Time-domain single photon-excited autofluorescence lifetime for label-free detection of T cell activation

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique, capable of label-free assessment of the metabolic state and function within single cells. The FLIM measurements of autofluorescence were recently shown to be sensitive to the functional state and subtype of T cells. Therefore, autofluorescence FLIM could improve cell manufacturing technologies for adoptive immunotherapy, which currently require a time-intensive process of cell labeling with fluorescent antibodies. However, current autofluorescence FLIM implementations are typically too slow, bulky, and prohibitively expensive for use in cell manufacturing pipelines. Here we report a single photon-excited confocal whole-cell autofluorescence system that uses fast field-programmable gate array-based time tagging electronics to achieve time-correlated single photon counting (TCSPC) of single-cell autofluorescence. The system includes simultaneous near-infrared bright-field imaging and is sensitive to variations in the fluorescence decay profile of the metabolic coenzyme NAD(P)H in human T cells due to the activation state. The classification of activated and quiescent T cells achieved high accuracy and precision (area under the receiver operating characteristic curve, AUC = 0.92). The lower-cost, higher acquisition speed, and resistance to pile-up effects at high photon flux compared to traditional multiphoton-excited FLIM and TCSPC implementations with similar SNR make this system attractive for integration into flow cytometry, sorting, and quality control in cell manufacturing.

Inflammatory CD4/CD8 double-positive human T cells arise from reactive CD8 T cells and are sufficient to mediate GVHD pathology

“…6, A to E). This system is able to detect the autofluorescence of NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) and FAD (flavin adenine dinucleotide) at a single-cell resolution and use their ratios to determine the optical redox ratio (ORR) of the cell population as a measurement of metabolic activity ( 61 , 62 ). This analysis revealed that the DPT population had elevated ORR, indicative of high metabolic activity (Fig.…”

Label free metabolic imaging to enhance the efficacy of Chimeric Antigen Receptor T cell therapy

“…Further research should explore the effects of media composition and cytokines on CAR T cells with an intact TCR and validate the relationship between OMI measurements and in vivo potency across various CAR T cell and tumor models. Additionally, the current two-photon laser scanning OMI system can be adapted into other configurations such as a single-photon microscope, microfluidic devices ( 51 ), or flow cytometer ( 52, 53 ) to achieve faster speed, automation, and a smaller footprint to better integrate into CAR T manufacturing workflows. These technical efforts will expand the throughput and scope of OMI as a non-invasive, sensitive tool to support the translation of CAR T cell therapy for solid tumors.…”

Label free metabolic imaging to enhance the efficacy of chimeric antigen receptor T cell therapy

“…Further research should explore the effects of media composition and cytokines on CAR T cells with an intact TCR and validate the relationship between OMI measurements and in vivo potency across various CAR T cell and tumor models. Additionally, the current two-photon laser scanning OMI system can be adapted into other configurations such as a flow cytometer, microfluidic devices (63), or single-photon microscope (64,65) to achieve faster speed, automation, and a smaller footprint to better integrate into CAR T manufacturing workflows. These technical efforts will expand the throughput and scope of OMI as a noninvasive, sensitive tool to support the translation of CAR T cell therapy for solid tumors.…”