Name: peptide:mhc tetramer-based enrichment of epitope-specific t cells
Uploaded: 2012-10-22T13:00:00
Description: Watch this Scientific Journal Video about Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells at JoVE.com

लेखकों के लिए इस प्रोटोकॉल के विकास में मदद के लिए आंद्रे हान और लॉरेंस येन तकनीकी सहायता के लिए और जेनकींस प्रयोगशाला के सदस्यों को धन्यवाद करना चाहते हैं.

1. Lymphoid ऊतक से सेल अलगाव <ol><li> बर्फ के ठंडे (EHAA + 10% FBS, कलम / strep, gentamycin, 2 मिमी एल glutamine, 55 मिमी 2-mercaptoethanol) cEHAA या अन्य समकक्ष टी सेल मध्यम 1 मिलीग्राम एक 60 मिमी संस्कृति का एक छोटा सा वर्ग युक्त पकवान, जोड़ें 100 सुक्ष्ममापी नायलॉ?...

<ol>
	<li>Knutson, K. L., dela Rosa, C., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+analysis+of+T-cell+immunity.">Laboratory analysis of T-cell immunity.</a> Front Biosci. 11, 1932-1944 (2006).</li><li>Kearney, E. R., Pape, K. A., Loh, D. Y., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+peptide-specific+T+cell+immunity+and+peripheral+tolerance+induction+in+vivo.">Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo.</a> Immunity. 1, 327-339 (1994).</li><li>Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+epitope-specific+T+cells.">Tracking epitope-specific T cells.</a> Nat Protoc. 4, 565-581 (2009).</li><li>Hataye, J., Moon, J. J., Khoruts, A., Reilly, C., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+and+memory+CD4++T+cell+survival+controlled+by+clonal+abundance.">Naive and memory CD4+ T cell survival controlled by clonal abundance.</a> Science. 312, 114-116 (2006).</li><li>Marzo, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+T+cell+frequency+dictates+memory+CD8++T+cell+lineage+commitment.">Initial T cell frequency dictates memory CD8+ T cell lineage commitment.</a> Nat Immunol. 6, 793-799 (2005).</li><li>Davis, M. M., Altman, J. D., Newell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+the+repertoire:+broadening+the+scope+of+peptide-MHC+multimer+analysis.">Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis.</a> Nature reviews. Immunology. 11, 551-558 (2011).</li><li>Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+CD4(+)+T+cell+frequency+varies+for+different+epitopes+and+predicts+repertoire+diversity+and+response+magnitude.">Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.</a> Immunity. 27, 203-213 (2007).</li><li>Seah, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linear+range+for+accurately+quantifying+antigen-specific+T-cell+frequencies+by+tetramer+staining+during+natural+immune+responses.">The linear range for accurately quantifying antigen-specific T-cell frequencies by tetramer staining during natural immune responses.</a> European Journal of Immunology. 41, 1499-1500 (2011).</li><li>Obar, J. J., Khanna, K. M., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+naive+CD8++T+cell+precursor+frequency+regulates+primary+and+memory+responses+to+infection.">Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection.</a> Immunity. 28, 859-869 (2008).</li><li>Daniels, M. A., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+for+CD8+in+T+cell+receptor+binding+and+activation+by+peptide/major+histocompatibility+complex+multimers.">Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.</a> J Exp Med. 191, 335-346 (2000).</li><li>Pittet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha+3+domain+mutants+of+peptide/MHC+class+I+multimers+allow+the+selective+isolation+of+high+avidity+tumor-reactive+CD8+T+cells.">Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.</a> Journal of Immunology. 171, 1844-1849 (2003).</li><li>Choi, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+avidity+antigen-specific+CTL+identified+by+CD8-independent+tetramer+staining.">High avidity antigen-specific CTL identified by CD8-independent tetramer staining.</a> Journal of Immunology. 171, 5116-5123 (2003).</li><li>Chu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+optimizes+the+number+and+function+of+MHCII-restricted+CD4++T+cell+clones+in+the+naive+polyclonal+repertoire.">Positive selection optimizes the number and function of MHCII-restricted CD4+ T cell clones in the naive polyclonal repertoire.</a> Proc Natl Acad Sci U S A. 106, 11241-11245 (2009).</li><li>Chu, H. H., Moon, J. J., Kruse, A. C., Pepper, M., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Selection+and+Peptide+Chemistry+Determine+the+Size+of+Naive+Foreign+Peptide-MHC+Class+II-Specific+CD4++T+Cell+Populations.">Negative Selection and Peptide Chemistry Determine the Size of Naive Foreign Peptide-MHC Class II-Specific CD4+ T Cell Populations.</a> J Immunol. 185, 4705-4713 (2010).</li><li>Legoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+TCR+reactivity+and+HLA+phenotype+on+naive+CD8+T+cell+frequency+in+humans.">Impact of TCR reactivity and HLA phenotype on naive CD8 T cell frequency in humans.</a> J Immunol. 184, 6731-6738 (2010).</li><li>Alanio, C., Lemaitre, F., Law, H. K., Hasan, M., Albert, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+human+antigen-specific+naive+CD8++T+cells+reveals+conserved+precursor+frequencies.">Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies.</a> Blood. 115, 3718-3725 (2010).</li><li>Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+Epitope-Specific+Naive+CD4++T+Cells+Correlates+with+Immunodominance+in+the+Human+Memory+Repertoire.">Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire.</a> Journal of Immunology. 188, 2537-2544 (2012).</li><li>Jenkins, M. K., Chu, H. H., McLachlan, J. B., Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+composition+of+the+preimmune+repertoire+of+T+cells+specific+for+Peptide-major+histocompatibility+complex+ligands.">On the composition of the preimmune repertoire of T cells specific for Peptide-major histocompatibility complex ligands.</a> Annu Rev Immunol. 28, 275-294 (2010).</li><li>Matechak, E. O., Killeen, N., Hedrick, S. M., Fowlkes, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-specific+T+cells+can+develop+in+the+CD8+lineage+when+CD4+is+absent.">MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent.</a> Immunity. 4, 337-347 (1996).</li><li>Burchill, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linked+T+cell+receptor+and+cytokine+signaling+govern+the+development+of+the+regulatory+T+cell+repertoire.">Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire.</a> Immunity. 28, 112-121 (2008).</li><li>Pepper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+routes+of+bacterial+infection+induce+long-lived+TH1+memory+cells+and+short-lived+TH17+cells.">Different routes of bacterial infection induce long-lived TH1 memory cells and short-lived TH17 cells.</a> Nature Immunology. 11, 83-89 (2010).</li></ol>

pMHC टेट्रामर आधारित सेल संवर्धन विधि इस प्रोटोकॉल द्वारा प्रस्तुत अंतर्जात टी सेल repertoires से मिलान विशेष टी कोशिकाओं का अध्ययन करने के लिए एक शक्तिशाली उपकरण है. मिलान विशेष टी कोशिकाओं के उनके TCRs की क्षमत?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; अभिकर्मक</strong</td&gt;<td<strong&gt; विक्रेता</strong</td&gt;<td<strong&gt; कैटलॉग संख्या</strong</td&gt;</tr&gt;<tr&gt;<td&gt; पीई या APC संयुग्मित pMHC टेट्रामर (या multimer)</td&gt;<td&gt; अन्वेषक द्वारा निर्मित, NIH टेट्रामर कोर से प्राप्त, या वाणिज्यिक स्रोतों से खरीदा</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; विरोधी पीई संयुग्मित चुंबकीय microbeads</td&gt;<td&gt; Miltenyi</td&gt;<td130-048-801&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; विरोधी APC संयुग्मित चुंबकीय microbeads</td&gt;<td&gt; Miltenyi</td&gt;<td130-090-855&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; रास चुंबकीय स्तंभों</td&gt;<td&gt; Miltenyi</td&gt;<td130-042-401&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; MidiMACS या चुंबक QuadroMACS</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-302 या 130-090-976</td&gt;</tr&gt;<tr&gt;<td&gt; सेल गिनती मोती</td&gt;<td&gt; जीवन टेक्नोलॉजीज</td&gt;<td&gt; पीसीबी-100</td&gt;</tr&gt;</tbody&gt;</table

peptide:mhc tetramer-based enrichment of epitope-specific t cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

चित्रा 1 प्रतिनिधि pMHCII टेट्रामर समृद्ध और अनुभवहीन चूहों से तिल्ली लिम्फ नोड के नमूने के प्रवाह cytometry भूखंडों को दर्शाया गया है, जबकि 2 चित्रा पहले प्रासंगिक पेप्टाइड + CFA के साथ प्रतिरक्षित चू...

Watch this Scientific Journal Video about Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells at JoVE.com

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

MHC tetramers और चुंबकीय microbeads मिलान विशेष टी कोशिकाओं की कम आवृत्ति आबादी को अलग करने और उन्हें प्रवाह cytometry द्वारा विश्लेषण: इस प्रोटोकॉल पेप्टाइड के उपयोग का वर्णन करता है. इस विधि अंतर्जात से ब्याज की टी सेल की आबादी के प्रत्यक्ष अध्ययन के लिए सक्षम बनाता है<em&gt; Vivo में</em&gt; प्रयोगात्मक सिस्टम.

मिलान विशेष टी कोशिकाओं की MHC tetramer आधारित संवर्धन: पेप्टाइड

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

माउस टी कोशिकाओं में टी सेल रिसेप्टर्स की रेट्रोवायरल transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

प्राथमिक मानव भोले और स्मृति टी कोशिकाओं से प्रेरित विनियामक टी कोशिकाओं की पीढ़ी

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

टी lymphocytes की ओर प्रेरित pluripotent स्टेम सेल की निर्देशित भेदभाव

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

piggyBac Transposon System Modification of Primary Human T Cells

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प्राथमिक मानव टी कोशिकाओं की piggyBac Transposon सिस्टम संशोधन

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

गुर्दे से डबल नकारात्मक αβ टी कोशिकाओं के अलगाव

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

टी कोशिकाओं की व्युत्पत्ति<em&gt; इन विट्रो</em&gt; माउस भ्रूणीय स्टेम कोशिकाओं से

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

परिधीय रक्त से मानव Alloantigen विशेष टी कोशिकाओं की पीढ़ी

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

साइटोकाइन कब्जा प्रणाली का उपयोग करते हुए नैदानिक ​​अनुप्रयोगों के लिए Cytomegalovirus-विशेष की स्वचालित सेल संवर्धन टी कोशिकाओं

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

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Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

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भोले सीडी 4 + टी कोशिकाओं से मानव सीडी 4 + Foxp3 + प्रेरित विनियामक टी कोशिकाओं (iTregs) एक TGF-β युक्त प्रोटोकॉल का उपयोग की इन विट्रो भेदभाव में

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...