Phenotypic and functional characterization of CD4-2⁺ T cells in large yellow croaker Larimichthys crocea

CD4⁺ T cells, also known as helper T (Th) cells, are crucial regulators of the adaptive immune system, orchestrating and coordinating immune responses that protect the host against a broad spectrum of pathogenic threats. Two CD4 co-receptor genes, cd4-1 and cd4-2, have been identified in teleost species, including the large yellow croaker Larimichthys crocea. In this study, we developed a monoclonal antibody (mAb) that specifically recognizes croaker CD4-2, enabling the identification and characterization of CD4-2⁺ T lymphocytes. Croaker CD4-2⁺ T cells were found predominantly distributed across both systemic and mucosal immune tissues, with the exception of peripheral blood, and were notably more abundant than IgM⁺ B cells in the liver, gut, head kidney (HK) and gills. Moreover, elevated transcription levels of ifng, il2, il17a/f3, and il6 in sorted HK CD4-2⁺ T cells suggest their potential for functional differentiation into distinct Th lineages. Functional studies demonstrated that CD4-2⁺ T cells in the HK and spleen exhibited robust proliferative responses upon in vitro stimulation with T cell mitogens, including Phytohemagglutinin-L and Concanavalin A. More importantly, following infection with the protozoan parasite Cryptocaryon irritans, pronounced proliferations of CD4-2⁺ T cells were observed in the HK, spleen and gills, whereas the cellular expansion and activation were detected exclusively in the spleen, underscoring the pivotal role of the spleen in the induction and activation of CD4-2⁺ T cells in large yellow croaker. This study provides an in-depth characterization of CD4-2⁺ T cells in large yellow croaker and highlights their critical role in immune activation and anti-parasitic defense, offering valuable insights into their potential applications in immunological research and disease control in aquaculture.