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.