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

Abstract

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.

Time-Resolved Single-Cell Assay for Measuring Intracellular Reactive Oxygen Species upon Exposure to Ambient Particulate Matter

“…Microfluidic Device Design and Fabrication. The design of the microfluidic device is shown in Figure 1 (an AutoCAD drawing of the design and images of the real device can be seen in Figure S1) and a detailed description is provided in He et al 31 Briefly, the device consists of four cell trap arrays (Figure 1a), where each cell trap array contains one main channel connected head-to-end with repetitive cell-trapand-chamber units in between two adjacent channels. Each cell-trap-and-chamber unit (Figure 1a Note that the abovementioned device dimensions are optimized from He et al 31 in order to obtain a better capture performance for the cell line used in this study.…”

“…A cell suspension (1 × 10 7 cells/mL) was then loaded into the front cell traps of the device by a gravity-driven flow. The cell loading mechanism has been provided in He et al 31 By gently pushing a syringe connected to the inlet manually, a pressure was applied through culture media for a few seconds to push cells into the cell chambers. The cells were cultured overnight by supplying cell culture media at a low flow rate of ∼0.1 μL/ min.…”

“…The design of the microfluidic device is shown in Figure (an AutoCAD drawing of the design and images of the real device can be seen in Figure S1) and a detailed description is provided in He et al Briefly, the device consists of four cell trap arrays (Figure a), where each cell trap array contains one main channel connected head-to-end with repetitive cell-trap-and-chamber units in between two adjacent channels. Each cell-trap-and-chamber unit (Figure a) consists of a front cell trap ( L : 15 × W : 15 × H : 20 μm), a front restriction ( L : 10 × W : 10 × H : 7 μm), a cell chamber ( L : 20 × W : 40 × H : 20 μm), and two back restrictions ( L : 10 × W : 20 × H : 4 μm).…”

“…Each cell-trap-and-chamber unit (Figure a) consists of a front cell trap ( L : 15 × W : 15 × H : 20 μm), a front restriction ( L : 10 × W : 10 × H : 7 μm), a cell chamber ( L : 20 × W : 40 × H : 20 μm), and two back restrictions ( L : 10 × W : 20 × H : 4 μm). Note that the abovementioned device dimensions are optimized from He et al in order to obtain a better capture performance for the cell line used in this study.…”

The DISC1–Girdin complex – a missing link in signaling to the T cell cytoskeleton

“…1B). These organelles were subsequently identified as mitochondria, which are known to accumulate at the immunological synapse in a microtubuledependent manner (He et al, 2019;Maccari et al, 2016;Quintana et al, 2007). Isoform L concentrated around the MTOC in unstimulated Jurkat cells but also became localized to the synapse after stimulation with SEE-coated Raji cells (Fig.…”