2024 AIChE Annual Meeting
Metabolic Kinetics of T Cell Activation with Soluble Antibodies and Antibody-Coated Magnetic Beads
Human T cell activation is a crucial area of study as it provides insights into the adaptive immune response and T cell cytotoxicity. T cell activation requires rapid changes in T cell metabolism to support proliferation and effector functions. This study investigates differences in T cell metabolism kinetics between two activation methods: antibodies (CD3/CD28) that are soluble vs. coated onto magnetic beads (Dynabeads). The Dynabeads are hypothesized to more closely mimic the mechanical features of antigen-presenting cells. Studies were performed in human plasma like medium (HPLM), which is designed to mimic the metabolic profile of human plasma. First, we collected a sample of healthy human blood and isolated the CD3+ T cell population. T cell metabolism was imaged using two-photon fluorescence lifetime imaging microscopy (FLIM) of an autofluorescent metabolic co-enzyme before activation and over three days post-activation. Specifically, FLIM measured the fluorescence lifetime of the reduced form of nicotinamide adenine dinucleotide (phosphate), or NAD(P)H, a crucial metabolic coenzyme in cells. NAD(P)H exists in two conformations: a free form and a protein-bound form, the bound conformation of NAD(P)H has a longer lifetime than the free conformation. Therefore, NAD(P)H FLIM measures T cell metabolism related to NAD(P)H protein-binding activity. Since NAD(P)H is autofluorescent, this technique is a label-free method to probe cellular behavior. Following baseline imaging, the T cells were divided into two groups: one activated with the soluble antibodies and the other activated with the Dynabeads. Single cell segmentation was performed using Cellpose to generate masks, followed by automatic manual corrections using Napari. SPCImage was used to extract the lifetime decay parameters including the short and long lifetime and their relative weights. The mean lifetime was also calculated, which is the weighted average of the short and long lifetime. The mean lifetime of NAD(P)H decreased progressively each day following activation, indicating decreases in the proportion of bound NAD(P)H with respect to free NAD(P)H, for both soluble antibody and Dynabead groups. This suggests that activation in HPLM with Dynabeads and soluble antibodies produce similar changes in T cell metabolism over three days. Future research should explore whether these trends persist with different cell culture mediums to determine if Dynabeads and soluble antibody activation results in similar metabolic changes across other conditions.