Research ArticleTUMOR IMMUNOLOGY

RIG-I activation is critical for responsiveness to checkpoint blockade

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Science Immunology  13 Sep 2019:
Vol. 4, Issue 39, eaau8943
DOI: 10.1126/sciimmunol.aau8943
  • Fig. 1 Anti–CTLA-4–mediated systemic antitumor immunity requires tumor cell–intrinsic RIG-I signaling.

    (A) Treatment scheme 1: WT mice were implanted with either WT or RIG-I–deficient (RIG-I−/−) B16.OVA cells. Recipients were injected intraperitoneally with anti–CTLA-4 or isotype control antibodies. (B) Tumor growth and (C) overall survival of mice bearing either WT or RIG-I−/− tumors. Data show survival of n = 35 mice per group that were pooled from five independent experiments. (D) Treatment scheme 2: WT mice were bilaterally challenged with WT or RIG-I−/− B16.OVA cells. Right-sided tumors were induced with more tumor cells to facilitate a faster growth dynamic in comparison with left-sided tumors. Recipients were treated with anti–CTLA-4 as described above. Some mice were additionally injected with the RIG-I ligand 3pRNA into the right-sided (local) tumor. (E and F) Tumor growth of 3pRNA-injected (E) and noninjected (distant) (F) B16.OVA tumors. All tumor growth curves show mean tumor volume ± SEM of n = 12 to 15 individual mice. (G) Overall survival of treated mice bearing WT or RIG-I−/− B16.OVA tumors. All data were pooled from at least three independent experiments. (H and I) WT mice were implanted with WT or RIG-I−/− tumor cells. Recipient mice were injected intraperitoneally with anti–CTLA-4 or isotype control antibodies. (H) Tumor growth of n = 6 to 12 mice per group bearing CT26 colon carcinomas. Data are representative of two independent experiments. (I) Tumor growth in n = 14 to 15 mice per group bearing Panc02 pancreatic carcinomas. Data were pooled from two independent experiments. All tumor growth curves show mean tumor volume ± SEM.

  • Fig. 2 Tumor cell–intrinsic RIG-I signaling facilitates cross-presentation of tumor-associated antigen by CD103+ DCs and subsequent priming of CD8+ T cells.

    (A) WT mice bearing bilateral WT or RIG-I−/− B16.OVA tumors were treated with a one-sided intratumoral 3pRNA administration or its vehicle in vivo-jetPEI. Frequency of tumor cells undergoing programmed cell death was analyzed by TUNEL 24 hours later. (B to D) WT mice bilaterally inoculated with WT or RIG-I−/− B16.OVA cells were repeatedly treated with anti–CTLA-4 ± intratumoral 3pRNA as described in Fig. 1D. Analyses were performed on day 15 after tumor induction. (B) Cross-presentation of the processed OVA peptide-epitope SIINFEKL in the context of MHC-I on CD103+ DCs in the lymph node draining the 3pRNA-injected tumor (TdLN) determined by flow cytometry. (C) Frequency of H-2Kb–SIINFEKL Tetramer+ CD8+ T cells (carrying an OVA-specific T cell receptor) in peripheral blood. All data give values of individual mice (and group means presented as bars) that were pooled from at least two independent experiments. (D) Frequency of tumor-infiltrating CD8+ T cells analyzed by immunohistochemistry; representative tumor sections after hematoxylin and eosin and anti–CD8 (red, arrows) staining from one of two independent experiments. Magnification, ×20. Scale bars, 50 μm. N, highly necrotic area. (E and F) WT and Batf3-deficient (Batf3−/−) mice were bilaterally inoculated with WT B16.OVA cells and were treated with anti–CTLA-4 ± intratumoral 3pRNA as described in Fig. 1D. (E) Overall survival of tumor-bearing mice and (F) the frequency of H-2Kb–SIINFEKL Tetramer+ CD8+ T cells in peripheral blood on day 15 after tumor induction. All data give values of individual mice + group mean as bar that were pooled from at least two independent experiments.

  • Fig. 3 Host IFN-I contributes to efficacy of anti–CTLA-4 therapy.

    (A) Treatment scheme 1: WT and IFN-α receptor 1–deficient mice (Ifnar1−/−) were bilaterally inoculated with WT B16.OVA cells and were repeatedly treated with anti–CTLA-4 ± intratumoral 3pRNA as described in Fig. 1D. (B) Overall survival of n = 4 to 8 tumor-bearing animals per group that were pooled from two independent experiments. (C) Mice bearing bilateral WT or RIG-I−/− B16.OVA tumors were treated with a one-sided intratumoral 3pRNA administration or its vehicle in vivo-jetPEI. Concentration of IFN-I within the tumor microenvironment 24 hours later determined by enzyme-linked immunosorbent assay. (D) Frequency of H-2Kb–SIINFEKL Tetramer+ CD8+ T cells in peripheral blood on day 15 after tumor induction in WT and Ifnar1−/− mice bearing WT tumors that were treated as described in (A). (E) WT mice were bilaterally inoculated with WT or IRF3/7-deficient (IRF3/7−/−) B16.OVA cells and were repeatedly treated with anti–CTLA-4 ± 3pRNA as described in Fig. 1D. (F) Overall survival and (G) frequency of H-2Kb–SIINFEKL Tetramer+ CD8+ T cells in peripheral blood on day 15 after tumor induction in treated mice bearing WT or IRF3/7−/− B16.OVA tumors. All data give values of n = 10 individual mice and were pooled from at least two independent experiments. ns, not significant.

  • Fig. 4 Caspase-3–mediated tumor cell death is crucial for anti–CTLA-4 efficacy.

    (A and B) WT, RIG-I−/−, caspase-3–deficient (caspase-3−/−), and MLKL-deficient (MLKL−/−) B16.OVA cells were transfected with 3pRNA in vitro and were harvested at the indicated time points. Cell viability determined by (A) annexin V and Live/Dead or (B) cleaved caspase-3 and Live/Dead staining by flow cytometry. Representative data are from one of three independent experiments. (C) WT B16.OVA and murine L-929 fibroblasts were transfected with 3pRNA, and induction of necroptotic cell death by phosphorylated MLKL (pMLKL) was analyzed by Western blot. The combination of TNF-α, SMAC mimetics, and the pan-caspase inhibitor Z-VAD-FMK was used as a positive control to induce necroptosis. (D) WT mice bearing bilateral WT or RIG-I−/− B16.OVA tumors were treated with a one-sided intratumoral 3pRNA administration or its vehicle in vivo-jetPEI as described for Fig. 2A. Frequency of tumor cells expressing cleaved caspase-3 24 hours later. Data give values of individual mice + group mean as bar that were pooled from two independent experiments. (E) Treatment scheme: WT mice were bilaterally inoculated with WT or caspase-3−/− B16.OVA cells and were treated with anti–CTLA-4 ± intratumoral 3pRNA as described in Fig. 1D. (F) Frequency of H-2Kb–SIINFEKL Tetramer+ CD8+ T cells in peripheral blood on day 15 after tumor induction. Tumor growth of (G) 3pRNA-injected and (H) non-injected (distant) B16.OVA tumors. All tumor growth curves show mean tumor volume ± SEM of n = 10 individual mice. (I) Overall survival of treated mice bearing WT or caspase-3−/− B16.OVA tumors. All figure panels give data of n = 10 individual mice per group that were pooled from two independent experiments.

  • Fig. 5 Host RIG-I signaling is additionally required for systemic tumor control after anti–CTLA-4 therapy.

    (A) Treatment scheme 1: WT and MAVS-deficient (Mavs−/−) mice were bilaterally inoculated with WT B16.OVA cells and were treated with anti–CTLA-4 ± intratumoral 3pRNA as described in Fig. 1D. (B) Tumor growth of 3pRNA-injected and (C) distant WT B16.OVA tumors. (D) Frequency of H-2Kb–SIINFEKL Tetramer+ CD8+ T cells in peripheral blood on day 15 after tumor induction. (E) Overall survival in WT and Mavs−/− recipient mice bearing WT B16.OVA tumors. All figures give data of n = 9 or 10 individual mice per group that were pooled from two independent experiments. (F) Treatment scheme 2: WT and Mavs−/− mice were bilaterally inoculated with RIG-I−/− B16.OVA cells and were treated with anti–CTLA-4 ± intratumoral 3pRNA as described in Fig. 1D. (G) Overall survival in WT and Mavs−/− recipient mice bearing RIG-I−/− B16.OVA tumors. Data give survival of n = 4 to 6 individual mice per group and are representative of two independent experiments.

  • Fig. 6 Tumor cell–intrinsic STING signaling is dispensable for anti–CTLA-4–mediated antitumor immunity.

    (A) Treatment scheme 1: WT and STING-deficient (Stinggt/gt) mice were bilaterally inoculated with WT B16.OVA cells and were treated with anti–CTLA-4 ± intratumoral 3pRNA as described in Fig. 1D. (B) Tumor growth of 3pRNA-injected and (C) distant WT B16.OVA tumors. (D) Overall survival in WT and Stinggt/gt recipient mice bearing WT B16.OVA tumors. All figures give data of n = 11 to 13 individual mice per group that were pooled from two independent experiments. (E) Treatment scheme 2: WT mice were inoculated with WT or STING-deficient (STING−/−) B16.OVA cells and were treated with anti–CTLA-4 as described in Fig. 1A. (F) Frequency of H-2Kb–SIINFEKL Tetramer+ CD8+ T cells in peripheral blood was determined on day 15 after tumor induction. All data give values of individual mice + group mean as bar that were pooled from two independent experiments. (G) Overall survival in mice bearing WT or STING−/− B16.OVA tumors that were either treated with anti–CTLA-4 or isotype control antibodies. All data are pooled from at least two independent experiments.

  • Fig. 7 High expression of DDX58 in human melanoma correlates with prolonged survival and durable response to anti–CTLA-4 immunotherapy.

    (A) Overall survival in 456 patients with melanoma from TCGA by expression of DDX58 (RIG-I) or TMEM173 (STING) in tumor samples. (B) Heat map of DEGs in tumors with high versus low expression of DDX58. (C) KEGG pathway enrichment analysis of DEGs. Fraction of DEGs in pathway is indicated by circle size, significance of enrichment by color. NOD, nucleotide-binding oligomerization domain. (D) DDX58 and TMEM173 expression in tumor samples from 18 patients with durable clinical response to anti–CTLA-4 treatment versus nonresponders. Data give values from individual patients + geometric mean. (E) Overall survival in 18 patients with melanoma undergoing anti–CTLA-4 immunotherapy by expression of DDX58 or TMEM173 in tumor samples.

  • Fig. 8 Response to anti–PD-1 does not require tumor cell–intrinsic RIG-I pathway activity.

    (A to C) WT mice were implanted with WT or RIG-I–deficient (RIG-I−/−) B16.OVA cells in the right flank. Recipient mice were injected intraperitoneally with anti–PD-1 or isotype control antibodies. (A) Tumor growth and (B) overall survival of mice bearing WT or RIG-I−/− tumors. Data show survival of n = 24 to 30 mice per group that were pooled from four independent experiments. (C) Frequency of H-2Kb–SIINFEKL Tetramer+ CD8+ T cells in peripheral blood. (D) Retrospectively analyzed overall survival in 51 patients with melanoma undergoing anti–PD-1 immunotherapy by gene expression of RIG-I (DDX58) in pretherapy tumor samples. (E) Overall survival of patient cohort from (D) additionally stratified to prior exposure to ipilimumab (Ipi-naïve versus Ipi-progressive). (F) RIG-I (DDX58) expression levels in pretherapy tumor samples in patients with clinical response to anti–PD-1 treatment in comparison with nonresponders. Data give values from individual patients + geometric mean and are shown for the complete patient cohort and Ipi-naïve and Ipi-progressive patients. (G) WT mice were implanted with WT or RIG-I−/− B16.OVA cells and were treated intraperitoneally with anti–PD-1 and anti–CTLA-4 antibodies. Data show overall survival of n = 5 to 20 tumor-bearing mice per group that were pooled from two independent experiments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/39/eaau8943/DC1

    Materials and Methods

    Fig. S1. Characterization of RIG-I–deficient B16.OVA clones.

    Fig. S2. Tumor cell–intrinsic RIG-I signaling promotes localized cross-presentation of tumor-associated antigen by CD103+ DCs in TdLNs.

    Fig. S3. Tumor cell–intrinsic RIG-I deficiency is associated with reduced TIL frequencies and decreased expression of proteins involved in T cell lytic function.

    Fig. S4. Anti–CTLA-4–mediated antitumor immunity does not rely on tumor cell–derived IFN-I.

    Fig. S5. Melanoma cell–intrinsic STING signaling induces IFN-I production but not programmed cell death.

    Fig. S6. Local RIG-I activation renders poorly immunogenic tumors susceptible to checkpoint inhibition.

    Fig. S7. Antitumor synergy between CTLA-4 blockade and local RIG-I activation is not restricted to melanoma.

    Fig. S8. Proposed model: Tumor-intrinsic RIG-I signaling promotes checkpoint inhibitor–mediated anticancer immunity.

    Table S1. Single-gene data murine tumor RNA-seq (Excel).

    Table S2. Low RIG-I–encoding DDX58 expression in melanoma biopsies is an independent risk factor for death.

    Table S3. Raw data file (Excel).

    References (5357)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Characterization of RIG-I–deficient B16.OVA clones.
    • Fig. S2. Tumor cell–intrinsic RIG-I signaling promotes localized cross-presentation of tumor-associated antigen by CD103+ DCs in TdLNs.
    • Fig. S3. Tumor cell–intrinsic RIG-I deficiency is associated with reduced TIL frequencies and decreased expression of proteins involved in T cell lytic function.
    • Fig. S4. Anti–CTLA-4–mediated antitumor immunity does not rely on tumor cell–derived IFN-I.
    • Fig. S5. Melanoma cell–intrinsic STING signaling induces IFN-I production but not programmed cell death.
    • Fig. S6. Local RIG-I activation renders poorly immunogenic tumors susceptible to checkpoint inhibition.
    • Fig. S7. Antitumor synergy between CTLA-4 blockade and local RIG-I activation is not restricted to melanoma.
    • Fig. S8. Proposed model: Tumor-intrinsic RIG-I signaling promotes checkpoint inhibitor–mediated anticancer immunity.
    • Legend for table S1
    • Table S2. Low RIG-I–encoding DDX58 expression in melanoma biopsies is an independent risk factor for death.
    • References (5357)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Single-gene data murine tumor RNA-seq (Excel).
    • Table S3. Raw data file (Excel).

    Files in this Data Supplement:

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