Autofluorescence lifetime imaging classifies human B and NK cell activation state

Jeremiah M Riendeau ^1,2 Rebecca L Schmitz ¹ Kelsey E Tweed ^1,2 Peter Rehani ¹ Kayvan Samimi ¹ Dan Pham ^1,2 Isabel Jones ¹ Elizabeth M Maly ¹ Emmanuel Contreras Guzman ¹ Matthew H Forsberg ³ Ankita Shahi ³ Lucia Hockerman ¹ Jose M Ayuso ^2,4 Christian M Capitini ^3,4 Alex Walsh ¹ Melissa C Skala ^1,2,4*

• ¹ Morgridge Institute for Research, Madison, Wisconsin, United States
• ² Department of Biomedical Engineering, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
• ³ Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States
• ⁴ Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States

New non-destructive tools with single-cell resolution are needed to reliably assess B cell and NK cell function for applications including adoptive cell therapy and immune profiling. Optical metabolic imaging (OMI) is a label-free method that measures the autofluorescence intensity and lifetime of the metabolic cofactors NAD(P)H and FAD to quantify metabolism at a single-cell level. Here, we demonstrate that OMI can resolve metabolic changes between primary human quiescent and IL-4/anti-CD40 activated B cells and between quiescent and IL-12/IL-15/IL-18 activated NK cells. We found that stimulated B and NK cells had an increased proportion of free compared to protein-bound NAD(P)H, a reduced redox state, and produced more lactate compared to control cells. The NAD(P)H mean fluorescence lifetime decreased in the stimulated B and NK cells compared to control cells. Random forest models classified B cells and NK cells according to activation state (CD69+) based on OMI variables with an accuracy of 93%. Our results show that autofluorescence lifetime imaging can accurately assess B and NK cell activation in a label-free, non-destructive manner.