32 results on '"Matthew B. Dong"'
Search Results
2. Human Gene Age Dating Reveals an Early and Rapid Evolutionary Construction of the Adaptive Immune System
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Li Zhang, Jonathan J Park, Matthew B Dong, Deanna Arsala, Shengqian Xia, Jianhai Chen, Dylan Sosa, Jared E Atlas, Manyuan Long, and Sidi Chen
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Genetics ,Ecology, Evolution, Behavior and Systematics - Abstract
T cells are a type of white blood cell that play a critical role in the immune response against foreign pathogens through a process called T cell adaptive immunity (TCAI). However, the evolution of the genes and nucleotide sequences involved in TCAI is not well understood. To investigate this, we performed comparative studies of gene annotations and genome assemblies of 28 vertebrate species and identified sets of human genes that are involved in TCAI, carcinogenesis, and aging. We found that these gene sets share interaction pathways, which may have contributed to the evolution of longevity in the vertebrate lineage leading to humans. Our human gene age dating analyses revealed that there was rapid origination of genes with TCAI-related functions prior to the Cretaceous eutherian radiation and these new genes mainly encode negative regulators. We identified no new TCAI-related genes after the divergence of placental mammals, but we did detect an extensive number of amino acid substitutions under strong positive selection in recently evolved human immunity genes suggesting they are coevolving with adaptive immunity. More specifically, we observed that antigen processing and presentation and checkpoint genes are significantly enriched among new genes evolving under positive selection. These observations reveal evolutionary processes of TCAI that were associated with rapid gene duplication in the early stages of vertebrates and subsequent sequence changes in TCAI-related genes. The analysis of vertebrate genomes provides evidence that a "big bang" of adaptive immune genes occurred 300-500 million years ago. These processes together suggest an early genetic construction of the vertebrate immune system and subsequent molecular adaptation to diverse antigens.
- Published
- 2023
3. Supplementary Tables from CRISPR-GEMM Pooled Mutagenic Screening Identifies KMT2D as a Major Modulator of Immune Checkpoint Blockade
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Sidi Chen, Youssef Errami, Paul Clark, Cun Liao, Kristin Kim, Zhiyuan Chu, Leilei Niu, Yujing Cheng, Yaying Du, Xiaoya Zhang, Xiaoyun Dai, Matthew B. Dong, Paul A. Renauer, Feifei Zhang, Lupeng Ye, Zhigang Bai, Lvyun Zhu, Ryan D. Chow, and Guangchuan Wang
- Abstract
A supplementary table of a multi-tab excel file including source data (non-NGS) and statistics
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- 2023
4. Supplementary NGS Datasets from CRISPR-GEMM Pooled Mutagenic Screening Identifies KMT2D as a Major Modulator of Immune Checkpoint Blockade
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Sidi Chen, Youssef Errami, Paul Clark, Cun Liao, Kristin Kim, Zhiyuan Chu, Leilei Niu, Yujing Cheng, Yaying Du, Xiaoya Zhang, Xiaoyun Dai, Matthew B. Dong, Paul A. Renauer, Feifei Zhang, Lupeng Ye, Zhigang Bai, Lvyun Zhu, Ryan D. Chow, and Guangchuan Wang
- Abstract
Supplementary Datasets Data 1: Sample metadata from AAV-mTSG liver screen. Data 2: Filtered sgRNA-level mutation frequencies Data 3: Gene-level average mutation frequencies Data 4: Gene-level mutation frequencies, averaged by treatment group Data 5: Results of t-tests comparing mutation frequencies: aPD1 vs PBS, or aCTLA4 vs PBS Data 6: Summary of KMT2D mutations in pan-cancer cohort Data 7: Correlation between KMT2D expression and macrophage abundance in TCGA cohorts Data 8: Correlation between KMT2D and GZMB expression in TCGA cohorts Data 9: Correlation between KMT2D and GZMA expression in TCGA cohorts Data 10: Correlation between KMT2D and CD14 expression in TCGA cohorts Data 11: Composite list of genes positively correlated with KMT2D across TCGA cohorts Data 12: Composite list of genes negatively correlated with KMT2D across TCGA cohorts Data 13: Summary of exome sequencing analysis Data 14: Association between KMT2D mutation status and total mutation burden in TCGA cohorts Data 15: Differential accessibility analysis by DESeq, sgKmt2d vs Vector (-IFN-γ) Data 16: Motif analysis of regions more accessible in sgKmt2d vs Vector Data 17: Motif analysis of regions less accessible in sgKmt2d vs Vector Data 18: Differential accessibility analysis by DESeq, sgKmt2d + IFN-γ vs - IFN-γ Data 19: Differential accessibility analysis by DESeq, Vector + IFN-γ vs - IFN-γ Data 20: List of genomic regions in each of the six clusters Data 21: Motif analysis of C1 regions Data 22: Motif analysis of C4 regions Data 23: Motif analysis of C3 regions Data 24: Motif analysis of C5 regions Data 25: Differential expression analysis of RNAseq, sgKmt2d vs Vector Data 26: DAVID gene ontology analysis of genes upregulated in sgKmt2d vs Vector Data 27: DAVID gene ontology analysis of genes downregulated in sgKmt2d vs Vector Data 28: High-confidence predicted neoantigens in sgKmt2d or Vector samples Data 29: Differential expression analysis of transposable elements, sgKmt2d vs Vector
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- 2023
5. Supplementary Data from CRISPR-GEMM Pooled Mutagenic Screening Identifies KMT2D as a Major Modulator of Immune Checkpoint Blockade
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Sidi Chen, Youssef Errami, Paul Clark, Cun Liao, Kristin Kim, Zhiyuan Chu, Leilei Niu, Yujing Cheng, Yaying Du, Xiaoya Zhang, Xiaoyun Dai, Matthew B. Dong, Paul A. Renauer, Feifei Zhang, Lupeng Ye, Zhigang Bai, Lvyun Zhu, Ryan D. Chow, and Guangchuan Wang
- Abstract
Supplementary data file contains Supplementary Methods and Supplementary Figures S1-S11 and related figure legends
- Published
- 2023
6. Data from CRISPR-GEMM Pooled Mutagenic Screening Identifies KMT2D as a Major Modulator of Immune Checkpoint Blockade
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Sidi Chen, Youssef Errami, Paul Clark, Cun Liao, Kristin Kim, Zhiyuan Chu, Leilei Niu, Yujing Cheng, Yaying Du, Xiaoya Zhang, Xiaoyun Dai, Matthew B. Dong, Paul A. Renauer, Feifei Zhang, Lupeng Ye, Zhigang Bai, Lvyun Zhu, Ryan D. Chow, and Guangchuan Wang
- Abstract
Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy in several cancer types. However, only a fraction of patients will respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMM) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types. KMT2D encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in patients with cancer. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. In addition, Kmt2d-mutant cells exhibited increased protein turnover and IFNγ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human were characterized by increased immune infiltration. These data demonstrate that Kmt2d deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, and also highlight the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment.Significance:ICB is ineffective in the majority of patients. Through direct in vivo CRISPR mutagenesis screening in GEMMs of cancer, we find Kmt2d deficiency sensitizes tumors to ICB. Considering the prevalence of KMT2D mutations, this finding potentially has broad implications for patient stratification and clinical decision-making.This article is highlighted in the In This Issue feature, p. 1775
- Published
- 2023
7. CTLA-4 tail fusion enhances CAR-T anti-tumor immunity
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Xiaoyu Zhou, Hanbing Cao, Shao-Yu Fang, Ryan D. Chow, Kaiyuan Tang, Medha Majety, Meizhu Bai, Matthew B. Dong, Paul A. Renauer, Xingbo Shang, Kazushi Suzuki, Andre Levchenko, and Sidi Chen
- Subjects
Article - Abstract
Chimeric antigen receptor (CAR) T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here, utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail (CT), we reprogram CAR function and substantially enhance CAR-T efficacyin vivo. CAR-T cells with monomeric, duplex, or triplex CTLA-4 CTs (CCTs) fused to the C-terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of pro-inflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen, and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior anti-tumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.
- Published
- 2023
8. Cas12a/Cpf1 knock-in mice enable efficient multiplexed immune cell engineering
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Matthew B. Dong, Kaiyuan Tang, Xiaoyu Zhou, Johanna Shen, Krista Chen, Hyunu R. Kim, Jerry Zhou, Hanbing Cao, Erica Vandenbulcke, Yueqi Zhang, Ryan D. Chow, Andrew Du, Kazushi Suzuki, Shao-Yu Fang, Medha Majety, Xiaoyun Dai, and Sidi Chen
- Subjects
Article - Abstract
SummaryCas9 transgenic animals have drastically accelerated the discovery of novel immune modulators. But due to its inability to process its own CRISPR RNAs (crRNAs), simultaneous multiplexed gene perturbations using Cas9 remains limited, especially by pseudoviral vectors. Cas12a/Cpf1, however, can process concatenated crRNA arrays for this purpose. Here, we created conditional and constitutive LbCas12a knock-in transgenic mice. With these mice, we demonstrated efficient multiplexed gene editing and surface protein knockdown within individual primary immune cells. We showed genome editing across multiple types of primary immune cells including CD4 and CD8 T cells, B cells, and bone-marrow derived dendritic cells. These transgenic animals, along with the accompanying viral vectors, together provide a versatile toolkit for a broad range ofex vivoandin vivogene editing applications, including fundamental immunological discovery and immune gene engineering.
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- 2023
9. Tumor immunology CRISPR screening: present, past, and future
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Matthew B. Dong, Kaiyuan Tang, Xiaoyu Zhou, Jingjia J. Zhou, and Sidi Chen
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Gene Editing ,Cancer Research ,Oncology ,Neoplasms ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Immunotherapy ,CRISPR-Cas Systems ,Article - Abstract
Recent advances in immunotherapy have fundamentally changed the landscape of cancer treatment by leveraging the specificity and selectivity of the adaptive immune system to kill cancer cells. These successes have ushered in a new wave of research aimed at understanding immune recognition with the hope of developing newer immunotherapies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technologies and advancement of multiomics modalities have greatly accelerated the discovery process. Here, we review the current literature surrounding CRISPR screens within the context of tumor immunology, provide essential components needed to conduct immune-specific CRISPR screens, and present avenues for future research.
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- 2022
10. Simultaneous and sequential multi-species coronavirus vaccination
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Lei Peng, Zhenhao Fang, Paul A. Renauer, Andrew McNamara, Jonathan J. Park, Qianqian Lin, Xiaoyu Zhou, Matthew B. Dong, Biqing Zhu, Hongyu Zhao, Craig B. Wilen, and Sidi Chen
- Abstract
Although successful COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging for development of multi-species coronavirus vaccines. Here we developed prototype LNP-mRNA vaccine candidates against SARS-CoV-2 (Delta variant), SARS-CoV and MERS-CoV, and test how multiplexing of these LNP-mRNAs can induce effective immune responses in animal models. A triplex scheme of LNP-mRNA vaccination induced antigen-specific antibody responses against SARS-CoV-2, SARS-CoV and MERS-CoV, with a relatively weaker MERS-CoV response in this setting. Single cell RNA-seq profiled the global systemic immune repertoires and the respective transcriptome signatures of multiplexed vaccinated animals, which revealed a systemic increase in activated B cells, as well as differential gene expression signatures across major adaptive immune cells. Sequential vaccination showed potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrated the feasibility, antibody responses and single cell immune profiles of multi-species coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights on optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species.One sentence summaryMultiplexed mRNA vaccination in simultaneous and sequential modes provide broad and potent immunity against pathogenic coronavirus species.
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- 2022
11. A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy
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Lupeng Ye, Jonathan J. Park, Lei Peng, Quanjun Yang, Ryan D. Chow, Matthew B. Dong, Stanley Z. Lam, Jianjian Guo, Erting Tang, Yueqi Zhang, Guangchuan Wang, Xiaoyun Dai, Yaying Du, Hyunu R. Kim, Hanbing Cao, Youssef Errami, Paul Clark, Alexey Bersenev, Ruth R. Montgomery, and Sidi Chen
- Subjects
Receptors, Chimeric Antigen ,Proline ,Physiology ,Gain of Function Mutation ,Neoplasms ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Cell Biology ,CD8-Positive T-Lymphocytes ,Molecular Biology ,Article - Abstract
Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8(+) T cells, and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knock-in or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression reshaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together these findings provide a system for identification of GOF immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy.
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- 2022
12. Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9.
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Linda M Lee, Ming Ji, Meenal Sinha, Matthew B Dong, Xin Ren, Yanli Wang, Clifford A Lowell, Sankar Ghosh, Richard M Locksley, and Anthony L DeFranco
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Medicine ,Science - Abstract
Excessive type 2 helper T cell responses to environmental antigens can cause immunopathology such as asthma and allergy, but how such immune responses are induced remains unclear. We studied this process in the airways by immunizing mice intranasally with the antigen ovalbumin together with either of two Toll-like receptor (TLR) ligands. We found the TLR5 ligand flagellin promoted a type 2 helper T cell response, whereas, a TLR9 ligand CpG oligodeoxyribonucleotide (ODN) promoted a type 1 helper T cell response. CpG ODN induced mRNA encoding interleukin (IL)-12 p40, whereas, flagellin caused IL-33 secretion and induced mRNAs encoding IL-1 and thymic stromal lymphopoietin (TSLP). By using mice deficient in the TLR and IL-1R signaling molecule, myeloid differentiation primary response 88 (MyD88), in conventional dendritic cells (cDCs) and alveolar macrophages (AMs), and by cell sorting different lung populations after 2 hours of in vivo stimulation, we characterized the cell types that rapidly produced inflammatory cytokines in response to TLR stimulation. CpG ODN was likely recognized by TLR9 on cDCs and AMs, which made mRNA encoding IL-12. IL-12 was necessary for the subsequent innate and adaptive interferon-γ production. In contrast, flagellin stimulated multiple cells of hematopoietic and non-hematopoietic origin, including AMs, DCs, monocytes, and lung epithelial cells. AMs were largely responsible for IL-1α, whereas lung epithelial cells made TSLP. Multiple hematopoietic cells, including AMs, DCs, and monocytes contributed to other cytokines, including IL-1β and TNFα. MyD88-dependent signals, likely through IL-1R and IL-33R, and MyD88-independent signals, likely from TSLP, were necessary in cDCs for promotion of the early IL-4 response by CD4 T cells in the draining lymph node. Thus, the cell types that responded to TLR ligands were a critical determinant of the innate cytokines produced and the character of the resulting adaptive immune response in the airways.
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- 2016
- Full Text
- View/download PDF
13. High-content CRISPR screening
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Christoph Bock, Paul Datlinger, Florence Chardon, Matthew A. Coelho, Matthew B. Dong, Keith A. Lawson, Tian Lu, Laetitia Maroc, Thomas M. Norman, Bicna Song, Geoff Stanley, Sidi Chen, Mathew Garnett, Wei Li, Jason Moffat, Lei S. Qi, Rebecca S. Shapiro, Jay Shendure, Jonathan S. Weissman, and Xiaowei Zhuang
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General Medicine ,General Biochemistry, Genetics and Molecular Biology ,Article - Abstract
CRISPR screens are a powerful source of biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications and species. In pooled CRISPR screens, various genetically encoded perturbations are introduced into pools of cells. The targeted cells proliferate under a biological challenge such as cell competition, drug treatment or viral infection. Subsequently, the perturbation-induced effects are evaluated by sequencing-based counting of the guide RNAs that specify each perturbation. The typical results of such screens are ranked lists of genes that confer sensitivity or resistance to the biological challenge of interest. Contributing to the broad utility of CRISPR screens, adaptations of the core CRISPR technology make it possible to activate, silence or otherwise manipulate the target genes. Moreover, high-content read-outs such as single-cell RNA sequencing and spatial imaging help characterize screened cells with unprecedented detail. Dedicated software tools facilitate bioinformatic analysis and enhance reproducibility. CRISPR screening has unravelled various molecular mechanisms in basic biology, medical genetics, cancer research, immunology, infectious diseases, microbiology and other fields. This Primer describes the basic and advanced concepts of CRISPR screening and its application as a flexible and reliable method for biological discovery, biomedical research and drug development — with a special emphasis on high-content methods that make it possible to obtain detailed biological insights directly as part of the screen.
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- 2022
14. Systems immune profiling of variant-specific vaccination against SARS-CoV-2
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Lei Peng, Jonathan J. Park, Zhenhao Fang, Xiaoyu Zhou, Matthew B. Dong, Qiancheng Xiong, Chenxiang Lin, and Sidi Chen
- Abstract
Lipid-nanoparticle(LNP)-mRNA vaccines offer protection against COVID-19. However, multiple variant lineages caused widespread breakthrough infections. There is no report on variant-specific vaccines to date. Here, we generated LNP-mRNAs specifically encoding wildtype, B.1.351 and B.1.617 SARS-CoV-2 spikes, and systematically studied their immune responses in animal models. All three LNP-mRNAs induced potent antibody responses in mice. However, WT-LNP-mRNA vaccination showed reduced neutralization against B.1.351 and B.1.617; and B.1.617-specific vaccination showed differential neutralization. All three vaccine candidates elicited antigen-specific CD8 and CD4 T cell responses. Single cell transcriptomics of B.1.351-LNP-mRNA and B.1.617-LNP-mRNA vaccinated animals revealed a systematic landscape of immune cell populations and global gene expression. Variant-specific vaccination induced a systemic increase in reactive CD8 T cell population, with a strong signature of transcriptional and translational machineries in lymphocytes. BCR-seq and TCR-seq unveiled repertoire diversity and clonal expansions in vaccinated animals. These data provide direct systems immune profiling of variant-specific LNP-mRNA vaccination in vivo.
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- 2021
15. Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2
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Lei Peng, Paul A. Renauer, Arya Ökten, Zhenhao Fang, Jonathan J. Park, Xiaoyu Zhou, Qianqian Lin, Matthew B. Dong, Renata Filler, Qiancheng Xiong, Paul Clark, Chenxiang Lin, Craig B. Wilen, and Sidi Chen
- Subjects
COVID-19 Vaccines ,SARS-CoV-2 ,Liposomes ,Vaccination ,Immunity ,Animals ,COVID-19 ,Humans ,Nanoparticles ,RNA, Messenger ,Antibodies, Neutralizing ,General Biochemistry, Genetics and Molecular Biology - Abstract
Lipid nanoparticle (LNP)-mRNA vaccines offer protection against COVID-19; however, multiple variant lineages caused widespread breakthrough infections. Here, we generate LNP-mRNAs specifically encoding wild-type (WT), B.1.351, and B.1.617 SARS-CoV-2 spikes, and systematically study their immune responses. All three LNP-mRNAs induced potent antibody and T cell responses in animal models; however, differences in neutralization activity have been observed between variants. All three vaccines offer potent protection against in vivo challenges of authentic viruses of WA-1, Beta, and Delta variants. Single-cell transcriptomics of WT- and variant-specific LNP-mRNA-vaccinated animals reveal a systematic landscape of immune cell populations and global gene expression. Variant-specific vaccination induces a systemic increase of reactive CD8 T cells and altered gene expression programs in B and T lymphocytes. BCR-seq and TCR-seq unveil repertoire diversity and clonal expansions in vaccinated animals. These data provide assessment of efficacy and direct systems immune profiling of variant-specific LNP-mRNA vaccination in vivo.
- Published
- 2021
16. Multiplexed LNP-mRNA vaccination against pathogenic coronavirus species
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Lei Peng, Zhenhao Fang, Paul A. Renauer, Andrew McNamara, Jonathan J. Park, Qianqian Lin, Xiaoyu Zhou, Matthew B. Dong, Biqing Zhu, Hongyu Zhao, Craig B. Wilen, and Sidi Chen
- Subjects
Vaccines, Synthetic ,COVID-19 Vaccines ,SARS-CoV-2 ,Vaccination ,COVID-19 ,Viral Vaccines ,Antibodies, Viral ,Antibodies, Neutralizing ,General Biochemistry, Genetics and Molecular Biology ,Liposomes ,Spike Glycoprotein, Coronavirus ,Middle East Respiratory Syndrome Coronavirus ,Animals ,Humans ,Nanoparticles ,RNA, Messenger ,mRNA Vaccines - Abstract
Although COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging on development of multispecies coronavirus vaccines. Here we develop prototype lipid nanoparticle (LNP)-mRNA vaccine candidates against SARS-CoV-2 Delta, SARS-CoV, and MERS-CoV, and we test how multiplexing LNP-mRNAs can induce effective immune responses in animal models. Triplex and duplex LNP-mRNA vaccinations induce antigen-specific antibody responses against SARS-CoV-2, SARS-CoV, and MERS-CoV. Single-cell RNA sequencing profiles the global systemic immune repertoires and respective transcriptome signatures of vaccinated animals, revealing a systemic increase in activated B cells and differential gene expression across major adaptive immune cells. Sequential vaccination shows potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrate the feasibility, antibody responses, and single-cell immune profiles of multispecies coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights into optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species.
- Published
- 2022
17. Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity
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Zhigang Bai, Guangchuan Wang, Li Shen, Xiaoya Zhang, Charles S. Fuchs, Lupeng Ye, Hanghui Ye, Jonathan J. Park, Youssef Errami, Xiaoyun Dai, Matthew B. Dong, Ryan D. Chow, Paul A. Renauer, Sidi Chen, and Lvyun Zhu
- Subjects
Male ,0301 basic medicine ,medicine.medical_treatment ,Genetic Vectors ,Immunology ,Injections, Intralesional ,Biology ,Gene delivery ,Cancer Vaccines ,Article ,Mice ,03 medical and health sciences ,Lymphocytes, Tumor-Infiltrating ,0302 clinical medicine ,Immune system ,Antigen ,Cancer immunotherapy ,Antigens, Neoplasm ,Cell Line, Tumor ,Neoplasms ,Tumor Microenvironment ,medicine ,Animals ,Humans ,Immunology and Allergy ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Antigen Presentation ,Tumor microenvironment ,Cancer ,Genetic Therapy ,Immunotherapy ,Dependovirus ,medicine.disease ,Combined Modality Therapy ,Coculture Techniques ,Gene Expression Regulation, Neoplastic ,Disease Models, Animal ,HEK293 Cells ,030104 developmental biology ,Cancer research ,Female ,T-Lymphocytes, Cytotoxic ,030215 immunology - Abstract
Immunotherapy has transformed cancer treatment. However, current immunotherapy modalities face various limitations. In the present study, we developed multiplexed activation of endogenous genes as an immunotherapy (MAEGI), a new form of immunotherapy that elicits antitumor immunity through multiplexed activation of endogenous genes in tumors. We leveraged CRISPR activation (CRISPRa) to directly augment the in situ expression of endogenous genes, and thereby the presentation of tumor antigens, leading to dramatic antitumor immune responses. Deploying this as a cell-based vaccination strategy showed efficacy in both prophylactic and therapeutic settings. Intratumoral adeno-associated virus delivery of CRISPRa libraries elicited strong antitumor immunity across multiple cancer types. Precision targeting of mutated gene sets eradicated a large fraction of established tumors at both local and distant sites. This treatment modality led to alterations in the tumor microenvironment, marked by enhanced T cell infiltration and antitumor immune signatures. Multiplexed endogenous gene activation is a versatile and highly scalable strategy to elicit potent immune responses against cancer, distinct from all existing cancer therapies. CRISPR activation (CRISPRa) can target select genes and, rather than being used to delete them, can be used to activate their expression. Chen and colleagues use a CRISPRa-based approach to drive the expression of multiple endogenous genes in tumors and presentation of the antigens encoded, thus enhancing antitumor immune responses.
- Published
- 2019
18. In vivo CRISPR screening in CD8 T cells with AAV–Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma
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Quanjun Yang, Sidi Chen, Xiaoyun Dai, Lupeng Ye, Guangchuan Wang, Matthew B. Dong, Jianjian Guo, Ryan D. Chow, Lei Peng, Jonathan J. Park, Youssef Errami, and Yaying Du
- Subjects
Male ,Adoptive cell transfer ,medicine.medical_treatment ,T cell ,Protein Disulfide-Isomerases ,Biomedical Engineering ,Transposases ,Receptors, Cell Surface ,Bioengineering ,CD8-Positive T-Lymphocytes ,Biology ,N-Acetylglucosaminyltransferases ,Immunotherapy, Adoptive ,Applied Microbiology and Biotechnology ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,Antigen ,Antigens, CD ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Cytotoxic T cell ,Guide RNA ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Membrane Proteins ,Immunotherapy ,Dependovirus ,Xenograft Model Antitumor Assays ,Lymphocyte Activation Gene 3 Protein ,Neoplasm Proteins ,3. Good health ,Cell biology ,medicine.anatomical_structure ,Molecular Medicine ,Female ,CRISPR-Cas Systems ,Glioblastoma ,030217 neurology & neurosurgery ,CD8 ,RNA, Guide, Kinetoplastida ,Biotechnology - Abstract
Targeting membrane proteins could improve the efficacy of T cell-based immunotherapies. To facilitate the identification of T cell targets, we developed a hybrid genetic screening system where the Sleeping Beauty (SB) transposon and single guide RNA cassette are nested in an adeno-associated virus (AAV). SB-mediated genomic integration of the single guide RNA cassette enables efficient gene editing in primary murine T cells as well as a screen readout. We performed in vivo AAV-SB-CRISPR screens for membrane protein targets in CD8+ T cells in mouse models of glioblastoma (GBM). We validated screen hits by demonstrating that adoptive transfer of CD8+ T cells with Pdia3, Mgat5, Emp1 or Lag3 gene editing enhances the survival of GBM-bearing mice in both syngeneic and T-cell receptor transgenic models. Transcriptome profiling, single cell sequencing, cytokine assays and T cell signaling analysis showed that Pdia3 editing in T cells enhances effector functions. Engineered PDIA3 mutant EGFRvIII chimeric antigen T cells are more potent in antigen-specific killing of human GBM cells.
- Published
- 2019
19. In vivo profiling of metastatic double knockouts through CRISPR–Cpf1 screens
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Guangchuan Wang, Adan Codina, Li Shen, Hyunu Ray Kim, Ryan D. Chow, Sidi Chen, Youssef Errami, Lupeng Ye, and Matthew B. Dong
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CRISPR/Cpf1 ,Sequence analysis ,Computational biology ,Biology ,Biochemistry ,Article ,Metastasis ,Gene Knockout Techniques ,Mice ,03 medical and health sciences ,Bacterial Proteins ,In vivo ,CRISPR-Associated Protein 9 ,Cell Line, Tumor ,medicine ,Animals ,Clustered Regularly Interspaced Short Palindromic Repeats ,Neoplasm Metastasis ,Molecular Biology ,Gene ,Gene knockout ,030304 developmental biology ,Gene Editing ,Trans-activating crRNA ,0303 health sciences ,Sequence Analysis, RNA ,Cell Biology ,Endonucleases ,medicine.disease ,Phenotype ,CRISPR-Cas Systems ,Biotechnology - Abstract
Systematic investigation of the genetic interactions that influence metastatic potential has been challenging. Here we developed massively parallel CRISPR–Cpf1/Cas12a crRNA array profiling (MCAP), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions that drive metastasis. Massively parallel Cpf1 array profiling (MCAP) targets genes and gene pairs that are candidate drivers of metastasis in cancer. In vivo profiling of single and double gene knockouts enables quantitative mapping of the genes’ contribution to metastatic phenotypes.
- Published
- 2019
20. CRISPR-GEMM pooled mutagenic screening identifies KMT2D as a major modulator of immune checkpoint blockade
- Author
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Zhiyuan Chu, Paul A. Renauer, Leilei Niu, Lupeng Ye, Xiaoyun Dai, Lvyun Zhu, Matthew B. Dong, Yujing Cheng, Guangchuan Wang, Zhigang Bai, Yaying Du, Youssef Errami, Cun Liao, Xiaoya Zhang, Paul R. Clark, Ryan D. Chow, Kristin Kim, Sidi Chen, and Feifei Zhang
- Subjects
0301 basic medicine ,Antigen presentation ,Mutagenesis (molecular biology technique) ,Biology ,medicine.disease_cause ,Chromatin remodeling ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,medicine ,CRISPR ,Animals ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Immune Checkpoint Inhibitors ,Tumor microenvironment ,Mutation ,Cancer ,medicine.disease ,Immune checkpoint ,Neoplasm Proteins ,DNA-Binding Proteins ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Cancer research - Abstract
Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy in several cancer types. However, only a fraction of patients will respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMM) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types. KMT2D encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in patients with cancer. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. In addition, Kmt2d-mutant cells exhibited increased protein turnover and IFNγ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human were characterized by increased immune infiltration. These data demonstrate that Kmt2d deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, and also highlight the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment.Significance:ICB is ineffective in the majority of patients. Through direct in vivo CRISPR mutagenesis screening in GEMMs of cancer, we find Kmt2d deficiency sensitizes tumors to ICB. Considering the prevalence of KMT2D mutations, this finding potentially has broad implications for patient stratification and clinical decision-making.This article is highlighted in the In This Issue feature, p. 1775
- Published
- 2020
21. Nonstructural protein 1 of SARS-CoV-2 is a potent pathogenicity factor redirecting host protein synthesis machinery toward viral RNA
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Swapnil C. Devarkar, Yong Xiong, Shenping Wu, Ivan B. Lomakin, Sidi Chen, Lei Peng, Yingxia Hu, Jonathan J. Park, Matthew B. Dong, and Shuai Yuan
- Subjects
Messenger RNA ,NSP1 ,Internal ribosome entry site ,viruses ,Gene expression ,Protein biosynthesis ,virus diseases ,RNA ,Translation (biology) ,Biology ,Ribosome ,Cell biology - Abstract
SummaryThe COVID-19 pandemic affects millions of people worldwide with a rising death toll. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), uses its nonstructural protein 1 (Nsp1) to redirect host translation machinery to the viral RNA by binding to the ribosome and suppressing cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the transcriptome in human cells. The changes include repression of major gene clusters in ribosomal RNA processing, translation, mitochondria function, cell cycle and antigen presentation; and induction of factors in transcriptional regulation. We further gained a mechanistic understanding of the Nsp1 function by determining the cryo-EM structure of the Nsp1-40S ribosomal subunit complex, which shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the cryo-EM structure of the 48S preinitiation complex (PIC) formed by Nsp1, 40S, and the cricket paralysis virus (CrPV) internal ribosome entry site (IRES) RNA, which shows that this 48S PIC is nonfunctional due to the incorrect position of the 3’ region of the mRNA. Results presented here elucidate the mechanism of host translation inhibition by SARS-CoV-2, provide insight into viral protein synthesis, and furnish a comprehensive understanding of the impacts from one of the most potent pathogenicity factors of SARS-CoV-2.HighlightsORF screen identified Nsp1 as a major cellular pathogenicity factor of SARS-CoV-2Nsp1 broadly alters the gene expression programs in human cellsNsp1 inhibits translation by blocking mRNA entry channelNsp1 prevents physiological conformation of the 48S PIC
- Published
- 2020
22. AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma
- Author
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Phillip A. Sharp, Florian I. Schmidt, Sensen Zhang, Randall Jeffrey Platt, Feng Zhang, Lupeng Ye, Murat Gunel, Matthew B. Dong, Sidi Chen, Ryan D. Chow, Youssef Errami, Paul A. Renauer, Guangchuan Wang, Michael A. Q. Martinez, Mark W. Youngblood, Christopher D. Guzman, Kaya Bilguvar, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Biology, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Koch Institute for Integrative Cancer Research at MIT, Sharp, Phillip A, and Zhang, Feng
- Subjects
0301 basic medicine ,Male ,Mutant ,DNA Mutational Analysis ,medicine.disease_cause ,Article ,03 medical and health sciences ,Gene Knockout Techniques ,Mice ,Suppression, Genetic ,medicine ,Temozolomide ,PTEN ,CRISPR ,Animals ,Humans ,Gene Knock-In Techniques ,Gene ,Cells, Cultured ,Genetics ,Mutation ,biology ,Brain Neoplasms ,General Neuroscience ,Dependovirus ,eye diseases ,3. Good health ,Dacarbazine ,030104 developmental biology ,biology.protein ,Homeobox ,Female ,CRISPR-Cas Systems ,Glioblastoma ,Transcriptome ,medicine.drug - Abstract
A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo.
- Published
- 2017
23. Nonstructural Protein 1 of SARS-CoV-2 Is a Potent Pathogenicity Factor Redirecting Host Protein Synthesis Machinery toward Viral RNA
- Author
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Yingxia Hu, Yong Xiong, Lei Peng, Swapnil C. Devarkar, Shenping Wu, Jonathan J. Park, Qi Shen, Matthew B. Dong, Shuai Yuan, Ivan B. Lomakin, and Sidi Chen
- Subjects
viruses ,Viral Nonstructural Proteins ,Biology ,Ribosome ,Article ,03 medical and health sciences ,0302 clinical medicine ,Chlorocebus aethiops ,Protein biosynthesis ,Nsp1 ,Animals ,Humans ,RNA, Messenger ,Cricket paralysis virus ,Vero Cells ,Molecular Biology ,cell viability ,030304 developmental biology ,Ribosome Subunits, Small, Eukaryotic ,0303 health sciences ,NSP1 ,Messenger RNA ,SARS-CoV-2 ,transcriptome alteration ,Cryoelectron Microscopy ,COVID-19 ,virus diseases ,RNA ,Translation (biology) ,Cell Biology ,biology.organism_classification ,Cell biology ,Internal ribosome entry site ,ribosome ,Protein Biosynthesis ,cryo-EM ,RNA, Viral ,translation inhibition mechanism ,030217 neurology & neurosurgery - Abstract
The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3′ region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2., Graphical Abstract, Highlights • ORF screen identified Nsp1 as a major cellular pathogenicity factor of SARS-CoV-2 • Nsp1 broadly alters the gene expression programs in human cells of lung origin • Nsp1 inhibits translation by blocking mRNA entry channel on the 40S ribosome • Nsp1 prevents physiological conformation of the 48S preinitiation complex (PIC), Yuan et al. used functional and cryo-EM studies to show that SARS-CoV-2 Nsp1 significantly reduces cell viability, induces extensive transcriptome alteration, and blocks host mRNA access to the ribosome. These results help understand how Nsp1 suppresses host gene expression and its broad impact as a comorbidity-inducing factor.
- Published
- 2020
24. Despite Increased Type 1 IFN, Autoimmune Nonobese Diabetic Mice Display Impaired Dendritic Cell Response to CpG and Decreased Nuclear Localization of IFN-Activated STAT1
- Author
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Yongge Zhao, Chie Hotta-Iwamura, M. Jubayer Rahman, Alan D. Guerrero, Matthew B. Dong, Kameron B. Rodrigues, Yi Chen, Kristin V. Tarbell, and Gwendoline Rahir
- Subjects
0301 basic medicine ,Regulatory T cell ,Blotting, Western ,Immunology ,Active Transport, Cell Nucleus ,chemical and pharmacologic phenomena ,Nod ,Biology ,Real-Time Polymerase Chain Reaction ,Article ,Monocytes ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Mice, Inbred NOD ,medicine ,Animals ,Immunology and Allergy ,Cell Lineage ,Oligonucleotide Array Sequence Analysis ,NOD mice ,Cell Nucleus ,CD86 ,Microscopy, Confocal ,Innate immune system ,Monocyte ,TLR9 ,hemic and immune systems ,Dendritic Cells ,Dendritic cell ,Flow Cytometry ,Immunity, Innate ,Mice, Inbred C57BL ,Diabetes Mellitus, Type 1 ,STAT1 Transcription Factor ,Self Tolerance ,030104 developmental biology ,medicine.anatomical_structure ,Oligodeoxyribonucleotides ,Toll-Like Receptor 9 ,Interferon Type I ,030215 immunology - Abstract
Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF dendritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reflect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1−/− mice also display a defect in CpG-induced CD86 upregulation compared with B6 Rag1−/−, indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-α/β receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti–IFN-α/β receptor Ab is added. IFN-α–induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c+ cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but diminished cDC type 1 IFN response.
- Published
- 2016
25. Convergent Identification and Interrogation of Tumor-Intrinsic Factors that Modulate Cancer Immunity In Vivo
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Dhanpat Jain, Adan Codina, Jesse Rinehart, Roy S. Herbst, Kerou Zhang, Jonathan J. Park, Guangchuan Wang, Hanghui Ye, Youssef Errami, Lupeng Ye, Marcus Bosenberg, Matthew B. Dong, Alan Chang, Paul A. Renauer, Ryan D. Chow, Brandon M. Gassaway, Rong Fan, Sidi Chen, and Li Shen
- Subjects
0303 health sciences ,Tumor microenvironment ,Histology ,Gene Expression Profiling ,Cell Biology ,Biology ,medicine.disease_cause ,Article ,Pathology and Forensic Medicine ,Cell biology ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Neoplasms ,Proteome ,Cancer cell ,medicine ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Carcinogenesis ,030217 neurology & neurosurgery ,030304 developmental biology ,Cancer immunology - Abstract
Summary The genetic makeup of cancer cells directs oncogenesis and influences the tumor microenvironment. In this study, we massively profiled genes that functionally drive tumorigenesis using genome-scale in vivo CRISPR screens in hosts with different levels of immunocompetence. As a convergent hit from these screens, Prkar1a mutant cells are able to robustly outgrow as tumors in fully immunocompetent hosts. Functional interrogation showed that Prkar1a loss greatly altered the transcriptome and proteome involved in inflammatory and immune responses as well as extracellular protein production. Single-cell transcriptomic profiling and flow cytometry analysis mapped the tumor microenvironment of Prkar1a mutant tumors and revealed the transcriptomic alterations in host myeloid cells. Taken together, our data suggest that tumor-intrinsic mutations in Prkar1a lead to drastic alterations in the genetic program of cancer cells, thereby remodeling the tumor microenvironment.
- Published
- 2018
26. Systematic Immunotherapy Target Discovery Using Genome-Scale In Vivo CRISPR Screens in CD8 T Cells
- Author
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Stanley Z. Lam, Yaying Du, Xiaoyun Dai, Christopher D. Guzman, Xiaoyu Zhou, Sidi Chen, Jonathan J. Park, Donald R. Lannin, Guangchuan Wang, Hyunu Ray Kim, Lupeng Ye, Jingjia J. Zhou, Matthew B. Dong, Roy S. Herbst, Paul A. Renauer, Ryan D. Chow, Lvyun Zhu, Krista Y. Chen, Youssef Errami, and Johanna Shen
- Subjects
Male ,Adoptive cell transfer ,medicine.medical_treatment ,Programmed Cell Death 1 Receptor ,Breast Neoplasms ,Biology ,CD8-Positive T-Lymphocytes ,Article ,General Biochemistry, Genetics and Molecular Biology ,Transcriptome ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Immune system ,Cancer immunotherapy ,Cell Line, Tumor ,medicine ,CRISPR ,Animals ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,030304 developmental biology ,Mice, Knockout ,0303 health sciences ,Genome ,NF-kappa B ,Immunotherapy ,RNA Helicase A ,Cell biology ,Cytokines ,Female ,CRISPR-Cas Systems ,Immunologic Memory ,030217 neurology & neurosurgery ,RNA Helicases ,Genetic screen ,RNA, Guide, Kinetoplastida - Abstract
CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings, and identified regulators of tumor infiltration and degranulation. The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8 T cells against triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.
- Published
- 2018
27. Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR–mediated direct in vivo screening
- Author
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Xiaoyun Dai, Lupeng Ye, Matthew B. Dong, Guangchuan Wang, Phillip A. Sharp, Sidi Chen, Christopher D. Guzman, Randall Jeffrey Platt, Ryan D. Chow, Feng Zhang, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Biology, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, McGovern Institute for Brain Research at MIT, Koch Institute for Integrative Cancer Research at MIT, Zhang, Feng, Sharp, Phillip A, Platt, Randall Jeffrey, and Chen, Sidi
- Subjects
0301 basic medicine ,Carcinogenesis ,viruses ,Genomics ,Biology ,medicine.disease_cause ,Molecular Inversion Probe ,Genome ,03 medical and health sciences ,Mice ,Neoplasms ,medicine ,CRISPR ,Animals ,Clustered Regularly Interspaced Short Palindromic Repeats ,Genes, Tumor Suppressor ,Genetic Testing ,Gene ,Research Articles ,Cancer ,Genetics ,Multidisciplinary ,Cas9 ,RNA ,SciAdv r-articles ,Dependovirus ,3. Good health ,030104 developmental biology ,Liver ,Mutation ,Research Article - Abstract
Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas somemutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct highthroughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adenoassociated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice. All mice that received the AAV-mTSG library developed liver cancer and diedwithin 4 months.We usedmolecular inversion probe sequencing of the sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence of multiple variants in driving liver tumorigenesis in immunocompetent mice. AAV-mediated autochthonous CRISPR screens provide a powerful means for mapping a provisional functional cancer genome atlas of tumor suppressors in vivo., Damon Runyon Cancer Research Foundation (DRG-2117-12; DFS-13-15), Melanoma Research Foundation (412806, 16-003524), St. Baldrick’s Foundation (426685), American Cancer Society (IRG 58-012-54), Cancer Research Institute (New York, N.Y.), Breast Cancer Alliance, Cancer Research Institute (New York, N.Y.) (Clinic and Laboratory Integration Program), American Association for Cancer Research (499395), United States. Department of Defense (W81XWH-17-1-0235), National Cancer Institute (U.S.) (1U54CA209992), National Cancer Institute (U.S.) (5P50CA196530-A10805), National Cancer Institute (U.S.) (4P50CA121974-A08306), National Institutes of Health (U.S.) (R01-CA133404), National Institutes of Health (U.S.) (R01-GM034277), National Institutes of Health (U.S.) (CCNE), Skolkovo Foundation, Casimir-Lambert Fund, National Institutes of Health (U.S.) (grant 1R01-HG009761), National Institutes of Health (U.S.) (grant R01-MH110049), National Institutes of Health (U.S.) (grant DP1-HL141201), Howard Hughes Medical Institute, New York Stem Cell Foundation, Simons, Paul G. Allen Family, Vallee Foundations, Poitras Center for Affective Disorders Research at MIT, Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT
- Published
- 2018
28. Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing
- Author
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Xiaoyun Dai, Randall Jeffrey Platt, Phillip A. Sharp, Feng Zhang, Guangchuan Wang, Ryan D. Chow, Christopher D. Guzman, Sidi Chen, Lupeng Ye, and Matthew B. Dong
- Subjects
Genetics ,0303 health sciences ,Cas9 ,Transgene ,Cancer ,Biology ,medicine.disease ,Molecular Inversion Probe ,3. Good health ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,030220 oncology & carcinogenesis ,medicine ,CRISPR ,Gene ,030304 developmental biology ,Subgenomic mRNA - Abstract
High-resolution, high-throughput direct in vivo screening of functional genetic factors in native tissues has long been challenging. Adeno-associated viruses (AAV) are powerful carriers of transgenes and have been shown to mediate efficient genome editing in various organs in mice. Here, we developed a new technological approach, Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing (PASTAS), and demonstrated its application for directly mapping functional cancer driver variants in the mouse liver in an autochthonous manner. Intravenous delivery of an AAV-CRISPR library targeting a set of the most frequently mutated tumor suppressor genes into fully immunocompetent conditional Cas9 knock-in mice consistently generated highly complex autochthonous liver tumors. The molecular landscapes of these genetically diverse tumors were mapped out by deep direct readout of Cas9-generated variants at predicted sgRNA cut sites using molecular inversion probe sequencing. Co-occurrence and correlation analyses as well as validation with lower complexity minipools further confirmed the potency of various co-mutated drivers. The PASTAS method can be applied to virtually any gene sets, any cancer types, or any type of in vivo genetic studies other than cancer.
- Published
- 2017
29. Direct in vivo mapping of functional suppressors in glioblastoma genome
- Author
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Randall Jeffrey Platt, Paul A. Renauer, Florian I. Schmidt, Sensen Zhang, Ryan D. Chow, Fan Zhang, Sidi Chen, Youssef Errami, Murat Gunel, Kaya Bilguvar, Lupeng Ye, Matthew B. Dong, Mark W. Youngblood, Christopher D. Guzman, Phillip A. Sharp, Guangchuan Wang, and Michael A. Q. Martinez
- Subjects
Zinc finger ,Genetics ,0303 health sciences ,Mutation ,biology ,Genomics ,medicine.disease_cause ,Phenotype ,3. Good health ,03 medical and health sciences ,0302 clinical medicine ,030220 oncology & carcinogenesis ,biology.protein ,medicine ,PTEN ,CRISPR ,Epigenetics ,Gene ,030304 developmental biology - Abstract
Glioblastoma (GBM) is one of the deadliest cancers, with limited effective treatments and single-digit five-year survival1-7. A causative understanding of genetic factors that regulate GBM formation is of central importance8-19. However, a global, quantitative and functional understanding of gliomagenesis in the native brain environment has been lacking due to multiple challenges. Here, we developed an adeno-associated virus (AAV) mediated autochthonous CRISPR screen and directly mapped functional suppressors in the GBM genome. Stereotaxic delivery of an AAV library targeting significantly mutated genes into fully immunocompetent conditional Cas9 mice robustly led to gliomagenesis, resulting in tumors that recapitulate features of human GBM. Targeted capture sequencing revealed deep mutational profiles with diverse patterns across mice, uncoveringin vivoroles of previously uncharacterized factors in GBM such as immune regulatorB2m,zinc finger proteinZc3h13,transcription repressorCic,epigenetic regulatorsMll2/3andArid1b,alongside canonical tumor suppressorsNf1andPten. Comparative cancer genomics showed that the mutation frequencies across all genes tested in mice significantly correlate with those in human from two independent patient cohorts. Co-mutation analysis identified frequently co-occurring driver combinations, which were validated using AAV minipools, such asMll2, B2m-Nf1,Mll3-Nf1andZc3h13-Rb1. Distinct fromNf1-oncotype tumors,Rb1-oncotype tumors exhibit undifferentiated histopathology phenotype and aberrant activation of developmental reprogramming signatures such asHomeoboxgene clusters. The secondary addition ofZc3h13orPtenmutations drastically altered the gene expression profiles ofRb1mutants and rendered them more resistant to the GBM chemotherapeutic temozolomide. Our study provides a systematic functional landscape of GBM suppressors directlyin vivo, opening new paths for high-throughput molecular mapping and cancer phenotyping.
- Published
- 2017
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30. Flow cytometric gating for spleen monocyte and DC subsets: differences in autoimmune NOD mice and with acute inflammation
- Author
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Kristin V. Tarbell, Matthew B. Dong, and M. Jubayer Rahman
- Subjects
0301 basic medicine ,Immunology ,Inflammation ,Autoimmunity ,Plasmacytoid dendritic cell ,Nod ,Cell Separation ,Biology ,medicine.disease_cause ,Monocytes ,Article ,Autoimmune Diseases ,Immunophenotyping ,03 medical and health sciences ,Interferon-gamma ,0302 clinical medicine ,Mice, Inbred NOD ,medicine ,Immunology and Allergy ,Animals ,Phosphorylation ,Antigen-presenting cell ,NOD mice ,Cluster of differentiation ,Monocyte ,Proteins ,Dendritic Cells ,Flow Cytometry ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,medicine.anatomical_structure ,Phenotype ,Oligodeoxyribonucleotides ,Toll-Like Receptor 9 ,Acute Disease ,Female ,medicine.symptom ,Protein Processing, Post-Translational ,Biomarkers ,Spleen ,030215 immunology - Abstract
The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR–ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation. We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3 DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice.
- Published
- 2015
31. Interleukin-2-mediated inhibition of dendritic cell development correlates with decreased CD135 expression and increased monocyte/macrophage precursors
- Author
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Yongge Zhao, Alan D. Guerrero, Kristin V. Tarbell, Matthew B. Dong, and Annie W. Lau-Kilby
- Subjects
Interleukin 2 ,STAT3 Transcription Factor ,Immunology ,Bone Marrow Cells ,Mice, Transgenic ,Biology ,Monocytes ,Mice ,Proto-Oncogene Proteins ,Gene expression ,medicine ,Immunology and Allergy ,Animals ,Phosphorylation ,Receptor ,Myeloid Progenitor Cells ,Bcl-2-Like Protein 11 ,Monocyte ,Gene Expression Profiling ,Macrophages ,Membrane Proteins ,hemic and immune systems ,Dendritic cell ,Original Articles ,Dendritic Cells ,Cell biology ,medicine.anatomical_structure ,BCL2L11 ,Gene Expression Regulation ,fms-Like Tyrosine Kinase 3 ,Cytokines ,Interleukin-2 ,Apoptosis Regulatory Proteins ,Ex vivo ,CD8 ,medicine.drug - Abstract
We have previously shown that interleukin-2 (IL-2) inhibits dendritic cell (DC) development from mouse bone marrow (BM) precursors stimulated with the ligand for FMS-like tyrosine kinase 3 receptor (Flt3L), and have provided evidence that this inhibition occurs at the monocyte DC precursor stage of DC development. Here, we explored the mechanism of IL-2-mediated inhibition of DC development. First, we showed that these in vitro cultures accurately model DCs that develop in vivo by comparing gene and protein expression of the three main Flt3L-induced DC subsets from the BM, CD11b(+) and CD24(+) conventional DCs (cDCs) and plasmacytoid DCs (pDCs) with their respective ex vivo spleen DC subsets (CD11b(+), CD8(+) and pDCs). Next, gene expression changes were quantified in Flt3L DC subsets that developed in the presence of IL-2. These changes included increased expression of Bcl2l11, which encodes the apoptosis-inducing protein Bim, and decreased expression of Flt3 (CD135), the receptor that initiates DC development. Interleukin-2 also significantly reduced Flt3 protein expression on all three Flt3L DC subsets, and attenuated Flt3L-induced STAT3 phosphorylation in DCs. Based on these data, we hypothesized that decreased Flt3 signalling may divert BM precursors down monocyte and macrophage lineages. Indeed, addition of IL-2 led to increases in Flt3(-) cells, including cKit(+) Ly6C(+) CD11b(-) populations consistent with the recently identified committed monocyte/macrophage progenitor. Therefore, IL-2 can inhibit DC development via decreased signalling through Flt3 and increased monocyte/macrophage development.
- Published
- 2014
32. Diacylglycerol Kinase ζ Limits B Cell Antigen Receptor–Dependent Activation of ERK Signaling to Inhibit Early Antibody Responses
- Author
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Xiao-Ping Zhong, Robert Brink, Matthew L. Wheeler, Anthony L. DeFranco, and Matthew B. Dong
- Subjects
MAPK/ERK pathway ,T-Lymphocytes ,Inbred Strains ,Inbred C57BL ,Lymphocyte Activation ,Biochemistry ,Mice ,Receptors ,Extracellular Signal-Regulated MAP Kinases ,Receptor ,Cancer ,Mice, Knockout ,B-Lymphocytes ,biology ,Blotting ,Kinase ,breakpoint cluster region ,Flow Cytometry ,Adoptive Transfer ,Antigen ,Second messenger system ,lipids (amino acids, peptides, and proteins) ,Antibody ,Western ,Diacylglycerol Kinase ,MAP Kinase Signaling System ,Knockout ,Blotting, Western ,Plasma Cells ,bcl-X Protein ,Receptors, Antigen, B-Cell ,Mice, Inbred Strains ,Article ,Diglycerides ,Vaccine Related ,Animals ,Molecular Biology ,Cell Proliferation ,Diacylglycerol kinase ,Prevention ,Inflammatory and immune system ,B-Cell ,Cell Biology ,Molecular biology ,Mice, Inbred C57BL ,Immunoglobulin M ,Antibody Formation ,biology.protein ,Muramidase ,Immunization ,Biochemistry and Cell Biology - Abstract
Signaling downstream of the B cell antigen receptor (BCR) is tightly regulated to enable cells to gauge the strength and duration of antigen-receptor interactions and to respond appropriately. We investigated whether metabolism of the second messenger diacylglycerol (DAG) by members of the family of DAG kinases (DGKs) played a role in modulating the magnitude of signaling by DAG downstream of the BCR. In the absence of DGKζ, the threshold for BCR signaling, measured as activation of the Ras-extracellular signal-regulated kinase (ERK) pathway, was markedly reduced in mature follicular B cells, which resulted in enhanced responses to antigen in vitro and in vivo. Inhibition of DAG signaling by DGKζ limited the number of antibody-secreting cells that were generated early in response to T cell-independent type 2 antigens, as well as to T cell-dependent antigens. Furthermore, the effect of loss of DGKζ closely resembled the effect of increasing the affinity of the BCR for antigen during the T cell-dependent antibody response. These results suggest that the magnitude of DAG signaling is important for translating the affinity of the BCR for antigen into the amount of antibody produced during the early stages of an immune response.
- Published
- 2013
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