Research ArticleCancer

Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of dendritic cells and tumor antigen–specific CTLs

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Science Translational Medicine  20 Sep 2017:
Vol. 9, Issue 408, eaan4220
DOI: 10.1126/scitranslmed.aan4220
  • Fig. 1. PVSRIPO causes cytopathogenicity in cancer cell lines.

    Melanoma (DM6 and DM440), prostate (LNCaP and DU145), and breast cancer (MDA-MB231 and SUM149) cell lines were infected with PVSRIPO at multiplicities of infection (MOIs) of 0.1 or 10. (A) Lysates were collected at the denoted hpi and tested by immunoblot for markers of direct viral cytotoxicity (eIF4G cleavage), host cell demise (PARP cleavage), viral translation (viral proteins P2/2BC/2C), and the innate antiviral response [p-eIF2α(S51)]. Global reduction of host (tubulin) and viral proteins at later time points reflects gross sample loss upon lytic destruction of cells. (B) Cells were infected (MOI, 0.1) and harvested (48 hpi) for analysis of MHC class I and CD155 expression by flow cytometry (light gray, uninfected cells stained with isotype control; dark gray, uninfected cells stained for MHC or CD155; red, infected cells stained for MHC or CD155). All experiments were repeated twice, and representative results are shown.

  • Fig. 2. PVSRIPO oncolysis releases tumor antigens, dsRNA, and DAMPs.

    DM6 and MDA-MB231 cells were infected with PVSRIPO. (A) Silver stain was performed on oncolysates/corresponding supernatants. (B) Immunoblots from oncolysates/corresponding supernatants for tumor antigens (MART1/CEA), DAMPs (HSPs and HMGB1), HSP60 (mitochondrial), Na+,K+-ATPase (membrane-associated), PARP (nucleus), and tubulin (cytoplasm). (C) Immunoblot for dsRNA was performed from oncolysates/corresponding supernatants of DM6 and MDA-MB231 cells (48 hpi); Ponceau S stain serves as the loading control. Loss of total protein late during PVSRIPO infection corresponds with cell lysis. All experiments were repeated twice, and a representative series is shown.

  • Fig. 3. PVSRIPO-induced oncolysate mediates DC activation.

    Human DCs were generated, and iDCs were treated as indicated. iDCs and mDCs were used as controls, as indicated in the figure. (A and B) Supernatant from untreated or PVSRIPO-infected (MOI, 0.1; 48 hpi) DM6 melanoma cells was either unfiltered or filtered through a 100-kDa cutoff filter. iDCs were treated with the resulting supernatants (24 hours). Flow cytometry (A) and enzyme-linked immunosorbent assay (ELISA) (B) were used to assess DC activation/maturation phenotype, viability, and proinflammatory cytokine production. (C and D) Human DCs were treated with DM6 oncolysate produced as in (A) or PVSRIPO at a titer equal to the amount detected in DM6 oncolysate. Supernatants were assessed for cytokine production by ELISA (C) and cell phenotype by flow cytometry (D). (A and D) For representative flow cytometry data, see fig. S2. Experiments were repeated three times with cells from two donors; error bars denote SEM, and asterisks denote significant (P < 0.05) analysis of variance (ANOVA) protected Tukey’s post hoc test compared to mock controls.

  • Fig. 4. PVSRIPO infection of DCs is sublethal, is marginally productive, and induces sustained proinflammatory cytokine production.

    (A and B) Analysis of percent lysis (as measured by LDH release) (A) and viral progeny (B) after PVSRIPO infection of DCs compared to PVSRIPO infection of DM6 and MDA-MB231 cells. Asterisks denote statistical significance comparing pooled cancer cell line data to DC values by ANOVA protected Tukey’s post hoc test; error bars depict SEM. (C) Dose-dependent PVSRIPO infection (viral protein 2C), type I IFN responses, and lack of cytotoxicity (PARP and eIF4G) in primary human DCs shown by immunoblot. (D and E) iDCs were untreated [mock (M)] or treated with PVSRIPO (PV) (MOI, 10), LPS (100 ng/ml), poly(I:C) (pIC) (10 μg/ml), or maturation CC. Cells and supernatant were harvested at the designated intervals. (D) Cell lysates were analyzed by immunoblot for the IFN response and DC activation proteins. (E) ELISA was used to measure IFN-β, TNF-α, IL-12, and IL-10 in DC supernatants after treatment. Data represent cumulative cytokine release at the designated time point. The mean of two experiments is shown for each time point. Asterisks denote PVSRIPO-mediated cytokine production that is significant compared to all other groups using ANOVA protected Tukey’s post hoc test. Repeat assays and magnified view of these data are presented in fig. S3.

  • Fig. 5. PVSRIPO-mediated APC activation occurs in immunosuppressive conditions.

    (A and B) DCs were cultured in the presence of AIM V or DM6CM (24 hours) and untreated (mock) or treated with PVSRIPO (MOI, 10) or poly(I:C) (10 μg/ml) (48 hours). (A) Cells were analyzed for activation markers by flow cytometry. Data bars represent the mean of two independent experiments, and error bars denote SEM. Asterisks depict significance as determined by ANOVA protected Tukey’s post hoc test. For representative flow cytometry data, see fig. S4. (B) Supernatant from (A) was tested for proinflammatory cytokine production by ELISA. (C and D) DM6 cells cultured alone or with DCs were mock-treated or infected with PVSRIPO (MOI, 10). (C) Supernatant was tested for lytic release of MART1 by immunoblot; cell pellets were tested for markers of DM6 cells (MART1) and DC activation [CD40, TAP1, p-STAT1(Y701), and STAT1]. (D) Supernatants from (C) were assessed for proinflammatory cytokine production. (E and F) Negatively selected human monocytes were differentiated with MCSF (25 ng/ml) or MCSF + IL-10, TGF-β, and IL-4 (all at 20 ng/ml) for 7 days. Cells were infected with PVSRIPO (MOI, 10) or treated with combined poly(I:C) (10 μg/ml) and LPS (100 ng/ml) as shown. Cell lysates were retained for immunoblot (E), and supernatants were used for ELISA (F). (B, C, and E) Experiments were repeated three times, and representative data are shown. (D and F) Data bars represent the mean of two independent experiments, and error bars indicate SEM.

  • Fig. 6. PVSRIPO oncolysate–pulsed DCs generate tumor antigen–specific CTL immunity in vitro.

    Primary human DCs coincubated with SUM149, MDA-MB231, LNCaP, or DM6 oncolysate stimulate tumor antigen–specific T cell responses in vitro. (A) Schema of the assay. (B) T cells were cocultured with oncolysate-pulsed autologous DCs, and the stimulated effector T cells were then harvested and tested in a CTL assay against the corresponding tumor cells (red bars), autologous DCs transfected with RNA that encodes for a relevant tumor antigen (black bars; positive control), or autologous DCs transfected with RNA that encodes for an irrelevant tumor antigen (white bars; negative control). Each bar represents mean % specific lysis and SD of triplicate samples. Statistical significance comparing autologous DCs expressing either the relevant or irrelevant tumor antigen for each panel in (B) was calculated using paired two-tailed Student’s t test. A probability of less than 0.05 (P < 0.05) is considered statistically significant. Panel SUM149 DC targets, P = 0.04; panel LNCaP DC targets, P = 0.0008; panel MDA-MB231 DC targets, P = 0.01; panel DM6 DC targets, P = 0.01. EGFR, epidermal growth factor receptor; PSA, prostate-specific antigen.

  • Fig. 7. mRIPO therapy restricts tumor growth and produces antigen-specific antitumor immunity.

    (A) Cytopathogenicity profile of mRIPO in B16-F10.9-OVA-CD155 cells. (B and C) Subcutaneous B16-F10.9-OVA-CD155 tumors were injected with either control (DMEM) or mRIPO (20 μl) when they reached a volume of ~50 to 100 mm3. Tumor volume was measured (n = 11 per group) on the days indicated (B); mice were euthanized when tumors reached 1000 mm3 (C). Data are from two pooled assays. Tumor growth curves were compared using multiple t tests with Holm-Sidak multiple comparison posttest; P ≤ 0.005 starting on day 6. Comparison of survival curves between the two groups was performed using the log-rank test (Mantel-Cox test), P < 0.0001. Median survival day: control, 14; mRIPO, 24. (D) Tumor-draining inguinal lymph nodes were harvested from mice (n = 4) 7 days after treatment with DMEM or mRIPO and restimulated with antigen-expressing cells for 5 days. Restimulated cells were tested for lytic activity against B16-F10.9-OVA cells or EL4 cells electroporated with RNA encoding GFP (control), TRP-2 (melanoma antigen), or OVA. (E) Supernatant from the CTL assay (D) was tested for markers of T cell activation and lytic activity by ELISA. (F) Tumor-bearing mice were treated with DMEM or mRIPO, and spleens were harvested 14 days after treatment (n = 4). Splenocytes from individual mice were cocultured with B16-F10.9-OVA-CD155 cells (5:1 ratio; 48 hours); supernatant was tested as in (E). (G) Tumor-draining inguinal lymph nodes were harvested after treatment with DMEM or mRIPO and individually restimulated in vitro. After 5 days, restimulated cells were analyzed for CD4 and CD8 T cells by flow cytometry. TRP-2–specific response was assessed using an H-2Kb TRP-2 tetramer (right panel). TRP-2–specific responses were compared using Student’s t test, with P < 0.05 considered significant. Figure S8D shows representative flow cytometry analyses of T cells and TRP-2–specific T cells (out of the four tested per group).

  • Fig. 8. mRIPO elicits neutrophil influx followed by DC and T cell infiltration into tumors.

    B16-F10.9-OVA-CD155 tumors were implanted subcutaneously, and when the tumor volume reached ~100 mm3, they were injected with DMEM (control) or mRIPO. Tumors were harvested after injection as indicated, digested to single-cell suspensions, and analyzed by flow cytometry. (A) Analysis of the percentage of CD45.2+ immune cells in the tumor after DMEM (control) or mRIPO treatment. Each bar represents three mice analyzed individually. (B) Cytokine concentrations in tumor homogenates. (C to E) Analysis of tumor-infiltrating neutrophils (C), DCs (D), and T cells (E) at the indicated days after mRIPO injection. (F) Longitudinal analysis of neutrophil, DC, CD4 T cell, and CD8 T cell infiltration is depicted as a percentage of total live cells in the tumor. Each bar represents three mice analyzed individually. Error bars represent SEM. The flow cytometry gating strategy is shown in figs. S9 and S10.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/408/eaan4220/DC1

    Materials and Methods

    Fig. S1. PVSRIPO cytotoxicity in malignant glioma cell lines.

    Fig. S2. Flow cytometry analysis of DC phenotype in Fig. 3.

    Fig. S3. Analysis of PVSRIPO infection of DCs.

    Fig. S4. Flow cytometry analysis of DC phenotype in Fig. 5.

    Fig. S5. Analysis of viral replication in DC/DM6 cocultures and PVSRIPO infection of human monocytes.

    Fig. S6. Tumor antigen–specific CTL induction by PVSRIPO oncolysate–pulsed DCs in vitro.

    Fig. S7. IFN-β production by mRIPO-infected and LPS-treated mouse DCs.

    Fig. S8. Analysis of B16-F10.9-OVA-CD155 tumor homogenate and tumor-draining lymph nodes after mRIPO treatment.

    Fig. S9. Analysis of tumor-infiltrating immune cells, myeloid cells, and monocytes/macrophages.

    Fig. S10. Analysis of tumor-infiltrating immune cells.

    Fig. S11. PVSRIPO-mediated tumor cytotoxicity, DC infection/stimulation, and antitumor immunity.

    Table S1. Relevant functions of cytokines/proteins present in DM6 melanoma-conditioned AIM V medium.

    References (6178)

  • Supplementary Material for:

    Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of dendritic cells and tumor antigen–specific CTLs

    Michael C. Brown, Eda K. Holl, David Boczkowski, Elena Dobrikova, Mubeen Mosaheb, Vidya Chandramohan, Darell D. Bigner, Matthias Gromeier,* Smita K. Nair*

    *Corresponding author. Email: smita.nair{at}duke.edu (S.K.N.); grome001{at}mc.duke.edu (M.G.)

    Published 20 September 2017, Sci. Transl. Med. 9, eaan4220 (2017)
    DOI: 10.1126/scitranslmed.aan4220

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. PVSRIPO cytotoxicity in malignant glioma cell lines.
    • Fig. S2. Flow cytometry analysis of DC phenotype in Fig. 3.
    • Fig. S3. Analysis of PVSRIPO infection of DCs.
    • Fig. S4. Flow cytometry analysis of DC phenotype in Fig. 5.
    • Fig. S5. Analysis of viral replication in DC/DM6 cocultures and PVSRIPO infection of human monocytes.
    • Fig. S6. Tumor antigen–specific CTL induction by PVSRIPO oncolysate–pulsed DCs in vitro.
    • Fig. S7. IFN-β production by mRIPO-infected and LPS-treated mouse DCs.
    • Fig. S8. Analysis of B16-F10.9-OVA-CD155 tumor homogenate and tumor-draining lymph nodes after mRIPO treatment.
    • Fig. S9. Analysis of tumor-infiltrating immune cells, myeloid cells, and monocytes/macrophages.
    • Fig. S10. Analysis of tumor-infiltrating immune cells.
    • Fig. S11. PVSRIPO-mediated tumor cytotoxicity, DC infection/stimulation, and antitumor immunity.
    • Table S1. Relevant functions of cytokines/proteins present in DM6 melanoma-conditioned AIM V medium.
    • References (6178)

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