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Peer-reviewed

Suppression of Human Dendritic Cells by Regulatory T Cells

调节性 T 细胞抑制人树突状细胞

QH Qing Huang *
AL Avery J. Lam *
DB Dominic A. Boardman *
ND Nicholas A. J. Dawson
ML Megan K. Levings

 (*contributed equally to this work) 发布: 2021年11月05日第11卷第21期 DOI: 10.21769/BioProtoc.4217 浏览次数: 3606

评审: Scott McCombSaskia F. ErttmannAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Science Translational Medicine, featuring study using the protocol.

Aug 2020

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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) search Dendritic cells (树突状细胞) search CD80 (CD80) search CD86 (CD86) search Chimeric antigen receptor (嵌合抗原受体) search

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

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文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

免疫学 > 免疫细胞分化 > T 细胞

细胞生物学 > 基于细胞的分析方法 > 流式细胞术

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