Abstract

CD4-1 + T cells serve as the main source of IL-21 upon stimulation with inactivated A. hydrophila. • Co-immunization with IL-21 and inactivated A. hydrophila vaccine improves antibacterial protection, demonstrating its function as a molecular adjuvant in fish. • CD4-1 + T cell depletion significantly reduces vaccine-elicited specific IgM and compromises immune protection, confirming their essential helper role. • IL-21 partially rescues survival in CD4-1 + T cell-depleted fish but cannot restore antibody levels, indicating that full humoral responses require CD4-1 + T cells. Although CD4 + T cells are known to play a crucial helper role in mammalian humoral immunity, the function of early-evolved CD4-1 + T cells in teleost fish for assisting B cell-mediated antibody production remain to be elucidated. Using grass carp ( Ctenopharyngodon idella ) as a model, this study demonstrated that CD4-1 + T cells play a critical role in vaccine-induced antibody production and antibacterial immunity. We confirmed that inactivated Aeromonas hydrophila vaccine significantly induced interleukin-21 (IL-21) expression in grass carp head kidney leukocytes (HKLs), and identified CD4-1 + T cells as the primary cellular source of IL-21. In vitro experiments showed that IL-21 effectively promoted B cell differentiation into plasma cells and enhanced IgM secretion, which synergized with A. hydrophila to exert a more pronounced effect. Meanwhile, our in vivo studies demonstrated that IL-21 possess molecular adjuvant function when co-immunized with A. hydrophila . Depletion of CD4-1 + T cells reduced A. hydrophila -elicited specific IgM levels and antibacterial immune protection, confirming their essential helper function. Injection of IL-21 into CD4-1 + T cell depletion fish can partially improve the survival rate but could not restore antibody levels, indicating that while IL-21 has direct immunoenhancing effects, CD4-1 + T cells are essential for a full antibody