The Expression of CD28 and Its Synergism on the Immune Response of Flounder (Paralichthys olivaceus) to Thymus-Dependent Antigen

Abstract

CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its expression profiles were characterized in flounder (Paralichthys olivaceus); variations of CD28+ cells after being stimulated with different types of antigens and the function of the CD28 costimulatory pathway on T-cell activation were investigated in vitro. fCD28 consists of four exons and three introns, and the full-length cDNA of fCD28 was 675-bp encoded 224 amino acids. The conserved motif (121TFPPPF126) binding to the CD80/86 ligand exists in the Ig-superfamily homology domain. The high expression of fCD28 is in gills, PBLs, head kidney, and spleen. CD28+ cells were co-localized with CD4+ T lymphocytes but not on IgM+ B lymphocyte cells. Moreover, the expression of CD28 was significantly varied in flounder after being stimulated by keyhole limpet hemocyanin (KLH) at both the transcriptional and cellular levels, while no significant differences were observed between lipopolysaccharide (LPS) stimulation and the control group. Notably, treatment of PBLs cultured in vitro with CD28 molecule-specific antibody (anti-CD28 Abs) and PHA produced more cell colonies and stimulated the proliferation of cultured leukocytes compared to PHA stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 ± 1.35%), CD4+ T lymphocytes (18.32 ± 2.15%), and CD28+/CD4+ double-positive cells (6.24 ± 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of interleukin-2 (IL-2) and nuclear factor of activated T cells (NFAT) in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation.

Molecular and Functional Analyses of the Primordial Costimulatory Molecule CD80/86 and Its Receptors CD28 and CD152 (CTLA-4) in a Teleost Fish

“…This result was further confirmed by the demonstration that CD80/86, CD28, and CD152 in grass carp are all membrane molecules expressed on the cell surface, which is necessary for the signaling between the costimulatory molecules. Moreover, the existence of the conserved B7 binding sites in the grass carp CD28 (YPPPF) and CD152 (FPPPY) further supports the conclusion that the binding activities of CD80/86 to CD28 and CD152 are specific ( 18 – 21 ).…”

“…The conserved 123 YPPPF 127 motif is responsible for binding the CD80/86 and exists in the Ig-like domain of grass carp CD28. In mammalian CD28, the YXXM motif is present in the cytoplasmic tail, which binds the Src homology 2 domain of p85, an adaptor subunit of PI3K ( 21 ). However, in grass carp and zebrafish CD28, the conserved YXXM motif has not been found, which is replaced by a similar motif YXXXM ( Figure 2C ).…”

“…Contrastingly, the CD28 and CD152 gene structures are consistent across teleost species, whereby the CD28 and CD152 genes in grass carp and other known teleost fish are all comprised of 4 exons and 3 introns. In mammalian CD28, the YXXM motif in the cytoplasmic tail were confirmed for binding the Src homology 2 domain of p85, an adaptor subunit of PI3K ( 21 ). However, in grass carp and zebrafish CD28, the conserved YXXM motif has not been found, which is replaced by a similar motif YXXXM.…”

Characterization of Co-Stimulatory Ligand CD80/86 and Its Effect as a Molecular Adjuvant on DNA Vaccine Against Vibrio anguillarum in Flounder (Paralichthys olivaceus)

“…The sequence encoding the extracellular domain except the signal peptide was amplified by polymerase chain reaction (PCR) and then embedded into pET-28a Vector (TaKaRa, Japan). After affinity purification of recombinant CD80/86 protein with a 6-histidine tag at the N-terminal end using His TrapTM HP Ni-Agarose (GE Healthcare China, Beijing, China), a total of 2.8 mg of recombinant protein was used to immunize New Zealand white rabbits (∼1 kg) and Balb/C mice (~30 g) ( 40 ). The specific procedure was followed as previously described.…”

“…Western blotting was conducted as previously described ( 40 ). After SDS-PAGE, rCD80/86 and whole-cell protein extracts from PBLs were electrophoretically transferred onto a PVDF membrane (Merck Millipore).…”