Cytotoxic CD8α+ leucocytes have heterogeneous features in antigen recognition and class I MHC restriction in grouper

CD8 is a membrane glycoprotein found primarily on the surface of T lymphocytes such as cytotoxic T lymphocytes (CTL), natural killer cells (NK) and γδ T lymphocytes. It helps T lymphocytes to kill the infected cells that presents microbial antigen on the cell surface. However, analysis of fish cellular immunity has been limited because of the lack of CD8 antibodies in grouper. In this present study, we cloned full-length CD8α cDNAs from orange-spotted grouper (Epinephelus coioides), an important fish species economically. The deduced protein of CD8α contained 227 amino acid residues in length and included one signal peptide, Ig superfamily V domain, hinge region, transmembrane domain, cytoplasmic tail and conserved binding motif associated with tyrosine kinase p56(lck). The molecular weight of the mature protein was estimated at 22.5 kDa and pI at 9.55. Phylogenetically, the predicted grouper CD8α protein was similar to CD8α from other marine fish species in which the identity was 50-60%. Real-time PCR revealed that CD8α transcript was constitutively expressed in thymus, head kidney, gill, spleen, gut and peripheral blood leucocyte (PBL); and the highest expression in thymus. CD8α transcript in the spleen of fish injected with nervous necrosis virus (NNV) was significantly up-regulated at 4 days post-injection compared to the untreated fish. Rabbit antiserum prepared against recombinant CD8α protein was able to recognize specifically the subset lymphocytes which have a diameter of 7 μm, a high nucleus/cytoplasm ratio and a ring-shaped cytoplasm. The cytotoxicity of CD8α(+) lymphocytes at one-week post-NNV infection was enhanced significantly against NNV-infected autologous fin cells in comparison with NNV-infected allogeneic or RSIV-infected autologous fin cells. Flow cytometry analysis revealed that both the number and mean fluorescence intensity (MFI) of CD8α(+) PBL were significantly increased at 7 days post-NNV infection. The specific cytotoxicity and MHC class I restriction of the lymphocytes sorted by rCD8α antibody are properties that can be attributed to CTL. In addition, low level of cytotoxicity was found in PBL against allogeneic targets as well as CD8α(+) effectors killed autologous targets nonspecifically, implicated presence of cytotoxic T subsets, possibly nonspecific cytotoxic cells (NCC) and γδ T lymphocytes, without MHC class I restriction. In conclusion, grouper cytotoxic CD8α(+) PBL have heterogeneous features in specific antigen recognition and class I MHC restriction.