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1.
Nature ; 630(8016): 412-420, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38839950

RESUMO

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.


Assuntos
Autorrenovação Celular , Células-Tronco Hematopoéticas , Proteínas Nucleares , Animais , Feminino , Humanos , Masculino , Camundongos , Células Cultivadas , Endocitose , Endossomos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Sangue Fetal/citologia , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/embriologia , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Análise da Expressão Gênica de Célula Única
2.
Sci Transl Med ; 16(733): eadh8162, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38324638

RESUMO

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.


Assuntos
Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Animais , Humanos , Camundongos , Éxons , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo
3.
Nat Biomed Eng ; 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-38062131

RESUMO

Pluripotent stem cells (PSCs) are a promising source of allogeneic T cells for off-the-shelf immunotherapies. However, the process of differentiating genetically engineered PSCs to generate mature T cells requires that the same molecular elements that are crucial for the selection of these cells be removed to prevent alloreactivity. Here we show that antigen-restricted mature T cells can be generated in vitro from PSCs edited via CRISPR to lack endogenous T cell receptors (TCRs) and class I major histocompatibility complexes. Specifically, we used T cell precursors from RAG1-/-RAG2-/-B2M-/- human PSCs expressing a single TCR, and a murine stromal cell line providing the cognate human major histocompatibility complex molecule and other critical signals for T cell maturation. Possibly owing to the absence of TCR mispairing, the generated T cells showed substantially better tumour control in mice than T cells with an intact endogenous TCR. Introducing the T cell selection components into the stromal microenvironment of the PSCs overcomes inherent biological challenges associated with the development of T cell immunotherapies from allogeneic PSCs.

4.
Proc Natl Acad Sci U S A ; 120(21): e2221116120, 2023 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-37192158

RESUMO

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.


Assuntos
Neoplasias , Precursores de RNA , Masculino , Humanos , Precursores de RNA/metabolismo , Processamento Alternativo , Leucócitos Mononucleares/metabolismo , Receptores de Antígenos de Linfócitos T , Epitopos de Linfócito T , Imunoterapia , Antígenos de Neoplasias , Peptídeos/metabolismo , Neoplasias/genética , Neoplasias/terapia
5.
Cell Rep ; 42(3): 112241, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36906850

RESUMO

Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. Understanding how CARs affect T cell differentiation from PSCs is important for this effort. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of PSCs to T cells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of T cell differentiation to the innate lymphoid cell 2 (ILC2) lineage in ATOs. T cells and ILC2s are closely related lymphoid lineages with shared developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during lymphoid development enriched for ILC2-primed precursors at the expense of T cell precursors. We applied this understanding to modulate CAR signaling strength through expression level, structure, and presentation of cognate antigen to demonstrate that the T cell-versus-ILC lineage decision can be rationally controlled in either direction, providing a framework for achieving CAR-T cell development from PSCs.


Assuntos
Células-Tronco Pluripotentes , Linfócitos T , Imunidade Inata , Linfócitos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Imunoterapia Adotiva/métodos , Antígenos CD19 , Receptores de Antígenos de Linfócitos T/metabolismo
6.
Cell ; 186(7): 1398-1416.e23, 2023 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-36944331

RESUMO

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.


Assuntos
Imunodeficiência Combinada Severa , Linfócitos T , Humanos , Animais , Camundongos , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Edição de Genes , Camundongos SCID , Complexo CD3 , Receptores de Antígenos de Linfócitos T/genética
7.
Proc Natl Acad Sci U S A ; 119(31): e2203410119, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35878026

RESUMO

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.


Assuntos
Fosfatase Ácida , Antígenos de Neoplasias , Receptores de Antígenos de Linfócitos T , Fosfatase Ácida/metabolismo , Antígenos de Neoplasias/metabolismo , Epitopos , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Humanos , Leucócitos Mononucleares , Neoplasias/imunologia , Peptídeos , Receptores de Antígenos de Linfócitos T/metabolismo
8.
J Clin Invest ; 132(16)2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35653193

RESUMO

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.


Assuntos
Síndromes de Imunodeficiência , Purina-Núcleosídeo Fosforilase , Animais , Autoimunidade , Humanos , Camundongos , Nucleosídeos de Purina , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Linfócitos T , Receptor 7 Toll-Like
10.
Curr Protoc ; 2(4): e403, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35384408

RESUMO

The generation of T cells is a complex, carefully orchestrated process that occurs in the thymus. The ability to mimic T cell differentiation in vitro has opened up avenues to better understand different stages of thymopoiesis but has also enabled the in vitro production of mature T cells suitable for immunotherapy. Among existing protocols, the artificial thymic organoid (ATO) system has been shown to be the most efficient at producing mature conventional T cells. In this serum-free model, human or murine hematopoietic stem and progenitor cells (HSPCs) are combined with a murine stromal cell line expressing a Notch ligand in a 3D cell aggregate. In ATOs, although only simple medium changes are required throughout the cultures, HSPCs differentiate into T cells with kinetics and phenotypes similar to those of endogenous thymopoiesis. This article describes protocols for the generation of ATOs from human and murine HSPCs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expansion and preparation of MS5-hDLL4 or MS5-mDLL4 cells Basic Protocol 2: Isolation of human hematopoietic stem and progenitor cells (HSPCs; CD34+ cells) Support Protocol 1: Transduction of human HSPCs (CD34+ cells) Basic Protocol 3: Production of thymic progenitors and mature T cells from human HSPCs in artificial thymic organoids (ATOs) Support Protocol 2: Phenotype analysis of human ATO cells by flow cytometry Basic Protocol 4: Isolation of murine HSPCs (Lin- Sca1+ cKit+; LSK) and hematopoietic stem cells (LSK CD150+ CD48-) Basic Protocol 5: Production of thymic progenitors and mature T cells from murine HSPCs in ATOs Support Protocol 3: Phenotype analysis of murine ATO cells by flow cytometry Alternate Protocol: Generation of ATOs from single HSPCs.


Assuntos
Células-Tronco Hematopoéticas , Organoides , Animais , Diferenciação Celular , Hematopoese , Humanos , Camundongos , Linfócitos T
11.
Cell Stem Cell ; 29(4): 515-527.e8, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35278370

RESUMO

Unlimited generation of chimeric antigen receptor (CAR) T cells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR T cell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αß T cell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory T cells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αß-positive CAR T cell characteristics. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR T cells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also mediated potent antitumor activity in vivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR T cells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.


Assuntos
Células-Tronco Pluripotentes Induzidas , Receptores de Antígenos Quiméricos , Animais , Diferenciação Celular , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Organoides/metabolismo , Receptores de Antígenos Quiméricos/metabolismo
12.
Front Immunol ; 13: 833636, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35185925

RESUMO

The establishment of an "interferon (IFN) signature" to subset SLE patients on disease severity has led to therapeutics targeting IFNα. Here, we investigate IFN signaling in SLE using multiplexed protein arrays and single cell cytometry by time of flight (CyTOF). First, the IFN signature for SLE patients (n=81) from the Stanford Lupus Registry is determined using fluidigm qPCR measuring 44 previously determined IFN-inducible transcripts. IFN-high (IFN-H) patients have increased SLE criteria and renal/CNS/immunologic involvement, and increased autoantibody reactivity against spliceosome-associated antigens. CyTOF analysis is performed on non-stimulated and stimulated (IFNα, IFNγ, IL-21) PBMCs from SLE patients (n=25) and HCs (n=9) in a panel identifying changes in phosphorylation of intracellular signaling proteins (pTOF). Another panel is utilized to detect changes in intracellular cytokine (ICTOF) production in non-stimulated and stimulated (PMA/ionomycin) PBMCs from SLE patients (n=31) and HCs (n=17). Bioinformatic analysis by MetaCyto and OMIQ reveal phenotypic changes in immune cell subsets between IFN-H and IFN-low (IFN-L) patients. Most notably, IFN-H patients exhibit increased STAT1/3/5 phosphorylation downstream of cytokine stimulation and increased phosphorylation of non-canonical STAT proteins. These results suggest that IFN signaling in SLE modulates STAT phosphorylation, potentially uncovering possible targets for future therapeutic approaches.


Assuntos
Interferon Tipo I/fisiologia , Interleucinas/fisiologia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Fator de Transcrição STAT1/metabolismo , Adulto , Feminino , Citometria de Fluxo , Humanos , Interferon Tipo I/análise , Interleucinas/análise , Masculino , Pessoa de Meia-Idade , Fosforilação , Transdução de Sinais , Análise de Célula Única
13.
Cell Rep Med ; 2(11): 100449, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34841295

RESUMO

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.


Assuntos
Células Alógenas/imunologia , Engenharia Celular , Células-Tronco Hematopoéticas/imunologia , Imunoterapia , Células T Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Células Alógenas/metabolismo , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Antígenos HLA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Células T Matadoras Naturais/metabolismo , Fenótipo , Receptores de Antígenos Quiméricos/metabolismo , Transcriptoma/genética
14.
Front Immunol ; 12: 716661, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394122

RESUMO

Although metabolic pathways have been shown to control differentiation and activation in peripheral T cells, metabolic studies on thymic T cell development are still lacking, especially in human tissue. In this study, we use transcriptomics and extracellular flux analyses to investigate the metabolic profiles of primary thymic and in vitro-derived mouse and human thymocytes. Core metabolic pathways, specifically glycolysis and oxidative phosphorylation, undergo dramatic changes between the double-negative (DN), double-positive (DP), and mature single-positive (SP) stages in murine and human thymus. Remarkably, despite the absence of the complex multicellular thymic microenvironment, in vitro murine and human T cell development recapitulated the coordinated decrease in glycolytic and oxidative phosphorylation activity between the DN and DP stages seen in primary thymus. Moreover, by inducing in vitro T cell differentiation from Rag1-/- mouse bone marrow, we show that reduced metabolic activity at the DP stage is independent of TCR rearrangement. Thus, our findings suggest that highly conserved metabolic transitions are critical for thymic T cell development.


Assuntos
Diferenciação Celular , Metabolismo Energético , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Animais , Evolução Biológica , Biomarcadores , Linhagem Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfopoese , Metaboloma , Metabolômica/métodos , Camundongos , Organoides , Timócitos/imunologia , Técnicas de Cultura de Tecidos
15.
N Engl J Med ; 384(21): 2002-2013, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33974366

RESUMO

BACKGROUND: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODS: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTS: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONS: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).


Assuntos
Agamaglobulinemia/terapia , Terapia Genética/métodos , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Lentivirus/genética , Imunodeficiência Combinada Severa/terapia , Adenosina Desaminase/deficiência , Adolescente , Criança , Pré-Escolar , Terapia Genética/efeitos adversos , Humanos , Lactente , Contagem de Linfócitos , Intervalo Livre de Progressão , Estudos Prospectivos , Transplante Autólogo
16.
Adv Sci (Weinh) ; 8(4): 2002500, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33643791

RESUMO

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet ß-cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic ß-cells via the αvß3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.

17.
Cell Rep ; 33(4): 108320, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33113379

RESUMO

We report a serum-free, 3D murine artificial thymic organoid (M-ATO) system that mimics normal murine thymopoiesis with the production of all T cell stages, from early thymic progenitors to functional single-positive (CD8SP and CD4SP) TCRαß and TCRγδ cells. RNA sequencing aligns M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATOs initiated with Rag1-/- marrow produce the same differentiation block as seen in the endogenous thymus, and Notch signaling patterns in M-ATOs mirror primary thymopoiesis. M-ATOs initiated with defined hematopoietic stem cells (HSCs) and lymphoid progenitors from marrow and thymus generate each of the downstream differentiation stages, allowing the kinetics of T cell differentiation to be tracked. Remarkably, single HSCs deposited into each M-ATO generate the complete trajectory of T cell differentiation, producing diverse TCR repertoires across clones that largely match endogenous thymus. M-ATOs represent a highly reproducible and efficient experimental platform for the interrogation of clonal thymopoiesis from HSCs.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Timo/fisiologia , Animais , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Camundongos
18.
J Immunol ; 205(9): 2423-2436, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32989093

RESUMO

Neonatal life marks the apogee of murine thymic growth. Over the first few days after birth, growth slows and the murine thymus switches from fetal to adult morphology and function; little is known about the cues driving this dramatic transition. In this study, we show for the first time (to our knowledge) the critical role of vascular endothelial growth factor (VEGF) on thymic morphogenesis beyond its well-known role in angiogenesis. During a brief window a few days after birth, VEGF inhibition induced rapid and profound remodeling of the endothelial, mesenchymal and epithelial thymic stromal compartments, mimicking changes seen during early adult maturation. Rapid transcriptional changes were seen in each compartment after VEGF inhibition, including genes involved in migration, chemotaxis, and cell adhesion as well as induction of a proinflammatory and proadipogenic signature in endothelium, pericytes, and mesenchyme. Thymocyte numbers fell subsequent to the stromal changes. Expression patterns and functional blockade of the receptors VEGFR2 and NRP1 demonstrated that VEGF mediates its pleiotropic effects through distinct receptors on each microenvironmental compartment of the developing mouse thymus.


Assuntos
Timo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Endotélio/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Pericitos/metabolismo , Timócitos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
19.
Blood Adv ; 4(12): 2611-2616, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32556283

RESUMO

The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαß+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.


Assuntos
Células-Tronco Hematopoéticas , Linfopenia , Antígenos CD34 , Diferenciação Celular , Humanos , Organoides
20.
Nature ; 576(7786): 281-286, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31776511

RESUMO

Limited knowledge of the mechanisms that govern the self-renewal of human haematopoietic stem cells (HSCs), and why this fails in culture, have impeded the expansion of HSCs for transplantation1. Here we identify MLLT3 (also known as AF9) as a crucial regulator of HSCs that is highly enriched in human fetal, neonatal and adult HSCs, but downregulated in culture. Depletion of MLLT3 prevented the maintenance of transplantable human haematopoietic stem or progenitor cells (HSPCs) in culture, whereas stabilizing MLLT3 expression in culture enabled more than 12-fold expansion of transplantable HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Similar to endogenous MLLT3, overexpressed MLLT3 localized to active promoters in HSPCs, sustained levels of H3K79me2 and protected the HSC transcriptional program in culture. MLLT3 thus acts as HSC maintenance factor that links histone reader and modifying activities to modulate HSC gene expression, and may provide a promising approach to expand HSCs for transplantation.


Assuntos
Autorrenovação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação Proteica
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