OCs protect MM cells against T-cell–mediated cytotoxicity by upregulating expression of multiple co-inhibitory molecules. (A) OCs and MM-specific CTLs were generated from the same healthy donor. CTLs were cocultured with target cells (KMS28-BM) in the absence or presence of OCs with or without the PD-L1 inhibitor (10 μg/mL)/IDO inhibitors (1-methyl-dl-Trp, 1 mM). After 4 hours, cytotoxicity was evaluated by measuring LDH activity in the supernatants. Shown is mean ± SD of the 3 representative independent experiments. (B) Proliferation of T cells stimulated by anti-CD2/CD3/CD28 beads (T:Bead ratio of 1:1) in the absence or presence of autologous OCs for 5 days was measured with CFSE dilution assay. (C-D) CD14⁺ monocytes were cocultured with RANKL and M-CSF for 14 days, and OCs were identified by TRAP staining. OCs were also cocultured with IFN-γ (20 IU/mL) for 12 hours. Protein expression in monocytes and OCs were determined by immunoblotting (C) and immunofluorescence (D). (E) T cells stimulated by anti-CD2/CD3/CD28 beads (T:Bead ratio of 1:1). Expression of PD-1, CD200R, Tim-3, and BTLA were examined by flow cytometry. (F) OCs were cultured from PBMCs or BM mononuclear cells from MM patients without CD14 selection. Levels of inhibitory molecules were significantly higher in OCs than in MM cells. (G) IHC analysis of 2 representative BM specimens from MM patients shows PD-L1 expression (brown) on OCs. Original magnification: ×20 (×100 in insets). (H) The expression of PD-L1, IDO, Galectin-9, and CD200 in CD138⁺ cells, CD138⁻ cells, and OCs from the same patient was determined by immunoblotting and real-time qRT-PCR. *P < .05; **P < .001; by unpaired 2-sided Student t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICOSL, inducible T-cell co-stimulator ligand; IHC, immunohistochemistry; PB, peripheral blood.