The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

注:此协议需要使用外周血样本的志愿者。所有的研究以人类为对象进行审查和批准由人类研究伦理审查委员会，以确保符合赫尔辛基宣言（2013年）和健康保险流通与责任法案1996。 1.隔离从全血中T细胞<ol><li>开始之前，温密度梯度介质至室温。等分4毫升密度梯度介质的成2-4无菌的15毫升锥形离心管（1管将用于各10ml的稀释血液总体积）。 </li><li>获得10-20毫升血液中1-2肝素钠喷涂（绿顶）静脉血液采集管。收集人体标本经过培训的人员的监督下，根据机构审查委员会批准的协议。 </li><li>擦拭用70％乙醇在管的外侧。小心地取下音响的顶部LLED收集管，倒出血液进入无菌50ml离心管中。 </li><li>加10毫升无菌磷酸盐缓冲盐水（PBS），以每血液收集管。收集PBS，加入到滗全血，轻轻混匀。 </li><li>加入25μl的人类T细胞富集鸡尾酒/2毫升总量。在室温下孵育20分钟。 </li><li>用10毫升吸管，层高达10毫升1:1的稀释血液轻轻放在密度梯度介质的顶部。注意不要打乱密度梯度介质的表面上。 </li><li>离心分层的血液，并在1200×g离心20分钟，密度梯度介质在20℃。 </li><li>从离心机中取出试管，小心地不扰乱密度梯度介质和白细胞的密度梯度介质和稀释的血浆之间的界面处的层之间的界面。使用5ml的吸量管，小心收集的白细胞层并转移到新的无菌的50ml锥形桶Ë。 </li><li>加入PBS带来的PBL收集到50毫升容量，轻轻混匀。 </li><li>离心600×g离心5分钟，在20℃。倒出。 </li><li>重悬沉淀的细胞在10ml无菌的流式细胞分选缓冲液（含1％BSA无菌过滤的PBS）中。样品10微升细胞悬浮液，稀释的1点10分（加至90微升）台盼蓝计数使用血球细胞（预期产量1 - 4×10 6个细胞的全血/管）。 <ol><li>除去1毫升等分试样，转移至5ml管，并保持在冰上的T细胞不通过四聚体标记的分析。置于冰上细胞悬液。 </li></ol></li></ol>同种异体抗原 - 特异性T细胞的2磁性富集<ol><li>在无菌排序缓冲区100的稀释:同种抗原的pMHC四〜1。 </li><li>离心细胞悬浮液（9毫升从步骤1.11），在600×g离心5分钟，在20℃。倒出。 </li><li>加入50微升稀释的同种抗原的pMHC四聚体，以沉淀细胞（多达10 7个细胞）。轻柔吹打混匀。转移到灭菌的5毫升管。孵育30分钟，在室温下进行。 </li><li>加2 ml排序缓冲区。离心600×g离心5分钟，在20℃。倒出。 </li><li>将细胞再悬浮于100μl的排序缓冲器。加10微升生物素选择鸡尾酒，孵育15分钟，在室温下进行。 </li><li>添加5μl的磁性纳米粒子的孵育在室温下10分钟。 </li><li>添加2.5毫升排序缓冲区和吸液轻轻混匀。去除100微升的等分试样，转移至5ml管，并保持在冰上预富集细胞的分析。 </li><li>放置5毫升管含有细胞悬浮液中的细胞分离磁体。帽应该是松散的管之上。孵育5分钟，在室温下进行。 </li><li>轻轻取下管帽。握住管体一起，倒出管内容到一个新的5毫升管。不要敲击或摇晃试管内容除去最后一滴液体。 </li><li>从磁体取出管。加入2毫升冷排序缓冲区，并通过吸液轻轻混匀。样品10微升细胞悬浮液，稀释1:2（加至10微升）台盼蓝使用血细胞计数器计数细胞。 </li></ol> 3，准备T细胞的单细胞流式细胞仪细胞分选的<ol><li>加冷排序缓冲区细胞的所有管道（未标记的四聚体，四聚体标记的未充实，四聚体标记的富集），使音量至3ml。 </li><li>离心600×g离心5分钟，在4℃，澄清缓冲来恢复细胞。 </li><li>重悬在25微升冷排序缓冲区沉淀的细胞。加入5微升人Fc块。在冰上孵育20分钟。 </li><li>准备抗体流式细胞分选。混合15微升样缓冲液，加入15μl抗CD5，加入15μl抗CD14，和15微升抗CD19。 </li><li>加入20μl抗体混合物对细胞的每管中。在冰上孵育20分钟。 </li><li>加入2毫升冷排序缓冲器的每个管。 </li><li>离心600×g离心5分钟，在4℃，澄清缓冲来恢复细胞。 </li><li>重悬的细胞以1 -2×10 6个细胞/在排序缓冲器ml的浓度。 </li><li>等份加入100μl的人T细胞培养基（Iscove氏DMEM培养基补充了2mM谷氨酰胺，10mM的HEPES，50μg/ ml的庆大霉素，50μM2-巯基乙醇，10％热灭活人AB血清和2.5纳克/ ml重组人IL-2）到96孔圆底细胞培养板的各孔中。保持在冰上。 </li></ol>由单细胞流式细胞分选4.隔离四聚体标记的T细胞<ol><li>建立流式细胞仪门控参数来识别同种抗原的pMHC四聚体标记的T细胞（ 图2.A）。使用预富集四聚体标记的部分，建立门控战略来消除双峰，门上的淋巴细胞，排除CD19表达B细胞和CD14表达单核细胞，并确定T细胞CD5表达。不使用标有四聚体作为荧光减一控制采样，建立门识别四聚体结合T细胞（与CD5 + CD14 0％ - CD19 -从没有被标记的四聚体的细胞样本应属于这扇大门内） 。 注意:门控策略是不充分严格将导致非T细胞或T细胞中未抗原特异性的隔离，而过于严格的选通策略会降低T细胞中分离的数量。 </li><li>编程板参数为单细胞类。直接分拣机放置1 CD5 + CD14 - CD19 -四聚体+细胞在每口井。指示板设置为包含1阴性对照孔（无细胞分类入井）和1个阳性对照孔（100 CD5 + CD14 - CD19 -四-细胞）每行中（ 图2.B </strong&gt;）。 </li><li>使用所建立的流式细胞仪设门和板的设置，分离单个的四聚体结合的T细胞直接注入96孔板中使用流式细胞仪的细胞分选器。 </li></ol> 5，文化和扩大同种异体抗原特异性T细胞克隆<ol><li>细胞分选，离心板在600×g离心5分钟，在20℃。完成后地方培养板上，在37℃下在6％CO 2的培养箱中培养。 </li><li>涡流刺激抗CD3 /抗CD28微珠30秒重悬珠子。 </li><li>计算所需的收集T细胞的刺激活化珠的体积（0.5微升活化微珠/孔）。转移刺激珠计算量灭菌的5毫升管。加入1 ml的人T细胞培养基中并涡旋。 </li><li>将管微珠悬浮体。帽应该是松散的管之上。孵育在室温下搅拌2分钟。 </li><li>轻轻取出盖从管。握住管体一起，澄清管内容到一个新的5毫升管。不要敲击或摇晃试管内容。 </li><li>从磁体取出管。添加人T细胞培养基（100μl的培养基中/ 0.5微升微珠的最初添加的）。 </li><li>等份加入100μl活化珠悬浮液至96孔板的各孔中。在6％CO 2的培养箱中孵育培养板在37℃下。 </li><li>监测细胞经显微镜检查每天的增长。培养第7天（ 图3.A） -小簇增殖细胞可在显微镜后5中观察到。 注:T细胞培养物，在这一点上的可视化是很困难的，并且不存在可观察到的细胞团在此阶段可能并不指示缺乏生长的T细胞。 </li><li>在后细胞分离14天，进料培养物通过仔细去除100微升的媒体离培养的顶部，并替换为100μ;升新鲜的人T细胞培养基中。继续在6％CO 2的培养箱中孵育培养板在37℃下。 </li><li>监测细胞通过显微镜检查和培养液的颜色的评估日生长。介质更改后第3天 - 微观2大细胞群体应该是可见的。宏观上，细胞沉淀应成为在14日内介质的变化可见。 </li><li> 28天以下的细胞分离，通过镜检确定增长的克隆。转移生长阳性的96孔板中培养至含有200μl的人T细胞培养基中的48孔组织培养板的各个孔中的200μl的体积。 </li><li>加入100μl含12.5微升刺激抗CD3 /抗CD28微珠（其制备如5.2节中所述 - 5.6）。在6％CO 2的培养箱中孵育培养板在37℃下。 </li><li>监察显微镜前培养中生长的日常胺化和培养基的颜色的评估。当T细胞培养物达到&gt; 2×10 6 / ml的（通常在3-5天后再次刺激后，可通过该介质，开始的当天推定回转草黄色），转移至培养在24孔组织培养板的孔中并加入500μl人类T细胞中。 </li><li>在10-14天随访再刺激，收集200微升T细胞培养用流量来评估术的pMHC四聚体结合分析（使用在第3和4.1中描述的标签和门控策略）同种抗原特异性。 </li></ol> 6.长期再刺激培养的T细胞克隆和<ol><li>不断地重新刺激和扩大克隆性T细胞培养物具有所需的特异性可以是每14天。收集的T细胞培养物至无菌5毫升管中在14天之后的最后的抗CD3 / CD28微珠刺激。合并相同的T细胞克隆的多口井在单一管中，最多为2.5毫升的体积。 </li&gt; <li>添加培养基以使终体积至2.5毫升，通过移液轻轻混匀。 </li><li>放置5毫升管含有T细胞培养到细胞分离磁铁，以从培养物中除去旧的微珠。帽应该是松散的管之上。孵育5分钟，在室温下进行。 </li><li>轻轻取下管帽。保持所述管和磁铁一起，滗出细胞悬浮液到一个新的5毫升管。不要敲击或摇晃管内容物去除最后一滴液体。 </li><li>添加培养基以使终体积到4ml。样品10微升进行计数使用血球细胞。 </li><li>恢复T细胞通过离心，在600×g离心5分钟，在室温下进行。 </li><li>弃去上清液。重悬的T细胞在10 6个细胞/ ml的人T细胞培养基中。转移1ml等分至24孔组织培养板的各孔中。 </li><li>再刺激T细胞，加入含有12.5微升刺激抗CD3 / 100μl的媒体NTI-CD28微珠（ - 5.6在5.2节所述）。在6％CO 2的培养箱中在37℃下。 </li></ol>

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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This diversity in the T cell response impairs the ability to examine the biochemistry of pMHC ligand recognition driving alloreactive T cell responses. The standard solution to this challenge has been to generate clonal T cell lines through repeated stimulation with a given allogeneic cell line and limiting dilution analysis. While this approach is capable of generating clonal T cell lines with a single specificity, it may not reflect the original composition of the oligoclonal response of naive T cells. Additionally, this approach does not directly identify the specific pMHC alloantigen recognized (even narrowing the response to a single HLA molecule still presents the challenge of finding the stimulatory peptide from among the estimated 2x104 endogenous peptides presented under steady-state conditions), complicating biochemical characterization of T cell ligand recognition in alloreactivity.
The protocol presented here utilizes fluorescently-labeled pMHC multimers, magnetic bead enrichment, and flow cytometry cell sorting to identify and isolate human T cells specific for individual allogeneic pMHC ligands. This technique has become a powerful tool for examining antigen-specific T cell responses12. Our increasing understanding of the highly peptide-specific nature of T cell recognition of allogeneic pMHC complexes7,8 has enabled us to adapt this approach to the study of alloreactive T cells in both mice14 and humans15. This approach uniquely enables examination of the composition of the naive T cell populations that comprise the initial oligoclonal alloreactive response. Furthermore, combination of the pMHC multimer labeling approach with in vitro tissue culture facilitates generation of clonal T cell cultures that can be used to dissect the biochemistry of allogeneic pMHC ligand recognition, a question that cannot adequately be addressed via studying polyclonal T cell responses and is significantly impaired by traditional T cell cloning methods. 
Using this approach, it can be expected that T cells specific for a given alloantigen pMHC should be identifiable for isolation and culture. However, the efficacy of this approach is dependent on multiple variables. First, the frequency of T cells from an immunologically naive donor that recognize a single alloantigen pMHC is relatively low (on the order of 1/103&#8211; 1/106) and varies between individuals. The ability to isolate pMHC-specific T cells depends on their abundance, as not all isolated cells will successfully expand to generate clonal cultures. The efficiency of the protocol can be influenced by several factors including the nature of the peptide used and the viability of the cells during preparation and isolation. The success of this technique also requires identification of allogeneic pMHC ligands capable of being re-folded into fluorescently-labeled multimers from recombinantly expressed HLA molecules and synthesized peptides. While there are multiple descriptions in the literature of identified peptides presented by specific HLA alleles, not all peptide re-fold in solution equally well. The efficacy of pMHC re-folding depends on several factors, including peptide length, solubility, and affinity for the MHC peptide binding pocket16. The ability of pMHC multimers to label antigen-specific T cells is also dependent on the peptide used. The affinity of the peptide for the MHC as well as the affinity of the TCR for the pMHC complex influences the binding of the fluorophore-labeled pMHC multimer. Weak binding can result in low shifts in fluorescence intensity, which can lead to difficulty in identifying antigen-specific cells by flow cytometry. These are significant technical hurdles that can impair the efficient isolation of alloantigen-specific T cells. In addition to these technical limitations, a significant biological caveat to this approach should be noted. T cell binding of pMHC tetramers is limited to TCRs with relatively high ligand affinity. This feature results in excluding T cells with low affinity for a ligand from identification and analysis. This is significant, as these low-affinity T cells are capable of substantial contribution to primary T cell responses17. However, even with these limitations, the approach described here is an efficient tool for the generation of clonal human T cells with defined antigen specificity. While the focus presented here is identification and isolation of human T cells specific for allogeneic pMHC ligands, the T cell identification, isolation, and culture techniques can be easily adapted to other pMHC specificities.

T淋巴细胞是适应性免疫系统的关键部件。 T细胞是负责不仅直接通过各种效应机制介导保护性免疫反应的病原体，还积极地维持免疫自身耐受性和指导其他细胞的应答，在免疫系统中。这些功能是通过一些综合信号，包括T细胞受体（TCR）的连接，细胞因子和趋化因子和代谢产物1定向。这些信号的，T细胞受体是特别重要的，因为它提供了特有的特异性，它定义在适应性免疫的T细胞的作用。 TCR的相互作用，在一个高度特异和敏感的态度提出了MHC（人类同源基因HLA）分子（的pMHC复合物），以提供启动T细胞的效应功能的信号线性肽抗原。 TCR互动与配体的pMHC的生化指标不仅提供了特异性对于T细胞的活化，同时也有在随后的T细胞的功能2质的影响。因此，研究T细胞功能往往需要研究克隆T细胞定义的抗原特异性的反应。 人T细胞区室，含有约10 12αβT细胞，包含大约7月10日至八月10日明显αβTCRs3-4。这种多元的曲目为识别肽繁多，从潜在的病原体，将必要的保护性免疫T细胞应答的机会。据估计，T细胞响应一个给定的外源抗原通过自MHC呈现的频率为10 -4的数量级上- 10-7在没有给该抗原5前的免疫应答。幼稚T细胞的剧目是由胸腺选择形状，确保认识自我MHC呈递的抗原肽和极限反应能力ivity对自身抗原肽，最大限度地提高调解的保护性免疫2的潜在效用。然而，违反了此设计的反应性，相对较大的频率，10 -3 - 10 -4，T细胞从免疫幼稚个体对刺激作出反应与同种异体细胞，识别两者的外来MHC分子，以及内源性肽它们呈现6。的同种异基因的pMHC配体的识别是结构上类似于识别通过自MHC呈现外源抗原的; TCR的使得与两个同种异体MHC分子以及所呈现的肽7临界生物化学相互作用。 T细胞以从的pMHC的多样性同种异体刺激的结果的响应的健壮本质络合物本同种异体细胞8的表面上。据估计，每MHC呈现约2×10 4个不同的内源性肽抗原9。这家B响应于同种异体刺激readth是临床相关的病理，如同种异体移植物排斥或移植物抗宿主病（GVHD）的显著方面，从T细胞的同种异体反应性所致。 研究的人T细胞的同种异体反应性的反应时检查下列刺激同种异体细胞幼稚T细胞的多克隆反应传统上依赖。反复刺激具有相同的同种异基因的细胞系结合的有限稀释分析能够产生单克隆T细胞定义的识别同种异体的HLA 10的。然而，这种方法是有问题的用于检查响应个体同种异体的pMHC配位体，如内源性的pMHC的大量不同的曲目络合物存在对于给定的同种异基因的HLA刺激T细胞的广泛的剧目。这种散装人口刺激和有限稀释方法需要大量克隆的筛选以分离的T细胞与所需reactiVITY针对单一的pMHC配体。此外，T细胞响应的个体同种异体的pMHC配体的频率相对低的幼稚T细胞群，其中介绍的障碍有效地产生人T细胞克隆响应于一个给定的抗原之一。 鉴定与多克隆群的抗原特异性T细胞的分离已经通过荧光团标记的pMHC多聚体11上的显影启用。该方法利用装入重组的可溶性生物素化的MHC分子，其通过结合到链霉亲和标记的荧光团标记的特异性肽抗原。的pMHC的多聚化增加了亲和力，补偿TCR的固有的低（微米）可溶性的pMHC配体的亲和力。标记的细胞可被识别和分离通过流式细胞术。但是，这种方法仍然由幼稚T细胞群中的抗原特异性T细胞，这是典型的低频率限制震级小于准确识别和量化大多数流式细胞仪的限制令。为了解决此限制，对的pMHC四聚体标记和随后的磁珠富集为四聚体标记的细胞的方法，已经开发了12。这种方法已经证明可靠检测，计数和低频抗原特异性T细胞的分离。 这个协议描述了一种有效的协议对人T细胞克隆的特异性个体同种异体的pMHC配体响应的产生。该协议适用的pMHC（HLA）的多聚体标记和富集对同种异体抗原-特异性的人T细胞的流式细胞术细胞分选和一个可靠的方法在体外培养的人T细胞的分离，使生产的从单个分选的细胞的T细胞克隆（概述图1）。

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Sodium heparin venous blood collection tube 16 x 100 mm</td>
			<td style="width:223px;">Becton, Dickenson and Company</td>
			<td style="width:119px;">366480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Lymphoprep</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">7801</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:273px;">Rosette Sep Human T Cell Enrichment Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">15061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dulbecco&#39;s PBS, 1x without Ca or Mg</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fluorophore-labeled pMHC tetramer</td>
			<td style="width:223px;">NIH Tetramer Facility</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Biotin Selection Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Selection magnet</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:273px;">TruStain FcX Human Fc blocking solution</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">422301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD5 PE-Cy7 (clone UCHT2)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">300621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD14 FITC (clone HCD14)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">325603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD19 FITC (clone HIB19)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">302205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Iscove&#39;s DMEM, without b-ME or L-glutamine</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">15-016-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin sulfate (50 mg/ml)</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">GT-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human AB serum, male donor</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">HS-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2</td>
			<td style="width:223px;">Peprotech</td>
			<td style="width:119px;">AF 200-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Dynabeads Human T-Activator CD3/CD28</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">11131D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Media</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cell sorting buffer</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS, pH 7.4</td>
			<td style="width:223px;">1 L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BSA</td>
			<td style="width:223px;">10g</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA (0.5 M)</td>
			<td style="width:223px;">2 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human T Cell Culture Medium</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Iscove&#39;s DMEM</td>
			<td style="width:223px;">351.6 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Heat-inactivated human AB serum</td>
			<td style="width:223px;">40 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES (1 M)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax (100 x)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin (50 mg/ml)</td>
			<td style="width:223px;">0.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-mercaptoethanol (14.3 M)</td>
			<td style="width:223px;">1.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2 (1 mg/ml)</td>
			<td style="width:223px;">1 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human alloantigen-specific t cells from peripheral blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

这个协议描述了克隆人T细胞培养物的产生与通过磁珠富集和单细胞的流式细胞术分选策略定义的同种异体抗原特异性。 图1提供了该方法的概要。 <img alt="图1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> 图1:协议概述这里描述的协议提供了用于产生从外周血同种异体抗原-特异性的人T细胞克隆的可靠方法。单T细胞的分离和建立细胞培养过程预计需时约4.5小时。扩展的T细胞克隆，需要多轮的非特异性T细胞的刺激和培养，每次服用14天。克隆培养物可以用于同种异体抗原特异性隔离28天之后进行测试，并且培养物可以进一步扩展为通过反复圆钢额外的测试刺激DS。 同种异体抗原 - 特异性T细胞的预期产量将取决于供体，所用的同种抗原，和反应性T细胞的供体的相应频率。平均健康供体将具有4.5 - 10.0×10 6个白细胞的血液/毫升，其中大约20％的T淋巴细胞图2示出了测量的T细胞的结合由HLA-DR4（HLA-DRB1 * 04的结果为:01。 ）阴性供体到一个特定的同种异体抗原，氨基酸残基的HLA-A2的蛋白（DTQFVRFDSDAASQRMEPR，A2 30-48）由II类分子HLA-DR4 13给出的30-48。 <img alt="图2" src="/files/ftp_upload/52257/52257fig2highres.jpg" /> 图2:流式细胞术，用于产生单细胞培养物分选同种异体抗原的四聚体标记的细胞。 A2的30-48 / HLA-DR4 tetrame A.流式细胞仪识别和隔离从HLA-DR4阴性供体的r-标记的T细胞。 T细胞用磁珠富集了四聚体标记的细胞通过流式细胞术进行排序，门控单峰CD5 + CD14 - CD19 -四聚体+淋巴细胞，如图所示。四聚体未标记的细胞被用作荧光增减一控制，建立选通的四聚体+细胞。B.四聚体+ T细胞分选至含有100μl的人T细胞培养基中在96孔圆底组织培养板中。该板设置包括阳性和阴性对照孔中所指示的。 从2.0×10 7白细胞起始数，我们发现四千七百七十六分之一T细胞同种异体抗原特异性细胞的频率: 1.1×10 6四聚体富集单元×0.992（单峰）×0.744（淋巴细胞）×0.477（CD5 + CD14 - CD19 - ）×0.0034（四聚体+ T细胞）/ 2.0×10 7外周血白细胞X 0.314（预富集CD3 +） 从开始2.0×10 7白细胞我们能够隔离88个人四聚体标记的T细胞进行培养。后2轮的抗CD3 / CD28微珠刺激和培养所描述的，我们确定了2个阳性生长培养物通过显微镜检查（生长单个细胞的分离和培养在图3.A所示后第10天的代表性的例子）。如所描述的克隆的培养物扩展到1ml，和同种异体抗原特异性通过检查A2 30-48 / HLA-DR4的四聚体（ 图3.B）的结合进行评估。 <img alt="图3" src="/files/ftp_upload/52257/52257fig3highres.jpg" /> 图3.评价T细胞的培养。答镜检96孔板的T细胞培养物。 T细胞培养10的有代表性的例子单个T细胞通过流式细胞仪分选分离后几天。细胞用0.5微升抗CD3 / CD28微珠/孔（实施例由箭头表示）在200微升的人T细胞培养基的体积。通过流式细胞术检测的pMHC的分析克隆性T细胞培养物B. T细胞的同种异体抗原特异性四标签。流的克隆性T细胞培养物中细胞计数分离CD5 + CD14 - CD19 - A2 30-48 / HLA-DR4的四聚体+细胞进行检查，以了解A2 30-48 / HLA-DR4四聚体4 周的体外培养后的约束力。四聚体未标记的细胞被用作荧光增减一控制，建立选通的四聚体+细胞。

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.9K 次观看.  University of California, San Diego. This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

视频: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

协同刺激信号封锁的同种抗原刺激诱导人外周血单个核细胞同种异体抗原特异性无能

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

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 ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.