Clinical significance of the induction of macrophage differentiation by the costimulatory molecule B7-H3 in human non-small cell lung cancer.

B7-H3, a member of the B7 family of molecules, is expressed in certain types of human cancer and is important in tumor development and progression. Although several studies have reported that the expression of B7-H3 is correlated with poor outcomes in patients with cancer, its exact role in cancer remains unknown. In the present study, the expression levels of B7-H3 in the pathological specimens of 105 patients treated for non-small cell lung cancer (NSCLC) were examined by immunohistochemistry. A high expression level of B7-H3 was observed in 46.9% of the 105 NSCLC tissue specimens. These patients demonstrated a more advanced tumor grade and a shorter survival time. In addition, we also examined the levels of tumor-associated macrophages (TAMs) in NSCLC tissues and observed that the levels were positively correlated with the expression of B7-H3, and that higher levels of macrophages were associated with lower levels of infiltrating T cells and a shorter survival time. These results demonstrated that TAMs are important in the evasion of tumor immune surveillance in NSCLC. Furthermore, through knockdown of B7-H3 by RNA interference, we observed that soluble B7-H3 was capable of inducing macrophages to express higher levels of macrophage mannose receptor (MMR) and lower levels of human leukocyte antigen (HLA)-DR, as well as higher levels of interleukin-10 (IL-10) and lower levels of IL-1ß in vitro. These observations are characteristic of an anti-inflammatory/reparatory (alternative/M2) phenotype. Therefore, our data suggests that B7-H3 proteins are involved in the progression of NSCLC by inducing the development of monocytes into anti-inflammatory cells.

Generation and characterization of two novel monoclonal antibodies produced against human TLT-2 molecule.

Trem-like transcript 2 (TLT-2), one of the TREM family members, which is expressed on B cells, T cells, and macrophages, plays a critical role in immune response mechanism. In this study, two novel mouse anti-human TLT-2 monoclonal antibodies (MAbs) were prepared using hybridoma technology and their immunological characteristics were determined. The results showed that the two MAbs (clones 10F5 and 8C10) were both IgG1 (κ) and bound specifically to human TLT-2. Furthermore, 10F5 and 8C10 seemed to recognize a different site (epitope) of TLT-2 by competition assay. MAb 10F5 was proven in Western blot analysis to specifically bind to denatured TLT-2 protein while both MAbs were proven in dot blot analyses and immunofluorescence to specifically bind to natural TLT-2 protein. In addition, crosslinking of TLT-2 with MAb 8C10 markedly blocked TLT-2 positive signal and T cell proliferation. Taken together, these two monoclonal antibodies might be of great value as tools for further exploration of the expression and function of TLT-2.

[Splicing factor SC35 regulated the expression of B7-H3 in vitro].

AIM: To explore the mechanisms by which splicing factor SC35 regulates the costimulatory molecule B7-H3 expression in vitro through bioinformatic and molecular biological methods. METHODS: We screened some regulatory proteins which might take part in regulating B7-H3 expression using bioinformatic methods. Then RNA interference (RNAi) and real-time PCR were performed to test if these proteins took part in the regulation. RESULTS: Splicing factor SC35, SRP40 and SF50 might play an important role in the regulation of B7-H3 expression. In PHA-activated T cells, B7-H3 was upregulated obviously and at the same time SC35 was also upregulated. When we suppressed SC35 expression using RNAi, we found B7-H3 was also downregulated. CONCLUSION: Splicing factor SC35 might take part in the regulation of B7-H3 expression, which could help understand B7-H3 biological function.

[Expression of mouse B7-H3-Fc fusion protein and characterization of its bioactivity].

AIM: To obtain mouse B7-H3-Fc fusion protein and to investigate its biological function and effects on T lymphocyte activation. METHODS: The genes coding extracellular domain of mouse B7-H3 and the Fc fragment of human IgG1 were amplified from pMD19-T/mouse B7-H3 and pMD19-T/human IgG1 vectors by PCR. The two genes were combined with mouse B7-H3-Fc fragment by overlap PCR. Then the resulting gene fragment was inserted into eukaryotic vector pIRES2-EGFP after digested with EcoR I and Bgl II to construct the recombinant vector pIRES2-EGFP/B7-H3-Fc. The recombinant vector was transfected into CHO cells with LipfectAMINE™ 2000, and the cells were further selected with G418. The collected supernatant of the transfected cell line cultured in serum-free media was ultrafiltrated and concentrated, then purified by Protein G column. The expression of mouse B7-H3-Fc fusion protein was confirmed by Western blot. Effects of fusion protein on T cells proliferation and cytokine production in vitro was studied by methods of CCK8 and ELISA. RESULTS: The results showed that the transfected CHO cell line secreting mouse B7-H3-Fc fusion protein was constructed successfully. In vitro, mouse B7-H3-Fc fusion protein obviously promoted the proliferation of T cells in a dose-dependent manner. CONCLUSION: A transfected CHO cell line stably expressing mouse B7-H3-Fc fusion protein has been obtained and the B7-H3-Fc fusion protein stimulates the proliferation of T cells and cytokines production in vitro.

A role of brassinosteroids in early fruit development in cucumber.

Brassinosteroids (BRs) are essential for many biological processes in plants, however, little is known about their roles in early fruit development. To address this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, cell division, and expression of cyclin and cyclin-dependent kinases (CDKs) genes were examined in two cucumber cultivars that differ in parthenocarpic capacity. The application of EBR induced parthenocarpic growth accompanied by active cell division in Jinchun No. 4, a cultivar without parthenocarpic capacity, whereas Brz treatment inhibited fruit set and, subsequently, fruit growth in Jinchun No. 2, a cultivar with natural parthenocarpic capacity, and this inhibitory effect could be rescued by the application of EBR. RT-PCR analysis showed both pollination and EBR induced expression of cell cycle-related genes (CycA, CycB, CycD3;1, CycD3;2, and CDKB) after anthesis. cDNA sequences for CsCycD3;1 and CsCycD3;2 were isolated through PCR amplification. Both CsCycD3;1 and CsCycD3;2 transcripts were up-regulated by EBR treatment and pollination but strongly repressed by Brz treatment. Meanwhile, BR6ox1 and SMT transcripts, two genes involved in BR synthesis, exhibited feedback regulation. These results strongly suggest that BRs play an important role during early fruit development in cucumber.