Lack of a contact requirement for direct antibacterial activity of lymphocyte subpopulations in ginbuna crucian carp

• CD8α+ cells killed pathogenic Edwardsiella tarda, Streptococcus iniae more efficiently than Escherichia coli.
• Supernatants from all three types of lymphocytes showed microbicidal activity.
• Both sensitized and non-sensitized lymphocytes non-specifically killed target bacteria without the need of contact.
• There are two different mechanisms in the direct bacterial killing by lymphocytes in ginbuna.

Cytotoxic T lymphocytes (CTL) recognize and kill cells infected with viruses, intracellular bacteria and tumors with MHC restriction and antigen specificity. In addition to these activities, recent studies in mammals have suggested that CTL can exhibit direct microbicidal activity. In our previous study we documented direct antibacterial activity of CD4+ T cells and sIgM+ cells as well as CD8α+ T cells from immunized fish. However, we also found weak non-specific killing activity of lymphocytes against bacteria. In the present study we further analyzed the weak killing activity of lymphocytes, increasing the effector cell to target bacteria ratio from 10:1 to 103:1. Sensitized and non-sensitized effector lymphocytes (CD8α+, CD4+ and sIgM+) separated by MACS were incubated with target bacteria. CD8α+ T cells from Edwardsiella tarda-immunized ginbuna crucian carp killed 98%, 100% and 70% of E. tarda, Streptococcus iniae and Escherichia coli, respectively. CD8α+ T cells from non-immunized fish showed similar but slightly lower killing activity than sensitized cells. CD4+ and sIgM+ lymphocytes also showed high killing activity against E. tarda and S. iniae as found for CD8α+ T cells, although the activity was lower against E. coli. Supernatants from all three types of lymphocytes showed microbicidal activity, although the activity was lower than that evoked by effector lymphocytes. Furthermore, the presence of a membrane between effectors and targets did not affect the killing activity. The present results suggest that both sensitized and non-sensitized lymphocytes non-specifically killed target bacteria without the need of contact. The major difference between the present and previous experiments is the E:T ratio. We suspect that there are two different mechanisms in the direct bacterial killing by lymphocytes in ginbuna.