Study On Immunoregulatory Function Of Neuropilin-1 Positive T Cells

Background:Neuropilin-1(Nrp-1/NP1/Npn-1)gradually becomes one hot spot in immunology and oncobiology in recent years.Nrp-1 has been found to be expressed on some immunocytes such as DC(Dendritic Cells)and T cell,etc. It was thought to be one new surface marker of CD4~+CD25~+ regulatory T cells(CD4~+CD25~+Treg),but the characteristics of Nrp-1~+T cells,such as immunoregulatory function,were rarely reported.Nrp-1 is also expressed on many kinds of tumor cells and plays an important role in the tumor biological behaviors such as proliferation and metastasis.As Treg plays an important role in tumor immunity and is one hot spot at present studies,it's worthy to explore the relation between Treg and tumor immunity by a bridge effect of Nrp-1.Objective:Distribution characteristics of Nrp-1 on lymphocytes and T cells,the relation between Nrp-1~+T cell and classical CD4~+CD25~+Treg and their comparison of immunoregulatory function in tumor immunity.To verificate whether Nrp-1 can be one new surface marker of Treg and accordingly whether Nrp-1~+T cell is one new type of Treg,the potential mechanism of immunoregulatory function of Nrp-1~+T cell in tumor immunity was also initially analysed.Methods:By using flow cytometry,distribution characteristics of Nrp-1 on lymphocytes and T cells,its relation with classical CD4~+CD25~+Treg was analysed.Culture method of mice B16-F10-1uc-G5 melanoma cells in vitro was established,function of Nrp-1 and its ligands in proliferation of melanoma cells was analysed by flow cytometry after labelled by CFDA-SE and preincubated with blocking antibodies.By advanced living image system,immunoregulatory function of Nrp-1~+T cell on NK' killing melanoma cells in vitro was studied and was compared with that of classical CD4~+CD25~+Treg.Regulatory function of Nrp-1~+T cell in tumor immunity of B16-F10-1uc-G5 melanoma was analysed in vivo.Results:Proportion of Nrp-1~+T cells in CD4~+CD25~+Treg was significantly higher than that in CD4~+CD25-T cells in mice splenic lymphocytes(P＜0.01). Proportion of CD4~+CD25~+Treg in Nrp-1~+T cells was higher than that in Nrp-1~-T cells(P＜0.01).Tumor cells number/tumor volume had fine linear correlation with photons number.The proliferation index of mice B16-F10-1uc-G5 melanoma cells decreased after Nrp-1 and VEGF165 were blocked and the difference was significant between blocking and nonblocking group(P＜0.01). In study of killing effect of NK cells on melanoma cells in vitro,the melanoma cells number of Nrp-1 group was significantly higher than that of CD4~+CD25~+Treg group at every time spot(P＜0.01).The tumor volume of the Nrp-1 group was larger than that of the control group(P＜0.01).Conclusion:Nrp-1~+T cell is one brand-new subgroup of T cells which has close relation with classical CD4~+CD25~+Treg while is different both in quantity and function.Both in vitro and in vivo bioluminescence imaging are easily handling,direct-viewing and the result is reliable.Both Nrp-1 and VEGF165 are essential for promotion of proliferation of mice B16-F10-1uc-G5 melanoma cells. Nrp-1~+T cell can supress the immune effect aimed at mice B16-F10-1uc-G5 melanoma cells both in vitro and in vivo.It posseses more powerful negtive immunoregulatory function compared with classical CD4~+CD25~+Treg and this negtive role might be played through mechanisms concerned with interactions of Nrp-1~+T/Teff(Effective T cells),Nrp-1~+T/NK,Nrp-1~+T/DC or even Nrp-1~+T/melanoma cells directly.Nrp-1 may be one new surface marker of Treg and accordingly Nrp-1~+T cell may be one new type of Treg which plays its negtive immunoregulatory function through multiple mechanisms.