Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 30
Filter
1.
Nat Biomed Eng ; 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38858586

ABSTRACT

Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites, PASSIGE with eeBxb1 led to an average targeted-gene-integration efficiencies of 23% (4.2-fold that of wild-type Bxb1). Notably, integration efficiencies exceeded 30% at multiple sites in primary human fibroblasts. PASSIGE with evoBxb1 or eeBxb1 outperformed PASTE (for 'programmable addition via site-specific targeting elements', a method that uses prime editors fused to recombinases) on average by 9.1-fold and 16-fold, respectively. PASSIGE with continuously evolved recombinases is an unusually efficient method for the targeted integration of genes in mammalian cells.

2.
J Reprod Immunol ; 163: 104244, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38555747

ABSTRACT

Implantation and maintenance of pregnancy involve intricate immunological processes that enable the developing fetus to coexist with the maternal immune system. Progesterone, a critical hormone during pregnancy, is known to promote immune tolerance and prevent preterm labor. However, the mechanism by which progesterone mediates these effects remains unclear. In this study, we investigated the role of the non-classical progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling at the maternal-fetal interface. Using JEG3 cells, a trophoblast model cell line, we observed that progesterone stimulation increased the expression of human leukocyte antigen-C (HLA-C) and HLA-G, key molecules involved in immune tolerance. We also found that progesterone upregulated the expression of the transcription factor ELF3, which is known to regulate trophoblast-specific HLA-C expression. Interestingly, JEG3 cells lacked expression of classical progesterone receptors (PRs) but exhibited high expression of PGRMC1, a finding we confirmed in primary trophoblasts by mining sc-RNA seq data from human placenta. To investigate the role of PGRMC1 in progesterone signaling, we used CRISPR/Cas9 technology to knockout PGRMC1 in JEG3 cells. PGRMC1-deficient cells showed a diminished response to progesterone stimulation. Furthermore, we found that the progesterone antagonist RU486 inhibited ELF3 expression in a PGRMC1-dependent manner, suggesting that RU486 acts as a progesterone antagonist by competing for receptor binding. Additionally, we found that RU486 inhibited cell invasion, an important process for successful pregnancy, and this inhibitory effect was dependent on PGRMC1. Our findings highlight the crucial role of PGRMC1 in mediating the immunoregulatory effects of progesterone at the maternal-fetal interface.


Subject(s)
Membrane Proteins , Progesterone , Receptors, Progesterone , Trophoblasts , Humans , Receptors, Progesterone/metabolism , Female , Pregnancy , Progesterone/metabolism , Progesterone/pharmacology , Membrane Proteins/metabolism , Membrane Proteins/genetics , Trophoblasts/metabolism , Trophoblasts/immunology , Placenta/immunology , Placenta/metabolism , Signal Transduction/immunology , Maternal-Fetal Exchange/immunology , Embryo Implantation/immunology
3.
Curr Stem Cell Rep ; 8(4): 206-218, 2022.
Article in English | MEDLINE | ID: mdl-36406259

ABSTRACT

Purpose of Review: Human pluripotent stem cells have the potential to revolutionize the treatment of inborn and degenerative diseases, including aging and autoimmunity. A major barrier to their wider adoption in cell therapies is immune rejection. Genome editing allows for tinkering of the human genome in stem and progenitor cells and raises the prospect for overcoming the immune barriers to transplantation. Recent Findings: Initial attempts have focused primarily on the major histocompatibility barrier that is formed by the human leukocyte antigens (HLA). More recently, immune checkpoint inhibitors, such as PD-L1, CD47, or HLA-G, are being explored both, in the presence or absence of HLA, to mitigate immune rejection by the various cellular components of the immune system. Summary: In this review, we discuss progress in surmounting immune barriers to cell transplantation, with a particular focus on genetic engineering of human pluripotent stem and progenitor cells and the therapeutic cell types derived from them.

4.
Front Immunol ; 12: 655122, 2021.
Article in English | MEDLINE | ID: mdl-34408743

ABSTRACT

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.


Subject(s)
Gene Editing/methods , Interleukin-2 Receptor alpha Subunit/genetics , Receptors, Interleukin-6/genetics , T-Lymphocytes, Regulatory/metabolism , Blood Buffy Coat/cytology , CRISPR-Cas Systems/genetics , Forkhead Transcription Factors/metabolism , Gene Knockdown Techniques , HEK293 Cells , Healthy Volunteers , Humans , Immunotherapy, Adoptive/methods , Primary Cell Culture , RNA, Guide, Kinetoplastida/genetics , Time Factors
5.
Proc Natl Acad Sci U S A ; 118(9)2021 03 02.
Article in English | MEDLINE | ID: mdl-33622787

ABSTRACT

HLA-C arose during evolution of pregnancy in the great apes 10 to 15 million years ago. It has a dual function on placental extravillous trophoblasts (EVTs) as it contributes to both tolerance and immunity at the maternal-fetal interface. The mode of its regulation is of considerable interest in connection with the biology of pregnancy and pregnancy abnormalities. First-trimester primary EVTs in which HLA-C is highly expressed, as well as JEG3, an EVT model cell line, were employed. Single-cell RNA-seq data and quantitative PCR identified high expression of the transcription factor ELF3 in those cells. Chromatin immunoprecipitation (ChIP)-PCR confirmed that both ELF3 and MED1 bound to the proximal HLA-C promoter region. However, binding of RFX5 to this region was absent or severely reduced, and the adjacent HLA-B locus remained closed. Expression of HLA-C was inhibited by ELF3 small interfering RNAs (siRNAs) and by wrenchnolol treatment. Wrenchnolol is a cell-permeable synthetic organic molecule that mimics ELF3 and is relatively specific for binding to ELF3's coactivator, MED23, as our data also showed in JEG3. Moreover, the ELF3 gene is regulated by a superenhancer that spans more than 5 Mb, identified by assay for transposase-accessible chromatin using sequencing (ATAC-seq), as well as by its sensitivity to (+)-JQ1 (inhibitor of BRD4). ELF3 bound to its own promoter, thus creating an autoregulatory feedback loop that establishes expression of ELF3 and HLA-C in trophoblasts. Wrenchnolol blocked binding of MED23 to ELF3, thus disrupting the positive-feedback loop that drives ELF3 expression, with down-regulation of HLA-C expression as a consequence.


Subject(s)
DNA-Binding Proteins/genetics , Enhancer Elements, Genetic , Feedback, Physiological , HLA-C Antigens/genetics , Proto-Oncogene Proteins c-ets/genetics , Transcription Factors/genetics , Trophoblasts/immunology , Abortion, Legal , Adamantane/pharmacology , Azepines/pharmacology , Cell Line , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/immunology , Female , Gene Expression Regulation, Developmental/immunology , HLA-B Antigens/genetics , HLA-B Antigens/immunology , HLA-C Antigens/immunology , Humans , Immunity, Maternally-Acquired , Indoles/pharmacology , Mediator Complex/genetics , Mediator Complex/immunology , Mediator Complex Subunit 1/genetics , Mediator Complex Subunit 1/immunology , Pregnancy , Pregnancy Trimester, First , Primary Cell Culture , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-ets/antagonists & inhibitors , Proto-Oncogene Proteins c-ets/immunology , RNA, Small Interfering/genetics , RNA, Small Interfering/immunology , Regulatory Factor X Transcription Factors/genetics , Regulatory Factor X Transcription Factors/immunology , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/immunology , Triazoles/pharmacology , Trophoblasts/cytology , Trophoblasts/drug effects
6.
Cell Rep ; 32(2): 107894, 2020 07 14.
Article in English | MEDLINE | ID: mdl-32668238

ABSTRACT

Understanding the root causes of autoimmune diseases is hampered by the inability to access relevant human tissues and identify the time of disease onset. To examine the interaction of immune cells and their cellular targets in type 1 diabetes, we differentiated human induced pluripotent stem cells into pancreatic endocrine cells, including ß cells. Here, we describe an in vitro platform that models features of human type 1 diabetes using stress-induced patient-derived endocrine cells and autologous immune cells. We demonstrate a cell-type-specific response by autologous immune cells against induced pluripotent stem cell-derived ß cells, along with a reduced effect on α cells. This approach represents a path to developing disease models that use patient-derived cells to predict the outcome of an autoimmune response.


Subject(s)
Diabetes Mellitus, Type 1/pathology , Models, Biological , Pluripotent Stem Cells/pathology , Animals , Cytotoxicity, Immunologic , Diabetes Mellitus, Type 1/immunology , Endoplasmic Reticulum Stress , Glucagon-Secreting Cells/pathology , Humans , Insulin-Secreting Cells/pathology , Lymphocyte Activation/immunology , Mice , T-Lymphocytes/immunology
8.
Cell Stem Cell ; 27(1): 147-157.e7, 2020 07 02.
Article in English | MEDLINE | ID: mdl-32413331

ABSTRACT

Although susceptibility to cardiovascular disease (CVD) is different for every patient, why some patients with type 2 diabetes mellitus (T2DM) develop CVD while others are protected has not yet been clarified. Using T2DM-patient-derived human induced pluripotent stem cells (hiPSCs), we found that in patients protected from CVD, there was significantly elevated expression of an esterase, arylacetamide deacetylase (AADAC), in vascular smooth muscle cells (VSMCs). We overexpressed this esterase in human primary VSMCs and VSMCs differentiated from hiPSCs and observed that the number of lipid droplets was significantly diminished. Further metabolomic analyses revealed a marked reduction in storage lipids and an increase in membrane phospholipids, suggesting changes in the Kennedy pathway of lipid bioassembly. Cell migration and proliferation were also significantly decreased in AADAC-overexpressing VSMCs. Moreover, apolipoprotein E (Apoe)-knockout mice overexpressing VSMC-specific Aadac showed amelioration of atherosclerotic lesions. Our findings suggest that higher AADAC expression in VSMCs protects T2DM patients from CVD.


Subject(s)
Atherosclerosis , Diabetes Mellitus, Type 2 , Induced Pluripotent Stem Cells , Animals , Cell Proliferation , Cells, Cultured , Humans , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle
9.
Circ Res ; 126(3): 330-346, 2020 01 31.
Article in English | MEDLINE | ID: mdl-31739742

ABSTRACT

Rationale: Genome-wide association studies have identified genetic loci associated with insulin resistance (IR) but pinpointing the causal genes of a risk locus has been challenging. Objective: To identify candidate causal genes for IR, we screened regional and biologically plausible genes (16 in total) near the top 10 IR-loci in risk-relevant cell types, namely preadipocytes and adipocytes. Methods and Results: We generated 16 human Simpson-Golabi-Behmel syndrome preadipocyte knockout lines each with a single IR-gene knocked out by lentivirus-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. We evaluated each gene knockout by screening IR-relevant phenotypes in the 3 insulin-sensitizing mechanisms, including adipogenesis, lipid metabolism, and insulin signaling. We performed genetic analyses using data on the genotype-tissue expression portal expression quantitative trait loci database and accelerating medicines partnership type 2 diabetes mellitus Knowledge Portal to evaluate whether candidate genes prioritized by our in vitro studies were expression quantitative trait loci genes in human subcutaneous adipose tissue, and whether expression of these genes is associated with risk of IR, type 2 diabetes mellitus, and cardiovascular diseases. We further validated the functions of 3 new adipose IR genes by overexpression-based phenotypic rescue in the Simpson-Golabi-Behmel syndrome preadipocyte knockout lines. Twelve genes, PPARG, IRS-1, FST, PEPD, PDGFC, MAP3K1, GRB14, ARL15, ANKRD55, RSPO3, COBLL1, and LYPLAL1, showed diverse phenotypes in the 3 insulin-sensitizing mechanisms, and the first 7 of these genes could affect all the 3 mechanisms. Five out of 6 expression quantitative trait loci genes are among the top candidate causal genes and the abnormal expression levels of these genes (IRS-1, GRB14, FST, PEPD, and PDGFC) in human subcutaneous adipose tissue could be associated with increased risk of IR, type 2 diabetes mellitus, and cardiovascular disease. Phenotypic rescue by overexpression of the candidate causal genes (FST, PEPD, and PDGFC) in the Simpson-Golabi-Behmel syndrome preadipocyte knockout lines confirmed their function in adipose IR. Conclusions: Twelve genes showed diverse phenotypes indicating differential roles in insulin sensitization, suggesting mechanisms bridging the association of their genomic loci with IR. We prioritized PPARG, IRS-1, GRB14, MAP3K1, FST, PEPD, and PDGFC as top candidate genes. Our work points to novel roles for FST, PEPD, and PDGFC in adipose tissue, with consequences for cardiometabolic diseases.


Subject(s)
Adipocytes/metabolism , Insulin Resistance/genetics , Quantitative Trait Loci , Adaptor Proteins, Signal Transducing/genetics , Cell Line , Dipeptidases/genetics , Follistatin/genetics , Genome-Wide Association Study/methods , Humans , Insulin Receptor Substrate Proteins/genetics , Loss of Function Mutation , Lymphokines/genetics , MAP Kinase Kinase Kinase 1/genetics , PPAR gamma/genetics , Platelet-Derived Growth Factor/genetics
10.
Proc Natl Acad Sci U S A ; 116(21): 10441-10446, 2019 05 21.
Article in English | MEDLINE | ID: mdl-31040209

ABSTRACT

Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage "don't-eat me" signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.


Subject(s)
Genetic Engineering/methods , Pluripotent Stem Cells/immunology , CRISPR-Cas Systems , Cell Line , Gene Knockout Techniques , Genes, MHC Class I , Genes, MHC Class II , Humans
11.
Front Immunol ; 10: 2730, 2019.
Article in English | MEDLINE | ID: mdl-31921098

ABSTRACT

To establish a healthy pregnancy, maternal immune cells must tolerate fetal allo-antigens and remain competent to respond to infections both systemically and in placental tissues. Extravillous trophoblasts (EVT) are the most invasive cells of extra-embryonic origin to invade uterine tissues and express polymorphic Human Leucocyte Antigen-C (HLA-C) of both maternal and paternal origin. Thus, HLA-C is a key molecule that can elicit allogeneic immune responses by maternal T and NK cells and for which maternal-fetal immune tolerance needs to be established. HLA-C is also the only classical MHC molecule expressed by EVT that can present a wide variety of peptides to maternal memory T cells and establish protective immunity. The expression of paternal HLA-C by EVT provides a target for maternal NK and T cells, whereas HLA-C expression levels may influence how this response is shaped. This dual function of HLA-C requires tight transcriptional regulation of its expression to balance induction of tolerance and immunity. Here, we critically review new insights into: (i) the mechanisms controlling expression of HLA-C by EVT, (ii) the mechanisms by which decidual NK cells, effector T cells and regulatory T cells recognize HLA-C allo-antigens, and (iii) immune recognition of pathogen derived antigens in context of HLA-C.


Subject(s)
HLA-C Antigens/immunology , Immune Tolerance , Immunity , Maternal-Fetal Exchange/immunology , Placenta/immunology , Placenta/metabolism , Alleles , Decidua/immunology , Decidua/metabolism , Female , Gene Expression Regulation , HLA-A Antigens/genetics , HLA-A Antigens/immunology , HLA-B Antigens/genetics , HLA-B Antigens/immunology , HLA-C Antigens/genetics , Humans , Immune Tolerance/genetics , Immunity/genetics , Immunomodulation , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Pregnancy , Promoter Regions, Genetic , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Trophoblasts/metabolism
12.
Stem Cell Reports ; 11(2): 348-362, 2018 08 14.
Article in English | MEDLINE | ID: mdl-29983385

ABSTRACT

Zika virus (ZIKV) and dengue virus (DENV) are two closely related flaviviruses that lead to different clinical outcomes. The mechanism for the distinct pathogenesis of ZIKV and DENV is poorly understood. Here, we investigate ZIKV and DENV infection of macrophages using a human pluripotent stem cell (hPSC)-derived macrophage model and discover key virus-specific responses. ZIKV and DENV productively infect hPSC-derived macrophages. DENV, but not ZIKV, infection of macrophages strongly activates macrophage migration inhibitory factor (MIF) secretion and decreases macrophage migration. Neutralization of MIF leads to improved migratory ability of DENV-infected macrophages. In contrast, ZIKV-infected macrophages exhibit prolonged migration and express low levels of pro-inflammatory cytokines and chemokines. Mechanistically, ZIKV disrupts the nuclear factor κB (NF-κB)-MIF positive feedback loop by inhibiting the NF-κB signaling pathway. Our results demonstrate the utility of hPSC-derived macrophages in infectious disease modeling and suggest that the distinct impact of ZIKV and DENV on macrophage immune response may underlie different pathogenesis of Zika and dengue diseases.


Subject(s)
Cell Differentiation , Dengue Virus/immunology , Dengue/immunology , Macrophages/cytology , Macrophages/immunology , Pluripotent Stem Cells/cytology , Zika Virus Infection/immunology , Zika Virus/immunology , Biomarkers , Cell Movement/immunology , Cells, Cultured , Cytokines/genetics , Host-Pathogen Interactions/immunology , Humans , Immunophenotyping , Macrophages/metabolism , Macrophages/virology , Pluripotent Stem Cells/metabolism , Virus Replication/immunology
13.
Biol Reprod ; 96(4): 831-842, 2017 Apr 01.
Article in English | MEDLINE | ID: mdl-28340094

ABSTRACT

During pregnancy, fetal extravillous trophoblasts (EVT) play a key role in the regulation of maternal T cell and NK cell responses. EVT display a unique combination of human leukocyte antigens (HLA); EVT do not express HLA-A and HLA-B, but do express HLA-C, HLA-E, and HLA-G. The mechanisms establishing this unique HLA expression pattern have not been fully elucidated. The major histocompatibility complex (MHC) class I and class II transcriptional activators NLRC5 and CIITA are expressed neither by EVT nor by the EVT model cell line JEG3, which has an MHC expression pattern identical to that of EVT. Therefore, other MHC regulators must be present to control HLA-C, HLA-E, and HLA-G expression in these cells. CIITA and NLRC5 are both members of the nucleotide-binding domain, leucine-rich repeat (NLR) family of proteins. Another member of this family, NLRP2, is highly expressed by EVT and JEG3, but not in maternal decidual stromal cells. In this study, transcription activator-like effector nuclease technology was used to delete NLRP2 in JEG3. Furthermore, lentiviral delivery of shRNA was used to knockdown NLRP2 in JEG3 and primary EVT. Upon NLRP2 deletion, Tumor Necrosis Factor-α (TNFα)-induced phosphorylation of NF-KB p65 increased in JEG3 and EVT, and more surprisingly a significant increase in constitutive HLA-C expression was observed in JEG3. These data suggest a broader role for NLR family members in the regulation of MHC expression during inflammation, thus forming a bridge between innate and adaptive immune responses. As suppressor of proinflammatory responses, NLRP2 may contribute to preventing unwanted antifetal responses.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Gene Expression Regulation, Developmental/physiology , HLA-C Antigens/metabolism , NF-kappa B/metabolism , Trophoblasts/metabolism , Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins , Cell Line , Cytokines/genetics , Cytokines/metabolism , Gene Deletion , Genes, MHC Class I/genetics , HLA-C Antigens/genetics , Humans , NF-kappa B/genetics , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction
14.
Trends Immunol ; 38(4): 272-286, 2017 04.
Article in English | MEDLINE | ID: mdl-28279591

ABSTRACT

During pregnancy, semiallogeneic fetal extravillous trophoblasts (EVT) invade the uterine mucosa without being rejected by the maternal immune system. Several mechanisms were initially proposed by Peter Medawar half a century ago to explain this apparent violation of the laws of transplantation. Then, three decades ago, an unusual human leukocyte antigen (HLA) molecule was identified: HLA-G. Uniquely expressed in EVT, HLA-G has since become the center of the present understanding of fetus-induced immune tolerance. Despite slow progress in the field, the last few years have seen an explosion in our knowledge of HLA-G biology. Here, we critically review new insights into the mechanisms controlling the expression and function of HLA-G at the maternal-fetal interface, and discuss their relevance for fetal tolerance.


Subject(s)
HLA-G Antigens/metabolism , Maternal-Fetal Relations , Pregnancy/immunology , Transplantation Tolerance , Trophoblasts/immunology , Animals , Female , HLA-G Antigens/immunology , Histocompatibility , Humans , Isoantigens/immunology
15.
Proc Natl Acad Sci U S A ; 113(21): 5999-6004, 2016 May 24.
Article in English | MEDLINE | ID: mdl-27162338

ABSTRACT

Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and ß2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.


Subject(s)
Antigen Presentation , Biomarkers, Tumor/immunology , Gene Expression Regulation, Neoplastic/immunology , Histocompatibility Antigens Class I/immunology , Intracellular Signaling Peptides and Proteins/immunology , Neoplasm Proteins/immunology , Neoplasms/immunology , Trans-Activators/immunology , Transcriptional Activation/immunology , Tumor Escape , ATP Binding Cassette Transporter, Subfamily B, Member 2/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 2/immunology , Biomarkers, Tumor/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/immunology , Female , Histocompatibility Antigens Class I/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Neoplasm Proteins/genetics , Neoplasms/genetics , Neoplasms/mortality , Neoplasms/pathology , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/immunology , Trans-Activators/genetics , beta 2-Microglobulin/genetics , beta 2-Microglobulin/immunology
16.
Proc Natl Acad Sci U S A ; 113(19): 5364-9, 2016 May 10.
Article in English | MEDLINE | ID: mdl-27078102

ABSTRACT

HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal-fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.


Subject(s)
Enhancer Elements, Genetic/immunology , Gene Expression Regulation, Developmental/immunology , HLA-G Antigens/immunology , Histocompatibility, Maternal-Fetal/immunology , Maternal-Fetal Exchange/immunology , Pregnancy/immunology , Trophoblasts/immunology , Enhancer Elements, Genetic/genetics , Female , Gene Expression Regulation, Developmental/genetics , HLA-G Antigens/genetics , Histocompatibility, Maternal-Fetal/genetics , Humans , Immunogenetic Phenomena/genetics , Maternal-Fetal Exchange/genetics , Placenta/immunology
17.
Arterioscler Thromb Vasc Biol ; 36(1): 15-8, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26543098

ABSTRACT

OBJECTIVE: To create isogenic human pluripotent stem cell-derived macrophages with and without ABCA1 expression as a model for reverse cholesterol transport. APPROACH AND RESULTS: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) genome-editing system was used to introduce frameshift mutations into the coding sequence of ATP-binding cassette, subfamily A, member 1. Individual human pluripotent stem cell clones with deleterious mutations were identified, expanded, and differentiated into mature macrophages with a cytokine-based, feeder-free differentiation protocol. Wild-type cells demonstrated effective cholesterol efflux to apoAI acceptor, whereas ABCA1(-/-) cells displayed significantly reduced efflux ability and increased expression of proinflammatory cytokines. CONCLUSIONS: Human pluripotent stem cell-derived macrophages capable of reverse cholesterol transport can be rapidly generated and genetically edited with CRISPR/Cas9. Introduction of homozygous frameshift mutations results in loss of ABCA1 expression in differentiated macrophages and subsequent reduction of cholesterol efflux capability. This facile genome-editing approach and differentiation protocol pave the way for future studies of the molecular determinants of reverse cholesterol transport and other macrophage properties.


Subject(s)
ATP Binding Cassette Transporter 1/metabolism , Cholesterol/metabolism , Genome, Human , Macrophages/metabolism , Pluripotent Stem Cells/metabolism , ATP Binding Cassette Transporter 1/genetics , Apolipoprotein A-I/metabolism , Biological Transport , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , Cell Differentiation , Cell Line , Cytokines/metabolism , Frameshift Mutation , Gene Expression Regulation , Genotype , Humans , INDEL Mutation , Inflammation Mediators/metabolism , Phenotype
18.
Science ; 350(6258): 334-9, 2015 Oct 16.
Article in English | MEDLINE | ID: mdl-26472910

ABSTRACT

The maintenance of immune homeostasis requires regulatory T cells (T(regs)). Given their intrinsic self-reactivity, T(regs) must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3(+) CD4 and Qa-1-restricted CD8 T(regs) results in defective regulatory activity and autoimmunity in mice. Helios-deficient T(regs) develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 T(regs) also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes T(regs) in the face of inflammatory responses provides a genetic explanation for a core property of T(regs).


Subject(s)
Autoimmunity/immunology , CD8-Positive T-Lymphocytes/immunology , DNA-Binding Proteins/biosynthesis , T-Lymphocytes, Regulatory/immunology , Transcription Factors/biosynthesis , Animals , Autoimmunity/genetics , DNA-Binding Proteins/genetics , Forkhead Transcription Factors/immunology , Gene Expression , Kidney/immunology , Liver/immunology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pancreas/immunology , STAT5 Transcription Factor/metabolism , Transcription Factors/genetics
19.
Cell Stem Cell ; 15(5): 643-52, 2014 Nov 06.
Article in English | MEDLINE | ID: mdl-25517468

ABSTRACT

Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.


Subject(s)
CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Deletion , Hematopoietic Stem Cells/metabolism , Animals , Antigens, CD34/metabolism , Cell Lineage , Cells, Cultured , Gene Targeting , Genetic Loci , Genome, Human/genetics , Hematopoietic Stem Cells/cytology , High-Throughput Nucleotide Sequencing , Humans , Mice , RNA Editing/genetics , RNA, Guide, Kinetoplastida/metabolism , Receptors, CCR5/metabolism
20.
Methods Enzymol ; 546: 273-95, 2014.
Article in English | MEDLINE | ID: mdl-25398345

ABSTRACT

The rapid advancement of genome-editing techniques holds much promise for the field of human gene therapy. From bacteria to model organisms and human cells, genome editing tools such as zinc-finger nucleases (ZNFs), TALENs, and CRISPR/Cas9 have been successfully used to manipulate the respective genomes with unprecedented precision. With regard to human gene therapy, it is of great interest to test the feasibility of genome editing in primary human hematopoietic cells that could potentially be used to treat a variety of human genetic disorders such as hemoglobinopathies, primary immunodeficiencies, and cancer. In this chapter, we explore the use of the CRISPR/Cas9 system for the efficient ablation of genes in two clinically relevant primary human cell types, CD4+ T cells and CD34+ hematopoietic stem and progenitor cells. By using two guide RNAs directed at a single locus, we achieve highly efficient and predictable deletions that ablate gene function. The use of a Cas9-2A-GFP fusion protein allows FACS-based enrichment of the transfected cells. The ease of designing, constructing, and testing guide RNAs makes this dual guide strategy an attractive approach for the efficient deletion of clinically relevant genes in primary human hematopoietic stem and effector cells and enables the use of CRISPR/Cas9 for gene therapy.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CRISPR-Cas Systems , Gene Deletion , Genetic Engineering/methods , Genetic Therapy/methods , Hematopoietic Stem Cells/metabolism , Antigens, CD34/analysis , Cell Separation/methods , Cells, Cultured , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Targeting/methods , Genome, Human , Humans
SELECTION OF CITATIONS
SEARCH DETAIL
...