IL-21 controls CD8 ⁺ T cell immunity and antibacterial defense in fish via STAT3 signaling

CD8 ⁺ T cells are indispensable effectors of adaptive immunity. While cytokine-mediated regulation of CD8 ⁺ T cell responses has been extensively characterized in mammals, the underlying mechanisms in fish remain largely unexplored. Here, we identified and characterized an interleukin-21 (IL-21) gene from Nile tilapia ( Oreochromis niloticus). In vitro stimulation with IL-21 significantly upregulated the transcription and expression of key CD8 ⁺ T cell effector molecules, including Granzyme B and interferon-gamma (IFN-γ), in splenic leukocytes, indicating a crucial role of IL-21 in modulating CD8 ⁺ T cell function in teleosts. Using a monoclonal antibody (mAb) against Nile tilapia IL-21, we further demonstrated that activated CD4-1 ⁺ T cells are the primary producers of this cytokine in fish. Intraperitoneal injection of recombinant IL-21 into Edwardsiella piscicida-infected Nile tilapia enhanced both proliferation and apoptosis of CD8 ⁺ T cells. Moreover, IL-21 elevated the expression of Granzyme B, IFN-γ, and IL-2, and boosted CD8 ⁺ T cell cytotoxicity, thereby strengthening their effector functions. In contrast, blockade of IL-21 signaling with the mAb severely impaired CD8 ⁺ T cell responses. Mechanistically, Nile tilapia IL-21 strongly induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), which was required for IL-21-mediated CD8 ⁺ T cell proliferation in vitro. Inhibition of STAT3 phosphorylation in vivo attenuated CD8 ⁺ T cell responses and antibacterial immunity, underscoring its functional importance. Collectively, our findings establish IL-21 as a pivotal regulator of CD8 ⁺ T cell immunity in fish and provide evolutionary insight into conserved cytokine pathways shaping adaptive immune responses.