Thymosin α1 Augments CD8⁺ T-Cell Activation and Reverses Exhaustion In Vitro

Thymosin alpha 1 (Tα1) is a thymic peptide hormone secreted by the thymus gland with known immunomodulatory properties, yet its specific effects on human CD8⁺ T-cell function remain incompletely understood. This study investigates the influence of Tα1 on CD8⁺ T-cell proliferation, activation, cytokine secretion, and exhaustion status in vitro.

Human CD8⁺ T-cells were cultured and treated under four conditions: untreated (negative control), CD3/CD28 stimulation (positive control), Tα1 treatment, and combined CD3/CD28 + Tα1 stimulation. Proliferation was measured using carboxyfluorescein succinimidyl ester (CFSE)-based flow cytometry. Surface expression of activation markers (CD69, CD25, HLA-DR) and exhaustion markers (PD-1, TIM-3, LAG-3) was analyzed by flow cytometry. Cytokine secretion (IL-2, IFN-γ, TNF-α, IL-10) was assessed using a multiplex bead-based assay. T-cell exhaustion was induced by repeated CD3/CD28 stimulation before Tα1 treatment.

Tα1 alone moderately increased proliferation and activation of CD8⁺ T-cells, while the combination of Tα1 and CD3/CD28 significantly enhanced the proliferation index and surface expression of CD69, CD25, and HLA-DR compared with individual treatments. Cytokine secretion of IL-2, IFN-γ, TNF-α, and IL-10 was elevated in the combination group, indicating enhanced effector function. In the exhaustion model, CD8⁺ T-cells exhibited overexpression of PD-1, TIM-3, and LAG-3, which was significantly reduced upon Tα1 treatment, suggesting a partial reversal of the exhausted phenotype.

Tα1 promotes functional activation of CD8⁺ T-cells and mitigates exhaustion marker expression following chronic stimulation. These findings suggest that Tα1 could potentially serve as a supportive agent in T-cell-based immunotherapies by enhancing activation and partially reversing exhaustion in vitro, warranting further in vivo validation.