The authors wish to thank Jennifer Strange and Greg Bauman for their assistance with Flow Cytometry analysis and sorting. This work was supported by the NIH Grant Number 2P20 RR020171 from the NCRR and by University of Kentucky startup funds to F.M.; G.I.E. acknowledges the support of the Presidential Graduate Fellowship and the Kentucky Opportunity Fellowship.

1. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) from Buffy Coat
<ol>
 <li>Acquire one unit of buffy coat from hospital or nearby blood center location. Our blood center provides us with about 40-60 mL of buffy coat per unit obtained from normal blood donors.</li>
 <li>Pour blood into an autoclaved 500 mL glass bottle containing sterile PBS to dilute buffy coat. The final volume of PBS + buffy coat should be 250 mL.</li>
 <li>Fill ten 50 mL conical tubes each with 20 mL Lymphoprep solution.</li>
 <li>Gently overlay the Lymphoprep solution with 25 mL of the diluted buffy coat, being careful not to disturb the Lymphoprep solution.</li>
 <li>Spin tubes for 30 minutes at room temperature at 500 x g. Make sure to turn off the centrifuge's brake, so as to not disturb the lymphocyte fraction.</li>
 <li>Collect the PBMCs at the interface between the Lymphoprep and the plasma-medium layers with a pipette. Aspirate the plasma-medium off until about 1 mL still covers the buffy coat layer containing the PBMCs. Use a 10 mL pipette to transfer PBMCs to a new 50 mL tube.</li>
 <li>Wash cells twice with PBS and then resuspend cells in 50 mL of cold RPMI for counting on a hemocytometer. Typical recovery is 8 x 108 - 1 x 109 PBMCs.</li>
</ol>
This procedure can be scaled down for smaller volumes of blood. Dilution of whole blood samples is 1:1 in PBS.
2. Magnetic Negative Selection of Total CD4+ T Cells, CD4+ CD45RA+ na&iuml;ve T Cells or CD4+ CD45RO+ Memory T Cells from PBMCs using EasySep Enrichment Kits (Stem Cell Technologies)
Steps to follow for 1 x 108 to 4.25 x 108 PBMCs.
<ol>
 <li>Resuspend PBMCs to a final concentration of 5 x 107 per mL in PBS containing 2% FBS and 1mM EDTA. Move cells to a fresh 14 mL polypropylene round bottom tube.
 <ol class="lalpha">
 <li>Isolation of total human CD4+ T cells by negative selection: add human CD4+ T Cell Enrichment Cocktail of antibodies (50 &mu;L per mL of PBMCs), mix and incubate for 10 minutes at room temperature.</li>
 <li>Isolation of human na&iuml;ve T cells by negative selection: add 50 &mu;L of anti-CD45RO antibody per mL of PBMC cell suspension, mix and incubate for 15 minutes at room temperature. Add enrichment cocktail provided with the kit (50 &mu;L of per mL of PBMCs), mix and incubate at room temperature for 10 minutes.</li>
 <li>Isolation of human memory T cells by negative selection: add human Memory CD4+ T Cell Enrichment Cocktail of antibodies (50&nbsp;&mu;L per mL of PBMCs), mix and incubate for 10 minutes at room temperature.</li>
 </ol>
 </li>
 <li>Mix magnetic particles well to equally distribute them throughout the solution. Do not vortex nanoparticles from na&iuml;ve kit.</li>
 <li>Add the magnetic particles (100 &mu;L per mL of PBMCs for total CD4+ and na&iuml;ve T cell selection, 50 &mu;L per mL of PBMCs for memory T cell selection). Mix by gently pipetting 2-3 times and incubate at room temperature for 10 minutes for na&iuml;ve T cell selection or 5 minutes for memory or total CD4+ T cell selection.</li>
 <li>Add PBS containing 2% FBS and 1 mM EDTA to bring the volume up to 10 mL per tube. Mix by gently pipetting 2-3 times before placing uncapped tube into silver EasySep magnet for 5, 10 or 2.5 minutes for total CD4+ T cells, na&iuml;ve and memory subsets, respectively.</li>
 <li>With tube still in EasySep magnet, pour liquid into new 50 mL tube to isolate cells of interest.</li>
 <li>Repeat steps 2.5 and 2.6 for better recovery.</li>
</ol>
3. Cell Culture Conditions to Induce Regulatory T Cells
<ol>
 <li>The day before steps 1 and 2, coat tissue culture plates by first diluting anti-CD3 antibody (OKT3 clone) to a concentration of 1 &mu;g/mL in sterile PBS. For a 6 well plate add 2 mL of PBS + anti-CD3 per well, for a 12 well plate add 1 mL or for a 24 well plate add 500 &mu;L per well. Keep the coated plate at 4 &deg;C until use.</li>
 <li>Prepare polarization medium by adding 10% heat-inactivated Fetal Bovine Serum (FBS),100 U/mL penicillin, 100 &mu;g/mL streptomycin and 50 &mu;M &beta;-mercaptoethanol to RPMI-1640 pre-supplemented with 2.06 mM Glutamax-I and 25 mM HEPES buffer. Next, add 2 ng/mL TGF-&beta; and 5 ng/mL IL-2. Here, one may add ligands or inhibitors of interest to the media. Resuspend cells from step 2 at a concentration of 2 x 106 cells/mL of polarization medium.</li>
 <li>Pre-warm plates at 37 &deg;C and aspirate the PBS out of anti-CD3-coated wells before adding cells. Add 4 mL per well of cell suspension for a 6 well plate, 2 mL of cells for a 12 well plate or 1 mL of cells for a 24 well plate. Fill up empty wells with PBS to reduce evaporation of media. Incubate for 3 days at 37 &deg;C / 5% CO2.</li>
 <li>On day 3 post plating, spin down plate in a centrifuge for 5 minutes at 500 x g. Without disturbing T cells on bottom of the plate, remove half of media and replace with fresh media. Alternatively, if the well becomes overcrowded with cells (concentration above 3 x 106/mL), split the volume equally into another anti-CD3 coated plate and add fresh media back up to original volume. Incubate for two to three more days at 37 &deg;C / 5% CO2.</li>
</ol>
4. Fluorescence-Activated Cell Sorting (FACS) of Three Populations of T Cells
<ol>
 <li>Prepare FACS washing buffer by adding 0.5% (w/v) bovine serum albumin (BSA) and 2 mM EDTA to sterile PBS. Place buffer at 4 &deg;C to get cold.</li>
 <li>Remove cells from cell culture well and rinse each well with 2 mL of PBS to ensure complete cell recovery. Place all cells in 50 mL polypropylene tubes and centrifuge at 500 x g for 10 minutes at 4 &deg;C.</li>
 <li>Count the cells on a hemocytometer to determine cell density.</li>
 <li>Place 3-5 x 105 cells per tube in four separate 5 mL round bottom polystyrene tubes for compensation controls. Place remaining cells in 50 mL polypropylene tubes, no more than 35 x 106 cells per tube.</li>
 <li>Spin down cells for 5 minutes at 4 &deg;C and aspirate media. Resuspend cells to be sorted in 90 &mu;L of cold washing buffer per 1 x 106 cells. Resuspend in 100 &mu;L of cold washing buffer the compensation control tubes.</li>
 <li>To the cells to be sorted, combine 2 &mu;L of anti-CD25-PE, 3 &mu;L of anti-CD45RA-PE-Cy5 and 1 &mu;L of anti-CD127-APC per 1 x 106 cells. Add no antibody to the first compensation control tube, 2 &mu;L of anti-CD25-PE to the second tube, 3 &mu;L of anti-CD45RA-PE-Cy5 to the third tube and1 &mu;L of anti-CD127-APC to the fourth tube. Incubate all tubes on ice for 45 minutes in the dark.</li>
 <li>Add 10 mL of cold washing buffer to the cells to be sorted and 1 mL to the compensation control tubes. Centrifuge all cells at 500 x g for 5 minutes at 4 &deg;C.</li>
 <li>Aspirate buffer and resuspend cells at a concentration of 1 x 107 cells per mL of washing buffer. Add 1.5 &mu;L of DNase II per mL of cells before filtering through a 40 &mu;M nylon cell strainer. Move cells to multiple 5 mL round bottom polypropylene tubes with no more than 3.5 mL per tube. Resuspend compensation control cells in 300 &mu;L of washing buffer.</li>
 <li>Set compensation on MoFlo flow cytometer to minimize cross detection by the PE, APC and PE-Cy5 filters. Set gates to sort iTregs (CD25HiCD127-/LOWCD45RA- cells), na&iuml;ve (CD25-CD45RA+) and memory (CD25-CD45RA-) T cells into 5 mL round bottom polystyrene tubes containing 1 mL newborn calf serum.</li>
</ol>
5. Suppression Assay
<ol>
 <li>Make suppression assay media by adding 100 U/mL of penicillin, 100 &mu;g/mL of streptomycin, 5 ng/mL of IL-2 and 2 ng/mL of TGF-&beta; to AIM-V.</li>
 <li>The day of the assay, purify heterologous CD4+ T cells from buffy coat as indicated in steps 1 and 2. These will be the target cells for the suppression assay and does not contain CD25+ Treg cells, as can be seen in Figure 2, Day 0.</li>
 <li>Label cells with CellTrace kit as per manufacturer's instructions, except using only 1 &mu;L of 5 mM stock solution per mL of cells instead of 2 &mu;L. Keeping out from direct light, add 18 &mu;L of the DMSO supplied by the CellTrace kit to one vial of CFSE to make a 5 mM stock solution. Resuspend the required number of target cells (to a maximum of 1 x 107) in prewarmed PBS + 0.1% (w/v) BSA to a final concentration of 1&nbsp;x&nbsp;106 cells/mL. Add 1 &mu;L of 5 mM CFSE per mL of cells and incubate in a 37 &deg;C water bath for 5 minutes. Add 5 volumes of complete, ice cold RPMI with 10% FBS to quench staining and incubate on ice for 5 minutes. Wash cells twice more with cold complete RPMI and resuspend 1 x 105 cells per 100 &mu;L of suppression assay media.</li>
 <li>Treg suppression inspector beads are at a stock concentration of 2 x 107 beads/mL. Pellet a number of beads equal the total number of cells per experiment by quick centrifugation in an eppendorf tube. Wash beads once with RPMI and re-pellet. After aspiration of RPMI, resuspend beads so that the appropriate amount of beads per well are in 8 &mu;L of suppression assay media.</li>
 <li>To a 96 well round bottom tissue culture plate, add CFSE-stained cells (1 x 105 cells/mL), inspector beads and polarized and sorted cells (1&nbsp;x&nbsp;105 cells/mL) in fresh suppression assay media to a desired target (CFSE stained):effector (sorted) ratio in a final volume of 200 &mu;L. All conditions are set in triplicates.</li>
 <li>Prepare the first of two control conditions by adding 100 &mu;L of CFSE stained cells, 8 &mu;L of inspector beads and 1 x 105 of fresh, unstained cells in 92 &mu;L suppressor assay medium per well. Prepare the second control with the same cellular components as above but without Treg inspector beads.</li>
 <li>Cover plate in aluminum foil and incubate at 37 &deg;C / 5% CO2 for five days.</li>
 <li>In the dark, collect cells from each well by pipetting and place in a 5 mL round bottom polystyrene tube. Centrifuge cells at 500 x g for 5 minutes at 4 &deg;C, aspirate media, and resuspend in 300 &mu;L cold FACS washing buffer from step 4. Analyze the first 3 x 104 CFSE+ events from the live lymphocyte gate representing target cells in a histogram with Cell Quest software.</li>
</ol>
6. Representative Results
Example of flow cytometric pseudocolor dot plots over a five-day time-course monitoring iTreg differentiation based on the relative co-expression of CD25 with FoxP3, CTLA-4 and CD45RA can be seen in Figure 2. The histogram in Figure 3 shows a successful suppression assay in which sorted iTregs (CD25HiCD45RA- CD127-/LOW cells) are the only subset from a five-day culture that has acquired regulatory/suppressor ability. Figure 4 shows the induction of Tregs from a na&iuml;ve T cell pool (top panels) and a memory T cell pool (bottom panels), after five-day culture in standard iTreg medium. CFSE staining of initial cells demonstrate that either na&iuml;ve (top right panel) or memory T cells (bottom right panel) differentiate to iTregs (highest FoxP3-expressing cells) only after several rounds of cell division.
<img alt="Figure 1" src="/files/ftp_upload/3738/3738fig1.jpg" /> 
Figure 1. Schematic of experimental procedure. PBMCs are separated out of human peripheral blood via gradient centrifugation before magnetic negative selection of CD4+CD25- T cells. After five to six days in culture, cells undergo FACS and are co-incubated with heterologous CFSE labeled target cells to measure suppressor activity.
<img alt="Figure 2" src="/files/ftp_upload/3738/3738fig2.jpg" /> 
Figure 2. Purified human primary CD4+CD25- T cells are cultured in iTreg medium. An aliquot of cells is collected just after isolation (day 0) and at days 1, 3 and 5 of cell culture to monitor the progress of CD45RA, FoxP3, CTLA-4 and CD25 markers. The iTreg profile corresponds to CD45RA-, FoxP3Hi, CTLA-4Hi and CD25Hi (highlighted in the in-graph window).
<img alt="Figure 3" src="/files/ftp_upload/3738/3738fig3.jpg" /> 
Figure 3. Purified CD4+ CFSE labeled cells (1 x 105/well) are cultured with Treg suppression inspector beads in the presence of sorted na&iuml;ve, memory or iTreg cells (3 x 104/ well). After five days, cells are harvested and the CFSE profile of the stained cells is analyzed by flow cytometry. The presence of iTreg cells completely abolishes the proliferation of CD4+ T cells. Numbers are indicative of percentage of CFSE-labeled cells that have undergone division.
<img alt="Figure 4" src="/files/ftp_upload/3738/3738fig4.jpg" /> 
Figure 4. Purified human primary na&iuml;ve (CD4+CD25-CD45RA+) and memory (CD4+CD25-CD45RO+) T cells are stained with CFSE and cultured in iTreg medium. After five days, cells are phenotyped and the cell division rates estimated. As indicated in Figure 2, the iTreg subset differentiated from both subsets corresponds to CD45RA-CD25Hi FoxP3Hi and CTLA-4Hi (latter two not shown). Comparative CFSE staining profiles identify iTregs (here as the highest expressing FoxP3 T cells) as the most proliferative cells during the five-day culture.

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The authors have nothing to disclose.

While Treg transfer holds enormous therapeutic promise in combating autoimmunity graft rejection and other immune or inflammatory-mediated disorders, methods for their efficient generation and stable maintenance have not yet been developed. As only 1-5% of circulating human T cells are Tregs, their controlled expansion and differentiation overcomes this paucity as a major deterrent of implementation of Tregs into the clinic. On the other hand, as we learned from the TGN1412 trial13 , it is scientifically and e...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Lymphoprep</td>
 <td>Axis-Shield</td>
 <td>1114547</td>
 <td>Keep at room temperature</td>
 </tr>
 <tr>
 <td>Refrigerated Bench-Top Centrifuge</td>
 <td>Eppendorf</td>
 <td>5810R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bright-Line Hemocytometer</td>
 <td>Hausser Scientific</td>
 <td>3110</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EasySep Human CD4+ T Cell Enrichment Kit</td>
 <td>Stem Cell Technologies</td>
 <td>19052</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EasySep Human Memory CD4+ T Cell Enrichment Kit </td>
 <td>Stem Cell Technologies</td>
 <td>19157</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EasySep Human Na&iuml;ve CD4+ T Cell Enrichment Kit</td>
 <td>Stem Cell Technologies</td>
 <td>19155</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>The Big Easy EasySep Magnet</td>
 <td>Stem Cell Technologies</td>
 <td>18001</td>
 <td>Silver</td>
 </tr>
 <tr>
 <td>5 mL Round Bottom Polystyrene Tubes</td>
 <td>BD Falcon</td>
 <td>352008</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 mL Polypropylene Centrifuge Tubes</td>
 <td>VWR</td>
 <td>89004-368</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>14 mL Polypropylene Round-Bottom Tubes</td>
 <td>BD Falcon</td>
 <td>352059</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL Polypropylene Centrifuge Tubes</td>
 <td>VWR</td>
 <td>89004-364</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human anti-CD3 Antibody</td>
 <td>Bio X Cell</td>
 <td>BE0001-2</td>
 <td>Clone: OKT3</td>
 </tr>
 <tr>
 <td>24 Well Cell Polystyrene Culture Plate</td>
 <td>BD Falcon</td>
 <td>353047</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96 Well Round Bottom Tissue Culture Plate</td>
 <td>Grenier Bio-One</td>
 <td>650180</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RPMI 1640 with Glutamax-I and HEPES Buffer</td>
 <td>Gibco</td>
 <td>72400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Gibco</td>
 <td>16000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>Sigma</td>
 <td>M7522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Recombinant Human TGF-&beta;1</td>
 <td>eBioscience</td>
 <td>14-8348-62</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Recombinant Human IL-2</td>
 <td>eBioscience</td>
 <td>14-8029-63</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin (BSA)</td>
 <td>MP Biomedicals Inc.</td>
 <td>810531</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M EDTA</td>
 <td>Amresco</td>
 <td>E177</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse Anti-Human CD25-PE</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-024</td>
 <td>Clone: 4E3</td>
 </tr>
 <tr>
 <td>Mouse Anti-Human CD45RA-PE-Cy5</td>
 <td>eBioscience</td>
 <td>15-0458-42</td>
 <td>Clone: HI100</td>
 </tr>
 <tr>
 <td>Mouse Anti-Human CD127-APC</td>
 <td>Miltenyi Biotec</td>
 <td>130-094-890</td>
 <td>Clone: MB15-18C9</td>
 </tr>
 <tr>
 <td>DNase II</td>
 <td>MP Biomedicals Inc.</td>
 <td>190370</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MoFlo Flow Cytometer</td>
 <td>Beckman Coulter</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FlowJo Software</td>
 <td>Tree Star, Inc.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell Quest Pro Software</td>
 <td>BD Biosciences</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Newborn Calf Serum </td>
 <td>Gibco</td>
 <td>16010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AIM-V</td>
 <td>Gibco</td>
 <td>0870112</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CellTrace CFSE Cell Proliferation Kit</td>
 <td>Invitrogen</td>
 <td>C34554</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Treg Suppression Inspector Beads</td>
 <td>Miltenyi Biotec</td>
 <td>130-092-909</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>40 &mu;M Nylon Cell Strainer</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
</table>

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.

Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃ¯ve and Memory T Cells at JoVE.com

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.

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 ...

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JoVE Journal

Immunology and Infection

23.9K Views.  University of Kentucky. We describe a method for generating regulatory, memory and naïve 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.

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

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 ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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 ...

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 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

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

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

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.