Computational and Microfluidic Platforms to Investigate the Role of Ca²⁺ and ROS in T Cell Activation with Single-Cell Resolution

Abstract:

As a key component of the adaptive immune response, T cell lymphocytes are widely studied but often difficult to isolate and visualize for experimentation with single-cell resolution. Intracellular signaling upon activation of the T cell receptor is necessary for proper immune function. The resulting cytosolic calcium (Ca²⁺) concentration has been shown to oscillate, differentially encoding downstream transcription factors. Additionally, activation also requires the concurrent signaling of hydrogen peroxide (H₂O₂), with implications on protein and channel functions between these signaling networks. However, the direct mechanisms and connections are difficult to analyze due to the fast, dynamic signaling and subcellular localization. Frequency response analysis, originally developed in control engineering for discerning complex, interconnected networks that are difficult to interrogate with bulk measurements, has been shown to be useful for analyzing biological systems and helps with identifying dominant interactions within the network. We utilize this technique to probe intracellular Ca²⁺ dynamics in the frequency domain to better investigate the relationship between H₂O₂ and Ca²⁺. To enable single-cell studies of intracellular T cell signaling dynamics using frequency response analysis, we developed complementary computational and microfluidic tools necessary for single-cell trapping, stimulation, imaging, and analysis. This novel platform provides a systematic approach for analyzing T cell signaling in the frequency domain and is applicable for assessing many biological questions. 

Stimulation with oscillatory H₂O₂ solutions identified specific input frequencies that facilitate entrainment of Ca²⁺ signaling and we observe heterogeneous responses within the population, illustrating the necessity of single-cell analysis to understand the realm of potential responses. Jurkat T cells were found to respond robustly to input oscillations of 2.78 mHz frequency, corresponding to a period of 6 minutes. We extended this analysis by switching the input and output signals such that cells were exposed to oscillatory Ca²⁺ solutions and localized intracellular H₂O₂ was measured using two variants of the reporter protein, HyPer. Ca²⁺ stimulated H₂O₂ dynamics vary depending on location of the reporter within the cell and this difference in signaling dynamics suggests altered regulatory mechanisms for Ca²⁺-H₂O₂ crosstalk dependent on subcellular localization. We also report the first investigation of the downstream transcriptional response using smFISH analysis following oscillatory stimulation with cytoplasmic Ca²⁺signaling. These findings uphold our previous natural frequency result of approximately 2.78 mHz as our smFISH response was maximal at this frequency, connecting the functional consequence with upstream frequency-based signaling. 

In summary, this thesis developed experimental and computational techniques to robustly deliver oscillatory stimulation to cells, monitor the response of various reporters, and analyze dynamic single-cell traces, highlighting a previously unexplored domain of Ca²⁺ signaling in T cell lymphocytes.