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背景 T细胞恶性肿瘤是一种血液学癌症，儿童和成人都有很高的复发率和死亡率，目前尚无有效的或针对性的治疗方法。对于初次治疗后复发的患者，挽救性化疗方案诱导20-40%的患者缓解。同种异体干细胞移植具有相关的风险和毒性，是唯一的治疗方法。 T细胞表达嵌合抗原受体（CAR）是一种有前途的肿瘤免疫治疗方法。这种靶向治疗在B细胞白血病和淋巴瘤患者中显示了巨大的潜力，可以诱导缓解甚至长期无复发生存。然而，一些挑战限制了Car-T细胞对抗T细胞恶性肿瘤的临床发展： 1. T效应细胞和T细胞恶性肿瘤之间靶抗原的共同表达导致Car-T细胞自相残杀。 2. 在不受恶性细胞污染的情况下收获足够数量的自体T细胞，技术上具有挑战性，而且成本高昂。 3. 当来自同种异体供体的基因修饰的Car-T细胞注入免疫受损的HLA匹配或不匹配的受体时，可能导致危及生命的移植物抗宿主病（GVHD）。 方法 作者构建了一种抗自相残杀的“现成”Car-T（或UCART7），它针对CD7+T细胞恶性肿瘤，并且通过CRISPR/CAS9基因编辑，敲除了CD7和T细胞受体α链（TRAC）的表达。 UCART7对CD7+的恶性T细胞能够有效靶向和杀伤，而无明显的效应器T细胞自相残杀。TRAC缺失阻断了TCR介导的信号传导，允许安全使用异基因T细胞作为Car-T的来源，而不会诱导危及生命的GVHD，也不会有被CD7缺失的恶性细胞污染的风险。 结果 UCART7对人T细胞急性淋巴细胞白血病（T-ALL）细胞株和原发性T-ALL在体外和体内均具有疗效，但未诱导异基因GVHD。抗自相残杀、耐受同种异体的“现成”Car-T是一种治疗复发和难治性T-ALL和非霍奇金T细胞淋巴瘤的策略，无需自体T细胞。  UCART7 kills primary patient T-ALL blast in vitro Primary blasts obtained from three individual patients with CD7+ T-ALL were labeled with 150 nM CFSE. Labeled cells were co-incubated at a 1:1 ratio with either CD7ΔCART7, UCART7, or their respective CD19 controls in triplicate for 24 hours prior to FACS analysis. Accucount florescent beads were used to determine actual cell counts. Data were collected using a Gallios cytometer. (a) representative FACS plots. (b) CD7ΔCAR7 and UCART7 effectively killed T-ALL blasts relative to CD7ΔCAR19 and UCART19 (n=4). Data were compared using one-way ANOVA, followed by ad-hoc multiple comparisons for between-group differences, and a logarithm transformation was also performed. p values < 0.05 considered significant, *p ≤ 0.05, ** p ≤ 0.01, ***p≤ 0.001, **** p ≤ 0.0001.  UCART7 kills primary patient T-ALL blast in vivo without inducing xenogeneic GvHD NSG were engrafted with 1×106 PDX DFCI12 cells on day 0 followed by infusion of 2×106 UCART7, UCART19, TRACΔ or WT T on day +1. Mice were assessed for GvHD, and blood and splenocytes analyzed by FACS 6 weeks post CAR-T injection (a) Representative flow cytometry plots of blood analysis presented to show both tumor and T cells (b) Percentage of tumor cells out of total mouse and human CD45 cells in the blood (WT n=6, UCART7 n=6, UCART19 n=6, TRACΔ n=4 or PDX only n=5) and spleens (n=3). Unlike TRACΔ T cells, WT T cells clear tumor through an alloreactive GvL effect (blood, p <0.0001; spleen, p = 0.0033). UCART7 is effective at clearing PDX relative to UCART19 (blood, p <0.0001; spleen p <0.0001). (c) Clinical GvHD scores, graded according to Cooke, et al43 (n=3 mean±s.d.). Unlike TRACΔ T cells, WT T cells induce GvHD. Representative images of mice following infusion of WT T cells, TRACΔ T cells, UCART7, and UCART19. *p ≤ 0.05, ** p ≤ 0.01, ***p≤ 0.001, **** p ≤ 0.0001. 在本实验中，作者利用Bio-Rad ZE5流式细胞仪进行了一系列实验： 1. 确定CD7被敲除:  2. CD7ΔCAR-T体外杀伤白血病T细胞  3. Car-T 表型鉴定多色流式方案:   参考文献： An ‘off-the-shelf’ fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies Leukemia. 2018 September ; 32(9): 1970–1983. doi:10.1038/s41375-018-0065-5. 如果您对我们的产品和技术感兴趣，希望申请仪器试用，可通过扫描下方二维码，留下您的联系方式，我们会尽快和您取得联系。 本产品仅限科研使用，不作为临床诊断。 Bio-Rad是Bio-Rad Laboratories, Inc.在特定区域的商标 这是一个广告 |
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