我们感谢西蒙博士的英语编辑。这项研究得到了干杯研究所 (ISCIII) 的赠款的支持 (PI14/00526;PI17/01395;CP11/00145;CPII16/00022), 米格尔 Servet 计划和基金会拉蒙 Areces;Fondo Europeo de 收复与发展区域 (菲德) 共同出资。CNIC 得到经济、工业和竞争力部 (经济) 和 CNIC 基金会的支持, 是 Severo 奥乔亚卓越中心 (SEV-2015-0505)。RTF 是由基金会拉蒙 Areces 和 CNIC, VZG 由 ISCIII, BHF 由 Investigación 疗养院医院 12 de Octubre (imas12) 和 JMG G ISCIII 的 Servet 计划和 imas12。

根据西班牙和欧洲准则, 基金会 Investigaciones Cardiovasculares 卡洛斯 III (CNIC) 和 Comunidad 国立马德里批准了实验程序。小鼠在特定病原体游离 (SPF) 条件下繁殖, 并通过二氧化碳 (CO2) 吸入进行安乐死。
1. 从胫骨和股骨中分离小鼠骨髓细胞
注: C57BL/6 同类系小鼠菌株携带的差异白细胞标记Ptprca, 一般认为 CD45.1 或 Ly5.1, 而野生型 C57BL 菌株携带Ptprcb 等位基因, 称为 CD45.2 或 Ly5.2。CD45.1 和 CD45.2 变异可以用抗体的流式细胞术来区分。CD45.1、CD45.2 和 CD45.1/CD45.2 小鼠可作为细胞来源或接受移植, 允许通过流式细胞仪追踪不同细胞群。优先使用年龄和性别匹配的雄性或雌性小鼠低于12周的年龄。
<ol><li>
股骨和胫骨的制备
	<ol>
<li>弄死老鼠使用由机构动物保育委员会批准的议定书。</li>
		<li>用70% 乙醇喷洒动物表面, 对后肢进行消毒。</li>
		<li>使用无菌剪刀, 镊子和手术刀。用手术刀, 做一个切口在皮肤和去除皮肤从老鼠的远端部分包括皮肤覆盖后四肢。剥下小腿肌肉周围的皮肤, 完全去除腿部的皮肤 (图 2A, 2B)。</li>
		<li>用手术刀将股四头肌从股骨中分离出来。Disarticulate 髋关节不折断股骨头。用手术刀从胫骨上取出肌肉 (图 2C, 2D)。将股骨与胫骨分开, 不折断骨末端。</li>
		<li>保持骨骼在一个培养皿中含有10% 胎牛血清 (血清) 和1% 青霉素/链霉素在冰冷的1x 罗斯威尔公园纪念研究所 (RPMI) 1640 中等。</li>
	</ol>
</li>
	<li>
单元格隔离 注: 所有后续步骤必须在一个文化罩和无菌材料, 以避免污染。<ol>
<li>在无菌培养皿中, 用手术刀小心翼翼地切断每根骨头的近端和远端。</li>
		<li>反复冲洗的骨骼与总容积10毫升的温暖完整的 RPMI 培养基 (RPMI + 10% 血清, 2 毫米 EDTA, 1% 青霉素/链霉素, 20 毫米 HEPES, 55 µM 2-巯基乙醇, 1 毫米丙酮酸钠, 2 毫米 l-谷氨酰胺)。用25克针附着在1毫升注射器上, 从两端冲洗骨头。</li>
		<li>将 effluate 转移到装有70µm 尼龙网过滤器的50毫升锥形管上。通过温和搅拌和吹打, 小心地去除碎片和细胞企业集团。</li>
		<li>在室温下, 将细胞悬浮在 250 x g处进行10分钟的离心。</li>
		<li>并用重悬细胞颗粒在1毫升的冷红细胞裂解缓冲 (0.15 米 NH4Cl + 1 毫米 KHCO3 + 0.1 毫米 EDTA, 调整 pH 值7.2 与 1 N HCl, 0.4 µm 无菌过滤, 并储存在4°c)。用人工震动在冰上维持5分钟。</li>
		<li>添加10毫升的冷缓冲 (1x 磷酸缓冲盐水 (PBS) + 2 毫米 EDTA + 2% 牛血清白蛋白), 以灭活和冲洗红细胞裂解缓冲液。</li>
		<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
		<li>洗涤细胞与25毫升完全 RPMI 培养基和离心机在 250 x g 5 分钟在4°c。</li>
		<li>并用重悬5毫升完全 RPMI 培养基中的细胞。混合50µL 的细胞悬浮1:1 与台盼蓝。在显微镜下确定计数室中的细胞数。使用公式计算单元格数: 细胞/mL = [(非染色细胞 count/4) x 2 x 1万]12。 注: 此方法产生 40 x 106到 60 x 106骨髓细胞每未感染8到12周老 C57BL/6 老鼠。</li>
		<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
	</ol>
</li>
</ol>2. DC 分化、成熟和抗原加载
<ol><li>
BM 细胞采集
	<ol>
<li>在6井超低附着面板上播种完整的培养基, 每毫升中等 106细胞, 通常在5毫升每井。分化的巨噬细胞将附着在培养板上。12小时后, 转移上清, 其中主要含有单核细胞, 以清洁6井板, 丢弃旧6井板与粘附巨噬细胞。</li>
		<li>为了促进细胞分化, 培养非黏附细胞在 5 x 105细胞每毫升在典型地5毫升每 RPMI 媒介包含 20 ng/毫升商业上获得的重组小鼠 GM 脑脊液在37°c 和5% 二氧化碳 (CO2) 和每日观察。</li>
		<li>每3天, 旋转下来悬浮细胞和收集的黏附细胞与5毫米 EDTA 在 PBS 和旋转下来。结合和并用重悬在 5 x 105细胞/毫升的新鲜培养基含有 20 ng/毫升 rm-脑脊液。</li>
		<li>在8天, 在非组织培养处理的无菌培养皿中, 以 5 x 105细胞/毫升为单位, 在含有 20 ng/毫升的并用重悬和 20 ng/毫升脂多糖 (LPS) 的培养基上采集附着体和分离细胞, 以诱导高 MHC II。, CD80 和 CD86 表达。</li>
		<li>用流式细胞术检查 DC 分化, 如步骤2.2 所示。</li>
	</ol>
</li>
	<li>
流式细胞术的分化和成熟分析。
	<ol>
<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。重复此步骤两次。</li>
		<li>阻断 fc 受体通过孵化悬浮细胞颗粒在小鼠 Fc 受体阻滞剂 (纯化的抗小鼠 CD16/CD32 IgG2b抗体) 15 分钟, 4 摄氏度根据制造商的指示。</li>
		<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
		<li>对于每个探针, 并用重悬25万细胞在300µL 流式细胞仪缓冲 (1x PBS + 2 毫米 EDTA + 2% BSA) 在4°c。</li>
		<li>将30µL 的细胞溶液转移到96井板上。</li>
		<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
		<li>在制造商的指示之后, 丢弃上清, 并在每个井中添加30µL 的含抗体缓冲液。用抗体在4摄氏度孵育细胞20分钟。 注: 通常用于 DCs、单核细胞和巨噬细胞的标记为 CD11b、CD64、CD115、CD11c、MHCII、CD80 和 CD86 (图 3)。</li>
		<li>离心样品在 250 x g 5 分钟在4°c 和并用重悬在200µL 每井的冷流式细胞术缓冲器包含标记排除死的细胞 (例如,碘碘化物, 4′-, 6-diamidino-2-苯基吲哚 (DAPI) 或胺反应染料) 在制造商推荐的浓度13。</li>
		<li>通过将样本转移到流式细胞术管并进行流式细胞术分析, 在0、3、6和9天排除死细胞, 从而监测细胞分化。</li>
	</ol>
</li>
	<li>
直流抗原加载。
	<ol>
<li>在8天, 离心机样品在 250 x g为5分钟在4°c 和并用重悬药丸在200µL 媒介 (RPMI + 10% 没有抗生素的血清)。重复此步骤两次。</li>
		<li>用10µg/毫升 OVAp (323-339) 孵化 DCs;ISQAVHAAHAEINEAGR) 每 1 x 107 DCs 在1毫升中等 (RMPI + 1%) 在塑料管为至少30分钟在37°c。使用非 OVAp 加载 DCs 作为负控制条件。</li>
		<li>离心样品在 250 x g 5 分钟在4°c 和并用重悬小球在200µL 完全 RPMI 媒介。重复此步骤两次。</li>
		<li>离心样品在 250 x g 5 分钟在4°c 和并用重悬小球在200µL 媒介 (RPMI + 10%) 在 2 x 106细胞/毫升。</li>
	</ol>
</li>
</ol>3. OTII 小鼠天真 CD4 T 细胞的分离
注: 此方法产生约 100 x 106 spleenocytes 每未感染8至12周老 C57BL/6 老鼠。男性和女性都可以使用。CD4 T 细胞可与脾或淋巴结分离;CD4 T 细胞分别占到这些器官中大约25% 和50% 的细胞。在无菌条件下执行以下步骤。
<ol><li>从 OTII 转基因小鼠中分离腹股沟、腋、臂、颈、肠系膜淋巴结和脾脏 (图 4), 并将其转移到含有10毫升完整 RPMI 培养基的塑料培养皿中。</li>
	<li>用2毫升完整的 RPMI 培养基冲洗细胞过滤器, 从50毫升管中取出。将淋巴结和脾脏转移到70µm 细胞过滤器放置在50毫升管。融汇使用注射器柱塞 (图 5), 并添加中等至15毫升。</li>
	<li>离心样品在 250 x g 5 分钟在4°c 和并用重悬颗粒在15毫升完全 RPMI 媒介。重复此步骤两次。</li>
	<li>对于脾细胞悬浮, 并用重悬细胞颗粒, 并溶解在步骤1.2.5 中所示的红细胞。</li>
	<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
	<li>用25毫升的完全 RPMI 培养基和离心机在 250 x g处冲洗细胞, 在 RT 上为5分钟。</li>
	<li>并用重悬5毫升培养基中的细胞。混合50µL 的细胞悬浮1:1 与台盼蓝。用显微镜下的计数室确定细胞数。使用公式计算单元格数: 细胞/mL = [(非染色细胞 count/4) x 2 x 1万]12。</li>
	<li>重复步骤2.2.2。</li>
	<li>按照制造商的指示, 孵化细胞与1:800 稀释生物素化抗体 CD8α, IgM, B220, CD19, MHCII (I Ab), CD11b, CD11c, CD44, CD25 和 DX5 30 分钟的冰, 以后来阴性选择 CD4 T 细胞。</li>
	<li>按照制造商的指示, 根据电池的数量和珠子的容量, 计算所需的链亲和素涂层磁性微球的数量和隔离 CD4 T 细胞使用磁性细胞分离器和适当的分离列。</li>
	<li>并用重悬5毫升完整的 RPMI 培养基中的细胞, 并保持它们在冰上, 而计数活细胞如步骤3.7 所述。</li>
	<li>提取 2 x 105细胞, 并检查收集细胞的纯度使用抗 CD4, CD3, B220 和 CD8 荧光抗体在流式细胞仪使用制造商推荐抗体稀释。</li>
	<li>为了染色孤立的天真 CD4/OTII T 细胞, 并用重悬 106细胞500µL 缓冲 (1x PBS + 2 毫米 EDTA + 2% BSA)。准备500µL 缓冲器 (1x PBS + 2 毫米 EDTA + 2% BSA), 其中包含双最终制造商推荐浓度的关键细胞示踪染料。通过缓慢地将细胞示踪液添加到细胞悬浮液中, 混合这两种溶液。在37摄氏度孵化细胞5分钟。</li>
	<li>用5毫升的完全 RPMI 培养基和离心机在 250 x g 处清洗细胞, 在4摄氏度时为5分钟。重复此步骤两次。并用重悬在 1 x 107细胞/毫升的1毫升完全 RPMI 培养基中的细胞。</li>
</ol>4.体内活化、增殖和 Th1 分化试验
注: 生命细胞示踪染料羧基双乙酸琥珀酰亚胺酯 (CFSE) 和其他琥珀酰胺酯基染料, 它们在不同波长激发和发出荧光, 被广泛用于评估流动的淋巴细胞增殖。细胞仪由于其荧光强度高, 寿命长, 变异性低, 毒性低14。
<ol><li>过继转输转移静脉 (静脉注射) 1 x 106活细胞示踪染料染色的天真 CD4/OTII T 细胞100µL 的 PBS 每只老鼠。标记的 T 细胞可以过继转输转移的尾静脉或复古轨道注射15;在这两种情况下, 细胞会回到淋巴器官 (图 6A)。</li>
	<li>一天后, 通过过继转输转移 10万 LPS 成熟的 OVAp 加载 DCs (s.i.) 在脚垫 (图 6B), 对接受者老鼠提出挑战。</li>
	<li>从接受者小鼠那里收获腘淋巴结, 并处理它们, 直到获得单细胞悬浮后, 与先前在3.1 和3.2 中描述的步骤相同。这样做2天后免疫后, 以测量 CD4/OTII T 细胞活化, 并在5天测量增殖加上 Th1 分化。</li>
	<li>为了分析2天的 T 细胞活化, 用荧光抗体对 CD4、CD69 和 CD25 (可选 CD45.1 和 CD45.2) 染色细胞, 并确定 C69 人口中CD25 + CD4+ T 细胞的百分比。用流式细胞仪分析 (图 7)。</li>
	<li>为了检查5天的 T 细胞增殖, 染色细胞使用适当的荧光抗体对 CD4 和可选的 CD45.1 和 CD45.2。用流式细胞仪分析 CD4 种群中生命细胞示踪染料信号的衰减情况 (图 8)。在这些研究中可以确定几个参数, 例如, 细胞分裂的数量由标记与关键细胞示踪染料, 每个荧光峰中分裂细胞的百分比和总细胞的分裂细胞百分比人口。</li>
	<li>为了确定5天的 Th1 分化, 40万个96井板中的200µL 的 RPMI 培养基中有1个µM ionomycin 和 10 ng/毫升佛波12酯13醋酸酯 (PMA), 在孵化箱6摄氏度的37小时。在过去的4小时, 添加3–10µg/毫升 brefeldin A 阻断细胞因子分泌的培养基。</li>
	<li>离心板5分钟在 250 x g在4°c。通过快速反演96井板, 丢弃上清液。</li>
	<li>重复步骤2.2.2。</li>
	<li>在30µL (1x PBS + 2 毫米 EDTA + 2% BSA) 中孵化15分钟, 在4摄氏度处染色细胞膜, 其中含有 CD4 的抗体, 并可选择在制造商推荐的稀释上 CD45.1 和 CD45.2。通过添加150µL 缓冲细胞 (1x PBS + 2 毫米 EDTA + 2% BSA) 和离心板为5分钟在 250 x g在4°c 和放弃上清。</li>
	<li>通过在 RT 中添加100µL 2% paraformaldehyde/1%sucrose 20 分钟来修复细胞. 离心机板材为5分钟在 1000 x g和4°c。放弃上清。</li>
	<li>Permeabilize 细胞增加50µL 胞内通透缓冲 (1x PBS + 0.1% BSA, 0.01 米 HEPES, 0.3% 皂苷) 和孵育30分钟在4摄氏度。</li>
	<li>增加150µL 的 PBS 和离心机5分钟在 1000 x g在 RT. 丢弃上清。</li>
	<li>在缓冲 (PBS + 0.1% 皂苷) 中加入30µL 的荧光共轭抗 IFNγ抗体, 在 RT 中孵化30分钟, 从而染色细胞内细胞因子。</li>
	<li>通过添加150µL 的缓冲 (PBS + 0.1% 皂苷) 来洗涤细胞。将板材离心5分钟, 1000 x g在 RT 上, 然后丢弃上清。重复此步骤两次。</li>
	<li>用流式细胞仪分析细胞数量 (图 8)。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Differentiation+of+Naive+CD4(+)+T+Cells:+A+Tool+for+Understanding+the+Development+of+Atherosclerosis.">In Vitro Differentiation of Naive CD4(+) T Cells: A Tool for Understanding the Development of Atherosclerosis.</a> Methods in Molecular Biology. 1339, 177-189 (2015).</li><li>Toribio-Fernandez, 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=Lamin+A/C+augments+Th1+differentiation+and+response+against+vaccinia+virus+and+Leishmania+major.">Lamin A/C augments Th1 differentiation and response against vaccinia virus and Leishmania major.</a> Cell Death &amp; Disease. 9 (1), 9 (2018).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Transduction+of+Bone+Marrow+Progenitor+Cells+to+Generate+T-cell+Receptor+Retrogenic+Mice.">Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.</a> Journal of Visualized Experiments. (113), (2016).</li><li>Cruz-Adalia, A., 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=Conventional+CD4(+)+T+cells+present+bacterial+antigens+to+induce+cytotoxic+and+memory+CD8(+)+T+cell+responses.">Conventional CD4(+) T cells present bacterial antigens to induce cytotoxic and memory CD8(+) T cell responses.</a> Nature Communications. 8 (1), 1591 (2017).</li><li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., 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=The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo.">The use of fluorescent target arrays for assessment of T cell responses in vivo.</a> Journal of Visualized Experiments. (88), e51627 (2014).</li><li>Cruz-Adalia, A., Ramirez-Santiago, G., Torres-Torresano, M., Garcia-Ferreras, R., Veiga Chacon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Cells+Capture+Bacteria+by+Transinfection+from+Dendritic+Cells.">T Cells Capture Bacteria by Transinfection from Dendritic Cells.</a> Journal of Visualized Experiments. (107), e52976 (2016).</li><li>Cossarizza, A., 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=Guidelines+for+the+use+of+flow+cytometry+and+cell+sorting+in+immunological+studies.">Guidelines for the use of flow cytometry and cell sorting in immunological studies.</a> European Journal of Immunology. 47 (10), 1584-1797 (2017).</li><li>Quah, B. J., 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+and+improved+methods+for+measuring+lymphocyte+proliferation+in+vitro+and+in+vivo+using+CFSE-like+fluorescent+dyes.">New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.</a> Journal of Immunological Methods. 379 (1-2), 1-14 (2012).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Animals (NY). 40 (5), 155-160 (2011).</li><li>Warth, S. C., Heissmeyer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+transduction+of+naive+CD4+T+cells+to+study+Treg+differentiation.">Adenoviral transduction of naive CD4 T cells to study Treg differentiation.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Liou, H. L., Myers, J. T., Barkauskas, D. S., Huang, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+the+mouse+popliteal+lymph+node.">Intravital imaging of the mouse popliteal lymph node.</a> Journal of Visualized Experiments. (60), (2012).</li></ol>

作者没有什么可透露的

该协议允许对 BM 衍生 DCs 的能力进行表征, 以调节天真 CD4 T 细胞的活化、增殖和分化。此外, 它还可以用来评估 CD4 T 细胞的易感性, 由 BM 衍生 DCs。通过该协议, 可以在体内测量这些事件的变化。
根据调查的假设, 可以使用 T 细胞和 DCs 的几种组合。例如, 你可以分析敲或敲出特定基因的后果, 或者 CD4 T 细胞活化、增殖或 Th1 分化的特定刺激的效率。这些过程可以在 DCs 或 CD4 T 单元格或两个单元格类型上执行。因此, 野生型或基因修饰小鼠的 CD4 T 细胞可以与野生型小鼠的 DCs 相结合, 反之亦然。
该协议适用于使用流式细胞仪和抗体对抗 CD45.1 和 CD45.2, 以区分不同细胞群的来源。事实上, CD45.1/CD45.1 和 CD45.2/CD45.2 小鼠可以被用作任何一种细胞类型的来源, 或作为任何组合收养的接受者。例如, CD4 T 细胞可以从 CD45.1/CD45.1/OTII 小鼠获得, 而 CD45.2/CD45.2 小鼠可以是 BM 的起源为 DC 世代。在这个实验中, 两种细胞类型都可以过继转输转移到 CD45.1/CD45.2 小鼠身上。此外, CD4/OTII T 细胞从 CD45.1/CD45.1 型小鼠和 CD45.2/CD45.2 型两个小鼠可以结合在相同或不同的 CD45.1/CD45.2 三型接受者比较的行为和第二类 CD4 T 细胞群体的竞争和非竞争条件, 分别。
该方法描述了 GM-CSF BM 源 DCs 的生成。但是, 可用于 DC 的成熟和分化的替代协议;例如, 木瓜蛋白酶可以代替 LPS 或 fms 相关的酪氨酸激酶3配体 (Flt3-L) 而不是 GM-脑脊液, 允许研究表型不同的 DCs 能够激活 CD4 T 细胞自适应免疫能力。同样的目的, DCs 的抗原加载和表达可以直接与小鼠分离。这项协议与其他9的结合, 允许操纵天真的 CD4/OTII T 细胞的病毒感染16之前, 他们的收养转移到接受鼠。BM 也可以与逆转录9转基因, 研究控制细胞修改对 DCs 的影响, 然后再使用该协议。
此外, 该协议可适用于活体显微学研究17使用荧光蛋白表达细胞。例如, CD4/OTII 小鼠可以与 gfp 或樱桃表达小鼠交叉, 以获得 gfp 或樱桃/OTII CD4 小鼠。这些小鼠可以被用作 CD4 T 细胞的来源, 并且可以根据需要在活体实验中与来自樱桃或 GFP 表达小鼠的 BM 源 dc 结合使用。

CD4 T 细胞和抗原呈现细胞 (apc), 如 DCs 是需要的调解人免疫的微生物病原体1,2。在外周淋巴器官中, CD4 T 细胞在 apc3、4、5所提出的特异抗原识别后激活。活化 CD4 T 细胞增殖和分化为独特的特定效应细胞, 是必要的发展一个正确的适应性免疫应答6,7。控制这些过程是至关重要的, 以产生适当的适应性防御, 杀死病原体而不产生有害的组织损伤8。Th 细胞是根据表面分子、转录因子和效应细胞因子的表达或产生而定义的, 对病原体的反应执行必要和精确的功能1。细胞 Th1 细胞子集表达转录因子 T 打赌和细胞因子干扰素γ (IFNγ) 和参与宿主防御细胞内病原体1。量化 CD4 t 细胞的活化、增殖和 Th1 分化是评估 t 细胞在免疫应答中发挥作用的有用手段。
该协议能够在体内分析体外生成的 BM 衍生 DCs 的能力, 以调节天真 CD4 T 细胞的活化、增殖和 Th1 分化。该议定书还有助于评估幼稚 CD4 T 细胞被激活、诱导增殖和 Th1 分化的能力 (图 1)。这个多才多艺的协议绕过无法具体地操作或选择被研究的细胞人口在纯粹体内协议。利用基因修饰小鼠5的 BM, 或通过治疗或基因操作分离的 bm 细胞9, 可以研究不同分子和处理对 DCs 的影响。同样, t 细胞反应可以探索通过获得 t 细胞为领养转移从不同的来源或在几操作以后3,8,10。
该协议的主要优点有两个方面。用流式细胞仪分析 T 细胞活化、增殖和 Th1 分化;这与体内研究相结合, 从而避免了在体外可能发生的改变, 包括细胞类型和其他仅在完好器官中发现的因素11。
使用重要染料是一种广泛使用的技术来跟踪细胞增殖, 同时避免使用放射性。用这些试剂进行增殖的测量是基于细胞分裂后的染料稀释。此外, 这些染料可以在多波长检测, 并易于分析的流式细胞术与多种荧光抗体或标记。通过流式细胞术分析 T 细胞的活化、增殖和 Th1 分化, 我们强调了该协议的实用性。

<table>
	<tbody>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20,824,365</td>
			<td>5 L</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11000-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11253-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>10020-00</td>
			<td>Box of 100 blades</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>SIGMA</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>LONZA</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute medium (RPMI)</td>
			<td>GIBCO</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>25-gauge needle</td>
			<td>BD Microlance 3</td>
			<td>300600</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Novico</td>
			<td>N15663</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m nylon web filter</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood lysis buffer</td>
			<td>SIGMA</td>
			<td>R7757</td>
			<td>100 Ml</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SIGMA</td>
			<td>ED2SS-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A7906</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>SIGMA</td>
			<td>302643-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture-plates</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Cambrex</td>
			<td>BE17-737E</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Merck</td>
			<td>8-05740-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>LONZA</td>
			<td>BE13-115E</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>LONZA</td>
			<td>BE17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF)</td>
			<td>Prepotech</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>lipopolysaccharide (LPS)</td>
			<td>SIGMA-ALDRICH</td>
			<td>L2018</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well-plate</td>
			<td>Costar</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>v450 anti-mouse CD11b antibody</td>
			<td>BD Biosciences</td>
			<td>560455</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD64 antibody</td>
			<td>BioLegend</td>
			<td>139303</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD115 antibody</td>
			<td>BioLegend</td>
			<td>135505</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD11c antibody</td>
			<td>BioLegend</td>
			<td>117305</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse MHCII antibody</td>
			<td>BioLegend</td>
			<td>125507</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD86 antibody</td>
			<td>BioLegend</td>
			<td>105011</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD80 antibody</td>
			<td>BioLegend</td>
			<td>104713</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry tubes</td>
			<td>Zelian</td>
			<td>300800-1</td>
			<td>PS 12X75 5mL</td>
		</tr>
		<tr>
			<td>OTII Ovoalbumine peptide</td>
			<td>InvivoGen</td>
			<td>323-339</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD8&#945; antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0081-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated IgM antibody</td>
			<td>BioLegend</td>
			<td>406503</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated B220 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0452-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD19 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0193-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated MHCII (I-Ab) antibody</td>
			<td>BioLegend</td>
			<td>115302</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11b antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0112-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11c antibody</td>
			<td>BioLegend</td>
			<td>117303</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD44 antibody</td>
			<td>BioLegend</td>
			<td>103003</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated DX5 antibody</td>
			<td>BioLegend</td>
			<td>108903</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin-coated magnetic microbeads</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic cell separator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>QuadroMACS Separator</td>
		</tr>
		<tr>
			<td>Separation columns</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD4 antibody</td>
			<td>BioLegend</td>
			<td>100408</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD3 antibody</td>
			<td>BioLegend</td>
			<td>100235</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD8 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0081-U025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Violet Tracer</td>
			<td>Thermofisher</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa647 anti-mouse CD69 antibody</td>
			<td>BioLegend</td>
			<td>104518</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCPCY5.5 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>65-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>PECY7 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>60-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45.2 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0454</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>SIGMA-ALDRICH</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12 Myristate 13 Acetate (PMA)</td>
			<td>SIGMA</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A (BD GolgiPlug)</td>
			<td>BD</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Millipore</td>
			<td>8-18715-02100</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular permeabilization buffer</td>
			<td>eBioscience</td>
			<td>00-8333</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFNg antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-7311-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block (anti-mouse CD16/CD32)</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.SJL CD45.1 mice</td>
			<td>The Jackson Laboratory</td>
			<td>002014</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482; Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td>649225</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Solution</td>
			<td>Thermofisher</td>
			<td>62248</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo mouse model to measure na&#239;ve cd4 t cell activation, proliferation and th1 differentiation induced by bone marrow-derived dendritic cells

量化的天真 CD4 t 细胞活化, 增殖, 并分化为 t 帮助 1 (Th1) 细胞是一个有用的方法来评估 t 细胞在免疫应答中发挥的作用。本协议描述骨髓 (BM) 祖细胞的体外分化, 以获得粒细胞巨噬细胞集落刺激因子 (GM-脑脊液) 衍生树突状干细胞 (DCs)。该协议还介绍了蛋白蛋白肽 (OVAp) 负载的 GM-脑脊液衍生 DCs 和天真的 CD4 T 细胞 OTII 转基因小鼠的移植, 以分析体内活化、增殖和 Th1 分化的转移 CD4 T 细胞。这个协议绕过了纯粹的体内方法的限制, 因为无法专门操作或选择所研究的细胞群体。此外, 本议定书允许在体内环境中进行研究, 从而避免对可能发生在体外的功能性因素的改变, 包括细胞类型和其他仅在完整器官中发现的因素的影响。该协议是一个有用的工具, 以产生变化的 DCs 和 T 细胞, 修改适应性免疫反应, 有可能提供重要的结果, 以了解许多免疫相关疾病的起源或发展。

图 1说明了本协议中描述的步骤。图 2展示了小鼠 BM 细胞的分离和培养。在这些培养中加入了 GM-csf 和 LPS, 可使 dcs 的体外生成和成熟.图 3说明了流式细胞术对所获得 dcs 的分化和成熟的分析. OVAp 负载的 GM-脑脊液 BM 基 dcs分离的重要细胞微量染料染色 CD4/OTII T 细胞过继转输转移到小鼠身上。图 4显示了用于隔离 CD4/OTII T 细胞的淋巴结的位置。图 5显示了一个50毫升管装有70µm 尼龙过滤器和注射器柱塞用于融汇淋巴结和脾脏。图 6显示静脉注射的 CD4/OTII T 细胞进入复古眶丛和 OVAp 注射的都安的 GM-脑脊液 BM。CD4/OTII T 细胞分布到不同的淋巴器官, 而 gm-脑脊液 dcs 迁移到腘淋巴结, 其中 gm-csf dcs 激活幼稚的 CD4/OTII T 细胞的介绍 OVAp。天真的 CD4/OTII T 细胞然后增殖和分化为 Th1 表型。图 7说明了从膜 CD69 和 CD25 表达的分析中对天真 CD4/OTII T 细胞活化的量化。图 8显示了 CD4/OTII T 细胞增殖的量化。图 9说明了 CD4/OTII T 细胞分化为 Th1 表型的量化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58118/58118fig1.jpg"> 图 1: 协议方案.1) 从小鼠股骨和胫骨中分离 BM 细胞。2) 培养的 BM 细胞与 gm-脑脊液生成 gm-脑脊液 bm-dcs. 3) 检查 DC 分化和成熟的 gm-csf-dcs 与 LPS。4) 检查直流成熟度。5) 用 OVAp 加载 DCs。6) 分离 CD4/OTII T 细胞形成小鼠脾。7) 用生命细胞示踪染料染色 CD4/OTII T 细胞。8) 检查隔离 CD4/OTII T 细胞的分离纯度和生命细胞示踪染料染色。9) 过继转输转移隔离 CD4/OTII T 细胞静脉注射给受体小鼠。10). 过继转输转移 OVAp 加载和成熟的 DCs 到受体小鼠。11) 检查 CD4/OTII T 细胞激活。12) 检查 CD4/OTII T 细胞的增殖和分化。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58118/58118fig2.jpg"> 图 2: 从小鼠股骨和胫骨的骨髓隔离过程.(A) 带剪刀的皮肤切口。(B) 清除肢体皮肤。(C) 带剪刀的肌肉切开术。(D) 用手术刀从肢体中取出肌肉。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58118/58118fig3.jpg"> 图 3: 流式细胞仪分析了 GM-脑脊液 BM 衍生 DCs 的成熟度.MHCII、CD80 和 CD86 在 lps 活化 (+ lps) 和非活化 (lps) 基因组中的表面表达. 数字显示任意单位 (天文单位) 的平均荧光强度。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58118/58118fig4.jpg"> 图 4: 小鼠腹股沟、腋、臂、颈、肠系膜淋巴结及脾的位置.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58118/58118fig5.jpg"> 图 5: 实验性设置为淋巴结和脾均质化.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58118/58118fig6.jpg"> 图 6: CD4/OTII T 细胞的领养转移和 OVAp 负载的 GM-脑脊液-DCs. (A) 静脉注射 CD4/OTII T 细胞进入复古眶丛。(B) 皮下注射 OVAp 负载的 GM-脑脊液 BM 都安。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58118/58118fig7.jpg"> 图 7:体内T 细胞活化的量化.流式细胞仪图和图形显示 CD69 在 CD4 T 细胞中的膜表达的分析和定量。CD45.1/CD45.2 小鼠被过继转输转移静脉注射与小鼠 CD45.1/CD4/OTII 和 CD45.1/CD4/OTII 细胞24小时之前, 在南卡罗来纳州收养转移 OVAp 负载的 GM-脑脊液 BM-DCs. 用流式细胞术检测2天后直流转导 T 细胞活化染色后, 荧光标记抗体的指示抗原。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58118/58118fig8.jpg"> 图 8:体内T 细胞增殖的量化.流式细胞仪图和图形显示了增殖 CD4 T 细胞中下降的关键细胞示踪染料染色的分析和量化。CD45.2 小鼠过继转输转移静脉注射与小鼠 CD45.1/CD4/OTII 细胞24小时之前, 在 OVAp 被装载的 GM-脑脊液 BM-DCs 的移植. 用流式细胞术检测5天后直流转移后的 T 细胞增殖显示荧光抗体。T 细胞增殖可以通过测量增殖细胞的百分比、每个除法峰中细胞的百分比, 或者通过计算图中所示的除法峰数来确定。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58118/58118fig9.jpg"> 图 9:在体内对 T 细胞分化为 Th1 表型的量化.流式细胞仪图和图形显示 IFNγ表达 CD4 T 细胞百分比的分析和量化。CD45.2 小鼠过继转输转移静脉注射与小鼠 CD45.1/CD4/OTII 细胞24小时之前, 在 OVAp 被装载的 GM-脑脊液 BM-DCs 的移植. 用流式细胞仪染色后, 在几天后直流转移的 T 细胞分化显示荧光抗体。t 细胞分化为 Th1 表型被确定为 IFNγ表达细胞占总 CD4 t 细胞和仅从增殖 CD4 t 细胞的百分比。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58118/58118fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃƒÂ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

在这里, 我们提出了一种在体内测定由 GM-脑脊液骨髓 (BM) 衍生树突状细胞 (CD4) 诱导的天真的 t 细胞 (t 细胞) 活化、增殖和 Th1 分化的方法。此外, 该协议还描述了 BM 和 t 细胞分离、dc 生成和 dc 和 t 细胞的移植。

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

an-vivo-mouse-model-to-measure-nave-cd4-t-cell-activation

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.3K 次观看.  Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC). 在这里, 我们提出了一种在体内测定由 GM-脑脊液骨髓 (BM) 衍生树突状细胞 (CD4) 诱导的天真的 t 细胞 (t 细胞) 活化、增殖和 Th1 分化的方法。此外, 该协议还描述了 BM 和 t 细胞分离、dc 生成和 dc 和 t 细胞的移植。

视频: An In Vivo Mouse Model to Measure Naïve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.