CXCL6-CXCR2 axis-mediated PD-L2+ mast cell accumulation shapes the immunosuppressive microenvironment in osteosarcoma

Abstract

Osteosarcoma (OS) is the most common primary bone malignancy and has a high propensity for local invasion and metastasis. The tumour microenvironment of OS is infiltrated by a large number of immune cells, which play a crucial role in its progression and prognosis. Mast cells are important innate immune cells in the tumour stroma and exhibit different phenotypes in diverse tumour microenvironments. However, the underlying mechanisms of mast cell accumulation and the phenotypic characteristics of mast cells in OS remain poorly understood. In this article, we found for the first time that mast cell accumulation in osteosarcoma tissue was modulated by the CXCL6-CXCR2 axis and that the number of infiltrating mast cells was significantly greater in tumour tissues than in adjacent nontumour tissues. These tumour-infiltrating mast cells express high levels of the immunosuppressive molecule PD-L2, and survival analyses revealed that patients in the PD-L2⁺ high-expression group had a worse prognosis. In vitro, mast cells were induced to express PD-L2 in a time- and dose-dependent manner using OS tissue culture supernatants to mimic the tumour microenvironment. Mechanistic studies revealed that tumour cell-derived G-CSF significantly induced mast cell PD-L2 expression by activating STAT3. Importantly, mast cells overexpressing PD-L2 inhibit tumour-specific CD8⁺ T-cell proliferation and tumour-killing cytokine secretion, which is reversed by blocking PD-L2 on mast cells. Therefore, our findings provide new insight into the immunosuppressive and tumorigenic roles of mast cells, as well as a novel mechanism by which PD-L2-expressing mast cells mediate immune tolerance.

Keywords: Immunosuppressive; Mast cells; Osteosarcoma; PD-L2.

Fig. 1

Mast cells accumulate in OS with disease progression and predict poor patient survival. (A) Representative analysis and Cumulative results of mast cell (CD45⁺ CD117⁺ FcεRI⁺ cell) gating on CD45⁺ cells in OST and ANT. (B) Representative analysis of tryptase⁺ (red) mast cell distributions in OST and ANT by immunofluorescence staining. Scale bars: 100 μm(C)Dot plots of surface molecule staining for mast cells gating in CD45⁺ cells among TNM stages with OS. Cumulative results from 54 OS patients were shown. (D)Kaplan-Meier plots for overall survival and disease-free survival by median mast cell percentage (4.185 %). The horizontal bars in panels A and C represent mean values. Each ring or dot in panels A and C represents 1 patient. OST, osteosarcoma tissues and ANT, adjacent non-tumor tissues. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Chemotaxis mediated by CXCL6-CXCR2 increases mast cell recruitment in OS. (A) OST and ANT mast cells stained for Ki-67. Red and blue histograms show Ki-67 staining respectively, while black represents isotype control. (B) Mast cell expression of CXCR2 molecule in OST and ANT. Red and blue histograms show CXCR2 staining respectively, while black represents isotype control. (C) Based on percentages of mast cells in CD45⁺ cells and CXCL6 concentrations in OST, a correlation between mast cells and CXCL6 was analyzed in OS. (D) CXCL6 concentration between autologous OST and ANT (n = 54) or between autologous OSCS and ANCS (n = 10) was analyzed. (E) Migration of tumor-infiltrating mast cells was assessed by transwell assay as described in Materials and methods and statistically analyzed (n = 3). (F) SCF concentration between autologous OST and ANT (n = 27). The horizontal bars in panels D and F represent mean values. Each dot in panels C, D, E and F represents 1 patient. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Expression of the immunosuppressive molecule PD-L2 is upregulated on mast cells infiltrated in osteosarcoma. (A) Representative analysis and statistics analysis of molecule PD-L2 on mast cells in OST and ANT (n = 3). (B) The expression of PD-L2 on hCBMCs after 12 h of exposure to 50 % autologous OSCS and ANCS. (C) HMC-1 cells stimulated by 50 % OSCS and autologous ANCS for 12 h were immunofluorescence stained for PD-L2. Red represents PD-L2, blue represents DAPI-stained nuclei. Scale bar is 50 μm. (D) PD-L2 expression exposed to 50 % OSCS for 3, 6, 12 h. (E) PD-L2 expression exposed to 10 %,20 %, 50 % OSCS for 12 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Mast cell PD-L2 expression is induced by OS-derived G-CSG via STAT3 pathway. (A) G-CSF production between OSCS and autologous ANCS was detected (n = 10). (B) An analysis conducted on the correlation between G-CSF concentration and PD-L2⁺ mast cells in OST. (C) PD-L2 expression on mast cells exposed for 12 h to OSCS and anti-G-CSF antibodies. (D) PD-L2 expression on mast cells exposed for 12 h to ANCS with recombinant G-CSF. (E) Western blots of mast cells treated with OSCS, ANCS, and OSCS with anti-G-CSF antibody or control IgG for 12 h revealed the presence of STAT3 and p-STAT3 (the uncropped version was referred in Supplementary Fig. 6). (F) PD-L2 expression on mast cells exposed for 12 h to OSCS with signaling pathway inhibitor AG490.

Fig. 5

Infiltrating mast cells in OS inhibit CD8⁺ T cell proliferation and antitumour immunity through PD-L2. (A and C) CFSE-labeled OS patients' peripheral CD8⁺ T cells were co-cultured with autologous mast cells from ANT or OST with anti-PD-L2 antibody or control IgG for 5 days. Representative and statistical analysis of CD8⁺ T cell proliferation and IFN-γ production were shown (n = 3). (B and D) A co-culture of CFSE-labeled peripheral CD8⁺ T cells and ANCS or OSCS-conditioned hCBMCs with or without anti-PD-L2 antibody was carried out for 5 days. Representative and statistical analysis of CD8⁺ T proliferation and IFN-γ production were shown (n = 3).

Fig. 6

Model for cross-talks among mast cells, tumor cells, and CD8⁺ T cells in the OS environment that results in PD-L2⁺ mast cell-mediated immunosuppression and tumor progression.

figs1

figs2

figs3

figs4

figs5

figs6