Regulations of Gata-3 on CD4+ T cell differentiation via its zinc finger domains in flounder (Paralichthys olivaceus).

CD4+ T cells are central to adaptive immunity, with GATA binding protein 3 (Gata-3) acting as a key transcription factor for Th subset differentiation. This study aimed to characterize Gata-3 in flounder (Paralichthys olivaceus) and investigate its role in CD4+ T cell differentiation via its zinc finger domains (ZnFs). Flounder Gata-3 contains two conserved C4-type ZnFs and showed peak expression in gills. Immunofluorescence assays suggested that Gata-3 located in gill leukocytes, co-localized with CD4-1+/CD4-2+ cells but not CD8+ or IgM+ cells. Under in vitro Th2 differentiation, the proportion of CD4+ cells increased from 15.77 ± 2.59% to 21.13 ± 1.92%. Concurrently, gata-3, jak3, stat6, il-10, and c-maf were significantly upregulated, while Th1-related genes (e.g., t-bet, ifn-γ) were significantly downregulated. Overexpression of Gata-3 in 293T cells significantly inhibited the promoters of t-bet and ifn-γ. Meanwhile, a recombinant plasmid of Gata-3 without the ZnFs was constructed and transfected into HINAE cells; this experiment that only gata-3 was significantly upregulated, while no significant changes were observed in the expression of t-bet, ifn-γ, stat4, or il-12rβ. Furthermore, two mutants of Gata-3 lacking the C- or N-terminal ZnF were constructed, but only the mutant lacking the C-terminal ZnF exhibited no inhibition on t-bet and ifn-γ promoters. In conclusion, Gata-3 promotes Th2 differentiation while inhibiting the Th1 pathway in flounder by directly repressing t-bet and ifn-γ, a process critically mediated by its C-terminal ZnF. This study provides new insights into the evolutionary conservation of T cell immune regulation in teleosts.