Role of the Innate Immune Response and Tumor Immunity Associated with Simian Virus 40 Large Tumor Antigen

We examined properties of the innate immune response against the tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag) following experimental pulmonary metastasis in naive mice. Approximately 14 days after mKSA tumor cell challenge, expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and RANTES was upregulated in splenocytes harvested from mice, as assessed by flow cytometry and antibody array assays. This response was hypothesized to activate and induce tumor-directed NK cell lysis since IL-2-stimulated NK cells mediated tumor cell destruction in vitro. The necessary function of NK cells was further validated in vivo through selected antibody depletion of NK cells, which resulted in an overwhelming lung tumor burden relative to that in animals receiving a control rabbit IgG depletion regimen. Interestingly, mice achieved increased protection from experimental pulmonary metastasis when NK cells were further activated indirectly through in vivo administration of poly(I:C), a Toll-like receptor 3 (TLR3) agonist. In a separate study, mice receiving treatments of poly(I:C) and recombinant SV40 Tag protein immunization mounted effective tumor immunity in an established experimental pulmonary metastasis setting. Initiating broad-based immunity with poly(I:C) was observed to induce a Th1 bias in the SV40 Tag antibody response that led to successful antitumor responses not observed in animals treated only with poly(I:C) or SV40 Tag. These data have direct implications for immunotherapeutic strategies incorporating methods to elicit inflammatory reactions, particularly NK cell-driven lysis, against malignant cell types that express a tumor-specific antigen such as SV40 Tag.Considerable interest has been directed toward the role innate immunity plays in reducing malignant growth and progression. Although the innate system by broad definition is not endowed with the antigen specificity and memory recall of adaptive immunity, natural killer (NK) cells are an innate effector population that shares most properties with the adaptive arm of the immune system, excluding receptor rearrangement (28). Interestingly, NK cells can be employed to directly target and destroy malignant cell types through diverse pathways that include tumor major histocompatibility complex class I (MHC-I) loss and upregulation of stress-inducible protein ligands for the NK cell activating receptor NKG2D (24, 29). Much effort is under way in human clinical trials to manipulate NK cell properties for directed therapies against cancer (13, 29).One strategy in eliciting innate immunity in general involves activating the Toll-like receptor (TLR) family, which are preferentially expressed by innate effectors such as NK cells, macrophages, and dendritic cells (DCs) (26). TLR ligands include a variety of pathogen-associated molecular patterns with differing downstream responses based on the cell type involved and specific TLR activated. In TLR-expressing cells, signal transduction pathways follow a MyD88-independent course to produce type I interferons (IFNs) (e.g., TLR3) or a MyD88-dependent pathway that results in the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6 and expression of costimulatory molecules such as CD40, CD80, and CD86 (e.g., TLR4 and TLR9) (2, 12, 23, 26). In the case of TLR3, activation by poly(I:C) causes DCs and additional accessory cells to secrete type I interferons and IL-12, activating NK cells and prompting NK cell secretion of IFN-γ among other effects (14, 20). Ultimately, modulation of TLR activation results in the generation of a range of cytokines that promote inflammation, Th1 bias, and NK cell-directed killing that can be utilized in a beneficial manner for tumor treatment strategies.TLR agonist incorporation alongside vaccine strategies has resulted in promising results in mouse models of cancer (12). Indeed, the TLR7 agonist imiquimod is an effective FDA-approved topical compound used to treat superficial basal-cell carcinoma and external genital warts (9). However, to our knowledge, modulating TLR activity while also incorporating recombinant simian virus 40 (SV40) large tumor antigen (Tag) protein immunizations in a therapeutic tumor setting has not been previously reported. SV40 Tag is a clinically relevant tumor-specific antigen that has been shown to be expressed by a number of human malignancies, including malignant pleural mesothelioma (MPM), and represents a potential target for immunotherapeutic strategies.Our laboratory has previously defined a unique role for antibody-dependent cell-mediated cytotoxicity (ADCC) reactions—specific against SV40 Tag—promoting cytotoxic T-lymphocyte (CTL) activity in response to neoantigens through cross-presentation of tumor cell debris in a model of experimental pulmonary metastasis (16, 17). In this report, we analyze the role of innate immunity in mediating tumor cell lysis during the early course of tumorigenesis in the absence of vaccination. Overall, we find that activated NK cells are necessary effector cells in achieving antitumor reactions and providing partial tumor immunity during the onset of tumorigenesis and that these functioning NK cells are likely activated in vivo due to inflammation as a result of tumor growth and progression. The burden of tumor challenge could be further reduced in naive animals with the indirect activation of NK cells using poly(I:C) as a TLR3 agonist prior to and during malignant dissemination. Interestingly, in an established pulmonary tumor setting, therapeutic treatment of mice with poly(I:C) and recombinant SV40 Tag resulted in enhanced protection that was not observed using poly(I:C) or SV40 Tag alone. One effect of instituting poly(I:C) treatment alongside SV40 Tag immunizations was a Th1 skewing of the SV40 Tag IgG antibody response that correlated with therapeutic tumor protection.Our results have direct implications for the prevention and treatment of malignancies, such as MPM, that express the SV40 Tag oncoprotein. Combining specific aspects of innate and adaptive immunity by targeting both NK cells and humoral activity against SV40 Tag, respectively, represents a novel and clinically significant immunotherapeutic strategy for potential use in patients.