Non-specific cytotoxic cell l (NCC) is a kind of important lymphocyte participating in the non-specific immune response in teleost. Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is a receptor molecule on the surface of NCC and plays an important role in mediating the activity of NCC. However, there are few reports on which signal molecule could transmit signals through NCCRP-1. In this study, yeast two-hybrid library of tilapia liver and head kidney was constructed, and a transferrin from Oreochromis niloticus (On-TF) with interaction protein sequence was obtained by screening the library with bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1). Then, the open reading frame (ORF) of On-TF was cloned, which had 2088 bp in length, encoding polypeptides of 695 amino acids. The deduced amino acid sequence was highly homologous to teleost and similar to mammalian TF, containing two TR_FER domains (25-343th aa and 344-686th aa) for binding iron ions. Furthermore, a point-to-point yeast two-hybrid method was used to further verify whether On-NCCRP-1 could bind to On-TF. The recombinant On-TF (rOn-TF) protein was purified by prokaryotic expression system. In vitro experiments showed that rOn-TF could up-regulate the expression of killing effector molecule of NCC by On-NCCRP-1, and rOn-TF-activated NCCs showed a significantly improved ability to kill FHM cells, indicating that rOn-TF could regulate the NCC signaling pathway through NCC receptor molecule On-NCCRP-1. The results provide a more theoretical basis for understanding the regulation mechanism of NCC activity.

The teleost non-specific cytotoxic cell (NCC), as the evolutionary precursors of NK cells, is an important cytotoxic cell population in the innate immune system of teleost. We have recently realized that costimulatory CD80/86 have conservation in structural and interactional features with its ligand CD28 in Nile tilapia (Oreochromis niloticus). However, the ability of CD80/86 to regulate NCC activity has not been fully investigated. In the present study, we first obtained the recombinant fusion CD80/86 protein from O. niloticus (rOn-CD80/86). Then, NCC incubation with rOn-CD80/86 resulted in a significant production of NCC effector cytokines, including tumor necrosis factor-alpha, cellular apoptosis susceptibility and NK-lysin. Furthermore, NCC treatment with rOn-CD80/86 could significantly improve the ability to kill kidney cells of Grass carp (CIK) and up-regulate the activities of caspase-1 and caspase-3 in CIKs. The yeast, two-hybrid assay showed that On-CD80/86 cannot directly interact with non-specific cytotoxic cell receptor protein-1 of O. niloticus (On-NCCRP-1). The single-cell RNA-Seq data of Nile tilapia head kidney lymphocytes analysis found On-CD28 did not exhibit expression on NCCs subsets. The above results suggest that costimulatory molecules On-CD80/86 is independent of On-NCCRP-1 and On-CD28 receptor in modulating NCC killing activity in vitro of Nile tilapia. The results also provide more insights into the mechanism of NCC activity regulation.