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>

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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 border="0" cellpadding="0" cellspacing="0" width="594">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="99">
			<td height="99" style="height:99px;width:164px;">Antigen of interest</td>
			<td style="width:104px;">variable</td>
			<td style="width:131px;">variable</td>
			<td style="width:196px;">At least 100 &#956;g to biotinylate easily; if using protein try to use protein that has been validated by ELISA. It must be carrier&#160; protein free.</td>
		</tr>
		<tr height="118">
			<td height="118" style="height:118px;width:164px;">Biotin for labeling; e.g. EZ link Sulfo-NHS-LC-Biotin</td>
			<td style="width:104px;">e.g. Thermo Scientific</td>
			<td style="width:131px;">e.g. 21335</td>
			<td style="width:196px;">Biotin is available in different formulations, such as those containing&#160; various length spacers, so the type used should be determined by the researcher</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Streptavidin-Alexa Fluor 647</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:131px;">S21374</td>
			<td style="width:196px;">Can obtain from other suppliers.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:164px;">Anti-Cy5/Anti-Alexa Fluor 647 Microbeads</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">130-091-395</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:164px;">LS Columns</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">130-042-401</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">MACS manual separators</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">variable</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:164px;">Formaldehyde</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;">Dilute to 2% with PBS; optional if downstream assay requires live cells</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:164px;">PBS without calcium and magnesium</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:164px;">Ficoll-Paque PLUS</td>
			<td style="width:104px;">GE Healthcare</td>
			<td style="width:131px;">17-1440-02</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:164px;">Whole blood in heparinized collection tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:164px;">FACS buffer (PBS + 1% BSA + 0.01% sodium azide)</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:164px;">Separation buffer (PBS + 0.5% BSA + 2mM EDTA)</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:164px;">50 mL conical tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:164px;">15 mL conical tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:164px;">1.5 mL Eppendorf tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:164px;">Surface marker&#160; reactive antibodies, Fc Block, live/dead discriminating stain, if needed</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:164px;">ELISPOT supplies, if needed</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></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

11.3K 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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

跨体内迟发型变态反应检测方法的抗原特异性调节

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

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 ...

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 ...