可视化抗原特异性CD4 + T细胞的MHC II类四聚四
Summary
此过程演示的纯化和体外扩增抗原特异性CD4 +从整体外周血T细胞及其可视化使用MHC II类四聚体。四聚体，允许直接与单个抗原特异性T细胞和定义MHC II类限制性的可视化。
Abstract
主要组织相容性复合体（MHC）Ⅱ类四聚体可以直接可视化的抗原特异性CD4 + T细胞流式细胞仪。此方法依赖于非常具体的肽载入MHC和相应的T细胞受体之间的互动。虽然单一的MHC /肽分子的亲和力低，交叉连接链霉亲和的MHC /肽复合物增加亲和力的互动，使染色试剂的使用。由于CD4 + T细胞（〜1 30万为一个单一的特异性）的频率相对较低，本试验利用体外扩增步骤，以提高其检测阈值。纯化外周血单个核细胞经Ficoll底图。然后由消极的选择，使用生物素抗体鸡尾酒和抗生物素标记的磁珠分离CD4 +细胞。使用贴壁细胞的CD4细胞的一小部分，作为抗原提呈细胞，CD4 + T细胞在媒体扩大，加入的抗原肽和IL – 2。孵化一小时，随后在37℃染色使用表面抗体，如抗CD4，抗CD3和抗CD25扩大细胞与相应的Ⅱ类四聚体染色。标签后，细胞可以直接通过流式细胞仪分析。扩大CD4 +细胞中的四聚体阳性细胞通常形成一个独特的人口。四聚体阳性细胞通常CD25 +调节性和经常的CD4高。由于背景的四聚体染色水平可以有所不同，应该始终是积极的染色结果相比，相同的细胞与一个不相关的四聚体染色。这一基本检测的多种变化是可能的。四聚体阳性细胞可能为进一步的表型分析，列入酶联免疫斑点或增殖实验，或其他二次检测进行排序。一些团体也展示了合作，使用四聚体的抗细胞因子或抗FOXP3抗体染色。
Protocol
Discussion
了解免疫的CD4 + T细胞的作用是极其重要的。然而，抗原特异性CD4 + T细胞可以用传统方法难以发现和隔离。相比之下，MHC II类四聚体，使所需的抗原特异性CD4 + T细胞的直接可视化。这个视频演示的分离，纯化，并在体外扩增的CD4 + T细胞，并随后将其可视化，使用四聚体。 II类四聚四是荧光蛋白偶联物的水溶性生物素化的II类的α/围绕一个荧光标记的链霉亲和的核心（图4）结合β二聚体组成。在本?…
Acknowledgements
我们感谢他们在拍摄和视频制作的关键作用迪克弗利和Leigh金博尔。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Class 2 Laminar flow biosafety cabinet | equipment | Thermo Forma | 1200 | Or equivalent |
| 50 mL conical Tube | equipment | VWR | 47747-182 | Or equivalent |
| 1X PBS (Ca/Mg free) | Reagent | HyClone | SH30028.02 | Or equivalent |
| Ficoll-paque PLUS | Reagent | GE Healthcare | 17-1440-03 | foil wrapped to protect from light |
| Pipet Aid XP | equipment | Drummond | 4-000-101 | Or equivalent |
| 10 mL pipet | equipment | Corning/Costar | CLS4492 | Or equivalent |
| Aerosolve Canisters | equipment | Beckman | 359481 | with 50 mL inserts |
| Centrifuge | equipment | Beckman | GS-6R | Or equivalent |
| Transfer pipets | equipment | Samco | 202-20S | Or equivalent |
| Pasteur pipets | equipment | VWR | 14672-200 | Or equivalent |
| Hemolytic Buffer | Reagent | N/A | N/A | 8.3 g/L NH4Cl, 1.0 g/L NaHCO3, 0.04 g/L disodium EDTA |
| Trypan blue | Reagent | Sigma | T6146 | 0.2% in PBS |
| Hemocytometer | equipment | VWR | 15170-208 | |
| 15 mL conical tube | equipment | VWR | 05-527-90 | Or equivalent |
| Running Buffer | Reagent | N/A | N/A | PBS + 2mM EDTA + 5g/L BSA |
| MACS CD4+ T Cell Isolation Kit II | Reagent | Miltenyi Biotec | 130-091-155 | Or equivalent |
| EasySep® Magnet | equipment | Stem Cell Technologies | 18000 | |
| 5 mL polypropylene tube | equipment | Falcon | 352063 | |
| RPMI 1640 | Reagent | Invitrogen | 22400-089 | with 25 mM HEPES |
| Pooled human serum | Reagent | N/A | N/A | drawn from healthy donors, heat inactivated and filtered |
| Pen Strep | Reagent | Invitrogen | 1570-063 | Or equivalent |
| 500mL 0.22 micron bottle top filter | equipment | Nalgene | 595-3320 | Or equivalent |
| 48-well plate | equipment | Costar | 3548 | Or equivalent |
| 37°C CO2 incubator | equipment | Sanyo Scientific | MCO-18AIC | Or equivalent |
| 20 mg/mL synthetic peptide | Reagent | N/A | N/A | Custom synthesis from any peptide vendor |
| 5 mL FACS tube | equipment | Falcon | 352008 | Polystyrene |
| Peptide loaded class II tetramer (as described) | Reagent | N/A | N/A | Prepared by the BRI tetramer core |
| Anti-CD3 | Reagent | BD pharmingen | 347344 | Or equivalent |
| Anti-CD4 | Reagent | eBioscience | 17-0049-73 | Or equivalent |
| Anti-CD25 | Reagent | eBioscience | 11-0259-73 | Or equivalent |
| Facs Calibur Flow Cytometer | equipment | BD Biosciences | 342975 | Or equivalent |
Referências
- Novak, E. J., Liu, A. W., Nepom, G. T., Kwok, W. W. MHC Class II tetramers permit detection and characterization of peptide-specific human CD4+ T cells proliferating to influenza A antigen. J. Clin. Invest. 104, R63-R67 (1999).
- Novak, E. J., Liu, A. W., Gebe, J. A., Falk, B., Nepom, G. T., Falk, B., Koelle, D. M., Kwok, W. W. Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens. J. Immunol. 166, 6665-6670 (2001).
