我们感谢 Mads Radmer 詹森的实际和学术建议;刘 Lindqvist 和 Kaare Svejstrup 提供技术专长;海琳 Bæk Juel, 乔纳森 Filskov, Signe t. 施密特, Solveig W Harpøth 为他们的技术援助;Gentofte 结核病门诊诊所的工作人员在 PPD 管理方面的培训;和所有参加这项研究的志愿者。该项目由欧洲联盟委员会 H2020 计划 (赠款编号 TBVAC2020 643381) 提供资金, 我们要感谢联盟伙伴进行富有成果的讨论。

下文所述的所有方法, 包括使用人类志愿人员, 已得到丹麦卫生研究伦理学委员会 (H-15002988) 和丹麦数据保护局 (jr. nr 2015-57-0102) 的批准。PPD 必须是经认证的产品, 供人使用, 并在制造商提供的正确剂量内进行管理。任何剂量或管理上的偏差可能需要额外的道德认证, 志愿者必须给予知情同意。
1. 结核菌素皮肤试验
<ol><li>获得志愿者的口头和书面知情同意。在注射前, 要确保志愿者理解并接受程序, 包括可能产生的不良影响。 注: 本议定书特有的纳入标准为年龄 > 18 年, 并有记录的卡介苗接种。纳入标准可能因研究问题而异 (也就是说, 附加的纳入标准可能是正尖沙咀的证据)。</li>
	<li>使用可供人类使用的 PPD 的现成解决方案 [20 个结核菌素单位 (TU)/毫升]。使用酒精拭子对瓶子的橡皮帽进行消毒。</li>
	<li>准备注塑现场<ol>
<li>找到志愿者前臂腹侧的注射部位。</li>
		<li>将手臂手掌放在平坦的表面上, 选择前臂上部第三的区域, 从肘关节处大约 5-10 厘米。远离疤痕, 静脉, 或受损的皮肤。</li>
		<li>使用酒精拭子消毒该区域。</li>
	</ol>
</li>
	<li>准备 PPD 解决方案<ol>
<li>使用1毫升无菌注射器与 27-30 克/短 (如12 毫米) 固定针。</li>
		<li>轻轻地混合并渴望0.1 毫升的 PPD 溶液。确保没有可见气泡。</li>
	</ol>
</li>
	<li>皮下 PPD 注射液 注意: 此步骤描述如何执行单个 ppd 沉积, 但可能会在同一臂中对 ppd 进行多次证言。然而, 这可能需要特定的道德认同。<ol>
<li>伸展的皮肤, 并指出针在 5-的角度对皮肤与斜面朝上向上。</li>
		<li>将针的尖端插入皮肤的真皮层, 并确保通过表皮几乎可见尖端。</li>
		<li>慢慢注射0.1 毫升的 PPD 溶液。 注: 如果正确管理, 将立即出现 6-10 毫米的苍白丘疹。丘疹在大约10分钟以后消失。</li>
		<li>当进行两个或更多的证词, 目的是最大的分离 (5-10 厘米) 的注射部位, 同时保持良好的距离, 从肘部和手腕地区。 注意: 这可以防止皮肤测试响应的潜在汇合, 并允许两个吸入腔的不间断定位。</li>
	</ol>
</li>
</ol>2. 皮肤反应的评估-天3
<ol><li>48-72 小时后, 注意皮肤反应的大小。测量硬化 (肿胀) 而不是反应的红斑 (红肿)。</li>
	<li>用手指触及硬肿胀, 然后用圆珠笔标记硬化的边缘。使用标尺测量硬化直径并且注意结果在毫米。 注: 对硬化尺寸的读数可指导吸入腔孔直径的选择。</li>
</ol>3. 吸塑诱导-天7
<ol><li>装配吸尘装置单元。 注: 吸入装置单位为抽吸吸塑用附腔的真空泵。此演示中使用的设备是自定义的, 但吸设备单元也可在商业上使用。泵需要在-20 至-40 帕的范围内进行调节, 并提供稳定可靠的负压。在使用该方法进行实验之前, 应先获得区域伦理认同。<ol>
<li>首先, 将底部孔板和窗板与腔室和连接软管装配在一起, 准备吸尘室。将室连接软管与真空泵紧密连接。 注: 分庭应包括一个底板与一个孔的吸塑感应, 这应该是适合与人的皮肤接触, 和一个顶部板与窗口, 允许视觉监测的水泡。</li>
	</ol>
</li>
	<li>确定孔板的最佳孔直径相对于皮肤反应的大小;孔的尺寸越大, 水泡越大。</li>
	<li>用酒精拭子对志愿者的孔板和皮肤进行消毒。</li>
	<li>将吸入室连接到皮肤上。<ol>
<li>请确保志愿者的手臂舒适地休息。</li>
		<li>确保吸入室底板的孔位于 PPD 反应之上, 使皮肤反应中心可见。</li>
		<li>用皮带松散地固定房间: 当施加负压时, 腔室会附着在皮肤上, 保持其位置。</li>
	</ol>
</li>
	<li>打开吸入装置, 并调整压力为-20 帕。</li>
	<li>30分钟后, 将负压增加至-25 帕。</li>
	<li>60分钟后, 进一步增加负压至-30 帕。</li>
	<li>保持-30 帕的压力, 直到水泡完全成型。确保本协议中提供的负面压力特定于定制的仪器。在其他设置中应用协议时, 可能需要稍微调整压力。 注: 吸塑感应相的范围从60到180分钟 (1-3 小时), 实际水泡形成在最近30分钟内发生. 起泡通常与瘙痒感有关。水泡可能发生作为单一或多次合并水泡。如果水泡破裂或出血发生, 建议通过缓慢释放压力来终止水泡诱导。</li>
	<li>水泡完全成型后, 释放压力, 并小心地从皮肤上取出吸入腔, 而不破裂水泡。</li>
	<li>在周围的皮肤区域应用一个软敷料, 并放置一个硬帽 (例如, 从50毫升塑料管) 在水泡。</li>
	<li>用不过敏的手术胶带和柔软的弹力绷带保护瓶盖。</li>
	<li>指导志愿者在 12-24 小时内避免水泡区过度的身体压力。</li>
</ol>4. 吸塑液的收获-天8
<ol><li>在8天 , 轻轻地去除保护敷料和消毒水泡与皮肤消毒喷雾。</li>
	<li>使用一个2毫升无菌注射器与23G 针, 以收获水泡内容。将针头插入吸塑屋顶的顶部/侧面, 慢慢地吸入液体。避免接触洞的地板, 但要确保收获所有的液体。</li>
	<li>在折叠的水泡上涂抹敷料。</li>
	<li>将水泡液转移到无菌管上。记下音量。</li>
	<li>用台式离心机在 600 x g 处旋转吸塑液4分钟。</li>
	<li>将上清液转移到另一管, 并在细胞培养基的500µL 中并用重悬细胞颗粒。 注意: 细胞现在已经准备好进行计数和进一步分析。上清液可以存储在-20 到-80 °c, 直到使用。</li>
</ol>5. 吸塑细胞和液体的分析
注: 为分析皮肤吸入水泡所获得的细胞和液体提供指导不属于本议定书的范围。然而, 为了提供本报告的代表性结果, 进行了细胞内染色流式细胞术和多路分析。下文简述了这一方法。
<ol><li>流式细胞仪<ol>
<li>刺激 1 x 105新鲜细胞从吸入水泡分离的悬浮从1卡介苗接种志愿者与10µg 或毫升 PPD 并且孵化细胞在37°c 和 5% CO2.包括静态细胞进行平行孵化。</li>
		<li>刺激悬浮液 1 x 106 PBMCs 获得从1卡介苗接种志愿者与10µg/毫升的 PPD 和孵化混合物在37°c 和 5% CO2.包括静态细胞进行平行孵化。</li>
		<li>2小时后, 用 Brefaldin A 和莫能治疗所有细胞, 并在37°c 孵育6小时。</li>
		<li>用 Live/Dead-stain-BV510、anti-CD4-AF780、anti-CD3-BV421、反 CD8 PerCP Cy5.5、anti-CCR7-PE、anti-CD45RA-BV786、anti-IFN-γ-AF700、anti-TNF-α-PE-Cy7 和 anti-IL-2-FITC 的面板染色细胞。</li>
		<li>在 PBMC 面板中包括 FMO 控件。</li>
		<li>用流式细胞仪进行流细胞分析, 并使用相关软件对结果进行分析。</li>
		<li>门上的细胞因子产生 CD3+CD4+CD8-亚群使用以下的浇口策略: 汗衫-> 可行的 CD3+ > CD4+CD8-> 干扰素-γ +, TNF α +, 或 IL-2+。</li>
		<li>利用以下门控策略对效应器记忆细胞进行闸门: 汗衫 > 可行 CD3+ > CD4+CD8 > CCR7-CD45RA。</li>
		<li>使用布尔浇口计算单个细胞因子剖面。</li>
	</ol>
</li>
	<li>细胞因子释放演示<ol>
<li>为了演示体内细胞因子释放, 使用多重分析来量化4名志愿者在解冻吸泡液中 IL-2、干扰素、TNF α、IL-10 和 IL-13 的含量。</li>
		<li>为演示细胞因子释放由吸入水泡前体从体内获得, 使用新鲜吸塑细胞和 PBMCs 获得1卡介苗接种志愿者。</li>
		<li>感染 PBMC 和吸入水泡细胞与100个集落形成单位 (CFU) 的山地车 H37Rv 和孵化他们4天。</li>
		<li>使用多重分析来量化 IL-2、干扰素γ、TNF α、IL-10 和 IL-13 在所有文化上清液中的水平。</li>
		<li>将测定计算范围以上的测量值调整到上限计算极限。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Kiistala, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suction+blister+device+for+separation+of+viable+epidermis+from+dermis.">Suction blister device for separation of viable epidermis from dermis.</a> Journal of Investigative Dermatology. 50 (2), 129-137 (1968).</li><li>Akbar, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+cutaneous+response+to+recall+antigen+in+humans+in+vivo.">Investigation of the cutaneous response to recall antigen in humans in vivo.</a> Clinical &amp; Experimental Immunology. 173 (2), 163-172 (2013).</li><li>Hatje, L. K., Richter, C., Blume-Peytavi, U., Kottner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blistering+time+as+a+parameter+for+the+strength+of+dermoepidermal+adhesion:+a+systematic+review+and+meta-analysis.">Blistering time as a parameter for the strength of dermoepidermal adhesion: a systematic review and meta-analysis.</a> British Journal of Dermatology. 172 (2), 323-330 (2015).</li><li>Smith, T. J., Wilson, M. A., Young, A. J., Montain, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suction+blister+model+reliably+assesses+skin+barrier+restoration+and+immune+response.">A suction blister model reliably assesses skin barrier restoration and immune response.</a> Journal of Immunological Methods. 417, 124-130 (2015).</li><li>Maranda, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+management+of+leukoderma+after+burn:+A+review.">Surgical management of leukoderma after burn: A review.</a> Burns: Journal of the International Society of Burn Injuries. 44 (2), 256-262 (2017).</li><li>Wilhelm, K. P., Wilhelm, D., Bielfeldt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+wound+healing:+an+emphasis+on+clinical+studiea.">Models of wound healing: an emphasis on clinical studiea.</a> Skin Research and Technology: Official Journal of the International Society for Bioengineering and the Skin (ISBS) and the International Society for Digital Imaging of Skin (ISDIS) and the International Society for Skin Imaging (ISSI). 23 (1), 3-12 (2017).</li><li>Maini, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+Human+Neutrophils+Acquired+from+Four+Experimental+Models+of+Inflammation.">A Comparison of Human Neutrophils Acquired from Four Experimental Models of Inflammation.</a> PLoS ONE. 11 (10), (2016).</li><li>Kenney, R. T., Rangdaeng, S., Scollard, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+blister+immunocytology.+A+new+method+to+quantify+cellular+kinetics+in+vivo.">Skin blister immunocytology. A new method to quantify cellular kinetics in vivo.</a> Journal of Immunological Methods. 97 (1), 101-110 (1987).</li><li>Carvalho, J. C. O., Palero, J. A., Jurna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+suction+blistering+in+human+skin+using+optical+coherence+tomography.">Real-time imaging of suction blistering in human skin using optical coherence tomography.</a> Biomedical Optics Express. 6 (12), 4790-4795 (2015).</li><li>Belson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+the+clinical+and+activated+T+cell+response+to+repeat+delayed-type+hypersensitivity+skin+challenges+in+human+subjects,+with+KLH+and+PPD,+as+a+potential+model+to+test+T+cell-targeted+therapies.">Characterisation of the clinical and activated T cell response to repeat delayed-type hypersensitivity skin challenges in human subjects, with KLH and PPD, as a potential model to test T cell-targeted therapies.</a> Inflammation Research: Official Journal of the European Histamine Research Society ... [et al.]. 65 (5), 389-404 (2016).</li><li>Vukmanovic-Stejic, M., Reed, J. R., Lacy, K. E., Rustin, M. H. A., Akbar, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mantoux+Test+as+a+model+for+a+secondary+immune+response+in+humans.">Mantoux Test as a model for a secondary immune response in humans.</a> Immunology Letters. 107 (2), 93-101 (2006).</li><li>Thakur, A., Pedersen, L. E., Jungersen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+markers+and+correlates+of+protection+for+vaccine+induced+immune+responses.">Immune markers and correlates of protection for vaccine induced immune responses.</a> Vaccine. 30 (33), 4907-4920 (2012).</li><li>CDC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> TB | Fact Sheets - Tuberculin Skin Testing for TB. , (2018).</li><li>Vukmanovic-Stejic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Varicella+Zoster-Specific+CD4+Foxp3++T+Cells+Accumulate+after+Cutaneous+Antigen+Challenge+in+Humans.">Varicella Zoster-Specific CD4+Foxp3+ T Cells Accumulate after Cutaneous Antigen Challenge in Humans.</a> The Journal of Immunology. 190 (3), 977-986 (2013).</li><li>Reed, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+erosion+in+memory+T+cells+induced+by+telomerase+inhibition+at+the+site+of+antigenic+challenge+in+vivo.">Telomere erosion in memory T cells induced by telomerase inhibition at the site of antigenic challenge in vivo.</a> The Journal of Experimental Medicine. 199 (10), 1433-1443 (2004).</li><li>Tatovic, D., Young, P., Kochba, E., Levin, Y., Wong, F. S., Dayan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine-Needle+Aspiration+Biopsy+of+the+Lymph+Node:+A+Novel+Tool+for+the+Monitoring+of+Immune+Responses+after+Skin+Antigen+Delivery.">Fine-Needle Aspiration Biopsy of the Lymph Node: A Novel Tool for the Monitoring of Immune Responses after Skin Antigen Delivery.</a> The Journal of Immunology. 195 (1), 386-392 (2015).</li><li>Bond, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmacytoid+dendritic+cells+infiltrate+the+skin+in+positive+tuberculin+skin+test+indurations.">Plasmacytoid dendritic cells infiltrate the skin in positive tuberculin skin test indurations.</a> Journal of Investigative Dermatology. 132 (1), 114-123 (2012).</li><li>Bell, L. C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Molecular+Dissection+of+the+Effects+of+HIV-1+in+Active+Tuberculosis.">In Vivo Molecular Dissection of the Effects of HIV-1 in Active Tuberculosis.</a> PLoS Pathogens. 12 (3), e1005469 (2016).</li><li>Tomlinson, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+innate+and+adaptive+human+immune+responses+to+mycobacteria+in+the+tuberculin+skin+test.">Transcriptional profiling of innate and adaptive human immune responses to mycobacteria in the tuberculin skin test.</a> European Journal of Immunology. 41 (11), 3253-3260 (2011).</li><li>Nayak, S., Acharjya, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mantoux+test+and+its+interpretation.">Mantoux test and its interpretation.</a> Indian Dermatology Online Journal. 3 (1), 2-6 (2012).</li><li>Vukmanovic-Stejic, M., Reed, J. R., Lacy, K. E., Rustin, M. H. A., Akbar, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mantoux+Test+as+a+model+for+a+secondary+immune+response+in+humans.">Mantoux Test as a model for a secondary immune response in humans.</a> Immunology Letters. 107 (2), 93-101 (2006).</li><li>Menzies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+of+Repeated+Tuberculin+Tests.">Interpretation of Repeated Tuberculin Tests.</a> American Journal of Respiratory and Critical Care Medicine. 159 (1), 15-21 (1999).</li><li>Pitzalis, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+migration+of+the+human+helper-inducer+memory+T+cell+subset:+confirmation+by+in+vivo+cellular+kinetic+studies.">Selective migration of the human helper-inducer memory T cell subset: confirmation by in vivo cellular kinetic studies.</a> European Journal of Immunology. 21 (2), 369-376 (1991).</li><li>Kool, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suction+blister+fluid+as+potential+body+fluid+for+biomarker+proteins.">Suction blister fluid as potential body fluid for biomarker proteins.</a> Proteomics. 7 (20), 3638-3650 (2007).</li><li>Flynn, J. L., Chan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+tuberculosis.">Immunology of tuberculosis.</a> Annual Review of Immunology. 19, 93-129 (2001).</li><li>Kagina, B. M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+T+cell+frequency+and+cytokine+expression+profile+do+not+correlate+with+protection+against+tuberculosis+after+bacillus+Calmette-Gu&#233;rin+vaccination+of+newborns.">Specific T cell frequency and cytokine expression profile do not correlate with protection against tuberculosis after bacillus Calmette-Gu&#233;rin vaccination of newborns.</a> American Journal of Respiratory and Critical Care Medicine. 182 (8), 1073-1079 (2010).</li><li>Garc&#237;a-Basteiro, A. L., Ruhwald, M., Lange, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+tuberculosis+vaccine+trials+under+financial+constraints.">Design of tuberculosis vaccine trials under financial constraints.</a> Expert Review of Vaccines. 15 (7), 799-801 (2016).</li><li>Luabeya, A. K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-in-human+trial+of+the+post-exposure+tuberculosis+vaccine+H56:IC31+in+Mycobacterium+tuberculosis+infected+and+non-infected+healthy+adults.">First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected healthy adults.</a> Vaccine. 33 (33), 4130-4140 (2015).</li><li>Tameris, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+candidate+TB+vaccine,+MVA85A,+induces+highly+durable+Th1+responses.">The candidate TB vaccine, MVA85A, induces highly durable Th1 responses.</a> PloS One. 9 (2), e87340 (2014).</li><li>Woodworth, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+vaccine+H56/CAF01+induces+a+population+of+circulating+CD4+T+cells+that+traffic+into+the+Mycobacterium+tuberculosis-infected+lung.">Subunit vaccine H56/CAF01 induces a population of circulating CD4 T cells that traffic into the Mycobacterium tuberculosis-infected lung.</a> Mucosal Immunology. 10 (2), 555-564 (2016).</li><li>Gideon, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+in+tuberculosis+granuloma+T+cell+responses+exists,+but+a+balance+of+pro-+and+anti-inflammatory+cytokines+is+associated+with+sterilization.">Variability in tuberculosis granuloma T cell responses exists, but a balance of pro- and anti-inflammatory cytokines is associated with sterilization.</a> PLoS Pathogens. 11 (1), e1004603 (2015).</li><li>Ruhwald, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+the+C-Tb+skin+test+to+diagnose+Mycobacterium+tuberculosis+infection,+compared+with+an+interferon+&#947;+release+assay+and+the+tuberculin+skin+test:+a+phase+3,+double-blind,+randomised,+controlled+trial.">Safety and efficacy of the C-Tb skin test to diagnose Mycobacterium tuberculosis infection, compared with an interferon &#947; release assay and the tuberculin skin test: a phase 3, double-blind, randomised, controlled trial.</a> The Lancet Respiratory Medicine. 5 (4), 259-268 (2017).</li><li>He, X., de Oliveira, V. L., Keijsers, R., Joosten, I., Koenen, H. J. P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+Isolation+from+Human+Skin+for+Phenotypic+Analysis+and+Ex+Vivo+Cell+Culture.">Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture.</a> Journal of Visualized Experiments. (110), e52564 (2016).</li><li>Koskela, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blister+fluid+and+serum+cytokine+levels+in+severe+sepsis+in+humans+reflect+skin+dysfunction.">Blister fluid and serum cytokine levels in severe sepsis in humans reflect skin dysfunction.</a> Acta Anaesthesiologica Scandinavica. 61 (1), 53-61 (2017).</li><li>Wada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+skin+blister+fluids+from+children+with+Epstein-Barr+virus-associated+lymphoproliferative+disease.">Characterization of skin blister fluids from children with Epstein-Barr virus-associated lymphoproliferative disease.</a> The Journal of Dermatology. 45 (4), 444-449 (2018).</li><li>Andersson, C., Rajala, T., S&#228;rkk&#228;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+models+for+epidermal+nerve+fiber+data.">Hierarchical models for epidermal nerve fiber data.</a> Statistics in Medicine. 37 (3), 357-374 (2018).</li><li>Ramshanker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Prednisolone+on+Serum+and+Tissue+Fluid+IGF-I+Receptor+Activation+and+Post-Receptor+Signaling+in+Humans.">Effects of Prednisolone on Serum and Tissue Fluid IGF-I Receptor Activation and Post-Receptor Signaling in Humans.</a> The Journal of Clinical Endocrinology &amp; Metabolism. 102 (11), 4031-4040 (2017).</li><li>Bouma, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL20+neutralization+by+a+monoclonal+antibody+in+healthy+subjects+selectively+inhibits+recruitment+of+CCR6++cells+in+an+experimental+suction+blister.">CCL20 neutralization by a monoclonal antibody in healthy subjects selectively inhibits recruitment of CCR6+ cells in an experimental suction blister.</a> British Journal of Clinical Pharmacology. 83 (9), 1976-1990 (2017).</li></ol>

作者没有什么可透露的。

这篇手稿描述了一个实用程序, 研究人体免疫记忆反应在体内, 利用皮肤抗原召回和细胞收获的吸入水泡诱导。尖沙咀被用作皮抗原沉积和 BCG 疫苗召回的一个例子。最后, 通过流细胞和多重细胞因子分析, 给出了 sb 标本的一个例子, 表明约三个细胞是效应记忆表型的抗原特异多功能 T 细胞。
该协议的关键步骤包括皮下注射技术、吸塑诱导和吸塑穿刺。首先, 正确的皮下沉积需要训练有素的人员。错误的沉积可能导致不理想的结果。PPD 通常是一种众所周知的安全的皮肤抗原, 但其组成可能不同制造商之间, 限制可比性13,20。这份报告提供了一个单一的皮下沉积2涂, 可选的两个平行证言 (2 x 2 涂), 这可能需要额外的道德批准。然而, 其他研究使用了 10 TU, 增加了强烈皮肤反应的可能性10,21。在诱导阶段, 负压小逐步增加会降低出血和水泡破裂的风险。从血液中感染红细胞或白血病的几率一般低2。无菌穿刺技术可防止微生物污染, 避免穿刺针与真皮吸尘地板之间的接触, 减少碎片或居民皮肤细胞的杂质。然而, 一些研究人员更喜欢用轧制压力对穿刺水泡10来收割锑液。可能有必要刺穿水泡内的隔膜。某人的技术本身是微创的;折叠 SBs 愈合没有疤痕和感染是非常罕见的。2然而, 有一定程度的亚色素沉着可能发生, 并应避免诱导某人在具有胶体疤痕史的人。
技术限制包括低总电池产量, 因此长期储存的选择有限。白细胞产量, 临床尖沙咀反应, 水泡大小和红细胞污染之间的关系已被彻底描述在别处2,10。在这里提出的卡介苗召回实验 (图 1) 中, 每个水泡的平均总产量为 5万, 这意味着使用这些细胞进行小规模实验, 特别是如果 SBCs 单独研究15。然而, 在某一样品中, 特定的 T 细胞种群在相对和绝对细胞计数中的 PBMC 样本中的数量大多超过了所发现的。在图 2所示的示例中, 在 10万 SB 细胞样本中, 三阳性 PPD 特定 T 细胞的数量超过了同一个捐献者 1 x 106 PBMCs 中发现的数字的 2x (未显示数据)。尖沙咀反应的视觉评分是评价结核记忆的最常用的临床方法。值得注意的是, 潜在的适应性免疫反应并不与临床皮肤反应2,21的同时峰值。并非所有卡介苗接种或结核分枝杆菌暴露的个人都将制定强有力的尖沙咀反应和分类战略, 并处理来自尖沙咀非应答者的样本, 以及在研究人群中预先存在的结核杆菌敏感度,需要在使用此方法启动召回试验之前考虑。此外, 在某人的抽样和视觉评分, 有一个潜在的理论偏见, 皮肤反应减少, 由于全球或皮肤相关的免疫力受损, 如所见, 例如, 在艾滋病毒感染和某些年龄组2, 13,18,20。此外, 对尖沙咀反应的免疫促进与反复试验是众所周知的20,22。然而, 当 TSTs 重复10时, 某人的细胞表型保持不变。这一观察支持了某人在细胞免疫反应的纵向研究中所起的作用。
对于 T 细胞位免疫学家, 某人召回允许获得高分数的抗原特异细胞。然而, 当细胞成分和细胞因子微环境随着时间的推移而变化时, 某人从 PPD 沉积取样的时间是至关重要的。在本议定书中, 水泡被诱导7天后的挑战和孤立的细胞主要是 CD4+ 效应记忆 T 细胞, 包括高分数的细胞与干扰素-γ, TNF α, 和 IL-2 的共同表达。这些观察与以前的研究是一致的, 描述了 T 细胞的活化、增殖和分化发生在 PPD 引皮皮肤2,15,21。在 PPD 敏感的个体中, 某人的研究表明早期皮肤反应是不特异的;然而, 已经在 PPD 挑战后的第一天, 反应成为主导的 CD4+ T 细胞 (中央记忆和效应记忆表型被描述), 并且, 在3天以后, 反应由高分数控制PPD 特异的细胞因子产生 CD4+ T 细胞2,8,10,15,21。这种自适应细胞因子反应仍可检测2周2,8,10,23。7天后尖沙咀似乎是最理想的时间点收集细胞因子产生的记忆 CD4+ T 细胞2。然而, 其他时间点, 当然, 可能是相关的, 取决于抗原和反应的兴趣。值得注意的是, 在一项研究中, 自适应 PPD 反应已被报告为较早的峰值, 在5天后尖沙咀10的炎症细胞因子分泌减少。
液中含有高浓度的促炎性细胞因子和其他蛋白, 表现为皮肤微环境的15、24。动力学研究表明, 肿瘤坏死因子α和干扰素-γ水平在3天后, IL-2 水平峰值后7天2,10, 可能反映的优势, 自适应反应在这个后期阶段。由于某人细胞在体内被激活, 它们表现出高度自发的细胞因子释放, 以及在 restimulation 上的特定释放的可能性 (图 3和4)。
本报告的重点是 CD4+ T 细胞免疫。如图 2所示, 大多数孤立的 t 细胞确实是 CD4+ 的, 而吸入水泡液中几乎没有 CD8+ t 细胞。这符合其他人的 PPD 召回研究报告低比例和较低的迁徙能力的 CD8+ t 细胞相比, CD4+ t 细胞2,8,10,23。进一步描述 CD8+ 的贡献是可取的;但是, 这超出了本报告的范围。
T 细胞被认为是重要的免疫控制结核分枝杆菌;然而, 很难确定从血液25,26的适应性免疫反应中反映出的保护的可靠关联。这一路障严重阻碍了新的结核病疫苗的开发, 因为目前没有替代大型和非常昂贵的药效试验27。结核病疫苗开发商通过评估血液中28、29的疫苗特定 T 细胞种群的微小和瞬变变化来确定疫苗的免疫原性。然而, 在血液中发现的循环抗原特异性 T 细胞的小部分是否相关 (即能够迁移到感染部位并代表 T 细胞丰富的微环境控制) 是值得怀疑的。山地车)27,30,31.
根据先前的研究和本文所提供的数据, 某人皮肤召回模型代表了研究疫苗特异 T 细胞的未开发的潜力。这一模型不仅能够召回几十年前产生的疫苗反应, 而且还允许对特定于组织的上下文15、18、19、21中的真实记忆潜能进行评估。新的, 具体的皮肤测试, 其中包括抗原也由候选亚单位结核疫苗, 提出了新的机会, 疫苗评估使用这个模型28,32。此外, transcriptomic 分析表明, 在 PPD 挑战皮肤中产生的细胞介导的免疫应答类似于在结核菌感染的肺18中发现的反应。
虽然皮肤穿孔活检也允许从皮肤的细胞收获和提供空间信息, 与 SB 抽样相比, 这种方法更具侵入性, 需要酶或机械加工来制备单细胞悬浮液33。细胞因子和细胞标记的测量在两种方法2,10之间是可比较的。
吸塑法已经被应用于医学研究的许多领域, 除了皮肤病学, 单独或结合全身或局部皮肤挑战。例子包括脓毒症, eb 病毒相关的淋巴疾病, 糖尿病神经病, 糖皮质激素摄入的影响, 和人类试验测试治疗抗体或 T 细胞靶向治疗模型10 ,34,35,36,37,38。
从治疗的角度来看, 某人的皮肤召回方法为研究中心 T 细胞记忆潜能提供了独特的优势, 从生物学和技术的角度来看, 皮肤似乎提供了一个相关的取样室2, 15,18,19,21。特别是, 与传统的, 被动抽样的循环 PBMCs, 人皮肤召回方法, 以研究的 T 细胞已证明有能力从淋巴结迁移, 以响应其相关的抗原, 并完成当地在体内的组织特异的扩张和分化15,18,19,21。
总之, 这里所展示的模型可能与人类自适应免疫和 T 细胞靶向剂 (即传染病疫苗或癌症研究) 领域的研究人员相关。当然, 在本议定书中应用的尖沙咀模型在结核病疫苗领域具有特殊的相关性。但是, 该模型的基本概念在其他研究领域也有很高的应用价值。

皮肤某人是人为诱发的水泡, 它允许从皮肤直接获得细胞和液体。采用真空法提高 SBs 的技术, 是皮肤病学领域的一个知名工具, 用于研究健康和疾病1、2、3、4、5、6,7,8. 本报告展示了如何将某一技术与皮肤抗原召回 (某人皮肤召回方法) 结合起来, 可以直接洞察体内的适应性免疫反应。
感应器背后的原理很简单: 轻真空被应用到皮肤的一个小区域。负压最终会迫使表皮与真皮分离, 造成局部水泡充满液体和细胞1,2,9。吸塑液可通过细针抽吸得到, 其含量可用于体外进一步研究。
近年来, 在人体免疫应答研究中, 除了受限于皮肤10、11的疾病外, 人们对该方法的兴趣越来越大。由于对淋巴结或胃肠粘膜组织的侵入性取样可能是不可接受和不道德的, 因此对人体自适应免疫的研究受到了来自外周血液取样的细胞和细胞因子的限制。一个例子是研究长寿命的人类记忆 T 细胞反应后接种12。在这种试验中, 相关 t 细胞的取样可能是一个巨大的挑战, 因为相关的细胞的免疫调节免疫力驻留在淋巴组织中, 只有非常有限数量的特定 T 细胞在外周血中循环。
某人的技术为研究记忆 T 细胞和其他特定细胞群体提供了一个独特的机会。在抗原的皮肤接种之后, 抗原特异的 T 细胞从他们的淋巴藏身之处被吸收到皮肤并且可以容易地被取样从某人。本文介绍了这种皮肤召回模型的方法和研究应用. 在 2013年2。一种常用的皮肤召回抗原是用于执行结核菌素皮肤试验 (尖沙咀)13的分枝杆菌的商用纯化蛋白衍生物 (ppd), 其中 PPD 被注入皮肤的真皮层。在对 PPD 有免疫记忆的个体中 (例如,结核分枝杆菌感染的个体或先前的卡介苗 Guérin (BCG) 疫苗), 抗原沉积导致召回反应与迁移到皮肤和体内克隆扩张的 PPD 特定的 CD4+ T 细胞2,11,14,15。因此, 高分数的 PPD 特异多功能 CD4+ T 细胞积累在皮肤准备为某人取样。这种方法收集的 T 细胞已被证明是健壮的, 足够丰富, 以一系列的免疫和长期的体外培养15为特征。因此, 皮肤召回模型和 SB 诱导可以证明是一种有价值的方法来研究在体内T 细胞反应的体外分析, 并增加知识, 这一方法可能有利于研究人员对细胞免疫学的兴趣和疫苗。
本报告提供第一个逐步指南, 如何诱导人皮肤吸入水泡在 PPD 注射皮肤。应用卡介苗接种疫苗的志愿者对皮肤抗原召回模型进行了验证。最后, 对人分离的细胞和细胞因子的相关体外分析进行了实证研究.用表型鉴定方法获得的 PPD 特异性 T 细胞的功能特征和作用特性在别处2、10、11、16、17、18,19. 本报告旨在讨论方法的实际和免疫学方面, 以减轻其他研究小组对这项技术的应用。

<table><tbody><tr><td>Gloves for laboratory use</td><td>Imtex, Denmark</td><td>1013</td><td></td></tr><tr><td>Desinfection spray&amp;nbsp;</td><td>Apotek, Denmark</td><td></td><td>82% alcohol, with glycerin, for human skin&amp;nbsp;</td></tr><tr><td>Alcohol swaps</td><td>Mediq, Denmark</td><td>1131892</td><td></td></tr><tr><td>PPD RT 23 &quot;SSI&quot; (2 T.U./0.1ml)</td><td>AJ Vaccines</td><td></td><td></td></tr><tr><td>&amp;nbsp;Myjector syringe with fixed needle&amp;nbsp;</td><td>Terumo</td><td>A236</td><td>1 ml/29G/12mm</td></tr><tr><td>Ruler&amp;nbsp;</td><td>not relevant</td><td></td><td>&amp;nbsp;for skin reaction evaluation</td></tr><tr><td>Ballpoint pen</td><td>not relevant</td><td></td><td>&amp;nbsp;for skin reaction evaluation</td></tr><tr><td>Portable Lung Suction Unit</td><td>Laerdal Medical, Denmark</td><td>78000011</td><td>NOTE.&amp;nbsp; Modified for ranges below -40kPa and supplied with pressure gauge&amp;nbsp; (ref. 1031107) by the Technical Dept. , Hvidovre Hospital</td></tr><tr><td>Negative Pressure Chamber Assembly, unheated with connecting tubing</td><td>Electronic Diversities, Finksburg, MD, USA</td><td></td><td></td></tr><tr><td>Orifice Plate, 47mm, 10mm Hole</td><td>Electronic Diversities, Finksburg, MD, USA</td><td></td><td></td></tr><tr><td>Negative Pressure Instrument Seal Kit</td><td>Electronic Diversities, Finksburg, MD, USA</td><td></td><td></td></tr><tr><td>Negative pressure Chamber Strap&amp;nbsp;</td><td>Electronic Diversities, Finksburg, MD, USA</td><td></td><td>12 inches</td></tr><tr><td>Micropore Surgical tape</td><td>&amp;nbsp;3M</td><td>1533-1</td><td>2.5cm x 9.1m</td></tr><tr><td>Cap from 15ml plastic tube</td><td>Sarstedt, Germany</td><td>62,547,254</td><td></td></tr><tr><td>Tubigrip bandage</td><td>M&amp;ouml;lnlycke Health Care, Sweden</td><td>1443</td><td></td></tr><tr><td>Cosmopor steril adhesive dressing</td><td>Hartmann</td><td>9008337</td><td>7.2 x 5 cm</td></tr><tr><td>2ml Syringe Concentric Luer Slip&amp;nbsp;</td><td>Becton Dickinson&amp;nbsp;</td><td>300185</td><td></td></tr><tr><td>Microlance 23G/30mm needle</td><td>Becton Dickinson</td><td>300700</td><td></td></tr><tr><td>Safe-Lock microcentrifuge tubes (autoclaved)</td><td>Eppendorf</td><td>&amp;nbsp;0030120086</td><td>Autoclaved</td></tr><tr><td>Minispin plus centrifuge</td><td>Eppendorf</td><td>5453000011</td><td></td></tr><tr><td>Gibco AIM V Medium</td><td>Life Technologies</td><td>12055-091</td><td></td></tr></tbody></table>

a suction blister protocol to study human t-cell recall responses in vivo

皮肤抗原召回模型允许研究人的记忆反应在体内。该模型结合皮肤吸泡 (锑) 诱导, 为罕见的抗原特异性 T 细胞提供可访问性, 代表细胞记忆反应以及细胞因子微环境的原位。
本报告描述了皮肤召回的实际程序, 某人的诱导, 和一个抗原特定的 T 细胞的收获。为举例说明该方法, 结核菌素皮肤试验用于抗原召回的个人谁, 在本研究之前, 接受了 Guérin 芽孢杆菌疫苗接种的结核分枝杆菌感染.最后, 给出了细胞样品的多重性和流性分析的例子, 说明了这种取样方法与血液分离的细胞相比, 抗原特异性多功能 CD4+ T 细胞的高分。
这里描述的方法是安全和微创的, 提供了一个独特的机会, 以研究先天和自适应免疫反应在体内, 并可能有利于一个广泛的社区的研究人员细胞介导免疫和人类记忆反应, 在疫苗开发的背景下。

八名健康的成人志愿者 (中位年龄:30 岁, 范围:26-43 岁) 与已记录的前卡介苗接种 (中位时间从卡介苗接种: 5.5 年, 范围: 1-30 年) 包括在内。参加者被挑战 intradermally 与2个 PPD 然后在3天的尖沙咀评估。SBs 在7天被诱导并且在8天收获了, 并且所有水泡由吸入水泡室提出了以10毫米孔直径。七人分别被给予2单独 ppd 接种同时在同一只胳膊跟随2平行某人归纳 (请参见关于在协议的步骤1.4 之下多 PPD 证言的笔记)。用密度梯度离心法对7天的血浆和 PBMCs 隔离进行了外周血的提取。从 SBs 中的等离子和流体上清液存储在摄氏-20 摄氏度。新鲜的某人细胞 (SBCs) 计数使用 nigrosine 染色和显微术。
临床尖沙咀反应和 SBC 产量如图 1所示。尖沙咀硬结的正中大小为10.25 毫米 (范围: 0-20 毫米), 每个水泡的中位细胞数是 5万 (范围: 1.5万-21万个细胞, 水泡的数量:15)。其中两名志愿者在 2 TSTs 中均无临床反应, 相应的低总细胞产量为1.5万细胞/水泡。细胞产量与尖沙咀的平均临床反应有关 (长矛的r = 0.643, p = 0.094)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57554/57554fig1.jpg"> 图 1: 吸塑细胞产量.(a) 本小组展示了在尖沙咀后7天内从 SBs 中分离出的 nigrosine 染色细胞的代表性显微镜。(b) 本小组显示8名卡介苗接种志愿者 (水泡数量 = 15) 与平均尖沙咀硬化 (mm) 和平均细胞产量 (n/水泡) 之间的关系。点表示单个平均测量。请注意, 2 点是重叠的, 因为2志愿者都有尖沙咀硬化0毫米和细胞产量1.5万。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57554/57554fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了演示细胞的流动情况, SBCs 是从一名43岁的志愿者那里获得的, 他曾在30年前接种过卡介苗, 并且没有接触过结核分枝杆菌. SBCs 在单一水泡的诱导下被隔绝了 (硬化: 1.4 mm/100,000 SBCs)。图 2显示了 SBCs与PBMCs 细胞内细胞因子染色的代表性地块。
在这个志愿者中, CD3+CD4+CD8-SBCs 的分数从静态细胞的67% 增加到 > 90%, 在体外restimulation 的 PPD, 而 CD3+CD4+CD8 PBMCs 的分数保持不变 (51%,图 2)。超过92% 的体外PPD-, 以及静态 CD3+CD4+CD8-SBCs, 是效应记忆类型 (CCR7-CD45RA-, 数据没有显示)。SBCs 与 PBMCs (33.1 对0.2%、静态样品减去) 相比, PPD 刺激诱发的特异性 CD3+CD4+CD8 细胞的总分数较高。在 PBMCs 中, 多功能 PPD 特异 CD3+CD4+CD8 细胞的分数均 < 0.05%。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57554/57554fig2.jpg"> 图 2: SBCs 的代表性流式细胞仪地块与PBMCs.a组和b组显示了 CD8+ 和 CD4+ 种群的代表性密度图 (a) 静态vs。ppd 刺激的 PBMCs 和 (c) SBCs. 小组b和d在 PPD 刺激的 CD3+CD4+CD8 细胞内细胞因子染色的慢块 (b) PBMCs 和 (d) SBCs.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57554/57554fig2large.jpg" target="_blank">请单击此处查看更大的此数字的版本.</a>
正如预期的那样, CD3+CD4+CD8-SBCs 在体内激活, 这说明了上调细胞因子 (12%) 中很大比例的红细胞染色, 主要细胞因子为 TNF α和干扰素γ。然而, 在 PPD 刺激下, 分泌细胞因子向三重或双阳性 IFN-γ+TNF-α+IL-2+ (17%) 和干扰素-γ + TNF α + (15%) 剖面 (图 3b, PBMC 剖面从同一捐赠者被包括用于比较 图 3a)。PPD 刺激效应器记忆 CD4+ T 细胞亚群 (CD3+CD4+CD8-CCR7-CD45RA) 的细胞因子表达谱与图2和3中的 CD3+CD4+CD8 种群 (未显示数据) 相比较。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57554/57554fig3.jpg"> 图 3: CD4+ PPD 刺激和静态 SBCs 与 PBMCs 的细胞因子图谱. 这些面板显示细胞因子产生 (a) CD3+CD4+CD8-PBMCs 细胞和 (b) CD3+CD4+CD8 SBCs 的个人概况。酒吧代表静态细胞 (白条) 抗原特异性细胞因子剖面的分数, 并在体外刺激与 PPD (有色棒)。
为了在皮肤检测的部位, 探索液体作为细胞因子微环境的信息来源, 从 SBs 诱发的7天后尖沙咀诱导的液体中测定细胞因子水平 (图 4a)。干扰素γ、TNF α和 IL-2 的中位数分别为 339 pg/毫升、19 pg/毫升、1 pg/毫升 (n = 6)。血浆的水平一般很低。从 SBs 中分离出来的细胞产生了高水平的促炎性细胞因子 (图 4b)。1名志愿者 SBCs 的滴定是在自体 PBMCs 的毒力结核的存在下培养4天的。干扰素-γ水平 > 30 倍以上的文化中含有 SBCs, 无论是否存在的 PBMCs。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57554/57554fig4.jpg"> 图 4: 细胞因子水平在某人液体, 血浆和山地-受感染的文化上清液.(a) 本小组显示6名卡介苗接种志愿者的上清液和血浆中的细胞因子水平。条形代表细胞因子水平的中位数;误差线表示范围。(b) 这个小组显示了上清液中的细胞因子水平, 从 4 平行600µl 的4天的文化 1 x 106 PBMCs (黑条), 1.75 x 105 SBCs (白条), 1 x 106 PBMCs 尖刺与0.5 或 1.75 x 105 SBCs (灰条) 感染结核分枝杆菌。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57554/57554fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo at JoVE.com

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

在这里, 我们提供了一个示范的吸入水泡皮肤召回模型。该模型允许一个简单的访问研究人体自适应免疫反应, 例如在疫苗开发的背景下。

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

a-suction-blister-protocol-to-study-human-t-cell-recall-responses

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

11.9K 次观看.  Statens Serum Institut. 在这里, 我们提供了一个示范的吸入水泡皮肤召回模型。该模型允许一个简单的访问研究人体自适应免疫反应, 例如在疫苗开发的背景下。

视频: A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

科研

免疫与感染

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

一种新的生理免疫原树突状细胞生成方法

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

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

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

医学

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

一种监测佐剂介导的细胞毒性 T 淋巴细胞生成的免疫学技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...