体外胸腺调节性T细胞祖细胞向成熟胸腺调节性T细胞的分化

David L. Owen; Michael A. Farrar

doi:10.21769/BioProtoc.3335

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Peer-reviewed

In vitro Differentiation of Thymic T_reg Cell Progenitors to Mature Thymic T_reg Cells

体外胸腺调节性T细胞祖细胞向成熟胸腺调节性T细胞的分化

DO David L. Owen

MF Michael A. Farrar email

发布: 2019年08月20日第9卷第16期 DOI: 10.21769/BioProtoc.3335 浏览次数: 4873

评审: Alka MehraChris TibbittAnonymous reviewer(s)

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Cover of Nature Immunology, featuring study using the protocol.

Feb 2019

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Abstract

Thymic T_reg cell differentiation occurs via a two-step process. Step one generates T_reg cell progenitors (T_regP) and is driven by strong TCR interactions with antigens presented in the thymus. Step two is initiated by activation of STAT5 via IL-2, or IL-15, leading to mature T_reg cells capable of emigrating from the thymus and mediating immune tolerance and homeostasis in peripheral tissues. Herein we describe an in vitro T_regP cell differentiation assay that models the second, cytokine dependent, step of thymic T_reg cell development. It can be utilized with relative ease to determine if a population of thymocytes represents a potential progenitor population for T_reg cells as well as test how different cytokines or chemical inhibitors modulate the differentiation of known T_regP cell populations into mature T_reg cells.

Keywords: Thymus (胸腺)

Thymic selection (胸腺选择)

Regulatory T (T_reg) cells (调节性T细胞)

T_reg cell development (调节性T细胞发育)

T cell tolerance (T细胞耐受)

Cytokine stimulation (细胞因子刺激)

STAT5 (STAT5)

Foxp3 (Foxp3)

Background

Regulatory T cells (T_reg) cells are a population of T cells that can suppress immune responses and are required to maintain immune tolerance and tissue homeostasis. While two broad classes of T_reg cells have been described, thymic T_reg cells (tT_reg) and peripheral T_reg cells (pT_reg), the majority of T_reg cells are generated during thymic selection (Asano et al., 1996). Early experiments describing autoimmune manifestations in thymectomized mice, which can be rescued by T_reg cell transfer, confirm the importance of the thymus in generating T_reg cells capable of maintaining immune homeostasis (Asano et al., 1996). The development of T_reg cells in the thymus is a two-step process (Burchill et al., 2008; Lio and Hsieh, 2008). The first step is TCR mediated and gives rise to CD25⁺Foxp3^- or CD25^-Foxp3^lo T_regP cells. The second step is mediated by contact of these progenitor populations with intrathymic cytokines leading to co-expression of CD25 and Foxp3 and generation of mature tT_reg cells.

The in vitro T_regP conversion assay models the second, cytokine dependent, step in tT_reg cell development. This assay builds on protocols initially described by work from Chyi Hsieh’s lab and modified by our own lab (Burchill et al., 2008; Lio and Hsieh, 2008). The technique described below provides a simple method to determine if an isolated thymic population represents a T_regP, characterized by independence from TCR stimulation and upregulation of tT_reg markers in response to cytokine. This assay has been performed with varied stimulation conditions previously, including stimulation with high concentrations of IL-2 (200 U/ml) (Tai et al., 2013) as well as IL-2 + IL-7 (Lio and Hsieh, 2008). The protocol we describe converts bona-fide T_regP to mature Treg cells robustly and reproducibly with small amounts of IL-2 (Mahmud et al., 2014; Owen et al., 2019). As such, investigators can narrow the application of IL-2 to concentrations that are more physiologically relevant. This assay has also been able to detect conversion of T_regP to mature T_reg cells with other cytokines like IL-7, IL-15 and IL-4, as well as combination of factors such as IL-2 with TNFRSF agonists (Mahmud et al., 2014). Thus, the in vitro T_regP conversion assay can be used to query various important stimulatory, or inhibitory, signals that may be encountered in vivo by developing T_regP.

Materials and Reagents

Pipette tips
0.22 μm 1,000 ml Stericup (Millipore Sigma, catalog number: SCGPU11RE)
0.22 μm 50 ml Steriflip (Millipore Sigma, catalog number: SCGP00525)
LS Columns (Miltenyi Biotec, catalog number: 130-042-101)
50 ml Falcon conical tube (Corning, catalog number: 352070)
15 ml CentriStar conical tube (Corning, catalog number: 430790)
5 ml Falcon Round Bottom test tube (Corning, catalog number: 352052)
96-well round bottom, non-TC treated plates (Sarstedt, catalog number: 82.1582.001)
CytoOne Non-treated 6-well plates (USA Scientific, catalog number: CC7672-7506)
Falcon 70 μm cell stainer (Corning, catalog number: 352350)
Plain and frosted glass slides (VWR International, catalog number: 48312-004)
Foxp3^GFP(B6.Cg-Foxp3tm2(EGFP)Tch/J) mice (THE JACKSON LABORATORY, catalog number: 006772)
The best T_regP cell yields come from younger (5-6 weeks old) female mice due to a generally large thymus. If younger mice are not available older mice will provide usable T_regP cells. However, the number of T_regP cells recovered could be reduced by 50% of that obtained from younger mice.
Recombinant Human IL-2 (R&D Systems, catalog number: 202-IL)
Anti-mouse CD4 (GK1.5) BV786 (1:200) (BD Biosciences, catalog number: 563331)
Anti-mouse CD8a (53-6.7) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-0081-U500)
Anti-mouse CD25 (PC61.5) PerCP-Cy5.5 (1:200) (Tonbo Biosciences, catalog number: 65-0251-U100)
Anti-mouse CD73 (eBioTY/11.8) eF450 (1:100) (eBioscience, catalog number: 48-0731-82)
Anti-mouse Ter119 (Ter-119) Biotin (1:200) (BD Biosciences, catalog number: 553672)
(Optional) antibodies:
1. Anti-CD11b (M1/70) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-0112-U500)
2. Anti-CD11c (N418) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-0114-U500)
3. Anti-NK1.1 (PK136) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-5941-U500)
4. Anti-γδTCR (eBioGL3) Biotin (1:200) (eBioscience, catalog number: 13-5711-85)
5. Anti-B220 (RA3-6B2) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-0452-U500)
Streptavidin APC-eF780 (1:200) (eBioscience, catalog number: 47-4317-82)
Viability Dye Ghost^TM Red 780 (1:1,000) (Tonbo Biosciences, catalog number: 13-0865-T100)
Streptavidin Microbeads (Miltenyi Biotec, catalog number: 130-048-101)
AccuCheck Counting Beads (Life Technologies, catalog number: PCB100)
Sodium azide (NaN₃) (Ricca Chemical, catalog number: 7144.8-16)
10x PBS (Fisher BioReagents, catalog number: BP399-20)
Ethylenediaminetetraacetic Acid (EDTA) (Fisher Scientific, catalog number: BP120-500)
Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150)
RPMI 1640 media (Corning, catalog number: 15-040-CV)
Penicillin-Streptomycin Solution (Corning, catalog number: 30-002-CL)
L-Glutamine (Corning, catalog number: 25-005-CL)
MEM Nonessential amino acids (Corning, catalog number: 25-025-CL)
2-mercaptoethanol (MP Bio, catalog number: 02194705-CF)
HEPES (Corning, catalog number: 25-060-CL)
Sodium Pyruvate (Corning, catalog number: 25-000-CL)
Sort Buffer (see Recipes)
FACS Buffer (see Recipes)
cRPMI (see Recipes)

Equipment

Pipettes
Sorvall Legend X1R Centrifuge (Thermo Scientific, catalog number: 75004260)
BD Biosciences LSR II (BD, model: LSR II)
BD Biosciences FACSAria II (BD, model: FACSAria II)
MACS Multistand (Miltenyi Biotec, catalog number: 130-042-303)
QuadroMACS Separator Magnet (Miltenyi, catalog number: 130-090-976)
Class II Biosafety Cabinet/Laminar Flow Hood
IncuSafe Incubator (Panasonic, catalog number: KM-CC17T0A)
Hemocytometer (Fisher, catalog number: 0267110)
Refrigerator (2-8 °C, Kenmore)
Panasonic -80 °C VIP Plus Freezer

Software

FlowJo 10 (10.5.3) (https://www.flowjo.com/)

Procedure

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如何引用

Owen, D. L. and Farrar, M. A. (2019). In vitro Differentiation of Thymic T_reg Cell Progenitors to Mature Thymic T_reg Cells. Bio-protocol 9(16): e3335. DOI: 10.21769/BioProtoc.3335.