Rgamma(c)(-/-) (T-, B-, and NK-cell-deficient) mice. In C57BL /6 mice, MAPCs were transiently detected only in the chest compared with long-term persistence in T- and B-cell-deficient mice. NK depletion reduced MAPC elimination. Because the lungs were the major uptake site after intravenous injection, intra-arterial injections were tested and found to result in more widespread biodistribution. Widespread MAPC biodistribution and long-term persistence were seen in irradiated recipients given allogeneic marrow and MAPCs; such MAPCs expressed MHC class I antigens in tissues. Our data indicate that the biodistribution and persistence of reporter gene-labeled MAPCs are maximized after intra-arterial delivery or host irradiation and that T cells, B cells, and NK cells contribute to in vivo MAPC rejection.
Assuntos
Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Transplante de Medula Óssea , Sobrevivência Celular , Células Matadoras Naturais/imunologia , Teste de Cultura Mista de Linfócitos , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Células-Tronco/imunologia , Linfócitos T/imunologia , Irradiação Corporal TotalRESUMO
The 2A-like sequences from members of the picornavirus family were utilized to construct a tricistronic vector bearing the human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes. The 2A-like sequences mediate a cotranslational cleavage event resulting in the release of each individual protein product. Efficient cleavage was observed and all three proteins were functional in vitro and in vivo, allowing for supratherapeutic IDUA enzyme levels and the coexpression of luciferase and DsRed2 expression, which enabled us to track gene expression.