Discussion
At present, the most effective method of immunotherapy for NB is adoptive transfer chimeric GD2 antigen receptors CTL 6, 16. However, some phase III clinical trials found that some children with high-risk NB underwent adoptive cell transfer immunotherapy of chimeric GD2 antigen receptors CTL still appeared recurrence and metastasis although the survival rates have improved. This suggested this kind of passive immunotherapy need improve its curative effect by adjusting the immunosuppressive microenvironment 4, 16.
In tumor immune microenvironment, accumulation of a variety of immunosuppressive cells including MDSC formed the main immunosuppressive factors and resulted in immune tolerance and disability of the immune system 3. MDSC is a group of innate immune cells originated from myeloid which play a negative immune regulative role in tumor progression 17, 18. Some studies suggested MDSC inhibit the body’s natural immune by inhibiting DC, accelerating polarization of macrophages to M2, decreasing interleukin-12 and reducing function of NK cells. on the other hand, MDSC inhibit T cells adoptive immunotherapy through high expression of arginase-1, inducible nitric oxide synthase and reactive oxygen species 4, 19-21. Therefore, MDSC induced tumor immune tolerance and became the main impediment to immunotherapy.
The role of MDSC in neuroblastoma still remain unclear although the mechanism of MDSC in other tumors has been explicated too much. In the present study, neuroblastoma Ag-specific CTLs were stained by CFSE. The cytoplasm with fluorescent protein were evenly distributed to the next generation of cells and the fluorescence intensity reduced half when cells proliferated continuously. So, the more generations of cell division, the weaker of cell fluorescence intensity. Therefore, in this study, the fluorescence intensity of CTL became weaker and the number of CTL increased if CTL cultivated without MDSC. Contrarily, cells proliferation decreased and the fluorescence intensity unchanged if CTL cultivated with MDSC. The result fully showed MDSC can inhibit proliferation of neuroblastoma Ag-specific CTL obviously.
In further study, the expressions of CD3ζ and CD62L in Ag-specific CTLs decreased significantly when CTL cultivated with MDSC, but the two proteins raised again when DOX administration. As a chain of CD3 molecules, CD3ζ play a key role in signal transmission inside and outside the cell 22. The increased expressive activity of ζ chain will promote TCR identified immune signal transmission to intracellular which lead to activation of T cells and produce amounts of cytokines, such as IFN-γ, IL2, etc 23. Moreover, CD62L is an important molecule involving the extravasation of lymphocytes from the blood and lymphatics, and their homing to lymph nodes and tumors24-26. Therefore, the results indicated DOX could targeted relieve the inhibitory role of MDSC on CD3ζ and CD62L in Ag-specific CTL which can promote activation and migration of Ag-specific CTL and effectively kill neuroblastoma cells.
In recent years, some researches have pointed out CTL produce cytotoxicity to target cells by two separate ways, namely Perforin way and PCD way mediated by Fas antigen molecules 27-29. Of them, Perforin way was a major way and played a significant role in antiviral, intracellular bacteria, tumor and immune pathology, etc29-31. During the course of Perforin way, CTL killed tumor meanwhile the cytokines IL-2 and IFN-γ were released. The more obvious of the killing effect, the higher concentrations of the cytokine, and vice versa. In the present study, the killing rate of NB cells and the levels of IL-2 and IFN-γ in supernatant were all decreased significantly when CTLs cultivated with MDSC. However, this inhibitory role of MDSC can be effectively reversed by DOX administration. Some researches pointed out doxorubicin can selectively eliminates MDSCs and promots the activity of immune effector cells and improves the therapeutic profile of adoptively transferred helper T lymphocytes13, 32, 33. So, in this study, we demonstrated the underlying mechanism of targeting inhibition of myeloid-derived suppressor cells by doxorubicin to enhance antigen-specific cytotoxic T lymphocytes killing neuroblastoma cells in vitro.