Abstract
Introduction: Myeloid differentiation primary response gene 88 (Myd 88) is an important adaptor molecule for the activation of NADPH oxidase and regulation of arginase-1, which are responsible for the suppressive function of myeloid-derived suppressor cells (MDSCs). Blockade of Myd88 signaling induces antitumor effects in mice by skewing the immunosuppressive function of myeloid-derived suppressor cells. As the PD-L1/PD-1 axis has been characterized as a potent inhibitor of immune activation, particularly through inhibition of effector T cell function, we characterized the effect of Myd88 on checkpoint expression on tumor-infiltrating MDSC/T cells in a murine model melanoma.
Methods: Pathogen-free 8-10-week-old WT(B6-background) and Myd 88−/− mice that been backcrossed to a C57BL/6 genetic background were challenged with 1 × 106 B16 (F1) tumor cells s.c. On day 14, mice were sacrificed and spleen and tumors were removed and digested into single-cell suspensions, blocked with anti-FcR mAbs and analyzed for surface and intracellular staining by flow cytometry. We analyzed CD11b+/Gr-1+hi/int myeloid cells subsets and T cells in the blood, spleen and tumors of mice by flow cytomery.
Results: The growth of B16 melanoma tumor was significantly slower in Myd 88−/− mice compared with WT mice. No significant difference between two groups was found in the frequency of absolute number of MDSC subsets and expression of PDL1 check-point marker on spleen-derived MDSC subsets. In contrast CD4(+) and C8(+) T cells residing in spleens of Myd88(-/-) mice showed increased expression of TNF-α/IFN-α and GrZB compared with T cells from wild-type mice following short-term activation with PMA/iono. Of note, the frequencies and absolute numbers of infiltrating CD11b+/Gr1+ MDSC in tumor-bearing Myd 88−/− mice were lower than those in WT mice. Also we found that viable CD11b+/Gr1+ MDSC subsets from WT mice expressed higher level of PD-L1 compared with MDSCs from Myd 88−/− mice in concordance with the reduced expression of PD-1 on tumor infiltrating CD4+ T cells in Myd 88−/− mice. Collectively, the profile of PD-L1 and PD-1 expression in tumor microenvironments is favorably altered to enhance adaptive immune response in myd 88 KO vs WT mice harboring B16 melanoma.
Conclusion: The results of this study provide further evidence that blocking Myd 88 signaling increases anti tumor immunity against melanoma, and that the enhanced immunity can be explained, in part, by reduction of expression PDL1/PD1 immune checkpoint molecules. Considering the importance of tumor-infiltrating MDSCs in regulating anti tumor immunity in the tumor microenvironment, our findings could provide insight into the design of new therapeutics targeting Myd 88. Further experiments are needed to show how alteration in profile of PDL1 checkpoint expression on MDSCs influences anti-tumor T cell responses.
Disclosures
No relevant conflicts of interest to declare.
The proportion of myeloid-derived suppressor cells in the spleen is related to the severity of the clinical course and tissue damage extent in a murine model of multiple sclerosis
