搜索

Efficient Isolation of Influenza Specific CTLs
流感特异性的杀伤性T细胞的高效分离   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Human antigen-specific CD8+ T-cell clones are valuable tools for dissecting CD8+ T-cell responses against antigens derived from infectious agents, cancer and self antigens. Here we describe a protocol for isolating human antigen-specific CD8+ T cells. This protocol uses surface capture of IFNγ to identify antigen responsive cells that are then cloned by single-cell sorting. Here we use CD8+ T-cell responses to influenza matrix protein (MP) as an example, but this approach can be applied to any antigen specificity.

Keywords: Cloning(克隆), CD8(CD8), CTL(CTL), Human(人类), T cell(T细胞)

Materials and Reagents

  1. 50 ml Falcon tubes
  2. Greiner CELLSTAR®, 96-well round-bottom tissue culture plates (Sigma-Aldrich, catalog number: M9311 )
  3. Corning® Costar® 24 and 48 well tissue culture plates (Sigma-Aldrich, catalog number: CLS3524 and CLS3548 )
  4. 10 ml tube
  5. Vials
  6. Peripheral blood
  7. EBV-transformed B-cell lines (HLA-A2)
  8. Ficoll (GE Healthcare, catalog number: 17-1440-03 )
  9. Phosphate buffered saline (DPBS) (Lonza, catalog number: 17-512F )
  10. Trypan blue solution (0.4%) (Sigma-Aldrich, Catalogue number: T8154 )
  11. RPMI 1640 (Sigma-Aldrich, catalog number: R8758 )
  12. Synthetic peptide(s) (Purar Chemicals)
  13. Influenza matrix peptide 58-66 (MP58-66; GILGFVFTL) (GL-Biochem)
  14. 5% pooled human serum
  15. Cytokines IL-2 (Peprotech, catalog number: AF-200-02 ), IL-15 (Peprotech, catalog number: AF-200-15 )
  16. Human IFNγELISA Kit (BioLegend, catalogue number: 430106 )
  17. Miltenyi IFN-γ secretion assay kit-PE (Miltenyi Biotec, catalog number: 130-054-202 )
  18. CD8-FITC (SK1) mAb (clone SK1) (Thermo Fisher Scientific, eBiosciencesTM, catalog number: 11-0087-42 )
  19. Fungizone (Thermo Fisher Scientific, catalog number: 15290-018 )
  20. Propidium iodide (Sigma-Aldrich, catalog number: 81845 )
  21. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
  22. Ionomycin (Sigma-Aldrich, catalog number: I0634 )
  23. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  24. HEPES (1 M) (Life technologies, GibcoTM, catalog number: 15630-080 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080”.
  25. Non-essential amino acids (NEAA) (Life Technologies, GibcoTM, catalog number: 11140-050 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050”.
  26. GlutaMAXTM (Life Technologies, GibcoTM, catalog number: 35050-061 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061”.
  27. UltraPureTM 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575-020 )
  28. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P0781 )
  29. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  30. Stock concentration of cytokines and peptides (see Recipes)
  31. Cell culture media (see Recipes)
  32. Staining buffer (see Recipes)
  33. Stock concentration of propidium iodide (PI) (see Recipes)
  34. Stock concentration of PMA and ionomycin (see Recipes)
  35. Stock concentration of fungizone (see Recipes)

Equipment

  1. Megafuge 40R centrifuge (ThermoFisher, catalog number: 75004503 )
  2. Neubauer chamber
  3. Freezing container, Nalgene® Mr. Frosty (Sigma-Aldrich, C1562 )
  4. Cell culture CO2 incubator (Panasonic Healthcare Co., model: MCO-20AIC ) with 5% CO2
  5. Flow cytometer (BD Biosciences, model: BD FACSAria III )
  6. Liquid nitrogen dewar

Procedure

  1. Collect approximately 25 ml of blood from HLA*A02:01 positive donor. Prepare peripheral blood mononuclear cells (PBMC) by centrifuging over 10 ml of Ficoll (20 °C, 805 x g, no brake). Collect PBMC from above the Ficoll and wash twice in DPBS. Count live PBMC using trypan blue exclusion on a Neubauer chamber. Dilute the PBMC to 2 x 106 cells/ml in RPMI 1640 supplemented with 5% pooled human serum. A total of 6.0 ml of cell suspension (12 x 106 cells) is used for peptide stimulation. PBMC were cultured (at 37 °C, 5% CO2) with 1 μM influenza matrix peptide 58-66 (MP58-66; GILGFVFTL) for 3 days in RPMI 1640 supplemented with 5% pooled human serum [The Australian Red Cross Blood Service (ARBCS), Melbourne, Australia]. Freeze remaining PBMC at 5 x 106 cells/vial and store in liquid nitrogen for later to use (step 3) as antigen presenting cells (APCs) in re-stimulation and screening step.
    Notes:
    1. Choose donors who have the appropriate HLA class I allele(s) for the peptide antigen.
    2. Other sera, such as foetal bovine serum, can be used. We prefer to use human serum because this results in a lower background response in the absence of peptide.
  2. After three days add IL-2 (final concentration of 20 U/ml) and IL-15 (final concentration of 5 ng/ml) to the cells. The culture is continued for a further 7 days making the total culture time 10 days.
  3. IFNγ-secreting CD8+ cells are detected after overnight restimulation with peptide (1 μM final concentration) and thawed, autologous, irradiated (60 Gy) PBMC (from step 1 above), by surface-capture staining. IFNγ secretion and capture was performed according to Miltenyi’s instructions. Autologous, irradiated PBMC are used at a 1:1 ratio with the PBMC culture with peptide and cytokines. After 1 h of culture at 37 °C/5% CO2 2 ml of ice cold staining buffer is added to each tube and cells are washed once at 515 x g for 5 min, at room temperature. Washed cells are resuspended with 90 μl of staining buffer and 10 μl of IFNγ capture reagent and incubated on ice for 5 min. Cells are then transferred to 50 ml Falcon tubes and warm (37 °C) culture media is added to dilute the cells to 1 x 105 cells/ml. The diluted cells are then incubated at 37 °C/5% CO2 for 60 min with occasional mixing. In parallel, a small sample of cells (~0.5 x 106) is ‘restimulated’ in the absence of peptide. After 1 h captured IFNγ was detected using a PE-labeled detection mAb. For IFNγ detection, the cells are diluted in ice-cold staining buffer and centrifuged at 515 x g for 5 min, at 4 °C. Cells are resuspended in 90 μl staining buffer for up to 5 x 106 cells. For staining, 10 μl of IFNγ detection antibody and 10 μl of CD8-FITC is added to stain for CD8. Tubes were incubated on ice for 10 min then the cells are washed once with ice-cold staining buffer and transferred to FACS tubes for sorting.
    Note: A small number of cells are restimulated in the absence of peptide to determine how many CD8+ IFNγ+ cells are present without restimulation. We expect to see >5x increase in the number of IFNγ+ cells after restimulation with peptide.
  4. Flow cytometry. Compensation settings were determined using single-colour stained samples. Propidium iodide (final concentration of 1 μg/ml) was added to exclude dead cells. Negative control sample (without peptide) and positive control sample (PMA/ionomycin-stimulated) can be used to set sorting gates. Doublets are excluded using a FSC-w (forward scatter width) vs FSC-h (forward scatter height) gate (Figure 1). A single CD8+, IFNγ+ cells is sorted into the 60 inner wells of a 96-well plate containing feeder cells [irradiated (60 Gy)] PBMC, anti-CD3 (OKT3, 30 ng/ml) and IL-2 (20 U/ml) and IL-15 (5 ng/ml) and fungizone, as described in Mannering et al. (2003).
    Notes:
    1. We prefer not to use the outer wells of the 96-well plate because these often become infected with fungus.
    2. Fungizone (final concentration of 2.5 μg/ml) is routinely included in the cultures. This markedly reduces the rate of fungal contamination in the plates.
  5. Plates containing the sorted IFNγ+, CD8+ cells, irradiated feeder cells, cytokines, anti-CD3 and fungizone are cultured (37 °C/5% CO2). After a week 50 μl of culture media containing 60 U/ml of IL-2 and 15 ng/ml of IL-15 is added to each well of the 96-well, round-bottom plate. This gives a final concentration of 20 U/ml of IL-2 and 5 ng/ml of IL-15.
    Each well is ‘fed’ again, with IL-2 and IL-15 in 50 μl of media, after another week of culture. This gives a final volume of 200 μl in each well.
    By ~14 days after sorting growing clones should start to become be visible in some of the wells.
    Notes:
    1. By this time the irradiated feeder cells should be dead.
    2. Growing clones are best identified using an inverted microscope.
    3. It is most convenient to use a felt-tipped pen to put a circle on the lid of the plate over the well(s) with growing clones.
  6. Transfer the growing clones to a 48-well plate. Add another 200 μl of culture media containing IL-2 (40 U/ml) and IL-15 (10 ng/ml) to give a final concentration of 20 U/ml of IL-2 and 5 ng/ml of IL-15 in a final volume of 400 μl.
  7. The clones are tested for antigen specificity by ELISA assay.
    Antigen specificity testing is done in duplicate-two wells with HLA-A2+ APC and peptide and two wells with APC, but no peptide. Each of the clones in the 48-well plate is resuspended in ~400 μl. One to two hundred microliters of this cell suspension is transferred to a 10 ml tube, washed in 10 ml of DPBS and then with 1.0 ml of culture media. For an ELISA assay, the washed clones are resuspended in 400 μl of culture media and dispense 100 μl into four wells in 96-well round-bottom plate containing APC (50,000 EBV transformed B cells/well) with (two wells) and without (two wells) peptide (1.0 μM). After overnight culture collect 100 μl of the supernatant and assay for IFNγ by ELISA (http://www.biolegend.com/human-ifn-gamma-elisa-max-standard-2226.html).
    Notes:
    1. We routinely use EBV-transformed B-cell lines that express the required HLA molecule, HLA-A2 in this case. Alternative APC, such as irradiated PBMC (60 Gy) or transfected cell lines can also be used.
    2. Other assay, such as 51Cr release, can also be used to screen clones. We prefer to use IFNγ secretion as the initial screen because all the responding cells should secrete this cytokine because they were selected based on IFNγ secretion.
    3. Clones that responded specifically to MP58-66 were grown to large numbers by restimulation with anti-CD3 (OKT3) and feeding with IL-2 and IL-4 as described in Ciantar and Mannering (2011).
  8. Following expansion the clones were re-tested for antigen specificity and stored in liquid nitrogen until required. To freeze the clones they centrifuged, (515 x g, 5 min, room temperature). After pouring off the supernantant the cells are resuspended in FCS (usually to 10 x 106 cells/ml). An equal volume of FCS containing 20% DMSO is slowly added while mixing gently. Aliquots of 1.0 ml (~5 x 106 cells/vial) are dispensed in vials suitable for freezing in liquid nitrogen. The vials are placed in a freezing container containing propanol and stored in a -80 °C freezer overnight. The next day the vials are transferred to a liquid nitrogen dewar and stored until required.
    Note: We have found that killing function of CD8+ T cells can improve if they are cultured in IL-2 (20 U/ml) overnight after being thawed.

Representative data



Figure 1. Cloning MP58-66 specific, IFNγ+ CD8+ T cells. The gating strategy for MP58-66 specific, IFNγ+ CD8+. A. Gating on lymphocytes based on forward (FSC) and side scatter (SSC); B. Exclusion of dead cells by propidium iodide staining, viable cells are PI negative; C. Exclusion of doublets by FSC-Height (FSC-H) and FSC-width (FSC-W) gating strategy; D. IFNγ+ CD8+ T cells when PBMC are cultured without peptide; E. The same gate with PBMC cultured with MP58-66 and re-stimulated with the same peptide.


Figure 2. Screening of antigen specificity for growing CD8+ T-cell clones. HLA-A2+ EBV cells were pulsed with either MP58-66, or without peptide. A sample of each growing clone was washed and incubated with these APCs overnight. The next day the concentration of IFNγ in the supernatant are determined by ELISA. Data represents mean ± standard deviation of duplicate cultures for each clone. The clones that secreted IFNγ in response to MP58-66 were selected for further expansion.


Figure 3. Re-screening of antigen specificity after expansion of CD8+ T-cell clones. Clones were incubated with HLA-A2+ EBV cells pulsed with, or without, MP58-66. After overnight incubation the IFNγ in the supernatant was measured by ELISA. The data represent the means ± standard deviation of triplicate samples for each clone.

Recipes

  1. Stock concentration of cytokines and peptides
    Prepared according to manufacturer’s instructions
  2. Staining buffer
    0.5% BSA and 2 mM EDTA in DPBS
  3. Cell culture media-RPMI with 5% pooled human serum (Table 1)

    Table 1. Preparation of T-cell culture medium


  4. Stock concentration of propidium iodide (1 mg/ml)
    Prepared by dissolving propidium iodide in sterile filtered water
  5. Stock concentration of PMA (0.1 mg/ml) and ionomycin (0.5 mg/ml)
    Prepared by dissolving in DMSO
  6. Stock concentration of fungizone (50 μg/ml)
    Prepared by dissolving fungizone into sterile filtered water

Acknowledgements

We thank Eleanora Tresoldi for technical assistance and blood donors for providing blood samples for research. SIM is supported by The Australian National Health and Medical Research Council (NHMRC, APP1061961, APP1087341) and a JDRF Career Development Award 5-CDA2014210-A-N. St Vincent’s Institute of Medical Research receives support from the Operational Infrastructure Support Scheme of the Government of Victoria. The authors have no conflict of interest to declare.

References

  1. Ciantar, J. P. and Mannering, S. I. (2011). An improved method for growing and analysing human antigen-specific CD4+ T-cell clones. Diabetes Metab Res Rev 27(8): 906-912.
  2. Mannering, S. I., Morris, J. S., Jensen, K. P., Purcell, A. W., Honeyman, M. C., van Endert, P. M. and Harrison, L. C. (2003). A sensitive method for detecting proliferation of rare autoantigen-specific human T cells. J Immunol Methods 283(1-2): 173-183.

简介

人抗原特异性CD8 + T细胞克隆是用于解剖来自感染剂,癌症和自身抗原的抗原的CD8 + T细胞应答的有价值的工具。 在这里我们描述了用于分离人抗原特异性CD8 + T细胞的方案。 该协议使用IFNγ的表面捕获来鉴定抗原反应性细胞,然后通过单细胞分选克隆。 这里我们使用CD8 + T细胞对流感基质蛋白(MP)的反应作为例子,但这种方法可以应用于任何抗原特异性。

关键字:克隆, CD8, CTL, 人类, T细胞

材料和试剂

  1. 50ml Falcon管
  2. Greiner CELLSTAR ,96孔圆底组织培养板(Sigma-Aldrich,目录号:M9311)
  3. 24孔和48孔组织培养板(Sigma-Aldrich,目录号:CLS3524和CLS3548)的孔中。
  4. 10ml管
  5. 小瓶
  6. 外周血
  7. EBV转化的B细胞系(HLA-A2)
  8. Ficoll(GE Healthcare,目录号:17-1440-03)
  9. 磷酸盐缓冲盐水(DPBS)(Lonza,目录号:17-512F)
  10. 台盼蓝溶液(0.4%)(Sigma-Aldrich,目录号:T8154)
  11. RPMI 1640(Sigma-Aldrich,目录号:R8758)
  12. 合成肽(Purar Chemicals)
  13. 流感基质肽58-66(MP 58-66; GILGFVFTL)(GL-Biochem)
  14. 5%汇集的人血清
  15. 细胞因子IL-2(Peprotech,目录号:AF-200-02),IL-15(Peprotech,目录号:AF-200-15)
  16. 人IFNγELISA试剂盒(BioLegend,目录号:430106)
  17. Miltenyi IFN-γ分泌测定试剂盒-PE(Miltenyi Biotec,目录号:130-054-202)
  18. CD8-FITC(SK1)mAb(克隆SK1)(Thermo Fisher Scientific,eBiosciences TM ,目录号:11-0087-42)
  19. Fungizone(Thermo Fisher Scientific,目录号:15290-018)
  20. 碘化丙啶(Sigma-Aldrich,目录号:81845)
  21. 佛波醇12-豆蔻酸酯13-乙酸酯(PMA)(Sigma-Aldrich,目录号:P8139)
  22. 离子霉素(Sigma-Aldrich,目录号:I0634)
  23. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  24. HEPES(1M)(Life technologies,Gibco TM ,目录号:15630-080)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15630-080"
  25. 非必需氨基酸(NEAA)(Life Technologies,Gibco TM ,目录号:11140-050)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:11140-050"
  26. GlutaMAX TM (Life Technologies,Gibco TM ,目录号:35050-061)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:35050-061"
  27. UltraPure TM 0.5M EDTA,pH 8.0(Thermo Fisher Scientific,Invitrogen TM,目录号:15575-020)
  28. 青霉素 - 链霉素(Sigma-Aldrich,目录号:P0781)
  29. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  30. 细胞因子和肽的储备浓度(参见配方)
  31. 细胞培养基(参见配方)
  32. 染色缓冲液(见配方)
  33. 碘化丙啶(PI)的储备浓度(见配方)
  34. PMA和离子霉素的储备浓度(参见配方)
  35. 除草剂的浓度(见配方)

设备

  1. Megafuge 40R离心机(ThermoFisher,目录号:75004503)
  2. Neubauer
  3. 冷冻容器,Nalgene ® Mr. Frosty先生(Sigma-Aldrich,C1562)
  4. 细胞培养CO 2培养箱(Panasonic Healthcare Co.,型号:MCO-20AIC),具有5%CO 2
  5. 流式细胞仪(BD Biosciences,型号:BD FACSAria III)
  6. 液氮杜瓦

程序

  1. 从HLA * A02:01阳性供体收集约25ml的血液。通过在10ml Ficoll(20℃,805×g,无制动)上离心制备外周血单核细胞(PBMC)。从上面的Ficoll收集PBMC和在DPBS中洗涤两次。使用台盼蓝排除在Neubauer腔上计数活PBMC。在补充有5%汇集的人血清的RPMI 1640中将PBMC稀释至2×10 6个细胞/ml。将总共​​6.0ml的细胞悬浮液(12×10 6个细胞)用于肽刺激。用1μM流感基质肽58-66(MP 58-66 GILGFVFTL)在RPMI中培养PBMC(在37℃,5%CO 2下)3天1640,补充有5%汇集的人血清[The Australian Red Cross Blood Service(ARBCS),Melbourne,Australia]。以5×10 6个细胞/瓶冷冻剩余的PBMC,并储存在液氮中,以供稍后在再刺激和筛选步骤中作为抗原呈递细胞(APC)使用(步骤3)。
    注意:
    1. 选择具有针对肽抗原的合适HLA I类等位基因的供体。
    2. 可以使用其他血清,例如胎牛血清。我们优选使用人血清,因为这导致在没有肽的情况下较低的背景响应
  2. 三天后,向细胞中加入IL-2(终浓度为20U/ml)和IL-15(终浓度为5ng/ml)。继续培养另外7天,使总培养时间为10天。
  3. 通过表面捕获染色,在用肽(1μM终浓度)和解冻的,自体的,辐射的(60Gy)PBMC(来自上述步骤1)的过夜再刺激后检测分泌IFNγ的CD8 +细胞。 IFNγ分泌和捕获根据 Miltenyi的说明。自体的,辐射的PBMC与PBMC培养物与肽和细胞因子以1:1的比例使用。在37℃/5%CO 2下培养1小时后,向每个管中加入2ml冰冷染色缓冲液,将细胞在515×g下洗涤5次,每次5分钟min。将洗涤的细胞用90μl染色缓冲液和10μlIFNγ捕获试剂重悬,并在冰上孵育5分钟。然后将细胞转移至50ml Falcon管中,加入温热(37℃)培养基以将细胞稀释至1×10 5个细胞/ml。然后将稀释的细胞在37℃/5%CO 2下孵育60分钟,偶尔混合。平行地,在不存在肽的情况下,细胞的小样本(〜0.5×10 6个)被"再刺激"。捕获1小时后,使用PE标记的检测mAb检测IFNγ。对于IFNγ检测,将细胞在冰冷的染色缓冲液中稀释,并在4℃下以515×g离心5分钟。将细胞重悬于90μl染色缓冲液中达5×10 6个细胞。为了染色,加入10μlIFNγ检测抗体和10μlCD8-FITC以染色CD8。将管在冰上孵育10分钟,然后将细胞用冰冷的染色缓冲液洗涤一次,并转移至FACS管进行分选。
    sup> + 单元格存在,没有重新刺激。在用肽再刺激后,我们预期IFNγ + 细胞数量增加> 5倍。
  4. 流式细胞术。使用单色染色样品测定补偿设置。加入碘化丙啶(终浓度为1μg/ml)以排除死细胞。阴性对照样品(无肽)和阳性对照样品(PMA /离子霉素刺激)可用于设置分选门。使用FSC-w(前向散射宽度)vs FSC-h(前向散射高度)门(图1)排除双峰。将单个CD8 +细胞,IFNγ+细胞分选到含有饲养细胞的96孔板的60个内孔中(照射(60Gy)] PBMC,抗CD3 (Mannering等人,(2003))中所描述的(OKT3,30ng/ml)和IL-2(20U/ml)和IL-15(5ng/ml) br /> 注意:
    1. 我们不喜欢使用96孔板的外孔,因为这些孔常常被真菌感染。
    2. 常规地在培养物中包含菌丝体(终浓度2.5μg/ml)。这显着降低了板中的真菌污染率。
  5. 培养含有分选的IFNγ+,CD8 +细胞,辐照的饲养细胞,细胞因子,抗CD3和真菌的平板(37℃/5%CO 2 )。一星期后,将含有60U/ml IL-2和15ng/ml IL-15的50μl培养基加入96孔圆底板的每个孔中。这使得终浓度为20U/ml的IL-2和5ng/ml的IL-15 在培养另一周后,在50μl培养基中用IL-2和IL-15再次"喂养"每个孔。这使每个孔的最终体积为200μl。
    在分拣后〜14天,生长的克隆应该开始变得在一些孔中可见 注意:
    1. 此时被辐照的饲养细胞应该死了。
    2. 最好使用倒置显微镜识别生长克隆。
    3. 最方便的是使用毡尖笔在生长克隆的孔上方的板的盖子上放置一个圆圈。
  6. 将生长的克隆转移到48孔板。加入另外200μl含有IL-2(40U/ml)和IL-15(10ng/ml)的培养基,使终浓度为20U/ml IL-2和5ng/ml IL-15最终体积为400μl
  7. 通过ELISA测定测试克隆的抗原特异性 抗原特异性测试在具有HLA-A2 + APC和肽的两个孔中以及具有APC但没有肽的两个孔中进行。将48孔板中的每个克隆重悬于〜400μl。一至二百微升的该细胞悬浮液转移到10ml试管中,在10ml DPBS中洗涤,然后用1.0ml培养基洗涤。对于ELISA测定,将洗涤的克隆重悬于400μl培养基中,并将100μl分配到含有APC(50,000个EBV转化的B细胞/孔)的96孔圆底板的四个孔中,其中(两个孔)和两孔)肽(1.0μM)。过夜培养后收集100μl上清液并通过ELISA测定IFNγ( http://www.biolegend.com/human-ifn-gamma-elisa-max-standard-2226.html )。
    注意:
    1. 我们常规使用表达所需HLA分子(在这种情况下为HLA-A2)的EBV转化的B细胞系。也可以使用可选的APC,例如辐射的PBMC(60Gy)或转染的细胞系。
    2. 其他测定,例如 Cr发布,也可用于筛选克隆。我们优选使用IFNγ分泌作为初始筛选,因为所有应答细胞应该分泌这种细胞因子,因为它们是基于IFNγ分泌选择的。
    3. 通过用抗CD3(OKT3)再刺激, IL-2和IL-4,如Ciantar和Mannering(2011)中所述
  8. 扩增后,重新测试克隆的抗原特异性并储存在液体中氮气直至需要。为了冷冻它们离心的克隆(515×g,5分钟,室温)。在倒出上清液之后,将细胞重悬于FCS中(通常至10×10 6个细胞/ml)。缓慢加入等体积的含有20%DMSO的FCS,同时轻轻混合。将1.0ml(〜5×10 6个细胞/小瓶)的等分试样分配在适于在液氮中冷冻的小瓶中。将小瓶置于含有丙醇的冷冻容器中,并在-80℃冷冻箱中贮存过夜。第二天将小瓶转移到液氮杜瓦瓶中并储存直至需要 注意:我们已经发现,如果CD8 + T细胞在IL-2/ml)解冻后过夜。

代表数据



图1.克隆MP 58-66 特异性IFNγ + CD8 + T细胞。 MP 58-66 特异性,IFNγ + CD8 + 的门控策略。 A.基于正向(FSC)和侧向散射(SSC)的淋巴细胞门控; B.通过碘化丙啶染色排除死细胞,活细胞为PI阴性; C.通过FSC高度(FSC-H)和FSC宽度(FSC-W)选通策略排除双峰;当没有肽培养PBMC时,IFNγ+ CD8 + T细胞; E.与用MP sub 58-66培养的PBMC相同的门并用相同的肽再刺激。


图2.筛选生长中的CD8 + T细胞克隆的抗原特异性。 HLA-A2 +或者没有肽脉冲的EBV细胞。一个洗涤每个生长克隆的样品并与这些APC一起温育过夜。第二天,通过ELISA测定上清液中IFNγ的浓度。数据表示每个克隆的重复培养物的平均值±标准偏差。选择响应MP sub 58-66分泌IFNγ的克隆用于进一步扩增。


图3.在CD8阳性+ T细胞克隆扩增后重新筛选抗原特异性。将克隆孵育与用或不用MP sub 58-66脉冲的HLA-A2 +/+ EBV细胞进行比较。孵育过夜后,通过ELISA测量上清液中的IFNγ。数据表示每个克隆的一式三份样品的平均值±标准偏差。

食谱

  1. 细胞因子和肽的储备浓度
    根据制造商的说明制备
  2. 染色缓冲区
    0.5%BSA和2mM EDTA的DPBS中
  3. 细胞培养基-RPMI与5%汇集的人血清(表1)

    表1. T细胞培养基的制备


  4. 碘化丙啶的浓度(1mg/ml)
    通过将碘化丙啶溶解在无菌过滤的水中来制备
  5. PMA(0.1mg/ml)和离子霉素(0.5mg/ml)的贮存浓度 通过溶解在DMSO中制备
  6. (50μg/ml)的浓度 通过将除虫菌素溶解于无菌过滤的水中来制备

致谢

我们感谢Eleanora Tresoldi的技术援助和献血者为研究提供血液样本。 SIM由澳大利亚国家卫生和医学研究委员会(NHMRC,APP1061961,APP1087341)和JDRF职业发展奖5-CDA2014210-A-N支持。圣文森特医学研究所得到维多利亚州政府运营基础设施支持计划的支持。作者没有声明利益冲突。

参考文献

  1. Ciantar,J.P。和Mannering,S.I。(2011)。 用于生长和分析人类抗原特异性CD4 + T细胞的改进方法细胞克隆。 Diabetes Metab Res Rev 27(8):906-912。
  2. Mannering,SI,Morris,JS,Jensen,KP,Purcell,AW,Honeyman,MC,van Endert,PM和Harrison,LC(2003)。< a class ="ke-insertfile"href ="http: www.ncbi.nlm.nih.gov/pubmed/14659909"target ="_ blank">用于检测稀有自身抗原特异性人T细胞增殖的灵敏方法。 J Immunol Methods 283 (1-2):173-183
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Trivedi, P. and Mannering, S. I. (2016). Efficient Isolation of Influenza Specific CTLs. Bio-protocol 6(12): e1847. DOI: 10.21769/BioProtoc.1847.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。