In Vivo Dynamics of Memory CD8⁺ T Cell Activation Using a Fluorescent Timer Protein of TCR Signaling

Introduction/Rationale: T cell activation is a dynamic process governed by the strength and duration of antigen recognition via the T cell receptor (TCR). The timing of in vivo TCR signaling– when and how strongly memory CD8⁺ T cells become reactivated in the inflamed brain following antigen exposure– is a fundamental mechanism that remains unclear. The Nr4a3-Tocky reporter (Tocky) mouse employs a fluorescent timer that shifts from blue to red after TCR-driven Nr4a3 activation, which allows researchers to track activation kinetics through two parameters: the Timer Angle (recency of T cell activation), and the Timer Intensity (strength of signaling).

Methods: To understand CD8+ T cell activation during this neuroinflammation, we generated memory CD8+ T cells by intracranial Theiler’s murine encephalomyelitis virus infection of these Tocky mice. After viral clearance, we reactivated memory CD8+ T cells by intravenously injecting their cognate antigen. Tissues were collected 0-72 hours post-injection and profiled by flow cytometry. An R pipeline was constructed to transform event-level flow data to quantify the TCR activation. Data were analyzed with a Metropolis–Hastings MCMC algorithm to model activation dynamics and estimate probabilistic transitions between biologically defined states (newly activated, persistent, arrested states).

Results: These empirical transition probabilities enabled Bayesian comparison of antigen-specific versus nonspecific CD8+ T cells. It was revealed that antigen-specific CD8⁺ T cells rapidly transition to a high-intensity activation state within 24 hours and maintain persistent signaling compared to nonspecific populations.

Conclusion: This establishes a biologically grounded, in vivo framework for modeling and comparing TCR signaling during neuroinflammation.