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 nylon mesh in the culture dish.</li>
 <li>Using the flat top of a closed 1.5 ml microfuge tube, gently mash the lymphoid tissue over the nylon mesh to liberate lymphocytes. Add another 1 ml of cEHAA and pipet up and down to work cells into a suspension. Transfer the cells through another piece of nylon mesh placed over the top of a 15 ml polypropylene centrifuge tube. Rinse the dish and mesh with another 1 ml of ice cold cEHAA, pooling the volumes into the same tube. Repeat to achieve a final cell suspension volume of 4 ml.</li>
 <li>Add cold sorter buffer (PBS + 2% FBS, 0.05% Azide) to a final volume of 15 ml and centrifuge the tube for 5 min at 300 x g, 4 &deg;C.</li>
 <li>Carefully aspirate the supernatant, making sure no droplets of liquid are left on the sides of the tube. Resuspend the cell pellet in Fc block (sorter buffer + 2.4G2 antibody) to a final volume equal to approximately twice that of the pellet itself. For example, the spleen and lymph nodes of a naive mouse usually produces a cell pellet of about 100 &mu;l. In this case, add 100 &mu;l of Fc block to bring the volume to 200 &mu;l. If a large degree of cell clumping has occurred, carefully remove the cell clump at this point with a pipet tip.</li>
</ol>
2. Tetramer Staining
<ol>
 <li>Add PE- or APC-labeled pMHC tetramer to a final concentration of 10 nM (or empirically optimized concentration).</li>
 <li>Mix and incubate for 1 hr at room temperature (or empirically optimized time and temperature).</li>
 <li>Add cold sorter buffer to a volume of 15 ml and centrifuge the tube for 5 min at 300 x g, 4 &deg;C. Keep samples on ice or at 4 &deg;C from now on.</li>
 <li>Carefully aspirate the supernatant, making sure no droplets of liquid are left on the sides of the tube. Resuspend the cell pellet in sorter buffer to a final volume of 200 &mu;l. For double tetramer enrichment, resuspend to a final volume of 150 &mu;l.</li>
</ol>
3. Magnetic Enrichment
<ol>
 <li>Add 50 &mu;l of Miltenyi anti-PE or anti-APC microbeads. For double tetramer enrichment, add 50 &mu;l of both.</li>
 <li>Mix and incubate for 20 min at 4 &deg;C.</li>
 <li>Add cold sorter buffer to a volume of 15 ml and centrifuge the tube for 5 min at 300 x g, 4 &deg;C.</li>
 <li>While waiting, set up a Miltenyi LS magnetic column on a MidiMACS or QuadroMACS magnet. Position an open 15 ml polypropylene centrifuge tube on a rack directly underneath the column.</li>
 <li>Add 3 ml of sorter buffer to the top of the column, allowing it to drain into the 15 ml tube.</li>
 <li>Place a 100 &mu;m nylon mesh square on top of the column.</li>
 <li>When cells have finished spinning, carefully aspirate the supernatant and resuspend the pellet in 3 ml of sorter buffer.</li>
 <li>Transfer the cell suspension through the nylon mesh onto the top of the column.</li>
 <li>When the cell suspension has completely drained into the column, rinse the original tube with another 3 ml of sorter buffer and transfer the buffer through the nylon mesh into the column, rinsing the mesh in the process. Discard the nylon mesh.</li>
 <li>When the buffer has completely drained into the column, add another 3 ml of sorter buffer to the column.</li>
 <li>Repeat step 10 for a total of 3 x 3 ml washes.</li>
 <li>Remove the column from the magnet and place over a new 15 ml polypropylene centrifuge tube.</li>
 <li>Add 5 ml of sorter buffer to the column.</li>
 <li>Immediately insert the plunger into the top of the column and in one continuous motion, push the plunger all the way down, forcing the buffer out the column into the tube.</li>
 <li>Centrifuge the tubes containing the eluted bound fraction and the flow-through unbound fraction for 5 min at 300 x g, 4 &deg;C.</li>
 <li>Carefully aspirate the supernatant from the bound fraction, making sure no droplets of liquid are left on the sides of the tube. Resuspend the cell pellet in sorter buffer to a final volume of exactly 95 &mu;l. Aspirate and resuspend the unbound fraction to a final volume of 2 ml.</li>
</ol>
4. Flow Cytometry
<ol>
 <li>For each fraction, remove 5 &mu;l and add to 200 &mu;l of counting beads (adjusted to a concentration of 200,000/ml in sorter buffer) in a 5 ml FACS tube. Set aside at 4 &deg;C for analysis later. To save time, beads can be pre-aliquoted to labeled tubes before the start of the experiment.</li>
 <li>Prepare a master mix of antibodies to stain surface markers on the cells (Table 1). To save time, this can be done prior to the start of the experiment.</li>
 <li>For the bound fraction, add a dose of antibody cocktail directly to the ~90 &mu;l of cells in the tube. If analysis of the unbound fraction is desired, transfer 90 &mu;l of cells to a 5 ml FACS tube and add a dose of antibody cocktail.</li>
 <li>Set up a panel of single-color compensation controls for the flow cytometer. For each fluorochrome to be used, mix 50 &mu;l of leftover unbound fraction cells in a 5 ml FACS tube with 1 &mu;l of anti-CD4 antibody conjugated to the corresponding fluorochrome. Remember to set up an unstained control as well.</li>
 <li>Vortex and incubate all samples for 30 min at 4 &deg;C.</li>
 <li>Add 5 ml of sorter buffer to each tube and centrifuge for 5 min at 300 x g, 4 &deg;C.</li>
 <li>For the bound fraction, carefully aspirate supernatant and resuspend the cells in 200 &mu;l of sorter buffer. Transfer the cells into a 1.2 ml FACS microtube. Rinse the tube with another 200 &mu;l of sorter buffer and pool into the same microtube. If cell clumps are apparent, pass the cells through a 50 &mu;m filter.</li>
 <li>For the unbound fraction and compensation controls, decant or aspirate the supernatant and resuspend in 2 ml sorter buffer.</li>
 <li>Set up the flow cytometer using the single-color compensation controls. Select side scatter-width (SSC-W) as an additional parameter to be recorded.</li>
 <li>Analyze the stained samples using a sequence of successive inclusion gates to identify CD4+ or CD8+ T cells as illustrated in Figures 1 and 2. For bound fractions, collect as many cells as possible up to a maximum of 2,000,000 total events. For unbound fractions, collect 1,000,000 total events. Keep the acquisition rate at or below 3,000 events per second.</li>
 <li>Using the same machine settings, analyze the counting bead samples. Collect 10,000 total events.</li>
 <li>Save all data as FCS files.</li>
</ol>
5. Data Analysis
<ol>
 <li>Analyze FCS data files for the counting bead samples using FlowJo software. Plot forward scatter by FITC and set a gate aroung the counting beads (see Figures 1 and 2). Determine the total number of counting beads detected in each sample. Subtract from the total number of collected events to determine the number of cell events collected.</li>
 <li>Calculate the total number of all cells in each sample using the equations outlined in Box 1.</li>
 <li>Analyze FCS data files for the stained bound and unbound fraction samples. Set up a sequence of successive inclusion gates to identify lymphoid+, side-scatter-widthlo, dump-, CD3+, CD4+ or CD8+, tetramer+ T cells in each sample as illustrated in Figures 1 and 2.</li>
 <li>Multiply the total number of cells in the sample by the frequencies of each of the inclusion gates used to define CD4+tetramer+ or CD8+tetramer+ cells to calculate the total number of epitope-specific T cells (Box 1).</li>
</ol>

<ol>
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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>

No conflicts of interest declared.

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

17.8K Views.  Massachusetts General Hospital and Harvard Medical School. 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.

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

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

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

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 ...

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.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Adoptive Transfer of P14 T-Cells into Tumor-Bearing Mice

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

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

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) tetramers.

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...