Current Protocols in Immunology最新文献_第3页

T Cell Receptor Engineered Lymphocytes for Cancer Therapy T细胞受体工程淋巴细胞用于癌症治疗

Current Protocols in Immunology Pub Date : 2020-05-20 DOI: 10.1002/cpim.97

Meagan R. Rollins, Ellen J. Spartz, Ingunn M. Stromnes

{"title":"T Cell Receptor Engineered Lymphocytes for Cancer Therapy","authors":"Meagan R. Rollins, Ellen J. Spartz, Ingunn M. Stromnes","doi":"10.1002/cpim.97","DOIUrl":"10.1002/cpim.97","url":null,"abstract":"T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single-cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor-reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Generation of human autologous dendritic cells from monocytesBasic Protocol 2: In vitro priming and expansion of human antigen-specific T cellsBasic Protocol 3: Cloning of antigen-specific T cell receptors based on single-cell VDJ sequencing dataBasic Protocol 4: Validation of T cell receptor expression and functionality in vitroBasic Protocol 5: Rapid expansion of T cell receptor–transduced T cells and human T cell clones","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"129 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.97","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37957954","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Multispecific, Multivalent Antibody-Based Molecules Engineered on the DART® and TRIDENTTM Platforms 基于DART®和TRIDENTTM平台的多特异性、多价抗体分子

Current Protocols in Immunology Pub Date : 2020-04-15 DOI: 10.1002/cpim.95

Ling Huang, Kalpana Shah, Bhaswati Barat, Chia-Ying K. Lam, Sergey Gorlatov, Valentina Ciccarone, James Tamura, Paul A. Moore, Gundo Diedrich

{"title":"Multispecific, Multivalent Antibody-Based Molecules Engineered on the DART® and TRIDENTTM Platforms","authors":"Ling Huang, Kalpana Shah, Bhaswati Barat, Chia-Ying K. Lam, Sergey Gorlatov, Valentina Ciccarone, James Tamura, Paul A. Moore, Gundo Diedrich","doi":"10.1002/cpim.95","DOIUrl":"https://doi.org/10.1002/cpim.95","url":null,"abstract":"Multispecific antibodies bind two or more different antigens and enable new therapeutic applications that cannot be replicated with conventional monoclonal antibodies, such as bridging different cells or bringing soluble proteins in close proximity. The DART and TRIDENT platforms enable the engineering of such antibodies. A DART molecule combines two independent antigen‐binding sites in a stabilized, diabody‐like structure. A DART molecule can be expressed with or without an Fc domain and thus can be tailored to have a long or short half‐life in vivo and to induce or ablate effector function. Linking two DART units or a DART unit and a Fab domain (the latter structure is called TRIDENT format) via an Fc domain creates a monospecific, bispecific, trispecific, or tetraspecific molecule with up to tetravalent targeting of antigens. This article focuses on the design of DART and TRIDENT molecules that target two or three different antigens. © 2020 by John Wiley & Sons, Inc.","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"129 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.95","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92296171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Issue Information 问题信息

Current Protocols in Immunology Pub Date : 2020-03-01 DOI: 10.1002/cpim.79

引用次数: 0

Antibody-Based CAR T Cells Produced by Lentiviral Transduction. 慢病毒转导产生的基于抗体的CAR - T细胞

Current Protocols in Immunology Pub Date : 2020-03-01 DOI: 10.1002/cpim.93

Sabrina Prommersberger, Michael Hudecek, Thomas Nerreter

{"title":"Antibody-Based CAR T Cells Produced by Lentiviral Transduction.","authors":"Sabrina Prommersberger, Michael Hudecek, Thomas Nerreter","doi":"10.1002/cpim.93","DOIUrl":"https://doi.org/10.1002/cpim.93","url":null,"abstract":"One promising approach to treat hematologic malignancies is the usage of patient-derived CAR T cells. There are continuous efforts to improve the function of these cells, to optimize their receptor, and to use them for the treatment of additional types of cancer and especially solid tumors. In this protocol, an easy and reliable approach for CAR T cell generation is described. T cells are first isolated from peripheral blood (here: leukoreduction system chambers) and afterwards activated for one day with anti-CD3/CD28 Dynabeads. The gene transfer is performed by lentiviral transduction and gene transfer rate can be verified by flowcytometric analysis. Six days after transduction, the stimulatory Dynabeads are removed. T cells are cultured in interleukin-2 conditioned medium for several days for expansion. There is an option to expand CAR T cells further by co-incubation with irradiated, antigen-expressing feeder cell lines. The CAR T cells are ready to use after 10 (without feeder cell expansion) to 24 days (with feeder cell expansion). © 2020 The Authors. Basic Protocol: Generation of CAR T cells by lentiviral transduction.","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"128 1","pages":"e93"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.93","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37719560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Purification of T Cell Populations T细胞群的纯化

Current Protocols in Immunology Pub Date : 2020-02-10 DOI: 10.1002/cpim.94

Ivan J. Fuss

引用次数: 2

Isolation and Analysis of Tumor‐Derived Exosomes 肿瘤衍生外泌体的分离与分析

Current Protocols in Immunology Pub Date : 2019-12-01 DOI: 10.1002/cpim.91

N. Ludwig, Chang‐Sook Hong, S. Ludwig, J. Azambuja, Priyanka Sharma, M. Theodoraki, T. Whiteside

{"title":"Isolation and Analysis of Tumor‐Derived Exosomes","authors":"N. Ludwig, Chang‐Sook Hong, S. Ludwig, J. Azambuja, Priyanka Sharma, M. Theodoraki, T. Whiteside","doi":"10.1002/cpim.91","DOIUrl":"https://doi.org/10.1002/cpim.91","url":null,"abstract":"A method for isolation of exosomes from tumor cell supernatants or cancer patients’ plasma is presented. Tumor‐derived exosomes (TEX) are defined as a subset of extracellular vesicles (EVs) sized at 30 to 150 nm and originating from multivesicular bodies (MVBs). The method utilizes size exclusion chromatography (SEC) for recovery of exosomes from cell‐line supernatants or cancer patients’ plasma. The recovered exosomes are morphologically intact, aggregate‐free, and functionally competent. Their molecular content parallels that of the parent tumor cells and they carry various immunoregulatory ligands known to modulate functions of immune cells. All exosomes isolated from tumor cell lines are TEX, while those isolated from plasma of cancer patients have to be fractionated into TEX and non‐TEX. Mini‐SEC allows for exosome isolation and recovery in quantities sufficient for molecular profiling, functional studies, and, in the case of plasma, further fractionation into TEX and non‐TEX. The mini‐SEC method can also be used for comparative studies of the exosome content in serial specimens of cancer patients’ body fluids. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.91","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42561826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 49

Isolation and Analysis of Mouse and Human Skin γδ T Cells 小鼠和人皮肤γδT细胞的分离与分析

Current Protocols in Immunology Pub Date : 2019-12-01 DOI: 10.1002/cpim.92

Shannon Gargas, Savannah Bshara-Corson, M. Cruz, J. Jameson

引用次数: 1

Issue Information TOC 发布信息TOC

Current Protocols in Immunology Pub Date : 2019-12-01 DOI: 10.1002/cpim.68

引用次数: 0

Characterization of Human Mucosal‐associated Invariant T (MAIT) Cells 人粘膜相关不变性T (MAIT)细胞的表征

Current Protocols in Immunology Pub Date : 2019-12-01 DOI: 10.1002/cpim.90

Michael N. T. Souter, L. Loh, Shihan Li, Bronwyn S. Meehan, N. Gherardin, D. Godfrey, J. Rossjohn, D. Fairlie, K. Kedzierska, D. Pellicci, Zhenjun Chen, L. Kjer-Nielsen, A. Corbett, J. McCluskey, S. Eckle

{"title":"Characterization of Human Mucosal‐associated Invariant T (MAIT) Cells","authors":"Michael N. T. Souter, L. Loh, Shihan Li, Bronwyn S. Meehan, N. Gherardin, D. Godfrey, J. Rossjohn, D. Fairlie, K. Kedzierska, D. Pellicci, Zhenjun Chen, L. Kjer-Nielsen, A. Corbett, J. McCluskey, S. Eckle","doi":"10.1002/cpim.90","DOIUrl":"https://doi.org/10.1002/cpim.90","url":null,"abstract":"Mucosal‐associated invariant T (MAIT) cells are a subset of unconventional T cells restricted by the major histocompatibility complex (MHC) class I–like molecule MHC‐related protein 1 (MR1). MAIT cells are found throughout the body, especially in human blood and liver. Unlike conventional T cells, which are stimulated by peptide antigens presented by MHC molecules, MAIT cells recognize metabolite antigens derived from an intermediate in the microbial biosynthesis of riboflavin. MAIT cells mediate protective immunity to infections by riboflavin‐producing microbes via the production of cytokines and cytotoxicity. The discovery of stimulating MAIT cell antigens allowed for the development of an analytical tool, the MR1 tetramer, that binds specifically to the MAIT T cell receptor (TCR) and is becoming the gold standard for identification of MAIT cells by flow cytometry. This article describes protocols to characterize the phenotype of human MAIT cells in blood and tissues by flow cytometry using fluorescently labeled human MR1 tetramers alongside antibodies specific for MAIT cell markers. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.90","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47161869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Characterization and Purification of Mouse Mucosal‐Associated Invariant T (MAIT) Cells 小鼠粘膜相关不变性T细胞(MAIT)的鉴定和纯化

Current Protocols in Immunology Pub Date : 2019-09-23 DOI: 10.1002/cpim.89

Zhenjun Chen, Huimeng Wang, C. D’Souza, H. Koay, Bronwyn S. Meehan, Zhe Zhao, Troi J Pediongco, M. Shi, Tianyuan Zhu, Bingjie Wang, L. Kjer-Nielsen, S. Eckle, J. Rossjohn, D. Fairlie, D. Godfrey, R. Strugnell, J. McCluskey, A. Corbett

引用次数: 11