Characterization of CD3+ T lymphocytes of Japanese flounder (Paralichthys olivaceus) and its response after immunization with formalin-inactivated Edwardsiella tarda

- The native CD3ε and CD3ε+ T lymphocytes were identified by anti-rCD3ε polyclonal antibodies.
- The tissues distribution of CD3+ T lymphocytes in Japanese flounder was determined.
- The response of CD3+ T lymphocytes against inactivated E. Tarda in Japanese flounder was investigated.

The CD3 complex is an important cell surface marker of T lymphocytes and essential for T lymphocytes activation in higher vertebrates. In the present work, the CD3ε of Japanese flounder (Paralichthys olivaceus) was recombinantly expressed in E. coli BL21 (DE3) and used as an immunogen to produce mouse anti-rCD3ε polyclonal antibodies, which could specifically recognize a 20 kDa protein in the membrane proteins of peripheral blood lymphocytes (PBL) of Japanese flounder by co-immunoprecipitation assay. Mass spectrometric analysis showed the 20 kDa protein was the native CD3ε of Japanese flounder. Both the flow cytometric analysis and double immunofluorescence assay (DIFA) showed that the CD3+ T lymphocytes could be identified specifically by the mouse anti-rCD3ε polyclonal antibodies, which didn't cross-react with the sIgM+ lymphocytes. Immunohistochemistry showed that CD3+ T lymphocytes could be detected in gill, skin, stomach, intestine, spleen, liver, head-kidney and mid-kidney. Flow cytometric analysis showed the percentages of CD3+ T lymphocytes in the PBL, spleen lymphocytes (SL) and head-kidney lymphocytes (HKL) of Japanese flounder increased rapidly after immunization with formalin-inactivated Edwardsiella tarda, and reached their peak levels at 5th day with 12.6%, 9.7% and 8.7%, respectively, and then decreased gradually. These results suggested that CD3+ T lymphocytes play important roles in mucosal and cell-mediated immunity, and the results would deepen our understanding on the roles of teleost T lymphocytes in the immune response.