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表达嵌合抗原受体的功能优良的T细胞的鉴定与选择性扩增

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发表于 2015-12-3 22:33:22 | 显示全部楼层 |阅读模式
Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors
Abstract
Background:
T cells expressing chimeric antigen receptors (CARs) have shown exciting promise in cancer therapy, particularly in the treatment of B-cell malignancies. However, optimization of CAR-T cell production remains a trial-and-error exercise due to a lack of phenotypic benchmarks that are clearly predictive of anti-tumor functionality. A close examination of the dynamic changes experienced by CAR-T cells upon stimulation can improve understanding of CAR–T-cell biology and identify potential points for optimization in the production of highly functional T cells.
Methods:
Primary human T cells expressing a second-generation, anti-CD19 CAR were systematically examined for changes in phenotypic and functional responses to antigen exposure over time. Multi-color flow cytometry was performed to quantify dynamic changes in CAR-T cell viability, proliferation, as well as expression of various activation and exhaustion markers in response to varied antigen stimulation conditions.
Results:
Stimulated CAR-T cells consistently bifurcate into two distinct subpopulations, only one of which (CARhi/CD25+) exhibit anti-tumor functions. The use of central memory T cells as the starting population and the resilience—but not antigen density—of antigen-presenting cells used to expand CAR-T cells were identified as critical parameters that augment the production of functionally superior T cells. We further demonstrate that the CARhi/CD25+ subpopulation upregulates PD-1 but is resistant to PD-L1-induced dysfunction.
Conclusions:
CAR-T cells expanded ex vivo for adoptive T-cell therapy undergo dynamic phenotypic changes during the expansion process and result in two distinct populations with dramatically different functional capacities. Significant and sustained CD25 and CAR expression upregulation is predictive of robust anti-tumor functionality in antigen-stimulated T cells, despite their correlation with persistent PD-1 upregulation. The functionally superior subpopulation can be selectively augmented by careful calibration of antigen stimulation and the enrichment of central memory T-cell type.
Keywords: Chimeric antigen receptor, CD19 CAR-T cell, T-cell immunotherapy, PD-1
表达嵌合抗原受体的功能优良的T细胞的鉴定与选择性扩增
摘要
背景:
细胞表达嵌合抗原受体(CAR-T)在癌症的治疗中表现出令人激动的前景,特别是在治疗B细胞恶性肿瘤。然而,CAR-T细胞的生产优化仍然是一个试验性质的,会有错误的经验,由于缺乏能够明确预测抗肿瘤功能的表型基准。CAR-T刺激后动态变化的严密测试能够增加对CAR-T细胞生物活性和识别点的理解,从而优化高效功能T细胞的生产。
方法:
表达二代靶向CD19 CAR的初级人类T细胞被系统的研究:随着时间变化,抗原逐渐暴露,表型和功能的变化。多色流式细胞仪量化CAR-T细胞活力,增殖能力以及检测不同抗原刺激条件下的各种活化和疲劳标记物的表达。
结果:
刺激的CAR-T细胞不断分化成两个不同的亚群,其中只有一个亚群(CARhi / CD25+)具有抗肿瘤作用。使用中央记忆T细胞作为初始种群,使用抗原递呈细胞而非抗原的密度作为增加功能优越T细胞增殖的临界参数值。我们进一步证明了,CARhi/CD25+亚群上调了PD-1,但是对PD-L1诱导的机制失调具有抵抗性。
结论:
CAR-T细胞在体外扩增用于过继细胞治疗的过程中,在扩增过程中经历了表型的动态变化,最终会分化为两种具有明显功能特性差异的亚群。虽然与PD-1持续性上调有关,对于抗原刺激的T细胞,明显和持续的CD25以及嵌合抗原表达上调意味着强大的抗肿瘤活性。通过仔细的抗原校准刺激以及中央记忆T细胞的增加,功能优越的亚群能够被选择性的扩增。
关键词:嵌合抗原受体,CD19 CAR-T细胞,T细胞免疫疗法,PD-1
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Identification and selective expansion of functionally superior T cells expressi.pdf

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