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Spleen tyrosine kinase (Syk) is a non-receptor protein tyrosine kinase, and it mediates downstream signaling of FcR-mediated immune responses. Our previous work revealed that the expression of Syk was significantly up-regulated in flounder mIgM+ B lymphocytes after phagocytosis of antiserum-opsonized Edwardsiella tarda , which suggested Syk might be involved in Ig-opsonized phagocytosis. In this paper, phospho-Syk (pSyk) inhibitor was used to investigate the potential role of phosphorylated Syk in FcR-mediated phagocytosis of IgM+ B cells. Indirect immunofluorescence assay (IFA) and Western blotting showed that the level of phosphorylated Syk in the mIgM+ B lymphocytes treated with pSyk inhibitor was significantly lower compared to the control group after stimulation with flounder antiserum. Flow cytometry analysis showed that after 3 h incubation with antiserum-opsonized E. tarda , the phagocytosis rates of mIgM+ B lymphocytes from peripheral blood, spleen and head kidney pre-treated with pSyk inhibitor were 48.1%, 40.1% and 43.6% respectively, which were significantly lower than that of the control groups with 58.7%, 53.2% and 57.4%, respectively. And likewise, after pSyk inhibitor treatment, the proportions of mIgM+ B lymphocytes with higher intracellular reactive oxygen species (ROS) levels in peripheral blood, spleen and head kidney decreased to 15.2%, 12.0% and 12.1% from the control level of 26.5%, 25.9% and 26.3%, respectively. Moreover, the expression of three genes affected by pSyk, including phospholipase Cγ1 (PLCγ1), phospholipase Cγ2 (PLCγ2) and phosphatidylinositol 3 kinase (PI3K) were found to be significantly down-regulated in pSyk inhibitor-treated mIgM+ B lymphocytes post phagocytosis. These results suggest that pSyk plays a key role in FcR-mediated phagocytosis and bactericidal activity of mIgM+ B lymphocytes, which promotes further understanding of the regulatory role of pSyk in teleost B cells phagocytosis. • The first study to find that the activated FcR could induce phosphorylation of Syk in flounder mIgM + B lymphocytes. • The pSyk played a key role in FcR-mediated phagocytosis and bactericidal activity of flounder mIgM+ B lymphocytes. • The pSyk was essential for ADCP-related downstream signaling in flounder mIgM+ B lymphocytes. [ABSTRACT FROM AUTHOR]

CD28 and CD80/86 are crucial co-stimulatory molecules for the T cell activation. Previous study illustrated that CD28 and CD80/86 present on T cells and antigen-presenting cells in flounder (Paralichthys olivaceus), respectively. The co-stimulatory molecules were closely associated with cell immunity. In this paper, recombinant protein of flounder CD80/86 (rCD80/86) and phytohemagglutinin (PHA) were added to peripheral blood leukocytes (PBLs) in vitro. Lymphocytes were significantly proliferated with CFSE staining, and the proportion of CD4+ and CD28+ lymphocytes significantly increased. In the meantime, genes related to the CD28-CD80/86 signaling pathway or T cell markers were significantly upregulated (p < 0.05). For further study, the interaction between CD80/86 and CD28 was confirmed. The plasmid of CD28 (pCD28-FLAG and pVN-CD28) or CD80/86 (pVC-CD80/86) was successfully constructed. In addition, pVN-ΔCD28 without the conserved motif "TFPPPF" was constructed. The results showed that bands of pCD28-FLAG bound to rCD80/86 were detected by both anti-FLAG and anti-CD80/86. pVN-CD28 complemented to pVC-CD80/86 showing positive fluorescent signals, and pVN-ΔCD28 failed to combine with pVC-CD80/86. The motif "TFPPPF" in CD28 played a crucial role in this linkage. These results indicate that CD28 and CD80/86 molecules interact with each other, and their binding may modulate T lymphocytes immune response in flounder. This study proved the existence of CD28-CD80/86 signaling pathway in flounder. • RCD80/86 and PHA induced lymphocyte proliferation and proportion of CD4+ cells in flounder. • RCD80/86 and PHA upregulated the expression of B7-CD28 signaling pathway genes, T cell markers, and cytokines. • PCD28-FLAG bound to rCD80/86, and pVN-CD28 complemented to pVC-CD80/86 in vitro. • Motif of "TFPPPF" in CD28 plays crucial role in the interaction between CD28 and CD80/86. [ABSTRACT FROM AUTHOR]

Interleukin-2 receptor (IL-2R), as the specific ligand of interleukin-2 (IL-2), binds to IL-2 and transmits signals and then can induce the proliferation of T lymphocytes in mammals. In this paper, the subunit of IL-2R in flounder (Paralichthys olivaceus), interleukin-2 receptor subunit gamma (IL-2Rγ) was cloned, and polyclonal antibodies (Abs) against its extracellular region were produced, then the expression of flounder IL-2Rγ (fIL-2Rγ) at transcriptional and cellular levels were characterized. Moreover, the interaction of flounder IL-2 (fIL-2) with fIL-2Rγ was investigated, and the variations on CD4+/IL-2Rγ+ cells in flounder after treatment with recombinant IL-2 (rIL-2), anti-IL-2Rγ Abs were detected, respectively. The results showed that fIL-2Rγ protein had a typical fibronectin type III (FN3) domain. The Abs could specifically recognize native fIL-2Rγ molecules at 39.9 kDa. FIL-2Rγ was localized on both T and B lymphocytes, and the percentages of CD4+/IL-2Rγ+ and IgM+/IL-2Rγ+ lymphocytes were high in spleen. In addition, pBiFC-VN173-IL-2Rγ plasmids could bind to pBiFC-VC155-IL-2 plasmids. The percentage of CD4+/IL-2Rγ+ lymphocytes was significantly decreased after blocking with anti-IL-2Rγ Abs both in vivo and in vitro. In the meantime, four T cell markers genes and six IL-2-IL-2R pathway genes were down-regulated in anti-IL-2Rγ Abs group. These results first demonstrated that fIL-2Rγ molecules were expressed on both T and B lymphocytes in flounder, and the bond between fIL-2Rγ and fIL-2 activated the CD4 T lymphocytes. This study gave a new sight into the exploration of IL-2R function on T lymphocytes proliferation in fish. • IL-2Rγ mRNA was highly expressed in peripheral blood leukocytes, spleen and head kidney in flounder. • IL-2Rγ molecules were localized on both T and B lymphocytes in flounder. • Flounder IL-2 could bind to flounder IL-2Rγ. • The addition of recombinant IL-2 significantly increased the percentage of CD4+/IL-2Rγ+ lymphocytes at 72 h. [ABSTRACT FROM AUTHOR]

Water temperature is an important factor for immune responses in poikilothermic fish. Especially, it has been known that adaptive immunity is more sensitive to temperature than innate immunity in fish. The optimal temperature for olive flounder (Paralichthys olivaceus) culture is known between 20 and 25 °C, and there are several papers reporting the low or no effectiveness of inactivated vaccines in olive flounder kept at low water temperatures. Previously, we had reported that a vaccine based on single-cycle viral hemorrhagic septicemia virus (VHSV) that was modified to produce the transmembrane and C-terminal cytoplasmic region-deleted G protein in host cells (rVHSV-GΔTM) induced significantly higher survival rates in olive flounder than a vaccine of rVHSV-ΔG that had no G gene in the genome. In the present study, we evaluated the availability of rVHSV-GΔTM as a protective vaccine that can be used in olive flounder at low water temperature periods. Olive flounder fingerlings were divided into 6 groups: group 1 and 2 were kept at 14 °C, group 3 and 4 were kept at 20 °C, and group 5 and 6 were kept at 14 °C for 1 week and then shifted to 20 °C. Fish in groups 1, 3, and 5 were intramuscularly (i.m.) immunized with 8.5 × 104 PFU/fish of rVHSV-GΔTM, and fish in groups of 2, 4, and 6 were i.m. Injected with L-15 alone. In the challenge test, the survival rates of fish immunized with rVHSV-GΔTM were significantly higher than those of control group fish that were injected with L-15 alone. Among three vaccination groups (group 1, 3, and 5), group 1 showed no mortality. The cumulative mortalities of group 3 and group 5 were both 25%. While fish in control groups (group 2, 4, and 6) showed 90–100% mortalities. The qPCR genome copy number of rVHSV-GΔTM in the kidney of fish immunized at 14 °C was clearly higher than that in fish immunized at 20 °C, which suggests that higher amount of secretory viral glycoprotein would be produced in fish vaccinated at 14 °C than at 20 °C. Olive flounder immunized with rVHSV-GΔTM at 14 °C showed the serum neutralization activity as high as fish immunized at 20 °C, suggesting that the humoral immune response of olive flounder was effectively induced at lower water temperature. These results suggest that VHSV vaccines based on single-cycle viruses can be used as prophylactic vaccines even at low water temperature period. [ABSTRACT FROM AUTHOR]

Vibrio anguillarum and Edwardsiella tarda are severe aquaculture pathogens shared similar epidemiological characteristics and susceptible to flounder ( Paralichthys olivaceus) . In our previous studies, recombinant(r) protein heat shock protein 33 (rHsp33) from V. anguillarum and outer membrane protein C (rOmpC) from E. tarda were proved to have protection against V. anguillarum and E. tarda , respectively. In this paper, the cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum , and the protection of divalent vaccine candidate (rHsp33 + rOmpC, rHC) against both V. anguillarum and E. tarda were evaluated. RHC, rHsp33, and rOmpC were vaccinated to flounder, respectively, and the percentages of surface immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBLs), serum IgM, specific antibodies against V. anguillarum or E. tarda , specific antibodies against rHsp33, rOmpC or rHC, the expression of immune-related genes and relative percent survival (RPS) against V. anguillarum or E. tarda were measured. The results showed that: RHC could induced the enhancement of sIg + cells and high levels of specific antibodies against both V. anguillarm and E. tarda ; Also a significant increase of specific antibodies against rHsp33, rOmpC or rHC, and up-regulation of gene expression of CD3, CD4-1, CD4-2, CD8α, CD8β and IgM in spleen, head-kidney, and hindgut, RPS of 70 ± 3.45% against V. anguillarum and 60 ± 1.48% against E. tarda , respectively. In addition, rHsp33 induced specific antibodies against E. tarda and rOmpC, and had a RPS of 43.3 ± 3.73% against E. tarda ; rOmpC could evoke specific antibodies against V. anguillarum and rHsp33, and had a RPS of 44 ± 1.27% against V. anguillarm ; The results demonstrated that there was cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum , rHC as a divalent vaccine can induce significant immune response and efficient protection against both E. tarda and V. anguillarum in flounder. [ABSTRACT FROM AUTHOR]

In acquired immunity, T lymphocytes regulate the immune responses of B lymphocytes, including the IgM + B lymphocyte counts and antibody production. In this paper, Cyclosporine A (CsA) and Rapamycin (RaPa) were used, and their inhibition on T lymphocytes and immune responses of B lymphocytes in flounder ( Paralichthys olivaceus ) were investigated. Flounder was injected with Keyhole Limpet Hemocyanin (KLH), a mixture of KLH and CsA (KLH + CsA), or a mixture of KLH and RaPa (KLH + RaPa). Then, the proportions of T and IgM + B lymphocytes (PT and PB) in peripheral blood leukocytes (PBL) were analysed by flow cytometry (FCM), total antibodies (TA) and KLH specific antibodies (KA) in serum were measured by ELISA, and expression of 9 immune-related genes in the spleen and kidneys were determined using q-PCR. On the other hand, the PBL culture was treated with Concanavalin A (ConA), a mixture of ConA and CsA, and a mixture of ConA and RaPa. Then the PT and PB were measured, and the cell proliferation was examined using the MTT method. The results showed that the PT peaked on the 5 th day in the KLH group, KLH + CsA group and KLH + RaPa group. The maximum inhibition rates (MIR) of CsA and RaPa were 27.44% ± 0.50% and 21.37% ± 2.06%, respectively. The PB peaked at the 5 th week, and the MIR of CsA and RaPa were 44.51% ± 1.36% and 33.3% ± 0.65%, respectively. The KA and TA peaked at the 5 th week. The MIR of CsA and RaPa on TA were 40.31% ± 1.59% and 32.96% ± 2.21%, respectively, and were 27.77% ± 2.02% and 23.41% ± 1.08% for KA, respectively. Nine immune-related genes had significantly lower expression in the KLH + CsA group and KLH + RaPa group compared to the KLH group. The proliferation of the PBL culture was inhibited by CsA or RaPa, and the inhibition rate of CsA and RaPa for PT was 18.14% ± 1.08% and 17.88% ± 1.02%, respectively, and the inhibition rates for PB were 3.03% ± 0.57% and 2.95% ± 0.53%, respectively. The results demonstrated that T lymphocytes counts were inhibited by CsA and RaPa, followed by suppression of IgM + B lymphocytes and antibody production, which suggests that T lymphocytes regulate the immune response of B lymphocytes in flounder. [ABSTRACT FROM AUTHOR]