Myeloid-Derived Suppressive Cell Expansion Promotes Melanoma Growth and Autoimmunity by Inhibiting CD40/IL27 Regulation in Macrophages

Abstract

The relationship between cancer and autoimmunity is complex. However, the incidence of solid tumors such as melanoma has increased significantly among patients with previous or newly diagnosed systemic autoimmune disease (AID). At the same time, immune checkpoint blockade (ICB) therapy of cancer induces de novo autoinflammation and exacerbates underlying AID, even without evident antitumor responses. Recently, systemic lupus erythematosus (SLE) activity was found to drive myeloid-derived suppressor cell (MDSC) formation in patients, a known barrier to healthy immune surveillance and successful cancer immunotherapy. Cross-talk between MDSCs and macrophages generally drives immune suppressive activity in the tumor microenvironment. However, it remains unclear how peripheral pregenerated MDSC under chronic inflammatory conditions modulates global macrophage immune functions and the impact it could have on existing tumors and underlying lupus nephritis. Here we show that pathogenic expansion of SLE-generated MDSCs by melanoma drives global macrophage polarization and simultaneously impacts the severity of lupus nephritis and tumor progression in SLE-prone mice. Molecular and functional data showed that MDSCs interact with autoimmune macrophages and inhibit cell surface expression of CD40 and the production of IL27. Moreover, low CD40/IL27 signaling in tumors correlated with high tumor-associated macrophage infiltration and ICB therapy resistance both in murine and human melanoma exhibiting active IFNγ signatures. These results suggest that preventing global macrophage reprogramming induced by MDSC-mediated inhibition of CD40/IL27 signaling provides a precision melanoma immunotherapy strategy, supporting an original and advantageous approach to treat solid tumors within established autoimmune landscapes. SIGNIFICANCE: Myeloid-derived suppressor cells induce macrophage reprogramming by suppressing CD40/IL27 signaling to drive melanoma progression, simultaneously affecting underlying autoimmune disease and facilitating resistance to immunotherapy within preexisting autoimmune landscapes.

Figure 1.. Disease status influenced survival and growth for IFN-sensitive melanoma cells in ARE mice.

(A) Experiment layout of melanoma models (B16, HcMel and M114433) and the cohorts of WT, ARE^−/−, ARE^+/−, ifnar^−/−ARE^+/−, ifnar^−/−ARE^−/−, and ifnar ^−/− mice (n=5-6 per group). (B-D) Survival studies for B16 and HcMel cells injected IV into separate cohorts of ARE mice (n=6 per group). (B). Survival curves (C-D) Number of metastasis per animal in lung (C) and (D) other organs from A. Each symbol represents the mean area in a slide containing all lung lobes. (E) Tumor growth curves of B16 after SQ injection separately in ARE mice and ifnar^−/−ARE mice. (F) Percentage of T (CD4⁺, CD8⁺) and myeloid (F4/80⁺ macrophage and Ly6G^hi neutrophils) populations within tumors in E, as determined by FACS. Data are shown as mean ± SEM of two or three independent experiments. Statistical significance, two-way ANOVA unless specified. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2.. Myeloid cells advance lupus nephritis and tumor growth in ARE^−/− mice.

(A-C) Age-matched ARE mice were left tumor free or IV challenged with melanoma cells. Cohorts were euthanized at day 21. (A) Representative kidney H&E images for tubular renal damage (arrows, right) and overall grade (B). Upper bar indicates SLE-disease activity. (C) Serum levels of indicated factors per genotype (n = 4-5 mice/group). (D-E) Representative IHC images (D) and summary quantitation (E) of high F4/80 staining in kidneys (top), lung metastases (Center), and primary tumors (bottom) from B16-bearing ARE^−/− mice. (F) Total number of positive cells per mm² stained for the ratio iNOS⁺/CD206⁺ cells from D. (G) Intratumoral phosphorylated STAT-1 and STAT3 protein expression, by immunoblotting (n=3 per group), 2 independent experiments. GAPDH used as loading control. (H) Pro-tumoral conditions assessed by digital measure of necrotic areas (left) and CD31⁺ staining (right). (I) Statistical comparisons for intratumoral levels of IL-1b and IL-12p70, as measured by MSD, in samples from G. Every dot represents 2 technical replicates from one mouse per genotype. For A and D, scale bars: 200 um (Original magnification, 10 X). Data are shown as mean ± SEM. Two-way ANOVA, * p < 0.05; **, p < 0.01; and ***, p < 0.0001.

Figure 3.. Melanoma impairs IFNγ-regulated IL-27/IL-27ra signaling in Ly6C⁺ monocytes.

(A) Nanostring analytics of IL-27p28 mRNA alone (left) or compared to IFNγ in bone marrow (BM), kidney (Ki), liver (li), lymph node (LN), spleen (Sp), and Thymus (Thy) from tumor-free ARE^−/− and WT mice (n=4-5 mice per genotype). (B-C) Individual serum levels of IL-27 (top) compared to IFNγ (bottom) levels in age matched tumor free (B), or in correlation (C) determined by MDS (pro-inflammatory panel) (n = 6 mice per genotype). (D-F) Representative dot plots (D) and statistical comparison of the percentage population (E) and the expression of IL-27ra (F) in Ly6C^hi monocytes examined by flow cytometry from D in B16-bearing and tumor-free ARE mice (n=5-6 per genotype). Pooled data in B and E from 2 independent experiments. Data are shown as mean ± SEM. Two-way ANOVA: *, p < 0.05; **, p < 0.01; ***, p < 0.0001, and ****, p < 0.00001

Figure 4.. Melanoma expands pre-existing PMN-MDSC in ARE mice.

(A-B) Summary quantitation of Ly6G^hi PMN (A) expressing IL-27ra (B) in tumor-free and B16-bearing WT and ARE mice. (C) RT-qPCR analysis show higher Arginase 1 (Arg1) mRNA than NOS2 mRNA in isolated CD11b⁺GR1⁺ cells from B16-bearing ARE^+/− mice compared to counterparts in WT mice. (n=3). (D) Summary quantitation of Ly6G⁺ cells within indicated tissues from tumor-bearing WT and ARE^+/− mice (n= 6 organs per genotype). (E-J) Statistical comparison of the percentage populations and expression (mean florescence intensity; MFI) of indicated markers in CD45.2⁺Ly6C^loLy6G^hi PMN from tumor-free ARE (E, F) and type 1 Ifnar-deficient ARE mice (H, I). (G, J) Representative histograms show that Ly6G⁺ cells inhibited proliferation of CD3-activated CD4⁺ T cells, as evidenced by overlay CFSE signaling (green) from control (grey), in ARE (G) and type 1 Ifnar-deficient ARE mice (J). Data are shown as mean ± SEM. Two-way ANOVA: *, p < 0.05; **, p < 0.01; ***, p < 0.0001, and ****, p < 0.00001

Figure 5.. Decreased CD40/IL-27 regulation on Ly6C^hi monocytes by MDSC promotes tumor progression in ARE mice.

Seven days post SQ challenge with B16 cells WT and ARE^+/− mice received either anti-Ly6G mAb, anti-IL-27p28 (E-H), or rmIL-27 (I-K) i.p. twice a week (arrow). (A-E) For anti-Ly6G mAb, (A) Tumor growth curves (n=4, per group). (B-C) Percentage population of TAMs (B) and CD45.2⁺Ly6C^hi Ly6G^lo monocytes (C), assessed by FACS. (D) Frequency of Ly6G^loLy6C^hi and CD11b⁺CD40⁺ population in blood, as determined by FACS. (E) Serum levels of IL-27 before (pre) and after (post) B16 challenge (n=4), as determined by MSD. (F-H) For anti-IL-27p28, (G) Tumor growth Curves (n = 5 per intervention). (G-H) Percentage of spleen LY6G⁺ and Ly6C^hi populations total (G) or expressing CD40⁺ (H), as determined by FACS. (I-K) For rmIL-27, (I) Tumor growth Curves (n = 3-4 per intervention). (J-K) Percentage of tumor infiltrating CD8⁺ and CD4⁺ T populations total (J) and spleen LY6G⁺ and Ly6C^hi (K), as determined by FACS. Data are shown as mean ± SEM. Two-way ANOVA was used, except for final tumor measurements (Two-tailed Student’s T test), * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6.. Agonist CD40 therapy delays melanoma growth in ARE mice.

Seven days post SQ challenge with B16 cells, WT and ARE^+/− mice received weekly i.p. injections of anti-CD40, anti-PD1, anti-PD1/CD40 or an isotype control (rat IgG2, 200 ug). (A) Tumor-growth curves. (B) cumulative quantification of F4/80⁺ IHC staining within whole tumor sections from A (n= 4-5). (C-D) Percentage of tumor immune populations of PMN (C), T cells (CD4⁺ and CD8⁺; D left) and monocytes (Ly6G^lo; D right) from A, assessed by FACS. (E) Intratumoral levels of IFNγ and IL-27 from A, as determined by MSD (n = 4-5 per group). (F) Immunoblots from spleen lysates showing protein expression of CD40 and indicated IFN signature genes. Side arrows show correct band size. (G) Statistical comparison of serum levels of IFNγ, IL-27, and IL-12p40 in treated groups. (H) HS for nephropathy in ARE^+/− mice. (I-J) Spleen size as % body weight (BW) (I) and percentage population of CD11b⁺Gr1⁺ MDSC, obtained by FACS (J). Unless indicated, pooled data from two independent experiments are shown as mean ± SEM. Two-way ANOVA: * p <0.05, ** p < 0.01, *** p < 0.001.

Figure. 7.. The CD40/IL27 signature associates with better response to ICB in human melanoma.

Transcriptomic data of melanoma tumors from a clinical study of CTLA4 were analyzed to generate signaling scores as described in Methods. (A), Correlation of IL27 scores (vertical axis) with IFNγ scores (horizontal axis) prior to treatment. (B) Correlation of the ratio of M2 macrophage subtype signature, as determined by CIBERTSORT. Data shown as the M2 macrophage signature over total tumor-associated macrophage (M2/All M) signatures. (C) Following the result in A, comparison of signaling scores (z-score) in non-responder patients (NR) and in responders (R) plus long-survival (LS) patients (R+LS). (D) Following the result in C, calculation of scores for CD40L signaling, which includes CD40, significantly improved correlation with IL27 scores only in the responsive (R+LS) group (right panel), but not in non-responders (NR) (left panel). (E) Cartoon details how PMN-MDSC inhibits IL-27 production and CD40 expression in ARE monocytes to induce M2-polarization and TME suppression.