PD-1 immune checkpoint blockade and cytokine IL-33 have shown significant therapeutic effects in tumor immunotherapy. These therapies promote CD8+ T cell activation, proliferation, and effector functions. However, there were few research about the combined therapy efficacy. In this study, we established B16-empty vector and B16-IL33 melanoma mouse models and treated with PD-1 monoclonal antibody. We reported that PD-1 blockade combined with cytokine IL-33 further inhibited tumor progression and prolonged the survival of tumor-bearing mice. Mechanistically, the combination therapy was found to further facilitate CD4+ and CD8+ T lymphocytes accumulation, and enhance the antitumor effects of CD4+or CD8+tumor-infiltrating lymphocytes by promoting type-1 immune response within the tumor microenvironment using flow cytometry and quantitative real time polymerase chain reaction. Thus, PD-1 blockade combined with IL-33 has application potential in tumor immunotherapy. Further, this study provides a new promising strategy and theoretical basis for tumor combination immunotherapy.

B7-H4 plays an important role in tumor immune evasion. In previous studies we have found that B7-H4 can translocate to the nucleus, and the exposure to PI3K inhibitor Ly294002 affects B7-H4 subcellular distribution. In this study we report the role of PI3K/Akt pathway in the B7-H4 subcellular distribution and the effect of PI3K/Akt inhibitors on B7-H4-mediated immunoresistance. The involvement of PI3K/Akt pathway in B7-H4 subcellular distribution was evident in experiments with wortmannin, while MDM2 inhibitor nutlin-3 and the mTOR inhibitor rapamycin were used to dissect the signaling downstream of Akt. Wortmannin and rapamycin demonstrated similar effects on B7-H4 subcellular distribution. Exposure to any of these inhibitors decreased levels of membrane B7-H4 while at the same time inducing its nuclear accumulation, while exposure to nutlin-3 had no effect on B7-H4 subcellular distribution. In the T cell proliferation assay, both wortmannin and rapamycin effectively inhibited B7-H4 WT/293 cells-mediated T cell proliferation while exerting no effect on Mock/293 cells. PI3K/Akt/mTOR plays a role in B7-H4 subcellular distribution, while MDM2 does not take part in it. Moreover, we show that wortmannin and rapamycin inhibit B7-H4-mediated tumor immunoresistance through regulating B7-H4 subcellular distribution. Taken together, these results suggest that PI3K/Akt/mTOR inhibitors might be used for adjuvant therapy aimed at inhibition of immune evasion.

PURPOSE:Wnt/β-catenin has emerged as an important signal pathway in renal cell carcinoma (RCC) pathogenesis. Frizzled 7 (Fzd7) is a member of Frizzled (Fzd) receptor family which binds with Wnt ligands and transduces canonical and non-canonical pathways. However, the expression of Fzd7 in human RCC is poorly investigated.METHODS:53 RCC tissues and peri-tumor tissues were collected from the patients treated with radical nephrectomy. The expression of Fzd7 was investigated by immunohistochemical staining. Three RCC cells were transfected with Fzd7shRNA and GFPshRNA to investigate the function of Fzd7 in RCC cells.RESULTS:The immunohistochemical analysis showed that Fzd7 protein expression level was significantly increased in RCC tissues when compared with peri-tumor tissues, which suggested that Fzd7 might be involved in the formation of tumors. However, the Fzd7 expression was not correlated with clinicopathological parameters. Three RCC cell lines: 786-O, Caki-1, and OS-RC-2 also expressed Fzd7. With Fzd7 expression being interfered by shRNA, the RCC cell proliferation was mildly decreased. Wnt3a could stimulate the RCC cells proliferation, but the stimulation was decreased when Fzd7 expression was interfered. Restoring the Fzd7 expression led to the proliferation stimulation effect of Wnt3a being restored.CONCLUSIONS:This paper suggests that Fzd7 may act as one of the molecules that take part in the course of RCC formation. Fzd7 can be activated by Wnt3a to stimulate cell proliferation.

OBJECTIVE To investigate the regulatory effect of phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway on the subcellular distribution of negative co-stimulatory molecule B7-H4. METHODS The HEK293 cells transfected stably with B7-H4, named B7-H4/HEK293, were treated with the PI3K/AKT specific inhibitor LY294002 and/or the nuclear export inhibitor leptomycin B (LMB). The subcellular localization of B7-H4 in B7-H4/HEK293 was observed by immunofluorescence and confocal laser scanning microscopy (CLSM), and the expression levels of B7-H4 in membrane, cytoplasm and nuclear were detected by Western blotting. RESULTS CLSM showed that LY294002 effectively induced the nuclear translocation of B7-H4 when compared with vehicle group. When the B7-H4/HEK293 cells were treated with LY294002 and LMB, more B7-H4 was translocated into nuclear. Western blotting demonstrated that after the PI3K/AKT signal pathway was inhibited by LY294002 for 24 hours, the levels of B7-H4 in cell membrane and cytoplasm decreased significantly (P<0.05), while the expression in nuclear increased significantly (P<0.05). CONCLUSION The PI3K/AKT signal pathway might inhibit the nuclear translocation of B7-H4.

Introduction: Inflammation plays a critical role in the progression of thoracic aortic aneurysms and dissections (TAAD). Inflammasomes are intracellular proteins that help regulate the inflammatory response. Activated by cellular stress signals, inflammasome molecules form multiprotein complexes and function as molecular platforms to mediate the activation of inflammatory caspases, the secretion of proinflammatory cytokines, and cellular injury. Within the subfamily of NLRP (nucleotide-binding oligomerization domain-like receptor proteins) inflammasomes, NLRP3 is the most fully characterized inflammasome. In this study, we tested the hypothesis that NLRP3 inflammasome expression is altered in human TAAD. Methods: Descending thoracic aortic tissues were obtained from 10 patients with thoracic aortic aneurysms caused by chronic dissection (TAD), 10 patients with thoracic aortic aneurysms without dissection (TAA), and 8 organ donors without aortic diseases. The protein expression of the NLRP3 inflammasome was detected by Western blot, immunohistochemistry, and immunofluorescence. Human aortic smooth muscle cells (SMCs) were treated with palmitic acid and NLRP3 siRNA, and the expressions of NLRP3 and MMP9 were detected by Western blot. Results: Western blot analysis revealed that the expression of NLRP3 was significantly greater in TAA and TAD aortas than in control aortas (Figure A). Similarly, immunohistochemistry staining revealed higher NLRP3 levels in TAAD tissue, especially in dedifferentiated vascular SMCs in the media (Figure B) and inflammatory cells in the adventitia. Double staining confirmed NLRP3 expression in macrophages and SMCs. in cultured aortic SMCs, palmitic acid induced the cleavage of MMP9 from its pro-form to the active form (Figure C); knocking down NLRP3 decreased MMP9 cleavage, suggesting a critical role of NLRP3 in MMP9 activation and tissue destruction. Conclusions: NLRP3 inflammasome levels are increased in TAAD. NLRP3 may play an important role in vascular inflammation and protease-mediated tissue destruction in TAAD.

FLT3, a transmembrane molecule, was found on hematopoietic stem/progenitor cells and leukemia cells and determined to be a promising target in leukemia diagnosis and therapy. In this study a functional anti-human FLT3, monoclonal antibody (MAb) 10G6, was obtained and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the FLT3 molecule expressed on a series of malignant cell lines. Furthermore, we demonstrated that MAb 10G6 inhibited the proliferation and migration ability and induced the apoptosis of SHI-1 cells that derived from a human monocytic leukemia. This functional anti-human FLT3 MAb provides a valuable tool for further study targeting the FLT3 on leukemia cells.

Engineered single chain antibodies have become a powerful source of immunotherapy against a wide range of diseases. Here, we present the generation of human CD28 single-chain antibody gene (CD28-ScFv), which contained variable fragments of heavy chain and light chain (VH and VL) of the anti-CD28 antibody, and a linking peptide (Gly4Ser)3 inserted in the middle of VH and VL. The fused gene CD28-ScFv was successfully expressed in BL21 (DE3) cells and confirmed by western blotting assay. The molecular weight of CD28-ScFv was 43 kDa and the major fraction was expressed as an insoluble body. By dissolving the insoluble bodies, renaturing in vitro and purifying with a Ni-NTA affinity column, highly purified expression products of CD28-ScFv were obtained. This product could recognize and bind to CD28+ positive T cells. The proliferation capacity of peripheral blood T cells was increased by purified CD28-ScFv. In this study, we improved orthodox renaturing techniques by combining the dilution renaturation with phase gradient dialysis. With this new method, highly purified CD28-ScFv products were developed and biological activity of the products was similar to that of the mouse monoclonal anti-human CD28 antibody.