Suppression of Human Dendritic Cells by Regulatory T Cells

Materials and Reagents

Materials

1. 5 ml polystyrene round-bottom tubes (Corning, catalog number: 352052)

2. TC-coated 6-well plates (Corning, catalog number: 353502)

3. TC-coated 12-well plates (Corning, catalog number: 353503)

4. 96-well U- and V-bottom plates (Corning, catalog numbers: 353077, 3894)

5. 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 229442)

6. 15 ml and 50 ml conical tubes (Corning, catalog numbers: 352096, 352070)

7. Sterile 1 ml or 3 ml syringe (BD, catalog numbers: 309628, 309657)

1. Lymphoprep^TM (STEMCELL Technologies, catalog number: 07801)

2. Dulbecco’s Phosphate Buffered Saline (DPBS; Gibco, catalog number: 14190), 1×

3. X-VIVO 15 (Lonza, catalog number: BEBP02-054Q)

4. Human serum (Wisent Bio Products, catalog number: 022210)

5. Penicillin/streptomycin (Gibco, catalog number: 15140-122)

6. GlutaMAX (Gibco, catalog number: 35050-061)

7. Sodium pyruvate (Gibco, catalog number: 11360-070)

8. Fetal bovine serum (Gibco, catalog number: 12483020)

9. Ethylenediaminetetraacetic acid solution (EDTA) (Sigma-Aldrich, catalog number: 03690)

10. EasySep Buffer (see Recipes)

11. Dendritic Cell Medium (see Recipes)

1. Stericup-GP Sterile Vacuum Filtration System (Millipore, catalog number: SCGPU05RE), 500 ml

2. EasySep Human CD14 Positive Selection Kit II (STEMCELL Technologies, catalog number: 17858)

3. Acridine Orange/Propidium Iodide (AO/PI; Nexcelom, catalog number: NEX-CS201065ML)

1. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (STEMCELL Technologies, catalog number: 78015), 20 μg/ml

2. Recombinant human interleukin (IL)-4 (STEMCELL Technologies, catalog number: 78045), 20 μg/ml

3. Recombinant human tumour necrosis factor (TNF)-α (eBioscience, catalog number: 14-8329-63), 20 μg/ml

4. Prostaglandin E2 (PGE2) (Tocris, catalog number: 2296),100 mM

5. Recombinant human IL-1β (STEMCELL Technologies, catalog number: 78041), 20 μg/ml

6. Recombinant human IL-6 (STEMCELL Technologies, catalog number: 78148), 20 μg/ml

7. Recombinant human interferon (IFN)-γ (eBioscience, catalog number: 14-8319-80), 20 μg/ml

8. Recombinant human IL-2 (Proleukin) (Novartis, DIN# 02130181)

1. Fc Receptor Binding Inhibitor Polyclonal Antibody (eBioscience, catalog number: 14-9161-73)

2. Fixable Viability Dye (FVD) (eBioscience, catalog number: 65-0865-18)

3. Anti-human CD3 (UCHT1) APC (BD Biosciences, catalog number: 564465)

4. Anti-human CD3 (UCHT1) BB515 (BD Biosciences, catalog number: 564465)

5. Anti-human CD4 (OKT4) APC (eBioscience, catalog number: 17-0048-42)

6. Anti-human CD4 (RPA-T4) BV711 (BioLegend, catalog number: 300558)

7. Anti-human CD8a (RPA-T8) BV711 (BioLegend, catalog number: 301044)

8. Anti-human CD11c (B-ly6) PE (BD Biosciences, catalog number: 555392)

9. Anti-human CD14 (M5E2) BV421 (BioLegend, catalog number: 301830)

10. Anti-human CD14 (M5E2) BV786 (BD Biosciences, catalog number: 563698)

11. Anti-human CD40 (5C3) PE-Cy7 (BD Biosciences, catalog number: 561215)

12. Anti-human CD56 (CMSSB) PE (eBioscience, catalog number: 12-0567-42)

13. Anti-human CD69 (FN50) BV785 (BioLegend, catalog number: 310932)

14. Anti-human CD70 (113-16) PerCP-Cy5.5 (BioLegend, catalog number: 355107)

15. Anti-human CD80 (L307.4) FITC (BD Biosciences, catalog number: 557226)

16. Anti-human CD83 (HB15e) BV421 (BioLegend, catalog number: 305324)

17. Anti-human CD86 (2331 (FUN-1)) APC (BD Biosciences, catalog number: 555660)

18. Anti-human CD86 (HA5.2B7) PerCP-Cy5.5 (Beckman Coulter, catalog number: B30647)

Equipment

1. Type II Biosafety cabinet (NuAire, model: LabGard ES NU-540)

2. Centrifuge, microcentrifuge (Eppendorf, models: 5810R and 5452)

3. STEMCELL EasySep^TM magnet (STEMCELL Technologies, catalog number: 18000)

4. Cell counter (Nexcelom, model: Cellometer Auto 2000)

5. 37°C incubator with 5% (v/v) CO₂ (Sanyo, model: MCO-18AIC)

6. Flow cytometer (BD LSRFortessa X-20; alternative instruments can be used)

Software

1. FlowJo software (BD Biosciences, v10.7)

Procedure

Overview

1. Day 0: Prepare peripheral blood mononuclear cells (PBMCs).

2. Day 0: Isolate CD14⁺ monocytes from PBMCs by positive selection.

3. Day 0: Differentiate monocytes into dendritic cells by culturing in the presence of GM-CSF and IL-4.

4. Day 3: Replenish GM-CSF and IL-4.

5. Day 5: Mature dendritic cells by adding TNF-α, PGE2, IL-1β, and IL-6 to the culture.

6. Day 6: Mature dendritic cells by adding IFN-γ to the culture.

7. Day 7: Confirm maturation of the DCs by flow cytometry and set up the suppression assay.

8. Day11: Collect cells, stain, perform flow cytometry, and analyse.

   
   

1. Day 0: Prepare PBMCs

   1. Prepare PBMCs: Either freshly isolated from blood or thawed PBMCs. PBMCs can be isolated from human peripheral blood by density gradient centrifugation using Lymphoprep^TM, according to the manufacturer’s protocol.

      Note: Using batch-frozen PBMCs from one donor to differentiate moDCs will reduce donor-to-donor variability. Expect 5-10% fresh/frozen PBMCs or 50-100% CD14⁺ cells to become moDCs. See Notes for further details.

   2. Optional: Set aside ~5 × 10³-10 × 10³ PBMCs for purity check (see Table 1, Figure 1, and Notes for further information).

      
      

      Table 1. Day 0 Purity Check Panel. Stain PBMCs in parallel, if desired. See Figure 1 for representative data.
      

      Marker	Dilution	Clone	Fluorophore
      Fc Receptor Binding Inhibitor (preincubate for 10 min and do not wash)	1:5	Polyclonal	N/A
      FVD	1:1,000	N/A	eF780
      CD3	1:100	UCHT1	BB515
      CD4	1:100	OKT4	APC
      CD8a	1:100	RPA-T8	BV711
      CD14	1:200	M5E2	BV421
      CD56	1:25	CMSSB	PE

      
      

      
      
      Figure 1. Day 0 CD14⁺ Purity Check. (A) Approximately 5 × 10³-10 × 10³ total PBMCs and (B) CD14-enriched cells were stained and analysed by flow cytometry to evaluate the purity of the CD14-enriched cells and the extent of CD3⁺ cell contamination. Contaminating T cells can affect downstream results if OKT3 (anti-CD3) is included during assays (see Notes). Cells were stained as described in Table 1 and in accordance with “Guidelines for the use of flow cytometry and cell sorting in immunological studies” (Cossarizza et al., 2019); they were acquired with a BD LSRFortessa X-20 and data analysed using FlowJo. The proportion of total live cells that were CD14⁺CD3^‒ (CD14⁺ cell purity) is shown on the left. From the live CD14^‒ cells, CD56 expression was analysed (middle) to identify NK/NKT cells (CD56^med/hi) and a subset of monocytes (CD56^lo). The contaminating live CD14^‒CD3⁺ T cells were further analysed for their expression of CD4 and CD8 (right). See Notes for more information.

      
      

2. Day 0: Isolate CD14⁺ monocytes using the STEMCELL EasySep Human CD14 Positive Selection Kit II, as per the manufacturer’s protocol and described below, unless stated otherwise:

   1. Resuspend PBMCs at 100 × 10⁶/ml in EasySep Buffer (0.1-2 ml). The total starting number of PBMCs should be between 10 × 10⁶ and 200 × 10⁶ cells. For x number of CD14⁺ monocytes, start with 10× (fresh) or ~20× (frozen) PBMCs.

   2. Transfer PBMCs to a 5 ml polystyrene round-bottom tube.

   3. Add 100 μl of Selection Cocktail per millilitre of cells and incubate 10 min at room temperature (RT).

   4. Vortex RapidSpheres for 30 s, add 100 μl of RapidSpheres per ml cells, mix tube by gentle rotation, and incubate for 3 min at RT.

   5. Top up tube with EasySep Buffer to 2.5 ml, mix by pipetting, place in an EasySep^TM magnet, and incubate for 3 min at RT.

   6. Gently pour tube while in magnet to discard supernatant. The round-bottom tube contains CD14⁺ cells (keep).

   7. Wash remaining RapidSpheres by removing the round-bottom tube from the magnet and topping up tube with 2.5 ml of EasySep Buffer. Mix by pipetting to wash layer off tube wall, replace the tube back in the magnet, and incubate for 3 min at RT. Pour to discard supernatant and pipette last drop from tube while inverted.

   8. Repeat Step B7 so that the tube of cells is incubated in the magnet a total of three times.

   9. Optionally repeat Step B7 two more times (total of five times on magnet) for potentially higher enrichment.

      Note: This step is not part of the original STEMCELL Technologies protocol.

   10. Transfer the cells from the round-bottom tube into a 15 ml conical tube by washing the walls of the round-bottom tube with ~3 ml of DPBS. Centrifuge at 450 × g for 5 min and discard supernatant.

   11. Resuspend the enriched CD14⁺ cells in Dendritic Cell Medium (see Recipes).

   12. Count cells (see protocols from Nexcelom Cellometer Auto 2000 for details; alternatively, a hemocytometer and trypan blue can be used).

   13. Optional: set aside ~5 × 10³-10 × 10³ monocytes for purity check (see Table 1, Figure 1, and Notes for further information).




3. Day 0: Culture and differentiate enriched CD14⁺ cells into dendritic cells

   1. Adjust cells to 2 × 10⁶ cells per millilitre with Dendritic Cell Medium.

   2. Plate 2 ml cells per well in a 6-well plate (4 × 10⁶ cells per well).

      Note: Plate multiple wells of cells as needed.

   3. Add GM-CSF (final concentration: 50 ng/ml) and IL-4 (final concentration: 100 ng/ml).

   4. Incubate cells at 37°C (5% v/v CO₂) for 3 days.

      Note: Cytokines can be stored at 4°C for ~1 week.




4. Day 3: Change Dendritic Cell Medium and replenish cytokines

   1. Change Dendritic Cell Medium: without disturbing the cells at the bottom of the well, collect 1.5 ml of medium into sterile 1.5 ml microcentrifuge tube, centrifuge at 450 × g for 5 min, discard the supernatant, resuspend the pelleted cells in 1.5 ml of fresh Dendritic Cell Medium, and re-plate into original well.

   2. Fully replenish the cytokines by adding fresh GM-CSF (final concentration: 50 ng/ml) and IL-4 (final concentration: 100 ng/ml), assuming that no cytokines from day 0 remain in the culture.

   3. Incubate at 37°C (5% v/v CO₂) for 2 days.

      Note: Cytokines can be stored at 4°C for ~1 week.

      Alternative medium change method: Centrifuge plate at 450 × g for 5 min, gently replace 1.5 ml of Dendritic Cell Medium, and fully replenish the GM-CSF and IL-4 as detailed above.




5. Day 5: Mature dendritic cells

   1. Collect cells: pipette up and down to detach cells and collect either into a 15 ml or 50 ml conical tube. Detach remaining cells using a 1 ml or 3 ml syringe rubber in gentle, one-way movements. Rinse well twice with DPBS to collect as many cells as possible.

   2. Wash cells by filling the conical tube with DPBS, centrifuge cells at 450 × g for 5 min, and discard the supernatant.

   3. Resuspend cells in fresh Dendritic Cell Medium, count cells, and adjust volume so that cells are at a final concentration of 1 × 10⁶ cells/ml in Dendritic Cell Medium.

   4. Add GM-CSF (final concentration: 50 ng/ml) and IL-4 (final concentration: 100 ng/ml).

   5. Plate cells in a new well by adding 2 ml of cells per well in a new 6-well plate (2 × 10⁶ cells per well) or 1 ml of cells per well in a new 12-well plate (1 × 10⁶ cells per well).

   6. Mature moDCs by adding TNF-α (final concentration: 50 ng/ml), PGE2 (final concentration: 1 μg/ml, ~2.837 µM), IL-1β (final concentration: 10 ng/ml), and IL-6 (final concentration: 100 ng/ml).

      Note: If immature moDCs are desired, skip this step. Keeping a minimum of 1 × 10⁶ immature moDCs (GM-CSF and IL-4 with no additional cytokines) is useful for evaluating the moDC maturation at the end (day 7, see Procedure G).

   7. Incubate at 37°C (5% v/v CO₂) for 1 day.




6. Day 6: Continue to mature dendritic cells

   1. Add IFN-γ (final concentration: 50 ng/ml).

      Note: Skip this step for immature moDCs.

   2. Incubate at 37°C (5% v/v CO₂) for 1 day.




7. Day 7: Confirm maturation of the DCs before setting up the suppression assay

   1. Collect cells by pipetting to resuspend and transfer to a 15 ml conical tube. Detach remaining cells using a 1 ml or 3 ml syringe rubber in gentle, one-way movements. Wash wells twice with DPBS and add this to the conical tube. Centrifuge cells at 450 × g for 5 min, resuspend in fresh Dendritic Cell Medium, count cells, and adjust cell concentration to 5 × 10⁵ cells/ml in Dendritic Cell Medium.

   2. Confirm moDC maturation by collecting ~5 × 10⁶-10 × 10⁶ mature moDCs and immature moDCs for staining for flow cytometry (see Table 2, Figure 2).

      
      

      Table 2. Day 7 moDC Maturation Check Panel. Immature moDC must be stained in parallel. See Figure 2 for representative data.
      

      Marker	Dilution	Clone	Fluorophore
      Fc Receptor Binding Inhibitor (preincubate for 10 min and do not wash)	1:5	Polyclonal	N/A
      FVD	1:1,000	N/A	eF780
      CD3	1:50	UCHT1	APC
      CD11c	1:50	B-ly6	PE
      CD14	1:100	M5E2	BV785
      CD80	1:50	L307.4	FITC
      CD83	1:50	HB15e	BV421
      CD86	1:50	HA5.2B7	PerCp-Cy5.5
      CD40	1:100	5c3	PECy-7
      HLA-DR	1:50	L243	BV510

      
      

      
      Figure 2. Day 7 phenotype of immature and mature moDCs. (A-C) Immature (black line) and mature (blue) moDCs were analysed by flow cytometry for expression of CD11c, CD14, CD80, CD86, CD40, CD83, and HLA-DR. Cells were stained as described in Table 2 and in accordance with “Guidelines for the use of flow cytometry and cell sorting in immunological studies” (Cossarizza et al., 2019); they were acquired with a BD LSRFortessa X-20 and data analysed using FlowJo. moDCs (gated as live, single cells) expressed similar levels of lineage markers (A) but upregulated expression of CD80, CD86, HLA-DR, CD40, and CD83 following maturation (B-C). MFI, geometric mean fluorescence intensity.

      
      

8. Day 7: Set up DC suppression assay

   1. Plate 100 µl moDC in a 96-well U-bottom plate (50,000 cells per well).

   2. Collect rested Tregs, count cells, and resuspend at 2.5 × 10⁶ cells per ml in Dendritic Cell Medium with 100 IU/ml IL-2.

      Note: Tregs should be expanded and rested using protocols established by the user’s lab. Details of how Tregs were expanded and rested for this protocol are provided in Dawson et al. (2020). Briefly, Tregs were isolated from peripheral blood and polyclonally expanded with artificial APCs in the presence of 1000 IU/ml IL-2 for 7 days. Tregs were then rested by culturing in fresh medium with 100 IU/ml IL-2 overnight. CAR Tregs were generated by lentivirally transducing polyclonal Tregs one day post-stimulation.

   3. Set up moDC and Treg co-culture in a 1-to-5 ratio (50,000 DCs to 250,000 Tregs per well): add 100 μl Treg cell suspension into the wells containing moDCs from Step H1.

   4. Control wells: set up one well of moDC alone and another well of Treg alone as controls in separate wells with a final volume of 200 µl per well.

   5. Co-culture cells in Dendritic Cell Medium with 50 IU/ml IL-2 at 37°C (5% v/v CO₂) for 4 days.




9. Day 11: Flow cytometry

   1. Collect cells: centrifuge the cell culture plate at 970 × g for 3 min, remove from centrifuge, and discard half the volume (100 μl) supernatant by pipetting. Resuspend the cells in the remaining supernatant and transfer into a new 96-well V-bottom plate. Wash the wells in the cell culture plate with 100 μl DPBS and add this to the respective wells in the V-bottom plate.

   2. Centrifuge the plate at 970 × g for 3 min, discard the supernatant, and resuspend the cells in 200 µl DPBS to wash.

   3. Centrifuge the plate at 970 × g for 3 min and discard the supernatant.

   4. Stain cells for analysis by flow cytometry using Table 3. See Figure 3 for example data.

      
      

      Table 3. Day 11 moDC Suppression Assay Panel. moDC-alone and Treg-alone controls must be stained in parallel. See Figure 3 for representative data.
      

      Marker	Dilution	Clone	Fluorophore
      Fc Receptor Binding Inhibitor (preincubate for 10 min and do not wash)	1:5	Polyclonal	N/A
      FVD	1:1,000	N/A	eF780
      CD4	1:100	RPA-T4	BV711
      CD69	1:50	FN50	BV785
      CD11c	1:50	B-ly6	PE
      CD70	1:50	113-16	PerCP-Cy5.5
      CD80	1:50	L307.4	FITC
      CD83	1:50	HB15e	BV421
      CD86	1:50	FUN-1	APC
      CD40	1:100	5c3	PE-Cy7
      HLA-DR	1:50	L243	BV510

      
      

      
      Figure 3. Day 11 analysis of CD80 and CD86 on mature moDCs following co-culture with Tregs. (A-B) Mature moDCs and Tregs were co-cultured for 4 days, and then cells were stained as described in Table 3 and in accordance with “Guidelines for the use of flow cytometry and cell sorting in immunological studies” (Cossarizza et al., 2019); they were acquired with a BD LSRFortessa X-20 and data analysed using FlowJo. (A) Gating strategy to identify mature moDCs. Live singlet cells were gated, and moDCs were identified as CD11c⁺CD4^‒ cells. (B) Expression of CD80 (left) and CD86 (right) following co-culture with Tregs. This assay was performed using HLA-A2⁺ moDCs co-cultured with either polyclonal ΔNGFR-transduced Tregs or HLA-A2-specific CAR-transduced Tregs. Compared to moDCs cultured alone (black line), polyclonal Tregs exert a moderate level of suppression (green), as determined by the decrease in CD80 and CD86 expression. This suppression is greater when co-cultures are performed with A2-CAR Tregs (blue). See Dawson et al. (2020) for more examples.

Notes

1. PBMC: Fresh and frozen PBMCs are similar and suitable for moDC differentiation. Frozen PBMCs yield slightly fewer CD14⁺ monocytes than fresh PBMCs. Using frozen PBMCs from a single donor can reduce donor-to-donor moDC variability. Fresh PBMCs can be prepared the day of (proceed to CD14⁺ isolation immediately) or the day before running the experiment [store overnight at 4°C in 25 ml 10% (v) fetal bovine serum-supplemented medium of choice (e.g., RPMI 1640) mixed with 25 ml DPBS in a 50 ml tube placed horizontally in a fridge].

2. CD14⁺ selection performance: Post-selection (3× magnet-isolation, as per manufacturer’s protocol), 90-95% are CD14⁺ cells and 0.5-5% are CD3⁺ T cells (see Figure 1). Detection of contaminating T cells can be obscured if FSC/SSC voltages and thresholds are set too low. T cell contamination persists to day 7 and can affect results from moDC-T cell co-cultures (e.g., if OKT3 is added). Thus, an anti-CD3 antibody can be added to the day 7 moDC maturation panel to evaluate purity since the cells have expanded (instead of day 0).

3. moDC lineage markers: moDCs should be CD11c⁺ and CD14^–. moDC maturation markers: mature moDCs should be CD80^hi, CD86^hi, HLA-DR^hi (Ag presentation and T cell co-stimulation), CD40⁺ (promote CD40L⁺ T cell maturation and cytokine secretion), and CD83⁺ (function less clear).

4. Co-culture: Confirmation of moDC maturation is important before setting up suppression co-culture.

Recipes

1. EasySep Buffer

   DPBS supplemented with 2% (v/v) fetal bovine serum, 1 mM EDTA. Contents were sterile-filtered with a Stericup-GP vacuum filtration flask.

2. Dendritic Cell Medium

   X-VIVO 15 supplemented with 5% (v/v) human serum, 1% (v/v) penicillin-streptomycin, 1% (v/v) GlutaMAX, 1% (v/v) sodium pyruvate. Contents were sterile-filtered with a Stericup-GP vacuum filtration flask.

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research (CIHR) FDN-154304 and TxCell. AJL and NAJD were supported by a CIHR doctoral award and MKL received a salary award from the BC Children's Hospital Research Institute. This method is derived from the original publication by Dawson et al. (2020) (DOI: 10.1126/scitranslmed.aaz3866).

Competing interests

The authors of this manuscript have received research funding from Sangamo Therapeutics (formerly TxCell SA) to partially support this work. MKL has also received research funding from Takeda, Bristol Myers Squibb, Pfizer, and CRISPR Therapeutics for work not related to this study.

Ethics

For all studies, healthy volunteers gave written informed consent according to protocols approved by the University of British Columbia Clinical Research Ethics Board and Canadian Blood Services.

References

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2. Dawson, N. A. J., Rosado-Sanchez, I., Novakovsky, G. E., Fung, V. C. W., Huang, Q., McIver, E., Sun, G., Gillies, J. and Speck, M. (2020). Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells. Sci Transl Med 12(557): eaaz3866.
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6. Wang, A. Y., Crome, S. Q., Jenkins, K. M., Medin, J. A., Bramson, J. L. and Levings, M. K. (2011). Adenoviral-transduced dendritic cells are susceptible to suppression by T regulatory cells and promote interleukin 17 production. Cancer Immunol Immunother 60(3): 381-388.