CD16 and Siglec expression refine the phenotypic heterogeneity of Steady State Myeloid Derived Suppressor Cells

Chris D St. Laurent¹Zeinab Jame-Chenarboo¹Alyssa E Beck²Stacy Stubblefield²Shiteng Duan³Darren Sigal^2*Matthew S Macauley^1*

• ¹University of Alberta, Edmonton, Canada
• ²Scripps Clinic, La Jolla, California, United States
• ³The Scripps Research Institute, La Jolla, California, United States

Background: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) mediate cancer immune suppression by promoting an immunosuppressive microenvironment that inhibits effective anti-tumor immunity. However, they are still a poorly understood, heterogeneous mix of neutrophil subsets. This study aims to determine the Siglec expression profile on several neutrophil subsets and assess their immunosuppressive ability. Methods: We identified CD16 high and CD16 low neutrophil subsets from the low-density fractions of human peripheral blood and compared them to high-density neutrophils. We profiled the expression of the entire family of Siglecs on these three key neutrophil populations under steady state conditions in healthy subjects as well as cancer patients. Moreover, the ability of these populations, isolated from healthy subjects, to suppress T cell proliferation was assessed. Results: Two distinct sub-populations were investigated within the low-density fraction of human peripheral blood (CD15 + CD66b + CD16 low and CD15 + CD66b + CD16 high ) and compared to highdensity neutrophils (CD15 + CD66b + CD16 high ). We found that in addition to CD33 (Siglec-3), Siglec-5/-14, -7, and -9, are differentially expressed on the low-density CD16 low and CD16 high subsets in both healthy, steady state subjects, and cancer patients. Upregulated expression of CD33 on the CD16 low cells led to the initial speculation that they are MDSCs. As the differential expression of Siglec-9 between these two populations was striking, we used CD16 and Siglec-9 double staining to quantify these populations, which demonstrated that the CD16 low Siglec-9 low population is greatly upregulated in cancer patients. The CD16 high low-density and high-density neutrophils, but not the CD16 low low-density neutrophils from healthy subjects, inhibited T cell proliferation, indicating that the CD16 low Siglec-9 low population are not MDSCs. Conclusions: These results demonstrate that Siglecs are differentially expressed on neutrophil subsets, and along with CD16, may be used to help further define what is a PMN-MDSC. Consistent with current observations by others, PMN-MDSCs may encompass an array of neutrophil subtypes, including low-density neutrophils, and point to the need for more work to precisely define the genetic signatures of PMN-MDSCs.