Protein phosphorylation in hemocytes of Fenneropenaeus chinensis in response to white spot syndrome virus infection.

Protein phosphorylation and dephosphorylation are the most common and important regulatory mechanisms in signal transduction, which play a vital role in immune defense response. Our previous study has found the level of tyrosine phosphorylation was significantly changed in the hemocytes of Fenneropenaeus chinensis upon white spot syndrome virus (WSSV) infection. In order to explore the relationship between protein phosphorylation and WSSV infection, the quantitative phosphoproteomics was employed to identify differential phosphorylated proteins in hemocytes of F. chinensis before and after WSSV infection, and elucidate the role of key differential phosphorylated proteins in WSSV infection process. The results showed that a total of 147 differential phosphorylated proteins were identified in the hemocytes, including 64 phosphorylated proteins and 83 dephosphorylated proteins, which were mostly enriched in pyruvate metabolism, TCA cycle, glycolysis, and ribosomal biosynthesis. Functional analysis of differential phosphorylated proteins showed that they were involved in cell apoptosis, cell phagocytosis, cell metabolism and antiviral infection. A total of 236 differential phosphorylation sites were found, including 91 modified sites in the phosphorylation proteins and 145 modified sites in the dephosphorylation proteins. Motif analysis showed that these phosphorylation sites could activate mitogen-activated protein kinase, P70 S6 kinase and other kinases in hemocytes. Moveover, the phosphorylation levels of eukaryotic protein initiation factor 4E binding proteins and histone H3 were further determined by ELISA and Western blotting, which both exhibited a significant increase post WSSV infection and reach their peak levels at 6 and 12 h, respectively. Moreover, we found that lactate, a metabolite closely related to pyruvate metabolism, TCA cycle and glycolysis, was significantly increased in the hemocytes after WSSV infection. This study revealed the protein phosphorylation response in hemocytes of F. chinensis to WSSV infection, which help to clarify the response characteristics and virus resistance mechanism of hemocytes in F. chinensis, and also facilitate further understanding of the interaction between WSSV and shrimp hemocytes.

Analysis and identification of tyrosine phosphorylated proteins in hemocytes of Litopenaeus vannamei infected with WSSV.

Previous studies have demonstrated that protein tyrosine phosphorylation plays an important role in WSSV infection. In the present work, in order to further elucidate the potential role of protein tyrosine phosphorylation in white spot syndrome virus (WSSV) infection. The expression variation of tyrosine phosphorylated proteins in hemocytes of shrimp (Litopenaeus vannamei) after WSSV infection were examined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linked immunosorbent assay (ELISA), and results showed that the level of protein tyrosine phosphorylation in hemocytes fluctuated significantly after WSSV infection and exhibited two peaks at 6 and 24 h post infection (hpi). Meanwhile, tyrosine phosphorylated proteins in hemocytes after WSSV infection were also detected by cell immunofluorescence, and results showed that the fluorescence intensity in hemocytes was altered with the course of WSSV infection and showed stronger fluorescent signals at 6 and 24 hpi compared to other time points. Furthermore, two dimensional gel electrophoresis (2-DE) and 2-DE western blotting were applied to identify the differentially expressed tyrosine phosphorylated proteins in hemocytes before and after WSSV infection. The result of 2-DE western blotting showed that there were nine tyrosine phosphorylated proteins in the hemocytes of healthy shrimp, whereas twenty-one tyrosine phosphorylated proteins were detected in the hemocytes of shrimp at 6hpi. Then, the differential tyrosine phosphorylated proteins were analyzed by Mass Spectrometry (MS), and eight of them were identified to be sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, actin-D, phosphopyruvate hydratase, beta-actin, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase. Moreover, the expression levels of sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, phosphopyruvate hydratase, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase were examined to be up-regulated post WSSV infection by quantitative real-time RT-PCR. Taken together, these results demonstrated that protein tyrosine phosphorylation was involved in the process of WSSV infection, which might play an important role in the immune response to WSSV infection in shrimp.

Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector/memory CD8 T cells in a poorly immunogenic tumor model.

Immunotherapies targeting the programmed death 1 (PD-1) coinhibitory receptor have shown great promise for a subset of patients with cancer. However, robust and safe combination therapies are still needed to bring the benefit of cancer immunotherapy to broader patient populations. To search for an optimal strategy of combinatorial immunotherapy, we have compared the antitumor activity of the anti-4-1BB/anti-PD-1 combination with that of the anti-PD-1/anti-LAG-3 combination in the poorly immunogenic B16F10 melanoma model. Pronounced tumor inhibition occurred only in animals receiving anti-PD-1 and anti-4-1BB concomitantly, while combining anti-PD-1 with anti-LAG-3 led to a modest degree of tumor suppression. The activity of the anti-4-1BB/anti-PD-1 combination was dependent on IFNγ and CD8(+) T cells. Both 4-1BB and PD-1 proteins were elevated on the surface of CD8(+) T cells by anti-4-1BB/anti-PD-1 cotreatment. In the tumor microenvironment, an effective antitumor immune response was induced as indicated by the increased CD8(+)/Treg ratio and the enrichment of genes such as Cd3e, Cd8a, Ifng, and Eomes. In the spleen, the combination treatment shaped the immune system to an effector/memory phenotype and increased the overall activity of tumor-specific CD8(+) CTLs, reflecting a long-lasting systemic antitumor response. Furthermore, combination treatment in C57BL/6 mice showed no additional safety signals, and only minimally increased severity of the known toxicity relative to 4-1BB agonist alone. Therefore, in the absence of any cancer vaccine, anti-4-1BB/anti-PD-1 combination therapy is sufficient to elicit a robust antitumor effector/memory T-cell response in an aggressive tumor model and is therefore a candidate for combination trials in patients.