Autophagy orchestrates the regulatory program of tumor-associated myeloid-derived suppressor cells

Abstract

Myeloid-derived suppressor cells (MDSCs) densely accumulate into tumors and potently suppress antitumor immune responses, promoting tumor development. Targeting MDSCs in tumor immunotherapy has been hampered by lack of understanding of the molecular pathways that govern MDSC differentiation and function. Herein, we identify autophagy as a crucial pathway for MDSC-mediated suppression of antitumor immunity. Specifically, MDSCs in patients with melanoma and mouse melanoma exhibited increased levels of functional autophagy. Ablation of autophagy in myeloid cells markedly delayed tumor growth and endowed antitumor immune responses. Notably, tumor-infiltrating autophagy-deficient monocytic MDSCs (M-MDSCs) demonstrated impaired suppressive activity in vitro and in vivo, whereas transcriptome analysis revealed substantial differences in genes related to lysosomal function. Accordingly, autophagy-deficient M-MDSCs exhibited impaired lysosomal degradation, thereby enhancing surface expression of MHC class II molecules, resulting in efficient activation of tumor-specific CD4+ T cells. Finally, targeting of the membrane-associated RING-CH1 (MARCH1) E3 ubiquitin ligase that mediates the lysosomal degradation of MHC II in M-MDSCs attenuated their suppressive function, and resulted in markedly decreased tumor volume followed by development of a robust antitumor immunity. Collectively, these findings depict autophagy as a molecular target of MDSC-mediated suppression of antitumor immunity.

Keywords: Autophagy; Cancer; Immunology; Tumor suppressors.

Figure 1. Enhanced autophagy in MDSCs from patients with melanoma.

(A) Gating strategy and frequencies of MDSCs (HLA-DR^–CD14^–CD33⁺CD15⁺) in PBMCs of healthy individuals (n = 18) and patients with melanoma (n = 17) (***P < 0.0001). (B) Representative confocal microscopy images for LC3 (red), LAMP-1 (green), p62 (silver white), and DAPI (blue), and Pearson’s correlation of LC3 versus p62 (***P < 0.0001) in sorted MDSCs from peripheral blood of healthy individuals (n = 4) and patients with melanoma (n = 4). Scale bar: 10 μM. One representative experiment of 3 is shown. Results are mean ± SEM. Statistical significance was obtained by unpaired Student’s t test.

Figure 2. Upregulation of the autophagy pathway in MDSCs from melanoma-bearing mice.

(A) Representative flow cytometric analysis and frequencies of total MDSCs (CD11c^–CD11b⁺Gr-1⁺) (n = 5 mice per group, ***P < 0.0001) and subsets from spleens of naive or B16-F10–inoculated mice (n = 4). (B) Representative immunofluorescence confocal images for LC3 (red), LAMP-1 (green), p62 (silver white), and DAPI (blue), and LC3 puncta/cell and p62 puncta/cell in sorted MDSCs from spleens and tumors of naive and B16-F10–inoculated mice (n = 4 mice per group) (LC3: ***P < 0.0001; p62: *P = 0.0459, **P = 0.0003, ***P < 0.0001). Scale bars: 10 μm. (C) MFI of pAkt (*P = 0.0483), pmTOR (^#P = 0.0515), and pS6 (**P = 0.0027) in MDSCs from spleens of naive or B16-F10 inoculated mice, n = 5 mice per group. (D) Representative immunofluorescence confocal images for pULK-1 (silver white), and DAPI (blue), and pULK-1 puncta/cell in sorted MDSCs from spleens and tumors of naive and B16-F10–inoculated mice (pULK-1 ***P < 0.0001). Scale bar: 10 μm; n = 5 mice per group. One representative experiment of 3 is shown. Results are mean ± SEM. Statistical significance was obtained by unpaired Student’s t test (A and C) or 2-way ANOVA (B and D).

Figure 3. Deficiency of autophagy pathway in the myeloid compartment attenuates tumor growth and enhances antitumor immune responses.

(A) Tumor volume (***P = 0.0005) and representative image of excised tumors of B16-F10–inoculated Atg5^ΔLysM and Atg5^fl/fl control mice. Representative results from 3 independent experiments are shown, n = 5 mice per group. (B) Tumor volume (**P = 0.0028) of LLC-inoculated Atg5^ΔLysM (n = 4) and Atg5^fl/fl control (n = 5) mice. Representative results from 3 independent experiments are shown. (C) Frequencies of CD4⁺, CD8⁺ T cells, and CD4⁺Foxp3⁺ Tregs (**P = 0.0032) in tdLNs of B16-F10–inoculated Atg5^ΔLysM and Atg5^fl/fl control mice, n = 8 mice per group. (D) Gating strategy and frequencies of CD45⁺ (*P = 0.0150, n = 4), CD4⁺ (**P = 0.0088, n = 6), CD8⁺ (n = 10), and CD4⁺Foxp3⁺ Tregs (***P = 0.0030, n = 11), in tumor sites of Atg5^ΔLysM and Atg5^fl/fl control mice. For C and D, representative results from 4 independent experiments are shown. (E) Representative digital slide scanner images of CD4⁺ T cells (red) and DAPI (blue) in tumor section from B16-F10–inoculated Atg5^ΔLysM and Atg5^fl/fl control mice are shown. Scale bar: 50 μm; n = 5 mice per group. (F) Representative flow cytometric analysis and frequencies of NK cells (*P = 0.0364) gated on CD45⁺ and CD3^–NK1.1⁺IFN-γ⁺ NK cells (***P = 0.0008) in tumor site, n = 4 mice per group. (G) Gating strategy and frequencies of CD8⁺IFN-γ⁺ T cells (*P = 0.0456) in tumor site. Representative results from 4 independent experiments are shown, n = 5 mice per group. Results are mean ± SEM. Statistical significance was obtained by unpaired Student’s t test.

Figure 4. Impaired suppressive function of tumor-infiltrating autophagy-deficient M-MDSCs from melanoma-bearing mice.

(A) Frequencies of MDSCs (CD11c^–CD11b⁺Gr-1⁺) (**P = 0.0036, n = 7) and DCs (CD11c⁺, n = 11) in spleens of B16-F10–inoculated Atg5^ΔLysM and Atg5^fl/fl control mice. (B) Representative flow cytometric analysis and frequencies of G-MDSCs (CD11c^–CD11b^hiLy6G⁺Ly6C^lo; ***P < 0.0001) and M-MDSCs (CD11c^–CD11b^hiLy6G^–Ly6C^hi; **P = 0.0067) in spleens of B16-F10–inoculated Atg5^ΔLysM and Atg5^fl/fl control mice (n = 10 mice per group). (C) Representative flow cytometric analysis and frequencies of tumor-infiltrating MDSCs (***P = 0.006) in B16-F10–inoculated Atg5^ΔLysM and Atg5^fl/fl control mice (n = 7 mice per group). (D) Frequencies of G-MDSCs and M-MDSCs (**P = 0.0050) in tumors of B16-F10–inoculated Atg5^ΔLysM and Atg5^fl/fl control mice (n = 8 mice per group). (E) Representative digital slide scanner images and percentages of CD206⁺ cells (*P = 0.0310) (red) and DAPI (blue) per tumor section isolated from B16-F10–inoculated Atg5^ΔLysM and Atg5^fl/fl control mice. Scale bar: 40 μm; n = 5 mice per group. (F) Representative histograms of CD4⁺ T cell proliferation and flow cytometric analysis of CD44 in CellTrace-labeled LNCs cultured with sorted M-MDSCs from tumors of Atg5^ΔLysM and control Atg5^fl/fl B16-F10–inoculated mice, n = 4 mice per group. For G and H, Ly6C⁺ cells from spleens of Atg5^ΔLysM and Atg5^fl/fl control B16-F10–inoculated mice were mixed with B16-F10 melanoma cells (3:1 ratio) and were s.c. injected into C57BL/6 mice (n = 8 mice per group). (G) Tumor volume (*P = 0.0082) and tumor weight (**P = 0.007) are shown. (H) Frequencies of CD4⁺ (*P = 0.0499) and CD8⁺ T cells from tdLNs. Results are mean ± SEM. Statistical significance was obtained by unpaired Student’s t test. Representative results from 3 independent experiments are shown.

Figure 5. Impaired lysosomal degradation and increased surface expression of MHC II molecules in autophagy-deficient M-MDSCs.

(A) Heat map of differentially expressed genes in M-MDSCs isolated from spleens of B16-F10–inoculated Atg5^ΔLysM and control mice (n = 3 mice per group). (B) Heat map of differentially expressed genes related to the lysosomal function in M-MDSCs isolated from the spleens of B16-F10 inoculated Atg5^ΔLysM and control mice (n = 3 mice per group). (C) MFI of lysosensor in M-MDSCs from spleen (*P = 0.0470) and tumor (**P = 0.0335) of B16-F10–inoculated Atg5^ΔLysM and control mice (n = 5 mice per group). (D) Percentage of protein degradation, using [³H] leucine, in M-MDSCs isolated from the spleens of B16-F10 inoculated Atg5^ΔLysM and control mice treated with lysosomal inhibitors (NH₄Cl and leupeptin or bafilomycin) or left untreated (n = 3 mice per group). *P = 0.0134, **P = 0.0084, ***P = 0.0195, ****P = 0.0128, ^#P = 0.0179, ^##P = 0.0088, ^###P = 0.0264. (E) Representative histograms for the expression of IA^b by M-MDSCs of spleen or tumor of Atg5^ΔLysM and control mice, n = 5 mice per group. (F) Representative confocal microscopy images for LAMP-1 (red), IA^b(green), DAPI (blue), and Pearson’s correlation of IA^b versus LAMP-1 (***P < 0.0001) in sorted M-MDSCs from splenocytes of B16-F10–inoculated Atg5^ΔLysM and control mice (n = 4 mice/group). Scale bar: 10 μm. (G) Representative histograms for the expression of IA^b by M-MDSCs isolated from spleens of B16-F10–inoculated Atg5^fl/fl mice after in vitro stimulation with TES in the presence of NH₄Cl or chloroquine. Geometric mean of IA^b (***P < 0.0001, *P = 0.048) and relative expression of Ciita and IA^b (***P < 0.0001) are shown, n = 8 mice per group. Results are mean ± SEM. Statistical significance was obtained by unpaired Student’s t test, or 2-way ANOVA (D and G). Representative results from 3 independent experiments are shown.

Figure 6. Sustained IA^b expression in autophagy-deficient tumor-derived M-MDSCs endows their immunogenic properties.

(A) Representative flow cytometric analysis of CellTrace-labeled OTII CD4⁺ T cells cultured with M-MDSCs of Atg5^ΔLysM and control B16-F10–inoculated mice in the presence of OVA peptide, n = 5 mice per group. (B) Gating strategy and frequencies of CD25⁺ (*P = 0.0236) and CD44⁺ (**P = 0.0116) OTII CD4⁺ T cells adoptively transferred in Atg5^ΔLysM and control tumor-bearing mice, n = 3 mice per group. (C) Relative March1 expression in M-MDSCs following transfection with siRNA for March1 or scramble si (**P = 0.0006, n = 3 mice per group). (D) Representative histograms and MFI for IA^b expression (*P = 0.0129) in M-MDSCs following transfection with siRNA for March1 or scramble si (n = 4 mice per group). For E and F, 4 × 10⁵ M-MDSCs transfected with scramble si or si-March1 were s.c. coinjected with 3 × 10⁵ B16-F10 cells in C57BL/6 mice (n = 7 mice per group). (E) Tumor volume (*P = 0.0044, **P = 0.017, ***P < 0.0001) and tumor weight (****P < 0.0001) are shown. (F) Numbers of CD45⁺ (**P = 0.0035), CD4⁺ (***P < 0.0001) and CD8⁺ (*P = 0.0307) T cells per 6 × 10⁵ tumor cells are depicted. One representative experiment of 3 is shown. Results are mean ± SEM. Statistical significance was obtained by unpaired Student’s t test.