流式细胞术 检测 接种乙型脑炎疫苗的儿童多功能T细胞
Summary
本协议结合了 离体 刺激和流式细胞术，以分析日本脑炎病毒（JEV）疫苗接种的儿童中外周血单核细胞（PBMC）中的多功能T细胞（TPF）谱。对JEV特异性TPF的检测方法和流式细胞术配色方案进行了测试，为类似研究提供参考。
Abstract
T细胞介导的免疫在控制黄病毒感染方面起着重要作用，无论是在接种疫苗后还是在自然感染后。T细胞的“质量”需要通过功能来评估，而更高的功能与更强大的免疫保护有关。在单细胞水平上可以同时产生两种或两种以上细胞因子或趋化因子的T细胞称为多功能T细胞（TPFs），其通过多种分子机制介导免疫应答，表达脱颗粒标志物（CD107a）并分泌干扰素（IFN）-γ、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-2或巨噬细胞炎症蛋白（MIP）-1α。越来越多的证据表明，TPFs与维持长期免疫记忆和保护密切相关，其增加的比例是保护性免疫的重要标志物，对有效控制病毒感染和再激活具有重要意义。该评估不仅适用于特异性免疫反应，也适用于交叉反应性免疫反应的评估。本文以乙型脑炎病毒（JEV）为例，对接种乙型脑炎接种患儿外周血单核细胞产生的JEV特异性TPFS的检测方法和流式细胞术配色方案进行了检测，为类似研究提供参考。
Introduction
日本脑炎病毒（JEV）是一种重要的蚊媒病毒，属于黄病毒科1中的黄病毒属。由于乙型脑炎（JE）造成的巨大疾病负担，许多亚太国家长期以来一直面临巨大的公共卫生挑战，但随着各类疫苗接种的增加，这种情况已得到显著改善2。自然感染或疫苗接种引起的适应性保护性免疫应答有助于预防和抗病毒调节。体液免疫和细胞介导免疫被归类为适应性免疫，前者的诱导一直被视为疫苗设计中的关键策略，尽管过去3的理解相对有限。然而，T细胞介导的免疫在限制黄病毒传播和病毒清除方面的作用越来越受到关注和广泛研究4。此外，T细胞免疫不仅在JEV特异性抗病毒反应中不可或缺，而且在交叉保护异源黄病毒继发感染方面也起着重要作用，这已在先前的研究中得到证实5。据推测，这种效应可能会绕过感染中潜在的抗体介导的增强效应5。值得注意的是，这种交叉反应性T细胞免疫很重要，特别是在没有针对黄病毒的疫苗和抗病毒药物的情况下。尽管已经进行了许多研究来确定T细胞在JEV感染中相对于CD4+和CD8 + T细胞的贡献6，7，但分泌细胞因子的各自谱系及其功能多样化仍未确定，这意味着辅助和杀伤性T细胞的确切功能的阐明受到阻碍。
它们的抗病毒防御规模决定了T细胞反应的质量。CD4+或CD8 + T细胞可以相容地赋予两种或多种功能，包括细胞因子分泌和脱颗粒，在单细胞水平8的特异性刺激下被表征为多功能T细胞（TPFs）。产生单个或多种细胞因子的CD4 + T细胞可能具有各种作用和免疫记忆。例如，IL-2+ IFN-γ+ CD4+ T细胞比IL-2+ CD4+ T细胞更容易形成长期有效的保护性反应9，这可以作为评估疫苗接种效果的重要参数。IL-2+ IFN-γ+ CD4 + T细胞的频率在长期不进展的获得性免疫缺陷综合征（AIDS）患者中增加，而由于IL-2对T细胞增殖的促进作用，艾滋病进展患者中CD4 + T细胞更倾向于单独产生IFN-γ10。此外，IL-2+ IFN-γ+ TNF-α +的一个子集被证明可以在体内长期存活并协同促进杀伤功能11。虽然CD8 + T细胞更有可能表现出细胞毒性活性，但一些CD4 + T细胞还具有细胞毒性活性，作为间接检测到表面CD107a分子的表达12。此外，某些T细胞亚群表达趋化因子MIP-1α，其通常由单核细胞分泌以参与T细胞介导的中性粒细胞募集13。同样，CD8+ TPFs也可用于表征上述标志物的多功能性。研究表明，初免-加强策略可以有效诱导长时间的TPF保护作用13，从而增强疫苗接种引起的保护作用。检查免疫系统的一个核心特征是记忆T细胞能够促进比幼稚T细胞更强，更快，更有效地应对继发性病毒攻击。效应记忆T细胞（TEM）和中央记忆T细胞（TCM）是重要的T细胞亚群，通常通过CD27 / CD45RO或CCR7 / CD45RA14的复合表达进行分化。TCM（CD27+ CD45RO+ 或 CCR7+ CD45RA-）倾向于定位在继发性淋巴组织中，而 TEM（CD27- CD45RO+ 或 CCR7- CD45RA–）定位于淋巴和外周组织15，16。TEM提供即时但不是持续的防御，而TCM通过在次级淋巴器官中增殖并产生新的效应物来维持反应17。因此，鉴于记忆细胞可以介导对病毒的特定和有效的回忆反应，关于这一多功能子集的贡献出现了问题。
随着流式细胞术技术的发展，同时检测10多个簇、表型和分化抗原的标志物已变得普遍，有利于更丰富地注释单个T细胞的功能免疫特征，以减少对T细胞表型的误解和理解困难。这项研究使用 离体 刺激和流式细胞术来分析接种JEV疫苗的儿童外周血单核细胞（PBMC）中的TPF 谱。应用这种方法，将扩大对疫苗接种诱导的短期和长期JEV特异性甚至交叉反应性T细胞免疫的理解。
Protocol
Representative Results
Discussion
该协议代表了一种可行的基于流式细胞术的检测方法，用于接种JEV疫苗SA14-14-2的儿童的PBMC中的TPF 谱。本研究使用接种疫苗和未接种疫苗的儿童的静脉血PBMC作为研究材料。随着JEV抗原对PBMC的刺激，那些扩增的抗原特异性TPFs可以通过多色流式细胞术抗体染色进行表征。与传统的酶联免疫点测定方法相比，流式细胞术的突出优势是尽可能多样化地呈现单细胞水平PBMC的功能特征，减少…
Declarações
The authors have nothing to disclose.
Acknowledgements
R.W.得到了中国国家自然科学基金（82002130），中国北京自然科学基金（7222059）的支持。ZD.X.获得CAMS医学科学创新基金（2019-I2M-5-026）的支持。
Materials
| anti-human CD28 | Biolegend | 302934 | Antibody |
| anti-human CD49d | Biolegend | 304339 | Antibody |
| APC anti-human MIP-1α | BD | 551533 | Fluorescent antibody |
| Automated cell counter | BIO RAD | TC20 | Cell count |
| BD FACSymphony A5 | BD | A5 | flow Cytometry |
| BUV395 anti-human CD4 | BD | 563550 | Fluorescent antibody |
| BUV737 anti-human CCR7 | BD | 741786 | Fluorescent antibody |
| BUV737 anti-human CD27 | BD | 612829 | Fluorescent antibody |
| BV421 anti-human CD8 | Biolegend | 344748 | Fluorescent antibody |
| BV480 anti-human CD45RA | BD | 566114 | Fluorescent antibody |
| BV480 anti-human CD45RO | BD | 566143 | Fluorescent antibody |
| BV605 anti-human CD107a | Biolegend | 328634 | Fluorescent antibody |
| BV650 anti-human CD3 | BD | 563999 | Fluorescent antibody |
| BV785 anti-human IL-2 | Biolegend | 500348 | Fluorescent antibody |
| Centrifuge Tube | BD Falcon | BD-35209715 | 15 mL centrifuge tube |
| Cytofix/Cytoperm Fixation/Permeabilization Solution Kit | BD | 554714 | Cell fixation and permeabilization |
| Density gradient medium | Dakewe | DKW-KLSH-0100 | Ficoll-Paque, human lymphocyte separation medium |
| FITC anti-human IFN-γ | Biolegend | 502506 | Fluorescent antibody |
| Gibco Fetal Bovine Serum | Thermo Fisher Scientific | 16000-044 | Fetal Bovine Serum |
| Gibco RPMI-1640 medium | Thermo Fisher Scientific | 22400089 | cell culture medium |
| High-speed centrifuge | Sigma | 3K15 | Cell centrifugation for 15 mL centrifuge tube |
| High-speed centrifuge | Eppendorf | 5424R | Cell centrifugation for 1.5 mL Eppendorf (EP) tube |
| Microcentrifuge tubes | Axygen | MCT-150-C | 1.5 mL microcentrifuge tube |
| PE anti-human TNF-α | Biolegend | 502909 | Fluorescent antibody |
| Phosphate Buffered Saline (PBS) | BI | 02-024-1ACS | PBS |
| Protein Transport Inhibitor (Containing Brefeldin A, GolgiPlug) | BD | 555029 | blocks intracellular protein transport processes |
| Protein Transport Inhibitor (Containing Monensin) | BD | 554724 | blocks intracellular protein transport processes |
| Round-bottom test tube | BD Falcon | 352235 | 5 mL test tube |
| Trypan Blue Staining Cell Viability Assay Kit | Beyotime | C0011 | Trypan Blue Staining |
| Zombie NIR Fixable Viability Dye | Biolegend | 423106 | Dead cell stain |
Referências
- Vanden Eynde, C., Sohier, C., Matthijs, S., De Regge, N. Japanese encephalitis virus interaction with mosquitoes: A review of vector competence, vector capacity and mosquito immunity. Pathogens. 11 (3), 317 (2022).
- Wang, R., et al. The epidemiology and disease burden of children hospitalized for viral infections within the family Flaviviridae in China: A national cross-sectional study. PLoS Neglected Tropical Diseases. 16 (7), 0010562 (2022).
- Wang, R., et al. Decreases in both the seroprevalence of serum antibodies and seroprotection against Japanese encephalitis virus among vaccinated children. Virologica Sinica. 34 (3), 243-252 (2019).
- Wang, R., et al. Neutralizing antibody rather than cellular immune response is maintained for nearly 20 years among Japanese encephalitis SA14-14-2 vaccinees in an endemic setting. Infection, Genetics and Evolution. 85, 104476 (2020).
- Wang, R., et al. T cell immunity rather than antibody mediates cross-protection against Zika virus infection conferred by a live attenuated Japanese encephalitis SA14-14-2 vaccine. Applied Microbiology and Biotechnology. 104 (15), 6779-6789 (2020).
- Redant, V., Favoreel, H. W., Dallmeier, K., Van Campe, W., De Regge, N. Japanese encephalitis virus persistence in porcine tonsils is associated with a weak induction of the innate immune response, an absence of IFNgamma mRNA expression, and a decreased frequency of CD4(+)CD8(+) double-positive T cells. Frontiers in Cellular and Infection Microbiology. 12, 834888 (2022).
- Jain, N., et al. CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function. PLoS Neglected Tropical Diseases. 11 (2), 0005329 (2017).
- Khakhum, N., Bharaj, P., Walker, D. H., Torres, A. G., Endsley, J. J. Antigen-specific antibody and polyfunctional T cells generated by respiratory immunization with protective Burkholderia DeltatonB Deltahcp1 live attenuated vaccines. NPJ Vaccines. 6 (1), 72 (2021).
- Weaver, J. M., et al. Increase in IFNgamma(-)IL-2(+) cells in recent human CD4 T cell responses to 2009 pandemic H1N1 influenza. PloS One. 8 (-), 57275 (2013).
- Boaz, M. J., Waters, A., Murad, S., Easterbrook, P. J., Vyakarnam, A. Presence of HIV-1 Gag-specific IFN-gamma+IL-2+ and CD28+IL-2+ CD4 T cell responses is associated with nonprogression in HIV-1 infection. Journal of Immunology. 169 (11), 6376-6385 (2002).
- Gui, L., et al. IL-2, IL-4, IFN-gamma or TNF-alpha enhances BAFF-stimulated cell viability and survival by activating Erk1/2 and S6K1 pathways in neoplastic B-lymphoid cells. Cytokine. 84, 37-46 (2016).
- Terahara, K., et al. Vaccine-induced CD107a+ CD4+ T cells are resistant to depletion following AIDS virus infection. Journal of Virology. 88 (24), 14232-14240 (2014).
- Tanyi, J. L., et al. Personalized cancer vaccine strategy elicits polyfunctional T cells and demonstrates clinical benefits in ovarian cancer. NPJ Vaccines. 6 (1), 36 (2021).
- Ammirati, E., et al. Effector memory T cells are associated with atherosclerosis in humans and animal models. Journal of the American Heart Association. 1 (1), 27-41 (2012).
- Rizk, N. M., Fadel, A., AlShammari, W., Younes, N., Bashah, M. The immunophenotyping changes of peripheral CD4+ T lymphocytes and inflammatory markers of class III obesity subjects after laparoscopic gastric sleeve surgery – A follow-up study. Journal of Inflammation Research. 14, 1743-1757 (2021).
- Zhang, Y., et al. Phenotypic and functional characterizations of CD8(+) T cell populations in malignant pleural effusion. Experimental Cell Research. 417 (1), 113212 (2022).
- Shin, H., Iwasaki, A. Tissue-resident memory T cells. Immunological Reviews. 255 (1), 165-181 (2013).
- Birnie, K. A., Noel, M., Chambers, C. T., Uman, L. S., Parker, J. A. Psychological interventions for needle-related procedural pain and distress in children and adolescents. Cochrane Database of Systematic Reviews. 10 (10), (2018).
- Lin, R. J., Liao, C. L., Lin, Y. L. Replication-incompetent virions of Japanese encephalitis virus trigger neuronal cell death by oxidative stress in a culture system. Journal of General Virology. 85, 521-533 (2004).
- Byford, E., Carr, M., Pinon, L., Ahearne, M. J., Wagner, S. D. Isolation of CD4+ T-cells and analysis of circulating T-follicular helper (cTfh) cell subsets from peripheral blood using 6-color flow cytometry. Journal of Visualized Experiments. (143), e58431 (2019).
- Zheng, X., et al. Immune responses and protective effects against Japanese encephalitis induced by a DNA vaccine encoding the prM/E proteins of the attenuated SA14-14-2 strain. Infection, Genetics and Evolution. 85, 104443 (2020).
- Zheng, X., et al. Complete protection for mice conferred by a DNA vaccine based on the Japanese encephalitis virus P3 strain used to prepare the inactivated vaccine in China. Virology Journal. 17 (1), 126 (2020).
- Lam, J. K. P., et al. Emergence of CD4+ and CD8+ polyfunctional T cell responses against immunodominant lytic and latent EBV antigens in children with primary EBV infection. Frontiers in Microbiology. 9, 416 (2018).
- Meckiff, B. J., et al. Imbalance of regulatory and cytotoxic SARS-CoV-2-reactive CD4(+) T cells in COVID-19. Cell. 183 (5), 1340-1353 (2020).
- Ning, R. J., Xu, X. Q., Chan, K. H., Chiang, A. K. Long-term carriers generate Epstein-Barr virus (EBV)-specific CD4(+) and CD8(+) polyfunctional T-cell responses which show immunodominance hierarchies of EBV proteins. Immunology. 134 (2), 161-171 (2011).
