Type III IFN interleukin-28 mediates the antitumor efficacy of oncolytic virus VSV in immune-competent mouse models of cancer

Abstract

Innate immune effector mechanisms triggered by oncolytic viruses may contribute to the clearance of both infected and uninfected tumor cells in immunocompetent murine hosts. Here, we developed an in vitro tumor cell/bone marrow coculture assay and used it to dissect innate immune sensor and effector responses to intratumoral vesicular stomatitis virus (VSV). We found that the type III IFN interleukin-28 (IL-28) was induced by viral activation of innate immune-sensing cells, acting as a key mediator of VSV-mediated virotherapy of B16ova melanomas. Using tumor variants which differentially express the IL-28 receptor, we showed that IL-28 induced by VSV within the tumor microenvironment sensitizes tumor cells to natural killer cell recognition and activation. These results revealed new insights into the immunovirological mechanisms associated with oncolytic virotherapy in immune-competent hosts. Moreover, they defined a new class of tumor-associated mutation, such as acquired loss of responsiveness to IL-28 signaling, which confers insensitivity to oncolytic virotherapy through a mechanism independent of viral replication in vitro. Lastly, the findings suggested new strategies to manipulate immune signals that may enhance viral replication, along with antitumor immune activation, and improve the efficacy of oncolytic virotherapies.

Figure 1

VSV activates bone marrow cytotoxicity against B16ova. A, in vitro coculture between tumor cells and bone marrow cells; NAb: neutralizing anti-VSV immune serum. B, light microscopy of cocultures (from A) of B16ova cells 24 h following treatment with (i) VSV MOI 0.1 (no bone marrow), (ii) VSV+NAb (no bone marrow), (iii) bone marrow+NAb, (iv) bone marrow cells alone (no VSV or Nab), or (v) bone marrow (BM)+VSV+NAb.

Figure 2

B16(LIF) cells are insensitive to VSV-activated bone marrow cytotoxicity. A and B, B16ova or B16(LIF) cells were cocultured with C57Bl/6 bone marrow with, or without, VSV as in Fig. 1A. Cell survival was assessed by crystal violet staining (A) or MTT assay (B). C, C57Bl/6 (8 mice/group) bearing 7-d subcutaneous (s.c.) B16(LIF) (i) or B16ova (ii) tumors were injected intratumorally every 2 d (three total) with 5 × 10⁸ pfu of heat-inactivated virus (HI) or VSV. Survival with time is shown. D, cDNA from B16ova or B16(LIF) cells was screened for IL-28R. A weak signal was detected in B16(LIF) cells with 20 additional cycles. **, P < 0.01; ***, P < 0.001; ns; non significant.

Figure 3

VSV-activated bone marrow cytotoxicity depends upon type I and III IFNs. A, B16ova cells were cocultured with bone marrow cells as in Fig. 1A, with or without anti-IL-28 antibody (15 ug/mL) upon infection with VSV. Twenty-four hours later, supernatants were harvested for viral titer and cell viability was assessed by crystal violet staining (A) and MTT assay (B). C, light microscopy of cocultures of B16ova cells with either C57Bl/6-derived bone marrow (i and iii) or bone marrow from IFN-α/βR KO mice (ii, iv, v, and vi) 48 h following treatment with VSV (MOI 0.1) +NAb (I and ii) or bone marrow+Nab (no VSV; iii and iv), or with bone marrow from IFN-α/βR KO mice +Nab (no VSV) with added IFN-α at 1 (v) or 100 (vi) U/mL. MTT assay of these results is shown in vii. D, the experiment in A was repeated with coculture of B16ova with C57Bl/6- or IFN-α/βR KO-derived bone marrow, with VSV +/− anti-IL-28 antibody. Twenty-four hours after VSV, supernatants were harvested for viral titer and cell viability was assessed by crystal violet. ***, P < 0.001.

Figure 4

GR1⁺ cells in bone marrow sense VSV. A, bone marrow from C57Bl/6 mice was left undepleted (none) or depleted of macrophages, NK, CD8, CD4, or GR1⁺ cells and cocultured with B16ova cells +/− VSV (MOI 0.1) as in Fig. 1A. Forty-eight hours after addition of VSV, supernatants were assayed for IL-28. B, C57Bl/6 (3 mice/group) bearing B16ova tumors were injected intratumorally with 5 × 10⁸ pfu of VSV (filled column) or PBS (opened column). Tumors (i) and draining lymph node (LN; ii and iii) were harvested at times shown and analyzed for (i) neutrophils (CD11b⁺GR1⁺F4/80⁻), CD11b⁺GR1⁺ (ii), or plasmacytoid dendritic cells (CD11b⁺GR1⁺PDCA⁺; iii). Infiltrates in tumors injected intratumorally with heat-inactivated VSV as a negative control were directly comparable with those tumors treated with PBS (data not shown). TM, tumor; DC, dendritic cells. C, C57Bl/6 bone marrow was left undepleted (i) or depleted of macrophages (ii) or GR1⁺ cells (iii), and cocultured with B16ova with VSV (MOI, 0.1) and neutralizing anti-VSV antibody (NAb) as in Fig. 1A. Forty-eight hours after the addition of VSV, cell survival was visualized or quantified by MTT assay (D). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5

IL-28 activates the NK recognition of B16ova. A and B, B16ova (A) or B16(LIF) (B) were cultured +/− recombinant IL-28 for 24 h and cDNA was screened for expression of NK ligands RAE, H60, and MULT-1. *, signal detected upon additional 20 cycles. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, NK cells were cocultured with B16ova (a–i) or B16(LIF) (a’-i’) +/− IL-28 or 48-h conditioned medium from C57Bl/6 bone marrow cells exposed to VSV (MOI, 0.1). Twenty-four hours later, supernatants were assayed for IFN-γ. D, mice (n = 8/group) bearing 4d B16ova s.c. tumors were depleted of NK cells by anti–asialo-GM-1 antibody or mock depleted (control IgG). VSV was injected intratumorally on days 7, 9, and 11. **, P < 0.01.

Figure 6

IL-28 signaling mediates the VSV therapy of B16ova tumors. A, freeze thaw lysates of in vitro cultured B16ova or B16ova infected with VSV (MOI, 0.1) 24 h previously or of 7d–established B16ova tumors from mice treated intratumorally with either PBS or VSV (5 × 10⁸ pfu) 24 h previously were assayed for IL-28. B, C57Bl/6 mice (n = 8/group) bearing 7d–established subcutaneous B16ova tumors were injected intratumorally at days 7, 9, and 11 with 5 × 10⁸ pfu of heat-inactivated virus (HI) or VSV along with anti–IL-28 or control antibody. C, C57Bl/6 mice (n = 8/group) bearing 7d–established B16(LIF) or B16(LIF)-IL-28R tumors were injected intratumorally with 5 × 10⁸ pfu of heat-inactivated VSV (HI-VSV) or VSV on days 7, 9, and 11. ***, P < 0.001.