CD8+ T细胞体外和体内抑制试验

Lu Xie; Guihuan Liu; Yanjun Liu; Yuming Yu

doi:10.21769/BioProtoc.4020

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Peer-reviewed

In vitro and In vivo CD8⁺ T Cell Suppression Assays

CD8+ T细胞体外和体内抑制试验

LX Lu Xie ^*

GL Guihuan Liu ^*

YL Yanjun Liu

YY Yuming Yu email

(*contributed equally to this work) 发布: 2021年05月20日第11卷第10期 DOI: 10.21769/BioProtoc.4020 浏览次数: 7412

评审: Luis Alberto Sánchez VargasMarieta RusevaAnonymous reviewer(s)

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Cover of BMC Immunology, featuring study using the protocol.

Apr 2020

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Abstract

CD8⁺CD28⁻ T suppressor cells (Ts) have been documented to promote immune tolerance by suppressing effector T cell responses to alloantigens following transplantation. The suppressive function of T cells has been defined as the inhibitory effect of Ts on the proliferation rate of effector T cells.³H-thymidine is a classical immunological technique for assaying T cell proliferation but this approach has drawbacks such as the inconvenience of working with radioactive materials. Labeling T cells with CFSE allows relatively easy tracking of generations of proliferated cells. In this report, we utilized antigen presenting cells (APCs) and T cells matched for human leukocyte antigen (HLA) class I or class II to study CD8⁺CD28^- T cell suppression generated in vitro by this novel approach of combining allogeneic APCs and γc cytokines. The expanded CD8⁺CD28^- T cells were isolated (purity 95%) and evaluated for their suppressive capacity in mixed lymphocyte reactions using CD4⁺T cells as responders. Here, we present our adapted protocol for assaying the Ts allospecific suppression of CFSE-labeled responder T cells.

Keywords: CD8⁺CD28^- T cells (CD8⁺CD28^-T 细胞)

Suppression (抑制)

Immune tolerance (免疫耐受)

Alloantigen specific tolerance (同种特异性免疫耐受)

Common gamma chain cytokines (常见γ链细胞因子)

Background

T regulatory cells (Tregs) with dedicated suppressor function play a crucial role in the homeostatic control of immunity in the periphery. Regulatory CD8⁺T cells have also been demonstrated to play an important role in neonatal tolerance and autoimmune diseases (Tang et al., 2005). There are two broad categories of immune regulation by Tregs: non-specific and antigen-specific. Non-specific immunosuppression potentially causes general immunosuppression and produces undesirable side effects, such as infectious diseases. These Tregs include CD8⁺CD25⁺, CD8⁺CD122⁺, CD45RC^low, and IL-2/GM-CSF-induced CD8⁺ Tregs. On the contrary, antigen-specific Tregs are primed during the immune response to foreign or self-antigens and subsequently specifically downregulate that immune response. These Tregs include CD8⁺CD28^-, CD8⁺CD75s⁺, plasmacytoid dendritic cell (DC2)-induced CD8⁺, CD8⁺CD45RC^high Tc1, and TCR peptide-specific CD8αα Tregs. CD8⁺CD28⁻ Tregs have been recently documented to play an important role in alloimmunity. In our previous studies, we have expanded large numbers of human CD8⁺CD28⁻Tregs in a relatively short period of time by stimulating CD8⁺ T cells with APCs following supplementation with the triple common gamma chain cytokines IL-2, IL-7, and IL-15 in vitro; however, the detailed characteristics of the expanded CD8⁺CD28⁻ Tregs were unclear. Moreover, the principal function of this population when transferred in vivo was yet to be examined. Measurement of suppression has been achieved through the co-culture of Tregs and T effector cells. Methods include the detection of cell proliferation, cytokine production, and activation markers (CD25 or CD134) (Long et al., 2017). CFSE-based co-culture has become the gold standard for proliferation assays and has been used successfully to assess the function of Tregs. Here, based on CFSE co-culture assays, we show that the in vitro-expanded CD8⁺CD28⁻Tregs maintain allospecific suppressive capacity both in vitro (Figure 1) and in vivo.

Figure 1. The main steps of the in vitro CD8⁺ T cell suppression assay protocol

Materials and Reagents

Heparin sodium anticoagulation tubes, 5 ml (JiangSu, YuLi)
Centrifuge tubes, 50 ml (Corning, catalog number: 430829)
Centrifuge tubes, 15 ml (Corning, catalog number: 430791)
24-well round-bottomed plates (Corning, catalog number: 3524)
96-well round-bottomed plates (Corning, catalog number: 3799)
MidiMACS separator (Miltenyi, catalog number: 130-042-302)
LS column (Miltenyi, catalog number: 130-042-401)
Disposable syringe with a needle (ShuangGe, China)
70-μm cell strainer (Biologix, catalog number: 15-1070)
NOG mice (Beijing Vital River Laboratory Animal Technology Co Charles River Laboratories)
Ficoll-Hypaque solution (Haoyang Biologiacal, TBD sciences, catalog number: LTS1077)
RPMI 1640, 500 ml (ThermoFisher, Gibco, catalog number: C11875500BT)
FBS Qualified Australia Origin (ThermoFisher, Gibco, catalog number: 10099141 C)
Bovine serum albumin, BSA (SIJIA, catalog number: N0008-1)
Phosphate-buffered saline, PBS (ThermoFisher, Gibco, catalog number: C10010500BT)
CD8 MicroBeads, human (Miltenyi, catalog number: 130-045-201)
CD28 MicroBeads, human (Miltenyi, catalog number: 130-093-247)
CD2 MicroBeads, human (Miltenyi, catalog number: 130-091-114)
CD4 MicroBeads, human (Miltenyi, catalog number: 130-045-101)
IL-2 (PeproTech, catalog number: AF-200-02-50)
IL-7 (PeproTech, catalog number: AF-200-07-50)
IL-15 (PeproTech, catalog number: AF-200-15-50)
10× RBC lysis buffer (ThermoFisher, Invitrogen, catalog number: 00-4300-54)
Trypan Blue solution 0.4%, liquid (MERCK,Sigma-Aldrich, catalog number: T8154-100ML)
7-AAD viability stain solution (ThermoFisher, Invitrogen, catalog number: 00-6993-50)
CFDA, SE (ThermoFisher, Invitrogen, catalog number: C1157)
Flow cytometry antibodies
1. AlexaFluor^TM 700 mouse anti-human CD3 monoclonal antibody, OKT3 (ThermoFisher, eBioscience, catalog number: 56-0037-42)
2. efluor 450 mouse anti-human CD8a monoclonal antibody, SK1 (ThermoFisher, eBioscience, catalog number: 48-0088-41)
3. APC-mouse anti-human CD28 monoclonal antibody, CD28.2 (ThermoFisher, eBioscience, catalog number: 17-0289-42)
4. APC-mouse anti-human CD4 monoclonal antibody, OKT4 (ThermoFisher,eBioscience, catalog number: 17-0048-42)
5. FITC mouse anti-human CD2 monoclonal antibody, RPA-2.10 (ThermoFisher, eBioscience, catalog number: 11-0029-42)
6. PE-mouse anti-human CD45 monoclonal antibody, HI30 (ThermoFisher,eBioscience, catalog number: 12-0459-42)
Fixable viability stain (FVS) 620 100 μg (BD Pharmingen, catalog number: 564996)
NaCl
KCl
Na₂HPO₄
KH₂PO₄
EDTA
1× PBS (pH 7.4) (see Recipes)
D-PBS (pH 7.4) (see Recipes)
1% BSA-PBS (see Recipes)
0.5% BSA-PBS (see Recipes)

Equipment

Centrifuge (Eppendorf, model: 5810R)
Electronic balance (Sartorius, model: BP61)
Microelectronic balance (OHAUS, model: AX124ZH)
Hemocytometer
Constant temperature water box
Incubator (Thermo, model: Thermo3111)
FACS LSRFortessa (BD)
Finnpipette (Eppendorf)
Magnetic stirrer (BG-stirrelDB)
Optical microscope (CONIC, XDS-1B)
Clean bench

Software

Flowjo vX.0.7
SPSS 20.0
GraphPad Prism 5.01

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Xie, L., Liu, G., Liu, Y. and Yu, Y. (2021). In vitro and In vivo CD8⁺ T Cell Suppression Assays. Bio-protocol 11(10): e4020. DOI: 10.21769/BioProtoc.4020.