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.
背景
嵌合抗原受体T细胞在肿瘤的治疗中，特别是在恶性B淋巴瘤的治疗中显示出令人兴奋的疗效。然而由于缺乏可显著预测抗瘤功能的标准，因此CAR-T的生产优化仍处于探索阶段。通过密切监测CAR-T细胞激活时的动态变化，可帮助我们理解T细胞的生物学特点并发现那些可以用于优化生产高效CAR-T细胞的潜在点。
方式
表达第二代anti-CD19的CAR分子的原代人类T细胞，被用于监测CAR-T在被抗原激活后表型的变化以及功能性的应答。多色流式细胞仪被用于定量监测CAR-T细胞在响应抗原时的增殖和存活力的动态变化，以及与与激活和耗竭有关的标记物的表达。
结果
经抗原激活的CAR-T细胞最终分化成两个不同的亚群，但只有CARhi/ CD25 +亚群表达出抗肿瘤的作用，中枢记忆T细胞被认为是生产功能优越T细胞的关键点。我们进一步证明CARhi/ CD25 +亚群会上调的PD-1的表达，但是会耐受PD-L1引起的免疫抑制。
结论：
CAR-T细胞在体外扩增经历了动态的表型改变，并产生了两个具有不同功能的细胞亚群。显著的CD25和CAR的表达量的上升预示着这些经抗体激活的T细胞将具备显著的抗肿瘤活性，与此同时也伴随着PD-1表达量的上升。这些具备优越功能的亚群细胞数量可通过调节抗原刺激和中枢记忆T细胞富集被选择性地扩大。
