Effect Of Self-expression Of Sirpα-fc Fusion Protein On T Cell Function

CD47 is a membrane protein, which is highly expressed on the tumor cell surface. When CD47 binds with SIRPα, a CD47 receptor on the macrophage cell surface, a “don’t eat me” signal will be released to help tumor cells escape immune surveillance. Furthermore, CD47 is also involved in regulating the function of activated lymphocytes, through inhibiting the activation, proliferation, and cytotoxicity of lymphocytes by interacting with Thrombospondin-1(TSP1). Therefore, to block CD47 can not only help to enhance the phagocytosis effect of macrophage cells but also improve the function of lymphocyte to kill tumor cells.In this study, we constructed a fusion protein(i.e. SIRPα-Fc, SF) by fusing the Fc region of human Ig G1 with the SIRPα mutant CV1, a peptide with a high affinity to bind CD47. To effectively express the SF fusion protein, an adenovirus-based expression system was developed by modifying the adenoviral fiber and designing a promoter with high activity within T cells. Subsequently, the SF expression system, Ad35-SF, was used to infect T cells(e.g. Jurkat T cell and CIK cell) to investigate the influence of CD47 blocking on the cell proliferation, and cytotoxicity on tumor cell in vitro. The main results and methods were listed as follows:(1) Construction of expression system for SF fusion proteinBy dual luciferase reporter system, we found the newly designed promoter TCEF1α(in) have the highest activity in Jurkat T cells; By immunofluorescence and flow cytometry analyse, we identified that the type 35 serotype adenoviral vector had higher efficiency to infect T cells than the type 5 correspondence.(2) Construction and identification of recombinatnt adenovirus Ad35-SFWe constructed the recombinant adenovirus Ad35-SF via molecular cloning technique and examined its correctness by PCR analysis. Results of Western Blotting and ELISA assay indicated that the SF fusion protein could be successfully expressed and secreted in HEK293 post adenoviral infection.(3)Detection of the impact of CD47-blocking on Jurkat T cell and CIK by self-expression of SF fusion proteinResults of Western Blotting and ELISA assay indicated that the infected Jurkat T cell could correctly and effectively express and secret SF fusion protein, with a protein weight around 48 k D and a concentration at 19.1 μg/ml 48 hours post infection. Similar results were also found when Ad35-SF was utilized to infect CIK cells. FACS analysis indicated that the proportion of CD47-positive cells was dramatically decreased from 97.06 % to 15.32 % eighteen hours post infection at MOI=1 and further decreased to 2.94 % and 1.73 %, if MOI was increased to 5 and 10, respectively. Such a CD47 blocking was also showed in CIK cells, that the CD47-positive proportion was decreased from 71.87 % to 9.3 % forty-eight hours post infection at MOI=5.CCK8 assays were used to detect the effect of CD47 blocking on Jurkat T cell proliferation as well as their capacity to kill cancer cells. The results indicated that, relative to the blank control, the proliferation of Jurkat T cells expressing SF protein was increased 29 %(p<0.01) and the killing effect to hepatocellular carcinoma cell line Hep3 B was increased 25.6 %(p<0.01) 48 hours post infection.In summary, these results indicated that the constructed adenovirus Ad35-SF can efficiently infect Jurkat T cells and express SF fusion protein and thereby blocking the cell surface marker CD47 in situ. Subsequently, either the proliferation or the capacity of Jurkat T cells to kill hepatocellular carcinoma cells can be significantly improved.