IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.     

IL-17E, a proinflammatory cytokine, has antitumor efficacy against several tumor types in vivo

Interleukin-17E (IL-17E) belongs to a novel family of cytokines that possess significant homology to IL-17. IL-17E has potent inflammatory effects in vitro and in vivo. Overexpression of IL-17E in mice results in a T helper-2 (Th2)-type immune response, which includes the expansion of eosinophils through the production of IL-5, and elevated gene expression of IL-4 and IL-13 in multiple tissues. In this study, we show that IL-17E has antitumor activity in vivo, a previously unrecognized function of IL-17E. Antitumor efficacy of IL-17E was examined in a variety of human tumor xenograft models, including melanoma, breast, lung, colon, and pancreatic cancers. Injection of recombinant IL-17E every other day resulted in significant antitumor activity in these tumor models. In addition, the combination of IL-17E with chemotherapy or immunotherapy agents showed an enhanced antitumor efficacy in human tumor xenograft models in mice as compared to either agent alone. Antitumor activity was demonstrated using different routes of administration, including intraperitoneal, intravenous, and subcutaneous injection. Anticancer activity was shown for both mouse and human forms of IL-17E, which have a high degree of sequence identity. Tumor-bearing mice treated with IL-17E showed a significant increase in serum levels of IL-5 and increased numbers of eosinophils in peripheral blood compared to the control group. Spleens isolated from IL-17E-treated mice showed a significant increase in eosinophils that correlated with antitumor activity of IL-17E in a dose–response manner. Finally, we demonstrate that B cells are necessary for IL-17E-mediated antitumor activity and that IL-17E was found to activate signaling pathways in B cells in vitro. Taken together, these data demonstrate that IL-17E has antitumor activity in vivo, and support further investigation of the potential clinical use of IL-17E as an anticancer agent.     

Synergistic engagement of an ineffective endogenous anti-tumor immune response and induction of IFN-gamma and Fas-ligand-dependent tumor eradication by combined administration of IL-18 and IL-2

IFN-gamma is a critical component of the endogenous and many cytokine-induced antitumor immune responses. In this study we have shown that the combination of IL-18 and IL-2 (IL-18/IL-2) synergistically enhances IFN-gamma production both in vitro and in vivo, and synergizes in vivo to induce complete durable regression of well-established 3LL tumors in >80% of treated mice. We have observed a nascent, but ineffective, host immune response against 3LL that depends on endogenous IFN-gamma and IL-12 production and the Fas/Fas ligand (Fas-L) pathway. The combined administration of IL-18/IL-2 engages this endogenous response to induce tumor regression via a mechanism that is independent of NK and NKT cells or IL-12, but is critically dependent on CD8(+) T cells, IFN-gamma, and the Fas/Fas-L pathway. These studies demonstrate the importance of IFN-gamma as well as the Fas/Fas-L pathway in both endogenous and cytokine-driven antitumor immune responses engaged by IL-18/IL-2 and provide preclinical impetus for clinical investigation of this potent anti-tumor combination.     

IL-12 and IL-10 expression synergize to induce the immune-mediated eradication of established colon and mammary tumors and lung metastasis

Preclinical studies demonstrated that certain cytokines are potentially useful for the induction of antitumor immune responses. However, their administration in clinical settings was only marginally useful and evoked serious toxicity. In this study, we demonstrate that the combination of autologous inactivated tumor cells expressing IL-12 and IL-10 induced tumor remission in 50-70% of mice harboring large established colon or mammary tumors and spontaneous lung metastases, with the consequent establishment of an antitumor immune memory. Mice treatment with tumor cells expressing IL-12 was only marginally effective, while expression of IL-10 was not effective at all. Administration of the combined immunotherapy stimulated the recruitment of a strong inflammatory infiltrate that correlated with local, increased expression levels of the chemokines MIP-2, MCP-1, IFN-gamma-inducible protein-10, and TCA-3 and the overexpression of IFN-gamma, but not IL-4. The combined immunotherapy was also therapeutically effective on established lung metastases from both colon and mammary tumors. The antitumor effect of the combined immunotherapy was mainly dependent on CD8+ cells although CD4+ T cells also played a role. The production of IFN-gamma and IL-4 by spleen cells and the development of tumor-specific IgG1 and IgG2a Abs indicate that each cytokine stimulated its own Th pathway and that both arms were actively engaged in the antitumor effect. This study provides the first evidence of a synergistic antitumor effect of IL-12 and IL-10 suggesting that a Th1 and a Th2 cytokine can be effectively combined as a novel rational approach for cancer immunotherapy.     

Subtherapeutic doses of interleukin-15 augment the antitumor effect of interleukin-12 in a B16F10 melanoma model in mice.

Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that exhibits antitumor activity in many experimental tumor models. In the present study, we investigated the ability of IL-15, a cytokine sharing many functions of IL-2, to modulate antitumor effectiveness of IL-12 against B16F10 melanoma in mice. In a model of locally growing tumor, intratumoral (i.t.) administration of IL-12, in three cycles of five consecutive daily injections (0.1 mug) followed by 2 days of rest, led to considerable delay of tumor development but no curative response was achieved. When combined with IL-12, subtherapeutic doses of IL-15 (0.4 mug) pontentiated the antitumor effects of IL-12 and induced complete tumor regressions in 50% of mice. Similar results were obtained in a model in which tumor-bearing mice were intravenously co-injected with melanoma cells to induce metastases. Combined administration of IL-12 and IL-15 yielded greater antitumor activity than injections of either cytokine alone and resulted in prolonged survival of mice bearing locally growing tumor and metastases. Studies of immunological parameters in mice treated with both IL-12 and IL-15 have shown enhanced NK activity (against YAC-1 cells) in the spleen and stimulation of both NK activity and specific anti-B16F10 cytotoxic effector cells in tumor-draining lymph nodes (LN). The strong antitumor effect of the IL-12 + IL-15 combination correlated with a high serum level of IFN-gamma in the treated mice. Moreover, increased expression of IL-15Ralpha was demonstrated in LN lymphocytes isolated from mice injected with IL-12. This result together with findings of other authors showing enhanced expression of IL-12 receptor by IL-15 [1] suggests that the augmentation of the antitumor effect during the course of IL-12/IL-15-based therapy could result from reciprocal upregulation of receptors by both cytokines and synergistic effects on IFN-gamma induction.     

Virulizin® induces production of IL-17E to enhance antitumor activity by recruitment of eosinophils into tumors

Virulizin has demonstrated strong antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. Our previous studies have demonstrated that macrophages, NK cells, and cytokines are important in the antitumor mechanism of Virulizin. Virulizin treatment of tumor bearing mice results in the expansion as well as increased activity of monocytes/macrophages and production of cytokines IL-12 and TNFalpha and activation of NK cells. In this study we show that the inflammatory cytokine IL-17E (IL-25) is induced by Virulizin treatment and is part of its antitumor mechanism. IL-17E is a proinflammatory cytokine, which induces a T(H)2 type immune response, associated with eosinophil expansion and infiltration into mucosal tissues. IL-17E was increased in sera of Virulizin-treated mice bearing human melanoma xenografts, compared to saline-treated controls, as shown by 2D gel electrophoresis and ELISA. Treatment of splenocytes in vitro with Virulizin resulted in increased IL-17E mRNA expression, which peaked between 24 and 32 h post-stimulation. Both in vitro and in vivo experiments demonstrated that B cells produced IL-17E in response to Virulizin treatment. Furthermore, Virulizin treatment in vivo resulted in increased blood eosinophilia and eosinophil infiltration into tumors. Finally, injection of recombinant IL-17E showed antitumor activity towards xenografted tumors, which correlated with increased eosinophilia in blood and tumors. Taken together, these results support another antitumor mechanism mediated by Virulizin, through induction of IL-17E by B cells, leading to recruitment of eosinophils into tumors, which may function in parallel with macrophages and NK cells in mediating tumor destruction.     

NHS-IL12 and bintrafusp alfa combination therapy enhances antitumor activity in preclinical cancer models

Combinatorial immunotherapy approaches are emerging as viable cancer therapeutic strategies for improving patient responses and outcomes. This study investigated whether two such immunotherapies, with complementary mechanisms of action, could enhance antitumor activity in murine tumor models. The immunocytokine NHS-IL12, and surrogate NHS-muIL12, are designed to deliver IL-12 and muIL-12, respectively, to the tumor microenvironment (TME) to activate NK cells and CD8⁺ T cells and increase their cytotoxic functions. Bintrafusp alfa (BA) is a bifunctional fusion protein composed of the extracellular domains of the TGF-β receptor II to function as a TGF-β “trap” fused to a human IgG1 antibody blocking PD-L1. With this dual-targeting strategy, BA enhances efficacy over that of monotherapies in preclinical studies. In this study, NHS-muIL12 and BA combination therapy enhanced antitumor activity, prolonged survival, and induced tumor-specific antitumor immunity. This combination therapy increased tumor-specific CD8⁺ T cells and induced immune profiles, consistent with the activation of both adaptive and innate immune systems. In addition, BA reduced lung metastasis in the 4T1 model. Collectively, these findings could support clinical trials designed to investigate NHS-IL12 and BA combination therapy for patients with advanced solid tumors     

IL-28 elicits antitumor responses against murine fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-gamma production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-gamma and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-gamma production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-gamma. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.     

Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing.

Bispecific Abs (bsAb) are promising immunological tools for the elimination of tumor cells in minimal residual disease situations. In principle, they target an Ag on tumor cells and recruit one class of effector cell. Because immune reactions in vivo are more complex and are mediated by different classes of effector cell, we argue that conventional bsAb might not yield optimal immune responses at the tumor site. We therefore constructed a bsAb that combines the two potent effector subclasses mouse IgG2a and rat IgG2b. This bispecific molecule not only recruits T cells via its one binding arm, but simultaneously activates FcgammaR+ accessory cells via its Fc region. We demonstrate here that the activation of both T lymphocytes and accessory cells leads to production of immunomodulating cytokines like IL-1beta, IL-2, IL-6, IL-12, and DC-CK1. Thus this new class of bsAb elicits excellent antitumor activity in vitro even without the addition of exogenous IL-2, and therefore represents a totally self-supporting system.     

The role of IL-21 in hematological malignancies

IL-21, the newest member of the common γ-chain family of cytokines, has pleiotropic biological effects through regulating a variety of immune cells. Recently, the role of IL-21 in the treatment of cancers has been widely investigated. Conducted phase I trials in metastatic malignant melanoma and renal cell carcinoma have shown that rIL-21 has a favorable antitumor activity. Expression of IL-21 and IL-21R has also been found in many types of hematological malignancies, such as chronic lymphocytic leukemia (CLL), multiple myeloma (MM) and lymphoma. Through binding with IL-21R, IL-21 induces activation of different JAK/STAT signal transduction pathways and regulates proliferation or apoptosis of tumor cells. In this review, we will discuss the expression of IL-21/IL-21R and its effect in different types of hematological malignancies.     

Reovirus activates human dendritic cells to promote innate antitumor immunity

Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-alpha, TNF-alpha, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-kappaB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-gamma production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-gamma) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus's direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.     

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.     

Pre-existing tumor-sensitized T cells are essential for eradication of established tumors by IL-12 and cyclophosphamide plus IL-12.

The antitumor immune response activated by IL-12, especially by a combination of cyclophosphamide and IL-12 (Cy+IL-12), is clinically significant in certain experimental tumor models, in that a number of well-established (10-20 mm in diameter) s.c. tumors are completely eradicated. Furthermore, Cy+IL-12 treatment is also able to eradicate well-established grossly detectable experimental lung metastases and advanced ascites tumors. Despite the dramatic antitumor effects seen in some tumor models, Cy+IL-12 fails to induce regression of other established tumors. Characterization of tumor immunogenicity shows that all tumors responding to IL-12 and Cy+IL-12 treatments are immunogenic tumors, in that an antitumor immune response is detectable in tumor-bearing hosts upon tumor establishment. In contrast, none of the nonimmunogenic tumor responds to IL-12 and Cy+IL-12 treatments. Analysis of cellular requirements for successful tumor rejection through an adoptive cell transfer approach reveals that the presence of tumor-sensitized, but not naive, T cells is essential for tumor rejection by IL-12 and Cy+IL-12. Transfer of these tumor-sensitized T cells must be conducted before, but not after, IL-12 treatment in order for tumor rejection to occur. The requirement of sensitized T cells is also tumor specific. In mice bearing immunogenic tumors, the presence of pre-existing tumor-sensitized T cells is demonstrated by adoptive cell transfer experiments using purified spleen T cells from these mice. Results from our study show that Cy+IL-12-based immunotherapy of cancer may be highly effective and that pre-existing tumor-sensitized T cells are essential for the success of the therapy.     

IL-2 complex recovers steroid-induced inhibition in immunochemotherapy for head and neck cancer

Background: A combination therapy with immune checkpoint inhibitors (ICIs) and platinum-based chemotherapy has become the first-line treatment for recurrent or metastatic head and neck squamous carcinoma (HNSCC). Although steroids are often used as anti-emetic medications during chemotherapy, their adverse effects on immune-combined chemotherapy are unclear in HNSCC.Methods: The effects of dexamethasone on tumor growth and immune cell population were evaluated in a mouse HNSCC model treated with PD-1 blockade combined with cisplatin. The effect of various doses of dexamethasone on cell proliferation, survival, surface markers, IFN-γ production, and antitumor effects in antigen-specific T cells was examined in vitro. The recovery of T cell dysfunction by IL-2 was assessed in vitro and in vivo.Results: In a mouse HNSCC model, dexamethasone showed limited antitumor effects on immunochemotherapy. Dexamethasone decreased the number of T cells and inhibited T cell differentiation into effector and central memory T cells. In the in vitro assessment, dexamethasone induced cell death, limited proliferation, and reduced the reactivity against HNSCC cell lines of antigen-specific T cells in a dose-dependent manner. The expression of inhibitory receptors on T cells was not affected by steroids. This inhibition was recovered by IL-2 and IL-2/anti-IL-2 complexes (IL-2 Cx) in vitro and in vivo, respectively.Conclusion: Our preclinical data indicate that dexamethasone diminishes the antitumor effects of immunochemotherapy in patients with HNSCC. IL-2 Cx recovered the inhibition of antitumor immunity by steroids and might be a potent immune adjuvant for patients who require steroids during PD-1 blockade and chemotherapy.     

Microenvironment-derived IL-1 and IL-17 interact in the control of lung metastasis

Inflammatory cytokines modulate immune responses in the tumor microenvironment during progression/metastasis. In this study, we have assessed the role of IL-1 and IL-17 in the control of antitumor immunity versus progression in a model of experimental lung metastasis, using 3LL and B16 epithelial tumor cells. The absence of IL-1 signaling or its excess in the lung microenvironment (in IL-1β and IL-1R antagonist knockout [KO] mice, respectively) resulted in a poor prognosis and reduced T cell activity, compared with WT mice. In IL-1β KO mice, enhanced T regulatory cell development/function, due to a favorable in situ cytokine network and impairment in APC maturation, resulted in suppressed antitumor immunity, whereas in IL-1R antagonist KO mice, enhanced accumulation and activity of myeloid-derived suppressor cells were found. Reduced tumor progression along with improved T cell function was found in IL-17 KO mice, compared with WT mice. In the microenvironment of lung tumors, IL-1 induces IL-17 through recruitment of γ/δ T cells and their activation for IL-17 production, with no involvement of Th17 cells. These interactions were specific to the microenvironment of lung tumors, as in intrafootpad tumors in IL-1/IL-17 KO mice, different patterns of invasiveness were observed and no IL-17 could be locally detected. The results highlight the critical and unique role of IL-1, and cytokines induced by it such as IL-17, in determining the balance between inflammation and antitumor immunity in specific tumor microenvironments. Also, we suggest that intervention in IL-1/IL-17 production could be therapeutically used to tilt this balance toward enhanced antitumor immunity.     

Antitumor and antimetastatic activity of IL-23

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 +/- 333 mm(3), then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8(+) T cells, but not CD4(+) T cells or NK cells, were crucial for the antitumor activity of IL-23.     

Interleukin 15 as a promising candidate for tumor immunotherapy

Interleukin 15 participates in the development of important immune antitumor mechanisms. It activates CD8(+) T cells, natural killer (NK) cells, NK T cells, and can promote the formation of antitumor antibodies. IL-15 can also protect T effector cells from the action of T regulatory cells and reverse tolerance to tumor-associated antigens. In pre-clinical studies IL-15 has been found to demonstrate potentiated antitumor effects following pre-association with IL-15Rα, or when used in combination with chemotherapy, adoptive therapy, monoclonal antibodies, and tumor vaccines. Although a clinical trial based on application of IL-15 in tumor patients has already begun, it is important to be aware of its potential side effects, including induction of autoimmunity and promotion of proliferation, survival, and dissemination of some tumor cells.     

Immunomodulatory effects of inactivated parapoxvirus ovis (ORF virus) on human peripheral immune cells: induction of cytokine secretion in monocytes and Th1-like cells

Inactivated parapoxvirus ovis (Orf virus; PPVO) recently displayed strong immunostimulating and modulating capacities in several animal models for acute and chronic virus infections through the induction of gamma interferon (IFN-gamma) as a key mediator of antiviral activity. The data presented in this work demonstrate that inactivated PPVO has strong effects on cytokine secretion by human immune cells, including the upregulation of inflammatory and Th1-related cytokines (IFN-gamma, tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6], IL-8, IL-12, and IL-18) as well as anti-inflammatory and Th2-related cytokines (IL-4, IL-10, and IL-1 receptor antagonist [IL-1ra]). Studies on the mechanism of action revealed virus particles to be the effective components of the preparation. The virus particles activate monocytes or other antigen-presenting cells (APC), e.g., plasmacytoid dendritic cells, through signaling over CD14 and a Toll-like receptor and the intracellular presence of certain PPVO-specific components. The activation of monocytes or APC is followed by the release of early proinflammatory cytokines (TNF-alpha, IL-6, and IL-8) as well as the Th1-related cytokines IL-12 and IL-18. Both IL-18 and IL-12 are involved in PPVO-mediated IFN-gamma release by T cells and/or NK cells. The proinflammatory response is accompanied by the induction of anti-inflammatory and Th2-related cytokines (IL-4, IL-10, and IL-1ra), which exert a limiting efffect on the inflammatory response induced by PPVO. We conclude that the induction of a natural immune response with physiologically significant amounts of different cytokines and with antiviral potential might provide advantages over existing antiviral immunotherapies.