Id1 suppresses anti-tumour immune responses and promotes tumour progression by impairing myeloid cell maturation

Abstract

A central mechanism of tumour progression and metastasis involves the generation of an immunosuppressive 'macroenvironment' mediated in part through tumour-secreted factors. Here we demonstrate that upregulation of the Inhibitor of Differentiation 1 (Id1), in response to tumour-derived factors, such as TGFβ, is responsible for the switch from dendritic cell (DC) differentiation to myeloid-derived suppressor cell expansion during tumour progression. Genetic inactivation of Id1 largely corrects the myeloid imbalance, whereas Id1 overexpression in the absence of tumour-derived factors re-creates it. Id1 overexpression leads to systemic immunosuppression by downregulation of key molecules involved in DC differentiation and suppression of CD8 T-cell proliferation, thus promoting primary tumour growth and metastatic progression. Furthermore, advanced melanoma patients have increased plasma TGFβ levels and express higher levels of ID1 in myeloid peripheral blood cells. This study reveals a critical role for Id1 in suppressing the anti-tumour immune response during tumour progression and metastasis.

Figure 1. Tumour-secreted factors favour BMDC differentiation towards high Id1-expressing MDSCs but not DCs.

Flow cytometry analysis of splenic populations from B16F10 melanoma-implanted mice (day 21 post implantation). (a) Frequency and absolute numbers of DCs (unpaired t-test **P<0.01). (b) Frequency and absolute numbers of MDSCs (unpaired t-test, ***P<0.001). Flow cytometry analysis of spleens from E0771 mammary adenocarcinoma-implanted mice (day 21 post implantation) for (c) DC absolute numbers and (d) MDSC absolute numbers compared with control mice (unpaired t-test, **P<0.01. (e) Id1 and Id3 mRNA levels in FACS-sorted splenic DC and MDSC populations, as determined by qPCR analysis, (n=6, one-way analysis of variance (ANOVA), *P<0.05, **P<0.01, ***P<0.001). (f) Id1 protein levels in lysates from naive and B16F10-bearing CD11b⁺ bead-sorted splenocytes as determined by western blot and densitometric analyses (unpaired t-test *P<0.01). (g) In vitro differentiation of Lin⁻ haematopoietic progenitors isolated from C57BL/6 mice, cultured for 6 days in the presence of B16F10 melanoma TCM (25% v/v), and analysed for DC and MDSC content by flow cytometry (n=6, ANOVA, ***P<0.001). (h) Id1 mRNA relative expression levels of day 6 Lin⁻ cells differentiated in the presence B16F10-conditioned media compared with control media, as determined by qPCR analysis (means±s.e.m., n=6, unpaired t-test, *P<0.05).

Figure 2. Deletion of the Id1 gene restores myeloid differentiation defects.

Flow cytometry analysis of spleens from WT and Id1^−/− mice that received daily injections of B16F10 melanoma-derived conditioned media (B16F10 CM) or control media for (a) absolute numbers of DCs (one-way analysis of variance (ANOVA), ***P<0.001) and (b) absolute numbers of MDSC levels (one-way ANOVA, *P<0.05). (c) Representative frequency plots of DCs and (d) Splenic MDSCs isolated from Id1^−/− mice injected daily with B16F10 melanoma-derived TCM or control media. (e) In vitro differentiation of Lin⁻ haematopoietic progenitors isolated from Id1^−/− mice, cultured for 6 days in the presence of B16F10 melanoma TCM (25% v/v) and analysed for DC and MDSC content by flow cytometry (n=6, ANOVA, ^**P<0.01). (f) Gene expression analysis of Id1^−/− and WT cells after 6 days of in vitro differentiation in the presence of TCM, as determined by qPCR analysis (means±s.e.m., n=6, one-way ANOVA, ^**P<0.01, *P<0.05). (g) Analysis of primary tumour volume from Id1^−/− and control BM chimeric mice following implantation of B16F10 melanoma cells (two-way ANOVA, ****P<0.0001). (h) Relative quantification of mCherry-labelled B16F10 melanoma cells in cryosections of lungs of Id1^−/− and control BM chimeric mice measured by mCherry qPCR analysis (unpaired t-test, *P<0.05). NS, not significant.

Figure 3. Id1 overexpression induces a DC/MDSC imbalance.

Flow cytometry analysis of spleens from mice transplanted with Id1-overexpressing and control vector-transduced Lin⁻ BM cells for (a) frequency and absolute numbers of DCs and (b) MDSCs (unpaired t-test, ^**P<0.01). (c) Representative percentages of MDSCs in GFP-positive splenocytes from mice transplanted with Id1-overexpressing Lin⁻ cells and control vector splenocytes. (d) In vitro differentiation of Lin⁻ haematopoietic progenitors from C57BL/6 mice transduced with lentiviral or control and Id1-overexpressing vectors overnight, cultured for 6 days and analysed for DC and MDSC content by flow cytometry (n=6, analysis of variance, ^**P<0.01). (e) Gene expression analysis of Lin⁻ cells transduced with Id1-overexpressing and control vectors after 6 days of in vitro differentiation by qPCR analysis (means±s.e.m., n=6, unpaired t-test, ^**P<0.01, *P<0.05; NS, not significant). Four independent experiments were performed.

Figure 4. Id1 overexpression leads to an immunosuppressive phenotype and T-cell suppression.

Flow cytometry analysis of spleens from mice transplanted with Id1-overexpressing and control vector-transduced Lin⁻ BM cells for (a) absolute numbers of regulatory T cells (T-regs; CD4⁺CD25⁺Foxp3⁺; unpaired t-test, ^***P<0.001), for (b) ROS production, as determined by mean fluorescence intensity levels of dichlorofluorescein (DCF), a ROS-sensitive dye (unpaired t-test, ^***P<0.001). (c) CD8⁺ antigen-specific T-cell proliferation functional assessment of GFP⁺ CD11b⁺ Gr1⁺ splenocytes from Id1-overexpressing and control vector animals co-cultured with OT-I splenocytes in the presence of OVA_257–264 peptide. (analysis of variance, ^**P<0.01, *P<0.05). (d) OT-I T-cell proliferation expressed as suppression induced by GFP⁺ CD11b⁺ Gr1⁺ splenocytes from Id1-overexpressing and control vector animals, relative to the no MDSC control wells. Data expressed as percentage T-cell suppression compared with no MDSC control (unpaired t-test, *P<0.05). (e) Analysis of splenocytes from Id1-overexpressing mice and OT-II CD4+ T-cell co-cultures in the presence of OVA_323–329 peptide for IFNγ levels (unpaired t-test, *P<0.05) and (f) IL-10 levels compared with splenocytes from control vector-treated mice and OT-II CD4⁺ T-cell co-cultures (unpaired t-test, *P<0.05). Four independent experiments were performed.

Figure 5. Id1-overexpressing BMDCs promote tumour growth and metastatic progression.

(a) Analysis of primary tumour volume from Id1-overexpressing mice and control vector mice following implantation of B16F10 melanoma cells (two-way analysis of variance, ^****P<0.0001). (b) Quantification of mCherry-labelled B16F10 melanoma cells in cryosections of lungs of BM Id1-overexpressing mice and control vector mice measured as red pixels per field (unpaired t-test, *P<0.05). (c) Macro- and (d) micrometastatic lesion formation in lungs from Id1-overexpressing mice and control vector mice; scale bar (50 μm) on top left panel applies to all panels. Flow cytometry analysis of splenocytes from BM Id1-overexpressing and control vector mice implanted with B16F10 melanoma cells for absolute numbers of (e) DCs (f) MDSCs (g) regulatory T cell (T-regs) and (h) ROS production (unpaired t-tests; NS, non-significant, *P<0.05, ^**P<0.01). Four independent experiments were performed.

Figure 6. Id1 is upregulated via a TGFβ-dependent mechanism and downregulates key genes involved in DC maturation.

(a) Id1 mRNA relative expression levels in day 6 Lin⁻ cells differentiated in the presence of 100 ng μl⁻¹ of murine recombinant proteins (VEGF, IL-6, BMP-7, -9 and -10, and TGFβ compared with Lin⁻ cells differentiated in control media, as determined by qPCR analysis (means±s.e.m., n=6, analysis of variance (ANOVA), ^****P<0.0001, ^**P<0.01, *P<0.05). (b) Id1 mRNA expression levels in day 6 Lin⁻ cells differentiated in the presence of B16F10 CM (abbreviation introduced in Fig 2) alone, with anti-TGFβ and anti-IgG compared with control media, as determined by qPCR analysis (means±s.e.m., n=6, ANOVA, ^***P<0.001, ^**P<0.01). (c) Irf8 mRNA relative expression levels of day 6 WT Lin⁻ cells differentiated in the presence of 100 ng μl⁻¹ of TGFβ, B16F10 CM alone, with anti-TGFβ and anti-IgG compared with control media, as determined by qPCR analysis (means±s.e.m., n=6, ANOVA, ****P<0.0001, ***P<0.001). (d) Irf8 mRNA relative expression levels of day 6 Id1^−/− Lin⁻ cells differentiated in the presence of 100 ng μl⁻¹ of TGFβ, B16F10 CM alone, with anti-TGFβ and anti-IgG compared with control media, as determined by qPCR analysis (means±s.e.m., n=6, ANOVA; NS, not significant). Four independent experiments were performed.

Figure 7. Advanced stage melanoma patients express higher levels of Id1 in the CD11B⁺ PBMC fraction and have elevated plasma TGFβ levels.

(a) qPCR analysis of ID1 (unpaired t-test, *P<0.05), (b) S100A8 (unpaired t-test, **P<0.01) and (c) S100A9 (unpaired t-test, **P<0.01) mRNA expression levels following isolation of CD11B⁺ PBMCs from metastatic melanoma patient blood samples (n=15) compared with healthy matched controls (n=7). (d) VEGFR1 mean fluorescence intensity levels in CD11B⁺CD14⁻HLA⁻CD33⁺ cells PBMCs from metastatic melanoma patient blood samples compared with healthy matched controls, determined by flow cytometry (unpaired t-test, ***P<0.001). (e) TGFβ plasma levels from metastatic melanoma patients compared with controls, measured by ELISA (unpaired t-test, **P<0.01).