这项研究得到新加坡国立大学卫生系统 (T1-2014 Oct-12 和 T1-2015 Sep-10) 赠款的支持。

所有在本议定书中使用的老鼠都是在 C57BL/6 的遗传背景下, 并被安置在新加坡国立大学特定的无病原体条件下。所有实验都使用新加坡国立大学机构动物保育和使用委员会批准的协议进行。
1. 试剂和材料制备
<ol><li>制备500毫升磷酸盐缓冲盐水 (PBS), 含2% 胎牛血清 (血清) 和1毫米乙二胺四乙酸 (EDTA)。 注意: 在整个过程中保持缓冲区在冰的最佳效果。</li>
	<li>准备50毫升完整的 RPMI 培养基, 含有 RPMI 1640, 10% 的血清和1% 青霉素-链霉素。使用包含50µM β巯基乙醇的完整 RPMI 培养基进行 THGM 细胞分化。在通风罩中加入β巯基乙醇。</li>
	<li>通过将 anti-CD3e 浓缩的库存稀释到 PBS 中的3微克/毫升, 制备7.5 毫升的 anti-CD3e 抗体混合物。涂层48井板材通过增加 150 ul 抗体混合物对每个井和孵化板材在37°c 至少 1 h, 或者在4°c 隔夜。</li>
	<li>消毒一双剪刀和镊子, 并保持他们在70% 的乙醇夹层解剖。</li>
</ol>2. 小鼠脾的制备
<ol><li>弄死老鼠 (6–8周老) 使用一个制度上批准的 CO2窒息或颈椎脱位。用70% 乙醇喷雾鼠标, 并将其装在其背面的聚苯乙烯块上。将鼠标转移到生物安全柜, 以执行以下步骤。</li>
	<li>用一把钳子握住皮肤, 用一把剪刀将左侧肋骨下面的皮肤切开4厘米。打开腹膜囊露出脾脏, 用无菌钳提取脾脏。</li>
	<li>在50毫升管上放置一个70微米的细胞过滤器, 用2毫升的缓冲器将其预湿。将脾放在细胞过滤器上 (2–3脾一个细胞滤网), 并浸渍使用5毫升注射器柱塞的末端。</li>
	<li>将过滤器和管多次冲洗成细胞隔离缓冲器, 直到所有细胞都冲入管内。丢弃细胞过滤器和离心细胞在 350 x g 5 分钟在4摄氏度。放弃上清。</li>
	<li>并用重悬5毫升冷 (4 °c) 氯化铵-钾 (ACK) 株溶藻缓冲器 (150 毫米 NH4氯) 的细胞颗粒; 10 毫米 KHCO3, 0.1 毫米 Na2EDTA; pH 7.2–7.4), 并将其轻轻地混合为2分钟. 添加10毫升完整的 RPMI 介质以中和应答缓冲, 并立即离心细胞在 350 x g 5 分钟在4摄氏度。离心后要丢弃上清液。</li>
</ol>3. 用磁性 CD4 微珠和细胞分离柱分离 CD4+ T 细胞
<ol><li>并用重悬细胞颗粒在5毫升细胞隔离缓冲。用预分离过滤器 (30 微米) 将细胞悬浮物过滤成新的15毫升管以去除任何碎片。</li>
	<li>采取 10 ul 的细胞悬浮, 并作出10x 稀释通过增加 90 ul 的缓冲。取 10 ul 的稀释细胞悬浮, 并混合它与 10 ul 的台盼蓝的细胞计数。计数 hemocytometer 中的细胞以确定存活细胞的产量。</li>
	<li>离心细胞在 350 x g 5 分钟在4°c 和放弃上清。</li>
	<li>并用重悬细胞在 90 ul 的缓冲每 107细胞。添加 10 ul 磁性 CD4 微珠每 107细胞。在4摄氏度孵化细胞15分钟。为获得最佳效果, 在孵化过程中, 每5分钟轻轻混合一次细胞悬浮。</li>
	<li>当细胞孵化时, 在磁性支架上放置一个分离柱。用2毫升的缓冲器预湿柱。</li>
	<li>在细胞/珠孵化结束时, 用5毫升的缓冲液冲洗细胞, 在4摄氏度时将其离心在 350 x g处5分钟。放弃上清。</li>
	<li>并用重悬1毫升的缓冲细胞, 将样品装入细胞分离柱中, 让它流过。使用15毫升离心管收集 CD4 细胞分数。添加1毫升的缓冲液, 在干燥之前将其冲洗干净。重复洗涤步骤2x。</li>
	<li>从磁支架中取出单元分离柱, 将其放在新的15毫升离心管上。将2毫升的细胞隔离缓冲器添加到细胞分离柱上, 并将柱塞牢固地应用于将细胞从柱上强行取出。</li>
	<li>离心率为 350 x克5 分钟, 在4摄氏度, 以获得 CD4+ 细胞。放弃上清。</li>
</ol>4. 荧光活化细胞分选和 THCD4+ 纯化天真的 CD4+CD25-CD44螺CD62Lhi)
<ol><li>并用重悬在500µL 缓冲液中获得 CD4+ 细胞颗粒。将细胞与含有 CD4-PerCp (2.8 µg/毫升)、CD44-APC (2.4 µg/毫升)、CD25-PE (2 µg/毫升) 和 CD62L-FITC (10 µg/毫升) 的荧光共轭抗体混合物 (表 1) 混合。在冰上孵化细胞以 20–30 min, 同时保护它们不受光照。 注意: 抗体的浓度在实验室中进行了优化。所列出的抗体的数量是来自于小鼠的细胞。</li>
	<li>孵化后, 用5毫升的缓冲液冲洗细胞, 将细胞在 350 x g处进行离心处理, 在4摄氏度时进行5分钟。</li>
	<li>丢弃上清液, 并用重悬500µL 的细胞。使用尼龙网格再次过滤单元格。</li>
	<li>将细胞悬浮液转移到用于细胞分类的转流管。涂型了500µL 缓冲器的采集器管。</li>
	<li>通过 CD4 的分类,获得 CD25CD44 CD62Lhi种群 (天真 CD4+ T 细胞)。门在 CD4+CD25-首先, 然后选择 CD44 CD62Lhi细胞从这个人群。</li>
	<li>将天真的 CD4和 T 细胞分数收集成一个新的15毫升离心管。离心细胞在 350 x g 5 分钟在4°c 和放弃上清。</li>
	<li>并用重悬在含有50µM β巯基乙醇的完整 RPMI 培养基中, CD4+ 的浓度为 106细胞/毫升的天真的 T 细胞。</li>
	<li>在48井板中吸入用于预涂的 anti-CD3e 抗体。种子 25万 (250 µL) 细胞在每个井与 IL-7 (2 ng/毫升), anti-CD28 (1 µg/毫升), 和反 IFNγ (10 µg/毫升) (表 1)。 注意: 细胞因子和抗体的浓度在实验室中被优化为 THGM 细胞分化。</li>
	<li>孵化细胞在37°c 与 5% CO2 3 天。在孵化过程中不要改变培养基。</li>
</ol>5. 小鼠 T 型转基因细胞体外生成的分析
<ol><li>用显微镜检查细胞分化后 3 d 的分化。收获细胞和离心他们在 350 x g 5 分钟在4摄氏度。用完整的 RPMI 介质冲洗细胞2x。 注意: 分化细胞的大小比天真的 CD4+ T 细胞大。</li>
	<li>并用重悬1毫升完整的 RPMI 培养基中的细胞。采取 10 ul 的细胞悬浮和混合它与 10 ul 的台盼蓝, 以确定的细胞数与 hemocytometer。将分化的细胞分成三药剂进行 restimulation 和分析。 注: 细胞内细胞因子染色和 ELISA 检测需要≥0.5 百万细胞, qPCR 分析需要≥100万细胞。</li>
	<li>
为细胞内细胞因子染色, restimulate 细胞与佛波 12-酯 13-醋酸盐 (100 ng/毫升) 和 ionomycin (1 微克/毫升) 存在蛋白质转运抑制剂 (1 µL/毫升) 为第4-6 h。
	<ol>
<li>用 anti-CD4 抗体 (PerCP 共轭, 0.2 毫克/毫升, 1:100 稀释; 或 FITC 共轭, 0.5 毫克/毫升, 1:100 稀释), 以30分钟的方法收割细胞并染色。</li>
		<li>用 200 ul 固定缓冲细胞固定20–60分钟。固定后, 用1毫升的通透缓冲液冲洗细胞2x。</li>
		<li>对 GM 脑脊液 (PE 共轭, 0.2 毫克/毫升, 1:100 稀释), IL-17A (FITC 共轭, 0.5 毫克/毫升, 1:100 稀释) 抗体进行细胞内染色;apc 共轭, 0.2 毫克/毫升, 1:100 稀释), IL-4 (apc 共轭, 0.2 毫克/毫升, 1:100 稀释), 和 IFNγ (apc 共轭, 0.2 毫克/毫升, 1:100 稀释;PE 共轭, 0.2 毫克/毫升, 1:100 稀释) 在黑暗中30分钟。</li>
	</ol>
</li>
	<li>qPCR 分析分化细胞的细胞因子基因表达, 激活细胞与板块绑定 anti-CD3e (3 微克/毫升) (预涂的板块, 如步骤1.3 所述)。在刺激后3小时, 利用苯酚 RNA 萃取试剂, 采集细胞以分离总 RNA, 为 qPCR 制备 cDNA。用于 qPCR 的引物和程序分别列在表 2和3中。</li>
	<li>为分析分化细胞的细胞因子蛋白分泌, restimulate 细胞与板块约束 anti-CD3e (3 微克/毫升) 为 24 h (预涂的板块, 如步骤1.3 所述)。收获细胞培养上清 IL-17 和 GM-脑脊液 ELISA, 以确定其浓度。</li>
</ol>

<ol>
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作者没有什么可透露的。

在这里我们描述了一个从小鼠天真的 CD4+ 细胞的体外TH基因分化的协议, 其次是对分化细胞的分析, 以验证该方法。注意, 脾脏和淋巴结均可用于幼稚的 CD4+ t 细胞纯化和 tH基因的分化。细胞内细胞因子染色与流式细胞术相结合的细胞因子表达表明, 在 gm (图 1A) 下, 约有55% 的神经细胞被诱导成为 gm-脑脊液表达细胞。与 th17 分化条件下的细...

CD4+ (tH) 细胞是免疫系统的重要组成部分, 在宿主防御微生物病原体、癌症监测和自身免疫性1、2、3中具有重要作用。在 t 细胞受体 (TCR) 激活, 天真的 CD4+ t 细胞可以被区分为t h1,t h2, th 17, 或调节 t (Treg) 细胞在不同的细胞因子环境传道2,4,5. 最近, 发现了一种主要生产 gm-CSF 的 th细胞的新子集, 并命名为 gm6。IL-7 通过激活信号传感器和转录激活剂 5 (STAT5) 来驱动 THGM 细胞的分化。这些细胞表达了大量的 GM-脑脊液, 同时低表达其他 TH细胞特征因子, 如 IFNγ和 IL-176。在 CD4+ T 细胞介导的 neuroinflammation7、8的发展中, 基因型脑脊液被发现发挥了关键作用。与 IFNγ或 IL-17-expressing 自身反应性 t 细胞相比, 转基因脑脊液表达 t 细胞转移到野生型 (小鼠) 导致早期疾病发作和更高的疾病严重性。此外, Csf2细胞没有诱导实验性自身免疫脑脊髓炎 (EAE) 后被过继转输转移到脑细胞受体, 而缺乏 IFNγ或 IL-17A 的 T 淋巴细胞保留了调解 EAE7的能力。此外, 使用中和抗体对 GM-CSF 的封锁改善了 EAE 疾病严重程度8。此外, 小鼠 t 细胞中 STAT5 的缺失导致了转基因一代的减少, 因此, 在小鼠对 EAE 发展6的抵抗力。这些发现强调了 GM-脑脊液表达 TH细胞在自身免疫性 neuroinflammatory 疾病中的重要性。因此, 建立一种从天真的 CD4+ t 细胞中鉴别 GM-脑脊液表达 tH细胞的方法, 对于研究自身免疫 neuroinflammation 和 T 细胞介导的免疫应答的发病机制具有重要意义。然而, 从小鼠天真的 CD4+ 有效地产生 TH基因电池的协议尚未建立。
在这里, 我们提出了一种方法, 区分小鼠 tHGM 细胞从天真的 CD4+ t 细胞。该协议描述了整个过程, 包括从小鼠中提取脾脏、单细胞悬浮液的制备、CD4 阳性选择、荧光活化细胞分选和 TH细胞。分化和分析。用细胞内细胞因子染色结合流式细胞仪对分化的 T 辅助细胞进行分析, 以确定单细胞水平的细胞因子表达, 采用定量实时 PCR 方法测定 mRNA 水平的细胞因子表达, 并通过 ELISA 对蛋白质水平的细胞因子表达进行评估。该方法可应用于 EAE 等不同条件下的转基因细胞生物学研究中, 其在发病机制中起着重要作用。

<table>
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>Biowest</td>
			<td>L0500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Heat Inactivated</td>
			<td>Capricorn</td>
			<td>FBS-HI-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>1st Base</td>
			<td>BUF-2040-10</td>
			<td>Diluted to 1x PBS in sterile water</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>1st Base</td>
			<td>BUF-1053-100ml-pH8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>10X ACK buffer</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td>diluted in sterile water</td>
		</tr>
		<tr>
			<td>cell strainer</td>
			<td>SPL Life Sciences</td>
			<td>93070</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-201</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>LS column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>Terumo</td>
			<td>SS+01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Eppendorf 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sony SY3200 cell sorter</td>
			<td>Sony</td>
			<td>Sony SY3200 cell sorter</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>210261</td>
			<td></td>
		</tr>
		<tr>
			<td>15m conical centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well cell culture plate</td>
			<td>Greiner Bio-One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD4 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0041-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE conjugated anti-mouse CD25 (IL-2Ra, p55)</td>
			<td>eBioscience</td>
			<td>12-0251-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human/mouse CD44 APC</td>
			<td>eBioscience</td>
			<td>17-0441-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD62L FITC</td>
			<td>eBioscience</td>
			<td>11-0621-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer-improved counting chamber</td>
			<td>Marienfeld</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone Trypan Blue Solution</td>
			<td>GE healthcare Life Sciences</td>
			<td>SV30084.01</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Nikon</td>
			<td>Nikon Elipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified&#160;anti-mouse CD3e&#160;antibody</td>
			<td>Biolegend</td>
			<td>100314</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified hamster anti-mouse CD28</td>
			<td>BD Biosciences</td>
			<td>553295</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat&#160;anti-mouse IFN&#947; &#160;</td>
			<td>eBioscience</td>
			<td>16-7312-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse IL-4 Antibody</td>
			<td>Biolegend</td>
			<td>504102</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-7 Protein</td>
			<td>R&#38;D system</td>
			<td>407-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-6</td>
			<td>R&#38;D system</td>
			<td>406&#8209;ML</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human TGF-beta</td>
			<td>R&#38;D system</td>
			<td>240-B-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Merck Millipore</td>
			<td>19-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>GolgiPlug protein transport inhibitor</td>
			<td>BD Biosciences</td>
			<td>51-2301KZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular Fixation&#38;Permeabilization buffer set</td>
			<td>eBioscience</td>
			<td>88-8824-00</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse GM-CSF PE</td>
			<td>eBioscience</td>
			<td>12-7331-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse IL-17A</td>
			<td>Biolegend</td>
			<td>506908</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFN-gamma</td>
			<td>Biolegend</td>
			<td>505810</td>
			<td></td>
		</tr>
		<tr>
			<td>GO Taq qPCR master mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse GM-CSF ELISA Ready-SET-Go!</td>
			<td>invitrogen</td>
			<td>88-7334-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A Uncouted ELISA</td>
			<td>invitrogen</td>
			<td>88-7371-22</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro differentiation of mouse granulocyte-macrophage-colony-stimulating factor (gm-csf)-producing t helper (thgm) cells

粒-巨噬细胞集落刺激因子 (gm-csf) 生产的 t 帮助器 (tHGM) 细胞是一个新发现的 t 辅助细胞子集, 主要分泌的 GM-脑脊液不产生干扰素 (干扰素) γ或白细胞介素 (IL)-17, 并发现在自身免疫性 neuroinflammation 中起重要作用。从单纯的 CD4+ t 细胞中分离出单纯的脾和 tH基因 CD4+ t 细胞, 是研究 t 细胞介导免疫和自身免疫性疾病的有效方法。这里我们描述了一种方法, 将小鼠天真 CD4+ t 细胞与 IL-7 促进的 tH基因细胞区分开来。用不同的方法分析细胞因子的表达, 包括细胞内细胞因子染色结合流式细胞术、定量实时聚合酶链反应 (PCR), 对分化结果进行评估, 并酶联免疫吸附测定法 (ELISA)。根据这里所描述的 THgm 分化协议, 大约55% 的细胞表达了 IFNα或 IL-17 的最小表达。通过对基因型脑脊液、IFNα和 IL-17在 mRNA 和蛋白质水平上表达的分析, 进一步证实了转基因脑脊液的主要表达。因此, 该方法可用于体外诱导单纯 CD4+ t 细胞与 th基因细胞的分离, 对转基因细胞生物学研究有一定的参考价值。

从两个8周大的雄性 C57BL/6 小鼠中分离出的朴素 CD4+ T 细胞分为三部分。在描述的协议之后, 细胞的一部分被区分成 THGM 细胞。另一部分是在 thgm 条件下培养的 anti-IL-4 抗体 (10 µg/毫升), 以测试 IL-4 封锁的影响, 在分化的 thgm。最后一部分是培养在 TH17 分化条件 (3 µg/毫升 anti-CD3e, 1 µg/毫升 anti-CD28, 10 ng/毫升 TGFβ, 30 ng/毫升 IL-6, 10 µg/毫升反 IFNγ...

Watch this Scientific Journal Video about In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells at JoVE.com

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

体外小鼠粒细胞-巨噬细胞集落刺激因子 (gm-CSF) 的分化--生成 t 辅助器 (tHgm) 细胞

在这里, 我们提出了一种区分小鼠粒细胞-巨噬细胞集落刺激因子产生的 t 型CD4+ t 细胞, 包括天真的 CD4+ t 细胞的分离, th基因的分化和差异性 TH基因细胞的分析。该方法可用于转基因细胞的调节和功能研究.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

in-vitro-differentiation-mouse-granulocyte-macrophage-colony

Research

JoVE Journal

Immunology and Infection

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

7.1K 次观看.  National University of Singapore. 在这里, 我们提出了一种区分小鼠粒细胞-巨噬细胞集落刺激因子产生的 t 型CD4+ t 细胞, 包括天真的 CD4+ t 细胞的分离, th基因的分化和差异性 TH基因细胞的分析。该方法可用于转基因细胞的调节和功能研究.

视频: In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM 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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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 ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

人正常记忆 b 细胞与长寿浆细胞体外分化模型

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...