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New non-destructive tools are needed to reliably assess lymphocyte function for immune profiling and adoptive cell therapy. Optical metabolic imaging (OMI) is a label-free method that measures the autofluorescence intensity and lifetime of metabolic cofactors NAD(P)H and FAD to quantify metabolism at a single-cell level. Here, we investigate whether OMI can resolve metabolic changes between human quiescent versus IL4/CD40 activated B cells and IL12/IL15/IL18 activated memory-like NK cells. We found that quiescent B and NK cells were more oxidized compared to activated cells. Additionally, the NAD(P)H mean fluorescence lifetime decreased and the fraction of unbound NAD(P)H increased in the activated B and NK cells compared to quiescent cells. Machine learning classified B cells and NK cells according to activation state (CD69+) based on OMI parameters with up to 93.4% and 92.6% accuracy, respectively. Leveraging our previously published OMI data from activated and quiescent T cells, we found that the NAD(P)H mean fluorescence lifetime increased in NK cells compared to T cells, and further increased in B cells compared to NK cells. Random forest models based on OMI classified lymphocytes according to subtype (B, NK, T cell) with 97.8% accuracy, and according to activation state (quiescent or activated) and subtype (B, NK, T cell) with 90.0% accuracy. Our results show that autofluorescence lifetime imaging can accurately assess lymphocyte activation and subtype in a label-free, non-destructive manner.

Teaser Label-free optical imaging can assess the metabolic state of lymphocytes on a single-cell level in a touch-free system.

Competing Interest Statement

RLS, KS, ECG, AJW, and MCS are inventors on patent applications related to this work filed by Wisconsin Alumni Research Foundation (WO2020047133A1, filed on 2019-08-28; US20210049346A1, filed on 2020-08-13; US20210354143A1, filed on 2021-05-17). CMC receives honorarium for advisory board membership with Bayer, Elephas Bio, Nektar Therapeutics, Novartis and WiCell, who had no input in the study design, analysis, manuscript preparation or decision to submit for publication. All other authors declare they have no competing interests.