miR-34a promotes the immunosuppressive function of multiple myeloma-associated macrophages by dampening the TLR-9 signaling

Abstract

Background: Promising outcomes have been observed in multiple myeloma (MM) with the use of immunotherapies, specifically chimeric antigen receptor T (CAR-T) cell therapy. However, a portion of MM patients do not respond to CAR-T therapy, and the reasons for this lack of response remain unclear. The objective of this study was to investigate the impact of miR-34a on the immunosuppressive polarization of macrophages obtained from MM patients.

Methods: The levels of miR-34a and TLR9 (Toll-like receptor 9) were examined in macrophages obtained from both healthy individuals and patients with MM. ELISA was employed to investigate the cytokine profiles of the macrophage samples. Co-culture experiments were conducted to evaluate the immunomodulatory impact of MM-associated macrophages on CAR-T cells.

Results: There was an observed suppressed activation of macrophages and CD4⁺ T lymphocytes in the blood samples of MM patients. Overexpression of miR-34a in MM-associated macrophages dampened the TLR9 expression and impaired the inflammatory polarization. In both the co-culture system and an animal model, MM-associated macrophages suppressed the activity and tumoricidal effect of CAR-T cells in a miR-34a-dependent manner.

Conclusion: The findings imply that targeting the macrophage miR-34a/TLR9 axis could potentially alleviate the immunosuppression associated with CAR-T therapy in MM patients.

Keywords: CAR‐T; TLR9; macrophages; miR‐34a; multiple myeloma.

FIGURE 1

Suppressed activation of macrophages and CD4+ T lymphocytes in the blood samples of MM patients. (A). ELISA analysis of TNF‐α、IL1β (M1‐type cytokines) and IL‐10 and TGF‐β1 (M2‐type cytokines) in the macrophages from the healthy controls and MM patients. (B). Western blot analysis of M2 marker CD206 and M1 marker iNOS in the macrophages isolated from the healthy controls and MM patent samples. (C). ELISA analysis of IL‐2 and IFN‐γ in the CD4⁺ T cells isolated from the healthy controls and MM patients. (D). Western blot analysis of the relative phosphorylation levels of LCK and ZAP70 in the CD4⁺ T cells isolated from the healthy controls and MM patients. (D) Data were summarized as mean ± SD from 10 clinical samples in each group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

The upregulation of miR‐34a in macrophages from MM patients suppresses TLR9 expression. (A). qRT‐PCR analysis of miR‐34a in the CD4⁺ and CD8⁺ T cells isolated from the healthy controls and MM patients. (B). qRT‐PCR analysis of miR‐34a in the macrophages isolated from the healthy controls and MM patients. (C). Bioinformatics prediction of the interaction sites between using the TargetScan resources, we found that there were potential interaction sites between miR‐34a and TLR9 mRNA 3'UTR using TargetScan database. (D). Dual luciferase reporter assay using WT and MUT reporter in the presence of miR‐NC (miRNA mimic control) or miR‐34a mimic. (E). qRT‐PCR and (F). Western blot analysis of TLR9 in the macrophages isolated from the healthy controls and the MM patients. (G). Western blot and qRT‐PCR analysis of TLR9 expression the transfection of miR‐34a mimic or miR‐NC. (H). Western blot and qRT‐PCR analysis of TLR9 expression the transfection of miR‐34a inhibitor or inh‐NC (inhibitor control). Data in A, B, and E were summarized as mean ± SD from 10 clinical samples in each group. Data in D, F, G and H were summarized from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

The miR‐34a dependent TLR9 downregulation mediates the inflammatory activation of macrophages. The macrophages isolated from the MM patients were transfected with miR‐NC or miR‐34a inhibitor. TLR9 agonist CpG ODN was also applied in the macrophages to examine the effect on the production of inflammatory cytokines. (A). qRT‐PCR analysis of miR‐34a expression levels. (B). Western blot analysis of TLR9, the phosphorylation levels of IKKβ and the protein levels of IkB‐α. (C). ELISA analysis of TNF‐α and IL1β in each experimental group. (D). RT‐qPCT analysis of M1 marker genes (CD86, iNOS, and IL‐6) and M2 marker genes (Arg1, CD163, and CD206). Data were summarized as mean ± SD from 3 independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

Macrophages from the MM patients dampen the activation of CAR‐T in a miR‐34a dependent manner. The macrophages from MM patients were transfected with miR‐34a inhibitor or treated with TLR9 agonist, and then introduced into co‐culture system with CAR‐T cells targeting MM cells. (A). CCK‐8 proliferation assay in the CAR‐T cells. (B). ELISA analysis of IFN‐γ、IL‐2 and TNF‐α. (C). Western blot analysis of TCR‐dependent signaling (phosphorylation of LCK and ZAP70). Data were summarized as mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Macrophages from the MM patients suppress the cytotoxic effect of CAR‐T cells on MM cells in a miR‐34a/TLR9‐dependent manner. CAR‐T cells after the co‐culturing with different groups of macrophages (with inhibitor NC, miR‐34a inhibitor or TLR9 agonist) were plated with the MM cell line MM.1S. (A). LDH cytotoxicity assay and (B) apoptosis analysis by flow cytometry. Data were summarized as mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

Macrophages from the MM patients suppress the anti‐tumorigenic effect of CAR‐T cells on MM cells. Nude mice were injected with MM.1S cells (control group), MM.1S/CAR‐T cells (CAR‐T group), MM.1S/CAR‐T cells and macrophages (CAR‐T + M group), MM.1S/CAR‐T cells, macrophages and miR‐34a inhibitor (CAR‐T + M + miR‐34a inh group) or MM.1S/CAR‐T cells, macrophages and TLR9 agonist (CAR‐T + M + CpG ODN group). (A). The images of xenograft tumors in each experimental group. (B). The summary of xenograft tumor weight in each experimental group. (C). TUNEL staining in the xenograft tumor tissues. The detection of CAR gene in the blood samples (E) and tumor tissues (F). Data in B, C, D, and E were summarized as mean ± SD from 5 xenograft samples in each experimental group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.