Molecular and cellular characterization of European sea bass CD3 epsilon(+) T lymphocytes and their modulation by microalgal feed supplementation

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3 zeta, gamma delta, and epsilon chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3e chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3 epsilon expression levels increased in head kidney leukocytes, confirming that CD3 epsilon T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3e cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3 epsilon(+) lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3 epsilon(+) lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3 epsilon(+) T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3 epsilon(+) T cells are involved in nutritional immune responses.