Dengue virus rNS3pro immunization is associated with the expansion of regulatory-like T-cell phenotypes in BALB/c mice.

Dengue virus (DENV) nonstructural protein 3 (NS3) is essential for viral replication and host immune evasion. While its protease domain (NS3pro) has been linked to T-cell anergy, its role in inducing regulatory T cells (Tregs) requires further exploration. To analyze the induction of diverse regulatory T-cell populations in BALB/c mice immunized with recombinant rNS3pro proteins from the four DENV serotypes. Mice were immunized with rNS3pro, and splenocytes were stimulated in vitro with homologous proteins. Cytokine expression and T-cell phenotypes-including markers for activation, exhaustion, and cytotoxicity-were analyzed via RT-PCR, ELISA, and flow cytometry. rNS3pro immunization induced a predominantly regulatory-associated response characterized by significant IL-10 expression. A marked expansion of CD4+CD25+ T cells (23.5% vs. 1.05% in controls) was observed, encompassing CD25+FoxP3+ regulatory-like T cells, alongside FoxP3-independent subsets expressing CD25+IL-10+ and CD25+TGF-β+. These cells co-expressed cytotoxicity-associated (FoxP3+Perforin+/GzB+) and co-inhibitory (CTLA-4+, PD-1+) markers. Effector CD8+ T-cell proliferation was significantly inhibited and could not be restored by exogenous IL-2. In conclusion, DENV rNS3pro immunization drives a robust expansion of multifaceted regulatory-like T-cell phenotypes. This immunomodulatory response correlates with compromised CD8+ effector proliferation, identifying the NS3 protease domain as a potential player in DENV immune evasion strategies.