3D live imaging and phenotyping of CAR-T cell mediated-cytotoxicity using high-throughput Bessel oblique plane microscopy

Zhaofei Wang, Jie Wang, Yuxuan Zhao, Jin Jin, Wentian Si, Longbiao Chen, Man Zhang, Yao Zhou, Shiqi Mao, Chunhong Zheng, Yicheng Zhang, Liting Chen () and Peng Fei ()
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Zhaofei Wang: Huazhong University of Science and Technology
Jie Wang: Huazhong University of Science and Technology
Yuxuan Zhao: Huazhong University of Science and Technology
Jin Jin: Huazhong University of Science and Technology
Wentian Si: Huazhong University of Science and Technology
Longbiao Chen: Huazhong University of Science and Technology
Man Zhang: Huazhong University of Science and Technology
Yao Zhou: Huazhong University of Science and Technology
Shiqi Mao: Huazhong University of Science and Technology
Chunhong Zheng: Peking University
Yicheng Zhang: Huazhong University of Science and Technology
Liting Chen: Huazhong University of Science and Technology
Peng Fei: Huazhong University of Science and Technology

Nature Communications, 2024, vol. 15, issue 1, 1-11

Abstract: Abstract Clarification of the cytotoxic function of T cells is crucial for understanding human immune responses and immunotherapy procedures. Here, we report a high-throughput Bessel oblique plane microscopy (HBOPM) platform capable of 3D live imaging and phenotyping of chimeric antigen receptor (CAR)-modified T-cell cytotoxicity against cancer cells. The HBOPM platform has the following characteristics: an isotropic subcellular resolution of 320 nm, large-scale scouting over 400 interacting cell pairs, long-term observation across 5 hours, and quantitative analysis of the Terabyte-scale 3D, multichannel, time-lapse image datasets. Using this advanced microscopy platform, several key subcellular events in CAR-T cells are captured and comprehensively analyzed; these events include the instantaneous formation of immune synapses and the sustained changes in the microtubing morphology. Furthermore, we identify the actin retrograde flow speed, the actin depletion coefficient, the microtubule polarization and the contact area of the CAR-T/target cell conjugates as essential parameters strongly correlated with CAR-T-cell cytotoxic function. Our approach will be useful for establishing criteria for quantifying T-cell function in individual patients for all T-cell-based immunotherapies.

Date: 2024
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DOI: 10.1038/s41467-024-51039-9

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