这项工作得到了密歇根大学综合癌症中心的威尔和珍妮·考德威尔捐赠研究基金的支持，部分得到了NIH授予CA82529和吉尔森·朗根博基金会的支持。

1. 阿尔德弗卢尔染色
<ol><li>细胞样本制备：准备肿瘤细胞的单细胞悬浮，无论是从培养的肿瘤细胞或从新收获的肿瘤样本。数数细胞并调整细胞悬浮到ALDEFLUOR检测缓冲区中1 x10 6 细胞/毫升的浓度。</li>
	<li>将四个 12 mm x 75 mm 聚苯乙烯试管标记为控制管，具体如下： #1： 未染色， #2： 阿尔德弗卢尔， #3： 阿尔德弗卢尔加 DEAB， 和#4： 7AAD.</li>
	<li>对于这些控制管，将 1ml 细胞悬浮放入#1和#4，但将 2 毫升放入管#2。将 5μl DEAB （ALDH 抑制剂） 添加到管#3中，并保持冰上。然后将 10 μl 激活的ALDEFLUOR基板加入管#2，混合并立即将1毫升的混合物转移到管#3。</li>
	<li>同时，在ALDEFLUOR测定缓冲区中，以1 x 106细胞/毫升的速度将每百万个细胞中激活的ALDEFLUOR基板添加到其他细胞（样本管）中。</li>
	<li>在 37 °C 水浴中将 4 个控制管和样品管孵育 30 分钟。</li>
	<li>孵化后，将 5μl 7AAD 添加到控制管#4中，并将 1 μl 7AAD 1 x 106 细胞添加到样品管中。在4°C孵育10分钟。</li>
	<li>在250 x g下将所有管子离心5分钟，并去除超自然物。</li>
	<li>在阿尔德弗卢尔分析缓冲区中恢复细胞。</li>
	<li>执行基于流细胞测量的排序。</li>
	<li>使用与DEAB视为负控制的ALDEFLUOR染色细胞和硫化物（PI）7AAD染色细胞设置分拣门，以进行生存控制。基于这些控制，建立了一个门来区分ALDEFLUOR®/ALDH高，D5和SCC7细胞。然后，这些门将用于对上面为ALDEFLUOR®/ALDH高 D5和SCC7细胞准备的所有样本细胞进行排序。</li>
</ol>2. 癌症干细胞莱萨特-脉冲丹德里特细胞（CSC-TPDC）疫苗的制备
<ol><li>使用二氧化碳 2和分离股骨和头骨，对小鼠（分别合成B6和C3H）进行安乐死。</li>
	<li>在室温下将骨头放入75%乙醇中1分钟。然后使用 HBSS 清洗骨头。</li>
	<li>切掉骨头，用21G针和10毫升注射器将细胞推入菜中。</li>
	<li>使用注射器吸气并吹击细胞，使单细胞悬架。</li>
	<li>离心后在1，500转米5分钟，丢弃超自然。然后添加 5 毫升 RBC 裂解缓冲器，在 37 °C 水浴中溶解 RBC 1 分钟。</li>
	<li>计算细胞数量，并将细胞悬浮量调整为100万个细胞/毫升的浓度，在全中等（CM）中含有10 ng/ml GM-CSF和10 ng/ml IL-4。</li>
	<li>培养细胞，并在3天后补偿CM加IL-4和GM-CSF。</li>
	<li>在第5天，收获不成熟的树突状细胞（DCs），并准备包含4.2毫升溶液C加1毫升OptiPrep密度梯度介质的直流隔离介质。</li>
	<li>将细胞颗粒悬浮在 5 毫升 CM 中。将细胞悬浮缓慢添加到隔离介质上。离心机在室温下为 2，000 rpm。</li>
	<li>收集CM和隔离介质之间的DC。计算细胞数量，并将细胞悬浮量调整为培养介质中浓度为 1000 万细胞/毫升，其中含有 10 ng/ml GM-CSF 和 10 ng/ml IL-4。</li>
	<li>通过将ALDH高 或未分类的D5或SCC7细胞悬浮在培养介质中，并接受四次快速冻解暴露，然后以∼100 x g旋转5分钟来收集岩气的膜部分，从而准备肿瘤解冻。</li>
	<li>为了准备 CSC-TPDC，脉冲 DCs 与自体 ALDH高细胞的解体。为了准备H-TPDC，脉冲DC与未分类的异质肿瘤细胞催化。直流与肿瘤细胞的对比是相同的。</li>
</ol>3. 疫苗接种和有效性评估
<ol><li>分别用2，500个D5 CSC-TPDC或D5 H-TPDC肿瘤细胞皮下（s.c）给普通B6小鼠接种疫苗。分别 用 5000个SCC7 CSC-TPDC和SCC7 H-TPDC肿瘤细胞接种正常的C3H动物.c。</li>
	<li>接种疫苗后，用异质D5肿瘤细胞（即20天后使用二氧化碳对小鼠实施安乐死）挑战B6小鼠。收获肺部并列举肺转移。</li>
	<li>在SCC7模型中，挑战C3H小鼠与未分类的SCC7肿瘤细胞 .c 在DC疫苗的对面。监测肿瘤大小。</li>
	<li>实验结束时，使用二氧化碳对B6和C3H小鼠实施安乐死。在第一次接种疫苗后的第34天，用二氧化碳对小鼠实施安乐死，同时通过无菌程序收集每组的脾脏。</li>
	<li>激活脾 T 和/或 B 细胞与固定的反 CD3 加上抗 CD28 mAb 在 CM 包含 hrIL-2 或 LPS 加上防 CD40 （FGK45） mAb 阿西特。</li>
	<li>激活后，收集超纳特并用于 ELISA 检测。</li>
	<li>对于 ELISA 检测，涂层板与抗体为IFNγ，GM-CSF 和 IgG 在 4 °C O/N.</li>
	<li>使用阻塞缓冲后，添加样品和标准，并在室温下孵化。</li>
	<li>清洗板并添加HRP检测抗体和TMB基板进行孵化。</li>
	<li>停止反应后 30 分钟内测量 ELISA 板读卡器上的吸收度，达到 450 nm。</li>
</ol>

<ol>
	<li>Shafee, N., Smith, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+contribute+to+cisplatin+resistance+in+Brca1/p53-mediated+mouse+mammary+tumors.">Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors.</a> Cancer Res. 68, 3243-3250 (2008).</li><li>Nandi, S., Ulasov, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+radiation+enhances+survivin-mediated+virotherapy+against+malignant+glioma+stem+cells.">Low-dose radiation enhances survivin-mediated virotherapy against malignant glioma stem cells.</a> Cancer Res. 68, 5778-5784 (2008).</li><li>Dallas, N. A., Xia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoresistant+colorectal+cancer+cells,+the+cancer+stem+cell+phenotype,+and+increased+sensitivity+to+insulin-like+growth+factor-I+receptor+inhibition.">Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition.</a> Cancer Res. 69, 1951-1957 (2009).</li><li>Yamauchi, K., Yang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cancer+metastasis+by+cyclophosphamide+pretreatment+of+host+mice:+an+opposite+effect+of+chemotherapy.">Induction of cancer metastasis by cyclophosphamide pretreatment of host mice: an opposite effect of chemotherapy.</a> Cancer Res. 68, 516-520 (2008).</li><li>Chang, A. E., Redman, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+trial+of+tumor+lysate-pulsed+dendritic+cells+in+the+treatment+of+advanced+cancer.">A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer.</a> Clin. Cancer Res. 8, 1021-1032 (2002).</li><li>Ito, F., Li, Q., Shreiner, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-CD137+monoclonal+antibody+administration+augments+the+antitumor+efficacy+of+dendritic+cell-based+vaccines.">Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines.</a> Cancer Res. 64, 8411-8419 (2004).</li><li>Kirk, C. J., Hartigan-O'Connor, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell-dependent+antitumor+immunity+mediated+by+secondary+lymphoid+tissue+chemokine:+augmentation+of+dendritic+cell-based+immunotherapy.">T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy.</a> Cancer Res. 61, 2062-2070 (2001).</li><li>Ning, N., Pan, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+vaccination+confers+significant+antitumor+immunity.">Cancer stem cell vaccination confers significant antitumor immunity.</a> Cancer Res. 72, 1853-1864 (2012).</li><li>Ginestier, C., Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR1+blockade+selectively+targets+human+breast+cancer+stem+cells+in+vitro+and+in+xenografts.">CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts.</a> J. Clin. Invest. 120, 485-497 (2010).</li><li>Carpentino, J. E., Hynes, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase-expressing+colon+stem+cells+contribute+to+tumorigenesis+in+the+transition+from+colitis+to+cancer.">Aldehyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer.</a> Cancer Res. 69, 8208-8215 (2009).</li><li>Charafe-Jauffret, E., Ginestier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cell+lines+contain+functional+cancer+stem+cells+with+metastatic+capacity+and+a+distinct+molecular+signature.">Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.</a> Cancer Res. 69, 1302-1313 (2009).</li></ol>

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免疫功能不强的宿主，如SCID小鼠，由于宿主内部缺乏适应性免疫力，排除了对CSC的免疫学评估。在这项研究中，我们评估了CSC在免疫能力宿主中的免疫原性，它可以更密切地模仿患者设置。与未经选用的肿瘤细胞利萨酸盐脉冲DC相比，富含浓缩的CS是免疫性的，当其利萨特作为疫苗加载到DC中时，可以诱导更有效的肿瘤保护免疫力。 从机械学上讲，保护是通过选择性地诱导CSC反应抗体和T细胞8 以及生产1型细胞因子， 如 IFNγ和GM-CSF而给予的。
目前大多数免疫疗法，包括树突状细胞基疫苗和采用T细胞转移，都旨在针对肿瘤分化抗原。因此，可能无法表达这些分化抗原的CSC可能逃脱这些免疫靶向。相比之下，专门针对癌症干细胞的CSC疫苗可能会破坏癌细胞的特殊群体，从而通过防止肿瘤复发和转移来提高疫苗的疗效。
在培养的肿瘤细胞和新收获的肿瘤中，我们根据高醛脱氢酶活性，通过流动细胞学识别出CSC富集人群。这种ALDH高 细胞可以通过流分拣来分离，用作脉冲直流以产生CSC-TPDC的抗原源。 与从培养肿瘤细胞中分离出来的ALDH高 细胞 与从新收获的肿瘤中分离出来的比较表明，抗CSC免疫8的诱导期没有显著差异。这些结果表明，有可能使用从培养肿瘤细胞或从新收获的肿瘤中分离出来用于临床应用的CSC。
为了在临床上相关，疫苗需要在治疗环境中检查。这些实验正在我们的实验室进行。

癌症干细胞对常规化疗和放疗具有相对的抗药性。另一方面，这种细胞群可能是传统癌症疗法1-4后导致癌症复发和进展的细胞。由于在癌症干细胞上缺乏分化肿瘤抗原的表达，癌症干细胞可能逃避目前癌症治疗的免疫干预，这些干预主要针对分化肿瘤细胞上的抗原。因此，制定专门针对和摧毁癌症干细胞的新战略可能有望提高当前癌症治疗的疗效。为此，我们从两个动物肿瘤（黑色素瘤D5和鳞状细胞癌SCC7）中分离出富含癌症干细胞（CSC）的群体，并利用它们作为抗原源脉冲抗原呈现细胞（树突状细胞，DC），以准备CSC-TPDC疫苗。然后，我们分别对CSC-TPDC疫苗在合成免疫能力宿主、B6小鼠和C3H小鼠中诱导的抗肿瘤免疫进行了评估。CSC-TPDC 诱导的抗肿瘤功效与传统的 DC 疫苗与来自未分类的异质肿瘤细胞 （H-TPDC） 的裂解剂进行了比较，该疫苗以前已被我们组5，6以及临床前研究和临床试验中的其他研究人员 7 使用。

<table border="0" cellpadding="0" cellspacing="0" width="1022">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">ALDHEFLOUR KIT</td>
			<td style="width:399px;">Stemcell Technologies</td>
			<td style="width:219px;">1700</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine IL-4</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">214-14</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine GM-CSF</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">315-03</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse IFN-&#611; ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555138</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse GM-CSF ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555167</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">OptiPrep Density Gradient Medium</td>
			<td style="width:399px;">Sigma Aldrich</td>
			<td style="width:219px;">D1556</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">BD OptEIA TMB Substrated Reagent Set</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555214</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;"></td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Equipment</td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">BD FACS Aria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>336834</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Kcjunior</td>
			<td style="width:399px;">Bio-Tek Instruments</td>
			<td style="width:219px;">176058</td>
		</tr>
	</tbody>
</table>

generation of a novel dendritic-cell vaccine using melanoma and squamous cancer stem cells

我们用ALDEFLUOR/ALDH作为标记，从穆林黑色素瘤D5合成物到C57BL/6小鼠和鳞状癌SCC7同基因到C3H小鼠，并利用细胞赖氨酸作为抗原来源来脉冲树突状细胞（DCs），确认了其免疫原性。与 DCs 脉冲与 ALDH高 CSC 裂解剂诱导明显更高的保护性抗肿瘤免疫力比 DCs 脉冲与未分类的整个肿瘤细胞裂解的裂解剂在两个模型和肺转移设置和 s .c。肿瘤生长设置，分别。这种现象是由于CSC疫苗引起的幽默以及细胞抗CSC反应。特别是，从接受CSC-DC疫苗的宿主分离出的脾脏细胞产生的IFNγ和GM-CSF量明显高于从接受未分类肿瘤细胞催化脉冲直流疫苗的宿主中分离的脾酸细胞。这些结果支持了开发一种基于 CSC 的自体治疗疫苗，用于辅助环境下临床使用的努力。

ALDEFLUOR/ALDH已被用作分离多种恶性肿瘤中干细胞的单一标记。我们使用ALDEFLUOR作为标记，在两个肿瘤模型D5和SCC7中识别出癌症干细胞丰富的人群。我们在穆林黑色素黑色素瘤 B16-D5 和鳞状细胞癌 SCC7 中检测到 ALDEFLUOR® 细胞。我们发现，ALDEFLUOR®细胞在培养D5和SCC7肿瘤细胞系中分别贡献了约0.5%和5.2%（图1）。已对已建立肿瘤的新鲜收获的肿瘤细胞进行了分析，以确认ALDEFLUOR®细胞的存在。如图1所示，体内的ALDEFLUOR®细胞分别有2.5%和4.2%来自体内的D5和SCC7肿瘤。这些分类D5和SCC7 ALDEFLUOR®/ALDH高种群的肿瘤原性和自我更新能力分别在合成免疫能力宿主、C57BL/6和C3H小鼠中进行了评估。
我们在动物研究和临床试验中都使用异质未分类肿瘤细胞催化物来脉冲DC（H-TPDC）。为了检查CSC的免疫原性，我们分离出ALDH高 CSC和脉冲直流与CSC的脉冲直流产生CSC-TPDC（图2），并使用H-TPDC作为常规的癌症疫苗控制，以测试CSC-TPDC是否对预防肿瘤生长有任何有益作用。
我们通过检查CSC-TPDC诱导的保护性抗肿瘤免疫来评估CSC的免疫原性。在D5模型中，天真的免疫能力小鼠接种了CSC-TPDC或H-TPDC（在同一个lysate：直流比）.c。对照组收到盐水（PBS）。上次接种疫苗一周后，小鼠接受未分类的D5肿瘤细胞静脉注射（即静脉注射）的挑战，3周后采集肺部以列举肺转移。如表1所示，与PBS组相比，用H-TPDC治疗的小鼠的肺转移较少。重要的是，在两项实验中，用CSC-TPDC治疗的小鼠的肺转移比H-TPDC疫苗组少得多。在SCC7模型中，普通的C3H动物接种了疫苗.c SCC7 CSC-TPDC和H-TPDC分别在右侧，随后挑战未分类的SCC7肿瘤细胞.c到左侧。与PBS组相比，H-TPDC对肿瘤生长具有适度的抗肿瘤免疫力。然而，与对照组和H-TPDC组（p<0.05，图3）相比，用CSC-TPDC治疗的小鼠的肿瘤生长有显著的抑制作用。这些结果表明，CSC可以用作比传统未分类肿瘤细胞更有效的抗原源来加载DC，以诱导保护性免疫力免受肿瘤细胞的挑战。
为了进一步了解观测到的CSC诱导的保护性抗肿瘤免疫机制，我们在实验结束时从接受DC疫苗接种的动物身上采集了脾脏。然后，通过抗 CD3/CD28/IL-2 或反 CD3/CD28/IL-2 + LPS/抗 CD40 激活脾脏细胞。然后收集培养超纳特，以检测细胞因子和抗体的表达。 接种D5 CSC-TPDC或SCC7 CSC-TPDC（图4）的动物脾细胞产生的IFNγ和GM-CSF显著增加。此外，LPS/抗CD40活性脾细胞从接种D5 CSC-TPDC或SCC7 CSC-TPDC接种的动物身上采集的IgG产量显著（p<0.05）显著高于D5 H-TPDC或SCC7 H-TPDC。这些抗体分别与D5和SCC7 CSC结合，这种结合可能导致CSC裂解在补充8的情况下。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig1highres.jpg" src="/files/ftp_upload/50561/50561fig1.jpg"> 图1。 在培养以及新收获的新鲜穆林D5黑色素瘤和SCC7鳞状细胞肿瘤中检测到ALDHFLUOR®/ALDH高 种群。 用50 mmol/L DEAB（一种特定的ALDH抑制剂）治疗的肿瘤细胞被用作负控制。
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig1highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig2highres.jpg" src="/files/ftp_upload/50561/50561fig2.jpg"> 图2。 生成树突状细胞为基础的癌症干细胞疫苗。 在CSC-TPDC和H-TPDC的制备中，骨髓衍生的树突状细胞分别用ALDH高 或未分类的肿瘤细胞催化物脉冲。
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig2highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig3highres.jpg" src="/files/ftp_upload/50561/50561fig3.jpg"> 图3。 CSC赖苏酸盐脉冲直流（CSC-TPDC）疫苗可以诱导更有效的保护性抗肿瘤免疫在 s.c。 SCC7肿瘤模型。
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig3highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig4highres.jpg" src="/files/ftp_upload/50561/50561fig4.jpg"> 图4。 更有力的全身细胞反应在免疫能力主机接种CSC-TPDC疫苗。 从接受H-TPDC或CSC-TPDC疫苗接种的动物身上采集了圈卵细胞，并按指示激活。然后收集培养超纳特，使用ELISA进行细胞因子检测。
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig4highres.jpg" target="_blank">单击此处查看更大的图像。</a>

Watch this Scientific Journal Video about Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells at JoVE.com

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

在合成免疫能力宿主中评估，基于癌症干细胞 （CSC） 的树突状细胞 （DC） 疫苗的抗肿瘤免疫力明显高于使用异质散装肿瘤细胞脉冲的传统 DC 疫苗。

使用黑色素瘤和鳞状癌干细胞生成新型支气干细胞疫苗

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

generation-novel-dendritic-cell-vaccine-using-melanoma-squamous

Research

JoVE Journal

Immunology and Infection

11.2K 次观看.  University of Michigan. 在合成免疫能力宿主中评估，基于癌症干细胞 （CSC） 的树突状细胞 （DC） 疫苗的抗肿瘤免疫力明显高于使用异质散装肿瘤细胞脉冲的传统 DC 疫苗。

视频: Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

科研

免疫与感染

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

鞘糖脂抗原的质谱分析

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

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 Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...