Dynamic Mitochondrial Migratory Features Associated with Calcium Responses during T Cell Antigen Recognition.

: A T cell clone is able to distinguish Ags in the form of peptide-MHC complexes with high specificity and sensitivity; however, how subtle differences in peptide-MHC structures translate to distinct T cell effector functions remains unknown. We hypothesized that mitochondrial positioning and associated calcium responses play an important role in T cell Ag recognition. We engineered a microfluidic system to precisely manipulate and synchronize a large number of cell-cell pairing events, which provided simultaneous real-time signaling imaging and organelle tracking with temporal precision. In addition, we developed image-derived metrics to quantify calcium response and mitochondria movement. Using myelin proteolipid altered peptide ligands and a hybridoma T cell line derived from a mouse model of experimental autoimmune encephalomyelitis, we observed that Ag potency modulates calcium response at the single-cell level. We further developed a partial least squares regression model, which highlighted mitochondrial positioning as a strong predictor of calcium response. The model revealed T cell mitochondria sharply alter direction within minutes following exposure to agonist peptide Ag, changing from accumulation at the immunological synapse to retrograde movement toward the distal end of the T cell body. By quantifying mitochondria movement as a highly dynamic process with rapidly changing phases, our result reconciles conflicting prior reports of mitochondria positioning during T cell Ag recognition. We envision applying this pipeline of methodology to study cell interactions between other immune cell types to reveal important signaling phenomenon that is inaccessible because of data-limited experimental design.