Melanoma treatment is being revolutionized by the development of effective immunotherapeutic approaches1, 2. These strategies include blockade of immune-inhibitory receptors on activated T cells; for example, using monoclonal antibodies against CTLA-4, PD-1, and PD-L1 (refs 3, 4, 5). However, only a subset of patients responds to these treatments, and data suggest that therapeutic benefit is preferentially achieved in patients with a pre-existing T-cell response against their tumour, as evidenced by a baseline CD8+ T-cell infiltration within the tumour microenvironment6, 7. Understanding the molecular mechanisms that underlie the presence or absence of a spontaneous anti-tumour T-cell response in subsets of cases, therefore, should enable the development of therapeutic solutions for patients lacking a T-cell infiltrate. Here we identify a melanoma-cell-intrinsic oncogenic pathway that contributes to a lack of T-cell infiltration in melanoma. Molecular analysis of human metastatic melanoma samples revealed a correlation between activation of the WNT/β-catenin signalling pathway and absence of a T-cell gene expression signature. Using autochthonous mouse melanoma models8, 9 we identified the mechanism by which tumour-intrinsic active β-catenin signalling results in T-cell exclusion and resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody therapy. Specific oncogenic signals, therefore, can mediate cancer immune evasion and resistance to immunotherapies, pointing to new candidate targets for immune potentiation.
At a glance
Gene Expression Omnibus
- The Society for Immunotherapy of Cancer consensus statement on tumour immunotherapy for the treatment of cutaneous melanoma. Nature Rev. Clin. Oncol. 10, 588–598 (2013) et al.
- Cancer immunotherapy comes of age. Nature 480, 480–489 (2011) , &
- Nivolumab plus ipilimumab in advanced melanoma. N. Engl. J. Med. 369, 122–133 (2013) et al.
- Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab. J. Clin. Oncol. 32, 1020–1030 (2014) et al.
- Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 363, 711–723 (2010) et al.
- Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment. Cancer Res. 69, 3077–3085 (2009) et al.
- An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol. Immunother. 61, 1019–1031 (2012) et al.
- Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nature Genet. 41, 544–552 (2009) et al.
- β-Catenin signaling controls metastasis in Braf-activated Pten-deficient melanomas. Cancer Cell 20, 741–754 (2011) et al.
- Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313, 1960–1964 (2006) et al.
- Frequent nuclear/cytoplasmic localization of β-catenin without exon 3 mutations in malignant melanoma. Am. J. Pathol. 154, 325–329 (1999) , , , &
- Up-regulation of PD-L1, IDO, and Tregs in the melanoma tumor microenvironment is driven by CD8+ T cells. Sci. Transl. Med. 5, 200ra116 (2013) et al.
- Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape. Cancer Res. 72, 917–927 (2012) et al.
- Melanomas resist T-cell therapy through inflammation-induced reversible dedifferentiation. Nature 490, 412–416 (2012) et al.
- Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting. Nature 482, 400–404 (2012) et al.
- Regulated expression of a tumor-associated antigen reveals multiple levels of T-cell tolerance in a mouse model of lung cancer. Cancer Res. 68, 9459–9468 (2008) , , , &
- Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8α+ dendritic cells. J. Exp. Med. 208, 2005–2016 (2011) et al.
- Batf3 deficiency reveals a critical role for CD8α+ dendritic cells in cytotoxic T cell immunity. Science 322, 1097–1100 (2008) et al.
- Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells. Nature Immunol. 10, 488–495 (2009) et al.
- Flt3L dependence helps define an uncharacterized subset of murine cutaneous dendritic cells. J. Invest. Dermatol. 134, 1265–1275 (2014) et al.
- CCR5 provides a signal for microbial induced production of IL-12 by CD8α+ dendritic cells. Nature Immunol. 1, 83–87 (2000) et al.
- PD-1 blockade enhances T-cell migration to tumors by elevating IFN-γ inducible chemokines. Cancer Res. 72, 5209–5218 (2012) et al.
- N-myc downstream-regulated gene 2, a novel estrogen-targeted gene, is involved in the regulation of Na+/K+-ATPase. J. Biol. Chem. 286, 32289–32299 (2011) et al.
- Activating transcription factor 3 (ATF3) represses the expression of CCL4 in murine macrophages. Mol. Immunol. 44, 1598–1605 (2007) , , &
- Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens. J. Exp. Med. 207, 1247–1260 (2010) et al.
- Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8+ T cells directly within the tumor microenvironment. J. Immunother. Cancer (2014) et al.
- Immune suppression and resistance mediated by constitutive activation of Wnt/β-catenin signaling in human melanoma cells. J. Immunol. 189, 2110–2117 (2012) et al.
- β-Catenin inhibits T cell activation by selective interference with linker for activation of T cells–phospholipase C-γ1 phosphorylation. J. Immunol. 186, 784–790 (2011) et al.
- Tumor-infiltrating lymphocytes: apparently good for melanoma patients. But why? Cancer Immunol. Immunother. 60, 1153–1160 (2011) , , &
- RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011) &
- ConsensusClusterPlus: a class discovery tool with confidence assessments and item tracking. Bioinformatics 26, 1572–1573 (2010) &
- ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010) , &
- The 1000 Genomes Project Consortium. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56–65 (2012)
- STRING 8—a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res. 37, D412–D416 (2009) et al.
- High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice. Curr. Biol. 8, 1169–1178 (1998) et al.
- Stabilization of β-catenin induces lesions reminiscent of prostatic intraepithelial neoplasia, but terminal squamous transdifferentiation of other secretory epithelia. Oncogene 21, 4099–4107 (2002) et al.
- A new mouse model to explore the initiation, progression, and therapy of BRAFV600E-induced lung tumors. Genes Dev. 21, 379–384 (2007) et al.
- Characterization of melanocyte-specific inducible Cre recombinase transgenic mice. Genesis 44, 262–267 (2006) et al.
- Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Genes Dev. 18, 937–951 (2004) , , , &
- Antigen recognition and allogeneic tumor rejection in CD8+ TCR transgenic/RAG−/− mice. J. Immunol. 159, 4665–4675 (1997) et al.
- Immunotype and immunohistologic characteristics of tumor-infiltrating immune cells are associated with clinical outcome in metastatic melanoma. Cancer Res. 72, 1070–1080 (2012) et al.
- Analyzing real-time PCR data by the comparative CT method. Nature Protocols 3, 1101–1108 (2008) &
- Generation of Th1-polarizing dendritic cells using the TLR7/8 agonist CL075. J. Immunol. 185, 738–747 (2010) et al.
Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Correlation between active β-catenin and CD8 T-cell infiltrate in human patients. (1,537 KB)
a, A continuous numerical score was generated using six β-catenin target genes (CTNNB1 score). Using this score, patients from the TCGA data set were grouped in high or low CTNNB1 score (centred on the average score) (low, 91 patients; high, 108 patients). Subsequent correlation analysis was performed using a Fisher’s exact test. b, Representative examples for CD8 and β-catenin staining in human needle biopsies used for analysis shown in Fig. 1d.
- Extended Data Figure 2: Tumour growth of genetically engineered mice. (424 KB)
a, Overall survival of all three models: BrafV600E/Pten−/− with 100% lethality and mean time to death of 31 days (n = 14), BrafV600E/CAT-STA with 85% lethality and mean time to tumour event of 93 days (n = 8), and BrafV600E/Pten−/−/CAT-STA with 100% lethality and mean time to tumour event of 36 days (n = 14). b, Tumour outgrowth of BrafV600E/Pten−/− (red) and BrafV600E/Pten−/−/CAT-STA (blue) tumours shown as mm3 at days after tamoxifen application (n = 10). c, Representative macroscopic pictures for tumour growth over time when tamoxifen was applied on the lower back of the mouse (see illustration). d, Gene array analysis of tumours isolated from GEMs (n = 4, Mann–Whitney U test). e, Histology slides showing representative examples for haematoxylin and eosin stain in all three mouse models (left, ×20, scale bars indicate 100 μm; right, ×100, scale bars indicate 20 μm). *P ≤ 0.05; NS, not significant.
- Extended Data Figure 3: T-cell infiltration of genetically engineered mice. (692 KB)
a, Representative images of immmunofluorescent staining against CD3 (red, left panel) and TRP1 (green, right panel) in all three tumour tissues (scale bar, 100 μm; ×4, ×10, ×20 with ×4 differential interference contrast (DIC) on top; nuclei Hoechst ×20 CD3 stain as shown in Fig. 1). b, Representative immmunofluorescent staining against CD3 (red, left panel) and TRP1 (green, right panel) in a highly pigmented area of BrafV600E/Pten−/− tumour tissues (scale bar, 100 μm; ×10, ×20 with ×10 DIC left) excluding that the lack of T cells is associated with increased pigmentation (nuclei Hoechst). c, Numbers of CD3+ T cells were counted within 13 different fields (0.5 mm × 1 mm) from two tumour samples. Mean of 12 T cells or 3.2 T cells per 0.5 mm2 in BrafV600E/CAT-STA or BrafV600E/Pten−/−/CAT-STA tumours, respectively, versus 100 T cells per 0.5 mm2 in BrafV600E/Pten−/− tumours. Data are given as mean with minimum and maximum, as well as individual values. Statistical analysis was performed using Mann–Whitney U test. ****P ≤ 0.0001.
- Extended Data Figure 4: Characterization of the T-cell infiltrate in BrafV600E/Pten−/−/CAT-STA mice. (314 KB)
a, Distribution of T-cell subsets in BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA tumours (n = 6). b, c, Representative flow cytometry plots to discriminate αβ-TCR T cells and γδ-TCR T cells (b), naive (CD62L+CD44−) and effector (CD62L−CD44+) T cells (pre-gated on CD3+CD8+ T cells), and one representative example of CD44/CD45RA staining (c). Quantification of naive (CD62L+CD44−CD45RA+), effector (CD62L−CD44+CD45RA−) and memory (CD62L+CD44+CD45RA−) T cells is indicated on the right (n = 6). d, Representative flow cytometry plots of FoxP3+ T regulatory cells (n = 6). e, Quantification and comparison of PD-1/Lag3 double-positive T cells in BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA tumours (n = 12). f, Representative flow cytometry of PD-1- and Lag3-positive T cells (pre-gated on CD3+CD8+ T cells) in BrafV600E/Pten−/− tumours. g, Il2 transcripts present in sorted CD3+ T cells from BrafV600E/Pten−/− tumours and spleen (n = 10). h, Ifng transcripts present in sorted CD3+ T cells from BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA mice (n = 10). i, Expression level of PD-L1 in whole tumour tissue from both mouse models assessed by qRT–PCR (n = 8). j, Flow cytometric analysis of PD-L1 expression of non-haematopoietic tumour cells (CD45−), CD45+CD11c+ dendritic cells (DC) and CD45+CD3+ T cells. Shown is a representative example as histogram (grey isotype, red BrafV600E/Pten−/−; blue, BrafV600E/Pten−/−/CAT-STA) with mean fluorescent intensity of n = 3 given each histogram (red, BrafV600E/Pten−/−; blue, BrafV600E/Pten−/−/CAT-STA). k, Percentage of Gr1+ cells within the CD11b+ fraction of the tumour immune cell infiltrate (n = 8; absolute numbers BrafV600E/Pten−/−: 1,047 ± 418 cells per gram tumour to BrafV600E/Pten−/−/CAT-STA: 739 ± 185 cells per gram tumour; P = 0.7429). All data are mean ± s.e.m., Mann–Whitney U test.*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001; NS, not significant.
- Extended Data Figure 5: Injection of Flt3 ligand-derived dendritic cells into tumours of BrafV600E/Pten−/−/CAT-STA mice is sufficient to overcome the lack of CD103+ dermal dendritic cells. (229 KB)
a, Expression level of Ifnb in CD45+CD11c+ sorted dendritic cells from tumours from BrafV600E/Pten−/− (open bars) and BrafV600E/Pten−/−/CAT-STA (filled bars) mice. FC, fold change. b, Expression level of Batf3, Irf8 and Itgae in sorted dendritic cells. Fold change is indicated in each graph (n = 8). c, Mean (± s.e.m.) tumour weight of BrafV600E/Pten−/−/CAT-STA assessed at the endpoint of the experiment depicted in Fig. 3e, after intra-tumoural injection of dendritic cells. d, Per cent of GFP+CD11c+ dendritic cells (DC) present at the tumour site after injections of Flt3 ligand-derived dendritic cells from actin–GFP mice. Depicted are the percentages detected in the tumour of both genotypes injected with either wild-type or actin–GFP dendritic cells as well as in the TdLNs for the actin–GFP injected mice (n = 4). All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.
- Extended Data Figure 6: Chemokine expression patterns indicate that CCL4 expression from tumour cells is directly inhibited by active β-catenin-signalling. (132 KB)
a, Expression of Ccl4 mRNA in established tumour cell lines BP and BPC (8 independent experiments). b, Amount of secreted CCL4 in 48 h conditioned BP and BPC tumour cell supernatants, assessed by ELISA (4 independent experiments). c, d, Control qRT–PCR for the experiment shown in Fig. 4e with Ifnb expression (c) and Ifng expression (d) (n = 6). ND, not detected. All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.
- Extended Data Figure 7: Active β-catenin signalling blocks CCL4 production in human melanoma cell lines. (165 KB)
a, Western blot on mel537 and mel888 showing stabilized β-catenin expression. b, Expression level of human ATF3 and human CCL4 in mel537 and mel888 (three independent experiments, duplicates per experiment). c, Expression level of β-catenin target genes in mel537 and mel888. d, ATF3-specific ChIP assay in mel537 and mel888 cell lines for the CCL4 gene locus (two independent experiments, duplicates per experiment). e, CCL4 secretion (left) and ATF3 transcription levels (right) after siRNA-mediated knockdown of CTNNB1 and ATF3 in mel537 and mel888 assessed by ELISA or qRT–PCR, respectively (two independent experiments, duplicates per experiment). All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.
- Extended Data Figure 8: β-Catenin target gene expression correlates inversely with markers for human BATF3-lineage dendritic cells and T cells. (163 KB)
Pearson correlation of CTNNB1 score with CD8Α (R2 = 0.214), THBD (R2 = 0.109) and IRF8 (R2 = 0.2374) (red indicates T-cell-signature high, blue indicates T-cell-signature low).
- Extended Data Figure 9: Graphical summary. (240 KB)
Left, tumour without active β-catenin signalling in which ATF3 transcription is not induced and thus CCL4 (red circles) is transcribed and secreted. Downstream CD103+ dendritic cells (DC) (blue) are attracted and subsequent activation of CD8+ T cells (green) is enabled. Right, tumour with active β-catenin signalling (green), which leads to induction of ATF3 transcription (red), which in turn leads, among others effects, to suppression of CCL4 transcription. This leads to an active escape from the anti-tumour immune response since dendritic cell recruitment is insufficient.
- Supplementary Information (3.3 MB)
This file contains Supplementary Tables 1-5 comprising:1 (a) Expression of T cell genes in segregated groups (b) Gene clusters after supervised hierarchical clustering (c) List of differentially expressed genes; 2 - Pearson correlation of β-catenin target genes and CD8a transcripts; 3 - Mutation analysis summary with (a) T cell signature low patients and (b) T cell signature high patients (c) Table summarizing potential pathway activators in patients with an active β-catenin signature; 4 - Mouse gene array data of differentially expressed genes (a) and a summary table focusing on chemokine expression (b); 5 - Detailed primer and antibody information (a) Genotyping primers (b) antibodies (c) qPCR primer/ probes (d) ChIP assay primer and (e) siRNA oligos.