Treg depletion-enhanced IL-2 treatment facilitates therapy of established tumors using systemically delivered oncolytic virus

Abstract

There are several roadblocks that hinder systemic delivery of oncolytic viruses to the sites of metastatic disease. These include the tumor vasculature, which provides a physical barrier to tumor-specific virus extravasation. Although interleukin-2 (IL-2) has been used in antitumor therapy, it is associated with endothelial cell injury, leading to vascular leak syndrome (VLS). Here, we demonstrate that IL-2-mediated VLS, accentuated by depletion of regulatory T cells (Treg), facilitates localization of intravenously (i.v.) delivered oncolytic virus into established tumors in immune-competent mice. IL-2, in association with Treg depletion, generates "hyperactivated" natural killer (NK) cells, possessing antitumor activity and secreting factors that facilitate virus spread/replication throughout the tumor by disrupting the tumor architecture. As a result, the combination of Treg depletion/IL-2 and systemic oncolytic virotherapy was found to be significantly more therapeutic against established disease than either treatment alone. These data demonstrate that it is possible to combine biological therapy with oncolytic virotherapy to generate systemic therapy against established tumors.

Figure 1. Interleukin-2 (IL-2) induces vascular leak and immune infiltration into tumors

(a) C57Bl/6 mice were seeded with B16 tumors subcutaneously (SC). After 10 days, (when tumors were typically 0.5–0.6cm in the longest diameter), the mice were injected intraperitoneally with rhIL-2 at 75,000U/injection three times a day for 3 days. On the fourth day, a single further injection of IL-2 was given. (b) Forty-eight hours after the final injection of phosphate-buffered saline (PBS) or IL-2 (in a), vascular leakage into the lungs was measured (n = 4; representative of two separate experiments). (c) C57Bl/6 mice treated with intraperitoneal injections of either IL-2 or PBS (as described in a; three/group) were killed 48 hours after the last injection. Immune cells positive for the markers shown are plotted as a percentage of CD45⁺ gated cells. NK cells, natural killer cells; NKT cells, NK T cells; rhIL-2, recombinant human IL-2.

Figure 2. Depletion of regulatory T cells (Treg) enhances activity of interleukin-2 (IL-2) activated natural killer (NK)/lymphokine-activated killer cells

(a) Nontumor-bearing C57Bl/6 mice were treated as described for Figure 1a, with the addition of an intraperitoneal injection of PC61 24 hours before the first intraperitoneal injection of IL-2 or phosphate-buffered saline (PBS). An additional group of mice received an injection of the NK cell-depleting asialo GM-1 antibody 24 hours before treatment with PC61 and then IL-2. Forty-eight hours after the final injection of IL-2 or PBS, vascular leakage into the lungs was measured. The results shown are from two separate experiments per treatment. (b) Splenocytes were recovered from mice treated as in a, and were plated with YAC cells (1 splenocyte effector:1 YAC target cell) in triplicate wells. After 48 hours, the surviving cells were counted and the result is expressed as a percentage of the controls (YAC + splenocytes from PBS-treated mice). (c) Splenocytes were recovered from mice treated as in a, and were plated with YAC cells (1:1). After 48 hours, the supernatants were assayed for interferon-γ (IFN-γ) using enzyme-linked immunosorbent assay (ELISA). (d) Splenocytes from the same samples as in c were also plated with B16 targets (1:1), and 48 hours later the supernatants were assayed for IFN-γ using ELISA. The results are representative of two separate experiments.

Figure 3. Regulatory T cells depletion combined with interleukin-2 (IL-2) permits tumor localization of vesicular stomatitis virus (VSV)

(a) C57Bl/6 mice (three/group) were seeded with subcutaneous (SC) B16 tumors. After 9 days, the mice received an intraperitoneal (IP) injection of PC61 or control immunoglobulin G (IgG). After a further 24hours, the mice were injected IP with phosphate-buffered saline (PBS) or with recombinant human (75,000U/injection three times a day for 3 days). On the fourth day, a single further injection of IL-2 was given. Two hours after this last injection of IL-2/PBS, the mice received an intravenous (IV) injection of 10⁸ plaque forming units (pfu) of VSV-GFP. (b–e) Thirty-six hours after in vivo virus delivery, the tumors were explanted and dissociated in vitro. The cells were left to adhere to the culture dish for 6–12 hours and then washed three times gently in PBS to remove nonadherent cells. These cultures were examined visually using phase microscopy (upper panels) or fluorescence for GFP expression (lower panels). The treatments received by the mice from which the tumors were explanted are shown between the panels. This protocol reproducibly generated cultures which were >98% positive for gp100, a B16-specific melanoma/melanocyte-associated antigen. This shows that GFP positivity is derived almost exclusively from recovered tumor cells rather than from infiltrating (nonadherent) immune cells [such as natural killer (NK) cells]. (f) Samples of the explanted B16 tumors obtained from a–e above were analyzed using flow cytometry for GFP. The results from a–f are representative of three separate experiments. GFP, green fluorescent protein. FSC, forward scatter.

Figure 4. Regulatory T cells depletion with interleukin-2 (IL-2) enhances viral delivery to systemic metastatic disease

(a and b) C57Bl/6 mice were left uninjected (nontumor bearing) or were injected intravenously (IV) with B16 cells to seed metastases in the lung. After 9 days, the mice received an intraperitoneal injection of PC61 or a control immunoglobulin G. One group received the natural killer (NK) cell-depleting asialo GM-1 antibody 24 hours before PC61 (no NK cell). After a further 24 hours, the mice were injected intraperitoneally with phosphate-buffered saline (PBS) or recombinant human IL-2 (75,000 U/injection for 10 injections). Two hours after this last injection of IL-2/PBS, the mice received 10⁸ plaque forming units (pfu) of VSV-GFP IV. After a further 36 hours the mice were killed, the lungs were removed, and viral titers were measured from freeze–thaw lysates of (a) the lungs or (b) hearts (two mice/group). One group of mice, that had been injected IV with 10⁸ pfu of VSV-GFP 3 weeks before the seeding of the B16 lung metastases, was treated with PC61/IL-2 and VSV (VSV, PC61/IL-2, preimmune). (c) Dissociated heart tissues and (d) spleen tissues from the mice described in were analyzed using flow cytometry for GFP expression. GFP, green fluorescent protein; VSV, vesicular stomatitis virus.

Figure 5. PC61/interleukin-2 (IL-2)-activated natural killer/lymphokine-activated killer cells facilitate intratumoral viral replication and spread

(a) Virus replication and spread throughout intact, dissociate B16 tumors was measured as described in Materials and Methods. Splenocytes recovered from mice treated with PC61/control immunoglobulin G (IgG)/± asialo GM-1 and then with phosphate-buffered saline (PBS) or with recombinant human IL-2 were added to the wells containing explanted B16 tumors along with vesicular stomatitis virus (VSV) [4 × 10⁸ plaque forming units (pfu)/well]. One set of tumors was treated with splenocytes from mice that had received PC61 + IL-2 with added EDTA (1 mmol/l). Virus titers from freeze–thaw lysates of the tumors 48 hours later are shown (pfu/mg tumor; three/group). (b) The experiment described in a was repeated, except that, after co-culture with splenocytes (from mice treated as shown over each panel) and VSV, tumors were dissociated in vitro and analyzed for the extent of viral spread/infection, using flow cytometry for green fluorescent protein expression, 24 hours after plating. Dissociated cultures were almost exclusively tumor cells as assessed by fluorescence-activated cell sorting for the melanoma marker gp100. (c) 5 × 10⁵ B16 cells were plated in vitro and 24 hours later, 5 × 10⁵ splenocytes from mice treated with PC61/IL-2/asialo-GM-1 as in a were added along with VSV at a density of 5 × 10⁴ pfu/well. After a further 48 hours, the supernatants and surviving cells were recovered. Virus titers from freeze–thaw lysates are shown as pfu/ml (three/group). (d) C57Bl/6 mice received an intraperitoneal injection of PC61 or control immunoglobulin G. One group of these mice received asialo GM-1 antibody 24 hours before treatment with PC61. After 24 hours, the mice were injected intraperitoneally with PBS or with rhIL-2 (75,000 U/injection for 10 injections). After a further 24 hours, splenocytes from these mice were recovered, cDNA was prepared, and the expression of the metalloproteinase-2 (MMP-2) gene was analyzed using PCR as shown. Equal loading of RNA was demonstrated using amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control. (e) The experiment described in a was repeated with 10⁷ splenocytes from mice treated with PBS, IL-2, PC61, or PC61/IL-2 added to wells containing explanted B16 tumors along with VSV at a density of 4 × 10⁸ pfu/well. One set of tumors (n = 2) was not treated with splenocytes; instead they were incubated with VSV (4 × 10⁸ pfu/well) along with recombinant MMP-2 (12.5 ng/ml). After a further 48 hours, the tumors were recovered from the wells and dissociated, and virus titers were determined from freeze–thaw lysates (shown as pfu/mg tumor; two/group).

Figure 6. Systemic oncolytic virotherapy in combination with regulatory T cells depletion–enhanced interleukin-2 (IL-2) therapy

(a) C57Bl/6 mice (six to seven per group) were seeded subcutaneously with B16 tumors. After 9 days, the mice received an intraperitoneal (IP) injection of PC61 or control immunoglobulin G (IgG). After a further 24 hours, the mice were injected IP with phosphate-buffered saline (PBS) or with recombinant human IL-2 (75,000 U/injection three times a day for 3 days). On the fourth day, a single further injection of IL-2 was given. Two hours after this last injection of IL-2/PBS, the mice received the first of two intravenous (IV) injections of 10⁸ plaque forming units (pfu) of VSV-GFP, followed 24 hours later by the second IV virus injection. (b) The survival of the mice treated as shown is plotted against the days elapsed after tumor seeding. (c) The lungs taken from the mice that had received the treatments described in a or b were examined 7 days after the final injection. Hematoxylin and eosin–stained sections, examined by two independent pathologists, showed no abnormalities in any group. Representative sections of lungs from mice treated with PBS or PC61/IL-2 are shown. (d) The experiment described in b was repeated in two groups of mice, both of which received PC61/IL-2 and VSV. One group also received an injection of the natural killer (NK) cell-depleting asialo GM-1 antibody 24 hours before treatment with PC61 (NK depleted). The survival of the mice treated as shown is plotted against the days elapsed after tumor seeding. GFP, green fluorescent protein; VSV, vesicular stomatitis virus.