Summary

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

Published: June 19, 2018
doi:

Summary

我们在这里提出一个标准免疫技术 (CFSE 染色的应用), 旨在快速监测佐剂介导的细胞毒性 T 淋巴细胞 (CTL) 生成在体内.这种对 CTL 容量的快速估计有助于开发预防性疫苗, 防止细胞内病原体和治疗性癌症疫苗的发展。

Abstract

对现代分单元疫苗的评估表明, 中中和抗体的产生是重要的, 但不足以辅助选择。因此, 迫切需要具有体液和细胞免疫刺激能力的佐剂, 能够促进细胞毒 T 淋巴细胞 (CTL) 反应。因此, 对诱导交叉启动并随后增强 CTL 生成的佐剂候选者的忠实监测是疫苗开发的关键步骤。在这里, 我们提出一个方法的应用, 使用 SIINFEKL 特定 (OT) T 细胞监测模型抗原卵清蛋白 (卵)在体内的交叉呈现, 在不同的佐剂候选者在场。该方法是一种快速试验, 用于选择具有最佳交叉启动能力的佐剂。CD8+ T 细胞的增殖是交叉引发的最有价值的标志, 同时也被认为是佐剂诱导的交叉表达的相关性。这一特点可以评估在不同的免疫器官, 如淋巴结和脾脏。CTL 生成的程度也可以监测, 从而对局部 (引流淋巴结主要) 的性质或系统性反应 (远端淋巴结和/或脾脏) 有深入的了解。这项技术进一步允许对检测药物进行多次修改, 以抑制特定的交叉表达通路, 并提供了可能用于不同品系的常规和转基因小鼠。总之, 我们在这里提出的应用将是有用的在工业或学术界的疫苗实验室开发或修改疫苗研究和发展的化学佐剂。

Introduction

细胞毒性 T 淋巴细胞 (CTL) 诱导疫苗是为抗击某些类型的癌症而开发的关键治疗措施1。CTL 对预防性疫苗对细胞内病原体2也很重要。此外, ctl 是少数的免疫防御机制之一的功能活跃的风险人群, 如新生儿3,4谁也依赖 CTL, 以抗击早期生命感染5。在这方面, 与不引起 CTL 反应的佐剂一起研制的呼吸道合胞病毒疫苗 (明矾) 导致6婴儿感染严重并发症导致的疫苗完全失败。免疫接种的这些负面影响可以通过 CD8 细胞反应 7逆转。我们以前已经证明, 一些干扰素基因 (蜇) 激动剂诱发的主要细胞因子 (I. 干扰素) 对这些佐剂8产生的 CTL 反应至关重要, 部分是通过测量在疫苗接种后的 T 细胞, 并将这些结果作为在延长接种计划9中观察到的 CTL 诱导能力的量度。用羧基琥珀酰亚胺酯 (CFSE)染料稀释法测定野生型 (CD8) C57BL/6 受体小鼠中的 OT I. T 细胞的增殖是一种对疫苗佐剂能力的稳健估计, 可产生SIINFEKL 的交叉启动 (卵清蛋白的免疫主导肽, 卵)。这种技术的变异被广泛用于评估 CD8 和 CD4 T 细胞的增殖. 例如, 它已被用于没有选定的细胞因子 (高鼠) 或测量疫苗的功效后, 抗原召回的动物。我们设计了一个简短的协议 (4 天的实验), 在 CFSE 染色的 CD8+ T 细胞被动转移后, 皮下 (南卡罗来纳州) 免疫由一剂量50µg 的无内毒素卵子辅以测试佐剂被管理(图 1)。疫苗接种后48小时随访结果提供了可靠的证据证明佐剂产生 CTL 反应的能力。通过这一策略, 可以评估免疫后引流淋巴结局部免疫反应的效力, 以及测量脾脏 (或远端淋巴结) CTL 活动的反应程度。

Protocol

所有在这项研究中使用的老鼠都是从 C57BL/6 的背景。所有的动物都被保存在无病原体条件下。所有实验都是根据德国动物保护法 (TierSchG BGBl) 的规范进行的。我是 1105;25.05.1998), 并获下萨克森动物实验伦理学委员会和国家办事处 (下萨克森国家消费者保护和食品安全办公室) 批准, 许可证号为 33.4-42502-04-13/1281 和162280。 1. CFSE 细胞染色及移植 注意: OT I 小鼠是 tr…

Representative Results

为了测试使用不同佐剂 (ADJ1 和 ADJ2) 的治疗方法, 我们通过流式细胞仪测量过继转输转移的 CD8+ T 细胞的增殖来评估 CTL 的生成能力 (图 2)。为此, 我们以前从引流淋巴结和脾脏中分离出细胞 (表 1)。通过测量淋巴结和脾脏中 CD8+ T 细胞的增殖, 我们能够证实在引流淋巴结中 ADJ2 的 CTL 生成能力 (图 3) 与 A…

Discussion

现代疫苗是理想的 composedof 纯化抗原和佐剂, 有可能添加一个交付系统, 如脂质体, 病毒样粒子, 纳米粒子或活载体。设计疫苗的一个关键方面是根据临床需要选择合适的佐剂。部分的范围可能涉及倾向于体液与细胞免疫应答 (或两者), 选举局部与系统性免疫应答 (或两者), 以及这种疫苗必须在目标人群中产生的记忆。辅助评价的一个关键方面是迅速确定其产生 CTL 的能力。本文提出了一种基于已知?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢我们的技术助理: U. Bröder 和 h. Shkarlet, 他在实验过程中帮助我们。这项工作部分是由欧盟赠款 (UniVax, 601738 号合同和 TRANSVAC2, 730964号合同) 和亥姆霍兹协会赠款 (海-IDR) 资助的。资金来源不影响研究设计, 生成手稿或决定提交出版。

Materials

BD LSR Fortessa Cell Analyzer BD Special Order Flow Cytometer
CFSE Molecular Probes C34554 Proliferation Dye
MojoSort Mouse CD8 T Cell Isolation Kit Biolegend 480007 Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.
LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation Molecular Probes L23105 Dead Cell Marker
CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7 eBioscience 25-0900-82 antibody
APC anti-mouse CD8a Antibody BioLegend 100712 antibody
BV421 Rat Anti-Mouse CD4 BD 740007 antibody
Z2 coulter Particle count and Size Analyzer Beckman Coulter 9914591DA Cell counter. Z2 Automated particle/cell counter
EndoGrade Ovalbumin (10 mg) Hyglos(Germany) 321000 Ovalbumin endotoxin free tested.
Cell Strainer 100µm nylon Corning 352360 Cell strainer (100 µm pore mesh cups).
Sample Vials Beckman Coulter 899366014 Sample vials for Z2 automated counter
C57BL/6 mice (CD90.2) Harlan (Rossdorf, Germany) Company is now Envigo
OT-I (C57BL/6 background, CD90.1) Harlan (Rossdorf, Germany) Inbreed at our animal facility. Company from where adquired is now Envigo
FACS tubes Fischer (Corning) 14-959-5 Corning Falcon Round-Bottom Polystyrene Tubes
Falcon 15 mL tubes Fischer (Corning) 05-527-90 Falcon 15mL Conical Centrifuge Tubes
PBS (500 mL) Fischer (Gibco) 20-012-027 Gibco PBS (Phosphate Buffered Saline), pH 7.2
Red lamp (heating lamp) Dirk Rossmann GmbH (Germany) 405096 Heating infrred lamp (100 wats)
IsoFlo (Isoflurane) Abbott Laboratories (USA) 5260.04-05. Isoflurane anesthesic (250 mL flask).
Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber Parkland Scientific V3000PK Isoflurane anesthesia machine.
RPMI 1640 medium Gibco (distributed by ThermoFischer) 11-875-093 Base medium with Glutamine (500 mL)
Pen-Strept antibiotic solution (Gibco) Gibco (distributed by ThermoFischer) 15-140-148 Gibco Penicillin-Streptomycin (10,000 U/mL)
Fetal Bobine Serum (Gibco) Gibco (distributed by ThermoFischer) 10082147 Fetal Bovine Serum, certified, heat inactivated, US origin
ACK Lysing Buffer (100 ml) Gibco (distributed by ThermoFischer) A1049201 Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also
Plastic Petri Dishes Nunc (distributed by ThermoFischer) 150340 60 x 15mm Plastic Petri Dish, Non-treated
Cell Clump Filter CellTrics (Sysmex) 04-004-2317 CellTrics® 50 μm, sterile

References

  1. Krishna, S., Anderson, K. S. T-Cell Epitope Discovery for Therapeutic Cancer Vaccines. Methods Mol Biol. 1403, 779-796 (2016).
  2. Pinchuk, I., et al. A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae. Journal of immunology. 174, 5729-5739 (2005).
  3. Zhang, J., Silvestri, N., Whitton, J. L., Hassett, D. E. Neonates mount robust and protective adult-like CD8(+)-T-cell responses to DNA vaccines. Journal of virology. 76, 11911-11919 (2002).
  4. Marchant, A., et al. Mature CD8(+) T lymphocyte response to viral infection during fetal life. J Clin Invest. 111, 1747-1755 (2003).
  5. Simmons, C. P., et al. Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses. Journal of immunology. 166, 1106-1113 (2001).
  6. Fulginiti, V. A., et al. Respiratory Virus Immunizationa Field Trial Of Two Inactivated Respiratory Virus Vaccines; An Aqueous Trivalent Paratnfluenza Virus Vaccine And An Alum-Precipitated Respiratory Syncytial Virus Vaccine1. American journal of epidemiology. 89, 435-448 (1969).
  7. Olson, M. R., Varga, S. M. CD8 T cells inhibit respiratory syncytial virus (RSV) vaccine-enhanced disease. Journal of immunology. 179, 5415-5424 (2007).
  8. Lirussi, D., et al. Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway. EBioMedicine. 22, 100-111 (2017).
  9. Ebensen, T., et al. Bis-(3′,5′)-cyclic dimeric adenosine monophosphate: strong Th1/Th2/Th17 promoting mucosal adjuvant. Vaccine. 29, 5210-5220 (2011).
  10. Hogquist, K. A., et al. T cell receptor antagonist peptides induce positive selection. Cell. 76, 17-27 (1994).
  11. Clarke, S. R., et al. Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection. Immunology and cell biology. 78, 110-117 (2000).
  12. Topham, D. J., Castrucci, M. R., Wingo, F. S., Belz, G. T., Doherty, P. C. The role of antigen in the localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza pneumonia. Journal of immunology. 167, 6983-6990 (2001).
  13. Le Bon, A., et al. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nature immunology. 4, 1009-1015 (2003).
  14. Otto, K., Bullock, G. . The Laboratory Mouse. , 555-569 (2004).
  15. Lim, J. F., Berger, H., Su, I. H. Isolation and Activation of Murine Lymphocytes. Journal of visualized experiments: JoVE. , (2016).
  16. Shimizu, S., Bullock, G. . The Laboratory Mouse. , 527-542 (2004).
  17. Breton, G., Lee, J., Liu, K., Nussenzweig, M. C. Defining human dendritic cell progenitors by multiparametric flow cytometry. Nature protocols. 10, 1407-1422 (2015).
  18. Kaminski, D. A., Wei, C., Rosenberg, A. F., Lee, F. E. -. H., Sanz, I. Multiparameter Flow Cytometry and Bioanalytics for B Cell Profiling in Systemic Lupus Erythematosus. Methods in molecular biology. 900, 109-134 (2012).
  19. Bayer, J., Grunwald, D., Lambert, C., Mayol, J. F., Maynadie, M. Thematic workshop on fluorescence compensation settings in multicolor flow cytometry. Cytometry. Part B, Clinical cytometry. 72, 8-13 (2007).
  20. Newrzela, S., et al. T-cell receptor diversity prevents T-cell lymphoma development. Leukemia. 26, 2499-2507 (2012).
  21. Iwasaki, N., et al. Allergen endotoxins induce T-cell-dependent and non-IgE-mediated nasal hypersensitivity in mice. J Allergy Clin Immunol. 139, 258-268 (2017).
  22. Tsuchiya, K., Siddiqui, S., Risse, P. A., Hirota, N., Martin, J. G. The presence of LPS in OVA inhalations affects airway inflammation and AHR but not remodeling in a rodent model of asthma. American journal of physiology. Lung cellular and molecular physiology. , L54-L63 (2012).
  23. Burgdorf, S., Scholz, C., Kautz, A., Tampe, R., Kurts, C. Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation. Nature. 9, 558-566 (2008).
  24. Last’ovicka, J., Budinsky, V., Spisek, R., Bartunkova, J. Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers. Cellular immunology. , 79-85 (2009).
  25. Oelke, M., et al. Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: comparison with the MHC-tetramer technology. Scand J Immunol. 52, 544-549 (2000).
  26. Wang, W., Golding, B. The cytotoxic T lymphocyte response against a protein antigen does not decrease the antibody response to that antigen although antigen-pulsed B cells can be targets. Immunology letters. 100, 195-201 (2005).
  27. O’Sullivan, D., et al. Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development. Immunity. 41, 75-88 (2014).
  28. Xu, H. C., et al. Type I interferon protects antiviral CD8+ T cells from NK cell cytotoxicity. Immunity. 40, 949-960 (2014).
  29. Volk, A., et al. Comparison of three humanized mouse models for adoptive T cell transfer. The journal of gene medicine. 14, 540-548 (2012).
  30. Safinia, N., et al. Humanized Mice as Preclinical Models in Transplantation. Methods Mol Biol. 1371, 177-196 (2016).
  31. Grover, A., et al. Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis. Immunology. 152, 150-162 (2017).

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Cite This Article
Lirussi, D., Ebensen, T., Schulze, K., Reinhard, E., Trittel, S., Riese, P., Prochnow, B., Guzmán, C. A. Rapid In Vivo Assessment of Adjuvant’s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development. J. Vis. Exp. (136), e57401, doi:10.3791/57401 (2018).

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