Cellular activities during a mixed leucocyte reaction in the teleost sea bass Dicentrarchus labrax

In this investigation a number of “in vitro” activities of sea bass peripheral blood leucocytes (PBL) against allogeneic PBL inactivated by irradiation were studied. Stimulator PBL were cultured with inactivated allogeneic PBL, and direct counting of lymphocytes was done after 2 weeks by immunofluorescence and flow cytometry using mAbs DLT15 and DLIg3 specific for T-cells and B-cells, respectively. In a one-way mixed leucocyte reaction (MLR), results showed an increase of T lymphocytes, whereas B lymphocytes had values similar to those in control PBL. The increase of T-cells in MLR cultures was also confirmed using RT-PCR by analyzing the expression of the T-cell receptor (β-subunit) mRNA. The addition of 5 μg/ml of cyclosporin A (CsA) to the MLR caused a significant decrease in T-cell proliferation. Leucocytes from MLR cultures displayed an enhanced cytotoxic activity against xenogeneic target cells with respect to control PBL, raising the possibility of the presence of cytotoxic-like T lymphocytes. Cellular activation of PBL was confirmed in 2 weeks MLR by measuring antibody-induced intracellular Ca++ mobilization with Fura-2 AM. This work represents the first direct quantitative determination of an “in vitro” T-cell activity in a teleost species.