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1.
Nature ; 630(8016): 412-420, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38839950

ABSTRACT

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.


Subject(s)
Cell Self Renewal , Hematopoietic Stem Cells , Nuclear Proteins , Animals , Female , Humans , Male , Mice , Cells, Cultured , Endocytosis , Endosomes/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Fetal Blood/cytology , Gene Knockdown Techniques , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Liver/cytology , Liver/metabolism , Liver/embryology , Mitochondria/metabolism , Nuclear Proteins/metabolism , Signal Transduction , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , Single-Cell Gene Expression Analysis
2.
Sci Transl Med ; 16(733): eadh8162, 2024 Feb 07.
Article in English | MEDLINE | ID: mdl-38324638

ABSTRACT

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.


Subject(s)
Gene Editing , Hematopoietic Stem Cell Transplantation , Animals , Humans , Mice , Exons , Gene Editing/methods , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism
3.
Nat Biomed Eng ; 2023 Dec 07.
Article in English | MEDLINE | ID: mdl-38062131

ABSTRACT

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.
Article in English | MEDLINE | ID: mdl-37192158

ABSTRACT

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.


Subject(s)
Neoplasms , RNA Precursors , Male , Humans , RNA Precursors/metabolism , Alternative Splicing , Leukocytes, Mononuclear/metabolism , Receptors, Antigen, T-Cell , Epitopes, T-Lymphocyte , Immunotherapy , Antigens, Neoplasm , Peptides/metabolism , Neoplasms/genetics , Neoplasms/therapy
5.
Cell Rep ; 42(3): 112241, 2023 03 28.
Article in English | MEDLINE | ID: mdl-36906850

ABSTRACT

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.


Subject(s)
Pluripotent Stem Cells , T-Lymphocytes , Immunity, Innate , Lymphocytes/metabolism , Pluripotent Stem Cells/metabolism , Cell Differentiation , Immunotherapy, Adoptive/methods , Antigens, CD19 , Receptors, Antigen, T-Cell/metabolism
6.
Cell ; 186(7): 1398-1416.e23, 2023 03 30.
Article in English | MEDLINE | ID: mdl-36944331

ABSTRACT

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.


Subject(s)
Severe Combined Immunodeficiency , T-Lymphocytes , Humans , Animals , Mice , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/therapy , Gene Editing , Mice, SCID , CD3 Complex , Receptors, Antigen, T-Cell/genetics
7.
Proc Natl Acad Sci U S A ; 119(31): e2203410119, 2022 08 02.
Article in English | MEDLINE | ID: mdl-35878026

ABSTRACT

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.


Subject(s)
Acid Phosphatase , Antigens, Neoplasm , Receptors, Antigen, T-Cell , Acid Phosphatase/metabolism , Antigens, Neoplasm/metabolism , Epitopes , HLA-A Antigens/metabolism , HLA-A2 Antigen , Humans , Leukocytes, Mononuclear , Neoplasms/immunology , Peptides , Receptors, Antigen, T-Cell/metabolism
8.
J Clin Invest ; 132(16)2022 08 15.
Article in English | MEDLINE | ID: mdl-35653193

ABSTRACT

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.


Subject(s)
Immunologic Deficiency Syndromes , Purine-Nucleoside Phosphorylase , Animals , Autoimmunity , Humans , Mice , Purine Nucleosides , Purine-Nucleoside Phosphorylase/genetics , Purine-Nucleoside Phosphorylase/metabolism , T-Lymphocytes , Toll-Like Receptor 7
9.
Rev. clín. esp. (Ed. impr.) ; 222(5): 266-271, Mayo 2022.
Article in Spanish | IBECS | ID: ibc-204736

ABSTRACT

Antecedentes: El propósito de este estudio fue conocer la práctica clínica de especialistas que atienden a pacientes con arteritis de células gigantes, para comprobar si siguen las recomendaciones para el diagnóstico y tratamiento de esta enfermedad e identificar áreas de mejora. Métodos: Encuesta transversal de prácticas clínicas realizada en 2019. Ciento sesenta y siete médicos (64% reumatólogos, 27% especialistas en medicina interna, 9% otros especialistas) que asistieron a un curso de actualización del tratamiento de la arteritis de células gigantes completó la encuesta. Comparamos la práctica clínica recogida en el estudio con las últimas recomendaciones aprobadas por la Liga Europea Contra el Reumatismo (EULAR). Resultados: Los médicos encuestados atendían a una mediana de 10 pacientes (rango intercuartílico 6-30) con arteritis de células gigantes en su práctica clínica. Como método de diagnóstico, los encuestados utilizaron biopsia de arteria temporal (84%), ecografía de arteria temporal (61%) u otras técnicas de imagen (37%). Como terapia de primera línea, los encuestados utilizaron glucocorticoides en dosis altas (al menos 40mg de prednisona o equivalente por día) (84%), glucocorticoides con metotrexato (7%) y glucocorticoides con tocilizumab (5%). Los fármacos más utilizados para la recaída fueron el metotrexato (37%) y tocilizumab (58%). Conclusión: Los resultados de esta encuesta indican que los médicos especialistas encuestados siguen las recomendaciones recientes de EULAR sobre diagnóstico y tratamiento de la arteritis de células gigantes (AU)


Background: The purpose of this study was to learn about the clinical practice of specialists who care for patients with giant cell arteritis, to verify whether they follow the diagnosis and treatment recommendations for this disease, and to identify areas for improvement. Methods: A cross-sectional survey on clinical practice in 2019. The survey was completed by 167 physicians (64% rheumatologists, 27% internal medicine specialists, and 9% other specialists) who attended a course on updating giant cell arteritis treatment. We compared the clinical practice collected in the study with the latest recommendations approved by the European League Against Rheumatism (EULAR). Results: The physicians surveyed cared for a median of 10 patients (interquartile range 6-30) with giant cell arteritis during their practice. As a diagnostic method, respondents used temporal artery biopsy (84%), temporal artery ultrasound (61%) or other imaging techniques (37%). As first-line therapy, respondents used high-dose glucocorticoids (at least 40mg of prednisone, or equivalent, per day) (84%), glucocorticoids with methotrexate (7%) and glucocorticoids with tocilizumab (5%). The most frequent drugs used for relapse were methotrexate (37%) and tocilizumab (58%). Conclusion: Our results indicate that the medical specialists surveyed follow the recent EULAR recommendations for giant cell arteritis diagnosis and therapy (AU)


Subject(s)
Humans , Practice Patterns, Physicians' , Giant Cell Arteritis , Cross-Sectional Studies , Surveys and Questionnaires , Giant Cell Arteritis/diagnosis , Giant Cell Arteritis/drug therapy , Glucocorticoids/therapeutic use , Methotrexate/therapeutic use
11.
Curr Protoc ; 2(4): e403, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35384408

ABSTRACT

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.


Subject(s)
Hematopoietic Stem Cells , Organoids , Animals , Cell Differentiation , Hematopoiesis , Humans , Mice , T-Lymphocytes
12.
Cell Stem Cell ; 29(4): 515-527.e8, 2022 04 07.
Article in English | MEDLINE | ID: mdl-35278370

ABSTRACT

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.


Subject(s)
Induced Pluripotent Stem Cells , Receptors, Chimeric Antigen , Animals , Cell Differentiation , Humans , Immunotherapy , Immunotherapy, Adoptive/methods , Induced Pluripotent Stem Cells/metabolism , Mice , Organoids/metabolism , Receptors, Chimeric Antigen/metabolism
13.
Front Immunol ; 13: 833636, 2022.
Article in English | MEDLINE | ID: mdl-35185925

ABSTRACT

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.


Subject(s)
Interferon Type I/physiology , Interleukins/physiology , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic/metabolism , STAT1 Transcription Factor/metabolism , Adult , Female , Flow Cytometry , Humans , Interferon Type I/analysis , Interleukins/analysis , Male , Middle Aged , Phosphorylation , Signal Transduction , Single-Cell Analysis
14.
Rev Clin Esp (Barc) ; 222(5): 266-271, 2022 05.
Article in English | MEDLINE | ID: mdl-34620581

ABSTRACT

BACKGROUND: The purpose of this study was to learn about the clinical practice of specialists who care for patients with giant cell arteritis, to verify whether they follow the diagnosis and treatment recommendations for this disease, and to identify areas for improvement. METHODS: A cross-sectional survey on clinical practice in 2019. The survey was completed by 167 physicians (64% rheumatologists, 27% internal medicine specialists, and 9% other specialists) who attended a course on updating giant cell arteritis treatment. We compared the clinical practice collected in the study with the latest recommendations approved by the European League Against Rheumatism (EULAR). RESULTS: The physicians surveyed cared for a median of 10 patients (interquartile range 6-30) with giant cell arteritis during their practice. As a diagnostic method, respondents used temporal artery biopsy (84%), temporal artery ultrasound (61%) or other imaging techniques (37%). As first-line therapy, respondents used high-dose glucocorticoids (at least 40 mg of prednisone, or equivalent, per day) (84%), glucocorticoids with methotrexate (7%) and glucocorticoids with tocilizumab (5%). The most frequent drugs used for relapse were methotrexate (37%) and tocilizumab (58%). CONCLUSION: Our results indicate that the medical specialists surveyed follow the recent EULAR recommendations for giant cell arteritis diagnosis and therapy.


Subject(s)
Giant Cell Arteritis , Cross-Sectional Studies , Giant Cell Arteritis/diagnosis , Giant Cell Arteritis/drug therapy , Glucocorticoids/therapeutic use , Humans , Methotrexate/therapeutic use , Surveys and Questionnaires
15.
Cell Rep Med ; 2(11): 100449, 2021 11 16.
Article in English | MEDLINE | ID: mdl-34841295

ABSTRACT

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.


Subject(s)
Allogeneic Cells/immunology , Cell Engineering , Hematopoietic Stem Cells/immunology , Immunotherapy , Natural Killer T-Cells/immunology , Neoplasms/immunology , Neoplasms/therapy , Allogeneic Cells/metabolism , Animals , Cell Line, Tumor , Gene Expression Profiling , HLA Antigens/metabolism , Hematopoietic Stem Cells/metabolism , Humans , Mice, Inbred NOD , Mice, SCID , Natural Killer T-Cells/metabolism , Phenotype , Receptors, Chimeric Antigen/metabolism , Transcriptome/genetics
16.
Front Immunol ; 12: 716661, 2021.
Article in English | MEDLINE | ID: mdl-34394122

ABSTRACT

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.


Subject(s)
Cell Differentiation , Energy Metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Thymocytes/cytology , Thymocytes/metabolism , Animals , Biological Evolution , Biomarkers , Cell Line , Computational Biology/methods , Gene Expression Profiling , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Humans , Lymphopoiesis , Metabolome , Metabolomics/methods , Mice , Organoids , Thymocytes/immunology , Tissue Culture Techniques
17.
J Med Radiat Sci ; 68(4): 426-434, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34263548

ABSTRACT

INTRODUCTION: Research indicates radiation therapy students are at risk of burnout. Peer Group Supervision (PGS) is a tool used to help reduce stress, increase reflective practice and help manage professional issues. This pilot study aimed to investigate the third-year New Zealand radiation therapy students' perceptions of participating in PGS. METHODS: In 2019, all 27 third-year radiation therapy students were introduced to PGS. At the end of the year, the students were invited to fill in a 14-item Clinical Supervision Evaluation Questionnaire (CSEQ), answer an open-ended question and provide demographic data. The CSEQ asks participants to indicate the extent to which they agree with 14 statements related to Purpose, Process and Impact of PGS. The open-ended question asked if there were anything else they would like to say about participating in PGS as a student. The study utilised both qualitative and quantitative methods. RESULTS: Of the 27 students invited, 22 responded to the questionnaire. Analysis of the CSEQ revealed that eight of the 22 students (36.4%) had a positive impression of PGS, 13 (59.1%) were neither positive nor negative, and one (4.6%) had a negative perception of PGS. The thematic data showed that the students perceived PGS to assist with stress management. They valued having scheduled time out to reflect on practice and appreciated the safety and trust established in the groups. CONCLUSION: Overall, the radiation therapy students responded positively to PGS. The students felt safe talking about clinical issues in their groups, and they perceive PGS to positively affect their stress management, resulting in new clinical insights and increased self-awareness. Further research is required to examine the long-term effects of PGS on patient care and if PGS can help reduce burnout for student radiation therapists.


Subject(s)
Peer Group , Students , Humans , Perception , Pilot Projects , Surveys and Questionnaires
18.
Semergen ; 47(4): 256-266, 2021.
Article in Spanish | MEDLINE | ID: mdl-34112594

ABSTRACT

Giant cell arteritis is a systemic vasculitis with significant intra and extracranial involvement that, with early diagnosis and treatment in primary care, can improve its prognosis as it is a medical emergency. Our working group on vascular diseases of the Spanish Society of Primary Care Physicians (SEMERGEN) proposes a series of recommendations based on current scientific evidence for a multidisciplinary approach and follow-up in primary care.


Subject(s)
Giant Cell Arteritis , Physicians, Primary Care , Humans , Primary Health Care , Prognosis
19.
N Engl J Med ; 384(21): 2002-2013, 2021 05 27.
Article in English | MEDLINE | ID: mdl-33974366

ABSTRACT

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.).


Subject(s)
Agammaglobulinemia/therapy , Genetic Therapy/methods , Genetic Vectors , Hematopoietic Stem Cell Transplantation , Lentivirus/genetics , Severe Combined Immunodeficiency/therapy , Adenosine Deaminase/deficiency , Adolescent , Child , Child, Preschool , Genetic Therapy/adverse effects , Humans , Infant , Lymphocyte Count , Progression-Free Survival , Prospective Studies , Transplantation, Autologous
20.
Adv Sci (Weinh) ; 8(4): 2002500, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33643791

ABSTRACT

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.

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