Figure 4. CD84 upregulates PD-L1 expression in MDSCs.

(A and B) Sorted primary M-MDSCs (CD14⁺, CD15^–, CD11b⁺, HLA-DR^–) (A) and G-MDSCs (CD15⁺, CD14^–, CD11b⁺, HLA-DR^–) (B) from BM aspirates of MM samples were analyzed for PD-L1 message by qPCR. (C–F) Sorted (mRNA) or whole BM (protein) primary M-MDSCs (LY6C⁺, LY6G^–, CD11b⁺, CD11C^–) (C and E), and G-MDSCs (LY6G⁺, LY6C^lo, CD11b⁺, CD11C^–) (D and F) from BM aspirates taken from 5TGM1-injected mice were incubated in the presence of anti-CD84 (B4) blocking or isotype control antibodies and analyzed for PD-L1 mRNA (C and D) and protein (E and F) by qPCR or FACS analysis, respectively (n = 4). Representative histograms are shown in E and F. (G and H) Sorted M-MDSCs (G) and G-MDSCs (H) were treated with anti-CD84 B4 inhibitory or control antibody and cocultured for 72 hours with WT whole spleens at a ratio of 1:4, and the percentage of dividing CD8⁺ T cells and IFN-γ was analyzed thereafter (n = 4–7, *P < 0.05). (I) LentiCRISPR-v2 CRISPR/Cas9 system was established in the THP1 cell line with constitutive KO of CD84 expression, and protein levels of CD84 and PD-L1 were determined by flow cytometry (n = 3). (J) THP1 cells were activated with anti-CD84 or control antibodies, and CD84, p-mTOR, pAKT, pS6, and PD-L1 expression levels were determined by Western blotting analysis. *P < 0.05, **P < 0.01 with either 1-tailed (B, F, E, and G) or 2-tailed unpaired or paired 2-tailed t test for pairwise comparisons.