The authors would like to acknowledge Dr. Laura Johnson and Dr. Richard Morgan for providing the CAR retroviral construct. The authors also thank Giao Ngyuen for her assistance with dosimetry for whole brain irradiation. This work was supported by an NIH NCI grant 1R01CA177476-01.

这个协议是基于其中10只小鼠用10 7 CAR t各自细胞进行了处理的实验设计。这意味着10 8 CAR T细胞，将需要;屈服应为5×10 7 -1×10 8被高估到帐户中生存能力的损失。以下协议进行缩放，以产生大约200×10 6个细胞。然后将细胞静脉内施用给雌性C57BL / 6小鼠用9天建立同基因的EGFRvIII阳性颅内肿瘤，从现有KR158B星形细胞瘤或B16黑色素瘤细胞系的开发。与之同时CAR T细胞的生成，荷瘤小鼠服用临床相应剂量的lymphodepletive TMZ（60毫克/公斤）和WBI（16.5戈瑞）的。 小鼠维护和无病原体条件在杜克大学医学中心（DUMC）下饲养。所有的动物实验是按照批准杜克大学机构的协议进行人的动物护理和使用委员会（IACUC）。 1.政府替莫唑胺对荷瘤小鼠天0到4： <ol><li>计算要注射的小鼠的总数和由0.5乘以此确定需要（ 例如，30只小鼠X0.5毫升=15毫升TMZ）TMZ溶液的体积和加入1.5毫升该体积占溢出（16.5毫升TMZ ）。 </li><li> 3毫克乘以这个总量/毫升，以确定冻干TMZ所需要的重量（ 例如，16.5×3 = 49.5毫克TMZ）。称量到50毫升锥形此。 注意：TMZ是通过吸入，食入，并与眼睛，皮肤接触和黏膜毒性。咨询与减少暴露风险的建议，机构的职业安全和/或环境健康和保健部门。 </li><li>由0.15乘以总体积来确定的二甲基亚砜的体积（DMSO）中所需的（ 例如，16.5 X0.15 =2.475毫升DMSO）中。过滤消毒的DMSO此体积（加上额外的2 ml至占溢出）通过0.2微米的过滤器进入无菌管中。添加2.475毫升无菌DMSO中至TMZ粉末。 </li><li>放置TMZ / DMSO溶液中的水的烧杯中在热板上在65℃下10-15分钟。一旦TMZ完全溶解，该溶液应成为颜色浅黄色;如果溶液变成粉红色，则TMZ降低，和一个新的解决方案应该用新鲜很多TMZ的制备。 </li><li>计算的无菌盐水中所需的总体积（0.85×16.5毫升=1​​4.025毫升盐水）乘以0.85的适当体积。加入15毫升无菌盐水以50ml锥形。而TMZ被溶解在DMSO中，热盐水至65℃。 </li><li>涡流的TMZ / DMSO溶液，以确保该TMZ粉末完全溶解，并慢慢加入温热生理盐水到TMZ / DMSO溶液。 </li><li>配制后立即完全加载15×1毫升结核菌素syringES与施用TMZ解决4天建立荷瘤小鼠。 </li><li>通过重复4天1这个过程总共5 TMZ施用。 </li></ol>荷瘤小鼠的2全脑照射天2至4： <ol><li>功率上的X射线照射，并允许它预热到全电压。确保适当的过滤器是到位，并输入正确的电压和电流设置（ 图2A）。 注：该辐照装置的剂量应建立预先确定合适的电压设定和网格布局（ 图2A，B）。 </li><li>计算的时间是必要的，以导致5.5戈瑞X射线照射的长度。例如，如果X射线被递送以2格雷/分钟的速度，然后2.75分钟是必要提供一个总的5.5戈瑞。输入适当的持续时间。 注： 表2鼠标WBI一个剂量第二临床等效于人类。在高级别胶质瘤中的背景下，60戈瑞分馏WBI（2戈瑞馏分，5天/周，连续6周）是护理的临床标准，并且在这里所用的鼠等价物是16.5戈瑞（5.5戈瑞×3）。 </li><li>通过加入2ml氯胺酮和1ml甲苯噻嗪以17毫升盐水，使得最终溶液为10毫克/毫升氯胺酮和1mg / ml的甲苯噻嗪制备氯胺酮/赛拉嗪用于全身麻醉的新鲜溶液。 </li><li>称重小鼠并管理10微升氯胺酮/赛拉嗪的腹膜内每克体重，使得动物接受氯胺酮的剂量为100毫克/公斤和10毫克/千克甲苯噻嗪。小鼠应充分镇静，并在氯胺酮/甲苯​​噻嗪管理2分钟明显呼吸。 </li><li>保持镇静动物眼用水分，轻轻擦在每只眼睛的少量人工泪液软膏。 </li><li>放置镇静的小鼠上，使得磁头被定位在接收中的最高的X射线的区域的网格张力（ 图2B，C）。从颈部与适当向下屏障体尺寸铅管阻断全身的X射线递送（ 图2D）。 </li><li>放置位置的小鼠的X射线束，使得激光表示的X射线束的焦点是在坐标（0,0）上的网格布局下。开始透视交付。 </li><li>当透视交付完成后，取出在一个温暖的加热垫的辐射和地方的动物。不要用有意识的动物容纳镇静剂动物直到动物重新获得足够的意识，保持胸骨recumbence。一旦动物能保持胸骨recumbence，将有意识的动物，早在其相应的笼子里。 </li><li>重复上天3和4，此过程总共三个分次剂量的辐射。 </li></ol> 3.转染 -1天： <ol><li>通过加入50ml的胎牛血清（FBS）的500毫升的Dulbecco并购准备D10媒体odified Eagle培养基（DMEM）中。 </li><li>通过加入5.5毫升L-谷氨酰胺，5.5毫升丙酮酸钠，5.5毫升非必需氨基酸，和5.5毫升青霉素/链霉素（青霉素/链霉素）至500ml的RPMI-1640培养基制备的T细胞培养基（TCM）的。接着，添加550微升2-巯基乙醇和550微升庆大霉素。最后，添加50毫升FBS。 </li><li>收获在体外培养的HEK293T细胞，并把以7.5×10 6个细胞/ D10在媒体ml的浓度。 </li><li>板10毫升D10媒体和加入1毫升HEK293T细胞悬浮液中的每一个16×10cm的聚D-赖氨酸（PDL）包被的板。 </li><li>孵育过夜，在37℃，5％的CO 2。 </li></ol>第0天： <ol start="6"><li>转染细胞替换之前用10毫升新鲜的D10至少30分钟的媒体。 </li><li>确定载体质粒所需的转染的量，PCL-生态矢量，和脂质体转染试剂通过板的总数乘以+ 1（16 + 1 = 17）14.1微克载体质粒（14.1×17 = 239.7微克），9.9微克的pCL-生态载体（9.9×17 = 168.3微克），和60微升脂质体转染试剂（60×17 = 1020微升）。所有试剂应该在室温下制备溶液之前。 </li><li>标签两管A和B管A，增加239.7微克载体质粒和168.3微克PCL-生态向量（1.5×17）毫升降低血清改进的Eagle培养基（RS-MEM）。在管B，加入1020微升脂质体转染试剂（1.5×17）毫升RS-MEM。 </li><li>孵育A和B分别为5分钟，在室温下。 </li><li>混合A和B一起轻轻（涡旋1-2秒或颠倒数次）并孵育20分钟，在室温下，以形成脂质/ DNA复合物。 </li><li>添加脂质/ DNA复合物滴至HEK293T细胞，10 cm培养皿。 </li><li>孵育在37℃，6-8小时或过夜（不超过24小时）。 </li><li>后6-8小时温育后，用12ml的新鲜TCM进行病毒生产更换介质。这镀金媒体将用于在第2天的病毒上清用于T细胞转导。 </li></ol> 4.脾切除和T细胞的制备第0天： <ol><li>倾10毫升医药到50ml锥形并置于冰上脾集合。 </li><li>通过CO 2窒息和次级断头牺牲动物的适当数目：将动物关在笼子里接收的CO 2在10-30％的笼体积/分钟的流速，每美国兽医医学协会准则（不超过5个动物可以是同时牺牲），直到呼吸终止，并在此后两分钟。从CO 2室中移除动物和斩首。 注：6-12周龄雌性C57BL / 6小鼠会产生约4.5-5×10 7脾健脾之一。这里，4个脾脏将收获约200×10 6个细胞。 </li><li>莱使得它的右侧朝上的鼠标和喷用70％的乙醇。随着镊子，抢薄下方左侧胸腔皮肤褶皱切一的切口略微用剪刀。剥开皮，小心抢薄用钳子腹膜折，切一小腔。 </li><li>脾是一个小的，长形，暗红色的器官，类似于一个扁平豆;微妙抢与镊子和消费税由切掉周围的结缔组织脾脏。将切除脾脏到含有冰10毫升中医的锥形。 </li><li>倒脾脏超过70微米的网状细胞过滤并通过用5毫升注射器的内部的钝端糖化以产生单细胞悬浮液分解。最多两个脾脏应该每个滤网进行分类。 </li><li>使用小体积中医仔细清洗下分解来收集任何剩余的脾细胞的过滤器。池中的所有分类脾脏成单细胞悬液。把最终体积50毫升中医一洗了二维自旋，在300×g离心10分钟。 </li><li>通过加入5ml 10×裂解缓冲液至45毫升无菌水制备的1×裂解缓冲液中的溶液。为了消除红血细胞，重悬沉淀在5ml每脾1×裂解缓冲液，在50毫升的锥形，轻轻吹打上下充分混合。如果超过5脾，用250毫升离心管。放置锥形或离心管在37℃水浴中5分钟。 </li><li>除去从水浴裂解反应，并添加医药以1：1的比例用裂解缓冲液中以中和反应。通过在300 XG旋转10分钟洗净。 </li><li>吸去上清液并充分重悬沉淀在中医（2毫升/脾，8毫升总计4脾脏）通过上下抽吸。 </li><li>通过加入10微升细胞悬浮液以190微升台盼蓝（1:20稀释）计数细胞。乘以20×10 4×总体积（8毫升）中的4网格正方形1得到的数，以获得总的细胞数目（ 例如，125细胞×20×10 4个</s达&gt; X8毫升= 200×10 6脾细胞）。 </li><li>稀释细胞以2×10 6个细胞/ ml，在中医的浓度，补充有2微克/ ml的伴刀豆球蛋白A（ConA刺激）和50IU / ml重组人白介素-2（的rhIL-2）。因此，对于200×10 6个脾细胞中，添加92毫升媒体8毫升细胞，200微克的ConA，和5000 IU的rhIL-2。 </li><li>加入2毫升细胞的24孔组织培养处理板各孔中，以使得4×10 6个细胞每孔（ 例如100毫升的细胞将需要大约4板）。 </li><li>孵育过夜，在37℃，5％的CO 2。 </li></ol> 5.转导第1天： <ol><li> 。预算非组织培养的数处理所需的转导24孔板乘以脾脏2收获的次数（需要为4脾脏8片）。 </li><li>接着，计算出的重组人纤连蛋白片段（RHFF）溶液至所需体积乘以需要外套板（总数井+ 3）×0.5 ML（8板×24井= 192 + 3 = 195）×0.5毫升=97.5毫升。 </li><li>通过将总体积25微克（97.5×25 = 2437.5微克）乘以制备含有RHFF以25微克/ ml的浓度97.5毫升磷酸盐缓冲盐水（PBS）。加入这一数额RHFF的97.5毫升的PBS。 </li><li>涂层的非组织培养处理的24孔板中加入每孔0.5毫升PBS / RHFF溶液。 </li><li>孵育过夜，在4℃。 </li></ol>第2天： <ol start="6"><li>倾倒的PBS / RHFF溶液从非组织培养处理过的24孔板中。 </li><li>加入1毫升/孔的2％牛血清白蛋白（BSA）的PBS，并在室温下孵育30分钟。 </li><li>紧紧颠覆板拆下BSA。加入2毫升的PBS洗涤。 </li><li>通过将媒体从每个HEK293T PDL板放入250毫升离心管中收集的病毒上清和自旋10分钟，在500×g。 </li><li>仔细转让苏病毒pernatant成新鲜250毫升离心管中，作为确保不打扰可能形成细胞沉淀。 </li><li>添加新鲜TCM到病毒上清，使得最终体积比所需RHFF包被的孔的量​​3毫升更多。例如，对于192 RHFF包被的孔，使病毒上清的量，以192 +3毫升=195毫升。 </li><li>在每口井的3倍 - 轻轻吹打上下2从孵化器，并重新悬浮培养脾细胞。转移到250毫升的锥形。如果使用多道移液器，使用转移将有助于加快这一步骤之前无菌水库。算如前所述和旋转，在300×g离心10分钟。 </li><li>添加的rhIL-2病毒上清以50IU / ml的浓度。例如，添加50×195 = 9,750 IU的rhIL-2195毫升病毒上清液。 </li><li>重悬在病毒上清的脾细胞以1×10 6个细胞/ ml的</li><li>加入1毫升/孔的脾细胞悬浮液，以RHFF包覆的24孔板。 </li><li>根据以下设置自旋90分钟：770×g离心，加速度= 4，制动/减速= 0时，32℃。 </li><li>准备用50 IU / ml的白细胞介素-2中医的解决方案。例如，通过加入万IU的rhIL-2制备200中医溶液。离心后加入1 ml的rhIL-2 /中的每个孔中。 </li><li>培养过夜，在37℃在5％CO 2。 </li></ol> 6. CAR T细胞培养和收获天3和4： <ol><li>如果T细胞达到&gt; 80％汇合，细胞可能会被分割（这通常发生在第3天或4天）。分裂的细胞，轻轻吸管上下各孔中的2-3倍，并移动1毫升从每个孔到一个新的24孔组织培养处理板的新井。然后，用50IU / ml的IL-2加入1毫升新鲜TCM到每个孔，使得最终体积为2毫升所有孔中。 </li><li>如果细胞不达到&gt; 80％汇合，通过缓慢地从每个顶部吹打关闭1毫升介质以及执行半媒体变化。避免ðisturbing细胞定居在底部，同时消除媒体。加入1毫升含50国际单位/毫升的rhIL-2的新鲜TCM。 </li></ol>第5天： <ol start="3"><li>重悬CAR T细胞轻轻吹打向上并在每个下降3倍井，并转移到250毫升的离心管中。旋转细胞在300×g离心10分钟。 </li><li>完全吸出上清，而不会干扰颗粒。用PBS洗一次，计数细胞如前所述，和洗涤用PBS第二时间。一个典型的汽车的T细胞产率是每孔约1×10 6个细胞（8板×24孔= 192×10 6个CAR T细胞）。 </li><li>重悬的细胞在PBS中的5×10 7 / ml的用于注射1×10 7中的200微克的体积的浓度（ 例如，对于192×10 6个汽车的T细胞，重悬洗涤沉淀在3.84毫升PBS）中。 </li></ol> 7. CAR T细胞当局荷瘤小鼠第5天： <ol><li> TRANSPO室温细胞在冰上动物设施用于静脉注射。负载500-1000微升到胰岛素注射器27日 - 一个31 G针，以确保所有气泡被排出。 </li><li>抢鼠标尾部的基础，并将在管限制器。 </li><li>拉拉紧与静脉朝上的尾部，并且通过插入其平行于所述尾静脉滑行针在皮肤下大约1-2毫米到静脉。慢慢地排出200微升进入静脉。如果针正确地插入静脉，体积会容易流入;否则就会出现阻力和针将需要从尾部取出并正确地重新插入。 </li></ol>

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The authors have no conflicts of interests to declare.

这里所描述的治疗时间表旨在模拟护理临床标准，并充分利用其影响汽车继治疗。 CAR T细胞的剂量，TMZ治疗方案，和放射治疗施用可以被修改，以提高在体内的T细胞活性，淋巴细胞缺失，以及肿瘤杀伤。 TMZ治疗方案可以增加以得到主机myeloablation和过继转移的细胞30的膨胀增加。此外，TMZ的lymphodepletive效果可以通过低剂量被概括（4 - 6 Gy）进行单级分的全身照射（TBI），从而绕过SO...

胶质母细胞瘤（GBM）是最常见的原发性恶性脑肿瘤，并且总是致命的。手术切除加上护理化疗和放疗的非特异性标准未能完全消除恶性细胞，导致少于15个月的患者一预后不佳这种疾病1。与此相反，免疫治疗提供了一种精确的方法用于特异性靶向肿瘤细胞，并因此具有作为一个非常有效的治疗平台抵押品毒性2-4的风险降低的潜力。 T细胞工程体外表达嵌合抗原受体（车）提供了一个通用的策略肿瘤免疫治疗。通过融合的抗体的胞外可变区与一种或多种细胞内的T细胞信号传导分子（多个），以代替全长主要组织相容性复合体的生成的CARs体（MHC）-restricted T细胞受体5。的抗体样抗原recogniti此模式上允许反应活性的抗原特异性T细胞识别并在没有MHC的响应肿瘤抗原，并且可以适合于几乎无限抗原剧目。 设计对多种肿瘤抗原的CAR T细胞表现出临床前功效和卓越的承诺在门诊6-9。具体而言，在GBM的上下文中，一辆汽车的T细胞平台靶向表皮生长因子受体变体III（EGFRvIII的），肿瘤特异性突变在细胞表面上表达的10，被证明延长生存胶质瘤的小鼠11。尽管他们的通用性，然而，汽车过继疗法的临床效益尚未完全实现，在部分肿瘤相关的免疫抑制和免疫逃避12-16以及在建立和维持抗原特异性T细胞在体内的挑战所致。借力护理（SOC）的标准与免疫治疗可以潜在地克服这几条升仿制品，从而在两者的临床前和临床的设置增强的功效。 SOC进行后切除GBM由大剂量替莫唑胺（TMZ），一个DNA烷化剂17，和全脑照射（WBI）1。这些治疗方法都是通过推测肿瘤MHC表达18-20上调及抗原的死肿瘤细胞17,19,21,22脱落，以协同与肿瘤疫苗。实际上，在加入的TMZ 20,23或18,24的WBI导致在临床前设置免疫为主的治疗增强抗肿瘤功效。此外，像许多非特异性的细胞毒性化学治疗剂，TMZ是已知的导致全身性淋巴细胞25,26，其可以被利用作为宿主调节过继疗法平台27-29的一种手段。 TMZ介导的淋巴细胞缺失已显示增强抗原特异性T细胞的频率和功能，导致一个ADOP的增加的效力略去治疗平台对颅内肿瘤的30。在CAR疗法的上下文中，淋巴细胞缺失用作宿主调节的通过减少内源性抑制性T细胞31的数目，并通过减少竞争因子33诱导的稳态增殖32，从而提高抗肿瘤活性11,34的装置。定GBM SOC与免疫平台之间的协同关系，评价新颖代管疗法和疫苗平台在SOC的上下文是用于绘制关于功效有意义的结论是至关重要的。 在这个协议中，我们概述了鼠EGFRvIII的特异性CAR T细胞一起TMZ和WBI的生成和静脉内给药于小鼠的EGFRvIII阳性颅内肿瘤（参见图1进行治疗的时间表）的方法。简言之，将汽车的T细胞是由逆转录病毒转导所作体外 。人胚胎肾（HEK）293T细胞用DNA /脂质复合物（包含CAR载体和PCL-生态质粒），以产生病毒，然后将其用于转导被收获并在平行培养的活化鼠脾细胞进行转染。在汽车发生的过程中，轴承的EGFRvIII阳性颅内肿瘤的鼠宿主施用分馏全脑的X射线照射和全身TMZ治疗的剂量相当于临床SOC。 CAR的T细胞，然后静脉内递送至lymphodepleted主机。 下面的过程是在七个不同的阶段中描述：（1）管理的替莫唑胺对荷瘤小鼠，（2）全脑荷瘤小鼠的辐射;（3）转染，（4）脾切除和T细胞的制备，（5 ）转导，（6）CAR T细胞培养和收获，以及荷瘤小鼠（7）CAR T细胞管理。这些阶段包括跨越6-7天，并同时执行多个步骤。

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Name of Material</td>
			<td style="width:172px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:449px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCL-Eco Retrovirus Packaging Vector</td>
			<td>Imgenex</td>
			<td>10045P</td>
			<td>Helper vector for generating CAR retrovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Concanavalin A</td>
			<td>Sigma Aldrich</td>
			<td>C2010</td>
			<td>Non-specific mitogen to induce T cell proliferation and viral transduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retronectin</td>
			<td>ClonTech/Takara</td>
			<td>T100B</td>
			<td>Facilitates retroviral transduction of T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>HEK293 culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>T cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Life technologies</td>
			<td>11058-021</td>
			<td>Transfection media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 mM L-Glutamine&#160;</td>
			<td>Life technologies</td>
			<td>25030-081</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Sodium Pyruvate</td>
			<td>Life technologies</td>
			<td>11360-070</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Life technologies</td>
			<td>11140-050</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">55 mM 2-Mercaptoethanol&#160;</td>
			<td>Life technologies</td>
			<td>21985-023</td>
			<td>Reducing agent to remove free radicals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin (50 mg/mL)&#160;</td>
			<td>Life technologies</td>
			<td>15750-060</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GemCell U.S. Origin Fetal Bovine Serum</td>
			<td>Gemini Bio Products</td>
			<td>100-500</td>
			<td>Provides growth factors and nutrients for in vitro cell growth&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA), Fraction V&#8212;Standard&#160;Grade&#160;</td>
			<td>Gemini Bio Products</td>
			<td>700-100P</td>
			<td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pharm Lyse (10X concentrate)&#160;</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td>Lyses red blood cells during splenocyte processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Sterile Cell Strainers</td>
			<td>Corning</td>
			<td>352350</td>
			<td>Filters away large tissue particles during splenocyte processing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm BioCoat Culture Dishes with Poly-D-Lysine&#160;</td>
			<td>Corning</td>
			<td>356469</td>
			<td>Promotes HEK293 cell adhesion to maximize proliferation after transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temozolomide</td>
			<td>Best Pharmatech</td>
			<td>N/A</td>
			<td>Lyophilized powder prepared on the day of administration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl Sulfoxide</td>
			<td>Sigma Life Sciences</td>
			<td>D2650</td>
			<td>Necessary for complete dissolution of temozolomide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td>Hospira</td>
			<td>IM 0132 (5/04)</td>
			<td>Solvent for temozolomide and ketamine/xylazine&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketathesia HCl</td>
			<td>Henry Schein Animal Health</td>
			<td>11695-0701-1</td>
			<td>Ketamine solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnaSed</td>
			<td>Lloyd Inc</td>
			<td>N/A</td>
			<td>Xylazine sterile solution 100 mg/mL&#160;</td>
		</tr>
	</tbody>
</table>

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.

通过转导与EGFRvIII的CAR逆转录病毒载体11中产生充足的T细胞。该载体，MSGV1，从SFGtcLuc_ITE4向量35，其中包含了鼠干细胞病毒（MSCV）长末端重复序列，扩展的gag区和包络剪接位显影（剪接供体，SD和剪接受体，SA），以及病毒包装信号（ψ）。含有人抗EGFRvIII的单链可变片段（scFv）139，在串联与鼠CD8TM的EGFRvIII的CAR，CD28，4-1BB，和CD3ζ胞内区域，被克隆到反转录病毒载体下游的NcoI位点（...

Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

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

generation-car-t-cells-for-adoptive-therapy-context-glioblastoma

Research

JoVE Journal

Immunology and Infection

21.3K 次观看.  Duke University. 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. 

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

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 of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

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

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

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

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

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

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

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 ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

肽：MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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

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

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

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

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

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.