BACKGROUND:Cancer immunotherapy using immune checkpoint blockade (ICB) has revolutionized cancer treatment. However, patients with multiple myeloma (MM) rarely respond to ICB. Accumulating evidence indicates that the complicated tumor microenvironment (TME) significantly impacts the efficacy of ICB therapy. Therefore, investigating how TME components in MM influence ICB treatment is urgent. METHODS:We employed two well-established murine myeloma models, 5TGM1 and Vk*MYC, by intravenously injecting 5TGM1 or Vk*MYC cells into mice, respectively, to determine ICB therapeutic efficacy in MM. Total mouse IgG or Ig2b ELISA or QuickGel split beta SPE kits and in vivo bioluminescent imaging were used to monitor MM tumor burden. Cytometry by time of flight (CyTOF) was used to quantify MM TME components. T cell proliferation and function were detected using flow cytometry. Peptide-Fc fusion proteins were used to deplete myeloid-derived suppressor cells (MDSCs). MMDTR, Foxp3DTR, CD4 KO and CD8 KO mice were used to elucidate the underlying mechanisms. Gene expression levels in human MM were analyzed using Gene Expression Omnibus public datasets. RESULTS:We found that programmed cell death protein 1 (PD-1) antibody treatment had a therapeutic effect in 5TGM1 mice; it was ineffective in Vk*MYC mice. CyTOF indicated that the bone marrow (BM) of both models was inflamed, suggesting that immune suppressive cells might be inhibiting the reactivation of T cells in the BM. We observed higher numbers of MDSCs, regulatory T (Treg) cells, and tumor-associated macrophage (TAMs) in myeloma BM compared with that of tumor-free mice. Specifically, depleting MDSCs, but not Treg cells or TAMs, sensitized Vk*MYC mice and enhanced the response of 5TGM1 mice to PD-1 ICB, which was dependent on CD8+ but not CD4+ T cells. MDSCs, especially M-MDSCs and CD84+ MDSCs, significantly inhibited the activation and cytotoxic cytokine production of CD8+ T cells in vitro. Moreover, database profiling of patient BM revealed a negative correlation between MDSCs signature genes and cytotoxic CD8+ T cell signature genes, with post-maintenance patients with myeloma displaying a higher ratio of cytotoxic CD8+ T cell to MDSCs signature genes compared with pretreated patients. CONCLUSION:Our study highlights the potential of MDSCs depletion in enhancing the sensitivity of patients with myeloma to PD-1 ICB therapy.

Detailed experimental procedures of cell culture and shRNA-mediated knockdown, collection of Serum samples, analysis of published single-cell sequencing data of human cancers,Flow Cytometry, in vitro generation of TAMs,immunofluorescence microscopy,microarray analysis, construction of mouse lymphoma and multiple myeloma model.

Fig. S1, relates to Figure 1: Lipid accumulation in mouse TAMs. Fig. S2, relates to Figure 2: Expression of scavenger receptors in control MΦs and TAMs. Fig. S3, relates to Figure 2: Knockdown of Cd36 decreases lipid accumulation in MФs. Fig. S4, relates to Figure 3: Expression of fatty acid β-oxidation-responsible genes and the glucose metabolism in indicated MΦs. Fig. S5, relates to Figure 5: Ki-67 expression and cell cycle progression in different murine tumor cells cocultured with control MΦs or TAMs. Fig. S6, relates to Figure 6: The phosphorylation of JAK-STAT6 pathway in human MΦs and the production of IL-4 and IL-13 in human and mouse tumor cells. Fig. S7. Clinical significance of CD36 expression in TAMs. Fig. S8., relates to Figure 7: Effect of CD36 deficiency on the infiltration and differentiation of MΦs in vivo.

Supplementary Table 1. Primer sequences used for qPCR and shRNA construction.

Supplementary Table 2. Microarray dataset information