Autofluorescence Imaging of T cell Activation

Abstract

Abstract T cells can have different activities based on receptor expression and cytokine production. Current methods to classify and assess immune cell behavior include flow cytometry and immunohistochemistry, which require immune cell labeling and tissue fixation. A non-invasive method for determining T cell behavior is needed to study immune cell behaviors in tumors and evaluate novel immunotherapies. Activated T cells require high rates of glycolysis to maintain immune activities. Therefore, we are developing optical metabolic imaging (OMI) to assess the metabolic profile of T cell subtypes and activation states using cells isolated from human blood. OMI probes the fluorescence intensity and lifetime of the metabolic coenzymes NAD(P)H and FAD, to quantitate the redox state of the cell through the optical redox ratio (NAD(P)H fluorescence intensity divided by the sum of NAD(P)H and FAD fluorescence intensity) and co-enzyme binding. Our results show that the optical redox ratio is increased in activated populations of unsorted T cells, and of CD8+ T cells, consistently across four different donors. Single-cell analysis of the unsorted, unactivated T cell populations revealed a small portion of cells in an activated state. Inter-donor heterogeneity highlights the variability of immune responses between patients. These results indicate that OMI is a powerful tool for assessing T cell subtype and behavior. OMI utilizes the autofluorescent properties of NAD(P)H and FAD, and thus is contrast agent free, non-damaging, and requires no genetic manipulation. Therefore, OMI can be used to image T cell interactions with tumors in time-course studies of tumor development or assess the efficacy of immunotherapy.