[methods in molecular biology] basic cell culture protocols volume 946 || in vitro generation of...

115

Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946,DOI 10.1007/978-1-62703-128-8_8, © Springer Science+Business Media, LLC 2013

Chapter 8

In Vitro Generation of Human T Regulatory Cells: Generation, Culture, and Analysis of FOXP3-Transduced T Cells

Alicia N. McMurchy and Megan K. Levings

Abstract

T regulatory cells (Tregs) suppress immune responses and therefore have potential to be used in the clinic as a cellular therapy for autoimmune disease and to prevent rejection of transplanted organs. Obtaining suf fi cient numbers of these cells for therapeutic use is a challenge, however, since there are currently no Treg cell-speci fi c markers, and they have a poor in vitro expansion potential. Tregs express high levels of FOXP3, a transcription factor that is critical for their function. We have shown that lentivirus-based overexpression of FOXP3 can reprogram naïve or memory human CD4 + T cells into cells which possess a phenotype and function similar to ex vivo Tregs. Here we will review the methodology involved in generating, expanding, and testing FOXP3-transduced cells and their ex vivo Treg counterparts.

Key words: T regulatory cells , Lentivirus , FOXP3 , Cellular therapy , Tolerance

T regulatory cells are immunosuppressive cells which regulate immune responses by inhibiting various cell types including antigen-presenting cells, B cells and T cells ( 1– 6 ) . Although there are many different types of Tregs, the best characterized are those which are CD4 + and constitutively express the IL-2 receptor alpha chain (CD25) ( 7 ) and the transcription factor FOXP3 ( 8– 10 ) , hereafter called “Tregs.” Experiments in mouse models have shown that adoptive transfer of Tregs, as an innovative cellular therapy, can suppress autoimmune disease ( 8, 11, 12 ) , graft rejection ( 13, 14 ) , and graft-versus-host disease ( 15, 16 ) and establish long-term and antigen-speci fi c tolerance. Thus, there is much excitement about the potential use of Tregs as a cellular therapy in humans.

1. Introduction

116 A.N. McMurchy and M.K. Levings

The major barrier to translating the use of Tregs in mice to humans is the lack of suitable methods to generate a large and homogenous population of cells in vitro since Tregs represent only ~3% of circulating CD4 + T cells and typically expand poorly in vitro. Moreover, there are no methods currently available to isolate pure populations of cells as no Treg-speci fi c cell surface molecules have been identi fi ed. Consequently, during in vitro expansion, small numbers of contaminating conventional T cells rapidly outgrow the Tregs, decreasing the suppressive capacity and hence the thera-peutic potential of the population ( 17, 18 ) . Furthermore, increasing evidence indicates that human FOXP3 + cells are a highly hetero-geneous population, with a signi fi cant fraction containing pro-in fl ammatory IL-17-producing cells ( 19– 21 ) . Therefore, better methods to generate and expand Tregs in vitro are required for the clinical translation of Treg cellular therapy.

We showed that when human CD4 + T cells are stably trans-duced with the transcription factor FOXP3, they acquire the phenotype and in vitro function of Tregs ( 22 ) . Thus, a large popu-lation of stably suppressive cells can be generated by transducing readily abundant naïve T cells with a lentivirus encoding FOXP3. A key feature of the lentivirus vector is the bidirectional promoter which allows for coordinate and constitutive expression of FOXP3 and a truncated form of the low af fi nity nerve growth factor receptor ( Δ LNGFR) as a cell surface marker of transduced cells. Transduced cells are puri fi ed based on Δ LNGFR expression and can be expanded and tested as necessary. A series of in vitro assays must be performed to con fi rm the Treg-like phenotype of FOXP3-transduced cells and should include testing of ex vivo expanded Tregs in parallel as a positive control. Key assays to con fi rm the expected phenotype of FOXP3-transduced T cells include analysis of expression of Treg-associated cell surface molecules, capacity to produce cytokines, and the ability to suppress the proliferation of CD4 + and/or CD8 + T cells.

Clones of antibodies used are listed in Table 1 .

1. Phosphate-buffered saline (PBS). 2. Ficoll-Paque. Store at room temperature or 4°C. 3. Ammonium chloride solution (0.8% ammonium chloride,

0.1 mM EDTA). Store aliquots at −20°C. Store thawed aliquots at 4°C for up to 2 weeks.

2. Materials

2.1. Isolation of Human Peripheral Blood Mononuclear Cells

1178 In Vitro Generation of Human T Regulatory Cells

1. Easy Sep Buffer: 2% fetal bovine serum in PBS. 2. Easy Sep CD4 + Negative Selection Kit (StemCell Technologies,

Vancouver, Canada). 3. Easy Sep Magnet (StemCell Technologies, Vancouver, Canada). 4. MACS buffer: PBS plus 2 mM EDTA and 0.5% fetal bovine

serum, degassed. Store at 4°C. 5. CD25 MicroBeads II (Miltenyi Biotec, Auburn, USA). 6. LS, MS, and LD columns (Miltenyi Biotec, Auburn, USA). 7. Midi MACS magnet and MACS stand (Miltenyi Biotec,

Auburn, USA). 8. CD45RO MicroBeads (Miltenyi Biotec, Auburn, USA). 9. Antibodies: anti-CD25, anti-CD45RO, anti-CD4. Ensure

anti-CD25 and anti-CD45RO antibodies recognize a different epitope than the antibody conjugated to the CD25 and CD45RO microbeads, respectively.

10. X-VIVO 15 supplemented with 5% AB human serum, 1× peni-cillin (100 U/mL), streptomycin (100 μ g/mL), and 1× Glutamax (2 mM). Store up to 1 month at 4°C. Add recombi-nant human rhIL-2 and rhIL-7 to the medium as required according to Subheading 3 . Do not refreeze IL-2 or IL-7.

1. Easy Sep Buffer as in Subheading 2.2.1 . 2. Easy Sep CD3 + Positive Selection Kit (StemCell Technologies,

Vancouver, Canada).

2.2. Isolation of Human CD4 + CD25 − CD45RO − T Cells and Human Antigen Presenting Cells from PBMCs

2.2.1. CD4 + CD25 − CD45RO − Cells

2.2.2. Antigen Presenting Cells

Table 1 Antibodies and clones

Antibody Clone

CD25 for purity check 4E3

CD45RO for purity check UCHL1

CD4 for purity check RPA-T4

CD25 M-A251

CD4 RPA-T4

LNGFR (CD271) ME20.4-1.H4 or C40-1457

FOXP3 236A/E7

IL-2 MQ1-17H12

IFN- γ 4S.B3

CD8 HIT8a

CD3 OKT3


3. Easy Sep Magnet as above. 4. X-VIVO 15 as in Subheading 2.2.1 , supplemented with con-

centrations of rhIL-2 and rhIL-7 indicated in Subheading 3 .

1. Anti-CD3 monoclonal antibody (OKT3). 2. Concentrated and titred lentivirus. Refer to ref. 22 and http://

tronolab.ep fl .ch for information on lentivirus production. Always transduce cells with the pCCL empty vector control and pCCL.FOXP3 virus in parallel. Store virus aliquots at −80°C. Avoid freeze-thawing, but if necessary, refreeze virus as soon as possible after use to prevent degradation.

3. X-VIVO 15 as in Subheading 2.2.1 , supplemented with con-centrations of rhIL-2 and rhIL-7 indicated in Subheading 3 .

4. Anti-LNGFR antibody. 5. 4% Formaldehyde in PBS.

1. PBE buffer: PBS + 5 mM EDTA. 2. MACSelect LNGFR MicroBeads (Miltenyi Biotec, Auburn,

USA). 3. LS columns (Miltenyi Biotec, Auburn, USA). 4. Midi MACS magnet and MACS stand. (Miltenyi Biotec,

Auburn, USA). 5. X-VIVO 15 as in Subheading 2.2.1 , supplemented with rhIL-2

as indicated in Subheading 3 .

1. Human peripheral blood mononuclear cells (PBMCs) isolated as described in Subheading 3.1 .

2. Epstein-Barr virus transformed JY cells, or equivalent lympho-blastoid cell line. Keep aliquots of JY cells frozen in liquid nitrogen. Keep cells in culture for no longer than 2 months before thawing a new vial. With each thaw, expand, and freeze down more cells for future use.

3. Phytohemagluttinin (PHA). Store stock dissolved in sterile distilled water at 1 mg/mL at −20°C.

4. Anti-LNGFR antibody. 5. X-VIVO 15 as in Subheading 2.2.1 . 6. FBS and 10% DMSO for freezing cells. 7. Cryovials for freezing cells. 8. Freezing container (optional).

1. PBS. 2. FACS buffer. PBS plus 1% FCS. Optional: Add sodium azide to

a fi nal concentration of 0.02% as a preservative. Store at 4°C. 3. Antibodies including: anti-CD25, anti-CD4, and anti-LNGFR.

2.3. Transduction of Human CD4 + CD25 − CD45RO − Cells with pCCL or pCCL.FP3 Lentivirus

2.4. Puri fi cation, Culture, and Restimulation of Transduced Cell Lines

2.4.1. Puri fi cation of Transduced Cell Lines

2.4.2. Culture and Restimulation of Transduced Cell Lines

2.5. Phenotypic and Functional Assays of Transduced Cells

2.5.1. CD25 Expression

http://tronolab.epfl.ch

http://tronolab.epfl.ch


1. PBS and FACS buffer as in Subheading 2.5.1 . 2. Antibodies including anti-CD4, anti-LNGFR, and anti-FOXP3

(236A/E7). 3. FOXP3 Fixation/Permeabilization Diluent and Concentrate

(eBioscience, San Diego, USA). Store at 4°C. Prepare fresh for each use by combining 1 part concentrate and 3 parts diluent.

4. 10× Permeabilization buffer (eBioscience, San Diego, USA). Store at 4°C. Prepare fresh for each use by diluting to 1× in distilled water.

1. Phorbol 12-myristate 13-acetate (PMA) dissolved in DMSO to 1 mg/mL. Store in aliquots at −80°C. Do not freeze–thaw.

2. Ionomycin dissolved in DMSO to 5 mg/mL and stored in aliquots at −80°C. Do not freeze/thaw.

3. Brefeldin A. Store stock at 10 mg/mL in DMSO at −20°C. Dilute 1 in 10 to 1 mg/mL before use.

4. FACS buffer as in Subheading 2.5.1 . 5. FOXP3 Fixation/Permeabilization Diluent and Concentrate

as in Subheading 2.5.2 . 6. 10× Permeabilization buffer as in Subheading 2.5.2 . 7. Antibodies including anti-CD4, anti-LNGFR, anti-IL-2, anti-

IFN- γ , and anti-FOXP3 (236A/E7).

1. PBMCs isolated as described in Subheading 3.1 . 2. PBS + 5% fetal bovine serum. 3. Carboxy fl uorescein diacetate succinimidyl ester (CFDA-SE).

Store dissolved in DMSO at a concentration of 5 mM in aliquots at −80°C. Do not freeze–thaw. CFDA-SE is converted to carboxy fl uorescein succinimidyl ester (CFSE) in the cyto-plasm of the cell.

4. Anti-CD3 monoclonal antibody (OKT3) or anti-CD3/anti-CD28-coated beads.

5. Anti-CD8 (conjugated to a fl urochrome other than FITC or Alexa 488).

1. To process a full donation of blood (450 mL), centrifuge whole blood at 600 × g for 25 min without brake (see Note 1). PBMCs are located at the interface (Buffy coat).

2. Remove the Buffy coat by pipetting carefully at the interface and transfer to a new tube (see Note 2). Dilute the Buffy coat

2.5.2. FOXP3 Expression

2.5.3. Intracellular Cytokine Staining

2.5.4. In Vitro Suppression Assay

3. Methods

3.1. Isolation of Human PBMCs from Whole Blood


1:1 with PBS. Alternatively, for smaller amounts of whole blood, dilute 1:1 in PBS directly without pre-enrichment for white blood cells.

3. Aliquot 15 mL of room temperature Ficoll-Paque into 50 mL Falcon tubes. Tilt the 50 mL Falcon 45° and slowly and care-fully layer 30 mL of the diluted buffy coat onto the Ficoll (see Note 3). Try to minimize mixing of the Ficoll and Buffy coat. Centrifuge tubes at 600 × g for 25 min without brake.

4. After the spin, the PBMCs will be at the Ficoll-plasma inter-face, with the red blood cells and granulocytes at the bottom of the tube. Carefully pipette at the interface, removing all PBMCs (see Note 4).

5. Dilute collected PBMCs 1:1 in 50 mL Falcon tubes with PBS, and centrifuge at 450 × g for 10 min.

6. Decant the supernatant (see Note 5) and collect the cells into one 50 mL Falcon tube. Top up the tube to 50 mL with PBS, and centrifuge at 450 × g for 5 min to wash out any remaining Ficoll.

7. Decant the supernatant. If there is red blood cell contamina-tion in the pellet, suspend the pellet in 5 mL room tempera-ture ammonium chloride solution and incubate at room temperature for 5 min (see Note 6) to lyse the red blood cells. Dilute the ammonium chloride with PBS to 50 mL after the 5 min incubation and centrifuge at 450 × g for 5 min.

8. Decant the supernatant and suspend the pellet again in 50 mL PBS. Centrifuge at 130 × g for 10 min to remove platelets.

9. Decant the supernatant and resuspend in PBS to count. Keep some PBMCs for isolation of antigen presenting cells (APCs), and use the rest to isolate CD4 + CD25 − CD45RO − cells (see Note 7).

1. Suspend PBMCs in Easy Sep Buffer at 5 × 10 7 cells/mL. Transfer cells to a 5 mL polystyrene tube if there are less than 1 × 10 8 cells or a 14 mL polystyrene tube for up to 4.25 × 10 8 cells.

2. Add enrichment cocktail from StemCell Easy Sep CD4 + Negative Selection Kit at 50 μ L/mL cells. Mix with a pipette and incubate at room temperature for 10 min.

3. Vortex the magnetic particles from the StemCell kit and add at 100 μ L/mL cells. Mix with a pipette and incubate at room temperature for 5 min.

4. Top up to 2.5 mL for less than 1 × 10 8 cells or 10 mL for 1–4.25 × 10 8 cells with Easy Sep Buffer. Mix gently with a pipette and place the tube without the cap into a small or large Easy Sep magnet, respectively. Incubate at room temperature for 5 min.

3.2. Isolation of Human CD4 + CD25 − CD45RO − Cells ( see Note 8 ) and Human APCs from PBMCs

3.2.1. CD4 + CD25 − CD45RO − Cells


5. Pour off the supernatant which contains CD4 + cells. Do not shake or blot off drops remaining at the lip of the tube.

6. Suspend CD4 + T cells in 90 μ L cold MACS buffer per 1 × 10 7 cells in a 15 mL Falcon tube.

7. Add 10 μ L of CD25 MicroBeads II per 1 × 10 7 cells. Incubate for 15 min at 4°C.

8. Top up to 10 mL with cold MACS buffer and centrifuge at 450 × g for 5 min. While the cells are spinning, prepare an LS column by placing it in a Midi MACS magnet on an MACS stand and washing with 3 mL of cold MACS buffer. Discard the fl ow-through.

9. Suspend cells well in 3 mL cold MACS buffer. Pass over the washed LS column. Once the 3 mL have passed through the column, wash three times with 3 mL cold MACS buffer. Keep the fl ow-through as CD25 − cells.

10. If a CD25 + Treg line is desired in parallel (see Note 9), elute the CD25 + cells by removing the column from the magnet and adding 5 mL of MACS buffer to the column. Immediately plunge the 5 mL through the column into a clean 15 mL tube using the plunger provided with the column. Remove the plunger from the column, add another 5 mL of MACS buffer to the column, and plunge again for a fi nal volume of 10 mL. Centrifuge eluted cells at 450 × g for 5 min. While cells are spinning, place an MS column in the magnet on the MACS stand and wash with 1 mL MACS buffer. Suspend cells in 1 mL cold MACS buffer, and add to the prewashed MS column. After addition of cells, rinse the column three times with 1 mL cold MACS buffer. Elute the cells by removing the column from the magnet and placing over a clean 15 mL tube. Add 1 mL of media to the column and plunge through with the plunger provided with the column. Remove the plunger and repeat the elution with another 1 mL of media. Keep the eluent as CD4 + CD25 + Tregs.

11. Count the CD25 − cells and suspend in 80 μ L cold MACS buffer per 1 × 10 7 cells.

12. Add 20 μ L CD45RO MicroBeads per 1 × 10 7 cells. Incubate for 15 min at 4°C.

13. Top up to 10 mL with cold MACS buffer and centrifuge at 450 × g for 5 min. While the cells are spinning, place an LD column in the magnet on the MACS stand and prewash with 2 mL MACS buffer. Discard the fl ow-through.

14. Suspend the cells in 2 mL cold MACS buffer and add the cells to the column. Wash the column two times with 1 mL and col-lect the fl ow-through as CD4 + CD25 − CD45RO − cells.

15. Check the purity of the isolated cells by fl ow cytometry with anti-CD4, anti-CD25, and anti-CD45RO antibodies.


16. Suspend the cells in X-VIVO 15 supplemented with 5% human serum, penicillin (100 U/mL), streptomycin (100 μ g/mL), and GlutaMAX (2 mM), as well as 100 U/mL rhIL-2 and 10 ng/mL rhIL-7 (see Note 10).

1. Suspend PBMCs at 1 × 10 8 /mL in Easy Sep Buffer in a 5 mL polystyrene tube (for up to 2 × 10 8 cells).

2. From a StemCell EasySep CD3 + Positive Selection Kit, add 100 μ L of positive selection cocktail per 1 mL of cells. Incubate at room temperature for 15 min.

3. Add 50 μ L of nanoparticles per 1 mL of cells and incubate at room temperature for 10 min.

4. Add Easy Sep Buffer to a fi nal volume of 2.5 mL. Mix and add to a small Easy Sep magnet for 10 min at room temperature.

5. Pour off the supernatant and keep this as CD3 − cells (APCs). 6. Suspend in the same medium plus cytokines as above in

Subheading 3.2.1 for CD4 + CD25 − CD45RO − cells.

Refer to Fig. 1 for a time line of the procedure from the activation and transduction of CD4 + cells to the assays for biological activity.

1. Plate 2–3 × 10 5 CD4 + CD25 − CD45RO − cells per well in a 24-well plate (or 10 5 in a 48-well plate). Add APCs at a 5:1 ratio of APCs:T cells.

2. Add anti-CD3 (OKT3) to a fi nal concentration of 1 μ g/mL, with a total of 1 mL fi nal volume for a 24-well plate or 0.5 mL fi nal volume for a 48-well plate.

3. Incubate overnight (16–18 h) at 37°C, 5% CO 2 . 4. Remove half the volume from each well and transfer to an

Eppendorf tube. Add pCCL.FP3 or pCCL control virus (see Note 11) to the Eppendorf at a multiplicity of infection of 10 (don’t count APCs in the calculation, only T cells). Mix gently and add media plus virus slowly and carefully back on top of the cells, placing the tip of the pipette at the edge of the well. Do not mix with the pipette, but swirl gently.

3.2.2. Antigen Presenting Cells

3.3. Transduction of Human CD4 + CD25 − CD45RO − Cells with pCCL or pCCL.FP3 Lentivirus

Fig. 1. Key time points in the generation, culture, and analysis of FOXP3-transduced T cells . The fi rst 20–26 days are outlined, which includes transduction of naïve T cells, puri fi cation of ΔLNGFR + cells, and restimulation and analysis of the cell lines.


5. Keep some cells untransduced and transduce some cells with pCCL control lentivirus as experimental controls.

6. After another 24 h, remove half the medium and replenish to dilute out the virus. Replace with fresh medium plus cytokines.

7. Monitor cell growth and split as required, keeping cells at approximately 1 × 10 6 /mL. Provide cells with fresh rhIL-2 and rhIL-7 every 2–3 days, as part of the splitting procedure or as a medium change. For a medium change, remove half the medium and add fresh medium containing 200 U/mL rhIL-2 for a fi nal concentration of 100 U/mL rhIL-2 and 20 ng/mL rh-IL-7 for a fi nal concentration of 10 ng/mL (assume cytokine consumption). Cease addition of rhIL-7 after ΔLNGFR puri fi cation (see below).

8. Six days after activation of the cells (5 days after transduction) the transduction ef fi ciency can be checked by staining with an anti-LNGFR antibody (see Note 12). Fix the cells with 4% formaldehyde to a fi nal concentration of 2% formaldehyde after staining and before reading by fl ow cytometry as a safety precaution against the possibility of live virus. An example of the transduction ef fi ciency analysis is shown in Fig. 2 .

Fig. 2. Transduction ef fi ciency and Δ LNGFR expression on puri fi ed cell lines. The left column shows an example of an average transduction ef fi ciency for pCCL control ( top ) and pCCL.FP3 ( bottom ) transduced cells. The right column shows ΔLNGFR expression after puri fi cation.


1. Between days 8 and 10 post-activation, purify the cells based on Δ LNGFR expression with MACSelect LNGFR MicroBeads. Wash the cells in PBE buffer and suspend in 160 μ L cold PBE plus 40 μ L MACSelect LNGFR MicroBeads in a 15 mL conical tube for up to 4 × 10 7 cells.

2. Incubate on ice for 15 min. 3. Top up to 10 mL with cold PBE and centrifuge at 450 × g for

5 min. While cells are spinning, place an LS column in a Midi MACS magnet on an MACS stand and prewash with 3 mL cold PBE buffer. Discard the fl ow-through.

4. Suspend the cells in 3 mL cold PBE and put over the pre-washed LS column.

5. Wash the column three times with 3 mL cold PBE buffer 6. Elute LNGFR + cells in X-VIVO 15 medium containing 100 U/

mL IL-2 by removing the column from the magnet, adding 3 mL of medium, and plunging the medium through the col-umn using the plunger provided with the column into a clean 15 mL tube. Repeat the elution step by removing the plunger from the column, adding another 3 mL of medium, and plung-ing again for a fi nal volume in the tube of 6 mL. Note the discontinuation of rhIL-7 in the medium since Tregs do not express the IL-7R α chain (CD127) and addition of IL-7 favors outgrowth of contaminating cells ( 22 ) . Culture as usual at approximately 1 × 10 6 cells/mL. An example of ΔLNGFR expression post-puri fi cation is shown in Fig. 2 .

1. Monitor the activation state of the cells by noting cell shape and clustering. When cells enter the resting phase (become small and round, stop proliferating), restimulate with a T cell feeder (see below). This will usually occur 10–13 days post-activation depending on the donor. Avoid restimulating within 48 h of the ΔLNGFR puri fi cation because the puri fi cation pro-cess can activate the cells, and restimulating them too soon after this process can lead to activation-induced cell death.

2. Prepare a 2× T cell feeder mixture according to the following recipe: 2 × 10 6 /mL irradiated (5,000 rad) allogeneic human PBMCs, prepared as described above (see Note 13), 2 × 10 5 /mL irradiated (7,500 rad) JY cells, 2 μ g/mL PHA, and 200 U/mL rhIL-2.

3. Plate cells in 0.5 mL in a 24-well plate with between 1 and 5 × 10 5 cells per well (2 × 10 5 per well is optimal). Add 0.5 mL 2× feeder on top.

4. Change medium after 2–3 days, adding fresh medium plus 200 U/mL rhIL-2, or split if necessary. Keep cells at approxi-mately 1 × 10 6 /mL as normal (see Note 14).

3.4. Puri fi cation, Culture, and Restimulation of Transduced Cell Lines

3.4.1. Puri fi cation of Transduced Cell Lines

3.4.2. Culture and Restimulation of Transduced Cell Lines


5. Check the phenotype and function of transduced cells 10–13 days after restimulation (see below). To keep cells in culture, restimulate again with a feeder mixture. Cells can also be frozen in FBS plus 10% DMSO at this point at densities between 1 × 10 5 and 5 × 10 7 cells/mL. Freeze cells by sus-pending them in cold FBS plus 10% DMSO and transferring to a cryovial. Freeze cells slowly by either incubating vials on ice for 25 min before transferring to −80°C or using a freezing container. Transfer frozen to cells liquid nitrogen the following day for maximal viability post-thaw.

6. Routinely check LNGFR expression of transduced cells since contaminating untransduced cells can outgrow FOXP3-expressing cells. If necessary (see Note 15), repurify ΔLNGFR + cells, but always wait until days 8–10 after restimulation and at least 2 days before the next restimulation and assays to avoid activation-induced cell death.

1. Before performing phenotypic and functional assays the cells should be rested overnight. Wash cells once with PBS and replate in medium lacking rhIL-2 at 2–3 × 10 6 /mL the night before the assays. In the morning, wash again with PBS and suspend in medium lacking rhIL-2.

2. Count cells and adjust the concentration to 1 × 10 6 cells/mL. Perform assays as described below.

1. Suspend 5 × 10 4 –1 × 10 5 cells in 25–50 μ L of FACS buffer and stain for CD25, CD4, and LNGFR in a V-bottom 96-well plate. Incubate at 4°C for 20–30 min.

2. Top up to 200 μ L with FACS buffer and centrifuge the plate at 980 × g for 3 min.

3. Suspend in 200 μ L FACS buffer and analyze on a fl ow cytom-eter. Expected results are shown in Fig. 3 .

1. Suspend 1–2 × 10 5 cells in 25–50 μ L of FACS buffer and stain for CD4 and LNGFR in a V-bottom 96-well plate. Stain for 20–30 min at 4°C (see Notes 19 and 20).

2. Wash cells by topping up to 200 μ L and centrifuging at 980 × g for 3 min. Prepare eBioscience Fixation/Permeabilization buffer and add 100 μ L per well. Incubate at 4°C for 30–60 min. Top up with PBS to 200 μ L and centrifuge at 980 × g for 3 min.

3. At this point, cells can be suspended in FACS buffer and left overnight at 4°C to continue the next morning, or the proce-dure can be continued immediately.

4. Suspend cells in 200 μ L eBioscience 1× Permeabilization buffer. Centrifuge at 980 × g for 3 min and wash again with Permeabilization buffer.

3.5. Phenotypic and Functional Assays of Transduced Cells ( see Note 16 )

3.5.1. CD25 Expression ( see Note 17 )

3.5.2. FOXP3 Expression ( See Note 18 )


5. Suspend cells in 25 μ L Permeabilization buffer and add anti-FOXP3 antibody. Incubate 30 min at room temperature.

6. Top up to 200 μ L with Permeabilization buffer and centrifuge at 980 × g for 3 min. Wash once more with FACS buffer. Suspend cells in 200 μ L FACS buffer and read results on a fl ow cytometer. Expected results are shown in Fig. 4 .

1. Remove 2 × 200 μ L of 1 × 10 6 /mL cells (or 2 × 100 μ L if cell number is limited and adjust the volume to 200 μ L with medium) for intracellular cytokine staining. Place each 200 μ L aliquot in one well of a 96-well round-bottom plate. One well will be stimulated with PMA and ionomycin and one well will be left as an unstimulated control. Also take extra cells from control transduced cells for fl ow cytometry compensation controls.

3.5.3. Intracellular Cytokine Staining ( See Note 21 )

Fig. 3. CD25 is upregulated on pCCL.FP3-transduced cells compared to pCCL control-transduced cells. CD25 expression is measured when cells are in the resting state, 10–13 days after initial activation or restimulation.

Fig. 4. FOXP3 and Δ LNGFR expression on pCCL.FP3-transduced cells and control cells. FOXP3 and ΔLNGFR expression when cells are in the resting state 10–13 days after restimulation.


2. Prepare a mixture of PMA and ionomycin as follows: Dilute stock PMA (1 mg/mL) 1 in 1,000 in X VIVO 15 media supple-mented with 5% human AB serum, 100 U/mL penicillin, 100 μ g/mL streptomycin, and 2 mM GlutaMAX. Using the same medium, dilute stock ionomycin (5 mg/mL) 1 in 100. Add 100 μ L of diluted PMA to 100 μ L of diluted ionomycin and mix. Add 4 μ L of this mixture to one of the two wells for each sample resulting in fi nal concentrations of 10 ng/mL for PMA and 500 ng/mL for ionomycin. Incubate at 37°C for 2 h.

3. Add 2 μ L of 1 mg/mL Brefeldin A to all wells for a fi nal concentration of 10 μ g/mL.

4. Incubate for 4 h. Transfer the cells to a V-bottom plate. Spin the plate at 980 × g for 3 min and shake out the supernatant.

5. Suspend the cells in 25–50 μ L of FACS buffer and stain for CD4 and LNGFR (see Notes 19 and 20). Incubate for 20–30 min at 4°C.

6. Top up each well to 200 μ L and centrifuge at 980 × g for 3 min to wash the cells. Prepare eBioscience Fixation/Permeabilization buffer and add 100 μ L per well. Incubate at 4°C for 30–60 min. Top up with PBS to 200 μ L and centrifuge at 980 × g for 3 min.

7. At this point, cells can be suspended in FACS buffer and left overnight at 4°C to continue the next morning, or the proce-dure can be continued immediately.

8. Suspend cells in 200 μ L eBioscience 1× Permeabilization buf-fer. Centrifuge at 980 × g for 3 min and wash again with Permeabilization buffer.

9. Suspend cells in Permeabilization buffer and add antibodies: anti-IL-2, anti-IFN- γ , and anti-FOXP3 (see Note 20). Incubate 30 min at room temperature.

10. Top up to 200 μ L with Permeabilization buffer and centrifuge at 980 × g for 3 min. Wash once more with FACS buffer. Suspend cells in FACS buffer and acquire data on a fl ow cytometer. Stimulated pCCL control transduced cells and untransduced cells should produce a signi fi cant amount of IL-2 and IFN- γ , while stimulated FOXP3-transduced cells should not produce much of either cytokine. Expected results are shown in Fig. 5 .

1. Isolate human PBMCs as described in Subheading 3.1 (see Note 22).

2. Suspend PBMCs in PBS plus 5% FBS to 1 × 10 6 /mL. 3. Label the PBMCs with CFSE by diluting stock CFDA-SE

(5 mM in DMSO) 1 in 100 in PBS plus 5% FBS. For each 1 mL of PBMCs, add 100 μ L of diluted CFDA-SE.

4. Incubate for 3.5 min at room temperature and wash with PBS + 5% FBS.

3.5.4. In Vitro Suppression Assay


5. Suspend cells in media lacking rhIL-2 and adjust the concen-tration to 1 × 10 6 /mL.

6. Plate cells in a 96-well round-bottom plate as described below. Negative control: 100 μ L PBMCs + 150 μ L medium Positive control: 100 μ L PBMCs + 100 μ L medium 1:1—100 μ L PBMCs + 100 μ L test cells 1:2—100 μ L PBMCs + 50 μ L test cells + 50 μ L medium 1:4—100 μ L PBMCs + 25 μ L test cells + 75 μ L medium 1:8—100 μ L PBMCs + 12.5 μ L test cells + 87.5 μ L medium 1:16—100 μ L PBMCs + 6.3 μ L test cells + 93.7 μ L medium 7. To all wells EXCEPT the negative control, add 50 μ L 5 μ g/

mL anti-CD3 for a fi nal concentration in the well of 1 μ g/mL (see Note 23).

Fig. 5. Cytokines are downregulated in pCCL.FP3-transduced cells compared to pCCL control-transduced cells. Cells are activated with PMA and Ionomycin for 6 h, with Brefeldin A added for the last 4 h. Following activation, pCCL.FP3 and expanded ex vivo CD4 + CD25 + T cells produce signi fi cantly less IL-2 and IFN- γ than pCCL control transduced cells.

Fig. 6. Ex vivo CD4 + CD25 + T cells suppress the proliferation of CD8 + responder cells. 1 × 10 5 human PBMCs are labeled with CFSE and cocultured with ex vivo CD4 + CD25 + cells at the indicated ratios in the presence of 1 μ g/mL anti-CD3. Four days later, cells are stained with anti-CD8 antibody and analyzed by fl ow cytometry. Analysis is done on gated on CD8 + T cells to ensure no CD4 + Tregs are included in the gate. The negative control contains PBMCs alone in the absence of anti-CD3, and the positive control contains PBMCs alone in the presence of anti-CD3.


8. Incubate at 37°C for 4 days. 9. Stain all samples with anti-CD8 and read by FACS (CFSE is in

the FITC channel.) Expected results for ex vivo CD4 + CD25 + T cells are shown in Fig. 6 . Using the proliferation platform of a fl ow cytometry software package, calculate the average number of divisions undergone by a cell in the starting popula-tion (de fi ned as division index or proliferation index, depending on the software package).

1. Alternatively, whole blood can be left overnight at room temperature to separate. Collect Buffy coat as normal the next day.

2. In a 250 mL conical tube, mark 1 cm above and below the interface. Remove and discard plasma to the top mark and care-fully pipette at the interface until the bottom mark is reached, transferring the Buffy coat to a new 250 mL conical tube.

3. Alternatively, 30 mL of diluted PBMCs can be added to the 50 mL falcon tube fi rst, and the Ficoll can be underlayed by slowly pipetting the Ficoll into the bottom of the tube.

4. Remove the cells from the Ficoll immediately, as Ficoll is toxic to the cells. Pipetting Ficoll with the cells cannot be avoided, but try to minimize Ficoll contamination by pipetting just above the cell layer in the plasma to favor plasma collection over Ficoll collection.

5. A high degree of Ficoll contamination may hinder pelleting of cells. The supernatant from this spin can be kept, diluted further with PBS, and recentrifuged in an attempt to recover more cells.

6. Do not incubate in the ammonium chloride solution for longer than 5 min, as a greater length of time will affect the white blood cells in addition to the red blood cells.

7. Expect the yield of APC from PBMCs to be about 15–30% and the yield of CD4 + CD25 − CD45RO − T cells from PBMCs to be about 2–10%. Keep in mind that fi ve times as many APCs as CD4 + CD25 − CD45RO − cells are required.

8. CD4 + CD25 − CD45RO − T cells are naïve cells which are most readily converted into Tregs. However CD4 + CD25 − or total CD4 + cells may also be transduced. Depleting CD25 + cells removes natural T regulatory cells from the transduced population.

9. Depending on their purity, a bead-sorted CD4 + CD25 + T cell line may serve as an adequate Treg cell line control, however the most reliable source of CD4 + Tregs for expansion can be

4. Notes


obtained by sorting the CD25 hi CD45RA + T cells from CD25-enriched T cells according to Hoffmann et al. ( 23 ) .

10. Addition of rhIL-7 is optional, but use it to obtain maximum transduction ef fi ciency.

11. Consult with your local biosafety of fi ce prior to commencing work with lentivirus to ensure adequate biosafety precautions are taken. Wear appropriate personal protective equipment and decontaminate all waste with 10% bleach before discarding.

12. The average transduction ef fi ciency for pCCL control lentivi-rus is 86 ± 12% (76–97%) and the average for pCCL.FP3 lenti-virus is 47 ± 18% (28–71%) ( 22 ) .

13. If possible, it is best to combine PBMCs from two different donors (1 × 10 6 /mL for each). Using two donors better ensures a successful stimulation in case one of the donors does not produce a strong allogeneic response. It is also preferable to use PBMCs that have been freshly prepared; however, a stock of PBMCs can be frozen in liquid nitrogen and thawed as needed for restimulations.

14. Cells will need to be split more often early after the stimulation (in the fi rst 6–7 days) and less often later on in the cycle. In the second week after the activation, as cells enter the resting phase, they should be kept denser (approximately 2 × 10 6 /mL). If cells appear to be dying in the second week, they can be washed to remove dead cells and replated at a higher density with fresh rhIL-2. In an average experiment, starting with 5 × 10 5 naïve T cells, approximately 1.5 × 10 7 pCCL.FP3-transduced T cells should be obtained after 20–26 days in culture (after the initial ΔLNGFR + puri fi cation and at the end of the fi rst restimulation) ( 22 ) .

15. Cells should be repuri fi ed if they are less than 85% ΔLNGFR + at 8–10 days after T cell receptor stimulation.

16. Lack of IL-2 and IFN- γ production and upregulation of CD25 can be observed after the fi rst round of expansion (10–13) days in culture, but suppressive capacity is not fully realized until after a second round of expansion. The assays can be conducted over 2 days, with the intracellular staining and CD25 expression performed 1 day and the FOXP3 expression and suppression assay performed another. It is useful to check FOXP3 expression on the same day that the suppression assay is set up so that the proportion of FOXP3-expressing cells at the time of the assay is known.

17. Cell surface markers in addition to CD25 can also be examined including CTLA4, CCR4, GITR, and CD127, but CD25 is the most robust and reliable. This assay should be performed when cells are in the resting state, when CD25 expression should be high in FOXP3-transduced cells and low in pCCL control and


untransduced cells. If CD25 is high in pCCL control or untrans-duced cells, they may still be activated, and the assay should be repeated at a later time point. Avoid giving cells fresh rhIL-2 the day before the assay to ensure cells are in the resting state.

18. The CD25 and FOXP3 stains can be combined; however, the permeablization step of FOXP3 intracellular staining can some-times interfere with CD25 and LNGFR surface staining, so it is also preferable to do surface stains alone in parallel to surface plus FOXP3 intracellular staining.

19. Also perform staining for fl ow cytometry controls including an unstained control, single-stained controls, and fl uorescence minus one controls. Treat controls the same way as samples throughout the procedure.

20. The FOXP3 fi xation/permeablization buffer can break apart conjugated monoclonal antibodies, so avoid using these when staining for FOXP3 and intracellular cytokines.

21. Cytokine production can also be determined by ELISA. 22. Autologous untransduced CD4 + cells cultured in parallel may

also be used as responders instead of freshly isolated PBMCs. Indeed, use of a cell line cultured in parallel may provide more accurate results than freshly isolated responders because freshly isolated T cells have different kinetics of activation compared with T cell lines. When analyzing data with CD4 + T cells as responders, gate out CFSE low T cells or label test cells with another cell proliferation dye to distinguish them from the responders.

23. Alternatively, stimulate PBMCs with anti-CD3/anti-CD28-coated beads. Perform a titration of beads to determine the optimum ratio of beads:cells that will result in at least 20% of the cells dividing at least once. Usually a ratio of 1:16 or 1:32 beads:cells results in good proliferation.

Acknowledgments

We thank Sarah E. Allan, Rosa Bacchetta, Maria Grazia Roncarolo, Mario Amendola, and Luigi Naldini for their contributions to the development of this protocol. Supported by the Roche Organ Transplant Research Foundation, CIHR (MOP-93793), and Stemcell Technologies, Inc. Core support for fl ow cytometry and virus production was funded by the Immunity and Infection Research Centre Michael Smith Foundation for Health Research (MSFHR) Unit. MKL holds a Canada Research Chair in Transplantation. ANM holds a Canada Vanier Scholarship, a MSFHR Junior Graduate Studentship, and a CIHR Transplantation Training Program award.


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