Development of a CD3ε mAb that can identify and in vitro activate T cells in large yellow croaker (Larimichthys crocea)

As a hallmark molecule of T cells, CD3ε forms a complex with T-cell receptor (TCR) to transduce antigen signals and drive T cell activation, playing a pivotal role in T cell-mediated immune response. However, the lack of specific monoclonal antibodies (mAbs) targeting CD3ε in fish has substantially impeded the study on adaptive immunity in these species. In this study, we constructed NIH/3T3 cells expressing CD3ε of the large yellow croaker (Larimichthys crocea) via retroviral transduction. Using these cells as immunogen to immune mice, we generated a mAb that specifically recognized a population of spleen leukocytes after cell fusion and screening. This identified population specifically expressed CD3ε, CD4-1 or CD8α, suggesting that it was T cell. Moreover, immunofluorescence demonstrated that the mAb could bind to the surface of some leukocytes, and it was detected as the IgG1 type. These results confirmed the specificity of this CD3ε mAb and its applicability to identify T cells in the large yellow croaker. Subsequently, we revealed the widespread distribution of CD3ε⁺ T cells in immune-related tissues including spleen, liver, head kidney, gill and peripheral blood by using this mAb. Upon PHA stimulation, the phosphorylation of mTORC1 and MAPK/ERK were enhanced in CD3ε⁺ T cells. More importantly, this CD3ε mAb could mimic antigenic signaling to induce T cell activation in vitro, since its treatment activated the mTORC1, MAPK/ERK and Ca²⁺ pathways which were crucial for T cell activation. Therefore, we generated a CD3ε mAb, which could not only identify but also in vitro activate T cells of the large yellow croaker, providing critical tools for investigating T-cell immune in teleost.