Research ArticleTUMOR MICROENVIRONMENT

Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity

See allHide authors and affiliations

Science Immunology  21 Jun 2019:
Vol. 4, Issue 36, eaaw2004
DOI: 10.1126/sciimmunol.aaw2004
  • Fig. 1 Necroptotic cells confer tumor control across multiple syngeneic flank tumor models.

    (A) Schematic of pro-death enzyme constructs and respective types of PCD induced downstream after enzyme activation with B/B homodimerizer. (B to F) Tumor growth of B6/J mice bearing (B and D) B16.F10-OVA, (C and E) LL/2-OVA, or (F) E.G7-OVA flank tumors after administration of apoptotic or necroptotic (B and C) autologous or (D to F) unmatched NIH 3T3 fibroblast cells. n = 10 to 16 mice per group. (G and H) Tumor growth of B6/J mice bearing (G) B16.F10 or (H) LL/2 flank tumors after administration of necroptotic NIH 3T3 cells. n = 9 to 14 mice per group. (I) Tumor growth of ipsilateral (“I,” treated) and contralateral (“C,” untreated) B16.F10-OVA tumors after administration of either apoptotic or necroptotic NIH 3T3 cells (left and middle panels) and survival curve of mice from the same experiment (right panel). n = 9 to 11 mice per group. **P < 0.01, ***P < 0.001, and ****P < 0.0001. Black arrows indicate intratumoral dying cell injections. Error bars represent SEM. Data are pooled from three to five independent experiments.

  • Fig. 2 Tumor control by necroptotic cells requires BATF3+cDC1 and CD8+leukocytes.

    (A) Day 12 B16.F10-OVA tumor volumes (left) and animal survival (right) of mice with varying Batf3 genotypes after necroptotic fibroblast injections. n = 4 to 12 mice per group. d.11, day 11. (B) B16.F10-OVA tumor growth (left) and animal survival (right) upon coadministration of necroptotic fibroblasts with depleting antibodies against either CD4+ or CD8+ leukocytes. n = 6 to 11 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Black arrows indicate intratumoral dying cell injections. Error bars represent SEM. Data are pooled from two to four independent experiments.

  • Fig. 3 Immune-mediated tumor control by necroptotic cells requires NF-κB activation within dying cells but not MLKL-mediated cell lysis and DAMP release.

    (A) Day 12 volumes of B16.F10-OVA tumors in various innate immune knock-out mice after necroptotic fibroblast injections on days 6, 8, and 10. n = 5 to 15 mice per group. (B) B16.F10-OVA tumor growth (left panel) and animal survival (right panel) upon coadministration of necroptotic fibroblasts with blocking α-CLEC9A antibody. n = 10 mice per group. (C and D) Tumor growth and overall survival after administration of lytic necrotic fibroblasts in (C) single or (D) contralateral B16.F10-OVA flank tumors. Tumor growth and survival curves for PBS, acCASP8, and acRIPK3 as presented in Fig. 1 and are also graphed for comparison. (E) B16.F10-OVA tumor growth curves after injection of necroptotic NIH 3T3 fibroblasts preincubated with the IκBα phosphorylation inhibitor BAY-117085, which prevents NF-κB activation in treated cells. n = 10 mice per group. (F) B16.F10-OVA tumor growth (left) and animal survival (right) after intratumoral injection of PBS, necroptotic fibroblasts, or MLKL−/− necroptotic fibroblasts. n = 7 to 9 mice per group. (G) Assessment of systemic inflammation via Luminex assay for inflammatory serum cytokines and chemokines 48 hours after intratumoral dying NIH 3T3 injection. DMXAA-injected mice were included as a positive control for systemic inflammatory cytokine production. The gray dashed line represents the limit of detection. n = 3 to 5 mice per group. (H) B16.F10-OVA tumor growth curves after intratumoral (IT), intraperitoneal (IP), distal subcutaneous (distal subQ), or intravenous (IV) injection of necroptotic fibroblasts. n = 7 to 9 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Black arrows indicate intratumoral dying cell injections. Error bars represent SEM. Data are pooled from two to five independent experiments. ns, not significant.

  • Fig. 4 Necroptosis promotes antitumor CD8+T responses and synergizes with ICB.

    (A) Absolute numbers of intratumoral CD8+ T cells with various phenotyping markers for proliferation (Ki67), effector function (GranzymeB, GzmB), and activation (CD44), normalized per gram of tumor tissue. (B) Quantification of the ratio of intratumoral activated (CD44hi, left) or tumor antigen–specific (SIINFEKL-H2Kb+, right) CD8+ T cells to immunosuppressive Foxp3+ CD25+ Treg, normalized per gram of tumor tissue. (C) Sample flow plots (left) and percentages (right) of overall activated CD8+ T cells in the tumor-draining (inguinal) lymph node. (D) Sample flow plots (left) and percentages (right) of OVA-specific and activated CD8+ T cells in the tumor-draining (inguinal) lymph node. (E) B16.F10-OVA tumor growth (left) and animal survival (right) after coadministration of necroptotic fibroblasts with the lymphocyte trafficking inhibitor FTY-720. n = 8 to 12 mice per group. (F) Tumor growth of ipsilateral (treated, left) and contralateral (untreated, right) B16.F10-OVA tumors after administration of necroptotic NIH 3T3 cells with FTY-720. n = 9 to 10 mice per group. (G) Survival curves of B16.F10-OVA tumor–bearing mice after coadministration of necroptotic fibroblasts with the ICB reagent α-PD-1 or IgG2a isotype. n = 8 to 14 mice per group. (H) Left: Schematic of tumor rechallenge experiments in mice from (G) that successfully cleared B16.F10-OVA tumors. Right: Survival of mice rechallenged with B16.F10-OVA cells on the same flank as initial tumor location. n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. All flow harvests performed 48 hours after dying cell injection. Error bars represent SEM. Data are pooled from two to four independent experiments.

  • Fig. 5 Exposure to necroptosis in the TME promotes antigen uptake and activation of tumor-associated APCs.

    (A) Absolute numbers of tumor-associated DC subsets 48 hours after intratumoral dying cell administration, normalized per gram of tumor tissue. (B) Intratumoral concentrations of DC-recruiting chemokines 24 hours after dying NIH 3T3 injection. The gray dashed line represents the limit of detection. n = 3 to 5 mice per group. (C) Experimental schematic of B16.F10-OVA tumor cells expressing zsGreen as a surrogate tumor antigen, allowing for gating on tumor APCs that have phagocytosed tumor antigen. (D) Percent of zsGreen+ tumor APCs after dying cell administration. (E) gMFI of CLEC9A receptor on subsets of tumor DC populations. n = 4 to 6 mice per group. (F) gMFI of PD-L1 on subsets of tumor APC populations and CD45 zsGreen+ tumor cells. n = 4 to 6 mice per group. (G) gMFI of the costimulatory marker CD80 on zsGreen+ subsets of tumor APC populations. n = 3 to 4 mice per group. (H) Quantification of previously activated OT-I T cell proliferation upon coculture with zsGreen+ tumor APC subsets sorted ex vivo from B16.F10-OVA-zsGreen tumors after dying cell injection. n = 3 technical replicates per group, using pooled cells from five mice per treatment group. (I) In vitro characterization of BMDMs cocultured with live or necroptotic B16.F10-zsGreen tumor cells and dextran-fluorophore beads, assessed for phagocytosis via dextran uptake (left) and expression of costimulatory marker CD80 in zsGreen+ BMDMs (middle) or dextran+ beads (right). n = 3 technical replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. All flow harvests performed 48 hours after dying cell injection. Error bars represent SEM. Data are representative plots from one to three independent experiments (E to I) or pooled from two to three independent experiments (A, B, and D).

  • Fig. 6 Engineered AAVs can be used to specifically induce necroptosis of tumor cells in vitro.

    (A) Schematic of AAVs used to transduce tumor cells to express engineered pro-death enzymes fused to a constitutively oligomerizing (co) recruitment domain under the control of a synthetic MND promoter, leading to subsequent induction of a corresponding PCD modality. (B and C) Validation and kinetics of AAV2.5 serotype transduction efficiency in B16.F10 cells in vitro. (B) Percent of GFP+ cells transduced with AAV2.5-eGFP control. (C) Percent cell death in cells transduced with various death-inducing constructs. n = 3 technical replicates per group. (D) Heatmap depicting relative expression values of NF-κB–dependent gene targets, chemokines, and cytokines via NanoString analysis of B16.F10 tumor cells compared to eGFP-transduced controls 10 hours after AAV2.5 transduction (1 × 1011 IFU). Data are representative plots from two independent experiments (B and C) or means of three technical replicates from one experiment (D).

  • Fig. 7 Administration of necroptosis-targeting AAVs in conjunction within vivo promotes durable tumor clearance.

    α-PD-1 (A) B16.F10-OVA tumor growth curves after intratumoral administration of 1 × 1011 IFUs of death-inducing AAVs or eGFP control AAV. n = 8 to 14 mice per group. (B) Intratumoral concentrations of inflammatory cytokines and chemokines 48 hours after intratumoral AAV injection. The gray dashed line represents the limit of detection. n = 3 to 4 mice per group. (C) Tumor growth of ipsilateral (I, treated) and contralateral (C, untreated) B16.F10-OVA tumors after AAV administration. n = 10 to 12 mice per group. (D) Survival curves of B16.F10-OVA tumor–bearing mice after coadministration of AAVs with isotype control antibody. n = 14 to 15 mice per group. (E) Survival curves of B16.F10-OVA tumor–bearing mice after coadministration of AAVs with α-PD-1. n = 13 to 16 mice per group. (F) B16.F10-OVA tumor growth upon coadministration of necroptosis-inducing coRIPK3 AAV with α-CD8+ depletion antibody. n = 8 to 10 mice per group. (G) B16.F10-OVA tumor growth in Batf3−/− or wild-type control mice after necroptosis-inducing AAV administration. n = 10 to 13 mice per group. (H) Left: Schematic of tumor rechallenge experiments in mice from (E) successfully clear B16.F10-OVA tumors. (H) Right: Survival of mice rechallenged with B16.F10-OVA cells on the same flank as initial tumor location. n = 8 to 10 mice per group. (I) Kaplan-Meier plot for overall survival of skin cutaneous melanoma patients in TCGA data set. Data are parsed on upper and lower quartiles (25%) of RIPK3 mRNA expression. n = 114 patients per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Black arrows indicate intratumoral AAV injections. Error bars represent SEM. Data are pooled from two to four independent experiments (A to H).

  • Fig. 8 Proposed model by which necroptotic cell death within the TME promotes antitumor immunity.

    RIPK1/RIPK3 activation in necroptotic cells produces NF-κB–dependent signals that promote CD103+ cDC1− and CD8+ leukocyte–mediated antitumor immunity, which synergizes with α-PD-1 to promote durable tumor clearance.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/36/eaaw2004/DC1

    Fig. S1. Necroptotic cells extend the survival of tumor-bearing mice.

    Fig. S2. BATF3+ and CD8+ leukocyte requirements for tumor control by necroptotic cells.

    Fig. S3. DAMP-independent tumor control by necroptotic cells is recapitulated in multiple syngeneic tumor models.

    Fig. S4. Gating strategies and quantification of T cell subsets in tumor and tdLN after dying cell administration.

    Fig. S5. Gating strategies and effects of dying cell administration on tumor APC subsets.

    Fig. S6. Characterization of transduction efficiency and cell death induction by engineered AAVs.

    Fig. S7. Tumor growth restriction and survival extension after administration of AAVs targeting tumor cell necroptosis in situ.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Necroptotic cells extend the survival of tumor-bearing mice.
    • Fig. S2. BATF3+ and CD8+ leukocyte requirements for tumor control by necroptotic cells.
    • Fig. S3. DAMP-independent tumor control by necroptotic cells is recapitulated in multiple syngeneic tumor models.
    • Fig. S4. Gating strategies and quantification of T cell subsets in tumor and tdLN after dying cell administration.
    • Fig. S5. Gating strategies and effects of dying cell administration on tumor APC subsets.
    • Fig. S6. Characterization of transduction efficiency and cell death induction by engineered AAVs.
    • Fig. S7. Tumor growth restriction and survival extension after administration of AAVs targeting tumor cell necroptosis in situ.

    Download PDF

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article