我们感谢 L. Brockmann 对手稿的严格审阅。这项工作得到了 NIH 1R01EB030352 和 UL1 TR001873 的支持。

所有动物程序均在机构动物护理和使用委员会（哥伦比亚大学，协议AC-AABQ5551和AC-AAAZ4470）的批准下使用体重在20-25克之间的6-8周龄雌性BALB / c或CF57BL / 6小鼠进行。这些动物是从商业来源获得的（见 材料表）。该协议围绕小鼠 T 细胞的"激活后天数"构建，病毒产生从第 -2 天开始。逆转录病毒在初始生产后可以储存在-80°C，并且对于该方案的未来使用，可以从步骤2开始，在第0天分离和激活T细胞。
1. 逆转录病毒载体生产
注意:通过将包装基因分离成两个单独的质粒，使病毒产物具有复制缺陷（参见 材料表），大大降低了重组事件和无意中产生具有复制能力的病毒的可能性。
<ol><li>在转染前一天（第 -2 天程序）准备 Phoenix Eco 细胞。<ol><li>使用 30 mL 培养基（Phoenix Eco 培养基，详见表 1）将大约 1 x 107 个细胞放入 15 cm TC 处理的平板或 T150 培养瓶中。</li>
		<li>在37°C孵育过夜。 18-24小时后，细胞应约70%汇合并均匀分布，以确保高病毒产量而不会过度生长。 注意:为获得最佳结果，请使用传代率低的细胞，并在接种前一天传代用于病毒生产。在常规培养过程中，不要让Phoenix Eco细胞过度生长。</li>
	</ol></li>
	<li>在还原血清培养基中制备含有脂质转染和增强子试剂、pCL-Eco （Gag/Pol） 和 pMSCV 表达质粒 （pMSCV_PGK_mGFP28z） 的转染混合物（参见 材料表）（第 -1 天程序）。 注意:共转染应以 pMSCV 和 pCL-Eco 的 1:1 比例进行。<ol><li>准备试管 A:每 15 cm 板将 105 μL 转染试剂稀释在 3.75 mL 还原血清培养基中，并通过涡旋或上下移液充分混合。</li>
		<li>准备管B:用90μL增强剂试剂将pMSCV表达质粒（21μg）和pCL-Eco（21μg）稀释到每15cm板3.75mL还原血清培养基中。通过上下移液充分混合。 注意:如果产生多种病毒产物，请设置含有 pCL-Eco 和增强剂试剂的预混液。</li>
		<li>将试管 A 加入试管 B 中，用移液管充分混合。在室温下孵育 10-20 分钟，总体积约为 7.5 mL。</li>
		<li>小心地从 Phoenix Eco 培养板中取出 10 mL 细胞培养基，并通过倾斜板和滴液将整个体积的转染混合物添加到剩余的培养基中。将细胞送回37°C培养箱。</li>
	</ol></li>
	<li>在转染后16-20小时（第0天程序）更换转染的Phoenix Eco细胞上的培养基。<ol><li>使用水或珠浴将不含β巯基乙醇的血清和-巯基乙醇（2-ME，参见 材料表）的鼠T细胞培养基（mTCM-病毒收获，如 表1所示）预热至37°C。</li>
		<li>倾斜平板并将移液器吸头放在底角，小心地取出Phoenix Eco培养基，如果可能的话，使用真空吸尘器。避免细胞过度干燥。</li>
		<li>通过在最慢的设置下移液 30 mL，将加热的 mTCM 病毒收获培养基轻轻加入倾斜板的侧面。将细胞送回37°C培养箱。 注意:此步骤会导致Phoenix Eco细胞从平板上脱落并降低病毒滴度。预热的培养基和轻柔的移液将最大限度地减少干扰。</li>
	</ol></li>
	<li>转染后48小时收获病毒上清液（第1天程序）。<ol><li>收获病毒上清液并通过0.45μmPVDF过滤器过滤（参见 材料表）以去除细胞和碎片。分装并在-80°C下冷冻病毒，如果使用新鲜病毒，则直接进行到步骤3的第1天。</li>
		<li>如前所述，通过流式细胞术测定病毒滴度（转导单位/mL）9。此步骤是可选的。<ol><li>通过在非组织培养 （TC） 处理的 96 孔板中小规模转导 1 x 105 个活化的小鼠 T 细胞，预涂有逆转录肾素（参见 材料表）并在最终体积为 100 μL 的 mTCM 病毒收获培养基中加载三倍连续稀释的逆转录病毒上清液来测定病毒滴度。 注意:有关 T 细胞分离、活化和转导的详细说明，请参阅下面的步骤 2 和步骤 3。</li>
			<li>通过流式细胞术在转导后 4-5 天测定 CAR 表面表达。</li>
			<li>根据公式10计算病毒滴度:（N x F x D）/V，其中N是转导的细胞数，F是CAR阳性细胞的频率，D是稀释因子，V是以mL为单位的转导体积，得到转导单位（TU）/mL。 注意:冻融时病毒滴度可能会降低;因此，理想情况下，病毒滴度是在冷冻并随后解冻的病毒上测定的。</li>
		</ol></li>
	</ol></li>
</ol>2. 小鼠T细胞分离
<ol><li>分离并激活小鼠 T 细胞（第 0 天程序）。 注意:这些步骤可以在室温（RT）或4°C下进行，并且应在无菌环境中进行。<ol><li>如前所述，从感兴趣的小鼠品系（例如，Balb / c，C57BL / 6）中收获脾脏11 ，并通过机械解离获得单细胞悬浮液。</li>
		<li>使用无菌注射器的背面，通过 70-100 μm 细胞过滤器将脾脏粉碎到 50 mL 锥形管中。</li>
		<li>将注射器放在一边后，用 5 mL T 细胞分离缓冲液（磷酸盐缓冲盐水，PBS，补充有 2% 胎牛血清，FBS）洗涤过滤器。</li>
		<li>重复糖化步骤，然后用 5 mL T 细胞分离缓冲液再次洗涤过滤器。用 T 细胞分离缓冲液将最终体积降至 50 mL。</li>
		<li>使用手动血细胞计数器或自动细胞计数器以 1:20 的比例稀释，然后以 1:1 的比例与台盼蓝混合，对活细胞进行计数。</li>
		<li>通过在4°C（或RT）下以450× g离心10分钟来沉淀细胞，如果使用推荐的T细胞分离试剂盒，则以1×108/ mL重悬脾细胞（参见 材料表）。 注意:在这个阶段，可以通过40-70μm过滤器重新过滤脾细胞以去除团块。</li>
	</ol></li>
	<li>按照制造商的说明通过阴性选择分离CD3 + T细胞（参见 材料表）。磁分离后，将分离物转移到 15 mL 锥形管中并进行最终活细胞计数。</li>
	<li>通过在4°C（或RT）下以450× g离心10分钟的方式沉淀分离的T细胞，并将其以1×106/ mL重悬于mTCM活化培养基中（表1）。</li>
	<li>通过以 25 μL/1 x 106 个 T 细胞和 100 U/mL IL-2 的比例加入小鼠抗 CD3/抗 CD28 单克隆抗体包被磁珠（参见材料表）来激活 T 细胞。</li>
	<li>将细胞置于37°C的培养箱中，并放置过夜。 注意:由于 T 细胞的体积小，可以通过以 1:10-1:20 的比例稀释并以 1:1 的比例与台盼蓝混合以使用血细胞计数器进行手动计数来实现更准确的活 T 细胞计数。纯度可以通过流式细胞术通过用荧光偶联的 αCD3 抗体对细胞进行染色来确定。每个脾脏将产生 7-10 x 106 个 T 细胞，具体取决于小鼠的年龄和品系。</li>
</ol>3. 小鼠T细胞转导
<ol><li>准备用于转导的板（第 0 天程序）。<ol><li>用0.5mL人纤连蛋白转导增强剂试剂（参见 材料表）预涂未经处理的无菌24孔板，终浓度为20-40μg/ mL，将其稀释在无菌PBS中，并在4°C下储存过夜。 注意:该步骤也可以在第 1 天通过用转导试剂包被未处理的 24 孔板并在室温下孵育 2 小时来执行。</li>
	</ol></li>
	<li>进行 T 细胞转导（第 1 天程序）。<ol><li>准备用于转导的预涂层板。从预包被的24孔板的每个孔中取出转导试剂，并在室温下用等体积的无菌过滤PBS + 2%牛血清白蛋白（BSA）（0.5mL）封闭30分钟。</li>
		<li>用 0.5-1 mL PBS 洗涤一次。</li>
	</ol></li>
	<li>将来自步骤1的0.5-1mL纯逆转录病毒或根据病毒滴度稀释到每个预包被孔中，并在2,000× g 和32°C下离心90分钟。</li>
	<li>向每个病毒加载的孔中加入1mL活化的T细胞，并在450× g 和32°C下离心10分钟。 将细胞送回37°C培养箱过夜。</li>
	<li>转导后24小时，除去1-1.5mL细胞培养基，并用1-1.5mL mTCM完全和10ng / mL重组人IL-7和IL-15代替（见 材料表）。将细胞返回至37°C培养箱（第2天程序）。 注意:在 离体 培养期间，必须每 48 小时向培养物中加入 10 ng/mL 细胞因子，并且 T 细胞不应稀释超过 1 x 106/mL。</li>
	<li>转导后48小时，将细胞从转导板转移到新鲜的24孔或6孔板中，并将细胞返回到37°C培养箱（第3天程序）。 注意:T细胞活力可以通过在转导后24小时至2天转移细胞来改善，具体取决于起始T细胞活力和病毒滴度。</li>
	<li>去除 T 细胞并确认 CAR 表达（第 5-6 天程序）。<ol><li>彻底重悬细胞以将活化的T细胞从αCD3 / CD28包被的珠子上解离（参见 材料表），并将细胞悬浮液放在磁铁上30秒。将细胞悬液转移到所需的 离体 培养容器中，并将其返回到37°C培养箱中。 注意:T细胞可以在培养瓶或深孔培养板中培养。建议在 6 孔规格板的深孔中，每孔在 30 mL mTCM 完全培养基中平板至少 5 x 106 个 T 细胞（参见 材料表）。</li>
		<li>通过流式细胞术测定 CAR 表达 8. 注意:GFP-CAR表达通过与100 ng/mL纯化的GFP孵育来测定。掺入 N 端表位标签将有助于检测替代 CAR 构建体。</li>
	</ol></li>
	<li>进行CAR-T细胞 的体外 培养（第7-10天程序）。<ol><li>在 离体 培养期间，通过去除 50% 的培养基并每 48 小时用新鲜的 mTCM-complete + 2x 10 ng/mL 的 IL-7 和 IL-15 代替来维持细胞。 注意:小鼠CAR-T细胞在活化后7天即可用于下游应用，由于解冻后活力差，不应冷冻保存。</li>
	</ol></li>
</ol>

在同基因模型中生成小鼠 CAR-T 细胞的简化方案

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没有声明利益冲突。

该方案描述了小鼠T细胞逆转录病毒转导以产生用于 体内 研究的CAR-T细胞所需的步骤和试剂。优化逆转录病毒转导条件可实现稳健的CAR表达，而无需通过超速离心或其他试剂进行病毒浓缩。但是，可以对这种方法进行多种修改。
虽然该协议描述了GFP特异性CAR的示例生成，但这些方法可以适应于生成靶向任何感兴趣的细胞外抗原的小鼠CAR-T细胞。本研究中使用的pMSCV_PGK逆转录病毒载体允许通过 NotI 和 BamHI 酶消化有效释放 GFP 特异性纳米抗体序列，并且可以用用户定义的抗原识别域（如 scFv、纳米抗体或配体结合域）替换（补充文件 1）。为了便于评估替代 CAR 表达，建议设计具有 N 端表位标签的 CAR，例如 Myc （EQKLISEEDL） 或 Flag （DYKDDDDK） 标签，可以使用荧光偶联抗体12 进行检测。此外，目前的研究证明了单个转基因的产生;然而，自裂解肽（2A序列）或内部核糖体进入位点（IRES）的掺入将使多个转基因的共同生产成为可能，包括增强体内T细胞功能的其他技术8,13,14。
尽管该协议的开发是为了通过省略超速离心（24,000×g，2小时，4°C）来消除对专用设备的需求，但值得注意的是，病毒上清液的浓度可以达到更高的病毒滴度并减少获得高CAR表达所需的病毒体积8。该方案还通过实现可重复的高病毒滴度（~2 x 107 TU / mL）和稳定的CAR表达来简化逆转录病毒载体的生产，而无需在使用前进行病毒滴度测定。然而，除了促进标准化外，在T细胞转导之前测定病毒滴度还可以通过减少不必要的高病毒浓度引起的潜在毒性来增加T细胞活力10,15。
建议补充 IL-7 和 IL-15 以促进 T 细胞扩增并保留 T 细胞记忆群;然而，重组人IL-2可用作减少体外培养所需试剂数量的替代方法16,17,18,19。尽管如此，这里概述的CAR-T细胞生成方法仍然受到小鼠T细胞扩增相对较差的限制。然而，通过在整个转导过程中在mTCM培养基中加入额外的补充剂和2-ME，可以进一步增强上述提高T细胞活力的措施20。虽然这些修饰可能会将 T 细胞扩增从 15 倍提高到 20 倍20，但它们可能会导致转导效率降低和 CAR 表达降低。
最终，小鼠CAR-T细胞的生产能够开发同源模型，以评估CAR-T细胞在完整免疫系统中的功能，从而促进对其效力、耐久性和安全性的特定评估。而免疫缺陷小鼠的模型允许对靶向人类肿瘤抗原的人类 T 细胞产品进行重要的临床前研究。这些互补模型系统的结合对于理解CAR-T细胞生物学的复杂性、优化新的CAR设计以及最终将临床前研究结果转化为有效的临床疗法是必不可少的。

表达嵌合抗原受体 （CAR） 的过继性 T 细胞疗法通过利用适应性免疫系统的力量特异性靶向和消除抗原阳性癌细胞，彻底改变了癌症免疫治疗领域1。虽然靶向B细胞恶性肿瘤的CAR-T细胞疗法的成功已经得到临床验证，但在动物模型中进行的临床前研究对于开发靶向实体瘤的新型CAR仍然至关重要。然而，到目前为止，在实体瘤适应症中已证明临床疗效有限，并且越来越明显的是，单个临床前模型无法准确预测活体药物的药效学和临床疗效2,3。因此，研究人员已开始扩大CAR-T细胞产品的临床前研究，分别包括人类和小鼠癌症的异种移植和同基因模型的平行评估。
与异种移植模型不同，异种移植模型将人类肿瘤和T细胞移植到免疫缺陷小鼠中，同基因模型能够在功能免疫系统的背景下检查CAR-T细胞反应。具体来说，携带同基因肿瘤的免疫能力小鼠提供了一个系统来研究过继转移的 T 细胞与环境特异性环境之间的相互作用——包括已知抑制肿瘤微环境 （TME） 中 T 细胞功能的肿瘤相关巨噬细胞 （TAM） 和调节性 T 细胞 （Tregs）4,5,6.此外，同基因模型提供了一个额外的平台来评估靶向、肿瘤外毒性以及 CAR-T 细胞与可能导致其他毒性（包括细胞因子释放综合征）的宿主因子的相互作用 7。
尽管有这些优势，但同基因CAR-T细胞研究的数量仍然有限。值得注意的是，同基因模型需要对来自同一小鼠品系的 CAR-T 细胞进行自体工程改造，因此由于缺乏高效的小鼠 T 细胞转导和离体扩增的方法，因此提出了额外的挑战 2,8。该协议概述了通过产生逆转录病毒载体和优化的T细胞转导来实现稳定CAR表达的方法。整个过程的示意图如图1所示。这种方法的使用证明了小鼠CAR-T细胞的有效逆转录病毒转导，并且无需通过超速离心实现高CAR表达，而无需病毒浓缩。除了其他转基因的共表达外，还讨论了改变CAR构建体抗原特异性的策略。

<table><tbody><tr><td>0.45 &amp;mu;m filters</td><td>MilliporeSigma</td><td>SLHVR33RS</td><td></td></tr><tr><td>1 mL syringe&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>14-955-450</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>05-408-135</td><td></td></tr><tr><td>10 mL syringe&amp;nbsp;</td><td>BD</td><td>14-823-16E</td><td></td></tr><tr><td>100 &amp;mu;m strainer</td><td>Corning</td><td>07-201-432</td><td></td></tr><tr><td>15 cm TC treated cell culture dishes</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>130183</td><td></td></tr><tr><td>15 mL conical tubes&amp;nbsp;</td><td>Falcon</td><td>14-959-70C</td><td></td></tr><tr><td>40 &amp;mu;m strainer&amp;nbsp;</td><td>Corning</td><td>07-201-430</td><td></td></tr><tr><td>50 mL conical tubes&amp;nbsp;</td><td>Falcon</td><td>14-959-49A</td><td></td></tr><tr><td>70 &amp;mu;m strainer</td><td>Corning</td><td>07-201-431</td><td></td></tr><tr><td>Attune NxT Flow Cytometer&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td></td><td></td></tr><tr><td>BALB/C, 6-8 week old&amp;nbsp;</td><td>Jackson Laboratory</td><td>651</td><td></td></tr><tr><td>B-Mercaptoethanol&amp;nbsp;</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>GOLDBIO</td><td>A-420-500</td><td></td></tr><tr><td>DMEM Medium</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS), without Calcium and Magnesium&amp;nbsp;</td><td>Gibco</td><td>14-190-250</td><td></td></tr><tr><td>DynaMag-2 Magnet&amp;nbsp;</td><td>Invitrogen</td><td>12-321-D</td><td></td></tr><tr><td>EasySep Magnet&amp;nbsp;</td><td>Stemcell Technologies</td><td>18000</td><td></td></tr><tr><td>EasySep Mouse T cell Isolation Kit</td><td>Stemcell Technologies</td><td>19851</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td>BD</td><td>BDB554657</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Corning</td><td>MT35011CV</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35-050-061</td><td></td></tr><tr><td>G-Rex6</td><td>Wilson Wolf</td><td>80240M&amp;nbsp;</td><td></td></tr><tr><td>HEPES Buffer Solution&amp;nbsp;</td><td>Gibco</td><td>15-630-080</td><td></td></tr><tr><td>Human recombinant IL-15&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-095-765</td><td></td></tr><tr><td>Human recombinant IL-2</td><td>Miltenyi Biotec</td><td>130-097-748</td><td></td></tr><tr><td>Human recombinant IL-7</td><td>Miltenyi Biotec</td><td>130-095-363</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Invitrogen</td><td>L3000008</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution&amp;nbsp;</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Mouse Anti-CD3 BV421</td><td>Biolegend</td><td>100228</td><td></td></tr><tr><td>Mouse Anti-CD3/CD28 Dynabeads</td><td>Gibco</td><td>11-453-D</td><td></td></tr><tr><td>Mouse Anti-CD4 BV605</td><td>BD</td><td>563151</td><td></td></tr><tr><td>Mouse Anti-CD44 APC&amp;nbsp;</td><td>Biolegend</td><td>103011</td><td></td></tr><tr><td>Mouse Anti-CD62L PE-Cy7</td><td>Tonbo</td><td>SKU 60-0621-U025</td><td></td></tr><tr><td>Mouse Anti-CD8 APC-Cy7</td><td>Tonbo</td><td>SKU 25-0081-U025</td><td></td></tr><tr><td>Nikon Ti2 with Prime 95B camera&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Non-treated 24 well plates&amp;nbsp;</td><td>CytoOne</td><td>CC7672-7524</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31-985-062</td><td></td></tr><tr><td>pCL-Eco</td><td>Addgene</td><td>#12371</td><td></td></tr><tr><td>Penicillin/Streptomycin Solution</td><td>Gibco</td><td>15-070-063</td><td></td></tr><tr><td>Phoenix Eco cells</td><td>ATCC</td><td>CRL-3214</td><td></td></tr><tr><td>pMDG.2</td><td>Addgene</td><td>#12259</td><td></td></tr><tr><td>pMSCV_PGK_GFP28z</td><td>N/A</td><td>Produced by R.LV.</td><td></td></tr><tr><td>Purified sfGFP</td><td>N/A</td><td>Produced by R.LV.</td><td></td></tr><tr><td>RetroNectin (&#039;transduction reagent&#039;)</td><td>Takara Bio</td><td>T100B</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>21875</td><td></td></tr><tr><td>Serological pipette 10 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>13-678-11E</td><td></td></tr><tr><td>Serological pipette 25 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>13-678-11</td><td></td></tr><tr><td>Serological pipette 5 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>13-678-11D</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11-360-070</td><td></td></tr><tr><td>TC-treated 24 well plates&amp;nbsp;</td><td>Corning</td><td>08-772-1</td><td></td></tr><tr><td>Trypan blue&amp;nbsp;</td><td>Gibco</td><td>15-250-061</td><td></td></tr></tbody></table>

efficient generation of murine chimeric antigen receptor (car)-t cells

利用嵌合抗原受体（CAR）-T细胞的工程细胞疗法在血液系统恶性肿瘤患者中取得了显着的疗效，目前正在开发用于治疗各种实体瘤。到目前为止，新型CAR-T细胞产品的初步评估主要在使用免疫缺陷小鼠的异种移植肿瘤模型中进行。选择这种方法是为了促进人类CAR-T细胞在实验环境中的成功植入。然而，肿瘤和CAR-T细胞来源于同一小鼠品系的同基因小鼠模型允许在功能免疫系统和综合肿瘤微环境（TME）的背景下评估新的CAR技术。此处描述的方案旨在通过提供逆转录病毒转导和 离体 T细胞培养的标准化方法来简化小鼠CAR-T细胞生成过程。本协议中描述的方法可应用于本研究中使用的方法以外的其他CAR构建体，以便在免疫能力系统中对新的CAR技术进行常规评估。

这里描述的方案旨在标准化小鼠T细胞转导产生小鼠CAR-T细胞的过程。 图 1 提供了所涉及步骤的详细说明。该过程始于 通过 将病毒成分共转染到 Phoenix Eco 细胞中来生产逆转录病毒载体。 图 2 显示了转染当天 Phoenix Eco 细胞的最佳密度图像。然后，在转染后24小时或本方案的"第0天"激活分离的T细胞，为第1天的转导做准备。转导后，CAR-T细胞在重组人IL-7和IL-15存在下培养，以实现足够的倍数扩增，同时保留干细胞记忆群。
根据流式细胞术测定，使用推荐的小鼠T细胞分离试剂盒（参见材料表）和该方案在转导前产生<98%的T细胞纯度（图3A）。此外，使用该方案和修饰的pMSCV逆转录病毒载体可以实现65%-75%的可重复CAR表达率，以表达来自PGK启动子的CAR构建体（图3B，C）。逆转录病毒滴度的测定显示滴度高达~2 x 107 TU/mL，CAR表达为74%，在pMSCV与pCL-Eco共转染后收获的病毒上清液为1/3rd（补充图1）。
最后，用 10 ng/mL 的 IL-7 和 IL-15 进行体外培养导致大约 15 倍的扩增，在单个脾脏激活后第 10 天产生 ~150 x 106 个 T 细胞（图 3D）。用IL-7和IL-15培养导致相当的CD8+和CD4 + T细胞频率（图3E）和保存的干细胞记忆群，在离体培养10天后由CD62L + CD44-表达确定（图3F）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65887/65887fig01.jpg"> 图 1:协议概述。 顶部面板:协议时间线的示意图概述。下图:逆转录病毒载体生产和小鼠 T 细胞转导的分步说明。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65887/65887fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65887/65887fig02.jpg"> 图 2:转染的最佳细胞密度。 用 pMSCV 和 pCL-Eco 转染前最佳密度的 Phoenix Eco 细胞的明场显微镜图像。使用10倍物镜拍摄。比例尺 = 200 μm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65887/65887fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65887/65887fig03.jpg"> 图3:小鼠CAR-T细胞生产和体外培养。 （A） 流式细胞术图显示小鼠 T 细胞分离后 BALB/c CD3+ 细胞的频率。（B） 用于小鼠 T 细胞中 GFP-CAR 表达的 pMSCV 逆转录病毒载体的示意图。（C） 流式细胞术直方图显示与纯化的 sfGFP 孵育后小鼠 T 细胞上的 CAR 表面表达。UT，未转导。（D） CAR-T 细胞在激活和重组人 IL-17 和 IL-15 培养后第 10 天的倍增。转导前激活约 8 x 106 个小鼠 T 细胞，并在激活后第 10 天达到约 1.28 x 108。（E） 流式细胞术图显示激活后第 10 天 CD8+ 和 CD4+ CAR-T 细胞的频率。（F） 流式细胞术图显示了由 CD62L 和 CD44 表面表达决定的 CD8+ T 细胞记忆群的频率。干细胞记忆（TSCM）的特征是CD62L+CD44-，中枢记忆（TCM）的特征是CD62L+CD44+，效应记忆（TEM）的特征是CD62L-CD44+。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65887/65887fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
表1:细胞培养基配方。 方案中使用的细胞培养基的配方详细信息。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65887/65887_Table%201.xlsx" target="_blank">请按此下载此表格。</a>
补充图1:CAR表面表达和逆转录病毒滴度。 （A） 流式细胞术直方图显示与纯化的 sfGFP 孵育后 BALB/c T 细胞上的 CAR 表面表达。逆转录病毒是通过用 pMSCV_PGK_mGFP28z 与 pCL-Eco 和 pMD2.G （I）、pCL-Eco （II） 或单独 （III） 联合转染 Phoenix Eco 细胞来生成的。如图所示，用稀释的逆转录病毒转导T细胞。（B） 由转导 T 细胞的 CAR 表面表达测定的逆转录病毒滴度。使用以下公式计算转导单位 （TU）/mL:（N∙F∙D）/V，其中 N 是转导的细胞数 （1 x 105），F 是 CAR+ 细胞的频率，D 是稀释因子，V 是以 mL （0.2 mL） 为单位的转导体积。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65887/Supplementary%20Figure%201.zip" target="_blank">请点击此处下载此文件。</a>
补充文件1:pMSCV表达载体序列。 pMSCV 表达载体中 GFP28z CAR 和侧翼序列（斜体）的序列，其中 NotI 和 BamHI 酶位点以蓝色突出显示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65887/Supplementary%20File%201.zip" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about Enhancing CAR-T Cell Function in Syngeneic Tumor Models at JoVE.com

作者聚焦:增强同基因肿瘤模型中的 CAR-T 细胞功能 

该方案简化了逆转录病毒载体的产生和小鼠T细胞的转导，促进了小鼠CAR-T细胞的高效生成。

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

我们的研究探索了工程细菌和CAR T细胞在实体瘤治疗中的有效组合。对我们来说，使用卵巢癌的同基因模型研究这两种细胞疗法如何在完整免疫系统的背景下发挥作用是很重要的。由于缺乏用于生成小鼠CAR T细胞的标准化方案，研究CAR T细胞在同基因肿瘤模型中的功能受到限制。使用传统方法在体外培养中转导小鼠T细胞特别困难。我们开发了该协议以简化小鼠CAR T细胞的生产，以便在同基因模型中进行研究，从而为评估新CAR技术的活性提供重要背景。

efficient-generation-of-murine-chimeric-antigen-receptor-car-t-cells

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

3.3K 次观看.  Columbia University. 我们的研究探索了工程细菌和CAR T细胞在实体瘤治疗中的有效组合。对我们来说，使用卵巢癌的同基因模型研究这两种细胞疗法如何在完整免疫系统的背景下发挥作用是很重要的。由于缺乏用于生成小鼠CAR T细胞的标准化方案，研究CAR T细胞在同基因肿瘤模型中的功能受到限制。使用传统方法在体外培养中转导小鼠T细胞特别困难。我们开发了该协议以简化小鼠CAR T细胞的?...

视频: 作者聚焦:增强同基因肿瘤模型中的 CAR-T 细胞功能 

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

科研

癌症研究

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

免疫与感染

<meta charset="utf8"></head><body>缺氧敏感 CAR-T 细胞用于选择性肿瘤的功能验证

Generation and Functional Verification of Hypoxia-Sensitive Chimeric Antigen Receptor-T Cells

Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating solid tumors remains challenging, as exemplified by the safety concerns that arise when CAR-T cells attack normal cells expressing the target antigens. Researchers have explored various approaches to enhance the tumor selectivity of CAR-T cell therapy. One representative strategy along this line is the construction of hypoxia-sensitive CAR-T cells, which are designed by fusing an oxygen-dependent degradation domain to the CAR moiety and are strategized to attain high CAR expression only in a hypoxic environment-the tumor microenvironment (TME). This paper presents a protocol for the generation of such CAR-T cells and their functional characterization, including methods to analyze the changes in CAR expression and killing capacity in response to different oxygen levels established by a mobile incubator chamber. The constructed CAR-T cells are anticipated to demonstrate CAR expression and cytotoxicity in an oxygen-sensitive manner, thus supporting their capability to distinguish between hypoxic TME and normoxic normal tissues for selective activation.

<meta charset="utf8"></head><body>作者聚焦:用于增强癌症免疫治疗的缺氧敏感 CAR-T 疗法的进展

Here we present a protocol for the generation and functional verification of hypoxia-sensitive chimeric antigen receptor (CAR)-T cells. This protocol presents the lentivirus-based generation of hypoxia-sensitive CAR-T cells and their characterization, including the validation of hypoxia-dependent CAR expression and selective cytotoxicity.

缺氧敏感的嵌合抗原受体-T 细胞的生成和功能验证

Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating ...

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

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

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

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

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

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

<meta charset="utf8"></head><body>用于免疫调节疗法的人 CAR Treg 的工程化和扩增

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

<meta charset="utf8"></head><body>人嵌合抗原受体调节性 T 细胞的产生

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

人嵌合抗原受体调节性 T 细胞的产生

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

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制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

缺氧敏感的嵌合抗原受体-T 细胞的生成和功能验证

缺氧敏感的嵌合抗原受体-T 细胞的生成和功能验证

缺氧敏感的嵌合抗原受体-T 细胞的生成和功能验证

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

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

人嵌合抗原受体调节性 T 细胞的产生

人嵌合抗原受体调节性 T 细胞的产生