Immune-mediated antitumor activity of reovirus is required for therapy and is independent of direct viral oncolysis and replication

Abstract

Purpose: Reovirus is a naturally occurring oncolytic virus in clinical trials. Although tumor infection by reovirus can generate adaptive antitumor immunity, its therapeutic importance versus direct viral oncolysis is undefined. This study addresses the requirement for viral oncolysis and replication, and the relative importance of antitumor immunity and direct oncolysis in therapy.

Experimental design: Nonantigen specific T cells loaded with reovirus were delivered i.v. to C57BL/6 and severe combined immunodeficient mice bearing lymph node and splenic metastases from the murine melanoma, B16ova, with assessment of viral replication, metastatic clearance by tumor colony outgrowth, and immune priming. Human cytotoxic lymphocyte priming assays were done with dendritic cells loaded with Mel888 cells before the addition of reovirus.

Results: B16ova was resistant to direct oncolysis in vitro, and failed to support reovirus replication in vitro or in vivo. Nevertheless, reovirus purged lymph node and splenic metastases in C57BL/6 mice and generated antitumor immunity. In contrast, reovirus failed to reduce tumor burden in severe combined immunodeficient mice bearing either B16ova or reovirus-sensitive B16tk metastases. In the human system, reovirus acted solely as an adjuvant when added to dendritic cells already loaded with Mel888, supporting priming of specific antitumor cytotoxic lymphocyte, in the absence of significant direct tumor oncolysis; UV-treated nonreplicating reovirus was similarly immunogenic.

Conclusion: The immune response is critical in mediating the efficacy of reovirus, and does not depend upon direct viral oncolysis or replication. The findings are of direct relevance to fulfilling the potential of this novel anticancer agent.

Fig. 1

In vitro B16ova tumor cells are resistant to reovirus oncolysis and replication. A, JAM-1 expression was assessed by flow cytometry. Gray, isotype control; black line, JAM-1. B, B16ova and B16tk cells were treated with serial dilutions of reovirus stock, and cell survival was determined at indicated time points by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data representative of three experiments. C, viral progeny following infection at 1 pfu per cell was determined after three cycles of freeze thaw lysis by plaque assay. B16ova cells were seeded in duplicate wells at 5 × 10⁵ and allowed to adhere overnight. D, lymphocytes from C57BL/6 mice were incubated with reovirus at different viral titer s at 4°C for 4 h, and seeded at a 1:1 ratio into B16 or B16ova cultures. Tumor cell death was assessed at 72 h after harvesting, by propidium iodide staining after gating out CD3+ lymphocytes. Data are mean values of duplicate wells ± SE, and representative of two experiments.

Fig. 2

Reovirus loaded T cells purge B16ova LN metastases in vivo and generate antitumor immunity, in the absence of reoviral replication. A, C57BL/6 mice were seeded (three per group) at 5 × 10⁵ B16ova or B16tk cells s.c. At 10 d, 2 × 10⁶ T cells loaded with 0.1 pfu reovirus were adaptively transferred. Tumor-draining lymph nodes were harvested at 2 and 4 d thereafter, and viral titer determined by plaque assay. Each titer represents an individual animal. B and C, C57BL/6 mice were seeded (n = 3 per group) with 5 × 10⁵ B16ova cells s.c. Ten days later, mice were treated with iv PBS, 2 × 10⁶ T cells, and 2 × 10⁶ T cells loaded with reovirus 0.1 pfu per cell (T-reo). 10 d posttherapy, tumor-draining LN and spleens were harvested, disaggregated, and 10⁶ cells seeded in six-well plates in G418-containing media to select for B16ova cells. B, after 7 d, the number of G418-resistant colonies were photographed. C, splenocytes recovered at day 10 after treatment were pulsed with tumor cell lysates or peptides as shown, in triplicates of 7.5 × 10⁵ cells. Forty-eight hours later, supernatants were assayed by ELISA for IFN-γ. Columns, means of triplicates; bars, SE.

Fig. 3

Reovirus fails to purge B16ova and B16tk metastases in SCID mice. SCID mice were seeded (three per group) with 5 × 10⁵ B16ova or B16tk cells s.c. Ten days later, mice were treated with 2 × 10⁶ T cells or 2 × 10⁶ T cells preincubated with reovirus 0.1 pfu per cell. Tumor-draining LN and spleen were harvested at 10 d posttreatment. Tumor burden was assessed by colony outgrowth in G418-containing media for B16ova (A), and puromycin-containing media for B16tk (B). Colonies were counted at 7 d. Columns, mean; bars, SE.

Fig. 4

Direct reovirus-induced oncolysis is not required to prime antitumor immunity in a human in vitro system. A, PKH-67–labeled Mel888 cells were seeded and allowed to adhere. Immature DC were added to the Mel888 monolayer after 48 h at a 1:1 ratio overnight, before gentle aspiration and pelleting of supernatant. DC were labeled with anti–CD11c-PE, and flow cytometry was done to determine uptake of Mel888-derived material, and enumerate the number of intact Mel888 cells. % shown represent the % of DC double-labeling for PKH-67. Representative of two experiments. B and C, Mel888 cells were seeded and cultured with DC as in A. Reovirus was added to aspirated Mel888-preloaded DC at 1 pfu/DC (Mel888-adj.reo). DC were cocultured with autologous PBMC (1:10-30 ratio), restimulated 1 wk later. CTL activity was assayed at 14 d following culture with Mel888 or irrelevant SKOV-3 cell targets by a ⁵¹Chromium cytotoxicity assay (B), and the CD107 degranulation assay (C). Points, means of triplicate wells; bars, SE (B). % shown in C are of CD8+ T cells. Results representative of four independent experiments. D, using HLA-A2+ve donors, cross-priming of MART-1reactive CD8+ T cells was assessed by pentamer analysis. % are of CD8+ T cells. Results representative of three independent experiments.

Fig. 5

Adjuvant replication-incompetent UV-treated reovirus also primes antitumor immunity. A, reovirus was UV irradiated in 100 μL volumes in 96-well plates, and titer assessed by an L929 plaque assay. Data are representative of two experiments. B, C, and D, reovirus treated with 480 mJ UV was added to Mel888-preloaded DC (Mel888-adj.UVreo) and assessed in priming assays done as in Fig. 4. Antitumor activity assessed by ⁵¹Chromium assay (B) and intracellular IFN-γ production (C) following coculture with Mel888 or irrelevant SKOV-3 cell targets. Points, means of triplicate wells; bars, SE. % shown in C are of CD8+ T cells. D, levels of IL-4, IL-10, and IFN-γ were determined by ELISA after 2 wk of cultures. Columns, means of triplicate wells; bars, SE. Results representative of three independent experiments.