Critical roles of CD4-1+ T cells and their secreted IL-21 in vaccine-induced humoral immunity in grass carp.

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 response. In conclusion, these results demonstrated the critical roles of CD4-1+ T cells and their secreted IL-21 in vaccine-induced humoral immunity in grass carp, providing a foundation for identifying Tfh-like cells in teleost fish and supporting the potential application of IL-21 as molecular adjuvant in fish vaccines.