Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

Abstract

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells.

Author Summary

The adaptive immune response to pathogen invasion requires the stimulation of lymphocytes by antigen-presenting cells. We hypothesized that investigating the dynamics of the T lymphocyte activation by monitoring intracellular calcium fluctuations might help explain the high specificity and selectivity of this phenomenon. However, the quantitative and exhaustive analysis of calcium fluctuations by video microscopy in the context of cell-to-cell contact is a tough challenge. To tackle this, we developed a complete solution named MAAACS (Methods for Automated and Accurate Analysis of Cell Signals), in order to automate the detection, cell tracking, raw data ordering and analysis of calcium signals. Our algorithm revealed that, when in contact with antigen-presenting cells, T lymphocytes generate oscillating calcium signals and not a massive and sustained calcium response as was originally thought. We anticipate our approach providing many more new insights into the molecular mechanisms triggering adaptive immunity.