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Method Article

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol

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DOI:

10.3791/55015

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December 30th, 2016

 ,  ,  , 
1Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory

* These authors contributed equally

In This Article

Summary

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

Abstract

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 naïve T cells using a range of Treg-inducing factors (TGF-β 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-β 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.

Introduction

CD4+CD25+FOXP3+ regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance, preventing autoimmunity and excessive inflammation1. The importance of Tregs is exemplified by the human disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), in which loss of Tregs due to mutations in the `master´ Treg transcription factor forkhead box P3 (FOXP3) leads to severe systemic autoimmune disease, lethal at an early age. However, Tregs act as a double-edged sword in the immune system as they can also hamper anti-tumor immunity in certain settings2. Therapeutic manipulation of Treg number and function is therefore subject to numerous clinical investigations. In cancer, depletion of Tregs can be desirable and some success of clinical approaches encourages further research3. In autoimmune and inflammatory diseases, in addition to therapeutic effects of Tregs in several mouse disease models, recent first in-man trials of adoptive Treg transfer to prevent graft-versus-host disease (GvHD)4-7 and to assess safety in treating type 1 diabetes8 showed very promising outcomes.

Naturally occurring Tregs (nTregs) comprise thymic-derived tTregs and peripherally induced pTregs, with non-redundant essential functions in maintaining health9-11. However, nTreg numbers are limited, encouraging the complementary approach of inducing Tregs (iTregs) in vitro from naïve T cell precursors12. Still stability of iTregs, presumably due to lack of demethylation in the so-called Treg-specific demethylated region (TSDR) in the FOXP3 gene locus13, remains a concern and several studies indicate that in vivo induced Tregs are more stable14.

To date, FOXP3 remains the best protein marker for Tregs but it is not absolutely specific because human conventional CD4+CD25- T cells transiently express intermediate levels of FOXP3 upon activation15,16. Although significant progress has been made in elucidating the regulation of FOXP3 expression, much remains to be discovered regarding the induction, stability and function of FOXP3 particularly in human cells. Despite differences to nTregs, in vitro induced FOXP3+ CD4+ T cells can be used as a model system to study molecular mechanisms of FOXP3 induction and as a starting point to develop protocols in the future that allow for generation of iTregs that are more similar to in vivo generated Tregs, which could be applicable for adoptive transfer strategies in the future.

There is no `gold standard´ protocol to induce human iTregs, and current protocols have been developed based on mimicking Treg-inducing conditions in vivo: interleukin 2 (IL-2) and transforming growth factor β (TGF-β) signaling are crucial for FOXP3 induction in vivo17, and all-trans retinoic acid (ATRA) — which is produced in vivo by gut-associated dendritic cells — is frequently used to enhance FOXP3 induction in vitro18-21. We have developed additional human Treg-inducing protocols using butyrate22, a gut microbiota-derived short-chain fatty acid that was recently shown to augment murine Treg induction23,24. We also recently established a new protocol for generation of iTregs with superior suppressive function in vitro by using a combination of TGF-β, ATRA and rapamycin22, the latter being a clinically approved mammalian target of rapamycin (mTOR) inhibitor that is known to promote FOXP3 maintenance during human Treg expansion25,26.

This method describes the reproducible in vitro generation of human CD4+FOXP3+ iTregs using a set of different conditions, and their subsequent phenotyping by flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to reveal protocol-specific patterns of expression of FOXP3 and other Treg signature molecules such as CD25, CTLA-4, EOS, as well as repression of IFN-γ and SATB1 expression22. The generated cell populations can be used for functional assays regarding suppressive activity or for molecular studies, either concerning general FOXP3 regulators or to study effects specific to certain compounds such as butyrate or rapamycin. Further understanding of molecular mechanisms driving Treg differentiation is highly relevant for future therapeutic approaches in autoimmunity or cancer to specifically target molecules involved in Treg generation and function.

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Protocol

Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from anonymized healthy donor buffy coats purchased from the Karolinska University Hospital, Sweden. Ethical permit for the experiments was obtained from the Regional Ethical Review Board in Stockholm (Regionala etikprövningsnämnden i Stockholm), Sweden (approval number: 2013/1458-31/1).

1. T Cell Isolation from Peripheral Blood

  1. PBMC Isolation
    1. Pre-lay 15 ml density centrifugation medium (such as Ficoll) solution in 50 ml tubes (5 tubes per buffy coat). Pre-warm to room temperature.
    2. Fill up buffy coat with phosphate buffered saline (PBS) (room temperature) to 180 ml. If fresh blood is used, dilute blood with equal volume of PBS.
    3. Tilt tube to the side and slowly overlay density centrifugation medium with ~35 ml diluted blood per tube. Add the blood very carefully, without any mixing of the density centrifugation medium phase and blood layer.
    4. Centrifuge 20 min at 1,150 x g at room temperature without brake. Operate the centrifuge without brake and if possible with low to medium acceleration to prevent mixing of the layers.
    5. Take out the white ring containing PBMCs between the density centrifugation medium and the plasma phase. Transfer to a new 50 ml tube (1 time with 5 ml pipette and 2 times with 2 ml plastic-Pasteur pipette). Take as little as possible plasma and density centrifugation medium. Do not touch the red erythrocyte pellet.
    6. Wash PBMCs. Split the PBMCs into 50 ml tubes (4 tubes per buffy coat). Fill each to 50 ml with PBS.
    7. Centrifuge 450 x g for 10 min at room temperature.
    8. Remove and discard supernatant, pool the cells into 1 tube with 10 ml RPMI/10% fetal calf serum (FCS) medium and resuspend well. If cell clumps are present, strain cells through a 40 µm cell strainer.
    9. Transfer cells in T175 cell culture flask for adherent cells. Rinse tubes with 40 ml medium and combine with cells (50 ml total). If needed, remove an aliquot for flow cytometry analysis (see step 1.4). Usually, CD4+ T cells comprise about 30-40% of cells in the lymphocyte gate, and naïve T cells comprise about 30 to 60% of CD4+ T cells depending on the donor.
    10. Incubate cells in laying flask for 45 to 90 min at 37 °C/5% CO2. This step will deplete monocytes by plastic adherence. It is not mandatory, but will increase the percentage T cell yield per amount of PBMCs in the following steps.
    11. Resuspend cells (in the 50 ml medium contained in the flask) and rinse cell layer on the bottom of the flask well. Distribute cells equally into two 50 ml tubes (the previous tubes may be re-used for the same donor). Rinse flask with 50 ml fresh RPMI/10% FCS medium, and transfer equally into the same 2 tubes.
      NOTE: PBMCs may be stored overnight at 4 °C with tubes laying on the side, or ideally, directly used for T cell isolation.
  2. nTreg Isolation by Magnetic-activated Cell Sorting
    1. Prepare isolation buffer: 0.5% (w/v) human serum albumin and 2 mM EDTA in PBS. Always keep buffer at 4 °C. Alternatively to human albumin, use bovine albumin.
    2. Resuspend and count PBMCs with an automatic or manual cell counter in the presence of trypan blue (do not count small platelets). If cell clumps are observed, strain cells through a 40 µm cell strainer.
    3. Centrifuge PBMCs at 450 x g for 10 min at room temperature.
    4. Resuspend cells (with 1 ml pipet) in 90 µl isolation buffer per 107 PBMCs to obtain a single cell suspension. Keep cells in the original 50 ml tubes (2 tubes per buffy coat).
    5. Add 2 µl CD25 beads per 107 cells, mix, and incubate for 15 min at 4 °C.
    6. Fill with PBS to 30 ml. Centrifuge at 450 x g for 10 min at 4 °C.
    7. Prepare LS columns (2 per buffy coat): equilibrate with 3 ml isolation buffer, and discard flow through. Tubes can be re-used to rinse further columns.
    8. Resuspend cells in 3 ml isolation buffer per tube (with 1 ml pipet). Transfer cells to LS column, and collect flow through in fresh 15 ml tubes.
    9. Rinse the 50 ml tubes with 2 ml Isolation buffer each. Transfer rinsing buffer to the columns.
    10. Wash column 2x with 3 ml Isolation buffer each. Always wait until the column stopped dripping, before adding any new liquid. Store flow through at 4 °C for later steps.
    11. Remove column from magnet. Elute 2x with 3 ml isolation buffer per column into 15 ml tube.
      NOTE: The eluate from 2 columns per donor can be combined. Do not dip the column into the liquid.
    12. Prepare and equilibrate new LS columns (1 column per buffy coat). Transfer the eluate from 1.2.11 to the column. Flow through can be discarded. After eluate has passed through the column, rinse tube and wash column 3x with 3 ml isolation buffer.
      NOTE: The second column is crucially needed to increase purity.
    13. Remove column from magnet. Elute CD25++ "nTregs" 2x with 3 ml Isolation buffer.
    14. Centrifuge at 450 x g for 10 min at 4 °C. Remove supernatant completely.
    15. Wash cells with 15 ml T cell culture medium, centrifuge at 450 x g for 10 min at 4 °C. Remove supernatant completely.
    16. Resuspend cells in 0.5 ml T cell culture medium, supplemented with 100 IU/ml IL-2, each. Transfer cells to 24-well plate. Rinse tube with 0.5 ml medium (+ 100 IU/ml IL-2) and combine into 24-well plate.
    17. Count nTregs (as in step 1.2.2). The yield is usually about 1-4 x 106 Tregs per buffy coat. Adjust density of Tregs to 1-2 x 106 cells/ml with T cell culture medium + 100 IU/ml IL-2.
    18. Incubate Tregs at 37 °C/5% CO2 until use.
  3. Naïve CD4+ T Cell Isolation by Magnetic-activated Cell Sorting
    NOTE: Take flow through from step 1.2.8-1.2.10 to proceed with cell isolation. Alternatively, if no nTregs were isolated, proceed directly from PBMCs.
    1. Combine 2 tubes per donor of Treg-depleted PBMCs in 50 ml tube. Fill with PBS to 50 ml at room temperature.
    2. Centrifuge PBMCs at 200 x g for 5-10 min at room temperature. Remove and discard supernatant. Resuspend cells in 50 ml PBS.
    3. Repeat step 1.3.2 twice.
      NOTE: The PBS washes are crucial for removal of platelets, which will not be removed with the following negative isolation kit. Platelets remain in the supernatant at this low-speed centrifugation. Platelet depletion can be monitored in the individual steps on a microscopic slide. The steps have to be performed with PBS and centrifugations at room temperature.
    4. Resuspend PBMCs in 50 ml PBS. Count cells as in step 1.2.2.
    5. Centrifuge PBMCs at 450 x g for 10 min at 4 °C. Discard supernatant and resuspend cells in 40 µl Isolation buffer (4 °C) per 107 cells.
    6. Add 10 µl of Naïve CD4+ T Cell Biotin-Antibody Cocktail II per 107 cells.
    7. Mix, and incubate for 10 min at 4 °C.
    8. Wash cells by adding 40 ml PBS (4 °C), and centrifuge at 450 x g for 10 min at 4°C. Remove supernatant and resuspend cells in 80 µl isolation buffer per 107 cells.
    9. Add 20 µl of anti-biotin microbeads per 107 cells. Mix well, and incubate for 15 min at 4 °C.
    10. Fill to 50 ml with cold PBS, and centrifuge 450 x g for 10 min at 4°C.
    11. Prepare LS columns (equilibrate with 3 ml isolation buffer). To avoid column overload, use one column for maximally 250 x 106 PBMCs, or less if PBMCs contain many red blood cells.
    12. Remove supernatant and resuspend cells in 3 ml isolation buffer. Transfer cells to LS column. Collect flow through, which contains the naïve CD4+ T cells, in 15 ml tubes.
    13. Rinse tube with 2 ml isolation buffer. Transfer to the column.
    14. Wash column 3x with 3 ml isolation buffer. Always wait until the column stops dripping, before adding any new liquid.
    15. Take the flow through (naïve CD4+ T cells) and centrifuge 450 x g for 10 min at 4 °C. Columns can be discarded.
    16. Remove supernatant completely. Wash cells with 15 ml medium, centrifuge 450 x g for 10 min at room temperature and remove supernatant completely.
      NOTE: Isolation buffer contains EDTA, which chelates calcium ions and thus impairs T cell activation. Before any stimulation assays, remove the isolation buffer completely and wash cells twice with 15 ml medium.
    17. Resuspend cells in T cell culture medium to 2-3 x 106 cells per ml. Rest cells in appropriate flask or well over night in the incubator. If cells are used directly for stimulation assays, wash them one more time with medium.
  4. Purity Control Staining
    1. Take ~50,000 cells (Tnaïve; nTreg; PBMCs) each, in 20 µl.
      NOTE: If both Tnaïve and nTreg panel are to be stained, take 2 samples each.
    2. Prepare 2x concentrated antibody premix (in PBS) as appropriate for the instrument, for example: Tnaïve panel: CD4-PerCP 1:5; CD45RA-FITC 1:10, CD45RO-PE 1:5, CD8-eFlour450 1:8. For Treg panel, replace CD45RO-PE with CD25-PE, 1:10.
      NOTE: Only certain CD25 antibodies, that recognize different epitopes than the CD25-microbeads, can be used to stain nTregs isolated with CD25 microbeads.
    3. Add 20 µl antibody premix to 20 µl cells. Also prepare single stainings for each fluorochrome (using a sample containing positive cells, e.g., PBMCs) and an unstained sample, for flow cytometry settings and compensation.
    4. Incubate for 15 min at room temperature in the dark.
    5. Fill each sample with 200 µl PBS, and centrifuge 450 x g for 5-10 min.
    6. Take up cells in fluorescence activated cell sorting (FACS) buffer (= isolation buffer without EDTA) and analyze by flow cytometry. The purity of naïve CD4+ T cells is usually >95% (see Figure 2).

2. Treg Induction Culture

  1. Preparation of Stimulation Media and Plates
    1. Prepare and pre-warm T cell culture medium: serum-free hematopoietic medium supplemented with 2 mM L-alanyl-L-glutamine.
    2. Prepare cytokine, stimulation antibody and compound stock concentrations.
      1. Prepare IL-2 stock solution: 400,000 IU/ml in sterile-filtered (with acid-resistant filter) 100 mM acetic acid (167 µg/ml if activity of the used lot is 2,400 IU per 1 µg; confirm with provider). Aliquot to sterilized low-protein binding 0.5 ml tubes, seal with Parafilm, freeze on dry ice and store at -80 °C for long-term storage or -20 °C for up to 3 months.
      2. Prepare TGF-β1 stock solution (for 10 µg vial, carrier-free): Centrifuge TGF-β1 powder (1,000 x g 3 min), add 100 µl of 4 mM HCl (sterile-filtered with acid-resistant filter) to prepare stock solution with 100 µg/ml, allow to dissolve completely; vortex. Aliquot 5 µl each to sterilized low-protein binding 0.5 ml tubes, seal with Parafilm, freeze on dry ice and store at -80 °C for up to 6 months.
      3. Prepare ATRA stock solution: Dissolve powder at 20 mg/ml (66.6 mM) in DMSO. Prepare stock solution of 10 mM by further dilution in DMSO and store aliquots, protected from light, at -80 °C for up to 1 year. ATRA is sensitive to light, heat and air.
      4. Prepare rapamycin stock solution: 1 mg/ml (1.11 mM) in DMSO, store in aliquots at -80 °C for up to 1 year.
      5. Prepare sodium butyrate stock solution: 0.908 M (0.1 mg/ml) stock in sterile ultra-pure H2O and sterile filter. Aliquot and store at -20 °C.
    3. Prepare 4x concentrated premixes (50 µl per well) in T cell culture medium as in Table 1.
    4. On the previous day, coat plates with anti-CD3 antibody. Coat desired number of wells from 96 U-well plate with 5 µg/ml anti-CD3 antibody in PBS, 65 µl/well. Wrap plate with foil and incubate overnight at 4 °C.
      NOTE: Do not use the margin wells of 96 well plates. Bigger wells may be used, but U-shaped wells facilitate use of the same cell numbers per area across experiments. If more cells are needed, pool the required amount of wells after culture. Usually, after 4-6 days of expansion, 2 wells each sample are sufficient for flow cytometry analysis and RNA analysis.
      1. On the day of plating, shortly before plating, remove anti-CD3 solution from wells. Fill wells with 200 µl/well PBS. Remove PBS. Repeat washing with 200 µl/well PBS. Remove PBS completely and use plates immediately.
    5. Prepare plates (do not use margin wells):
      1. For each well, add 50 µl of 4x anti-CD28/IL-2 premix (except for "unstimulated" cells, add T cell culture medium), 50 µl 4x TGF-β1 premix (for all iTregs) or 50 µl T cell culture medium for "stimulation only" mock control, 50 µl 4x ATRA, ATRA/rapamycin or butyrate premix (where applicable) or medium
      2. Fill all empty wells, including the margin wells, with 200 µl PBS. Pre-warm plates at 37 °C/5% CO2.
  2. Prepare Naïve T Cells for Plating
    1. After resting cells, transfer to 15 ml tube, rinse flask/wells with pre-warmed T cell culture medium and pool to tube. Fill tube to 15 ml with T cell culture medium.
    2. Centrifuge cells at 450 x g for 10 min at room temperature.
    3. Remove medium and take up cells in fresh, pre-warmed T cell culture medium to a density of 2.2-2.6 x 106/ml. Count cells as in step 1.2.2 and adjust density if needed.
    4. Add cells to prepared stimulation plates (see 2.1), 50 µl cells/well (110,000-130,000 cells/well). Each well should now contain a total volume of 200 µl.
    5. Wrap plates in aluminum foil to protect ATRA from light; incubate at 37 °C/5%CO2 for desired time periods.
  3. Monitor Treg Induction Culture
    1. Monitor Treg cultures by light microscopy (40X magnification). From about day 2 to 3, small clusters of proliferating cells should become visible, which merge into bigger clusters at later time points. Cultures grown with rapamycin generally grow less. See also Figure 3.
    2. At desired time points, take samples for RNA extraction or flow cytometry phenotyping (see below). For later time points with cell proliferation, 1 well each is sufficient, otherwise take 1 x 105-1 x 106 cells each for RNA and 3 x 105-1 x 106 cells for flow cytometry.
  4. Assessing FOXP3 Stability
    1. For assessing stability of the iTreg phenotype as described previously22,27, wash iTregs twice with T cell culture medium, and culture iTregs further in T cell culture medium either without or with anti-CD3 and anti-CD28 restimulation as described in 2.1 and 2.2. Add cytokines, such as 50 IU/ml IL-2, to study their influence on FOXP3 stability.
    2. At desired time points, take samples for RNA extraction, flow cytometry phenotyping (see below), and TSDR methylation analysis as described22,28.

3. Phenotypic Analysis of iTregs by qRT-PCR

  1. Preparation of Samples
    1. At desired time points, take 1 x 105 to 1 x 106 cells per sample for RNA extraction. Transfer cells to an RNase-free 1.5 ml tube, and centrifuge at 500 x g for 5 min.
    2. If needed, remove part of supernatant and freeze for enzyme-linked immunosorbent assay (ELISA) or similar analysis. Remove remaining supernatant completely from cells. Proceed directly to RNA extraction or freeze cell pellet on dry ice and store at -20 °C to -80 °C for up to 2 weeks.
    3. Extract RNA and perform qRT-PCR for FOXP3 and a housekeeping gene (such as RPL13A) according to standard protocols. In addition, measure expression of other Treg signature genes (for example `Treg up´ genes IL2RA, CTLA4, IKZF4, and `Treg down´ genes IFNG, SATB1).

4. Phenotypic Analysis by Flow Cytometry

NOTE: This protocol is optimized for staining in 96U well plate format with 3 x 105-1 x 106 cells. If there are less cells per well, start with several wells and pool as described below.

  1. Cell Restimulation (Optional)
    1. If staining for intracellular cytokines is desired, pulse cells with Phorbol 12-myristate 13-acetate (PMA)/Ionomycin in the presence of a protein transport inhibitor.
      NOTE: This procedure does not influence FOXP3 expression in above-described iTreg cultures, but other markers may change — for example, CD4 is downregulated.
    2. Prepare 10x Brefeldin A/PMA/Ionomycin premix (20 µl per well) in T cell culture medium: Brefeldin A stock solution 1:100, Ionomycin (stock 5 µg/µl) 1:1,300, PMA (stock 12.35 µg/µl, pre-dilute 1:1235) 1:100 of pre-diluted stock.
    3. 4 hours before staining, add 20 µl Brefeldin A/PMA/Ionomycin 10x mix per well to obtain end concentrations of 1:1,000 Brefeldin A, 0.5 µM (375 ng/ml) Ionomycin, 10 ng/ml PMA.
    4. Incubate for 4 hours at 37 °C/5%CO2
  2. Surface Staining
    NOTE: The panel suggested here is a suggestion; other panels may be used depending on the antigens of interest and instrument setup.
    1. Cool PBS on ice. Prepare FACS buffer (PBS with 0.5% human serum albumin, HSA) and cool on ice. BSA or FBS may be used as an alternative to HSA.
    2. Re-plate cells in staining plate: Resuspend cells with a pipette and transfer the cells into a new 96U well plate, leaving one well free around each well (to prevent spillover in later staining procedure). If less than desired cell numbers are present in the original well, remove part of the supernatant before resuspension, and pool several wells into a staining well.
    3. Centrifuge plate 400 x g for 10 min at room temperature.
    4. Remove the supernatant. Pour out supernatant into waste box, leave the plate upside down and immediately (without turning back the plate) tap the plate once on absorbent paper. Then immediately turn back the plate. Vortex plate to resuspend cells in remaining liquid.
    5. Add 25 µl surface staining premix (CD25-PE 1:20, CD4-PerCP 1:5 in FACS buffer) and resuspend.
      NOTE: Also include single stainings for each of the used fluorochromes with samples that contain positive cells for the respective antigen; and include an unstained sample. These will be needed for instrument compensation setup. Alternatively, use compensation beads.
    6. Incubate 30 min at 4 °C in the dark.
    7. Add 200 µl PBS, centrifuge plate 400 x g for 10 min at 4 °C and remove supernatant as described in step 4.2.4. Vortex plate.
    8. Repeat step 4.2.7 twice.
  3. Viability Staining
    NOTE: Viability staining is indispensable, since after fixation/permeabilization dead cells cannot sufficiently be excluded by fsc/ssc and may give rise to unspecific signals. A fixable viability dye has to be used.
    1. Resuspend cells in 130 µl viability stain premix (fixable viability dye-eFlour780 1:1,400 in PBS) and immediately resuspend cells with the multichannel pipet.
      NOTE: Also include a single staining for the viability dye containing dead cells, e.g., unstimulated T cells cultured for several days or kill cells by applying heat as by the manufacturers' instructions. These will be needed for compensation.
    2. Incubate 30 min at 4 °C in the dark.
    3. Centrifuge plate at 400 x g for 10 min at 4 °C. Remove supernatant as in step 4.2.4. Vortex plate.
    4. Fill with 200 µl FACS buffer. Repeat step 4.3.3.
    5. Fill with 200 µl PBS. Repeat step 4.3.3.
  4. Intracellular Staining with FOXP3 Staining Buffer Set
    1. For each well, prepare: 150 µl Fix/Perm buffer (dilute Fix/Perm concentrate 1:4 with diluent); 850 µl 1x permeabilization buffer (dilute 10x permeabilization buffer 1:10 with ultra-pure H2O).
    2. After vortexing, resuspend cells in 150 µl Fix/Perm buffer and immediately resuspend with pipette. It is important to immediately resuspend to avoid fixation of cell clumps.
      Caution: Fixation buffer contains paraformaldehyde and should be handled and discarded appropriately.
    3. Incubate 30 min at 4 °C in the dark.
    4. Add 100 µl cold PBS. Centrifuge at 850 x g for 10 min at 4 °C.
      NOTE: From this step onwards the centrifugation speed is increased to avoid loss of cells. After fixation, cells become invisible and care should be taken to remove supernatants as described in step 4.2.4 and immediately after the centrifugation is finished to minimize loss of cells.
    5. Remove supernatant as described in step 4.2.4. Vortex plate.
      NOTE: The staining may be paused at this step; in this case, wash the cells twice with 200 µl PBS; then take up in 250 µl PBS and store at 4 °C, protected from light. The cells can be stored for few days, then proceed with permeabilization. However, optimal results are achieved when directly continued to permeabilization.
    6. After vortexing, add 200 µl Permeabilization buffer. Centrifuge at 850 x g for 10 min at 4°C. Remove supernatant as described in step 4.2.4. Vortex plate.
    7. Add 200 µl Permeabilization buffer to resuspend.
      NOTE: Here, samples can be split into intracellular staining and isotype control sample (take away half of each sample). If cell numbers are limiting, a pooled isotype sample can be prepared (take away 10-20 µl of each sample and pool). Also, samples can be taken for Fluorescence-Minus-One (FMO) controls.
    8. Centrifuge at 850 x g for 10 min at 4 °C. Remove supernatant as described in step 4.2.4. Vortex plate.
    9. Resuspend pellet in 44 µl Permeabilization buffer plus 1 µl normal mouse serum (premixed) to block unspecific binding.
      NOTE: This applies if antibodies used in the following step are of murine isotype. If other antibodies, such as derived from rat, are used, include the appropriate serum for blocking (e.g., rat serum).
    10. Incubate at 4 °C for 15 min in the dark.
    11. Add antibodies:
      NOTE: Inquire the antibody concentrations for the specific Lots. If not done in step 4.2 with antibodies against surface antigens (e.g., CD4) with the respective fluorochromes, also include single stainings for each of the used fluorochromes with samples that contain positive cells for compensation.
      1. Add 15 µl antibody premix to samples (except to single stainings, unstained samples, isotype control samples and FMO controls): Premix per sample: 0.7 µl (0.35 µg) anti-Interferon gamma (IFN-γ)-FITC (if applicable after restimulation), 2.3 µl (0.115 µg) CTLA-4 Brilliant Violet 421, 2 µl (0.05 µg) Anti-FOXP3-APC, 10 µl PBS.
      2. To isotype control samples, add 15 µl isotype antibody premix, using the same amounts of antibody as for the antibodies used in 4.4.11.1: Premix per sample: 0.7 µl (0.35 µg) mouse IgG1 K isotype control FITC (if applicable), 0.58 µl (0.115 µg) mouse IgG2a-BV421 isotype, 0.5 µl (0.05 µg) mouse IgG1 K isotype control APC, 13.2 µl PBS.
      3. For FMO controls, add antibodies as in 4.4.11.1, replacing one antibody each with PBS.
    12. Incubate at 4 °C for 30 min in the dark.
    13. Add 200 µl Permeabilization buffer. Centrifuge, remove supernatant and vortex as in step 4.4.8. Repeat once.
    14. Resuspend cells in cold FACS buffer (volume according to the instrument used) and acquire, ideally immediately, on the flow cytometer.

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Results

Figure 1 shows a scheme of the experimental setup. Figure 2 shows a representative purity control staining for magnetically isolated naïve CD4+ T cells and nTregs.

Figure 3A shows the flow cytometry gating strategy and Figure 3B shows representative FOXP3 and CD25 flow cytometry stainings on day 6 of culture under the indicated iTreg or control condit...

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Discussion

The described protocol enables the robust induction of human CD4+FOXP3+ iTregs from human naïve CD4+ T cells. It includes a new protocol that we described recently, using a combination of TGF-β, ATRA and rapamycin, for induction of iTregs with superior in vitro suppressive function22. Compared to other published protocols, another advantage is the induction of different iTreg populations in parallel by different protocols, which enables the direct comparison of effects of certain ...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

Nina Nagel is gratefully acknowledged for technical assistance during the video shoot and experimental preparation. We thank Eva-Maria Weiss for help with the intracellular FOXP3 staining protocol and Elisabeth Suri-Payer and Nina Oberle for establishing the nTreg isolation protocol. Matilda Eriksson and Peri Noori are acknowledged for laboratory management.

Funding: A.S. was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme, the Dr. Åke Olsson Foundation and KI research foundations; A.S. and J.T. were supported by a CERIC (Center of Excellence for Research on Inflammation and Cardiovascular disease) grant, J.T. was supported by Vetenskapsrådet Medicine and Health (Dnr 2011-3264), Torsten Söderberg Foundation, FP7 STATegra, AFA Insurance and Stockholm County Council.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
All-trans retinoic acidSigma AldrichR2625-50MG  
anit-human Foxp3-APC clone 236A/E7eBioscience17-4777-42
anti-human CD25 microbeadsMiltenyi Biotec130-092-983
anti-human CD25-PEMiltenyi Biotec130-091-024
anti-human CD28 antibody, LEAF Purified Biolegend302914
anti-human CD3 Antibody, LEAF Purified Biolegend317315
anti-human CD45RA, FITCMiltenyi Biotec130-092-247
anti-human CD45RO PE clone UCHL1BD Biosciences555493
anti-human CD4-PerCP clone SK3; mIgG1BD Biosciences345770
anti-human CD8-eFluor 450 (clone OKT8), mIgG2aeBioscience48-0086-42 
anti-human CTLA-4 (CD152), clone BNI3, mIgG2ak, Brilliant violet 421BD Biosciences562743
anti-human IFN-g FITC clone 4S.B3; mIgG1keBioscience11-7319-81 
Brefeldin A-containing solution: GolgiPlugBD Biosciences555029
cDNA synthesis kit: SuperScript VILO(Reverse transcriptase) cDNA Synthesis KitInvitrogen11754-250
Density centrifugation medium: Ficoll-PaqueGE healthcare17-1440-03
DMSO 99.7%Sigma AldrichD2650-5X5ML
FBS, heat inactivatedInvitrogen10082-147
Fixable Viability Dye, eFluor 780  eBioscience65-0865-14 or 65-0865-18 
Foxp3 Staining Buffer SeteBioscience00-5523-00Caution: contains paraformaldehyde. Can be also bought in combined kit with antibody; 77-5774-40 Anti-Human Foxp3 Staining Set APC Clone: 236A/E7 Set
GlutaMAX (200 mM L-alanyl-L-glutamine)Invitrogen35050-061
human naive CD4 T cell isolation kit IIMiltenyi Biotec130-094-131
Human serum albumin 50 g/LBaxter1501057
Ionomycin from Streptomyces conglobatus >98%Sigma AldrichI9657-1MG
MACS LS-columnsMiltenyi Biotec130-042-401
mouse IgG1 K Isotype Control APC Clone: P3.6.2.8.1eBioscience17-4714-42
mouse IgG1 K Isotype Control FITC 50 μgeBioscience11-4714-81 
mouse IgG2a isotype control, Brilliant violet 421, clone MOPC-173BD Biosciences563464
Pasteur pipet plastic, individually packedSarstedt86.1172.001  
PMA Phorbol 12-myristate 13-acetateSigma AldrichP1585-1MG 
RapamycinEMD (Merck)553210-100UG
Recombinant Human IL-2, CFR&D202-IL-050/CF
Recombinant Human TGF-beta 1, CFRnD240-B-010/CF
RNA isolation kit: RNAqueous-Micro KitAmbionAM1931  
RPMI 1640 Medium Invitrogen72400-054 
Sodium butyrateSigma AldrichB5887-250MG 
T cell culture medium: X-Vivo 15 medium, with gentamicin+phenolredLonza04-418Q
TaqMan Gene Expression Assay, FOXP3 (Best Coverage) Applied Biosystems4331182; assay ID: Hs01085834_m1Caution: contains paraformaldehyde
TaqMan Gene Expression Assay, RPL13A (Best Coverage)Applied Biosystems4351370; assay ID: Hs04194366_g1  Caution: contains paraformaldehyde
TaqMan Gene Expression Master mixApplied Biosystems4369514

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Naive CD4 T CellsTGF beta ProtocolFlow Cytometry AnalysisqRT PCR AnalysisPeripheral Blood Mononuclear CellsMagnetic Bead IsolationFOXP3 ExpressionTreg DifferentiationCell Culture Incubation
In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol
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