Fig. 5. CD16/32 is a specific marker that defines bona fide immunosuppressive PMN-MDSCs.

(A and B) Violin plot showing the expression of the Fcgr3 (A) or Fcgr2b (B) gene in each granulocytic cluster infiltrating the wtIDH1 tumor (C7) or mIDH1 tumors (C1, C2, and C3). Fcgr3, but not Fcgr2b, is expressed at high level in both immunosuppressive granulocytic MDSCs clusters (C7 and C1). (C) Schematic of the in vitro T cell proliferation assay to analyze immune suppressive properties of CD45^high/CD11b⁺/Ly6G⁺/CD16/32^−/+. Sorted CD45^high/CD11b⁺/Ly6G⁺/CD16/32-positive or CD45^high/CD11b⁺/Ly6G⁺/CD16/32-negative cells were cocultured with CFSE-labeled splenocytes from Rag2/OT-1 transgenic mouse. Cultures were stimulated with 100 nM SIINFEKL peptide for 4 days, after which proliferation was analyzed by flow cytometry. (D) Representative flow plots showing CFSE staining of unstimulated splenocytes (T only), splenocytes undergoing rapid proliferation in response to SIINFEKL (T + SIIN), and the effect of SIINFEKL-induced T cell proliferation in the presence of CD45^high/CD11b⁺/Ly6G⁺/CD16/32-negative or CD45^high/CD11b⁺/Ly6G⁺/CD16/32-positive cells from the TME of mIDH1 tumors. (E) Quantitation analysis of the inhibitory potential of CD45^high/CD11b⁺/Ly6G⁺/CD16/32-negative or CD45^high/CD11b⁺/Ly6G⁺/CD16/32-positive cells sorted from TME of mIDH1 tumor. CD45^high/CD11b⁺/Ly6G⁺/CD16/32-positive cells inhibit T cell proliferation, whereas CD45^high/CD11b⁺/Ly6G⁺/CD16/32-negative cells did not suppress T cell proliferation. *P < 0.05, **P < 0.01, and ***P < 0.005, one-way ANOVA.