Bone marrow-derived mesenchymal stem cells inhibit CD8+ T cell immune responses via PD-1/PD-L1 pathway in multiple myeloma

Abstract

High expression of the inhibitory receptor programmed cell death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found to play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the programmed cell death 1 (PD-1)/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in multiple myeloma (MM) remains poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSC-mediated regulation of CD8⁺ T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased compared to that in normal controls (NC) (18·81 ± 1·61 versus 2·78± 0·70%; P < 0·001). Furthermore, the PD-1 expression on CD8⁺ T cells with NDMM patients was significantly higher than that in normal controls (43·22 ± 2·98 versus 20·71 ± 1·08%; P < 0·001). However, there was no significant difference in PD-1 expression of CD4⁺ T cells and natural killer (NK) cells between the NDMM and NC groups. Additionally, the co-culture assays revealed that BMSCs significantly suppressed CD8⁺ T cell function. However, the PD-L1 inhibitor effectively reversed BMSC-mediated suppression in CD8⁺ T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8⁺ T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8⁺ T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8⁺ T cells to promote the immune escape of MM.

Keywords: CD8+ T cells; PD-1/PD-L1; bone marrow mesenchymal stem cells (BMSCs); multiple myeloma (MM); pomalidomide.

Fig. 1

Culture and identification of bone marrow mesenchymal stem cells (BMSCs) from normal control (NC) and newly diagnosed multiple myeloma (NDMM) patients. BMSCs were isolated from the bone marrow of NC (n = 26) and NDMM patients (n = 48). (a) The morphology of BMSCs subsequent to being passaged for 72 h of normal controls (P3, ×40; scale bar: 100 μm). (b) The morphology of BMSCs subsequent to being passaged for 72 h of NDMM patients (P3, ×40; scale bar: 100 mm). The cell surface markers (c) CD34, (d) CD45, (e) CD73, (f) CD90 and (g) CD105 of BMSCs were detected by flow cytometer (FCM). Each experiment was performed at least three times. All the BMSCs used in subsequent experiments were third‐generation.

Fig. 2

Programmed cell death ligand 1 (PD‐L1) expression in bone marrow mesenchymal stem cells (BMSCs) and programmed cell death 1 (PD‐1) expression in natural killer (NK), CD4⁺ T and CD8⁺ T cells. (a) Representative flow cytometry scatter diagrams of PD‐L1 expression on BMSCs are shown in normal control or newly diagnosed multiple myeloma (NDMM) patients. (b) The dot diagrams represent the expression of PD‐L1 on BMSCs between normal control (NC) and NDMM groups. NDMM: n = 40, NC: n = 28. ***P < 0·001. (c,d) The representative flow cytometry scatter diagrams and the dot diagrams present the percentage of PD‐1⁺ cells in NK cells, CD4⁺ T cells and CD8⁺ T cells with NC and NDMM patients; n = 23. ***P < 0·001.

Fig. 3

Bone marrow mesenchymal stem cells (BMSCs) decrease the expression of perforin, granzyme B in CD8⁺ T cell while programmed cell death ligand 1 (PD‐L1) inhibitor counteracts BMSCs‐mediated inhibition of CD8⁺ T cells. Representative flow cytometry scatter diagrams of the perforin and granzyme B expression of CD8⁺ T cells in the three groups are shown. (a–d) The box‐plot represents the perforin and the granzyme B expression of CD8⁺ T cells from newly diagnosed multiple myeloma (NDMM) patients co‐cultured with BMSCs from NDMM patients or PD‐L1 inhibitors, respectively. (a) CD8⁺ T cells alone; (b) CD8⁺ T cells co‐cultured with BMSCs; (c) CD8⁺ T cells + BMSCs + PD‐L1 inhibitor; n = 11. **P < 0·01, ***P < 0·001.

Fig. 4

Effects of CD8⁺ T cells on multiple myeloma (MM) U266 cell apoptosis. MM U266 cells were co‐cultured with different treatment groups of CD8⁺ T cells (from autologous sources). (a) CD8⁺ T cells co‐cultured with bone marrow mesenchymal stem cells (BMSCs); (b) CD8⁺ T cells + bone marrow mesenchymal stem cells (BMSCs) + programmed cell death ligand 1 (PD‐L1) inhibitor; (c) CD8⁺ T cells + BMSCs + pomalidomide; (d) CD8⁺ T cells + BMSCs + PD‐L1 inhibitor + pomalidomide). After 72 h of co‐culture, apoptosis assay was performed at least three times to evaluate the effects of CD8⁺ T cells on the apoptosis of U266 cells. (a) Representative flow cytometry scatter diagrams of MM U266 cells in the four groups are shown. (b) The histogram represents the apoptosis of MM U266 cells in the four groups; n = 11. ***P < 0·001, n.s. = not significant.