Name: detection and enrichment of rare antigen-specific b cells for analysis of phenotype and function
Uploaded: 2017-02-16T15:00:00.000+00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function at JoVE.com

This work was supported by grants from the JDRF (1-2008-994, 27-2012-450) and the National Institutes of Health (R01DK096492-05, R21AI124488-01, T32OD012201, and F30OD021477).

1.人PBMC的分离<ol><li>收集使用肝素血液收集管30-50毫升的血液。 注:血液中所用的量取决于在感兴趣的抗原特异性B细胞的血液的特定的实验问题和频率。肝素化的血液可立即被处理或摇动轻轻在室温下过夜处理翌日。在处理的延迟对富集效率的影响非常小，并且与活力的最小损失有关。 </li><li>混合全血1:1用无菌，室温镁和钙自由磷酸盐缓冲的盐水（PBS）。 </li><li>在50毫升的无菌锥形管中，加入10毫升室温的密度梯度溶液中。存储在黑暗中打开的密度梯度瓶在4℃，但在使用前温热至室温。 </li><li>如果可能的话设置移液枪的缓凝和/或很少，但一致的，触发压力施加缓慢30-35层毫升稀释血液的密度梯度的顶部，小心不要混用两种。 注:放置针对50毫升的边缘吸移管的前端锥形同时加入稀释的血液，将有助于确保一致的稳定流动。 </li><li>离心机在20℃下与制动400×g离心30分钟，关闭。 </li><li>取出上层，其中包含血浆和血小板，小心不要去打扰下面的单核细胞层。 </li><li>使用无菌吸管，收集单核细胞层（血沉棕黄层）并放入一个单独的无菌50ml锥形管中。 </li><li>向细胞悬浮液中添加PBS中至50ml体积。离心在20°C带制动400 XG 10分钟。 </li><li>除去上清，悬浮沉淀的细胞在10ml PBS中。计数细胞并使用血球或自动细胞计数器测定生存力。 </li><li>重悬的细胞以10 7个细胞/ ml，在冰上的地方，直到准备好用于进一步处理。</li></ol> 2.细胞染色<ol><li>除去1-2×10 6个细胞的每个荧光补偿/需要的话，如果适用FMO控制。离心机在400×g离心10分钟，细胞的其余部分在4℃下与制动。 </li><li>在3-6×10 7个细胞/ ml的无菌过滤，冷FACS缓冲液（PBS + 1％牛血清白蛋白（BSA）+ 0.01％叠氮化钠）重悬细胞（在1.5ml的离心管中移除）。确保FACS缓冲是冷的，并保持它在冰上整个染色过程。 <ol><li>优化分析时除以细胞群为四分之三（AD）。使用级分A，以丰富的兴趣，使用馏分B和C的抗原特异性B细胞，以确定富集/染色的特异性（参见2.5和2.6以下），并使用组分D以确定抗原特异性细胞的频率和数目在未浓缩的人口，使产量计算和折叠式富集。 </li></ol></li><li>补充人体的FcγR再堵代理分数AD冰以防止抗体的Fc受体的结合。 注:按照适当的条件下制造商的说明。 </li><li>添加荧光标记的抗体细胞的利益表面抗原，以分数为AD在黑暗中冰30分钟，注意不要包括可以用交叉耦合磁性纳米粒子的抗远红外荧光染料抗体反应的荧光染料。 注意:这些包​​括抗体偶联物到近红外荧光染料Cy5标记，经Cy5.5和Cy7的。被添加任何活力污渍应与使用固定剂的兼容。如果不打算分析使用FACS的细胞，但确定抗体分泌频率，例如，通过ELISPOT，与抗体表面染色是不必要的。 </li><li>为了测试该试验的特异性，用未标记抗原的足够量的孵育馏分B在冰上30分钟，以使得广大的受体具有亲和至少10 -6 M被封锁。 注意:通常情况下，一个好起始浓度是50-100倍过量的抗原的量在2.6认为是足够的下面（ 例如 50-100微米）。这应该阻止任何B细胞具有高亲和性的未标记的抗原结合到生物素化的抗原，这是在下面的步骤2.6中加入，从而使富集的细胞的特异性的确认。本步骤可以在与上面步骤2.4一起完成。 </li><li>生物素化抗原的级分A，B和D孵育加在冰上30分钟。 注:生物素化抗原的浓度应滴定以确定需要确保足够的检测和来自非特异性旁观者细胞结合的细胞分离的最佳量。通常一个很好的起点浓度测试为1-5微米。 注:对于馏分A和D，这一步可以同时使用上述步骤完成2.4。对于馏分B，这一步应该在工序2.5（在betw清洗完成EEN步骤是多余的）。 </li><li>省略从组分C的生物素化抗原，以便确定B细胞中的协议的其他试剂，例如链霉远红荧光染料和磁性珠粒任何结合。 </li><li>在4℃在1ml每5×10 7个细胞的冷FACS缓冲液和离心洗细胞两次通过细胞悬浮液在400×g离心5分钟。 </li><li>最后一次洗涤后，稀释浓缩细胞悬浮液中每5×10 7个电池1毫升2-％甲醛和让坐在黑暗冰上5分钟。 注:细胞在这一点上的固定确保了生物素化抗原保持结合到BCR的过程的剩余。对活细胞 - 对于下游分析，如在ELISPOTs或过继转移，省略固定步骤。 </li><li>洗涤细胞用1毫升每5×10 7个细胞的冷FACS缓冲液和离心分离两次，在400×g离心在4℃下5分钟。悬浮在1毫升冷流式细胞仪每5×10缓冲7细胞。 </li><li>加1-2微克链霉远红荧光染料/毫升，并在冰上孵育在黑暗中20分钟。滴定链霉为每个应用程序的数量。 </li><li>在4℃下以每5×10 7个电池1毫升冷的FACS缓冲液，离心洗涤细胞两次，在400×g离心5分钟。组分D制剂在这一点完成，因为它不会使用磁珠进行富集。 注:此示例将被用于确定样品中的频率和抗原特异性B细胞的数量。这将被用于确定产量和折叠式富集富集实现。 </li></ol> 3.基于磁性纳米颗粒富集<ol><li>从样品交流重悬细胞于1ml每5×10 7个细胞，并通过冷分离缓冲液（PBS + 0.5％BSA + 2mM的EDTA）的通过40微米的过滤器，以消除可能堵塞列中的任何团块。 </li><li>添加抗的Cy5 /抗远红光荧光染料的纳米颗粒。佛ř最佳结果，纳米颗粒的浓度应该滴定每次使用，但一个好的起点是每5×10 7个细胞/ ml加入50μl悬浮液。旋转在黑暗中在4℃下10分钟。 </li><li>在4℃以400 xg离心用1ml冷的分离缓冲液洗两次，每次5分钟。悬浮细胞在1ml每5×10 7个细胞的冷分离缓冲液中。 </li></ol> 4.磁富集使用LS或LD列<ol><li>将三个LS或LD列在磁分离器的磁场，并添加3毫升分离缓冲液的润湿列（见产品说明，以确定哪些列的类型是最适合）。允许所有3毫升水穿过柱，收集后丢弃。 注意:虽然磁性纳米颗粒标记细胞的分离可以使用磁分离装置是市售的任何一种来完成，该实验室已与使用LS柱的最成功，在德产量，纯度和富集效率的有效值。 </li><li>将标有3支15mL的锥形管"分数A /（B）/（C）阴性"，为负选定单元格，列下。添加磁性粒子标记的细胞，以它们各自的柱，并允许整个体积通过。 </li><li>加上前3毫升冷的分离缓冲液中，并让此穿过柱并用2ml重复。收集负选定单元格，并预留冰上。 </li><li>从磁场中删除列和放置在标记为"份A /（B）/（C）的正面"，为阳性选择细胞的15毫升锥形管的顶部。 </li><li>用大约6毫升分离缓冲液的填充柱顶端，并立即通过该柱沉浸这个体积来收集已使用所提供的柱塞结合到磁场的细胞。 </li><li>为了提高纯度，在正选择的细胞可以进一步使用第二清洁列充实，重复PROCE杜热。 </li><li>在400 xg离心在4℃下旋转同时负，正选择的馏分为10分钟，并在所需的最终体积暂停。与下游的分析，如FACS，ELISPOT或过继转移继续。 </li></ol>

<ol>
	<li>de Wildt, R. 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=A+new+method+for+the+analysis+and+production+of+monoclonal+antibody+fragments+originating+from+single+human+B+cells.">A new method for the analysis and production of monoclonal antibody fragments originating from single human B cells.</a> J Immunol Methods. 207 (1), 61-67 (1997).</li><li>Edelman, G. M., Rutishauser, U., Millette, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fractionation+and+arrangement+on+fibers,+beads,+and+surfaces.">Cell fractionation and arrangement on fibers, beads, and surfaces.</a> Proc Natl Acad Sci U S A. 68 (9), 2153-2157 (1971).</li><li>Haas, W., Schrader, J. W., Szenberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new,+simple+method+for+the+preparation+of+lymphocytes+bearing+specific+receptors.">A new, simple method for the preparation of lymphocytes bearing specific receptors.</a> Eur J Immunol. 4 (8), 565-570 (1974).</li><li>Kenny, J. J., Merrill, J. E., Ashman, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-step+centrifugation+procedure+for+the+purification+of+sheep+erythrocyte+antigen-binding+cells.">A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.</a> J Immunol. 120 (4), 1233-1239 (1978).</li><li>Snow, E. C., Vitetta, E. S., Uhr, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+antigen-enriched+b+cells.+I.+Purification+and+response+to+thymus-independent+antigens.">Activation of antigen-enriched b cells. I. Purification and response to thymus-independent antigens.</a> J Immunol. 130 (2), 607-613 (1983).</li><li>Egeland, T., Hovdenes, A., Lea, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+of+antigen-specific+B+lymphocytes+by+means+of+immunomagnetic+particles.">Positive selection of antigen-specific B lymphocytes by means of immunomagnetic particles.</a> Scand J Immunol. 27 (4), 439-444 (1988).</li><li>Smith, M. 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=Loss+of+anergic+B+cells+in+prediabetic+and+new-onset+type+1+diabetic+patients.">Loss of anergic B cells in prediabetic and new-onset type 1 diabetic patients.</a> Diabetes. 64 (5), 1703-1712 (2015).</li><li>Hulbert, C., Riseili, B., Rojas, M., Thomas, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+specificity+contributes+to+the+outcome+of+diabetes+in+nonobese+diabetic+mice.">B cell specificity contributes to the outcome of diabetes in nonobese diabetic mice.</a> J Immunol. 167 (10), 5535-5538 (2001).</li><li>Woda, M., Mathew, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescently+labeled+dengue+viruses+as+probes+to+identify+antigen-specific+memory+B+cells+by+multiparametric+flow+cytometry.">Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry.</a> J Immunol Methods. 416, 167-177 (2015).</li><li>Nair, 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=Optimized+fluorescent+labeling+to+identify+memory+B+cells+specific+for+Neisseria+meningitidis+serogroup+B+vaccine+antigens+ex+vivo.">Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo.</a> Immun Inflamm Dis. 1 (1), 3-13 (2013).</li><li>Amanna, I. J., Slifka, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+rare+memory+B+cell+populations+by+two+independent+and+complementary+approaches.">Quantitation of rare memory B cell populations by two independent and complementary approaches.</a> Journal of immunological methods. 317 (1-2), 175-185 (2006).</li><li>Scheid, J. F., 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+method+for+identification+of+HIV+gp140+binding+memory+B+cells+in+human+blood.">A method for identification of HIV gp140 binding memory B cells in human blood.</a> Journal of immunological methods. 343 (2), 65-67 (2009).</li><li>Doucett, V. 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=Enumeration+and+characterization+of+virus-specific+B+cells+by+multicolor+flow+cytometry.">Enumeration and characterization of virus-specific B cells by multicolor flow cytometry.</a> Journal of immunological methods. 303 (1-2), 40-52 (2005).</li><li>Malkiel, 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=Checkpoints+for+Autoreactive+B+Cells+in+Peripheral+Blood+of+Lupus+Patients+Assessed+By+Flow+Cytometry.">Checkpoints for Autoreactive B Cells in Peripheral Blood of Lupus Patients Assessed By Flow Cytometry.</a> Arthritis Rheumatol. , (2016).</li><li>Pape, K. A., Taylor, J. J., Maul, R. W., Gearhart, P. J., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+B+cell+populations+mediate+early+and+late+memory+during+an+endogenous+immune+response.">Different B cell populations mediate early and late memory during an endogenous immune response.</a> Science. 331 (6021), 1203-1207 (2011).</li><li>Abts, H., Emmerich, M., Miltenyi, S., Radbruch, A., Tesch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD20+positive+human+B+lymphocytes+separated+with+the+magnetic+cell+sorter+(MACS)+can+be+induced+to+proliferation+and+antibody+secretion+in+vitro.">CD20 positive human B lymphocytes separated with the magnetic cell sorter (MACS) can be induced to proliferation and antibody secretion in vitro.</a> Journal of immunological methods. 125 (1-2), 19-28 (1989).</li></ol>

The authors declare they have no competing financial interests.

在这里，我们描述了一种新方法来完成分离和从人外周血抗原结合的B细胞的富集。该方法是容易适用于小鼠和其它组织，如脾脏和淋巴结肿大，是与细胞表型和功能（手稿准备中）的富集后分析兼容。 用户应该认识到许多的可能影响该过程的成功变量。从经验死细胞倾向于粘到磁性珠粒和"占用"荧光染料，如链霉亲和远红荧光染料，导致非特异性背景增加。关键是要开始此?...

限制稀释抗体分泌细胞前体的频率的分析已经表明，该反应的特定抗原的B细胞通常发生在0.05〜0.005％，在正常剧目的频率取决于接种的状态和存在于抗原表位的大小/数量。这些细胞的低频率，因此难以的免疫反应，如接种后或暴露于外来抗原或自身免疫的发展过程中发育过程研究在它们的状态的改变。先前，研究人员已经进行的使用的技术，从抗原包被的板或柱吸附剂，抗原包被的红细胞复位，对抗原反应性B细胞的分离荧光激活细胞分选1，2，3，4，5，6。 Thougħ这些技术已成功地鉴定和分离抗原反应性B细胞，结果在收率，纯度和可扩展性方面变化。最近我们开发了一种新的方法来检测二者并用的磁性纳米粒子丰富罕见的B淋巴细胞亚群。该方法能够富集具有相对高产量和纯度从大的起动种群，并与对抗原的反应分析兼容。通过从悬浮液中的细胞群富集，该方法消除了与抗原包被的板或列，并限制可以通过的几何结构相关的约束。最后，由于富集的细胞保持与抗原和荧光报道相关联的，它们可以被进一步通过FACS分选进行纯化。如本文所述，我们使用从与各种autoi受试者这种方法对于外周血破伤风类毒素反应性B前细胞的研究和人类受试者的免疫接种后，以及自身抗原反应的B细胞mmune疾病，包括1型糖尿病，格雷夫斯病，桥本氏病7。该方法同样适用于小鼠和人，并与从各种组织（手稿中制备）抗原反应性B细胞的分析相容。 在其基本形式，外周血单核细胞首先用抗体与细胞用于表型分析所需表面抗原的沿生物素化的抗原温育。此标记步骤之后是洗涤和固定，以及另外的链霉耦合到远红荧光染料用于检测生物素化的抗原结合的细胞（ 图1）。以前的研究已经确定了抗原特异性的B细胞以类似的方式，但使用抗原直接缀合到荧光染料8，9，10，11。虽然这是一个值得的做法，与链霉结合使用生物素标记抗原能够取得更大的信号放大（绑定和非绑定细胞从而更好的区分），特别是当抗原是小12，13，14。另外一个考虑是利用链霉代替抗生物素蛋白，因为链霉去糖基化，减少非特异性结合的。此外，我们用远红荧光染料作为荧光由于其光稳定性，量子产率（亮度），并且它的体积小（〜1.3 KD）。荧光蛋白，如藻红蛋白（〜250 KD）和别（〜105 KD）15不是最佳的，因为它们可能含有大量的抗原表位。单一表位组成，如远红荧光染料小的有机荧光染料的使用，减少了分离的细胞群的复杂性。 一旦细胞被生物素化一ntigen和远红色荧光染料的链霉亲和吸附，他们使用的是抗远红荧光染料缀合的磁性纳米粒子富集。单个纳米颗粒不被大多数流式细胞仪检测到的，因此，不必纯化之前通过FACS分选和下游测定16除去。抗原特异性B细胞的磁性选择丰富感兴趣的人口，消除了时间和排序使用流式细胞仪罕见的事件的成本。 下面，我们显示从破伤风类毒素特异性的B细胞的富集来自破伤风类毒素加强免疫后前七天受试者代表性结果。我们为了说明此方法的充实下列体内刺激急性抗原特异性B细胞的能力选择了这个特定的应用，例如，当用流式细胞仪结合，这种方法能够丰富和分化抗原特异性天真，内存，以及浆母B细胞和允许研究者跟随随时间在它们的频率的变化。此外，我们还包括另一种可能的下游分析， 例如 ELISPOT测定，这表明细胞保留分泌以下富集抗体的能力。该方法的另一个应用可能涉及富集细胞过继转移入宿主。我们以前曾表明细胞保持作为抗原呈递以下隔离和传输细胞抗原特异性T细胞的能力（数据未显示）。因此，有许多可能被耦合到所述的方法，其中一起通知抗原特异性免疫应答的理解可能下游测定。我们已经描述下面的方法，包括控制，以确定总收率，纯度，细胞特异性，和折叠式富集。

<table><tbody><tr><td>Antigen of interest</td><td>variable</td><td>variable</td><td>At least 100 &amp;mu;g to biotinylate easily; if using protein try to use protein that has been validated by ELISA. It must be carrier&amp;nbsp; protein free.</td></tr><tr><td>Biotin for labeling; &lt;em&gt;e.g.&lt;/em&gt; EZ link Sulfo-NHS-LC-Biotin</td><td>Thermo Scientific</td><td>21335</td><td>Biotin is available in different formulations, such as those containing&amp;nbsp; various length spacers, so the type used should be determined by the researcher</td></tr><tr><td>Streptavidin-Alexa Fluor 647</td><td>Invitrogen</td><td>S21374</td><td>Can obtain from other suppliers.</td></tr><tr><td>Anti-Cy5/Anti-Alexa Fluor 647 Microbeads</td><td>Miltenyi Biotech</td><td>130-091-395</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>MACS manual separators</td><td>Miltenyi Biotech</td><td>variable</td><td></td></tr><tr><td>Formaldehyde</td><td></td><td></td><td>Dilute to 2% with PBS; optional if downstream assay requires live cells</td></tr><tr><td>PBS without calcium and magnesium</td><td></td><td></td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>Whole blood in heparinized collection tubes</td><td></td><td></td><td></td></tr><tr><td>FACS buffer (PBS + 1% BSA + 0.01% sodium azide)</td><td></td><td></td><td></td></tr><tr><td>Separation buffer (PBS + 0.5% BSA + 2 mM EDTA)</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tubes</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tubes</td><td></td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>Surface marker reactive antibodies, Fc Block, live/dead discriminating stain, if needed</td><td></td><td></td><td></td></tr><tr><td>ELISPOT supplies, if needed</td><td></td><td></td><td></td></tr></tbody></table>

detection and enrichment of rare antigen-specific b cells for analysis of phenotype and function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

纯度，产量分析，并用流式细胞仪倍富集种群如上述那样必然包含未结合的链霉远红荧光染料，但被截留在基质污染细胞富集。这些杂质可以从富集的群体通过FACS分拣除去。估算富集群体的纯度，对活细胞栅极上了基于和侧向散射和/或活/死染色和分析远红光荧光染料+细胞频率。这后一种门控内的比例是指示纯度。这些细胞的?...

Watch this Scientific Journal Video about Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function at JoVE.com

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

描述了采用磁性纳米颗粒，以检测和富集的功能和表型分析抗原反应性B细胞中的简单而有效的方法。

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

detection-enrichment-rare-antigen-specific-b-cells-for-analysis

Research

JoVE Journal

Immunology and Infection

11.5K 次观看.  University of Colorado School of Medicine. 描述了采用磁性纳米颗粒，以检测和富集的功能和表型分析抗原反应性B细胞中的简单而有效的方法。 

视频: Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

肽：MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

科研

免疫与感染

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

可行的小鼠IL - 17分泌性T细胞的检测和隔离

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

生物学

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

利用磁性纳米粒子丰富和扩大罕见抗原特异性 t 细胞

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

工程学

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

生物工程

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

小鼠肺中抗原特异性 T 细胞的分离和表征

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

作者聚焦:使用四聚体检测小鼠肺中抗原特异性 T 细胞的优化方案

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...