Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors
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.
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.
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.
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
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。