CD8+ T 细胞活化的各向异性聚合物人工抗原呈现细胞的制备
Summary
在这里, 我们提出了一个协议, 以快速和可重复生成生物启发, 可生物降解的 articifical 抗原呈现细胞 (aAPC) 与可调谐的大小, 形状和表面蛋白呈现的 T 细胞扩张的体内或体内.
Abstract
人工抗原呈现细胞 (aAPC) 是一种有潜力的免疫调节平台, 由于其强大的刺激 T 细胞的能力。脱细胞基质提供了与基于细胞的 aAPC 的关键优势, 包括精确控制信号表示参数和 aAPC 表面的物理特性, 以调节其与 T 细胞的相互作用。由各向异性粒子 (特别是椭球粒子) 构造的 aAPC 在刺激 t 细胞时比球形对应体更有效, 因为可用于 t 细胞接触的增强的结合和更大的表面积, 以及减少非特异性摄取和增强药代动力学特性。尽管增加了对各向异性粒子的兴趣, 但即使是广泛接受的方法来生成各向异性粒子, 如薄膜拉伸, 也可能对实现和使用可重复具有挑战性。
为此, 我们描述了一种快速、标准化的可生物降解各向异性粒子基 aAPC 的协议, 其可调谐大小、形状和信号呈现用于 T 细胞在体内或体内的扩张, 以及方法表征其大小、形态和表面蛋白质含量, 并评估其功能。这种制造各向异性 aAPC 的方法是可扩展的, 可以重现, 因此非常适合为 “现成” 免疫疗法生成 aAPC。
Introduction
人工抗原呈现细胞 (aAPC) 已表明承诺作为免疫调节剂, 因为他们可以产生一个强大的抗原特异 T 细胞反应。这些平台的关键是它们能够有效地为 T 细胞活化提供关键信号。脱细胞 aAPC 是一种有吸引力的替代细胞的 aAPC, 因为它们更容易和成本更低的制造, 面临更少的挑战, 在扩大和翻译, 并减轻与细胞疗法相关的风险。脱细胞 aAPC 还允许对信号表示参数和表面物理特性进行高度控制, 这将与 T 细胞1连接。
aAPC 必须重述至少两个信号, 对于 T 细胞激活至关重要。信号1提供抗原识别和发生时 T 细胞受体 (TCR) 识别和参与的 MHC i 类或 II 轴承其同源抗原, 最终通过 TCR 复合信号。为了绕过抗原特异性要求, aAPC 系统经常承受竞赛 CD3 受体的单克隆抗体, 特异刺激 TCR 复合物。重组形式的 mhc, 特别是 mhc 多聚体, 也被用于 aAPC 的表面, 以提供抗原特异性2,3。信号2是指示 T 细胞活动的共刺激信号。为了提供 T 细胞活化所必需的刺激, CD28 受体通常用 aAPC 表面上的竞赛抗体刺激, 尽管共刺激的其他4-1BB 受体已经成功地瞄准了4。信号1和2蛋白通常固定在刚性颗粒表面, 以合成 aAPC。从历史上看, aAPC 是由各种材料制成的, 包括聚苯乙烯4、5和铁葡聚糖6。较新的系统利用生物降解聚合物 (聚乳酸-乙醇酸) (aAPC) 生成可以很容易地耦合到信号蛋白, 适合于体内直接管理, 并可促进持续释放封装细胞因子或可溶性因子, 以增加 T 细胞活化7,8。
除了存在必要的信号蛋白外, 在 aAPC/t 细胞相互作用期间, 受体在足够大的表面积上的参与对于 T 细胞活化是必不可少的。因此, aAPC 的物理参数 (如大小和形状) 会极大地改变其可用接触区域, 并影响其刺激 T 细胞的能力。微米级的 aAPC 在刺激 T 细胞方面比它们的纳米尺度对应9、10更有效。然而, 纳米 aAPC 可以有优越的生物分布和更好的引流到淋巴结, 可能提高其在体内的性能在微 aAPC11。形状是对基于粒子的 aAPC 系统感兴趣的另一个变量。在刺激 T 细胞时, 各向异性 aAPC 最近被证明比各向同性粒子更有效, 主要是由于增强了与靶细胞的相互作用以及减少了非特异细胞吸收。单元格优先绑定到椭球粒子的长轴, 曲率和平坦表面的半径越大, aAPC 和 T 细胞12之间的接触就越多。椭圆形颗粒的长轴也会抑制吞噬, 从而在体内施用12、13的情况下, 与球形颗粒相比, 循环时间增加。由于这些优点, 椭球粒子在体外和体内与球形颗粒相比, 能更好地调节抗原特异 T 细胞的扩张, 这在微 nanoscales12中观察到的效果, 13。制造各向异性粒子有多种策略, 但薄膜拉伸是一种简单、广泛接受的方法, 用于生成一系列不同的粒子形状14。合成后, 粒子被投射到薄膜中, 在一个或两个维度上拉伸, 温度高于粒子材料的玻璃过渡温度。然后将胶片溶解以检索粒子。尽管对各向异性粒子的兴趣越来越大, 但目前制造粒子基 aAPC 的方法大多限于各向同性系统, 改变颗粒形状的方法很难实现, 与某些 aAPC 合成不相容。策略, 缺乏精度和重现性15。我们的薄膜拉伸技术可以手动或以自动方式进行, 以快速生成由各种可生物降解聚合物合成的各向异性粒子, 在一个或两个维度中拉伸到所需的纵横比15。
基于我们以前的工作, 我们开发了一种可生物降解的粒子基方法, 结合可以扩展的薄膜拉伸技术, 快速生成 aAPC, 可调谐尺寸和形状, 以标准化的方式用于 T 细胞扩张, 例如体内或体内。我们的蛋白质共轭策略可用于将任何感兴趣的蛋白质与颗粒表面上的羧基基团进行耦合, 以达到所需的密度, 从而使该 aAPC 系统具有高度的灵活性。我们还描述了 aAPC 的大小、形态和表面蛋白含量的表征方法, 并对其体外功能进行了评价。该协议可以很容易地适应扩大免疫细胞的体内或体内的各种免疫治疗应用。
Protocol
Representative Results
Discussion
本协议详细介绍了一种用于精确生成各向异性聚合物粒子的通用方法。这里描述的薄膜拉伸技术是可扩展的, 高度重现性和成本低廉。产生各向异性粒子的替代技术受到许多限制, 包括高成本、低吞吐量和有限的粒子大小。薄膜拉伸方法也是有利的, 因为粒子在合成后被修改为各向异性, 因此, 与各种颗粒尺寸和合成技术兼容。图 1详细介绍了自动二维拉伸装置的设置。该装置?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
EBA (DGE-1746891) 和 KRR (DGE-1232825) 感谢 NSF 研究生研究奖学金计划的支持。RAM 感谢国家研究服务奖国立卫生研究院 NCI F31 (F31CA214147) 和大学科学家奖学金的成就奖励支持。作者感谢 NIH (R01EB016721 和 R01CA195503), 预防失明詹姆斯和卡罗尔免费催化剂奖的研究, 以及 JHU 彭博-Kimmel 癌症免疫治疗研究所的支持。
Materials
| Poly(vinyl alcohol), MW 25000, 88% hydrolyzed | Polysciences, Inc. | 02975-500 | |
| Glycerol | Sigma-Aldrich | G9012 | |
| Digital Thermometer | Fluke | N/A | Model name: Fluke 52 II |
| Immersion Temperature Probe | Fluke | N/A | Model name: Fluke 80PK 22 |
| Digital Hotplate & Stirrer | Benchmark Scientific | H3760-HS | |
| Multipoint stirrer | Thermo Fisher Scientific | 50093538 | |
| Resomer RG 504 H, Poly(D,L-lactide-co-glycolide) | Sigma-Aldrich | 719900 | |
| Dichloromethane | Sigma-Aldrich | D65100 | |
| Homogenizer | IKA | 0003725001 | |
| Sonicator | Sonics & Materials, Inc. | N/A | Model number: VC 505 |
| Sonicator sound abating enclosure | Sonics & Materials, Inc. | N/A | Part number: 630-0427 |
| Sonicator probe | Sonics & Materials, Inc. | N/A | Part number: 630-0220 |
| Sonicator microtip | Sonics & Materials, Inc. | N/A | Part number: 630-0423 |
| High speed centrifuge | Beckman Coulter | N/A | Model number: J-20XP (discontinued), alternative model: J-26XP |
| High speed centrifuge rotor | Beckman Coulter | 369691 | Model number: JA-17 |
| High speed polycarbonate centrifuge tubes | Thermo Fisher Scientific | 3118-0050 | 50 mL, screw cap |
| Rectangular disposable petri dish | VWR International | 25384-322 | 75 x 50 x 10 mm |
| Square disposable petri dish | VWR International | 10799-140 | 100 mm x 100 mm |
| LEAF Purified anti-mouse CD3ε Antibody | Biolegend | 100314 | |
| InVivoMab anti-mouse CD28, clone 37.51 | Bio X Cell | BE0015-1 | |
| N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride | Sigma-Aldrich | E6383 | |
| N-Hydroxysulfosuccinimide sodium salt | Sigma-Aldrich | 56485 | |
| MES | Sigma-Aldrich | M3671 | |
| Alexa Fluor 488 anti-mouse CD3 Antibody | Biolegend | 100212 | |
| APC anti-mouse CD28 Antibody | Biolegend | 102109 | |
| Corning 96 Well Solid Polystyrene Microplate | Sigma-Aldrich | CLS3915 | flat bottom, black polystyrene |
| Protein LoBind Tubes, 1.5 mL | Eppendorf | 22431081 | |
| RPMI 1640 Medium (+ L-Glutamine) | ThermoFisher Scientific | 11875093 | |
| Fetal Bovine Serum | Sigma-Aldrich | F4135 | Heat Inactivated, sterile-filtered |
| Ciprofloxacin | Sigma-Aldrich | 17850 | |
| 2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
| Recombinant Human IL-2 (carrier-free) | Biolegend | 589102 | |
| Sodium Pyruvate (100 mM) | ThermoFisher Scientific | 11360070 | |
| MEM Non-Essential Amino Acids Solution (100X) | ThermoFisher Scientific | 11140050 | |
| MEM Vitamin Solution (100X) | ThermoFisher Scientific | 11120052 | |
| CD8a+ T Cell Isolation Kit, mouse | Miltenyi Biotech | 130-104-075 | |
| CellTrace CFSE Cell Proliferation Kit | ThermoFisher Scientific | C34554 | |
| LS Columns | Miltenyi Biotech | 130-042-401 | |
| MidiMACS Separator | Miltenyi Biotech | 130-042-302 | |
| MACS Multistand | Miltenyi Biotech | 130-042-303 | |
| Flow Cytometer | Accuri C6 | ||
| Synergy 2 Multi-Detection Microplate Reader | BioTek | ||
| autoMACS Running Buffer | Miltenyi BIotech | 130-091-221 | |
| Cell Strainer | ThermoFisher Scientific | 22363548 | Sterile, 70 µm nylon mesh |
| ACK Lysing Buffer | ThermoFisher Scientific | A1049201 | |
| C57BL/6J (Black 6) Mouse | The Jackson Laboratory | 000664 | Male, at least 7 weeks old |
| U-Bottom Tissue Culture Plates | VWR | 353227 | Sterile, 96-well tissue culture treated polystyrene plates |
| 40 V DC Power Supply | Probotix | LPSK-4010 | |
| PTFE Coated Wire | Mouser | 602-5858-100-01 | This is for a 100 ft. spool but an equivalent wire will work |
| Stepper Motor Driver | Probotix | MondoStep5.6 | |
| IDC Connector Kit | Probotix | IDCM-10-12 | |
| Microcontroller | Probotix | PBX-RF | |
| 4A Fuses | Radio Shack | 2701026 | Equivalent fuses will work as well |
| DB25 Male to Male Cable | Probotix | DB25-6 | |
| USB-A to USB-B Cable | Staples | 2094915 | Equivalent cable will work as well |
| 8-Pin Amphenol Connectors Male and Female | Mouser | 654-97-3100A-20-7P and 654-97-3106A20-7S | |
| Stepper Motor | Probotix | HT23-420-8 | |
| Right Hand Lead Screw | Roton | 60722 | |
| Left Hand Lead Screw | Roton | 60723 | |
| Screws | McMaster Carr | 92196A151 | |
| Neoprene Rubber | McMaster Carr | 8698K51 | |
| Right Handed Flanged Lead Nut | Roton | 91962 | |
| Left Handed Flanged Lead Nut | Roton | 91963 | |
| Linux Control Computer | Probotix | LCNC-PC | Any computer with matching specification and Linux operating system will work |
| Corning bottle-top vacuum filter system | Sigma-Aldrich | CLS431097 | |
| Trypan Blue Solution, 0.4 % | ThermoFisher Scientific | 15250061 |
Referenzen
- Eggermont, L. J., Paulis, L. E., Tel, J., Figdor, C. G. Towards efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells. Trends in Biotechnology. 32 (9), 456-465 (2014).
- Maus, M. V., Riley, J. L., Kwok, W. W., Nepom, G. T., June, C. H. HLA tetramer-based artificial antigen-presenting cells for stimulation of CD4+ T cells. Clinical Immunology. 106 (1), 16-22 (2003).
- Oelke, M., et al. Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nature Medicine. 9 (5), 619-624 (2003).
- Rudolf, D., et al. Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells. Cancer Immunology, Immunotherapy. 57 (2), 175-183 (2008).
- Tham, E. L., Jensen, P. L., Mescher, M. F. Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptide-class I complexes and recombinant B7-Fc proteins. Journal of Immunological Methods. 249 (1-2), 111-119 (2001).
- Perica, K., et al. Magnetic field-induced T cell receptor clustering by nanoparticles enhances T cell activation and stimulates antitumor activity. ACS Nano. 8 (3), 2252-2260 (2014).
- Steenblock, E. R., Fadel, T., Labowsky, M., Pober, J. S., Fahmy, T. M. An artificial antigen-presenting cell with paracrine delivery of IL-2 impacts the magnitude and direction of the T cell response. The Journal of Biological Chemistry. 286 (40), 34883-34892 (2011).
- Zhang, L., et al. Paracrine release of IL-2 and anti-CTLA-4 enhances the ability of artificial polymer antigen-presenting cells to expand antigen-specific T cells and inhibit tumor growth in a mouse model. Cancer Immunology, Immunotherapy. 66 (9), 1229-1241 (2017).
- Mescher, M. F. Surface contact requirements for activation of cytotoxic T lymphocytes. The Journal of Immunology. 149 (7), 2402-2405 (1992).
- Steenblock, E. R., Fahmy, T. M. A comprehensive platform for ex vivo T-cell expansion based on biodegradable polymeric artificial antigen-presenting cells. Molecular Therapy. 16 (4), 765-772 (2008).
- Fifis, T., et al. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. The Journal of Immunology. 173 (5), 3148-3154 (2004).
- Sunshine, J. C., Perica, K., Schneck, J. P., Green, J. J. Particle shape dependence of CD8+ T cell activation by artificial antigen presenting cells. Biomaterials. 35 (1), 269-277 (2014).
- Meyer, R. A., et al. Biodegradable nanoellipsoidal artificial antigen presenting cells for antigen specific T-cell activation. Small. 11 (13), 1519-1525 (2015).
- Champion, J. A., Katare, Y. K., Mitragotri, S. Particle shape: a new design parameter for micro- and nanoscale drug delivery carriers. Journal of Controlled Release. 121 (1-2), 3-9 (2007).
- Meyer, R. A., Meyer, R. S., Green, J. J. An automated multidimensional thin film stretching device for the generation of anisotropic polymeric micro- and nanoparticles. Journal of Biomedical Materials Research Part A. 103 (8), 2747-2757 (2015).
- Ho, C. C., Keller, A., Odell, J. A., Ottewill, R. H. Preparation of monodisperse ellipsoidal polystyrene particles. Colloid and Polymer Science. 271 (5), 469-479 (1993).
- Shum, H. C., et al. Droplet microfluidics for fabrication of non-spherical particles. Macromolecular Rapid Communications. 31 (2), 108-118 (2010).
- Lan, W., Li, S., Xu, J., Luo, G. Controllable preparation of nanoparticle-coated chitosan microspheres in a co-axial microfluidic device. Lab on a Chip. 11 (4), 652-657 (2011).
- Yang, S., et al. Microfluidic synthesis of multifunctional Janus particles for biomedical applications. Lab on a Chip. 12 (12), 2097-2102 (2012).
- Zhou, Z., Anselmo, A. C., Mitragotri, S. Synthesis of protein-based, rod-shaped particles from spherical templates using layer-by-layer assembly. Advanced Materials. 25 (19), 2723-2727 (2013).
- Jang, S. G., et al. Striped, ellipsoidal particles by controlled assembly of diblock copolymers. Journal of the American Chemical Society. 135 (17), 6649-6657 (2013).
- Petzetakis, N., Dove, A. P., O’Reilly, R. K. Cylindrical micelles from the living crystallization-driven self-assembly of poly(lactide)-containing block copolymers. Chemical Science. 2 (5), 955-960 (2011).
- Rolland, J. P., et al. Direct fabrication and harvesting of monodisperse, shape-specific nanobiomaterials. Journal of the American Chemical Society. 127 (28), 10096-10100 (2005).
- Meyer, R. A., et al. Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation. Acta Biomaterialia. 72, 228-238 (2018).
