High expression of ID1 in monocytes is strongly associated with phenotypic and functional MDSC markers in advanced melanoma

Abstract

The efficacy of immunotherapies for malignant melanoma is severely hampered by local and systemic immunosuppression mediated by myeloid-derived suppressor cells (MDSC). Inhibitor of differentiation 1 (ID1) is a transcriptional regulator that was shown to be centrally involved in the induction of immunosuppressive properties in myeloid cells in mice, while it was overexpressed in CD11b⁺ cells in the blood of late-stage melanoma patients. Therefore, we comprehensively assessed ID1 expression in PBMC from stage III and IV melanoma patients, and studied ID1 regulation in models for human monocyte differentiation towards monocyte-derived dendritic cells. A highly significant elevation of ID1 was observed in CD33⁺CD11b⁺CD14⁺HLA-DR^low monocytic MDSC in the blood of melanoma patients compared to their HLA-DR^high counterparts, while expression of ID1 correlated positively with established MDSC markers S100A8/9 and iNOS. Moreover, expression of ID1 in monocytes significantly decreased in PBMC samples taken after surgical removal of melanoma metastases, compared to those taken before surgery. Finally, maturation of monocyte-derived DC coincided with a significant downregulation of ID1. Together, these data indicate that increased ID1 expression is strongly associated with expression of phenotypic and immunosuppressive markers of monocytic MDSC, while downregulation is associated with a more immunogenic myeloid phenotype. As such, ID1 may be an additional phenotypic marker for monocytic MDSC. Investigation of ID1 as a pharmacodynamic biomarker or its use as a target for modulating MDSC is warranted.

Keywords: Cancer; Immunosuppression; Melanoma; Myeloid cells.

Fig. 1

Expression of ID1 on monocytes coincides with known phenotypic characteristics of monocytic MDSC. a Flow cytometric analysis of PBMC from melanoma patients. Doublets were excluded and live PBMC were gated (not shown). Representative plots depicting the subpopulation of CD33⁺CD11b⁺CD14⁺ cells, indicating expression of ID1 plotted against markers commonly used for characterization of monocytic MDSC, with gates to indicate cells positive for ID1, HLA-DR, iNOS, and S100A8/9. b Flow cytometric analysis of CD33⁺CD11b⁺CD14⁺ cells within melanoma patient PBMC, indicating median fluorescence intensities in HLA-DR^high monocytes versus HLA-DR^low monocytic MDSC for ID1, S100A8/9, S100A9, iNOS, and IRF8. c Frequencies of cells positive for ID1, S100A8/9, and IRF8 with HLA-DR^hi and HLA-DR^low monocytes. **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

High expression of ID1 on monocytes correlates with expression of known phenotypic characteristics of monocytic MDSC. a Flow cytometric analysis of CD33⁺CD11b⁺CD14⁺ cells amongst melanoma patient PBMC. a Median fluorescence intensities of ID1 in monocytes comparing various subpopulations of cells within the CD33⁺CD11b⁺CD14⁺ gate: S100A8/9^low versus S100A8/9^high cells, S100A9^low versus S100A9^high cells, and iNOS^high versus iNOS^low cells. b Correlations between ID1 and either S100A8/9, S100A9 or iNOS. Depicted are r and p values of Spearman’s rank correlations. Black and red dots represent samples taken before and after surgery, respectively. **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

Flow cytometric analysis of PBMC from melanoma patients before and after surgical removal of melanoma metastases. Depicted are median fluorescence intensities of the indicated markers in monocytes, defined as CD33⁺CD11b⁺CD14⁺. *p < 0.05 **p < 0.01

Fig. 4

ID1 is downregulated during DC maturation. a Flow cytometric analysis of human monocytes and monocyte-derived dendritic cells. Density plots indicate CD80 and CD86 expression of monocytes and dendritic cells matured with various cocktails of immunostimulatory compounds, leading to well-described types of maturation. b Expression of ID1, and more established markers used for characterization of monocytic MDSC, during myeloid cell maturation to a fully immunogenic phenotype associated with the indicated models for differentiation of human monocytes to mature dendritic cells