Characterization of CD4-2+cell subsets and their responses in pearl gentian grouper (Epinephelus fuscoguttatus x E. lanceolatus)

CD4+ T cells (T helper cells, Th) are essential for triggering and maintaining both innate and adaptive immunity. Due to the variability in the molecular number, structure, and expression of the CD4 molecule in teleost fish and mammals, the subpopulation and differentiation of CD4+ T cells in fish is currently unclear. Here, we identified a CD4-2+ T cell subsets in the pearl gentian grouper (Epinephelus fuscoguttatus x E. lanceolatus), and preliminarily investigated its immune response dynamics. The CD4-2 sequence is highly homology in grouper species, CD4-2+ cells were characterized by a polyclonal antibody (Anti-CD4-2 Abs), CD4-2 Abs precisely examined recombinant protein and lysates natural protein, demonstrating the specific recognition of CD4-2 Abs. By using Abs specific for CD4-2, ZAP-70 and IgM, it was possible to observe the CD4-2+ cells, CD4-2+ ZAP-70+ cells and CD4-2+/ IgM+ in spleen and head kidney leukocytes. Cross-reactivity assay with other grouper species, CD4-2 Abs could recognize the leukocytes from all tested groupers. Flow cytometric analysis showed that both CD4-2+ cells and IgM+ cells could phagocytose fluorescent beads or FITC-labeled inactivated E. tarda, and that CD4-2+ cells were weaker than IgM+ cells. After immunization of inactivated-nervous necrosis virus (NNV), CD4-2+ cells give a significantly response on the third day, with a sudden decrease in cell proportions. Accordingly, these findings will contribute to a greater understanding of CD4-2+ cell function and immune responses. To our knowledge, this is the first demonstration of the presence of phagocytic CD4-2+ cell in teleost fish.