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Chromium-51 (51Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition
铬-51 (51Cr) 释放试验评估T淋巴细胞活性及对肿瘤细胞识别能力   

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本实验方案简略版
Cancer Research
May 2015

Abstract

Cytotoxic CD8+ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of antigen-specific cytotoxic T cells can be assessed functionally in the so-called chromium 51 (51Cr) release assay, which was developed almost 50 years ago in our institution (Brunner et al., 1968). Radioactively-labelled cells deficient for endogenous antigen presentation [e.g., transporter for antigen presentation (TAP)-deficient T2 cells] and stably transfected with the MHC of interest (e.g., HLA-A2+) are typically used as targets during this 4h assay. Alternatively, 51Cr-labelled virus-infected or tumor cell lines presenting immunogenic antigens endogenously can serve as target cells (e.g., for the assessment of tumor recognition).

In a peptide titration assay (section A), radioactively labelled target cells are pulsed with a serial dilution of the antigenic peptide and incubated at an effector (e.g., a CD8+ T cell clone) to target (51Cr -T2 cells) ratio (E:T) of 10:1 in a 96-well V-bottom plate for 4 h at 37 °C. In a tumor killing assay (section B), cytotoxic CD8+ effector cells are incubated at different ratios with the 51Cr-labelled target cell line (typically at E:T ratios of 30:1, 10:1, 3:1 and 1:1) in the presence or absence of the specific antigenic peptide (1 μM) and incubated for 4 h at 37 °C. At the end of the test, the amount of radioactivity release from the lysed target cells is determined in the supernatant using a liquid scintillation counter. The percentage of specific lysis, as well as the EC50 (i.e., 50% of maximal killing) and EMax values are then calculated, providing quantitative information about the antigen-specific functional avidity (i.e., the relative efficiency of T cell function based on antigen recognition via a defined TCR and maximal killing capacity of the analyzed T cells).

Keywords: CD8 T cell (CD8 T细胞), Cytotoxic assay (细胞毒性测定), Tumor killing assay (肿瘤的杀灭试验), Chromium-51 (铬-51), Functional avidity (功能性亲和力)

Materials and Reagents

  1. 96-well plates , V-shape (Corning, Costar®, catalog number: 3894 )
  2. 15 ml Falcon tubes (e.g., Fisher Scientific, catalog number: 14-959-53A )
  3. LumaPlate-96 (PerkinElmer, catalog number: 6006633 )
  4. Bassin or reservoir (e.g., Vitaris, catalog number: 4304-INT or equivalent)
  5. Antigen presenting target cells: e.g., human T2 (CRL-1992), mice P815 (ATCC, catalog number: TIB-64 ) or a characterized tumor cell line [e.g., Me275 (Zippelius et al., 2002)]
  6. Effector cells [(i.e., polyclonal or clonal population of antigen-specific cytotoxic CD8 T cells directly isolated from the blood of patients or healthy donors (Speiser et al., 2008)]
  7. RPMI 1640 (Thermo Fisher Scientific, Gibco®, catalog number: 61870010 )
  8. Fetal bovine serum (FBS) inactivated at 56 °C for 1 h (Thermo Fisher Scientific, Gibco®, catalog number: 10270106 )
  9. Antigenic peptide solution [e.g., NY-ESO-1157-165 peptide (SLLMWITQC)] dissolved in 1x PBS at initial concentration 1 mg/ml
    Note: For the controls, an irrelevant peptide solution at 1 mg/ml that is not recognized by the T cells (i.e., any other class I short peptide).
  10. 51Chromium solution (Na251CrO4) (2 ml vial containing 2 mCi at a concentration of 1 mCi/ml) (PerkinElmer, catalog number: NEZ030S002MC )
  11. 1 M HCl (Sigma-Aldrich, catalog number: 258148 or equivalent )

Equipment

  1. Multichannel pipette, 8 or 12 channels [e.g., Finnpipette F2 10-100 μl, 8 channels (Sigma-Aldrich, catalog number: Z678007 )]
  2. Water bath
  3. Incubator 37 °C, 5% CO2
  4. Standard laboratory desktop centrifuge for 15 ml sample tubes and 96-well plates
  5. Packard TopCount NXT reader (Perkin Elmer, USA)

Software

  1. Prism

Procedure

  1. Cytotoxic T Lymphocyte (CTL) Assay with peptide titration at an E:T ratio of 10:1
    This first section describes the different steps needed to perform a cytotoxic T lymphocyte (CTL) assay. Radioactive labelling of target cells with 51Cr (step A1). Preparation of the peptide dilution (step A2). Preparation of the effector CD8 T cells (step A3). Loading procedure into 96 wells plate (step A4). Determination of 51Cr radioactivity (step A5). Analysis of results (step A6).
    1. Radioactive labelling of T2 target cells with Chromium 51 (51Cr)
      1. Calculate the number of T2 target cells needed for the CTL assay. You will need 1,000 target cells for 1 well of a 96-well plate. Consider adding 20% more cells to account for volume loss during pipetting.
        Note: We usually label 0.5 x 106 T2 cells, which is enough for 500 wells.
      2. Harvest the correct number of T2 target cells in 15 ml Falcon tubes and spin them for 5 min at 530 x g at room temperature (RT).
      3. Wash the cells in 1 ml RT RPMI/10% FBS medium.
      4. Spin down, remove all liquid and incubate T2 cells with 50 μl of pure 51Cr solution (activity 1 mCi/ml) for 45 min at 37 °C in a water bath. Take the necessary precaution and protection when manipulating radioactive material (e.g., lead shields, rapid manipulation). Contact your radiosafety officer for detailed procedures.
      5. At the end of 51Cr labeling time, add 10 ml of RPMI/10%FBS and spin 5 min at 530 x g. Discard the supernatant with the required safety precautions (contact your radiosafety officer for details).
      6. Repeat the washing step with 10 ml of RPMI/10%FBS two more times.
      7. After the third wash, resuspend the cells at a concentration of 0.02 x 106/ml in RT RPMI/10% FBS.
      8. 50 μl (1,000 cells) of freshly labelled 51Cr cell solution is added at the last step (see step A4) in each well of a 96-V-bottom plate.
    2. Preparation of the peptide dilutions
      1. Dissolve the antigenic peptide of interest [here NY-ESO-1157-165 peptide (SLLMWITQC)] in PBS to prepare a stock solution of 1mg/ml (~1 mM) for a 10-AA peptide.
      2. From the stock solution, prepare a 500x dilution in RPMI/10% FBS to get a starting concentration of 2 μM for the following serial dilution steps. This represents a 2x concentrated solution of antigenic peptide, given the 1:2 dilution step occurring during the plating step (step A4).
        Note: To calculate the volume of antigenic peptide solution required for the serial dilutions, consider that each condition is tested in duplicate and that spare volume is needed. 200 μl [100 μl peptide per well x 2 (duplicates)] of peptide solution are required per T cell tested. Be sure to have at least 20% spare volume. Example for 4 CD8 T cells: 4 x [100 μl peptide x 2(duplicates)] = 800 μl required + spare volume → prepare 1 ml of initial 2 μM solution.
      3. Prepare 7 tubes (15 ml Falcon) with RPMI/10% FBS and make serial 10x-dilutions from the 2 μM peptide solution, making sure to thoroughly vortex the tube and to change the pipet tips each time.
        Note: Example for 4 CD8 T cells: 100 μl (2 μM) + 900 μl RPMI/10% FBS → 100 μl (0.2 μM)+ 900 μl RPMI/10% FBS 100 μl (0.02 μM) + 900 μl RPMI/10% FBS and so on.
      4. Pipette 100 μl of the 2x concentrated peptide solution per well of a 96-V-bottom plate, starting from the bottom (lowest concentration) to the top (highest concentration) using a 12-multichannel pipette and a bassin. This will avoid changing the tips each time.
      5. For controls, prepare one solution without peptide and one with an irrelevant peptide (e.g., a class I Flu-specific peptide) at the highest concentration (2 μM) for each T cell clone to be tested.
    3. Preparation of CD8 T cell clones (effector cells)
      1. Calculate the number of cells needed for the test:
        For each T cell clone, you will need 10,000 x 2(duplicates) = 20,000 x 8 dilutions = 160,000 cells and 40,000 cells for the controls w/o peptide and irrelevant peptide, making a total of 0.2 x 106 cells for each T cell clone to be tested.
      2. Accounting for volume loss, prepare 0.25 x 106 T cells and resuspend them in 1.25 ml RT RPMI/10% FBS (0.2 x 106 cells/ml).
      3. Add 50 μl of the prepared effector cells (= 10,000 cells/well) to each well that already contains 100 μl of the serial peptide dilutions.
    4. Steps to load the samples in assay plates (Figure 1)
      1. 1st Step: Peptide dilutions (100 μl) (step A2)
      2. 2nd Step: CD8 T cell clones (10,000 cells in 50 μl) (step A3)
      3. 3rd Step: 51Cr-labelled T2 (1,000 cells in 50 μl) (step A1)
      4. Controls: Spontaneous Release (SR): 4 wells with 150 μl of medium and 50 μl 51Cr- labelled T2. Total Release (TR): 4 wells with 150 μl of 1 M HCl and 50 μl 51Cr-labelled T2.
      5. When the plate is filled, incubate it for 4 h at 37 °C in an incubator with 5% CO2.


      Figure 1. Steps to load the CTL test samples into V-bottom 96-well plates. Step 1: Load 100 μl of the 2x peptide solution with a multichannel pipette, from low concentration (bottom, light blue) to high concentration (top, dark blue). Step 2: Individually add 50 μl of effector CD8 T cell clones in duplicate (here clones A and B), from bottom to top (black dots). You can test up to six clones per 96-well plate. Step 3: Load 50 μl of freshly 51Cr-labelled target cells into every well using a multichannel pipette. The control wells are prepared in the last plate of the experiment. Avoid touching the liquid present in the wells while introducing the different components of the test.

    5. Determination of 51Cr radioactivity
      At the end of the 4 h incubation, spin the plate for 2 min at 234 x g and transfer 50 μl of the supernatant from each well to a LumaPlate-96 using a multi-channel pipette. The amount of radiation emitted by 51Cr present in each supernatant is determined as Counts per Minute (CPM) using a TopCount NXT reader.
    6. Calculation of specific lysis: EC50 and EMax
      To calculate the amount of specific lysis that occurred in each well, use the following formula: 100x [experimental counts per minutes (cpm) - SR (average cpm)/(TR(average cpm) - SR (average cpm)] = % specific lysis.
      The antigen sensitivity and maximum killing capacity of each clone can be quantified as EC50 and EMax, respectively. EC50 is defined by the peptide concentration producing half maximal lysis of target cells and can be derived by fitting a dose response curve analysis using the Prism software for example (Figure 2). EMax is defined as the relative maximal lysis capacity of the effector T cells (Figure 2).


      Figure 2. The relative functional avidity of five NY-ESO-1157-165-reactive CD8 T cell clones expressing distinct αβTCRs assessed by measuring the lysis capacity of T2 target cells pulsed with graded concentrations of the natural NY-ESO-1157-165 peptide (SLLMWITQC). Also shown is an example of EC50 (i.e., the peptide concentration required to achieve 50% of maximal lysis) and EMax [i.e., maximal lysis at high (saturating) antigen dose)] for the clone producing highest maximal lysis (black round). Details can be found in Hebeisen et al., 2015.

  2. Tumor killing (TK) Assay with tumor target cell titration
    The following section describes the different steps needed to perform a tumor killing (TK) assay: Radioactive labelling of tumor target cells with 51Cr (step B1). Preparation of the effector CD8 T cells (step B2). Preparation of the peptide solutions (step B3). Loading procedure into 96-well plate (step B4). Determination of 51Cr radioactivity (step B5). Analysis of results (step B6).
    1. Radioactive labeling of tumor cell line with 51Cr
      1. Calculate the number of target cells (e.g., Melanoma cell line Me275) needed for the TK assay. You will need 1,000 target cells for 1 well of a 96-well plate. Consider adding 20% more cells to account for volume loss during pipetting.
        Note: We usually label 0.5 x 106 target cells, which is enough for 500 wells.
      2. Harvest the correct number of tumor target cells in 15 ml Falcon tubes and spin them for 5 min at 530 x g at room temperature (RT).
      3. Wash the cells in 1 ml RT RPMI/10% FBS medium.
      4. Spin down, remove all liquid and incubate the tumor cells with 50 μl of pure 51Cr solution (activity 1 mCi/ml) for 45 min at 37 °C in a water bath.
      5. After the 51Cr labeling, add 10 ml of RPMI/10% FBS and spin 5 min at 530 x g. Discard the supernatant with the required safety precautions (contact your radiosafety officer for detailed procedures).
      6. Repeat the washing step with 10 ml of RPMI/10%FBS two more times.
      7. After the third wash, resuspend the cells at a concentration of 0.02 x 106/ml in RT RPMI/10% FBS.
      8. 50 μl (1,000 cells) of this cell solution is added at the last step (see step B4) in each well of a 96-V-bottom plate.
    2. Preparation of T cell clones
      1. For the tumor killing assay done in duplicate, 0.1 x 106 T cells are needed per clone. If the test includes conditions with and without peptide pulsing on the target (see step B4), a minimum of 0.2 x 106 T cells are needed.
      2. CD8 T cell clones are counted and resuspended at a concentration of 0.6 x 106/ml in RT RPMI/10% FBS.
      3. From that solution, prepare a serial dilution directly in 96-V-bottom plates as follows:
        Add 75 μl (45,000 cells) in the first well for the first condition [effector (E) to target (T) ratio 30:1] and 50 μl of RPMI/10% FBS in the three wells located underneath. Remember to do it twice for the duplicate. Proceed by transferring 25 μl of the first well in the second, mix well by pipetting up and down, and proceed likewise for the successive dilutions. At the end, all the wells contain 50 ul of medium with the number of cells indicated in brackets.
        Note: A multi-channel pipet can be used to proceed entire 96-well plates rapidly.



    3. Peptide solution
      1. Dilute peptide stock solution (1 mM) in RPMI/10% FBS medium to make a 2x concentrated 2 μM solution. Final concentration per well with peptide will be 1 μM. For example, put 10 μl peptide stock solution (1 mM) in 5 ml RPMI/10% FBS (enough for 500 tests).
      2. Add either 100 μl RPMI/10% FBS (condition without peptide) or 100 μl 2x concentrated peptide solution to the appropriate wells.
        Note: Per 96-well plate, we prepare the “no peptide” condition in the top 4 lines (A to D) and a “+ peptide” condition in the 4 bottom lines (E to H).
    4. Steps to load the samples in assay plates (Figure 3)
      1. 1st Step: CD8 T cell clones serial dilution (50 μl) (step B2)
      2. 2nd Step: Solution with or without peptide (100 μl) (step B3)
      3. 3rd Step: 51Cr-labelled Me275 (1,000 cells in 50 μl) (step B1)
      4. Controls: Spontaneous Release (SR): 4 well with 150 μl of medium and 50 μl 51Cr-labelled T2 .Total Release (TR): 4 well with 150 μl of 1 M HCl and 50 μl 51Cr-labelled T2
      5. When the plate is filled, incubate the test samples for 4 h at 37 °C in an incubator with 5% CO2.


      Figure 3. Steps to load the TK test samples into V-bottom 96-well plates. Step 1: Perform the serial dilution of CD8 T cell clones to get 50 μl of cells in each wells. For each clone (here A and B), reproduce the serial dilutions in the two conditions, with or without peptide. Step 2: Add 100 μl of 2x peptide solution (red) or medium (blue) in the corresponding halves of the plate using a multichannel pipette. Step 3: Load 50 μl of freshly 51Cr-labelled target cells into every well using a multichannel pipette. The control wells are prepared in the last plate of the experiment. Avoid touching the liquid present in the wells while introducing the different components of the test.

    5. Determination of 51Cr radioactivity
      At the end of the 4 h incubation, spin the plate for 2 min at 234 x g and transfer 50 μl of the supernatant from each well to a LumaPlate-96 using a multi-channel pipette. The amount of radiation emitted by 51Cr present in each supernatant is determined as Counts per Minute (CPM) using a TopCount NXT reader.
    6. Calculation of specific lysis
      To calculate the amount of specific lysis that occurred in each well, use the following formula: 100x [(experimental counts per minutes (cpm) - SR (average cpm)/(TR (average cpm) - SR (average cpm)] = % specific lysis.
      The efficacy by which the effector CD8 T cell recognize and lyse tumor cells at the distinct effector to target ratios can be represented as shown in Figure 4 by using the Prism software.


      Figure 4. The relative tumor cell lysis capacity of five NY-ESO-1157-165-reactive CD8 T cell clones expressing distinct αβTCRs assessed using the melanoma cell line Me275 (A2pos/NY-ESO-1157-165pos) at the indicated effector:target ratios. For details, see Hebeisen et al., 2015.

Notes

  1. It is very important to have effector and target cells in very good conditions (Rubio-Godoy et al., 2001). Effector cells should be in a resting phase (around D12-D16 post-restimulation) to avoid high unspecific background killing. Target cells in bad condition (dying or dead) cannot be efficiently labelled with chromium, which leads to results that are difficult to interpret.
  2. Each new unknown target cell should be tested first for its capability to be labelled with chromium (4 h spontaneous and total release only).

Acknowledgments

This work was supported by the Department of Oncology of the University of Lausanne and the Ludwig Center for Cancer Research of the University of Lausanne.
The Chromium Release Assay has been developed by researchers of the Swiss Institute of Experimental Cancer Research (ISREC) and the Department of Biochemistry at the University of Lausanne (Switzerland) and was first published in 1968 (see Reference 1).

References

  1. Brunner, K. T., Mauel, J., Cerottini, J. C. and Chapuis, B. (1968). Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14(2): 181-196.
  2. Hebeisen, M., Schmidt, J., Guillaume, P., Baumgaertner, P., Speiser, D. E., Luescher, I. and Rufer, N. (2015). Identification of rare high-avidity, tumor-reactive CD8+ T cells by monomeric TCR-ligand off-rates measurements on living cells. Cancer Res 75(10): 1983-1991.
  3. Rubio-Godoy, V., Dutoit, V., Rimoldi, D., Lienard, D., Lejeune, F., Speiser, D., Guillaume, P., Cerottini, J. C., Romero, P. and Valmori, D. (2001). Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life. Proc Natl Acad Sci U S A 98(18): 10302-10307.
  4. Speiser, D. E., Baumgaertner, P., Voelter, V., Devevre, E., Barbey, C., Rufer, N. and Romero, P. (2008). Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen. Proc Natl Acad Sci U S A 105(10): 3849-3854.
  5. Zippelius, A., Pittet M. J., Batard P., Rufer N., de Smedt M., Guillaume P., Ellefsen K., Valmori D., Liénard D., Plum J., MacDonald H. R., Speiser D. E., Cerottini J.C. and Romero P. (2002). Thymic selection generates a large T cell pool recognizing a self-peptide in humans. J Exp Med 195(4): 485-94.

简介

细胞毒性CD8 + T细胞能够通过其T细胞受体(TCR)与主要组织相容性复合物(MHC)分子呈递的小免疫原性肽(抗原)之间的特异性相互作用特异性识别和杀死靶细胞。抗原特异性细胞毒性T细胞的抗原识别能力和体外裂解活性可以在所谓的铬51(<51> Cr)释放测定中功能性评估,其是几乎50年前在我们的机构中​​发展起来的(Brunner等人,1968年)。放射性标记的内源性抗原呈递缺陷的细胞[例如,用于抗原呈递(TAP)缺陷型T2细胞的转运蛋白],并用感兴趣的MHC稳定转染(例如 ,HLA-A2 sup + +)通常在这个4小时测定期间用作靶标。或者,内源性呈递免疫原性抗原的Cr标记的病毒感染或肿瘤细胞系可以作为靶细胞(例如,用于评估肿瘤识别)。
  在肽滴定测定(部分A)中,用抗原性肽的系列稀释物对放射性标记的靶细胞进行脉冲,并在效应物(例如,CD8 + T细胞克隆)与靶细胞( Cr-T2细胞)比(E:T)为10:1的混合物在96孔V型底板中37℃温育4小时。在肿瘤杀伤试验(B部分)中,将细胞毒性CD8 +效应细胞以不同的比率与支原体51-标记的靶细胞系(通常以E:T比例在特异性抗原肽(1μM)的存在或不存在的情况下为30:1,10:1,3:1和1:1),并在37℃下孵育4小时。在测试结束时,使用液体闪烁计数器在上清液中测定从裂解的靶细胞释放的放射性的量。然后计算特异性裂解的百分比以及EC 50(即最大杀死的50%)和EMax值,提供关于抗原特异性功能亲合力的定量信息(即,基于通过限定的TCR的抗原识别和分析的T细胞的最大杀伤能力的T细胞功能的相对效率)。

关键字:CD8 T细胞, 细胞毒性测定, 肿瘤的杀灭试验, 铬-51, 功能性亲和力

材料和试剂

  1. 96孔板,V形(Corning,Costar ,目录号:3894)
  2. 15ml Falcon管(例如,Fisher Scientific,目录号:14-959-53A)
  3. LumaPlate-96(PerkinElmer,目录号:6006633)
  4. Bassin或水库(,例如,Vitaris,目录号:4304-INT或等同物)
  5. 抗原呈递靶细胞:例如,人T2(CRL-1992),小鼠P815(ATCC,目录号:TIB-64)或表征的肿瘤细胞系例如 。,Me275(Zippelius等人,2002)]
  6. 效应细胞[即从患者或健康供体的血液中直接分离的抗原特异性细胞毒性CD8 T细胞的多克隆或克隆群体(Speiser等人,2008 )]
  7. RPMI 1640(Thermo Fisher Scientific,Gibco ,目录号:61870010)
  8. 在56℃灭活1小时的胎牛血清(FBS)(Thermo Fisher Scientific,Gibco ,目录号:10270106)
  9. 以1mg/ml的初始浓度溶解于1×PBS中的抗原肽溶液[例如,NY-ESO-1 157-165肽(SLLMWITQC)] 注意:对于对照,1mg/ml的不相关的肽溶液不被T细胞(即任何其它I类短肽)识别。
  10. (2ml小瓶,含有2mCi,浓度为1μl),1ml的溶液(Na 2 Cl 2, mCi/ml)(PerkinElmer,目录号:NEZ030S002MC)
  11. 1M HCl(Sigma-Aldrich,目录号:258148或等价物)

设备

  1. 多通道移液管,8或12个通道[例如,Finnpipette F210-100μl,8通道(Sigma-Aldrich,目录号:Z678007)]
  2. 水浴
  3. 培养箱37℃,5%CO 2
  4. 用于15ml样品管和96孔板的标准实验室台式离心机
  5. Packard TopCount NXT阅读器(Perkin Elmer,USA)

软件

  1. 棱镜

程序

  1. 用E:T比为10:1的肽滴定的细胞毒性T淋巴细胞(CTL)测定 该第一部分描述了进行细胞毒性T淋巴细胞(CTL)测定所需的不同步骤。 使用 51放射性标记靶细胞(步骤A1)。 肽稀释液的制备(步骤A2)。 效应CD8T细胞的制备(步骤A3)。 将装载程序装入96孔板(步骤A4)。 测定 Cr放射性(步骤A5)。 分析结果(步骤A6)。
    1. 用铬51( 51 Cr)放射性标记T2靶细胞,
      1. 计算CTL测定所需的T2靶细胞的数量。对于96孔板的1个孔,需要1,000个靶细胞。考虑增加20%的细胞以解决移液期间的体积损失。
        注意:我们通常标记0.5 x 10 6 T2细胞,这足以容纳500个孔。
      2. 在15ml Falcon管中收获正确数量的T2靶细胞,并在室温(RT)下在530×g下旋转5分钟。
      3. 在1ml RT RPMI/10%FBS培养基中洗涤细胞
      4. 旋转,去除所有液体,并与50μl的纯的51 Cr溶液(活性1mCi/ml)在水浴中37℃下孵育T2细胞45分钟。在操纵放射性材料(例如,导线屏蔽,快速操纵)时,采取必要的预防措施和保护措施。请联系您的无线电安全主管了解详细步骤。
      5. 51 Cr标记时间结束时,加入10ml RPMI/10%FBS并在530×g下旋转5分钟。 按照所需的安全预防措施丢弃上清液(有关详情,请联系您的无线电安全官员)
      6. 用10ml RPMI/10%FBS再重复洗涤步骤两次。
      7. 第三次洗涤后,以0.02×10 6/ml/ml的浓度在RT RPMI/10%FBS中重悬细胞。
      8. 在96-V底板的每个孔中的最后一个步骤(参见步骤A4)加入50μl(1,000个细胞)新鲜标记的51 Cr细胞溶液。
    2. 制备肽稀释液
      1. 将感兴趣的抗原肽[本文中为NY-ESO-1 157-165肽(SLLMWITQC)]溶解在PBS中,以制备10-AA肽的1mg/ml(〜1mM)的储备溶液 。
      2. 从储备溶液中,在RPMI/10%FBS中制备500x稀释液,以获得2μM的起始浓度用于以下连续稀释步骤。这表示在电镀步骤(步骤A4)期间发生1:2稀释步骤时抗原肽的2x浓缩溶液。
        注意:为计算连续稀释所需的抗原肽溶液的体积,请考虑每个条件一式两份进行测试,需要备用体积。每个测试的T细胞需要200μl[每孔100μl肽×2(一式两份)]肽溶液。确保至少有20%的备用卷。 4个CD8 T细胞的实施例:4×[100μl肽×2(重复)] =800μl所需的+备用体积→制备1ml的初始2μM溶液。
      3. 用RPMI/10%FBS制备7个管(15ml Falcon),并从2μM肽溶液制备系列10倍稀释液,确保每次彻底涡旋管并更换移液管吸头。
        注意:4个CD8 T细胞的实施例:100μl(2μM)+900μlRPMI/10%FBS→100μl(0.2μM)+900μlRPMI/10%FBS 100 μl(0.02μM)+900μlRPMI/10%FBS等。
      4. 使用12-多通道移液管和bassin从底部(最低浓度)开始至顶部(最高浓度),从96-V-底板的每孔中移取100μl2×浓缩肽溶液。这将避免每次更改提示。
      5. 对于对照,对于每个T细胞,以最高浓度(2μM)制备一种不含肽的溶液和一种具有不相关肽(例如I类Flu特异性肽)的溶液 克隆进行测试。
    3. CD8 T细胞克隆(效应细胞)的制备
      1. 计算测试所需的单元格数:
        对于每个T细胞克隆,对于不含肽和无关肽的对照,将需要10,000×2(重复)= 20,000×8稀释= 160,000个细胞和40,000个细胞,使得总共为0.2×10 6个 sup>细胞用于每个待测试的T细胞克隆
      2. 考虑体积损失,制备0.25×10 6个T细胞并将其重悬于1.25ml RT RPMI/10%FBS(0.2×10 6个细胞/ml)中。
      3. 向已经含有100μl系列肽稀释液的每个孔中加入50μl制备的效应细胞(= 10,000个细胞/孔)。
    4. 将样品装载在测定板中的步骤(图1)
      1. 1步骤:肽稀释液(100μl)(步骤A2)
      2. 2 <步骤:步骤:CD8T细胞克隆(10,000个细胞,50μl)(步骤A3)
      3. 3 步骤: 51 Cr标记的T2(1,000细胞,50μl)(步骤A1)
      4. 对照:自发释放(SR):具有150μl培养基和50μl51 Cr-标记的T2的4个孔。 总释放(TR):4个孔,150μl的1M HCl和50μl的 51 Cr标记的T2。
      5. 当板填充时,在37℃下在具有5%CO 2的培养箱中孵育其4小时。


      图1.将CTL测试样品装载到V-底96孔板中的步骤。步骤1:使用多通道移液管加载100μl的2x肽溶液,从低浓度(底部,浅蓝色)至高浓度(顶部,深蓝色)。步骤2:从底部到顶部单独添加50μl效应CD8 T细胞克隆(这里是克隆A和B)(黑点)。您可以每96孔板测试多达6个克隆。步骤3:使用多通道移液器将50μl新鲜的 51 Cr标记的靶细胞加载到每个孔中。在实验的最后一个板中制备对照孔。避免接触井中存在的液体,同时引入测试的不同组分
    5. 51 Cr放射性的测定
      在4小时孵育结束时,以234×g离心板2分钟,并使用多通道移液管将50μl来自每个孔的上清液转移至LumaPlate-96。使用TopCount NXT读数器将存在于每种上清液中的由 Cr发射的辐射量确定为每分钟计数(CPM)。
    6. 计算特异性裂解:EC50和EMax
      为了计算在每个孔中发生的特异性裂解的量,使用下式:100x [每分钟的实验计数(cpm)-SR(平均cpm)/(TR(平均cpm)-SR(平均cpm)裂解。
      每个克隆的抗原敏感性和最大杀伤能力可以分别定量为EC 50和EMax。 EC 50由产生靶细胞的半数最大裂解的肽浓度定义,并且可以通过使用例如Prism软件拟合剂量反应曲线分析来得到(图2)。 EMax定义为效应T细胞的相对最大裂解能力(图2)

      图2.通过测量用梯度浓度脉冲的T2靶细胞的裂解能力评估表达不同αβTCR的5种NY-ESO-1 157-165-反应性CD8 T细胞克隆的相对功能亲合力的 天然NY-ESO-1 157-165肽(SLLMWITQC)。还显示了EC50的实例(即对于产生最高最大裂解(黑色圆形)的克隆,最大裂解(最大裂解的50%)和最大裂解(即,高(饱和)抗原剂量的最大裂解)。细节可以在Hebeisen 等人。,2015年找到。

  2. 肿瘤杀伤(TK)用肿瘤靶细胞滴定测试
    以下部分描述了进行肿瘤杀伤(TK)测定所需的不同步骤:用 51 Cr放射性标记肿瘤靶细胞(步骤B1)。效应CD8 T细胞的制备(步骤B2)。肽溶液的制备(步骤B3)。将装载程序装入96孔板(步骤B4)。测定 Cr放射性(步骤B5)。分析结果(步骤B6)。
    1. 51 Cr放射性标记肿瘤细胞系
      1. 计算TK测定所需的靶细胞(例如,黑素瘤细胞系Me275)的数目。对于96孔板的1个孔,需要1,000个靶细胞。考虑增加20%的细胞以解决移液期间的体积损失。
        注意:我们通常会标记0.5 x 10 6 目标单元格,足以容纳500个井。 >
      2. 在15ml Falcon管中收获正确数目的肿瘤靶细胞,并在室温(RT)下以530×g离心5分钟。
      3. 在1ml RT RPMI/10%FBS培养基中洗涤细胞。
      4. 旋转,除去所有液体,并在50℃的水浴中用50μl的纯的51 Cr溶液(活性为1mCi/ml)孵育肿瘤细胞45分钟。
      5. 51 Cr标记后,加入10ml的RPMI/10%FBS并在530×g下旋转5分钟。按照所需的安全预防措施丢弃上清液(请联系您的无线电安全官员了解详细步骤)
      6. 用10ml RPMI/10%FBS再重复洗涤步骤两次。
      7. 在第三次洗涤后,以0.02×10 6个/ml的浓度在RT中重悬细胞 RPMI/10%FBS
      8. 在96-V底板的每个孔中的最后一步(见步骤B4)加入50μl(1,000细胞)的该细胞溶液。
    2. T细胞克隆的制备
      1. 对于一式两份进行的肿瘤杀伤测定,每个克隆需要0.1×10 6个T细胞。 如果测试包括在目标上具有和不具有肽脉冲的条件(参见步骤B4),则需要至少0.2×10 6个 T细胞。
      2. 计数CD8 T细胞克隆,并以0.6×10 6个/ml的浓度在RT RPMI/10%FBS中重悬。
      3. 从该溶液中,如下直接在96-V底板中制备连续稀释液:
        在第一个条件[效应器(E)到目标(T)比例30:1]的第一个孔中加入75μl(45,000个细胞)和50μl的RPMI/10%FBS,位于底部的三个孔中。记住对重复项做两次。继续转移25微升的第一个井在第二个,通过吸移上下混合井,并同样进行连续稀释。最后,所有孔含有50μl含有括号中所示细胞数的培养基。
        注意:多通道移液器可用于快速处理整个96孔板。



    3. 肽溶液
      1. 在RPMI/10%FBS培养基中稀释肽储备溶液(1mM)以制备2x浓缩的2μM溶液。使用肽的每孔终浓度为1μM。例如,将10μl肽储备溶液(1 mM)放入5 ml RPMI/10%FBS(足够500次测试)。
      2. 在适当的孔中加入100μlRPMI/10%FBS(无肽条件)或100μl2倍浓缩肽溶液。
        注意:每96孔板,我们在顶部4行(A至D)中制备"无肽"条件,在4条底部行(E至H)中制备"+肽"条件。
    4. 在测定板中装载样品的步骤(图3)
      1. 1 步骤:CD8 T细胞克隆系列稀释(50μl)(步骤B2)
      2. 2 <步骤:步骤:具有或不具有肽(100μl)的溶液(步骤B3)
      3. 3步骤:将51 Cr标记的Me275(1,000细胞,在50μl中)(步骤B1)
      4. 对照:自发释放(SR):用150μl培养基和50μl51 Cr标记的T2充满4孔。总释放(TR):用150μl1M HCl和50μl sup> 51 Cr标记的T2
      5. 当板被填充时,在37℃下在具有5%CO 2的培养箱中孵育测试样品4小时。


      图3.将TK测试样品加载到V-底96孔板中的步骤。步骤1:进行CD8 T细胞克隆的连续稀释,以在每个孔中获得50μl细胞。对于每个克隆(此处为A和B),在有或无肽的两种条件下再生系列稀释液。步骤2:使用多通道移液器,在板的相应的一半中加入100μl的2x肽溶液(红色)或培养基(蓝色)。步骤3:使用多通道移液器将50μl新鲜的 51 Cr标记的靶细胞加载到每个孔中。在实验的最后一个板中制备对照孔。避免接触井中存在的液体,同时引入测试的不同组分
    5. 51 Cr放射性的测定
      在4小时孵育结束时,以234×g离心板2分钟,并使用多通道移液管将50μl来自每个孔的上清液转移至LumaPlate-96。使用TopCount NXT读数器,将存在于每种上清液中的由 Cr发射的辐射量确定为每分钟计数(CPM)。
    6. 计算特异性裂解
      为了计算在每个孔中发生的特异性裂解的量,使用下式:100x [(每分钟的实验计数(cpm)-SR(平均cpm)/(TR(平均cpm)-SR(平均cpm)特异性裂解 效应CD8 T细胞识别和裂解肿瘤细胞在不同的效应物靶目标比率的功效可以表示如图4所示使用Prism软件。


      图4.使用黑色素瘤细胞系Me275(A2 sup-1)评估表达不同αβTCR的5种NY-ESO-1 157-165-反应性CD8 T细胞克隆的相对肿瘤细胞裂解能力。 pos /NY-ESO-1 157-165 pos )。详情请参阅Hebeisen et al 。,2015.

笔记

  1. 使效应细胞和靶细胞处于非常好的条件是非常重要的(Rubio-Godoy等人,2001)。效应细胞应该处于休眠期(在再次刺激后的D12-D16周围),以避免高度非特异性背景杀伤。靶细胞处于不良状态(死亡或死亡)不能用铬有效标记,这导致难以解释的结果。
  2. 每个新的未知靶细胞应首先测试其用铬标记的能力(仅4小时自发和完全释放)。

致谢

这项工作得到了洛桑大学肿瘤学系和洛桑大学癌症研究中心的支持。
铬释放测定由瑞士实验癌症研究所(ISREC)和洛桑大学生物化学系(瑞士)的研究人员开发,并于1968年首次出版(见参考文献1)。

参考文献

  1. Brunner,KT,Mauel,J.,Cerottini,JC and Chapuis,B.(1968)。  免疫淋巴细胞对51-Cr标记的同种异体靶细胞的体外溶解作用的定量测定等同抗体和药物的抑制。 Immunology 14(2):181-196。
  2. Hebeisen,M.,Schmidt,J.,Guillaume,P.,Baumgaertner,P.,Speiser,DE,Luescher,I. and Rufer,N.(2015)。  通过单体TCR-标记的罕见高亲和力,肿瘤反应性CD8 + T细胞的鉴定,对活细胞的配体解离速率测量。 Cancer Res 75(10):1983-1991。
  3. Rubio-Godoy,V.,Dutoit,V.,Rimoldi,D.,Lienard,D.,Lejeune,F.,Speiser,D.,Guillaume,P.,Cerottini,JC,Romero,P.and Valmori, (2001)。  ELISPOT干扰素-γ分泌与A2 /肽多聚体与TCR的结合揭示了CTL效应子功能与TCR-肽/MHC复合物半衰期的克隆间解离。 Proc Natl Acad Sci USA 98(18):10302-10307 。
  4. Speiser,DE,Baumgaertner,P.,Voelter,V.,Devevre,E.,Barbey,C.,Rufer,N。和Romero,P.(2008)。  未修饰的自身抗原触发具有比改变的抗原更强的肿瘤反应性的人类CD8 T细胞。 Acad Sci USA 105(10):3849-3854。
  5. Zippelius,A.,Pittet MJ,Batard P.,Rufer N.,de Smedt M.,Guillaume P.,Ellefsen K.,Valmori D.,LiénardD.,Plum J.,MacDonald HR,Speiser DE,Cerottini JC and Romero P.(2002)。  胸腺选择产生大 T cell pool recognition a self-peptide in humans.Vermat J Exp Med 199(4):485-94。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Baumgaertner, P., Speiser, D. E., Romero, P., Rufer, N. and Hebeisen, M. (2016). Chromium-51 (51Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition. Bio-protocol 6(16): e1906. DOI: 10.21769/BioProtoc.1906.
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