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Apr 2020
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In vitro and In vivo CD8+ T Cell Suppression Assays
CD8+ T细胞体外和体内抑制试验   

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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+, CD45RClow, 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+CD45RChigh 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+CD28Tregs 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+CD28Tregs 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

  1. Heparin sodium anticoagulation tubes, 5 ml (JiangSu, YuLi)

  2. Centrifuge tubes, 50 ml (Corning, catalog number: 430829)

  3. Centrifuge tubes, 15 ml (Corning, catalog number: 430791)

  4. 24-well round-bottomed plates (Corning, catalog number: 3524)

  5. 96-well round-bottomed plates (Corning, catalog number: 3799)

  6. MidiMACS separator (Miltenyi, catalog number: 130-042-302)

  7. LS column (Miltenyi, catalog number: 130-042-401)

  8. Disposable syringe with a needle (ShuangGe, China)

  9. 70-μm cell strainer (Biologix, catalog number: 15-1070)

  10. NOG mice (Beijing Vital River Laboratory Animal Technology Co Charles River Laboratories)

  11. Ficoll-Hypaque solution (Haoyang Biologiacal, TBD sciences, catalog number: LTS1077)

  12. RPMI 1640, 500 ml (ThermoFisher, Gibco, catalog number: C11875500BT)

  13. FBS Qualified Australia Origin (ThermoFisher, Gibco, catalog number: 10099141 C)

  14. Bovine serum albumin, BSA (SIJIA, catalog number: N0008-1)

  15. Phosphate-buffered saline, PBS (ThermoFisher, Gibco, catalog number: C10010500BT)

  16. CD8 MicroBeads, human (Miltenyi, catalog number: 130-045-201)

  17. CD28 MicroBeads, human (Miltenyi, catalog number: 130-093-247)

  18. CD2 MicroBeads, human (Miltenyi, catalog number: 130-091-114)

  19. CD4 MicroBeads, human (Miltenyi, catalog number: 130-045-101)

  20. IL-2 (PeproTech, catalog number: AF-200-02-50)

  21. IL-7 (PeproTech, catalog number: AF-200-07-50)

  22. IL-15 (PeproTech, catalog number: AF-200-15-50)

  23. 10× RBC lysis buffer (ThermoFisher, Invitrogen, catalog number: 00-4300-54)

  24. Trypan Blue solution 0.4%, liquid (MERCK,Sigma-Aldrich, catalog number: T8154-100ML)

  25. 7-AAD viability stain solution (ThermoFisher, Invitrogen, catalog number: 00-6993-50)

  26. CFDA, SE (ThermoFisher, Invitrogen, catalog number: C1157)

  27. Flow cytometry antibodies

    1. AlexaFluorTM 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)

  28. Fixable viability stain (FVS) 620 100 μg (BD Pharmingen, catalog number: 564996)

  29. NaCl

  30. KCl

  31. Na2HPO4

  32. KH2PO4

  33. EDTA

  34. 1× PBS (pH 7.4) (see Recipes)

  35. D-PBS (pH 7.4) (see Recipes)

  36. 1% BSA-PBS (see Recipes)

  37. 0.5% BSA-PBS (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5810R)

  2. Electronic balance (Sartorius, model: BP61)

  3. Microelectronic balance (OHAUS, model: AX124ZH)

  4. Hemocytometer

  5. Constant temperature water box

  6. Incubator (Thermo, model: Thermo3111)

  7. FACS LSRFortessa (BD)

  8. Finnpipette (Eppendorf)

  9. Magnetic stirrer (BG-stirrelDB)

  10. Optical microscope (CONIC, XDS-1B)

  11. Clean bench

Software

  1. Flowjo vX.0.7

  2. SPSS 20.0

  3. GraphPad Prism 5.01

Procedure

  1. Peripheral blood mononuclear cell (PBMC) isolation

    Isolate PBMCs from samples acquired in heparinized tubes from healthy volunteers. PBMCs are isolated by density gradient centrifugation using Ficoll-Hypaque solution.

    1. Dilute the blood sample 1:2 with the same volume of RPMI 1640.

    2. Add a volume of Ficoll-Hypaque solution equal to that of the blood sample in a 50-ml centrifuge tube.

    3. Add the diluted blood sample carefully and slowly to the surface of the separation fluid. Centrifuge at 800 × g for 20-30 min at room temperature.

      Note: The blood sample volume determines the centrifugal conditions; read the separation solution instructions.

    4. Carefully absorb the second annular opalescent lymphocyte layer into another centrifuge tube, add 10-20 ml RPMI 1640, mix well, and centrifuge the cell suspension at 580 × g for 10 min at room temperature.

    5. Aspirate the supernatant completely. Wash the cell pellet by adding 10 ml RPMI 1640 and centrifuging at 290 × g for 10 min at room temperature.

    6. Repeat Step A5.


  2. MACS separation

    CD8+ cells (A-CD8+) and CD4+ cells (A-CD4+) are isolated from PBMCs of individual A by positive selection using the MACS system. APCs are obtained by depletion of CD2+ cells from PBMCs of individual B (B-APCs) or individual I (I-APCs). Donors were selected according to their HLA-A, -B, and -DR compatibility or incompatibility based on the specific requirements of individual experiments. Several groups of individuals designated as A, B, and I, which are fully HLA-A, -B, and -DR mismatched were screened out from 130 volunteers and used for independent experiments.

    Note: Keep the cells and the buffer cold. Centrifuge at 4°C.

    1. Add 10 ml 0.5% BSA-PBS buffer to resuspend PBMCs (from Step A6) and determine the cell number.

    2. Centrifuge the cell suspension at 300 × g for 10 min. Aspirate the supernatant completely.

    3. Resuspend the cell pellet in 80 μl 0.5% BSA-PBS buffer per 107 cells.

    4. Add 20 μl appropriate Microbeads per 107 cells.

    5. Mix well and incubate for 20 min in the refrigerator (2-8°C).

    6. Wash the cells by adding 1-2 ml 0.5% BSA-PBS buffer per 107 cells and centrifuge at 300 × g for 10 min. Aspirate the supernatant completely.

    7. Resuspend up to 108 cells in 500 μl 0.5% BSA-PBS buffer.

      Note: For higher cell numbers, scale up the buffer volume accordingly.

    8. Place the LS column in the magnetic field of a MidiMACS separator.

      Note: Choose an appropriate MACS column and MACS separator according to the numbers of total and positive cells. For details, see the Microbead instructions.

    9. Prepare the column by rinsing with 3 ml 0.5% BSA-PBS buffer.

    10. Apply the cell suspension to the LS column.

    11. Collect the unlabeled cells that pass through and wash the column with 3 ml 0.5% BSA-PBS buffer. Collect the total effluent; this is the unlabeled cell fraction (such as CD2- cells).

    12. Wash the column by adding 3 ml 0.5% BSA-PBS buffer twice. Only add fresh 0.5% BSA-PBS buffer when the column reservoir is empty.

    13. Remove the column from the separator and place on a suitable collection tube.

    14. Pipette 5 ml buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column (such as CD8+ cells, CD4+ cells).

    15. Perform flow cytometry analysis to evaluate the purity of the sorted cell suspensions; the purity should be ≥97%.


  3. In vitro expansion of CD8+CD28 T cells

    A-CD8+ cells are isolated by positive selection using the MACS system according to Procedure B. B-APCs (CD2- cells) from HLA-A, -B, -DR mismatched individual B are isolated by depletion of CD2+ cells using CD2 microbeads.

    1. Centrifuge the A-CD8+ cells and B-APCs obtained above at 300 × g for 10 min. Aspirate the supernatant completely.

    2. Resuspend in 5 ml RPMI1640 and determine the cell number.

    3. Centrifuge the A-CD8+ cells and B-APCs at 300 × g for 10 min. Aspirate the supernatant completely.

    4. Resuspend the cells in cell culture medium (RPMI 1640 supplemented with 15% FBS) to achieve A-CD8+ at 2 × 106 cells/ml and B-APCs at 1 × 106 cells/ml.

    5. Seed the A-CD8+ cells (2 × 106/well) and B-APCs (1 × 106/well) onto 24-well flat-bottomed plates at a ratio of 2:1 in a total volume of 2 ml, in the presence of IL-2 at 20 U/ml, IL-7 at 50 ng/ml, and IL-15 at 50 ng/ml, in an incubator at 37°C and a humidified 5.5% CO2 atmosphere.

    6. Change semi-culture medium (1 ml) after 3 days. After 5 days and 7 days of coculture, mix well and divide one well into two.

    7. On day 9, harvest the cells into 50-ml centrifuge tubes. Add 10 ml 0.5% BSA-PBS buffer to wash the cells and determine the cell number.

    8. Centrifuge the cell suspension at 300 × g for 10 min at 4°C. Aspirate the supernatant completely.

    9. Resuspend the cell pellet in 40 μl 0.5% BSA-PBS buffer per 107 cells.

    10. Add 10 μl CD28-PE per 107 cells.

    11. Mix well and incubate for 10 min in the refrigerator (2–8°C).

    12. Wash the cells by adding 1-2 ml 0.5% BSA-PBS buffer per 107 cells and centrifuge at 300 × g for 10 min at 4°C. Aspirate the supernatant completely.

    13. Resuspend the cell pellet in 80 μl 0.5% BSA-PBS buffer per 107 cells.

    14. Add 20 μl anti-PE microbeads per 107 cells.

    15. Mix well and incubate for an additional 15 min in the refrigerator (2-8°C).

    16. Wash the cells by adding 1-2 ml 0.5% BSA-PBS buffer per 107 cells and centrifuging at 300 × g for 10 min at 4°C. Aspirate the supernatant completely.

    17. Resuspend up to 108 cells in 500 μl 0.5% BSA-PBS buffer.

      Note: For higher cell numbers, scale up the buffer volume accordingly.

    18. Place the LS column in the magnetic field of a MidiMACS separator.

      Note: Choose an appropriate MACS column and MACS separator according to the numbers of total and positive cells. For details, see the microbead instructions.

    19. Prepare the column by rinsing with 3 ml 0.5% BSA-PBS buffer.

    20. Apply the cell suspension to the column.

    21. Collect the unlabeled cells that pass through and wash the column with 3 ml 0.5% BSA-PBS buffer. Collect the total effluent; this is the unlabeled cell fraction (CD28- cells).

    22. Perform flow cytometry analysis to evaluate the purity of CD28- cell suspensions; the purity should be ≥95%.


  4. In vitro suppression assays

    Responder CD4+ cells are isolated from PBMCs of individual A (A-CD4+ T cells) by positive selection using CD4 microbeads. As stimulators, APCs cells from individual B (B-APCs) or from HLA-A, -B, -DR fully mismatched indifferent individual I (I-APCs) are isolated by depletion of CD2+ cells using CD2 microbeads.

    1. Centrifuge the A-CD4+ cell suspension (from Step B14) at 300 × g for 10 min at 4°C. Aspirate the supernatant completely.

    2. Wash the freshly isolated CD4+ cells with 10 ml PBS to remove any proteins. Centrifuge at 400 × g for 10 min at 4°C.

    3. Resuspend the cells in 1 ml PBS.

    4. Add CFSE working dilution to a final concentration of 0.5 μM at room temperature.

    5. Incubate for 7 min in the incubator (37°C); work gently and protect from light.

    6. Add 1 ml FBS and 9 ml RPMI 1640 to terminate the reaction, then centrifuge at 300 × g for 10 min. Aspirate the supernatant completely.

    7. Wash the cells once again with RPMI 1640. Aspirate the supernatant completely.

    8. Resuspend in 5 ml RPMI 1640 and count the cells.

      Note: After labeling with CFSE, perform flow cytometry analysis to ensure that the cells are stained.

    9. Centrifuge the A-CD8+CD28- cells, B-APCs, and I-APCs obtained above at 300 × g for 10 min. Aspirate the supernatant completely.

    10. Resuspend in 5 ml RPMI 1640 and count the corresponding cells.

    11. Centrifuge the A-CD8+CD28- cells, B-APCs, I-APCs, and A-CD4+CFSE at 300 × g for 10 min. Aspirate the supernatant completely.

    12. Resuspend the cell pellet in culture medium (RPMI 1640 supplemented with 15% FBS) to achieve A-CD4+CFSE at 1 × 106 cells/ml, B-APCs or I-APCs at 1 × 106 cells/ml, and A-CD8+CD28- cells at 2.5 × 105 cells/ml.

    13. Seed the A-CD4+CFSE cells (5 × 104/well), B- APCs or I-APCs (5 × 104/well), and CD8+CD28- cells (2.5 × 104/well) onto 96-well round-bottomed plates in a total volume of 200 μl cell culture medium containing 15% FBS.

      Note: Responder CD4+ T cells, stimulated with B-APCs or I-APCs, only served as positive controls (B-APCs were used as priming cells in vitro to expand CD8+CD28T cells from individual A, whereas I-APCs had never had immune recognition by CD8+CD28T cells from individual A during the in vitro expansion period).

    14. Change semi-culture medium (100 μl) after 3 days, 5 days, and 9 days of coculture.

    15. After 7 or 11 days of coculture, harvest, stain, and detect the CD4+ cells for CFSE dilution by flow cytometry.


  5. In vivo suppression assays

    1. Isolate the responder CD4+ T cells and APCs (Procedure A and B).

    2. Mix a total of 4 × 106 human CD4+ T cells with an equal number of APCs, either B-APCs or I-APCs, and combine with 2 × 106in vitro-expanded human CD8+CD28 T cells in a total volume of 1.5 ml PBS. The responder CD4+ T cells alone plus B-APCs are used in the same fashion as a positive control.

    3. Administer the cell mixture for each group to NOG mice by intraperitoneal injection.

    4. Sacrifice the mice on day 11 post-injection.

    5. Isolate the spleen and homogenize to generate a single-cell suspension.

    6. Centrifuge the cell suspension at 500 × g for 5 min at 4°C. Aspirate the supernatant completely.

    7. Add 3 ml 1× RBC lysis buffer to lyse the red cells, mix well, and incubate for 5 min at room temperature.

    8. Add 2 ml PBS, centrifuge at 350 × g for 10 min at room temperature. Aspirate the supernatant completely.

    9. Stain the cells and detect those expressing CD4+ by flow cytometry.

      Note: You can also detect human CD8+ and CD4+ cells in mouse spleen tissue by immunohistochemical techniques.


  6. Staining cells for analysis by flow cytometry

    Flow cytometry analysis is performed on cells harvested from the culture under different conditions (Procedures D and E).

    1. Place the cells to be analyzed into 12 × 75 mm polystyrene tubes.

    2. Wash the cells in 3 ml 1% BSA-PBS buffer at 350 × g for 10 min at 4°C.

    3. Discard the supernatant and quickly vortex to loosen the pelleted cells.

    4. Add 2 μl antibodies (such as anti-CD4), mix well, and incubate for 30 min at 4°C in the dark.

    5. Wash the cells in 3 ml 1% BSA-PBS buffer at 350 × g for 10 min at 4°C.

    6. Discard the supernatant and quickly vortex to loosen the pelleted cells.

    7. Add 3 μl 7-AAD Viability Staining Solution and incubate for 10 min at room temperature in the dark.

    8. Resuspend the cells in an appropriate volume of 1% BSA-PBS and detect by flow cytometry.

Data analysis

  1. Data analysis is performed using offline analysis software such as Flowjo. As shown in Figure 2, the identification of CD4+ T cells begins with the creation of gates to isolate the lymphocyte population (FSC-A; SSC-A), followed by rigorous doublet exclusion (FSC-W vs. FSC-H and SSC-W vs. SSC-A). Exclude cells that are positive for dead staining and then gate on the target population of cells.



    Figure 2. Gating strategy to identify CD4+ cells in suppression assays. A. Lymphocyte population identified in FSC-A vs. SSC-A plot. B-C. This is followed by doublet discrimination using FSC and SSC area and width plots. D. After staining with 7-AAD, live cells are then analyzed. E. Live CD4+ T cells are gated. F. The proliferation of CD4+ T cells was measured by CFSE dilution.


  2. The suppressive percentage is calculated as follows: % suppression = [1 – (% CD4+ T cell proliferation in the presence of CD8+CD28 T cells)/(% CD4+ T cell proliferation in the absence of CD8+CD28 T cells)] × 100.

  3. Statistical analysis is carried out using the SPSS version 22 software. For comparison of different groups, where appropriate, an independent-samples t-test and nonparametric test are used to determine statistical significance. Graphs are created using the GraphPad Prism version 5.01 software.

Recipes

  1. 1× PBS (pH 7.4)

    NaCl 8 g

    KCl 0.2 g

    Add ultrapure H2O to a total volume of 1 L

  2. D-PBS (pH 7.4)

    NaCl 8 g

    KCl 0.2 g

    Na2HPO4 1.15 g

    KH2PO4 0.2 g

    Add ultrapure H2O to a total volume of 1 L

  3. 1% BSA-PBS

    BSA 10 g

    NaCl 8 g

    KCl 0.2 g

    Na2HPO4 1.44 g

    KH2PO4 0.24 g

    Add ultrapure H2O to a total volume of 1 L

  4. 0.5% BSA-PBS

    EDTA 2 mM

    BSA 5 g

    NaCl 8 g

    KCl 0.2 g

    Na2HPO4 1.44 g

    KH2PO4 0.24 g

    Add ultrapure H2O to a total volume of 1 L

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China [No. 81270839, 81428007] and the Natural Science Foundation of Guangdong Province [2017A030313524]. The protocols outlined here were originally described in several papers from the Yu lab (Yu et al., 2011; Feng et al., 2018; Liu et al., 2020).

Competing interests

The authors declare no competing interests, financial or non-financial.

Ethics

All experiments using human cells were carried out in accordance with the recommendations of the Ethical Review Board of Southern Medical University (Guangzhou, China); all subjects gave written informed consent in accordance with the Declaration of Helsinki. All animal experiments were approved by the Guidelines for the Care and Use of Animals established by the Animal Care and Use Committee of Southern Medical University.

References

  1. Feng, F., Liu, Y., Liu, G., Zhu, P., Zhu, M., Zhang, H., Lu, X., Liu, J., Luo, X. and Yu, Y. (2018). Human CD8+CD28- T suppressor cells expanded by IL-15 in vitro suppress in an allospecific and programmed cell death protein 1-dependent manner. Front Immunol 9: 1442.
  2. Liu, G., Yu, Y., Feng, F., Zhu, P. and Liu, Y. (2020). Human CD8+CD28- T suppressor cells expanded by common gamma chain (gammac) cytokines retain steady allospecific suppressive capacity in vivo. BMC Immunol 21(1): 23.
  3. Tang, X. L., Smith, T. R. and Kumar, V. (2005). Specific Control of Immunity by Regulatory CD8 T Cells. Cell Mol Immunol 2(1):11-19.
  4. Long, A. E., Tatum, M., Mikacenic, C. and Buckner, J. H. (2017). A novel and rapid method to quantify Treg mediated suppression of CD4 T cells.J Immunol Methods 449: 15-22.
  5. Yu, Y., Zitzner, J. R., Houlihan, J., Herrera, N., Xu, L., Miller, J., Mathew, J. M., Tambur, A. R. and Luo, X. (2011). Common gamma chain cytokines promote rapid in vitro expansion of allo-specific human CD8+ suppressor T cells.PLoS ONE 6(12): e28948.

简介

[摘要] CD8 + CD28 - T抑制细胞(Ts)的已记录在案通过抑制与效应T细胞应答,以促进免疫耐受性同种异体抗原小号以下移植。所述suppressi已经的T细胞功能ħ作为被定义为在TS上的抑制效应的增殖率的效应T细胞。3 H-胸苷是用于测定T细胞增殖的经典免疫技术b UT这种方法也有缺点,如用放射性材料的工作的不便。大号abeling T细胞用CFSE允许相对容易轨道荷兰国际集团的增殖的细胞的后代。在本报告中,我们利用与人白细胞抗原(HLA)I类或II类匹配的抗原呈递细胞(APC)和T细胞,研究了通过异源APC和γc结合的新方法体外产生的CD8 + CD28 - T细胞抑制作用细胞因子。分离扩增的CD8 + CD28 - T细胞(纯度95%),并以CD4 + T细胞作为应答剂评估其在混合淋巴细胞反应中的抑制能力。ħ埃雷,我们提出我们适于协议用于测定的TS同种异体特异性CFSE标记的应答T细胞的抑制。

[背景] T调节细胞(Treg细胞)具有专用的抑制功能,在外围免疫力的稳态控制起到至关重要的作用。调节性CD8 + T细胞也已被证明在新生儿耐受性和自身免疫性疾病中起重要作用(Tang等,2005 )。有两大类免疫reg的ü升通货膨胀的Treg的小号:ñ的特异性和一个ntigen -具体。非特异性免疫抑制的潜在原因小号一般的免疫抑制和生产小号不希望的副作用,如感染性疾病。这些调节性T细胞包括CD8 + CD25 + ,CD8 + CD122 + ,CD45RC低,和IL-2 / GM-CSF-诱导ð CD8 + Treg的小号。相反,抗原-特异性调节性T细胞都准备在国外或自抗原的免疫应答,并随后明确下调的是免疫反应。这些调节性T细胞包括CD8 + CD28 - ,CD8 + CD75s + ,浆细胞样树突细胞(DC2)诱导的CD8 + ,CD8 + CD45RC高Tc1的,和TCR肽-特异性的TregCD8αα。CD8 + CD28 − T reg最近被证明可以播放 在同种异体免疫中发挥重要作用。在我们以往的研究中,我们已经扩大了大量人力的CD8 + CD28 -牛逼的REG通过刺激的时间相对较短的时间CD8 + T细胞的APC以下补充通货膨胀与该三重共同γ链细胞因子IL-2,IL-7 ,和IL-15体外; ħ H但是,在详细特征扩展的CD8 + CD28 - Ť REG小号不清楚。此外,当体内转移尚待研究时,其主要功能是种群。通过Tregs和T效应细胞的共培养已经实现了抑制作用的测量。在方法CLUDE的检测的细胞增殖,细胞因子产生,和活化标记(CD25或CD134) (朗等人,2017) 。基于CFSE -共-培养ħ作为成为黄金标准用于增殖测定和公顷š被成功地用于评估Treg的功能。在此,基于CFSE共培养测定法,我们显示了体外扩增的CD8 + CD28 - T reg s在体外(图1 )和体内均具有同种异体抑制能力。

˚F igure 1.的主要步骤的我Ñ体外CD8 + Ť Ç ELL小号uppression一个SSAY协议

关键字:CD8+CD28-T 细胞, 抑制, 免疫耐受, 同种特异性免疫耐受, 常见γ链细胞因子

材料和试剂

1.肝素钠一个nticoagu升通货膨胀管小号,加入5ml(江苏,宇立)     

2.离心机吨ubes ,50毫升(康宁,目录号:430829 )     

3.离心吨ubes ,15毫升(康宁,目录号:430791 )     

4. 24孔廿四底部ED板中(Corning ,目录号:3524 )     

5. 96孔圆-底ED板(Corning公司,目录号: 3799 )     

6. MidiMACS小号eparator (Miltenyi公司,目录号:130-042-302 )     

7.大号小号Ç olumn (Miltenyi公司,目录号:130-042-401 )     

8.一次性针头注射器(中国双葛)     

9. 70 -微米Ç ELL小号训练(Biologix ,目录号:15-1070 )     

10. NOG小鼠(北京生命河实验动物技术有限公司查尔斯河实验室) 

11.聚蔗糖-泛影葡胺小号olution(浩阳Biologiacal ,TBD科学,目录号:LTS1077) 

12. RPMI 1640,500毫升(Ť hermo ˚F isher ,GIBCO,目录号:C11875500BT) 

13. FBS Q ualified甲ustralia ø rigin (赛默飞,GIBCO,目录号:10099141 C) 

14.牛小号erum一个lbumin,BSA(思嘉,目录号:N0008-1) 

15.磷酸盐- b uffered小号艾琳,P BS (Ť hermo ˚F isher ,GIBCO,目录号:C10010500BT) 

16。    CD8 MicroBeads ,人类(Miltenyi ,目录号:130-045-201)


17.人CD28微珠(Miltenyi ,目录号:130-093-247) 

18.人CD2微珠(Miltenyi ,目录号:130-091-114) 

19.人CD4微珠(Miltenyi ,目录号:130-045-101) 

20.我L-2 (P易宝Ť ECH ,目录号:AF-200-02-50 ) 

21.我L-7 (P易宝Ť ECH ,目录号:AF-200-07-50 ) 

22. IL-15(P易宝Ť ECH ,目录号:AF-200-15-50 ) 

23. 10 × RBC升ysis b uffer (赛默飞,Invitrogen公司,目录号:00-4300-54) 

24.台盼蓝溶液0.4%,液体(MERCK,Sigma- Aldrich,目录号:T8154-100ML) 

25. 7-AAD生存力染色溶液(赛默飞,Invitrogen公司,目录号:00-6993-50) 

26。    CFDA,SE (ThermoFisher ,Invitrogen,目录号:C1157)


27. ˚F低Ç ytometry一个ntibodies 

的AlexaFluor TM 700的小鼠抗-人CD3米onoclonal一个ntibody ,OKT3(赛默飞,eBioscience公司,目录号:56-0037-42 )
efluor 450的小鼠抗-人CD8A米onoclonal一个ntibody ,SK1 (赛默飞,eBioscience公司,目录号:48-0088-41)
APC-小鼠抗-人CD28米onoclonal一个ntibody ,CD28.2 (赛默飞,eBioscience公司,目录号:17-0289-42)
APC-小鼠抗-人CD4米onoclonal一个ntibody ,OKT4 (赛默飞,eBioscience公司,目录号:17-0048-42 )
FITC鼠抗-人CD2米onoclonal一个ntibody ,RPA-2.10 (赛默飞,eBioscience公司,目录号:11-0029-42 )
PE-小鼠抗-人CD45米onoclonal一个ntibody,HI30 (赛默飞,eBioscience公司,目录号:12-0459-42)
28.可定影v iability小号覃(FVS )620 100 μ克(BD Pharmingen公司,目录号:564996) 

29.氯化钠 

30.氯化钾 

31.钠2 HPO 4 

32. KH 2 PO 4 

33. EDTA 

34. 1× PBS(pH 7.4 )(请参阅食谱) 

35. D-PBS (pH 7.4 )(参见食谱) 

36. 1%的BSA-PBS (请参阅食谱) 

37. 0.5%BSA-PBS (请参阅食谱) 



设备


离心机(Eppendorf,型号:5810R )
电子天平(Sartorius,型号:BP61)
中号icroelectronic天平(OHAUS ,型号:AX124ZH)
血细胞计数器
Ç onstant温度水箱
我ncubator (热,型号:热电3111)
F ACS LSRF ortessa (B D )
Finnpipette (埃彭多夫)
电磁搅拌器(B G- stirrel DB )
光学米icroscope (C ONIC,XDS-1B )
清洁b恩奇


软件


˚F lowjo v X 。0.7
SPSS 20.0
摹raphP广告棱镜5.01


程序


外周血单核细胞(PBMC)分离
从健康志愿者的肝素化试管中采集的样品中分离PBMC。的PBMC通过使用密度梯度离心法分离的Ficoll-Hypaque上小号olution。


用相同体积的RPMI 1640稀释血样1:2 。
添加的体积的Ficoll-Hypaque上SOLU和灰等于那个的血液样品在一个50 -毫升Ç entrifuge吨UBE。
将稀释的血液样本小心缓慢地添加到分离液的表面。离心机在800 ×g下进行20 -在室温下30分钟。
注意:Ť他的血液样品体积决定了离心条件; 阅读分离解决方案说明s 。


Ç arefully吸收第二环形乳白色淋巴细胞层到另一个离心管中,加入10 - 20毫升RPMI 1640,拌匀,和离心的580细胞悬液×克10分钟在室温下。
甲spirate的完全上清液。洗涤吨他通过加入10ml RPMI 1640和离心机细胞沉淀荷兰国际集团,在290 ×g下10分钟在室温下。
ř EPEAT小号TEP A5。


MACS小号eparation
CD8 +细胞(A-CD8 + )和CD4 +细胞(A-CD4 + )从个人A的PBMC中分离出由正选择使用的MACS系统。APC小号是获得由耗尽的CD2 +细胞从PBMC小号的个体B(B-的APC)或个人I(I-APC小号)。根据各个实验的具体要求,根据供体的HLA-A,-B和-DR相容性或不相容性选择供体。小号个体everal组小号指定为A,B,和I,这是完全HLA-A,-B和-DR不匹配呈S从130名志愿者creened出来并用于独立的实验。


注:K EEP的细胞,并在缓冲区冷。Ç entrifug向在4 ℃。


加入10 ml的 0.5%BSA-PBS缓冲液以重悬浮的PBMC(从小号TEP A6),并判断该细胞数目。
将细胞悬液以300 ×g离心10分钟。完全吸出上清液。
ř esuspend的在80细胞沉淀微升0.5%BSA-PBS每10个缓冲7细胞。
加入20个微升每10个适当微珠7细胞。
中号IX井和在冰箱孵育20分钟(2 - 8 ℃下)。
W¯¯灰的细胞中加入1 - 2毫升0.5%BSA-PBS每10个缓冲7细胞和离心机在300 ×g下10分钟。完全吸出上清液。
重悬高达10个8细胞在500微升0.5%BSA-PBS缓冲液中。
注:对于较高的细胞数量,扩大了相应的缓冲空间。


P花边的LS Ç olumn在的磁场MidiMACS小号eparator。
注意:卓ö本身适当的MACS Ç olumn和MACS小号根据数eparator小号总和阳性细胞。有关详细信息,请参见Microbead说明。


P repare的通过用3ml 0.5%BSA-PBS缓冲液漂洗柱。
应用的细胞悬液到LS列。
Ç ollect的穿过和洗涤未标记的细胞的用3ml柱0.5%BSA-PBS缓冲液中。收集的总流出物; 这是未标记的CE LL馏分(如CD2 -细胞)。
W¯¯灰的柱通过添加3毫升0.5%BSA-PBS缓冲液洗涤两次。当色谱柱容器为空时,仅添加新鲜的0.5%BSA-PBS缓冲液。
ř EMOVE的从分离器和地点列在合适的收集管中。
P ipette 5毫升缓冲液上柱。通过将柱塞牢固地推入色谱柱,立即冲洗出磁性标记的细胞(例如CD8 +细胞,CD4 +细胞)。
执行˚F低cytometr ý分析来评估的纯度的分选的细胞悬浮液; 纯度应≥ 97%。


CD8 + CD28 - T细胞的体外扩增
A-CD8 +细胞一个通过使用MACS系统根据阳性选择分离重新程序B. B-的APCs(CD2 -细胞)从HLA-A,- B,- DR错配个体乙隔离通过CD2的耗尽+细胞中使用CD2微珠。


将以上获得的A- CD8 +细胞和B-APC于300 ×g离心10分钟。完全吸出上清液。
重悬在5ml RPMI1640和确定的细胞数。
              以300 ×g离心A- CD8 +细胞和B-APC 10分钟。完全吸出上清液。
重悬的细胞培养物中的细胞MEDI微米(RPMI 1640补充有15%FBS)以实现A-CD 8 +在2× 10 6个细胞/ ml和B-的APCs以1 × 10 6细胞/毫升。
种子A-CD8 +细胞(2×10 6 /孔)和B-APC小号(1×10 6 /孔)ö n要24孔˚F LAT -底部ED板以2:1的比率在总体积的2毫升,在IL-2的存在下吨20U / ml时,IL-7在50纳克/毫升,和IL-15在50纳克/毫升,在37℃的培养箱中和加湿5.5%CO 2个气氛。
3天后更换半培养基(1毫升)。经过5天和7天共培养,拌匀,将一个好两成。
一天9,收获细胞成50 -毫升Ç entrifuge吨ubes。加入10 mL 0.5%BSA-PBS缓冲液洗涤所述细胞,并确定所述细胞数目。
离心机的在300细胞悬液×克10分钟在4℃下。完全吸出上清液。
重悬在40细胞沉淀微升0.5%BSA-PBS每10个缓冲7细胞。
添加10微升每10个CD28-PE 7细胞。
充分混合,然后在冰箱中(2 – 8 °C )孵育10分钟。
洗的加入1细胞- 2毫升0.5%BSA-PBS每10个缓冲7细胞和离心机在300 ×g下10分钟在4℃下。完全吸出上清液。
重悬在80细胞沉淀微升0.5%BSA-PBS每10个缓冲7细胞。
添加20微升一个NTI-PE米每10个icrobeads 7细胞。
拌匀孵育一个附加1 5中的冷藏库分钟(2 - 8 ℃下)。
洗的加入1细胞- 2毫升0.5%BSA-PBS每10个缓冲7细胞和离心机荷兰国际集团,在300 ×g下10分钟在4℃下。完全吸出上清液。
重悬高达10个8细胞在500微升0.5%BSA-PBS缓冲液中。
注:对于较高的细胞数量,扩大了相应的缓冲空间。


将在LS ç olumn中的磁场MidiMACS小号eparator。
注意:卓ö本身适当的MACS Ç olumn和MACS小号根据数eparator小号总和阳性细胞。有关详细信息,请参阅米icrobead说明。


制备的通过用3ml 0.5%BSA-PBS缓冲液漂洗柱。
应用的细胞悬液列。
收集的穿过未标记的细胞和洗涤的用3ml 0.5%BSA-PBS缓冲液柱。收集的总流出物; 这是未标记的细胞级分(CD28 -细胞)。
执行˚F低cytometr ý分析来评估的纯度CD28 -细胞悬浮液; 纯度昭ü LD是≥ 9 5 %。


体外抑制测定
通过使用CD4 m微珠进行阳性选择,从单个A的PBMC(A-CD4 + T细胞)中分离出响应者CD4 +细胞。作为刺激物,通过使用CD2微珠消耗CD2 +细胞,可以分离出来自个体B (B-APC)或来自HLA-A,-B,-DR完全错配的无关个体I(I-APC)的APC细胞。           

离心机的A-CD4 +细胞悬浮液(从小号TEP 300 B14)×G进行10分钟,在4℃下。完全吸出上清液。
用10 ml PBS洗涤新鲜分离的C D4 +细胞,以去除所有蛋白质。在4°C下以400 ×g离心10分钟。             
复查将细胞浸入1 ml PBS中。
甲DD CFSE工作稀释到一个0.5最终浓度μ中号在室温下。
我在恒温箱(37°C )中保温7分钟;工作轻轻地和保护的光。
用dd 1 ml FBS和9 ml RPMI 1640终止反应,然后以300 ×g离心10分钟。完全吸出上清液。
w ^灰细胞再次与RPMI 1640吸出的上清液完全。
重悬于5 ml RPMI 1640中并计数细胞。
注:后升与CFSE abeling,进行流量cytometr Ÿ分析恩确保细胞被染色。


Ç entrifuge的A-CD8 + CD28 -细胞,B-APC小号,一个第二I-APC小号获得上述在300 ×g下进行10分钟。完全吸出上清液。
重悬在5ml RPMI 1640 和计数的对应的细胞。
离心机在A - CD 8个+ CD28 -细胞 ,B-的APC,I-的APC ,和甲-CD4 + CFSE在300 ×g下10分钟。完全吸出上清液。
重悬在培养基中MEDI细胞沉淀微米(RPMI 1640补充有15%FBS )以实现A-CD4 + CFSE在1 ×10 6细胞/毫升,B-APC或I-的APCs在1×10 6个细胞/ ml,并甲- CD8 + CD28 -细胞在2.5×10 5细胞/ ml。
种子吨他A-CD4 + CFSE细胞(5×10 4 /孔),B- APC或I-的APCs(5×10 4 /孔),和CD8 + CD28 -细胞(2.5×10 4 /孔)ö n要96孔圆-底ED板在200的总体积微升细胞培养MEDI微米含15个%FBS。
注意:响应CD4 + T细胞,刺激的B-APC或I-的APC ,仅充当阳性对照(B-APC小号瓦特ERE用作引发细胞小号体外扩展的CD8 + CD28 -从单独的A T细胞,而I-APC小号从未有过免疫识别由CD8 + CD28 -从单个A的T细胞在体外EXPAN锡永时段)。


改变半培养基(1 00 μ升)后3天,5一天小号,并共培养9天。
经过7或11天共培养中,收获,染色,并检测CD4 +通过流式细胞术细胞CFSE稀释。


体内抑制测定
Isolat e是应答的CD4 + T细胞和的APCs(步骤A和B)。
将总量为4×10 6人CD4 + T细胞与相等数目的APCs ,要么B-APC或I-的APC,并用2×10结合6体外-扩增的人CD8 + CD28 - T细胞的共体积为1.5毫升的PBS。应答的CD4 +单独的T细胞和B-的APCs一个以相同的方式使用作为阳性对照重。
管理与c为每个组到NOG小鼠腹腔注射ELL混合物。
小号acrifice小鼠一天11注射后。
隔离的脾,并均化以产生一个单-细胞悬浮液。
离心机的500细胞悬液×克5分钟,在4 ℃下。完全吸出上清液。
添加3 ml的1 × RBC裂解缓冲液以裂解的红细胞,拌匀,以及我ncubate在室温下5分钟。
加入2 ml PBS,在室温下以350 ×g离心10分钟。完全吸出上清液。
染色细胞,并通过流式细胞仪检测表达CD4 +的细胞。
注:Y OU可也检测人类CD8 +和CD4 +细胞在米乌斯脾组织通过我mmunohistochemical技术小号。


染色细胞以通过流式细胞仪进行分析
流式细胞分析我S于收获的细胞进行从在不同条件下培养(步骤小号d和E)。


将要分析的细胞放入12 × 75 mm聚苯乙烯管中。
洗的细胞在3ml 1%BSA-PBS缓冲液中于350 ×g下10分钟,在4 ℃下。
丢弃上清液并迅速涡旋以使沉淀的细胞松动。
甲DD 2种微升抗体(诸如抗CD4),搅拌均匀,并温育30分钟,在4 ℃下在该暗。
洗的细胞3毫升1-%BSA-PBS缓冲液中,在350 ×g下10分钟,在4 ℃下。
丢弃上清液并迅速涡旋以使沉淀的细胞松动。
加入3μl7 -AAD活力染色溶液,并在黑暗中于室温下孵育10分钟。
              重悬的细胞中的适当体积的1%BSA-PBS一个第二通过检测流cytometr ÿ 。


数据分析


d ATA分析被执行使用离线分析软件等为˚F lowjo 。如图2所示,CD4的识别+ T细胞与开始的创建栅极以淋巴细胞隔离弹出ù特征研(FSC-A; SSC-A) ,其次是严格双峰排斥(FSC-W与FSC-H以及SSC-W与SSC-A)。排除死亡染色呈阳性的细胞,然后进入目标细胞群。






图2 。门控策略,以确定CD4 +细胞中抑制试验。A. Ly的米认定phocyte人口在FSC-A与SSC-A地块。公元前。接下来是使用FSC和SSC面积和宽度图进行双重识别。D.用7-AAD染色后,然后分析活细胞。E.活CD4 + T细胞被门控。F.通过CFSE稀释测量CD4 + T细胞的增殖。


的抑制百分比我S理论值如下小号:%抑制= [1 - ( %CD4 +在存在T细胞增殖的CD8 + CD28 - /(T细胞)%CD4 + T细胞增殖在缺乏CD8的+ CD28 - T细胞)] × 100。
统计分析我š进行使用SPSS软件版本22软件。对于不同组的比较,在适当情况下,一个独立样本吨-测试和非参数检验一个重新使用到determin Ë统计学意义。摹raphs一个重新科瑞版使用GraphPad Prism 5.01版软件。


菜谱


1 × PBS(pH 7.4 )
氯化钠          8克               

氯化钾0.2克                                                     

添加ü ltrapureħ 2 O操作一个总体积的1大号


D-PBS (pH 7.4 )
氯化钠8克


氯化钾             0.2克


Na 2 HPO 4         1.15克


KH 2 PO 4          0.2克                                                                       

添加ü ltrapureħ 2 O操作一个总体积的1升


1%BSA-PBS
牛血清白蛋白10克


氯化钠8克


氯化钾             0.2克


Na 2 HPO4 1.44克


KH 2 PO4 0.24克


添加ü ltrapureħ 2 O操作一个总体积的1升


0.5%牛血清白蛋白
EDTA 2毫米


牛血清白蛋白5克


氯化钠8克


氯化钾             0.2克


Na 2 HPO 4         1.44克


KH 2 PO 4          0.24克


添加ü ltrapureħ 2 O操作一个总体积的1升


致谢


这项工作得到了中国国家自然科学基金的资助。81270839,81428007],并在自然科学广东省的基础[2017A030313524。此处概述的协议最初是在Yu实验室的几篇论文中进行描述的(Yu等人,2011; Feng等人,2018; Liu等人,2020)。


比较è Ť ING利益


该作者宣称没有竞争的利益,金融或者非金融。


伦理


所有使用人体细胞的实验均按照南方医科大学伦理审查委员会(中国广州)的建议进行;根据赫尔辛基宣言,所有受试者均签署了知情同意书。所有动物实验均已获得南方医科大学动物护理和使用委员会制定的动物护理和使用指南的批准。


参考


冯锋,刘玉。,刘庚。,朱平。,朱敏。,张虹。,陆新霞,刘洁。,罗新霞,于玉(。)。(F. ,Liu,Y. ,Liu,G. ,Zhu,P. ,Zhu,M. ,Zhang,H. ,Lu,X. ,Liu,J. ,Luo,X. and 2018)。在体外通过IL-15扩增的人CD8 + CD28 - T抑制细胞以同种特异性和程序性细胞死亡蛋白1依赖性方式抑制。免疫前线9:1442。
Liu,G.,Yu,Y.,Feng F.,Zhu,P. and Liu,Y.(2020年)。通过共同的γ链(gammac)细胞因子扩增的人CD8 + CD28 - T抑制细胞在体内保持稳定的同种异体抑制能力。BMC免疫21(1):23。
Tang,XL,Smith,TR和Kumar,V。(2005)。通过调节性CD8 T细胞对免疫的特异性控制。细胞分子免疫学2(1):11-19。
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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.
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