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Aug 2020
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Suppression of Human Dendritic Cells by Regulatory T Cells
调节性 T 细胞抑制人树突状细胞   

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Abstract

Regulatory T cells (Tregs) suppress immune responses via a variety of mechanisms and can be used as a cellular therapy to induce tolerance. The function of Tregs is commonly assessed in vitro using assays that measure suppression of effector T cell proliferation and/or cytokine production. However, Tregs can also suppress the function of antigen presenting cells, creating a need for methodology to routinely measure this aspect of their function. This protocol describes a method to measure human Treg-mediated suppression of CD80 and CD86 expression on mature, monocyte-derived dendritic cells. Representative data show suppression mediated by polyclonal Tregs as well as antigen-specific Tregs generated using chimeric antigen receptor (CAR) technology. This method can be used in parallel to T cell suppression assays to measure the functional activity of human Tregs.

Keywords: Regulatory T cells (调节性T细胞 ), Dendritic cells (树突状细胞 ), CD80 (CD80), CD86 (CD86), Chimeric antigen receptor (嵌合抗原受体)

Background

Regulatory T cells (Tregs) are immunosuppressive cells that play a fundamental role in maintaining peripheral tolerance. Tregs inhibit the action of many immune cells, including effector T cells and antigen presenting cells (APC), via cell contact-dependent and contact-independent mechanisms. The suppressive function of Tregs is typically assessed in vitro by measuring their ability to inhibit the proliferation of polyclonally stimulated T cells. However, methods to measure how Tregs suppress APCs are limited.


One strategy Tregs use to inhibit the function of APCs is the removal of co-stimulatory molecules from the APC, thereby reducing their ability to stimulate effector T cells. Tregs achieve this by expressing CTLA-4, which binds CD80 and CD86 with a high affinity and allows the Treg to physically remove these molecules from the APC cell surface (Walker and Sansom, 2011). We have also previously reported the ability of human Tregs to suppress the expression of co-stimulatory molecules on both immature and mature monocyte-derived DCs (moDCs) (Wang et al., 2011).


This protocol describes a method to test Treg-mediated suppression of CD80 and CD86 and is modified from a previously published mouse-based protocol (Onishi et al., 2008). Our protocol focuses on the ability of human Tregs to suppress the expression of CD80 and CD86 by moDCs. In this assay, polyclonal Tregs transduced with a truncated nerve growth factor receptor (ΔNGFR) reporter can reduce CD80 and CD86 expression in moDCs. Furthermore, when using HLA-A2+ target moDCs, antigen-specific Tregs expressing an HLA-A2-specific chimeric antigen receptor (CAR) are more potent than polyclonal Tregs (Dawson et al., 2020; Fung et al., 2021).

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)


Media and Buffers
  1. LymphoprepTM (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)


Reagents
  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)


Cytokines
  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)


Antibodies
  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 EasySepTM 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.


Detailed Procedure
  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 LymphoprepTM, 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 (CD56med/hi) and a subset of monocytes (CD56lo). The contaminating live CD14CD3+ 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 EasySepTM 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 CD80hi, CD86hi, HLA-DRhi (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

  1. Cossarizza, A, Chang, HD, Radbruch, A, Acs, A, Adam, D, Adam-Klages, S, Agace, WW, Aghaeepour, N, Akdis, M and Allez, M. (2019). Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol 49(10): 1457-1973.
  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.
  3. Fung, V. C. W., Rosado-Sanchez, I. and Levings, M. K. (2021). Transduction of Human T Cell Subsets with Lentivirus. Methods Mol Biol 2285227-254.
  4. Onishi, Y., Fehervari, Z., Yamaguchi, T. and Sakaguchi, S. (2008). Foxp3+ natural regulatory T cells preferentially form aggregates on dendritic cells in vitro and actively inhibit their maturation. Proc Natl Acad Sci U S A 105(29): 10113-10118.
  5. Walker, L. S. and Sansom, D. M. (2011). The emerging role of CTLA4 as a cell-extrinsic regulator of T cell responses. Nat Rev Immunol 11(12): 852-863.
  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.

简介

[抽象的]调节性 T 细胞 (Tregs) 通过多种机制抑制免疫反应,可用作细胞疗法来诱导耐受。Tregs 的功能通常在体外使用测定效应 T 细胞增殖和/或细胞因子产生的抑制进行评估。然而,Tregs 也可以抑制抗原呈递细胞的功能,因此需要一种方法来常规测量其功能的这一方面。该协议描述了一种测量人类 Treg 介导的 CD80 和 CD86 表达对成熟单核细胞衍生树突细胞的抑制的方法。代表性数据显示由多克隆 Treg 以及使用嵌合抗原受体 (CAR) 技术生成的抗原特异性 Treg 介导的抑制。该方法可与 T 细胞抑制测定并行使用,以测量人类 Treg 的功能活性。


[背景]调节性 T 细胞 (Tregs) 是免疫抑制细胞,在维持外周耐受性方面发挥着重要作用。Tregs 通过细胞接触依赖性和非接触依赖性机制抑制许多免疫细胞的作用,包括效应 T 细胞和抗原呈递细胞 (APC)。通常通过测量Tregs抑制多克隆刺激 T 细胞增殖的能力在体外评估 Tregs 的抑制功能。然而,测量 Treg 如何抑制 APC 的方法是有限的。
Treg 用于抑制 APC 功能的一种策略是从 APC 中去除共刺激分子,从而降低它们刺激效应 T 细胞的能力。Tregs 通过表达 CTLA-4 实现这一点,CTLA-4 以高亲和力结合 CD80 和 CD86,并允许 Treg 从 APC 细胞表面物理去除这些分子(Walker 和 Sansom,2011)。我们之前还报道了人类 Tregs 抑制未成熟和成熟的单核细胞衍生 DCs (moDCs) 上共刺激分子表达的能力 (Wang et al ., 2011)。
该协议描述了一种测试 Treg 介导的 CD80 和 CD86 抑制的方法,并从先前发布的基于鼠标的协议(Onishi等人,2008 年)进行了修改。我们的协议侧重于人类 Tregs 抑制 moDCs 表达 CD80 和 CD86 的能力。在该测定中,用截短的神经生长因子受体 (ΔNGFR) 报告基因转导的多克隆 Treg 可以降低 moDC 中 CD80 和 CD86 的表达。此外,当使用 HLA-A2 +靶向 moDC 时,表达 HLA-A2 特异性嵌合抗原受体 (CAR) 的抗原特异性 Treg 比多克隆 Treg 更有效(Dawson等,2020;Fung等,2021)。

关键字:调节性T细胞 , 树突状细胞 , CD80, CD86, 嵌合抗原受体


材料和试剂

 
材料
5毫升聚苯乙烯圆底管(Corning,目录号:352052)
TC 涂层 6 孔板(Corning,目录号:353502)
TC 涂层 12 孔板(Corning,目录号:353503)
96 孔 U 型和 V 型底板(Corning,目录号:353077、3894)
1.5 ml 微量离心管(Fisher Scientific,目录号:229442)
15 ml 和 50 ml 锥形管(Corning,目录号:352096、352070)
无菌 1 ml 或 3 ml 注射器(BD,目录号:309628、309657)
 
媒体和缓冲区
Lymphoprep的TM (干细胞技术,目录号:07801)
Dulbecco 磷酸盐缓冲盐水(DPBS;Gibco,目录号:14190),1 ×
X-VIVO 15(Lonza,目录号:BEBP02-054Q)
人血清(Wisent Bio Products,目录号:022210)
青霉素/链霉素(Gibco,目录号:15140-122)
GlutaMAX(Gibco,目录号:35050-061)
丙酮酸钠(Gibco,目录号:11360-070)
胎牛血清(Gibco,目录号:12483020)
乙二胺四乙酸溶液(EDTA)(Sigma-Aldrich,目录号:03690)
EasySep 缓冲液(见配方)
树突细胞培养基(见食谱)
 
试剂
Stericup-GP 无菌真空过滤系统(Millipore,目录号:SCGPU05RE),500 ml
EasySep Human CD14 Positive Selection Kit II(STEMCELL Technologies,目录号:17858)
吖啶橙/碘化丙啶(AO/PI;Nexcelom,目录号:NEX-CS201065ML)
 
细胞因子
重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)(STEMCELL Technologies,目录号:78015),20 μ克/毫升
重组人白细胞介素(IL)-4(STEMCELL Technologies,目录号:78045),20 μg/ml
重组人肿瘤坏死因子(TNF)-α(eBioscience,目录号:14-8329-63),20 μg/ml
前列腺素 E2(PGE2)(Tocris,目录号:2296),100 mM
重组人 IL-1β(STEMCELL Technologies,目录号:78041),20 μg/ml
重组人 IL-6(STEMCELL Technologies,目录号:78148),20 μg/ml
重组人干扰素(IFN)-γ(eBioscience,目录号:14-8319-80),20 μg/ml
重组人 IL-2 (Proleukin) (Novartis, DIN# 02130181)
 
抗体
Fc受体结合抑制剂多克隆抗体(eBioscience,目录号:14-9161-73)
可固定活力染料(FVD)(eBioscience,目录号:65-0865-18)
抗人 CD3(UCHT1 )APC(BD Biosciences,目录号:564465)                                         
抗人 CD3(UCHT1)BB515(BD Biosciences,目录号:564465)
抗人 CD4(OKT4)APC(eBioscience,目录号:17-0048-42)             
抗人 CD4(RPA-T4)BV711 (BioLegend ,目录号:300558)                                                         
抗人 CD8a(RPA-T8)BV711(BioLegend ,目录号:301044)                                         
抗人 CD11c(B-ly6 )PE(BD Biosciences,目录号:555392)                                         
抗人 CD14(M5E2)BV421(BioLegend ,目录号:301830)                                         
抗人 CD14 (M5E2)BV786(BD Biosciences,目录号:563698)                                                        
抗人 CD40 (5C3 )PE-Cy7 (BD Biosciences,目录号:561215)                                                          
抗人 CD56 (CMSSB )PE(eBioscience,目录号:12-0567-42)                                                        
抗人 CD69 (FN50)BV785(BioLegend ,目录号:310932)                                                        
抗人 CD70 (113-16 )PerCP-Cy5.5(BioLegend,目录号:355107)                                                        
抗人 CD80(L307.4)FITC(BD Biosciences,目录号:557226)                                         
抗人 CD83 (HB15e )BV421 (BioLegend ,目录号:305324 )                                                          
抗人 CD86 (2331(FUN-1))APC (BD Biosciences,目录号:555660)                                                          
抗人 CD86 (HA5.2B7)PerCP-Cy5.5 (Beckman Coulter,目录号:B30647)                                                          
 
设备
 
II 型生物安全柜(NuAire,型号:LabGard ES NU-540)
离心机、微量离心机(Eppendorf,型号:5810R 和 5452)
STEMCELL EasySep TM磁铁(STEMCELL Technologies,目录号:18000)
细胞计数器(Nexcelom,型号:Cellometer Auto 2000)
37°C 培养箱,含 5% (v/v) CO 2 (三洋,型号:MCO-18AIC)
流式细胞仪(BD LSRFortessa X-20;可以使用替代仪器)


软件
 
FlowJo 软件(BD Biosciences,v10.7)
 
程序
 
概述
第 0 天:准备外周血单核细胞 (PBMC)。
第 0 天:通过正选择从 PBMC 中分离 CD14 +单核细胞。
第 0 天:通过在 GM-CSF 和 IL-4 存在下培养,将单核细胞分化为树突细胞。
第 3 天:补充 GM-CSF 和 IL-4。
第 5 天:通过向培养物中添加 TNF-α、PGE2、IL-1β 和 IL-6 使树突状细胞成熟。
第 6 天:通过向培养物中添加 IFN-γ 使树突状细胞成熟。
第 7 天:通过流式细胞术确认 DCs 的成熟并设置抑制检测。
第11天:收集细胞、染色、进行流式细胞术和分析。
 
详细程序
第 0 天:准备 PBMC
准备 PBMC:从血液中新鲜分离或解冻的 PBMC。根据制造商的方案,可以使用 Lymphoprep TM通过密度梯度离心从人外周血中分离 PBMC 。
注意:使用来自一位捐赠者的批量冷冻 PBMC 来区分 moDC 将减少捐赠者与捐赠者之间的变异性。预计 5-10% 新鲜/冷冻 PBMCs 或 50-100% CD14 +细胞成为 moDCs。有关更多详细信息,请参阅注释。
可选:留出 ~5 × 10 3 -10 × 10 3 PBMCs 用于纯度检查(参见表 1、图 1 和注释以获取更多信息)。


表 1. 第 0 天纯度检查面板。如果需要,并行染色 PBMC。有关代表性数据,请参见图 1 。
 
 
图 1. 第 0 天 CD14 +纯度检查。( A ) 大约 5 × 10 3 -10 × 10 3总 PBMC 和 ( B ) 富含 CD14 的细胞通过流式细胞术染色和分析,以评估富含 CD14 的细胞的纯度和 CD3 +细胞污染的程度。如果在检测过程中包含 OKT3(抗 CD3),污染 T 细胞会影响下游结果(见注释)。按照表 1 中所述并根据“免疫学研究中使用流式细胞术和细胞分选指南”(Cossarizza等人,2019 年)对细胞进行染色;它们是用 BD LSRFortessa X-20 采集的,并使用 FlowJo 分析数据。那是CD14总活细胞的比例+ CD3 - (CD14 +细胞的纯度)被示出在左侧。从活的 CD14 ‒细胞中,分析 CD56 表达(中)以识别 NK/NKT 细胞(CD56 med/hi )和单核细胞亚群(CD56 lo )。进一步分析了污染的活 CD14 ‒ CD3 + T 细胞的 CD4 和 CD8 表达(右图)。有关更多信息,请参阅注释。
 
第 0 天:使用 STEMCELL EasySep Human CD14 Positive Selection Kit II分离 CD14 +单核细胞,按照制造商的协议和如下所述,除非另有说明:
在 EasySep 缓冲液 (0.1-2 ml) 中以 100 × 10 6 /ml重悬 PBMC 。PBMC 的总起始数量应介于 10 × 10 6和 200 × 10 6细胞之间。对于 x 个 CD14 +单核细胞,从 10×(新鲜)或 ~20×(冷冻)PBMC 开始。
将 PBMC 转移到 5 ml 聚苯乙烯圆底管中。
每毫升细胞加入 100 μl Selection Cocktail,并在室温 (RT) 下孵育 10 分钟。
涡旋 RapidSpheres 30 秒,每毫升细胞加入 100 μl RapidSpheres,轻轻旋转混合管,并在室温下孵育 3 分钟。
用 EasySep 缓冲液将管加满至 2.5 ml,通过移液混合,置于 EasySep TM磁体中,并在室温下孵育 3 分钟。
在磁铁中轻轻倒入管子以丢弃上清液。圆底管含有 CD14 +细胞(保留)。
通过从磁铁上取下圆底管并用 2.5 ml EasySep 缓冲液加满管来清洗剩余的 RapidSpheres。通过移液将层从管壁上洗掉,将管放回磁铁中,并在 RT 中孵育 3 分钟。倒掉上清液,倒置时从管中吸取最后一滴。
重复步骤 B7,使细胞管在磁铁中共孵育 3 次。
可选地重复步骤 B7 两次(在磁铁上总共五次)以获得更高的富集度。
注意:此步骤不是原始 STEMCELL Technologies 协议的一部分。
通过用 ~ 3 ml DPBS 清洗圆底管壁,将细胞从圆底管转移到 15 ml 锥形管中。以 450 × g离心5 分钟并丢弃上清液。
在树突细胞培养基中重悬富集的 CD14 +细胞(参见食谱)。
计数细胞(有关详细信息,请参阅 Nexcelom Cellometer Auto 2000 的协议;或者,可以使用血细胞计数器和台盼蓝)。
可选:留出 ~5 × 10 3 -10 × 10 3单核细胞进行纯度检查(参见表 1、图 1 和注释以获取更多信息)。
 
第 0 天:培养并分化富集的 CD14 +细胞为树突细胞
使用树突细胞培养基将细胞调整为每毫升2 × 10 6 个细胞。
在 6 孔板中每孔加入 2 ml 细胞(每孔 4 × 10 6细胞)。
注意:根据需要接种多个细胞孔。
添加 GM-CSF(最终浓度:50 ng/ml)和 IL-4(最终浓度:100 ng/ml)。
在 37°C (5% v/v CO 2 )下孵育细胞3 天。
注意:细胞因子可以在 4°C 下储存约 1 周。
 
第 3 天:更换树突状细胞培养基并补充细胞因子
更换树突状细胞培养基:在不干扰孔底细胞的情况下,收集 1.5 ml 培养基到无菌 1.5 ml 微量离心管中,450 × g离心5 min,弃上清,将沉淀的细胞重悬于 1.5 ml 新鲜离心管中树突细胞培养基,并重新接种到原孔中。
通过添加新鲜的 GM-CSF(最终浓度:50 ng/ml)和 IL-4(最终浓度:100 ng/ml)来充分补充细胞因子,假设从第 0 天开始没有细胞因子留在培养物中。
在 37°C (5% v/v CO 2 )下孵育2 天。
注意:细胞因子可以在 4°C 下储存约 1 周。
替代培养基更换方法:45 0 × g离心板5 分钟,轻轻更换 1.5 ml 树突状细胞培养基,并如上文所述充分补充 GM-CSF 和 IL-4。
 
第 5 天:成熟的树突细胞
收集细胞:上下吸管以分离细胞并收集到 15 ml 或 50 ml 锥形管中。使用 1 ml 或 3 ml 注射器橡胶轻轻单向分离剩余细胞。用 DPBS 冲洗两次以收集尽可能多的细胞。
通过用 DPBS 填充锥形管来洗涤细胞,以 450 x g离心细胞5 分钟,并丢弃上清液。
在新鲜的树突细胞培养基中重悬细胞,计数细胞并调整体积,使细胞在树突细胞培养基中的最终浓度为 1 × 10 6 个细胞/ml。
添加 GM-CSF(最终浓度:50 ng/ml)和 IL-4(最终浓度:100 ng/ml)。
通过在新的 6 孔板中每孔添加 2 ml 细胞(每孔 2 × 10 6 个细胞)或在新的 12 孔板中每孔添加 1 ml 细胞(1 × 10 6 个细胞每口井)。
通过添加 TNF-α(终浓度:50 ng/ml)、PGE2(终浓度:1 μg/ml,~2.837 µM)、IL-1β(终浓度:10 ng/ml)和 IL-6(终浓度:100 ng/ml)。
注意:如果需要不成熟的 moDC,请跳过此步骤。保持至少 1 × 10 6未成熟的 moDC(GM-CSF 和 IL-4,没有额外的细胞因子)对于评估最后的 moDC 成熟(第 7 天,参见程序 G)很有用。
在 37°C (5% v/v CO 2 )下孵育1 天。
 
第 6 天:继续成熟的树突细胞
添加 IFN-γ(最终浓度:50 ng/ml)。
注意:对于未成熟的 moDC,请跳过此步骤。
在 37°C (5% v/v CO 2 )下孵育1 天。
 
第 7 天:在设置抑制检测之前确认 DCs 的成熟
通过移液重悬收集细胞并转移到 15 ml 锥形管中。使用 1 ml 或 3 ml 注射器橡胶轻轻单向分离剩余细胞。用 DPBS 洗两次井,并将其添加到锥形管中。将细胞以 450 × g离心5 分钟,重悬在新鲜的树突细胞培养基中,计数细胞,并在树突细胞培养基中调整细胞浓度至 5 × 10 5 个细胞/毫升。
通过收集 ~5 × 10 6 -10 × 10 6成熟的 moDCs 和未成熟的 moDCs 用于流式细胞术染色来确认 moDC 成熟(见表 2,图 2)。
 
表 2. 第 7 天 moDC 成熟度检查面板。未成熟的 moDC 必须平行染色。有关代表性数据,请参见图 2。
 
 
图 2.未成熟和成熟 moDC 的第 7 天表型。(AC)通过流式细胞术分析未成熟 (黑线) 和成熟 (蓝色) moDCs 的 CD11c、CD14、CD80、CD86、CD40、CD83 和 HLA-DR 的表达。如表 2 所述并根据“免疫学研究中流式细胞术和细胞分选的使用指南”(Cossarizza等人,2019 年)对细胞进行染色;它们是用 BD LSRFortessa X-20 采集的,并使用 FlowJo 分析数据。moDC(作为活的单细胞门控)表达相似水平的谱系标志物 ( A ),但在成熟后上调 CD80、CD86、HLA-DR、CD40 和 CD83 的表达 ( BC )。MFI,几何平均荧光强度。
 
第 7 天:设置 DC 抑制检测
在 96 孔 U 型底板(每孔 50,000 个细胞)中加入 100 µl moDC。
收集静止的 Tregs,计数细胞,并以每毫升2.5 × 10 6细胞重悬于含有 100 IU/ml IL-2 的树突细胞培养基中。
注意:Treg 应使用用户实验室建立的协议进行扩展和休息。Dawson 等人提供了有关如何扩展和休息此协议的 Treg 的详细信息。(2020)。简而言之,从外周血中分离出 Treg,并在 1000 IU/ml IL-2 存在下用人工 APC 多克隆扩增 7 天。然后通过在含有 100 IU/ml IL-2 的新鲜培养基中培养过夜使 Treg 静置。CAR Tregs 是在刺激后一天通过慢病毒转导多克隆 Tregs 产生的。
以 1 比 5 的比例设置 moDC 和 Treg 共培养(每孔 50,000 DC 至 250,000 Treg):将 100 μl Treg 细胞悬液添加到含有步骤 H1 中的 moDC 的孔中。
对照孔:单独设置一个 moDC 孔和另一个单独的 Treg 孔作为单独孔中的对照,每孔的最终体积为 200 µl。
将细胞在树突细胞培养基中与 50 IU/ml IL-2 在 37°C (5% v/v CO 2 )中共培养 4 天。
 
第 11 天:流式细胞术
收集细胞:将细胞培养板以 970 × g离心 3 分钟,从离心机中取出,并通过移液去除一半体积(100 μl)的上清液。将剩余的上清液中的细胞重新悬浮并转移到新的 96 孔 V 型底板中。用 100 μl DPBS 清洗细胞培养板中的孔,并将其添加到 V 型底板的各个孔中。
将板以 970 × g离心3 分钟,弃去上清液,并用 200 µl DPBS 重悬细胞进行洗涤。
将板以 970 × g离心3 分钟并丢弃上清液。
使用表 3 通过流式细胞术对细胞进行染色。示例数据参见图 3。
 
表 3. 第 11 天的 moDC 抑制分析面板。单独的 moDC 和单独的 Treg 对照必须平行染色。有关代表性数据,请参见图 3。
 
 
图 3. 与 Treg 共培养后成熟 moDC 上 CD80 和 CD86 的第 11 天分析。(AB)成熟的 moDCs 和 Tregs 共培养 4 天,然后按照表 3 中所述并根据“免疫学研究中使用流式细胞术和细胞分选指南”(Cossarizza等人, 2019); 它们是用 BD LSRFortessa X-20 采集的,并使用 FlowJo 分析数据。( A ) 识别成熟 moDC 的门控策略。活单线态细胞被门控,moDCs 被鉴定为 CD11c + CD4 -细胞。( B ) 与 Treg 共培养后 CD80(左)和 CD86(右)的表达。该测定使用与多克隆 ΔNGFR 转导的 Treg 或 HLA-A2 特异性 CAR 转导的 Treg 共培养的HLA-A2 + moDC 进行。与单独培养的 moDC(黑线)相比,多克隆 Treg 发挥中等水平的抑制作用(绿色),这由 CD80 和 CD86 表达的降低决定。当使用 A2-CAR Tregs(蓝色)进行共培养时,这种抑制作用更大。见道森等。(2020) 获取更多示例。
 
笔记
 
PBMC:新鲜和冷冻的 PBMC 相似,适用于 moDC 分化。冷冻 PBMC 产生的 CD14 +单核细胞比新鲜 PBMC略少。使用来自单个供体的冷冻 PBMC可以减少供体之间的 moDC 变异性。新鲜的 PBMC 可以在实验当天(立即进行 CD14 +分离)或实验前一天制备[在 4°C 下在 25 ml 10% (v) 胎牛血清补充培养基中储存过夜(例如, RPMI 1640) 与 25 ml DPBS 在水平放置在冰箱中的 50 ml 管中混合]。
CD14 +选择性能:选择后(3x 磁体隔离,根据制造商的协议),90-95% 为 CD14 +细胞,0.5-5% 为 CD3 + T 细胞(见图 1)。如果 FSC/SSC 电压和阈值设置得太低,则可能会掩盖污染 T 细胞的检测。T 细胞污染持续到第 7 天,并可能影响 moDC-T 细胞共培养的结果(例如,如果添加了 OKT3)。因此,可以将抗 CD3 抗体添加到第 7 天的 moDC 成熟面板中以评估纯度,因为细胞已经扩增(而不是第 0 天)。
moDC 谱系标记:moDC 应该是 CD11c +和 CD14 – 。moDC 成熟标志物:成熟的 moDCs 应该是 CD80 hi 、CD86 hi 、HLA-DR hi (Ag 呈递和 T 细胞共刺激)、CD40 + (促进 CD40L + T 细胞成熟和细胞因子分泌)和 CD83 + (功能不太清楚) )。
共培养:在建立抑制共培养之前,确认 moDC 成熟很重要。
 
食谱
 
EasySep 缓冲液
DPBS 补充了 2% (v/v) 胎牛血清、1 mM EDTA。内容物用 Stericup-GP 真空过滤瓶进行无菌过滤。
树突细胞培养基
X-VIVO 15 补充有 5% (v/v) 人血清、1% (v/v) 青霉素-链霉素、1% (v/v) GlutaMAX、1% (v/v) 丙酮酸钠。内容物用 Stericup-GP 真空过滤瓶进行无菌过滤。
 
致谢
 
这项工作得到了加拿大卫生研究院 (CIHR) FDN-154304 和 TxCell 的资助。AJL 和 NAJD 获得了 CIHR 博士奖的支持,MKL 获得了不列颠哥伦比亚省儿童医院研究所的工资奖。该方法源自 Dawson等人的原始出版物。(2020 年)(DOI:10.1126/scitranslmed.aaz3866)。
 
利益争夺
 
本手稿的作者已从 Sangamo Therapeutics(前身为 TxCell SA)获得研究资金,以部分支持这项工作。MKL 还获得了武田、百时美施贵宝、辉瑞和 CRISPR Therapeutics 的研究资助,用于与本研究无关的工作。
 
伦理
 
对于所有研究,健康志愿者根据不列颠哥伦比亚大学临床研究伦理委员会和加拿大血液服务机构批准的协议签署了书面知情同意书。


参考
Cossarizza A、Chang HD、Radbruch A、Acs A、Adam D、Adam-Klages S、Agace WW、Aghaeepour N、Akdis M、Allez M.等。(2019)。免疫学研究中流式细胞术和细胞分选的使用指南(第二版)。Eur J Immunol 49(10):1457-1973。
Dawson, NAJ, Rosado-Sanchez, I., Novakovsky, GE, Fung, VCW, Huang, Q., McIver, E., Sun, G., Gillies, J., Speck, M.等。(2020)。嵌合抗原受体共受体信号域在人类调节性 T 细胞中的功能作用。Sci Transl Med 12(557): eaaz3866。
Fung, VCW, Rosado-Sanchez, I. 和 Levings, MK (2021)。用慢病毒转导人 T 细胞亚群。方法 Mol Biol 2285:227-254。
Onishi, Y.、Fehervari, Z.、Yamaguchi, T. 和 Sakaguchi, S. (2008)。Foxp3 +天然调节性 T 细胞优先在体外在树突细胞上形成聚集体并积极抑制它们的成熟。Proc Natl Acad Sci USA 105(29): 10113-10118。
Walker, LS 和 Sansom, DM (2011)。CTLA4 作为 T 细胞反应的细胞外源调节剂的新兴作用。Nat Rev Immunol 11(12): 852-863。
Wang, AY, Crome, SQ, Jenkins, KM, Medin, JA, Bramson, JL 和 Levings, MK (2011)。腺病毒转导的树突细胞容易受到 T 调节细胞的抑制并促进白细胞介素 17 的产生。癌症免疫学免疫学60(3): 381-388。
 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Huang, Q., Lam, A. J., Boardman, D. A., Dawson, N. A. J. and Levings, M. K. (2021). Suppression of Human Dendritic Cells by Regulatory T Cells. Bio-protocol 11(21): e4217. DOI: 10.21769/BioProtoc.4217.
  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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