Structural characteristics and mucosal immune response of the interbranchial lymphoid tissue in the gills of flounder (Paralichthys olivaceus).

A specialized lymphoepithelial tissue termed the interbranchial lymphoid tissue (ILT) is recently identified in several fish species. However, the structural variation and mucosal immune functions of the ILT remain largely unknown. In this study, the anti-Zap-70 MAb was firstly determined to specifically recognize ZAP-70 protein, and CD4-1, CD4-2 and CD8β T-cells, but not IgM B cells, in peripheral blood leucocytes of flounder (Paralichthys olivaceus). Then we found that aggregates of Zap-70 cells were located in the epithelium covering the bottom of the interbranchial cleft and along the afferent and efferent edges of the filaments in a cross view, where a meshwork of epithelial cells containing diffused lymphoid cells was exhibited, confirming these structures as the ILT; In a sagittal view, Zap-70 cells were situated at the base of the filaments (here named as proximal ILT, pILT) and in the interlamellar epithelium (named as distal ILT, dILT). Also, a few IgM B cells were distributed at these sites. The lymphoepithelium within pILT and dILT was very thin with a low number of Zap-70 cells in premetamorphosis and postclimax larvae of flounder, and got thicker containing much more Zap-70 cells in juvenile and adult individuals. The aggregates of CD4-1/Zap-70, CD4-2/Zap-70, and CD8β/Zap-70 T-cell subsets were identified in the ILT. Post bath vaccination with inactivated Edwardsiella tarda and then intraperitoneal injection of EdU, the amounts of EdU and Zap-70 cells obviously increased at 3 d and 7 d, and co-localization of EdU/Zap-70 cells identified the presence of proliferative T cells; meanwhile, MHC class II-expressing cells were increased. These findings indicated that the ILT in gills of flounder was an important site for the induction of local T cell-mediated immunity, which would lead to a better understanding of mucosal immunity and defense mechanisms of teleost fish.