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Peptide Loading on MHC Class I Molecules of Tumor Cells
肿瘤细胞表面MHC I类分子结合肽实验   

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Abstract

MHC class I molecules present peptides to cytotoxic T cells allowing the immune system to scan for intracellular pathogens and mutated proteins. The generation of antigenic peptides is a multistep process that ends in the endoplasmic reticulum (ER). Only peptides with the right length and sequence will bind nascent MHC class I molecules in the ER. This protocol allows to detachment of the endogenous peptides bound to MHC class I molecules by preserving them for the binding of high affinity synthetic peptides. The complete dissociation of endogenous peptides by mild acid treatment as well as the binding of synthetic peptides to MHC class I molecules will be evaluated measuring HLA class I molecules express on the cell surface by flow cytometry. The mouse antibody W6/32 which recognizes β2m associated HLA-A, -B, -C, -E and -G heavy chains is suitable for this propose. Any tumor cell line that expresses surface HLA class I molecules is suitable for the assay. Another important aspect is to know the HLA class I typing of tumor cell line to allow selection of the known high affinity peptides.

Keywords: MHC class I(MHC I类), High affinity peptides(高亲和肽), Acid treatment(酸处理), MHC class I surface expression(MHC I类分子的表面表达), Tumor cells(肿瘤细胞)

Materials and Reagents

  1. T25 Flasks (Coring, Falcon®, catalog numbers: 353108 )
  2. T75 Flasks (Coring, Falcon®, catalog numbers: 353136 )
  3. 15 ml centrifuge tubes (Coring, Falcon®, catalog number: 352096 )
  4. 6 well plates (Coring, Falcon®, catalog number: 353046 )
  5. 1.5 ml Safe-Lock tubes (Eppendorf, catalog number: 0030120086 )
  6. 96 well plates (Coring, catalog number: 3799 )
  7. FACS tubes
  8. Tumor cell line (grown in flasks, in incubator at 37 °C and 5% CO2)
  9. RPMI 1640 (Euroclone, catalog number: ECM9106L )
  10. Fetal bovine serum (FBS) heat-inactivated for 1 h at 56 °C (Thermo Fisher Scientific, Gibco®, catalog number: 10270-106 )
  11. Penicillin/streptomycin (EuroClone, catalog number: ECB3001D )
  12. L-glutalmine (EuroClone, catalog number: ECB3000D )
  13. EDTA (Sigma-Aldrich, catalog number: E5134 )
  14. Phosphate buffered saline (PBS) (EuroClone, catalog number: ECB4004L )
  15. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
  16. Trypan-Blue
  17. Na2HPO4 (Sigma-Aldrich, catalog number: S5136 )
  18. Citric acid monohydrate (Sigma-Aldrich, catalog number: 251275 )
  19. mAb W6/32
  20. Beta 2-microglobulin (Sigma-Aldrich, catalog number: M4890 )
  21. Peptides (lyophilized, resuspended in DMSO at 10 mM) (Anaspec)
  22. Goat F(ab’)2 Fragment anti-mouse IgG (H+L)-FITC (Beckman Coulter, catalog number: PN IM0819 )
  23. Complete RPMI (see Recipes)
  24. 833 μM EDTA (see Recipes)
  25. FACS buffer (see Recipes)
  26. Acid buffer (see Recipes)

Equipment

  1. Centrifuge, used with maximal acceleration and deceleration (Eppendorf, model: 5810R )
  2. Incubator (5% CO2, 37 °C) (FormaTM Steri-CultTM CO2 Incubator-Thermo Fisher Scientific)
  3. Flow cytometer equipped with 2 lasers (8 detectors), interfaced with PC using DIVA Software version 6.1.3 (BD, model: FACSCanto II )

Software

  1. DIVA software version 6.1.3
  2. FlowJo software

Procedure

  1. Acid treatment of tumor cell lines
    1. Tumor cells are cultured in fresh RPMI medium at a cell density ≥ 0.5-2 x 106 cell/ml depending on the type of cells, in an incubator at 37 °C, 5% CO2.
    2. Wash adherent cells in PBS (5 ml) and detach cells by adding 833 μM EDTA (1 ml for T25 flask and 2 ml for T75 flask) for 3-5 min in an incubator at 37 °C, 5% CO2.
    3. Wash cells twice with PBS (2 x 5 ml in 15 ml tube) by centrifugation at 468 x g for 7 min and re-suspend them in cold FACS buffer.
    4. Count total viable cells with Trypan-Blue exclusion method.
    5. Harvest 5-10 x 106 cells with 5 ml PBS (at RT) in 15 ml tubes.
    6. Centrifuge cells at 468 x g for 7 min.
    7. Discard the supernatant and re-suspend the cells in 600 μl cold acid buffer by manual gentle shaking.
    8. Incubate for 1 to 3 min on ice (preliminary experiments should be performed for each cell line to detect the best condition to completely dissociate MHC class I complexes by preserving cell viability; the complete dissociation will be deduced with the cell surface expression of HLA class I molecules detected by staining the cells with the mAb W6/32).
    9. Immediately add 10 ml of complete RPMI (at RT).
    10. Centrifuge cells at 468 x g for 7 min.
    11. Discard supernatant and re-suspend the cells in 10 ml of complete RPMI.
    12. Centrifuge cells at 468 x g for 7 min.
    13. Discard supernatant and re-suspend the cells in 1 ml of complete RPMI containing beta 2-microglobulin (5 μg/ml).

  2. Pulsing with peptides
    1. Seed the cells in 6 well plate at a cell density of 1 x 106 cells/well. Prepare 1 well for the control (without peptide) and 1 well for each peptide to be tested and/or a mix of different peptides.
    2. Add 50 μM of peptide (stocked at 10 mM, single or a mix of different peptides).
    3. Incubate for 2 h at RT and for additional 2 h at 37 °C in incubator.

  3. Staining and FACS analysis
    1. Harvest cells in 1.5 ml Safe-Lock tubes and centrifuge at 468 x g for 7 min.
    2. Discard the supernatant and re-suspend the cells in 100 μl of cold FACS buffer.
    3. Distribute in a round-bottom 96 well plate (50 μl/well).
    4. Centrifuge the plate at 832 x g for 2 min at 4 °C.
    5. Discard the supernatant and re-suspend cells in 50 μl of cold FACS buffer containing mAb W6/32 (diluted 1:500) or in 50 μl of cold FACS buffer without mAb; mix carefully.
    6. Incubate for 25 min on ice.
    7. Add 150 μl of cold FACS buffer and centrifuge at 832 x g at 4 °C.
    8. Discard the supernatant and re-suspend cells in 50 μl of cold FACS buffer containing goat F(ab’)2 Fragment anti-mouse IgG (H+L)-FITC (diluted 1:100); mix carefully.
    9. Incubate for 25 min on ice in the dark.
    10. Add 150 μl of cold FACS buffer and centrifuge at 832 x g at 4 °C.
    11. Discard the supernatant and re-suspended cells in 150 µl of cold FACS buffer in FACS tubes and perform flow cytometric analysis. Viable human single cells will be selected by gating in forward scatter versus side scatter dot plot and the amount of cell surface MHC class I expression will be evaluated by mean fluorescence intensity (mfi) (Figure 1). Pulsing with high affinity peptides allows the recovery of surface MHC class I expression of acid-treated cells.
    12. Acquire data using DIVA software and analyse using FlowJo software.


      Figure 1. Recovery of peptide-MHC class I complexes on the surface of acid-treated DAOY cells pulsed with high affinity peptides. Flow cytometric analysis of MHC class I expressed on DAOY tumor cells left untreated (blue line) or pretreated with low pH buffer to allow dissociation of preformed peptide-MHC class I complexes (red line) and then pulsed with high affinity peptide (green line). Isotype-matched negative control antibody displayed as black line. 

Recipes

  1. Complete RPMI
    440 ml RPMI 1640
    50 ml FBS
    5 ml penicillin-streptomycin
    5 ml L-glutamine
  2. 833 μM EDTA
    Dissolve 7.3 g EDTA in 50 ml sterile water
    Adjust pH to 8.0
    Add 83.3 μl of this solution in 50 ml sterile PBS
    Filter sterilize through a 20 µm filter
    Store at 4 °C
  3. FACS buffer
    50 ml PBS
    1 g BSA
    4. Acid buffer
    Dissolve 2.52 g Na2HPO4 (263 mM) and 1.29 g Citric acid monohydrate (123 mM) in 50 ml sterile water
    Adjusted pH to 2.5

Acknowledgments

This protocol was adapted from Cifaldi et al. (2015). We acknowledge funding from Italian Ministry of Health (Rome, Italy) grant and the special project 5 x 1,000 Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan, Italy) grant.

References

  1. Cifaldi, L., Romania, P., Falco, M., Lorenzi, S., Meazza, R., Petrini, S., Andreani, M., Pende, D., Locatelli, F. and Fruci, D. (2015). ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors. Cancer Res 75(5): 824-834.

简介

MHC I类分子将肽呈递到细胞毒性T细胞,允许免疫系统扫描胞内病原体和突变蛋白。抗原肽的产生是以内质网(ER)结束的多步过程。只有具有正确长度和序列的肽将结合ER中的新生MHC I类分子。该方案允许通过保持结合MHC I类分子的内源肽与高亲和力合成肽的结合而脱离。通过温和酸处理以及合成肽与MHC I类分子的结合来评价内源肽的完全解离,通过流式细胞术测量在细胞表面上表达的HLA I类分子。识别β2m相关HLA-A,-B,-C,-E和-G重链的小鼠抗体W6/32适合于此提议。表达表面HLA I类分子的任何肿瘤细胞系适于测定。另一个重要方面是知道肿瘤细胞系的HLA I类分型以允许选择已知的高亲和力肽。

关键字:MHC I类, 高亲和肽, 酸处理, MHC I类分子的表面表达, 肿瘤细胞

材料和试剂

  1. T25烧瓶(Coring,Falcon ,目录号:353108)
  2. T75烧瓶(Coring,Falcon ®,目录号:353136)
  3. 15ml离心管(Coring,Falcon ,目录号:352096)
  4. 6孔板(Coring,Falcon ,目录号:353046)
  5. 1.5ml Safe-Lock管(Eppendorf,目录号:0030120086)
  6. 96孔板(Coring,目录号:3799)
  7. FACS管
  8. 肿瘤细胞系(在烧瓶中,在37℃和5%CO 2培养箱中生长)
  9. RPMI 1640(Euroclone,目录号:ECM9106L)
  10. 在56℃热灭活1小时的胎牛血清(FBS)(Thermo Fisher Scientific,Gibco 设备

    1. 离心机,使用最大加速和减速(Eppendorf,型号:5810R)
    2. 将培养箱(5%CO 2,37℃)(Forma TM Supter-Cult TM CO 2 Sub培养箱 - Thermo Fisher Scientific)
    3. 装有2个激光器(8个检测器)的流式细胞仪,使用DIVA软件版本6.1.3(BD,型号:FACSCanto II)

    软件

    1. DIVA软件版本6.1.3
    2. FlowJo软件

    程序

    1. 肿瘤细胞系的酸处理
      1. 根据细胞类型,在37℃,5%CO 2培养箱中,在新鲜RPMI培养基中培养肿瘤细胞,细胞密度≥0.5-2×10 6细胞/ml, 2 。
      2. 在PBS(5ml)中洗涤贴壁细胞并通过在37℃,5%CO 2下在培养箱中加入833μMEDTA(1ml用于T25烧瓶和2ml用于T75烧瓶)3-5分钟来分离细胞。 。
      3. 通过在468×g离心7分钟,用PBS(2×5ml,在15ml管中)洗涤细胞两次,并将它们重悬在冷的FACS缓冲液中。
      4. 用台盼蓝排除法计数总活细胞
      5. 在15ml管中用5ml PBS(在室温)收获5-10×10 6个细胞。
      6. 以468×g离心细胞7分钟。
      7. 弃去上清液,并通过手动轻轻摇动将细胞重悬在600μl冷酸缓冲液中。
      8. 在冰上孵育1至3分钟(应为每个细胞系进行初步实验以检测通过保持细胞活力来完全解离MHC I类复合物的最佳条件;完全解离将通过HLA I类的细胞表面表达来推导 通过用mAb W6/32染色细胞检测的分子)
      9. 立即加入10ml完全RPMI(室温)。
      10. 以468×g离心细胞7分钟。
      11. 弃去上清液并将细胞重悬于10ml完全RPMI中。
      12. 以468×g离心细胞7分钟。
      13. 弃去上清液并将细胞重悬在1ml含有β2-微球蛋白(5μg/ml)的完全RPMI中。

    2. 脉冲与肽
      1. 将细胞以1×10 6个细胞/孔的细胞密度接种在6孔板中。 准备1个孔用于对照(无肽),1个孔用于待测试的每种肽和/或不同肽的混合物。
      2. 加入50μM肽(以10mM储备,单一或不同肽的混合物)
      3. 在RT孵育2小时,在37℃孵育2小时。

    3. 染色和FACS分析
      1. 收获细胞在1.5毫升安全锁管和离心机在468×g下7分钟。
      2. 弃去上清液并将细胞重悬于100μl冷FACS缓冲液中
      3. 分布在圆底96孔板(50μl/孔)中
      4. 将板在832×g离心2分钟,在4℃
      5. 弃去上清液并将细胞重悬于50μl含有mAb W6/32(1:500稀释)的冷FACS缓冲液或50μl不含mAb的冷FACS缓冲液中; 小心混合。
      6. 在冰上孵育25分钟。
      7. 加入150μl冷FACS缓冲液,并在4℃下以832×g离心。
      8. 弃去上清液并将细胞再悬浮于含有山羊F(ab')2片段抗小鼠IgG(H + L)-FITC(稀释1:100)的50μl冷FACS缓冲液中; 小心混合。
      9. 在冰上在黑暗中孵育25分钟。
      10. 加入150μl冷FACS缓冲液,并在4℃下以832×g离心。
      11. 丢弃上清液和重悬浮的细胞在150μlFACS管中的冷FACS缓冲液,并执行流式细胞术分析。 通过在前向散射中选通来选择存活的人单个细胞 相对于侧散射点图,细胞表面MHC I类表达的量将通过平均荧光强度(mfi)评估(图1)。用高亲和力肽的脉冲允许恢复酸处理的细胞的表面MHC I类表达
      12. 使用DIVA软件获取数据,并使用FlowJo软件进行分析

        图1.在用高亲和力肽脉冲的酸处理的DAOY细胞表面上回收肽-MHC I类复合物。在未处理的DAOY肿瘤细胞上表达的MHC I类的流式细胞术分析(蓝线)或用低pH缓冲液预处理以允许预形成的肽-MHC I类复合物解离(红线),然后用高亲和力肽(绿线)脉冲。同种型匹配的阴性对照抗体显示为黑色。 

    食谱

    1. 完成RPMI
      440ml RPMI 1640
      50ml FBS
      5ml青霉素 - 链霉素 5ml L-谷氨酰胺
    2. 833μMEDTA
      将7.3g EDTA溶于50ml无菌水中
      将pH调节至8.0
      将83.3μl该溶液加入50ml无菌PBS中 通过20μm过滤器过滤灭菌
      存储在4°C
    3. FACS缓冲区
      50 ml PBS
      1 g BSA
      酸性缓冲液
      在50ml无菌水中溶解2.52g Na 2 HPO 4(263mM)和1.29g一水合柠檬酸(123mM)。 调节pH至2.5

    致谢

    此协议改编自Cifaldi等人。 (2015)。 我们承认意大利卫生部(意大利罗马)的资金和5 x 1,000意大利意大利安哥拉(意大利米兰)的特别项目。

    参考文献

    1. Cifaldi,L.,Romania,P.,Falco,M.,Lorenzi,S.,Meazza,R.,Petrini,S.,Andreani,M.,Pende,D.,Locatelli,F.and Fruci, 2015)。  ERAP1调节自然杀伤细胞功能,通过控制 抑制性受体的结合。 Cancer Res 75(5):824-834。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cifaldi, L., Locatelli, F. and Fruci, D. (2016). Peptide Loading on MHC Class I Molecules of Tumor Cells. Bio-protocol 6(18): e1932. DOI: 10.21769/BioProtoc.1932.
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