A monoclonal antibody against CD3ε enables characterization of T cell activation and metabolic reprogramming in Chinese seabass (Lateolabrax maculatus).

Abstract

T cells are essential components of adaptive immunity, while CD3ε, a key subunit of the T-cell receptor (TCR)-CD3 complex, serves as an important marker for T-cell identification and plays a critical role in TCR-mediated activation. However, the scarcity of monoclonal antibody (mAb) against CD3ε from teleosts has hindered the characterization of T cells in these species. In this study, mAbs against CD3ε of Chinese seabass (Lateolabrax maculatus), an economically important species in marine aquaculture, were developed and characterized. The coding sequence of CD3ε from L. maculatus (LmCD3ε) was cloned and expressed using a retroviral expression system to generate LmCD3ε-expressing cells for antibody development. Following mouse immunization and hybridoma fusion, the mAb secreted by hybridoma 1F5D9 was identified and its specificity for CD3ε+ T cells in Chinese seabass was validated by flow cytometry and semi-quantitative assay. Using this anti-LmCD3ε mAb, CD3ε+ T cells were detected in immune-related tissues, including the spleen, head kidney, liver, gills, and peripheral blood. Upon stimulation with phorbol 12-myristate 13-acetate and ionomycin (P+I), CD3ε+ T cells exhibited increased phosphorylation of ERK1/2 and NF-κB p65, demonstrating that anti-LmCD3ε mAb enables the identification and functional characterization of T cells in Chinese seabass. Furthermore, stimulation with anti-LmCD3ε mAb induced T-cell activation, accompanied by increased phosphorylation of ERK1/2 and mTOR, demonstrating its potential as a stimulatory reagent for investigating T-cell signaling in Chinese seabass. Additionally, this activation was also accompanied by glycolysis-associated metabolic changes, including increased expression of the glycolytic enzymes HKII and PKM, enhanced glucose uptake in CD3ε+ T cells, and elevated intracellular pyruvate levels. Collectively, these findings provide a useful immunological tool for T-cell identification and functional analysis in Chinese seabass and contribute to a better understanding of T-cell activation and metabolic regulation in teleost.