我想感谢我介绍了免疫分析和鼠标处理比安卡Mothé，Oseroff卡拉和玛丽 - 法国DelGuercio。在GA的分配器实验室工作的经费由美国国立卫生研究院授予1 RO1 - AI - 073558，GLRCE授予1 - U54 - AI - 057153和Bard美3829 - 06。

1。争取通过免疫目标特定效应的CTL（0天） <ol><li>在开始之前，决定在某个特定的效应:目标系统。在这个经典的例子在体内 CTL的检测，我们将使用纯化肽的免疫接种，以诱导特异性CD8 + T细胞。脉同源的靶细胞，创建一个特定的效应:目标系统，将使用相同​​的肽。或者，您也可以选择使用整个病原体或蛋白质来获得您的具体效应和目标。 </li><li>除污的工作表面内的一个生物安全罩，以确保注射的无菌。准备首先将50微克到100μL10％PBS /鼠标在2 mL试管中二甲基亚砜纯化肽肽的免疫接种。接下来，添加一个非常寒冷的不完全弗氏佐剂等量高达0.2毫升标记1毫米珠混合。从这一点来说，保持冰，直到注射的免疫原。安全盖的封口膜。 </li><li>使用设置最大RPM的混合3分钟的乳液，及时放置在冰乳液混合后珠打浆机。由此产生的乳液应该是类似的一致性，以蛋黄酱。允许乳液坐于4 ° C过夜，如果时间允许（长达一个星期），搅拌进一步指出如果任何分离。 </li><li>用1 ml注射器和针头20或20.5拉起乳液小心不要拉起任何珠（珠应大于针规，但仍然可以知道）。 </li><li>安全鼠标为基地的尾巴皮下注射。我们使用一个改性塑料50 ml锥形管，安全和注射用鼠标轻轻固定。注入0.2毫升乳液鼠标。 </li></ol> 2。收获靶细胞（8天） <ol><li>您将需要为你的目标细胞的天真同基因小鼠。根据AAALAC标准安乐死天真的小鼠。 </li><li>消毒，喷洒70％的乙醇，以尽量减少鼠标头发或其他外部污染物的污染的机会的鼠标。 </li><li>使用夹层剪刀，通过皮肤上的鼠标左侧小剪断。将皮瓣，发现腹膜囊和内脏器官。 </li><li>通过腹膜剪断，小心不要妥协任何底层的机关。脾脏是接近地表和颜色类似肝脏（暗红色），但体积更小，形状芸豆。感染小鼠的一般比未受感染的控制，特别是较大的脾脏。 </li><li>小心地拉出脾，照顾要轻柔，以免打破成多个部分脾脏为了恢复最大的手机号码。在冰上，立即放入15毫升中含10 MLS准备媒体锥形脾。继续余下的小鼠。 </li><li>倒入一个玻璃组织研磨机的15 mL锥形管（媒体和脾脏）的内容。研磨脾，使用大量的上臂力量。当所有的组织已经失去了它的颜色，你可以假设您已经收回了所有的结缔组织细胞。 </li><li>通过一个70微米的细胞过滤器中倒入50 mL锥形管磨床的内容。冲洗与新媒体的磨床和倒通过细胞过滤器。 </li><li>离心机在4 ° C，7分钟1300转。倒掉上清液。 </li><li>悬浮在准备裂解试剂和孵育7分钟，在37 ° 10 MLS。 </li><li>最多可添加50毫升新鲜的媒体和离心机，上面一样。 </li><li>悬浮在50毫升新鲜媒体和离心机，与上面相同（洗一步）。 *注:此时，您可以执行一个特定的细胞类型的磁性细胞分离（即巨噬细胞），如果一个纯粹的靶细胞的人口转移到受体小鼠的理想。此外，在这个时候，你可以引入到文化的实验性治疗，以研究治疗，对细胞的特异性杀伤的影响。 </li><li>在100μL数在96圆底组织文化对待板电池板1 × 10 7每口井。 *这是一种高浓度的细胞/孔，但因为检测是一种快速的，我们已经看到了在这个浓度的细胞的生存能力没有损失。 </li></ol> 3。靶细胞的治疗和标签<ol><li>脉冲适当的细胞和病原体的特异性多肽或无关的肽控制复制:添加1μg/ mL的肽。在4 ° C孵育1.5小时。最后30分钟，移至37℃培养箱。在这段时间内准备CFSE试剂。 </li><li>根据制造商的规格准备CFSE和稀释到工作浓度为0.5和25微米之间。 "低"的浓度10倍比"高浓度CFSE较低。例如，[低]添加10毫升PBS 0.5μL，CFSE和[高]，加入5μL，CFSE 10毫升PBS。热身准备CFSE至37℃前的标签。 </li><li>删除从孵化器和P细胞板ellet在室温条件下，7分钟1300转的高速离心机。弗里克去除上清液。 </li><li>悬浮细胞要么[低]或[高] CFSE加入适当的试剂200μL相应的井和15分钟37 ° C。 </li><li>卸下的孵化器，并再次颗粒细胞在室温下，7分钟1300转板。弗里克去除上清液。 </li><li>悬浮细胞，在200μL/孔回暖的新媒体和孵育30分钟，在37 ° C。 </li><li>卸下的孵化器和颗粒细胞在室温下，7分钟1300转板。弗里克去除上清液。 </li><li>在100μL/孔PBS悬浮细胞。 </li><li>继转移，添加一个[低]一个标记的细胞[高]标记每小鼠细胞。注射0.2毫升50％的具体目标和50％的不相关的细胞。 </li><li>不要忘记预留一些标记的细胞行为在数据分析和流式细胞仪上运行的非转移的控制（见图1）。 </li></ol> 4。继转移<ol><li>麻醉以前肽免疫小鼠（从本协议的＃1一部分），根据AAALAC标准。 </li><li>通过眼窝到受体小鼠的路线标记的靶细胞，注入0.2毫升。 </li><li>等待euthanizing受体小鼠前6小时或其他预定的最佳时间。 </li></ol> 5。收获标记的细胞 *注:步骤5.1通过5.11 2.1通过2.11的步骤相同<ol start="12"><li>到96圆底组织培养处理板计数细胞和盘开出100μL1 × 10 6每口井。 </li><li>加入100μL的2％多聚甲醛的终浓度为1％多聚甲醛，所有井修复细胞，恢复和非转移。 </li></ol> 6。流式细胞仪和数据分析<ol><li>流式细胞仪上运行的样品和分析数据。 </li></ol> 7。代表性的成果导致直方图的具体峰值降低抗原表位的特异性杀伤细胞（图1）的指标。使用下面的公式（在表1中的应用），以确定细胞特异性裂解的实际价值: 比率=不相关的百分比:抗原表位的特定百分比（[低]高峰:[高]峰）， 具体裂解百分比= [1 - （非转移的比例控制/实验的比例）] × 100 <img src="/files/ftp_upload/2250/2250fig1.jpg" alt="图1" /> 图1。 CFSE配置文件恢复BALB / c小鼠脾的数字表明，高或低的CFSE表型的细胞的百分比。 A）非转移控制。 [高] CFSE峰代表抗原表位的特定的靶细胞，而[低] CFSE峰代表无关肽脉冲的目标。二）CFSE标记的细胞恢复PBS免疫收件人鼠标（控制）。三）CFSE标记的细胞恢复从两个不同的肽受体小鼠，注意减少特定的抗原表位峰。 <img src="/files/ftp_upload/2250/2250fig2.jpg" alt="图2" /> 图2。分析数据的图形表示。三个数据点显示各组小鼠，PBS肽A和肽B。 <table border="1"><tbody><tr><td> 样品 </td><td> CFSE高％抗原表位脉冲 </td><td> CFSE低％不相关的 </td><td> 比低:高 </td><td> ％的表面抗原特异性杀伤 </td></tr><tr><td> PBS </td><td> 42.8 </td><td> 57.2 </td><td> 1.34 </td><td> 16.97 </td></tr><tr><td> PBS </td><td> 43.1 </td><td> 56.9 </td><td> 1.32 </td><td> 15.94 </td></tr><tr><td> PBS </td><td> 44.3 </td><td> 55.7 </td><td> 1.26 </td><td> 11.74 </td></tr><tr><td>肽A </td><td> 39.7 </td><td> 60.3 </td><td> 1.52 </td><td> 32.56 </td></tr><tr><td>肽A </td><td> 38.6 </td><td> 61.4 </td><td> 1.59 </td><td> 35.61 </td></tr><tr><td>肽A </td><td> 40.6 </td><td> 59.4 </td><td> 1.46 </td><td> 29.99 </td></tr><tr><td>肽乙</td><td> 38.7 </td><td> 61.3 </td><td> 1.58 </td><td> 24.68 </td></tr><tr><td>肽乙</td><td> 38.5 </td><td> 61.5 </td><td> 1.60 </td><td> 25.32 </td></tr><tr><td>肽乙</td><td> 39.3 </td><td> 60.7 </td><td> 1.54 </td><td> 22.76 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> PBS NT控制</td><td> 47.4 </td><td> 52.6 </td&gt; <td> 1.11 </td><td></td></tr><tr><td>佩普一个NT控制</td><td> 49.4 </td><td> 50.6 </td><td> 1.02 </td><td></td></tr><tr><td>佩普乙NT控制</td><td> 45.6 </td><td> 54.4 </td><td> 1.19 </td><td></td></tr></tbody></table> 表1。有代表性的数据分析。"新台币"非转移。

<ol>
	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

没有利益冲突的声明。动物实验机构在美国威斯康星 - 麦迪逊大学的动物护理和使用委员会（IACUC）规定的准则和法规的规定执行。

此法可修改进行调查，包括克隆扩增细胞的增殖能力，因为CFSE划分较为平等的后代 10,11 。它也可以使用CFSE标签探讨细胞迁移6,12的，虽然也有其他细胞的跟踪方法，可能更合适，这取决于你的假设，例如四聚体和生物发光13，这也可能与CFSE标签相结合，。 它重要的是要注意入流式细胞仪上的多渠道，CFSE出血，所以如果你想表面标记染色，PeCy7将是一?...

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<td>1 mm glass beads</td>
<td>&nbsp;</td>
<td>Biospec Products</td>
<td>11079110</td>
<td>&nbsp;</td>
<tr>
<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
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<td>96-well plate</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>3799</td>
<td>&nbsp;</td>
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<td>Adjulite Incomplete Freund's Adjuvant</td>
<td>&nbsp;</td>
<td>Pacific Immunology</td>
<td>n/a</td>
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<td>n/a</td>
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<td>PM992</td>
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<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>157-8</td>
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<td>&nbsp;</td>
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<td>10010-023</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>Syringe</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>309602</td>
<td>&nbsp;</td>
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<td>Vybrant CFSE Kit</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular Probes)</td>
<td>V12883</td>
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antigen specific in vivo killing assay using cfse labeled target cells

羧基荧光素二醋酸琥珀酰亚胺酯（CFSE）可用于轻松快捷地标签为利息细胞在体内的调查人口。这种标签经典被用来研究细胞增殖和迁移。在这里介绍的方法，我们已经缩短继转移，寻找生存和杀害的抗原表位的具体CFSE标记的靶细胞4-6后的时间表。一个CD8 + T细胞克隆特异性杀伤水平可以表明质量的响应，其数量可能会误导。特异性CD8 + T细胞可以成为与细胞因子的产生和杀害7,8下降随时间的功能用尽。此外，某些CD8 + T细胞克隆可能不杀以及其他不同的TCR特异性9。有效的细胞免疫（CMI）的，必须确定抗原产生的反应性T细胞不仅数量充足，而且功能强大的反应性T细胞。在这里，我们评估的两个肽特异性T细胞克隆BALB / c小鼠的特异性杀伤细胞％。

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

许多感染引起强烈的CTL反应，但偶尔，响应细胞的数量不关联控制病原体<sup&gt; 1</sup&gt;。 CTL的质量的措施之一是他们杀了专门的能力<sup&gt; 2</sup&gt;。 CFSE标签的靶细胞，可用于研究这种CTL反应质量<em&gt;在体内</em<sup&gt; 3,4</sup&gt;。

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.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

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JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

19.5K 次观看.  University of Wisconsin-Madison. 许多感染引起强烈的CTL反应，但偶尔，响应细胞的数量不关联控制病原体 1。 CTL的质量的措施之一是他们杀了专门的能力 2。 CFSE标签的靶细胞，可用于研究这种CTL反应质量在体内

视频: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

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 ...

科研

免疫与感染

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 ...

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 ...

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.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

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 ...

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 ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...