Background: Myeloid derived suppressor cells or MDSC are a heterogeneous population of bone marrow-derived cells that consist of myeloid progenitor and immature. Cancer-induced pro-inflammatory signals recruit MDSC from the bone marrow and maintain them in an undifferentiated state. MDSCs inhibit T cell proliferation via up regulation of iNOS and ROS thereby suppressing antitumor immune responses. MDSC have also been shown to promote tumor growth by stimulating angiogenesis and other mechanisms. TGF-b1 is a pleiotropic cytokine abundantly expressed in the tumor microenvironment with diverse effects on myeloid, lymphoid and tumor cells. The aim of this study is to determine the effect of TGF-b1 in the generation and function of MDSC, including its effects on T cell proliferation and tumor growth.
Methods: Ex vivo MDSC generation: Bone marrow progenitor cells were derived from WT C57bl/6 mice and co-cultured with MTEC (mouse tonsil epithelial cells transformed with HPV16 E6+E7 oncogenes and H-Ras ) tumor supernatants in the presence or absence of TGF-β1 for 5 days at 370C. Cells were then harvested, processed into single cell suspensions, and stained for MDSC surface markers, DAF-DA (to determine nitric oxide levels),iNOS and other functional markers analysis was performed by flow cytometry (FACS).
MDSC functional assay: A) T cell proliferation assay MDSCs were generated in the presence or absence of TGF-β1 with supernatants from MTEC cells and co-cultured with CFSE labeled T cells activated with anti CD3 and anti CD28 antibodies. T cell proliferation was measured by using CFSE dilution, which was analyzed by flow cytometry.
B) Effect of MDSC on tumor growth Control and TGF-β1 conditioned MDSC were co-cultured with MTEC tumor (grown as spheroids) for 72 hrs at the end of which histological sections of the spheroids were prepared and analyzed for tumor proliferation by Ki-67 staining. Sorted MDSCs were also co-cultured with T-hep3 cells grown in a monolayer (human oral cancer line) and tumor growth was determined by flow cytometry.
Results: While control MDSC suppressed T cell proliferation in a dose-dependent fashion, we observed that TGF-β1 primed MDSCs lost the ability to inhibit T cell proliferation. Further, TGF-β1 primed MDSC inhibited tumor growth in an ex vivo co-culture system. Histological sections of tumor spheroid / MDSC co-cultures revealed diminshed ki-67 expression in spheroids cultured with TGF-B1 conditioned MDSC compared to control. Upon further examining the cellular mechanism, it was seen that TGF-β1 treated MDSCs down regulate iNOS expression and produced decreased amounts of nitric oxide compared to their control counterparts, without altering the expression of other MDSC functional markers like arginase, PD-1 and PD-L1.
Conclusions: We conclude that TGF-β1 reprograms MDSC via an iNOS/NO dependent mechanism to a) T cell suppressive capacity and b) inhibit tumor cell growth. These observations have a direct translational implication wherein the inherent pro-tumor nature of MDSCs could potentially be reprogrammed with TGF-β1 and directed toward the tumor thereby suppressing tumor growth.
Citation Format: Padmini Jayaraman, Falguni Parikh, Rosemarie Krupar, Robin Parihar, Indu Varier, Andrew Sikora. TGF-beta1 primed myeloid derived suppressor cells decrease tumor growth and lose their ability to inhibit T cell proliferation via iNOS downregulation. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr PR005.
- ©2016 American Association for Cancer Research.