Generation and application of monoclonal antibodies against CD4-1 and CD8α for characterizing T cell subsets in grass carp (Ctenopharyngodon idella).

Mature T cells are functionally specialized through differential expression of CD4 or CD8, with CD4+ cells designated as helper T cells (Th cells) and CD8+ cells as cytotoxic T lymphocytes (CTLs). However, due to the lack of antibody tools, the functional mechanisms of T cell subsets in teleost remain poorly understood. In this study, we established NIH/3T3 cell lines expressing grass carp (Ctenopharyngodon idella) CD4-1 and CD8α through retroviral transduction, and used these as immunogens to generate monoclonal antibodies (mAbs) targeting the T cell co-receptors. Characterization results demonstrated that our mAbs exhibited high specificity in accurately identifying grass carp CD4-1+ and CD8α+ T cells. Subsequent applications revealed that CD4-1+ and CD8α+ T cells were widely distributed in immune-related tissues including spleen, head kidney, peripheral blood, and liver. Upon PHA stimulation, these T cell subsets showed enhanced phosphorylation levels of NF-κB p65 and MAPK/ERK signaling molecules. Furthermore, the proportion of CD4-1+ and CD8α+ T cells increased significantly in grass carp following infection with Edwardsiella piscicida. Overall, the mAbs developed in this study provide reliable tools for characterizing grass carp CD4-1+ and CD8α+ T cells and investigating their functions. These findings offer both technical support and theoretical foundations for understanding teleost T cell subset functionality and the evolution of adaptive immunity.