Tumor Infection by Oncolytic Reovirus Primes Adaptive Antitumor Immunity

Abstract

Purpose: Early clinical trials are under way exploring the direct oncolytic potential of reovirus.This study addresses whether tumor infection by reovirus is also able to generate bystander, adaptive antitumor immunity. Experimental Design: Reovirus was delivered intravenously to C57BL/6 mice bearing lymph node metastases from the murine melanoma, B16-tk, with assessment of nodal metastatic clearance, priming of antitumor immunity against the tumor-associated antigen tyrosinase-related protein-2, and cytokine responses. In an in vitro human system, the effect of reovirus infection on the ability of Mel888 melanoma cells to activate and load dendritic cells for cytotoxic lymphocyte (CTL) priming was investigated. Results: In the murine model, a single intravenous dose of reovirus reduced metastatic lymph node burden and induced antitumor immunity (splenocyte response to tyrosinase-related protein-2 and interleukin-12 production in disaggregated lymph nodes). In vitro human assays revealed that uninfected Mel888 cells failed to induce dendritic cell maturation or support priming of an anti-Mel888 CTL response. In contrast, reovirus-infected Mel888 cells (reo-Mel) matured dendritic cells in a reovirus dose-dependent manner. When cultured with autologous peripheral blood lymphocytes, dendritic cells loaded with reo-Mel induced lymphocyte expansion, IFN-g production, specific anti-Mel888 cell cytotoxicity, and cross-primed CD8 + Tcells specific against the human tumor-associated antigen MART-1. Conclusion: Reovirus infection of tumor cells reduces metastatic disease burden and primes antitumor immunity. Future clinical trials should be designed to explore both direct cytotoxic and immunotherapeutic effects of reovirus.

Reovirus Virotherapy Overrides Tumor Antigen Presentation Evasion and Promotes Protective Antitumor Immunity

“…Native B16 melanoma (B16) and Lewis lung carcinoma (LLC) were purchased from the American Type Culture Collection, whereas ovalbumin (ova)-expressing B16 (B16-ova; ref. Antibodies and reagents used in this study were purchased from different manufacturers as follows: eBioscience: Alexa 488-anti-CD11c, PE-anti-CD86, APCanti-CD40, APC-anti-CD80, PE-anti-mouse MHC class I molecule Kb bound to the peptide SIINFEKL (25-D1.16), unconjugated anti-mouse CD16/32, and unconjugated anti-mouse CD49d; Invitrogen: PE-anti-CD3, Alexa 488-IFN-γ, APC-anti-CD107a, unconjugated anti-mouse CD28, and 5-(and-6)-carboxyfluorescein diacetate (CFSE); BioLegend: PerCp-anti-MHC class II (H-2A/E) and PerCp-anti-CD90.2; BD Biosciences: PerCp-anti-CD8; GenScript: SIINFEKL (ova [257][258][259][260][261][262][263][264] ) and KAVYNFATM (LCMV gp [33][34][35][36][37][38][39][40][41] ) peptides.…”

“…B3Z is a genetically engineered T-cell hybridoma that bears ova-specific TCR and expresses β-galactosidase T-cell proliferation and IFN-γ/CD107a assay Lymphocytes were labeled with 1 μmol/L CFSE (32) and then stimulated with either SIINFEKL, tumor lysate (35), and control LCMV gp [33][34][35][36][37][38][39][40][41] peptide (5 μg/mL)-or UV reovirus [1 multiplicity of infection (MOI)]-pulsed BMDCs or concanavalin A (5 μg/mL), or left unstimulated. After 5 days of incubation, cells were harvested, stained with PE-anti-CD3 antibodies and analyzed.…”

“…Reovirus-initiated cytokine response in vitro has previously been reported (38,39); however, the comprehensive profile of such a response in different immune cell subsets or in vivo is still obscure. Using Antibody-based Quantitative (and Qualitative) Cytokine Array, we evaluated the expression of 60 cytokines following reovirus exposure of BMDCs, lymphocytes, and tumor cells in vitro or immunocompetent and immunocompromised mice in vivo.…”

Antitumor efficacy of oncolytic herpes simplex virus adsorbed onto antigen-specific lymphocytes

“…23,42 Infecting tumors with viruses induces antitumor immunity through tyrosinase-related protein 2 and interleukin 12. 43 Moreover, indirect tumor cell killing is induced by the activation of cytokine production and host antitumor immunity. 23,42 Virusadherent lymphocytes become a stimulus for IFN secretion and lead to suppression of tumor growth.…”

“…43,44 It is thought that long-term antitumor immune memory is initiated by this response. 45 Therefore, although we administered HSV-adherent tumor antigen-specific lymphocytes only once, 40% of the treated mice were completely cured.…”