The authors would like to thank Andre Han and Lawrence Yen for technical assistance, and members of the Jenkins lab for help in the development of this protocol.

1. Cell Isolation from Lymphoid Tissue
<ol>
 <li>Add 1 ml of ice cold cEHAA (EHAA + 10% FBS, pen/strep, gentamycin, 2 mM L-glutamine, 55 mM 2-mercaptoethanol) or other equivalent T cell medium, to a 60 mm culture dish containing a small square of 100 &mu;m nylon mesh and place on ice.</li>
 <li>Euthanize mouse.</li>
 <li>Remove the spleen and as many easily accessible lymph nodes as possible. These should include at least the inguinal, axillary, brachial, cervical, and mesenteric lymph nodes. Place them on top of the n...

<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>

The pMHC tetramer based cell enrichment method presented by this protocol is a powerful tool for studying epitope-specific T cells from endogenous T cell repertoires. The use of pMHC tetramers enables detection of epitope-specific T cells based directly on the ability of their TCRs to bind cognate pMHC ligands. The enrichment provides a level of sensitivity such that extremely rare populations of antigen-specific T cells can be detected from endogenous repertoires of T cells without any manipulation of their gene...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Vendor</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td>PE or APC conjugated pMHC tetramer (or multimer)</td>
 <td>Made by investigator, obtained from the NIH tetramer core, or purchased from commercial sources</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-PE conjugated magnetic microbeads</td>
 <td>Miltenyi</td>
 <td>130-048-801</td>
 </tr>
 <tr>
 <td>Anti-APC conjugated magnetic microbeads</td>
 <td>Miltenyi</td>
 <td>130-090-855</td>
 </tr>
 <tr>
 <td>LS magnetic columns</td>
 <td>Miltenyi</td>
 <td>130-042-401</td>
 </tr>
 <tr>
 <td>MidiMACS or QuadroMACS magnet</td>
 <td>Miltenyi</td>
 <td>130-042-302 or 130-090-976</td>
 </tr>
 <tr>
 <td>Cell counting beads</td>
 <td>Life Technologies</td>
 <td>PCB-100</td>
 </tr>
 </tbody>
 </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.

Figure 1 depicts representative flow cytometry plots of pMHCII tetramer enriched spleen and lymph node samples from naive mice, while Figure 2 depicts representative data for mice previously immunized with the relevant peptide+CFA. Serial gating removes autofluorescent and other unwanted events from the analysis of CD4+ T cell populations. The CD8+ T cell population serves as a useful internal negative control for pMHCII tetramer staining of CD4+ T cells. Note that bound fractions from t...

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

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

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 ...

peptidemhc-tetramer-based-enrichment-of-epitope-specific-t-cells

Research

JoVE Journal

Immunology and Infection

17.7K ビュー.  Massachusetts General Hospital and Harvard Medical School. エピトープ特異的T細胞の低周波の集団を区別し、フローサイトメトリーによって、それらを分析するためのMHCテトラマーと磁性マイクロビーズ：このプロトコルは、ペプチドの使用について説明します。この方法は、から対象の内因性T細胞集団の直接調査を可能に実験システム。

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

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry.  This method enables the direct study of endogenous T cell populations of interest from in vivo experimental...

analysis of simian immunodeficiency virus-specific cd8+ t-cells in rhesus macaques by peptide-mhc-i tetramer staining

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

a peptide-mhc-i tetramer staining technique to analyze siv-specific cd8+ memory t cells

This video demonstrates a method for characterizing CD8+ memory T cells specific to simian immunodeficiency virus (SIV) using peptide-major histocompatibility complex class I (pMHC-I) tetramers. A pMHC-I tetramer is a complex of four MHC-I molecules bound to SIV-specific peptides and conjugated to a fluorophore. These tetramers selectively bind to T cell receptors (TCRs) on SIV-specific CD8+ T cells, facilitating the characterization of these...

A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8+ Memory T Cells

The correct amount of titrated peptide MHC tetramer must be diluted enough master mix to stain all of the experimental tubes. Therefore, it is important to prepare a 15% excess of the master mix to account for pipetting...

simultaneous quantification of anti-vector and anti-transgene-specific cd8+ t cells via mhc i tetramer staining after vaccination with a viral vector

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy...

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

in situ mhc-tetramer staining and quantitative analysis to determine the location, abundance, and phenotype of antigen-specific cd8 t cells in tissues

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

visualizing antigen specific cd4+ t cells using mhc class ii tetramers

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.<br...

nanoparticle-based enrichment and expansion of antigen-specific cd8+ t cells

This video describes a protocol for isolating rare-antigen-specific CD8+ T cells using magnetic nanoparticle-based artificial antigen-presenting cells (aAPCs) functionalized with antigenic peptide-loaded MHC-Igs and anti-CD28 antibodies. Interaction of the aAPCs with CD8+ T cells specifically expressing the antigenic peptide-targeting T cell receptors, followed by their expansion in vitro, results in the enrichment and expansion of this CD8+ T cell...

Nanoparticle-Based Enrichment and Expansion of Antigen-Specific CD8+ T Cells

After counting, collect the isolated CD8-positive T cells by centrifugation, and resuspend the pellet in 100 microliters of PBS, supplemented with 0.5% bovine serum albumin and 2-millimolar...

evaluation of controlled t cell activation with a photoactivatable peptide mhc

This video demonstrates a method of decaging a photoactivatable peptide-major histocompatibility complex using UV irradiation. The decaging process exposes the native peptide sequence, enabling precise T cell...

Evaluation of Controlled T Cell Activation with a Photoactivatable Peptide MHC

Block bio-PLL-coated surfaces with blocking buffer consisting of HEPES-buffered saline with 2% BSA for 30 minutes at room temperature. Following the incubation, remove the blocking buffer from the bio-PLL-coated surfaces without allowing the wells to dry. Then, add streptavidin, and incubate at 4 degrees...

zika virus specific diagnostic epitope discovery

Zika Virus Specific Diagnostic Epitope Discovery

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious...

analysis of hbv-specific cd4 t-cell responses and identification of hla-dr-restricted cd4 t-cell epitopes based on a peptide matrix

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell...

automated cell enrichment of cytomegalovirus-specific t cells for clinical applications using the cytokine-capture system

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

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to...

generation of human alloantigen-specific t cells from peripheral blood

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC...

phosphopeptide enrichment: an antibody-based immunoprecipitation technique to separate specific phosphorylated peptides from complex peptide mixtures

After using P200 with a cut tip to transfer the 0.5 milligrams per milliliter stock of antibody bead slurry to the microfuge tube, add 450 microliters of ice-cold IP binding buffer to wash 50 microliters of 4G10 antibody bead slurry and 25 microliters of 27B10.4 antibody bead slurry in a microfuge tube. Centrifuge the tube at 100 x g and 4 degrees Celsius for 1 minute. After aspirating the supernatant, add an equal volume as the original slurry of IP binding buffer to resuspend the beads. Add...

Phosphopeptide Enrichment: An Antibody-based Immunoprecipitation Technique to Separate Specific Phosphorylated Peptides from Complex Peptide Mixtures

In this video, we demonstrate antibody-based immunoprecipitation of phosphopeptides from a peptide mixture. The enriched phosphopeptides are vacuum concentrated and stored for downstream...

identification of rare antigen-specific t cells from mouse lungs with peptide:major histocompatibility complex tetramers

Author Spotlight: Optimized Protocol for Detecting Antigen-Specific T Cells in Mouse Lungs Using Tetramers

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC)...

Isolation and Characterization of Antigen-Specific T Cells in Mouse Lungs

https://cloudfront.jove.com/CDNSource/teasers/66939_b.jpg

a high throughput mhc ii binding assay for quantitative analysis of peptide epitopes

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and...

quantification of proliferating human antigen-specific cd4+ t cells using carboxyfluorescein succinimidyl ester

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

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

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 presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral 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

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

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

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

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

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

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

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

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

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

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

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

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

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.