Tumor-associated macrophages regulate the function of cytotoxic T lymphocyte through PD-1/PD-L1 pathway in multiple myeloma

Abstract

Background: Tumor-associated macrophages (TAMs) are originated from circulating mononuclear cells in peripheral blood. They result from the recruitment of tumor cells and are a vital constituent of the tumor microenvironment. TAMs may be involved in the immunological escape of vicious clonal plasma cells (PC) in the bone marrow (BM) of sufferers with myeloma.

Methods: From March 2020 to January 2021, 28 healthy controls (HC) and 86 multiple myeloma (MM) (53 newly diagnosed MM [NDMM] and 33 remissions) patients were enrolled as objects of the study. The expression of TAMs in the BM, CSF1 on CD138 + cells, and CSF1R on macrophages were detected by the method of flow cytometry, and the expression of PD-1 on CD8 + T cells and PD-L1 on TAMs were also done. Bone marrow mononuclear cells (BMMNCs) were extracted and cultured into TAMs, CD8 + T cells were sorted by magnetic beads and cultured, a coculture system was established and different inhibitors were added. The expression of the perforin and granzyme B was detected by flow cytometry.

Results: The percentage of TAMs in NDMM group (61.49 ± 2.176%) increased when compared with remission (23.08 ± 1.699%, p < 0.001) and HC group (17.95 ± 1.865%, p < 0.001), and TAMs decreased after adding CSF1R inhibitor. Moreover, the expression of CSF1 on CD138 + cells increased significantly in NDMM group (17.090 ± 0.9156%) than remission (8.214 ± 0.5911% p < 0.001), and HC group (5.257 ± 0.6231%, p < 0.001), and CSF1R on macrophages increased significantly in NDMM group (58.78 ± 2.286%) than remission (20.74 ± 1.376%, p < 0.001) and HC group (17.42 ± 1.081%, p < 0.001). The expression of PD-1 on CD8 + T cells in NDMM group (32.64 ± 2.982%) increased than remission (20.35 ± 2.335% p < 0.01) and HC group (17.53 ± 1.349%, p < 0.001), and PD-L1 on TAMs also increased in NDMM group (50.92 ± 2.554%) than remission (20.02 ± 1.893%, p < 0.001) and HC group (13.08 ± 1.289%, p < 0.001). When CD8 + T cells were cocultured with TAMs, the perforin and granzyme B levels decreased significantly. However, the perforin and granzyme B levels were partly restored after adding CSF1R inhibitor and anti-PD-L1 antibody.

Conclusion: Our study shows that TAMs were increased in MM patients which can inhibit the function of cytotoxic T lymphocyte (CTL) through the PD-1/ PD-L1 signaling pathway and participate in the occurrence of immune escape of myeloma cells.

Keywords: CD8 + T cells; CSF1R; PD-1/PD-L1; TAMs; multiple myeloma.

FIGURE 1

NDMM patients expressed more TAMs and CSF1R inhibitor can decrease the production of TAMs. (A, B, C, D) Representative flow cytometry scatter diagrams of TAMs, CSF‐1, CSF1R in BM of MM and HC groups were shown. (E) The percentage of TAMs in NDMM group was remarkably higher than remission and HC group and the difference between remission and HC group was significant. (F) The expression of CSF1 on CD138 + cells in NDMM group was significantly increased than remission and HC group, and the difference between remission and HC group was significant. (G) The expression of CSF1R on macrophages in NDMM group was higher than remission and HC group, while there was no significant difference between remission and HC group. (H) The percentage of TAMs in NDMM group was decreased after adding CSF1R inhibitor. All the data were shown as mean ± SD. p values were obtained by using unpaired t‐test and p<0.05 was considered significant

FIGURE 2

The expression of PD‐1 on CD8 + T cells and PD‐L1 on TAMs in MM and HC groups were detected. (A, B) Representative flow cytometry scatter diagrams of PD‐1 on CD8 + T cells and PD‐L1 on TAMs of MM and HC groups were shown. (C) The expression of PD‐1 in NDMM increased significantly than remission and HC groups, but there was no significant difference between the remission and HC group. (d) The expression of PD‐L1 on TAMs was higher in NDMM group when compared to remission and HC group and there was a significant difference between remission and HC group. All the data were shown as mean ± SD. p values were obtained by using unpaired t‐test and p<0.05 was considered significant

FIGURE 3

CSF1/CSF1R signaling pathway promotes the formation of TAMs, then TAMs inhibit CTL function through PD‐1/PD‐L1 signaling pathway. The flow chart above shows the expression of perforin and granzyme B in five groups of CD8 + T cells. Statistical analysis showed the functional molecules of CD8 + T cells with TAMs (group B) were lower than CD8 + T cells alone (group A). CTL function was improved after adding CSF1R inhibitor (group D) or anti‐PD‐L1 antibody (group C). And CTL function was improved significantly after adding anti‐PD‐L1 antibody and CSF1R inhibitor together (Group E) and there was a distinction among groups E, C and D. All the data were shown as mean ± SD. p values were obtained by using unpaired t‐test and p<0.05 was considered significant

FIGURE 4

In the bone marrow microenvironment of MM patients, myeloma cells can secrete a large number of CSF1 molecules, recruit peripheral blood circulating monocytes and in situ macrophages to gather around the tumor, and combine with the CSF1R on the surface of macrophages. Under these circumstances, the phenotype and function of macrophages changed, which we called TAMs. High expression of PD‐L1 on TAMs combined with PD‐1 on CTL cells in the tumor microenvironment results in the immune escape of tumor cells. However, when CSF1/CSF1R pathway and PD‐1/PD‐L1 pathway were blocked, the function of effector T cells can be partially or completely restored, providing a new basis for targeted therapy