Pilot In Vitro Study of Dexamethasone-Induced Immunomodulation in Murine Splenic T Cells

Abstract

Glucocorticoids (GCs) are key hormones in stress response and chronic elevations from chronic stress or prolonged therapeutic use accelerates immune aging and dysfunction. To dissect their direct, time- and dose-dependent effects on T cells, we established an in vitro model in which murine splenocytes were cultured with graded concentrations of dexamethasone (a potent, synthetic GC) and analyzed by 22-color spectral flow cytometry over 24, 48, and 72 hours. Dexamethasone caused a progressive, dose-dependent decline in overall viability and absolute CD4⁺/CD8⁺ T cell counts, transiently increased proliferation (Ki-67) at 48 hours before inducing collapse and apoptosis, and modulated activation (CD69, CD25) and exhaustion (PD-1) markers. Memory and naïve T cell subsets were especially vulnerable, whereas low-dose dexamethasone selectively enriched regulatory (CD4⁺Foxp3⁺) and Th1 (CD4⁺Tbet⁺) subsets. Effector cytokine production (IFN-γ, TNF-α) was markedly suppressed in a dose- and time-dependent manner. This platform thus provides a robust tool for exploring GC-driven immunosenescence, GC resistance mechanisms, and strategies to restore T cell function after chronic GC exposure.