Tackling cancer cell dormancy: Insights from immune models, and transplantation.

Disseminated non-dividing (dormant) cancer cells as well as those in equilibrium with the immune response remain the major challenge for successful treatment of cancer. The equilibrium between disseminated dormant cancer cells and the immune system is reminiscent of states that can occur during infection or allogeneic tissue and cell transplantation. We discuss here the major competing models of how the immune system achieves a self nonself discrimination (pathogen/danger patterns, quorum, and coinhibition/tuning models), and suggest that taking advantage of a combination of the proposed mechanisms in each model may lead to increased efficacy in tackling cancer cell dormancy.

On antigen-specific signals, immune class regulation and energetics: Report III from the workshops on foundational concepts of immune regulation.

This is a report from a one-week workshop held in Athens, Greece in July of 2022. The workshop aimed to identify emerging concepts relevant to the fundamentals of immune regulation and areas for future research. Theories of immune regulation emphasize the role of T cell help or co-stimulation (signal 2). The workshop participants considered how new data on the characteristics of agonist antigens, the role of the antigen receptor signals (signal 1) in driving fate decisions, the effect of energetics on immunity and a better understanding of class-control in the immune response, may impact theories of immune regulation. These ideas were discussed in the context of tumour immunology, autoimmunity, pregnancy and transplantation. Here we present the discussions as a narrative of different viewpoints to allow the reader to join the conversation. These discussions highlight the evolving understanding of the nature of specific antigen recognition and how both antigen-specific and non-specific mechanisms impact immune responses.

The immune microenvironment in typical carcinoid lung tumour, a brief report of four cases.

Pulmonary typical carcinoid (TC) is a low-grade, rare lung cancer of neuroendocrine origin. Currently, there is very little information available about the immune cell composition in TC tumours. Here, we analysed by flow cytometry resected tumours from four never-smoker female patients with TC. Twelve distinct immune cell types were identified in TC tumours. The most abundant immune cells were CD8+ T cells, CD4+ T cells, B cells and macrophages, which represented 19.8%, 17.7%, 11.5% and 11% of all tumour-infiltrating CD45+ leucocytes, respectively. Natural killer (NK) cells (8.8%) and neutrophils (3.9%) were also common. Three types of dendritic cells (DCs) were identified (plasmacytoid DCs, CD1c  DCs, and CD141  DCs) which together constituted 1.4% of all immune cells in TC tumours. Small populations of basophils (1.2%), mast cells (0.8%) and eosinophils (0.6%) were also present. Notably, the percentage of leucocytes (of all living cells) was much lower in TC tumours compared to high-grade non-small cell lung cancer (NSCLC) tumours and also compared to non-cancerous lung tissue. We conclude that TC tumours are relatively non-inflammatory, although the immune landscape was found to be very complex.

Antibody combinations for optimized staining of macrophages in human lung tumours.

The analysis of tumour-associated macrophages (TAMs) has a high potential to predict cancer recurrence and response to immunotherapy. However, the heterogeneity of TAMs poses a challenge for quantitative and qualitative measurements. Here, we critically evaluated by immunohistochemistry and flow cytometry two commonly used pan-macrophage markers (CD14 and CD68) as well as some suggested markers for tumour-promoting M2 macrophages (CD163, CD204, CD206 and CD209) in human non-small cell lung cancer (NSCLC). Tumour, non-cancerous lung tissue and blood were investigated. For immunohistochemistry, CD68 was confirmed to be a useful pan-macrophage marker although careful selection of antibody was found to be critical. The widely used anti-CD68 antibody clone KP-1 stains both macrophages and neutrophils, which is problematic for TAM quantification because lung tumours contain many neutrophils. For TAM counting in tumour sections, we recommend combined labelling of CD68 with a cell membrane marker such as CD14, CD163 or CD206. In flow cytometry, the commonly used combination of CD14 and HLA-DR was found to not be optimal because some TAMs do not express CD14. Instead, combined staining of CD68 and HLA-DR is preferable to gate all TAMs. Concerning macrophage phenotypic markers, the scavenger receptor CD163 was found to be expressed by a substantial fraction (50%-86%) of TAMs with a large patient-to-patient variation. Approximately 50% of TAMs were positive for CD206. Surprisingly, there was no clear overlap between CD163 and CD206 positivity, and three distinct TAM sub-populations were identified in NSCLC tumours: CD163+ CD206+ , CD163+ CD206- and CD163- CD206- . This work should help develop macrophage-based prognostic tools for cancer.

Poly(I:C)-Encapsulating Nanoparticles Enhance Innate Immune Responses to the Tuberculosis Vaccine Bacille Calmette-Guérin (BCG) via Synergistic Activation of Innate Immune Receptors.

The attenuated live vaccine strain bacille Calmette-Guérin (BCG) is currently the only available vaccine against tuberculosis (TB), but is largely ineffective against adult pulmonary TB, the most common disease form. This is in part due to BCG's ability to interfere with the host innate immune response, a feature that might be targeted to enhance the potency of this vaccine. Here, we investigated the ability of chitosan-based nanoparticles (pIC-NPs) containing polyinosinic-polycytidylic acid (poly(I:C)), an inducer of innate immunity via Toll-like receptor 3 (TLR3), to enhance the immunogenicity of BCG in mouse bone marrow derived macrophages (BMDM) in vitro. Incorporation of poly(I:C) into NPs protected it against degradation by ribonucleases and increased its uptake by mouse BMDM. Whereas soluble poly(I:C) was ineffective, pIC-NPs strongly enhanced the proinflammatory immune response of BCG-infected macrophages in a synergistic fashion, as evident by increased production of cytokines and induction of nitric oxide synthesis. Using macrophages from mice deficient in key signaling molecules involved in the pathogen recognition response, we identified combined activation of MyD88- and TRIF-dependent TLR signaling pathways to be essential for the synergistic effect between BCG and NP. Moreover, synergy was strongly dependent on the order of the two stimuli, with TLR activation by BCG functioning as the priming event for the subsequent pIC-NP stimulus, which acted through an auto-/paracrine type I interferon (IFN) feedback loop. Our results provide a foundation for a promising new approach to enhance BCG-vaccine immunogenicity by costimulation with NPs. They also contribute to a molecular understanding of the observed synergistic interaction between the pIC-NPs and BCG vaccine.

High production of IL-12 by human dendritic cells stimulated with combinations of pattern-recognition receptor agonists.

The cytokine IL-12p70 is crucial for T helper 1 (Th1) polarization and the generation of type 1 immunity required to fight cancer and pathogens. Therefore, strategies to optimize the production of IL-12p70 by human dendritic cells (DCs) may significantly improve the efficacy of vaccines and immunotherapies. However, the rules governing the production of IL-12p70 remain obscure. Here, we stimulated pattern recognition receptors (PRRs) representing five families of PRRs, to evaluate their ability to elicit high production of IL-12p70 by monocyte-derived DCs. We used ten well-characterized agonists and stimulated DCs in vitro with either single agonists or 27 different combinations. We found that poly(I:C), which engages the RNA-sensing PRRs TLR3 and MDA5, and LPS which stimulates TLR4, were the only agonists that could elicit notable IL-12p70 production when used as single ligands. We identified six different combinations of PRR agonists, all containing either the TLR3/MDA5 agonist poly(I:C) or the TLR7/8 agonist R848, that could synergize to elicit high production of IL-12p70 by human DCs. Five of the six combinations also triggered high production of the antiviral and antitumor cytokine IFNß. Overall, the tested PRR ligands could be divided into three groups depending on whether they triggered production of both IL-12p70 and IFNß, only one of the two, or neither. Thus, combinations of PRR agonists were found to increase the production of IL-12p70 by human DCs in a synergistic manner, and we identified six PRR agonist combinations that may represent strong adjuvant candidates, in particular for therapeutic cancer vaccines.

Fungal polysaccharides from Inonotus obliquus are agonists for Toll-like receptors and induce macrophage anti-cancer activity.

Fungal polysaccharides can exert immunomodulating activity by triggering pattern recognition receptors (PRRs) on innate immune cells such as macrophages. Here, we evaluate six polysaccharides isolated from the medicinal fungus Inonotus obliquus for their ability to activate mouse and human macrophages. We identify two water-soluble polysaccharides, AcF1 and AcF3, being able to trigger several critical antitumor functions of macrophages. AcF1 and AcF3 activate macrophages to secrete nitric oxide and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Combined with interferon-γ, the fungal polysaccharides trigger high production of IL-12p70, a central cytokine for antitumor immunity, and induce macrophage-mediated inhibition of cancer cell growth in vitro and in vivo. AcF1 and AcF3 are strong agonists of the PRRs Toll-like receptor 2 (TLR2) and TLR4, and weak agonists of Dectin-1. In comparison, two prototypical particulate ß-glucans, one isolated from I. obliquus and one from Saccharomyces cerevisiae (zymosan), are agonists for Dectin-1 but not TLR2 or TLR4, and are unable to trigger anti-cancer functions of macrophages. We conclude that the water-soluble polysaccharides AcF1 and AcF3 from I. obliquus have a strong potential for cancer immunotherapy by triggering multiple PRRs and by inducing potent anti-cancer activity of macrophages.

Caspase activation counteracts interferon signaling after G2 checkpoint abrogation by ATR inhibition in irradiated human cancer cells.

Recent studies suggest that inhibition of the ATR kinase can potentiate radiation-induced antitumor immune responses, but the extent and mechanisms of such responses in human cancers remain scarcely understood. We aimed to assess whether the ATR inhibitors VE822 and AZD6738, by abrogating the G2 checkpoint, increase cGAS-mediated type I IFN response after irradiation in human lung cancer and osteosarcoma cell lines. Supporting that the checkpoint may prevent IFN induction, radiation-induced IFN signaling declined when the G2 checkpoint arrest was prolonged at high radiation doses. G2 checkpoint abrogation after co-treatment with radiation and ATR inhibitors was accompanied by increased radiation-induced IFN signaling in four out of five cell lines tested. Consistent with the hypothesis that the cytosolic DNA sensor cGAS may detect DNA from ruptured micronuclei after G2 checkpoint abrogation, cGAS co-localized with micronuclei, and depletion of cGAS or STING abolished the IFN responses. Contrastingly, one lung cancer cell line showed no increase in IFN signaling despite irradiation and G2 checkpoint abrogation. This cell line showed a higher level of the exonuclease TREX1 than the other cell lines, but TREX1 depletion did not enhance IFN signaling. Rather, addition of a pan-caspase inhibitor restored the IFN response in this cell line and also increased the responses in the other cell lines. These results show that treatment-induced caspase activation can suppress the IFN response after co-treatment with radiation and ATR inhibitors. Caspase activation thus warrants further consideration as a possible predictive marker for lack of IFN signaling.

Reactive Species from Two-Signal Activated Macrophages Interfere with Their Oxygen Consumption Measurements.

Metabolic modulation of macrophage activation has emerged as a promising strategy lately in immunotherapeutics. However, macrophages have a broad spectrum of functions and thus, understanding the exact metabolic changes that drive a particular immune response, is of major importance. In our previous work, we have reported a key role of nitric oxide (NO●) in two(2)-signal activated macrophages [M(2-signals)]. Further characterization using metabolic analysis in intact cells, showed that the basal and maximal respiration levels of M(2-signals) were comparable, with cells being unresponsive to the injections-inducd mitochondrial stress. Here, we show that excessive NO● secretion by the M(2-signals) macrophages, interferes with the oxygen (O2) consumption measurements on cells using the seahorse metabolic analyzer. This is attributed mainly to the consumption of ambient oxygen by NO● to form NO2- and/or NO3- but also to the reduction of O2 to superoxide anion (O2●-) by stray electrons from the electron transport chain, leading to the formation of peroxynitrite (ONOO-). We found that reactive species-donors in the absence of cells, produce comparable oxygen consumption rates (OCR) with M(2-signals) macrophages. Furthermore, inhibition of NO● production, partly recovered the respiration of activated macrophages, while external addition of NO● in non-activated macrophages downregulated their OCR levels. Our findings are crucial for the accurate metabolic characterization of cells, especially in cases where reactive nitrogen or oxygen species are produced in excess.

The Immune Landscape of Human Primary Lung Tumors Is Th2 Skewed.

Tumor-specific T helper (Th) cells have a central role in the immune response against cancer. However, there exist distinct Th cell subsets with very different and antagonizing properties. Some Th subsets such as Th1 protect against cancer, while others (Th2, T regulatory/Treg) are considered detrimental or of unknown significance (T follicular helper/Tfh, Th17). The Th composition of human solid tumors remains poorly characterized. Therefore, we established a four-color multiplex chromogenic immunohistochemical assay for detection of Th1, Th2, Th17, Tfh and Treg cells in human tumor sections. The method was used to analyze resected primary lung tumors from 11 patients with non-small cell lung cancer (NSCLC). Four microanatomical regions were investigated: tumor epithelium, tumor stroma, peritumoral tertiary lymphoid structures (TLS) and non-cancerous distal lung tissue. In tumor epithelium and stroma, most CD4+ T cells identified had either a Th2 (GATA-3+CD3+CD8-) or Treg (FOXP3+CD3+CD8-) phenotype, whereas only low numbers of Th1, Th17, and Tfh cells were observed. Similarly, Th2 was the most abundant Th subset in TLS, followed by Treg cells. In sharp contrast, Th1 was the most frequently detected Th subset in non-cancerous lung tissue from the same patients. A higher Th1:Th2 ratio in tumor stroma was found to be associated with increased numbers of intratumoral CD8+ T cells. The predominance of Th2 and Treg cells in both tumor stroma and tumor epithelium was consistent for all the 11 patients investigated. We conclude that human primary NSCLC tumors are Th2-skewed and contain numerous Treg cells. If human tumors are Th2-skewed, as our data in NSCLC suggest, reprogramming the type of immune response from a detrimental Th2 to a beneficial Th1 may be critical to increase the response rate of immunotherapy.

Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models.

The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/ß-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/ß-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/ß-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of ß-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFNγ- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome ß-catenin-mediated resistance to immune checkpoint blockade in melanoma.

Aiming for the Insulin-like Growth Factor-1 system in breast cancer therapeutics.

Despite the major discoveries occurred in oncology the recent years, breast malignancies remain one of the most common causes of cancer-related deaths for women in developed countries. Development of HER2-targeting drugs has been considered a breakthrough in anti-cancer approaches and alluded to the potential of targeting growth factors in breast cancer (BrCa) therapeutics. More than twenty-five years have passed since the Insulin-like Growth Factor-1 (IGF-1) system was initially recognized as a potential target candidate in BrCa therapy. To date, a growing body of studies have implicated the IGF-1 signaling with the BrCa biology. Despite the promising experimental evidence, the impression from clinical trials is rather disappointing. Several reasons may account for this and the last word regarding the efficacy of this system as a target candidate in BrCa therapeutics is probably not written yet. Herein, we provide the theoretical basis, as well as, a comprehensive overview of the current literature, regarding the different strategies targeting the various components of the IGF-1/IGF-1R axis in several pathophysiological aspects of BrCa, including the tumor micro-environment and cancer stemness. In addition, we review the rationale for targeting the IGF-1 system in the different BrCa molecular subtypes and in treatment resistant breast tumors with a focus on both the molecular mechanisms and on the clinical perspectives of such approaches in specific population subgroups. We also discuss the future challenges, as well as, the development of novel molecules and strategies targeting the system and suggest potential improvements in the field.

Both Type I and Type II Interferons Can Activate Antitumor M1 Macrophages When Combined With TLR Stimulation.

Triggering or enhancing antitumor activity of tumor-associated macrophages is an attractive strategy for cancer treatment. We have previously shown that the cytokine interferon-γ (IFN-γ), a type II IFN, could synergize with toll-like receptor (TLR) agonists for induction of antitumor M1 macrophages. However, the toxicity of IFN-γ limits its clinical use. Here, we investigated whether the less toxic type I IFNs, IFN-α, and IFN-ß, could potentially replace IFN-γ for induction of antitumor M1 macrophages. We measured in vitro the ability of type I and II IFNs to synergize with TLR agonists for transcription of inducible nitric oxide synthase (iNOS) mRNA and secretion of nitric oxide (NO) by mouse bone marrow-derived macrophages (BMDMs). An in vitro growth inhibition assay was used to measure both cytotoxic and cytostatic activity of activated macrophages against Lewis lung carcinoma (LLC) cancer cells. We found that both type I and II IFNs could synergize with TLR agonists in inducing macrophage-mediated inhibition of cancer cell growth, which was dependent on NO. The ability of high dose lipopolysaccharide (LPS) to induce tumoricidal activity in macrophages in the absence of IFN-γ was shown to depend on induction of autocrine type I IFNs. Antitumor M1 macrophages could also be generated in the absence of IFN-γ by a combination of two TLR ligands when using the TLR3 agonist poly(I:C) which induces autocrine type I IFNs. Finally, we show that encapsulation of poly(I:C) into nanoparticles improved its potency to induce M1 macrophages up to 100-fold. This study reveals the potential of type I IFNs for activation of antitumor macrophages and indicates new avenues for cancer immunotherapy based on type I IFN signaling, including combination of TLR agonists.

CD4+ T-cell-Mediated Rejection of MHC Class II-Positive Tumor Cells Is Dependent on Antigen Secretion and Indirect Presentation on Host APCs.

Tumor-specific CD4+ T cells have been shown to mediate efficient antitumor immune responses against cancer. Such responses can occur through direct binding to MHC class II (MHC II)-expressing tumor cells, or indirectly via activation of professional antigen-presenting cells (APC) that take up and present the tumor antigen. We have previously shown that CD4+ T cells reactive against an epitope within the Ig light chain variable region of a murine B-cell lymphoma can reject established tumors. Given the presence of MHC II molecules at the surface of lymphoma cells, we investigated whether MHC II-restricted antigen presentation on tumor cells alone was required for rejection. Variants of the A20 B lymphoma cell line that either secreted or intracellularly retained different versions of the tumor-specific antigen revealed that antigen secretion by the MHC II-expressing tumor cells was essential both for the priming and effector phase of CD4+ T-cell-driven antitumor immune responses. Consistent with this, genetic ablation of MHC II in tumor cells, both in the case of B lymphoma and B16 melanoma, did not preclude rejection of tumors by tumor antigen-specific CD4+ T cells in vivo These findings demonstrate that MHC class II expression on tumor cells themselves is not required for CD4+ T-cell-mediated rejection and that indirect display on host APC is sufficient for effective tumor elimination. These results support the importance of tumor-infiltrating APC as mediators of tumor cell killing by CD4+ T cells.Significance: Elimination of tumors by CD4+ T cells recognizing secreted tumor neoantigens can occur in the absence of tumor cell-intrinsic MHC II expression, highlighting the potential clinical relevance of indirect antigen recognition by tumor-infiltrating APC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4573/F1.large.jpg Cancer Res; 78(16); 4573-85. ©2018 AACR.

Structural characterization of bioactive heteropolysaccharides from the medicinal fungus Inonotus obliquus (Chaga).

The aim of this paper was to perform a comprehensive characterization of polysaccharides isolated from the interior (IOI) and exterior (IOE) parts of the fungus Inonotus obliquus. Pre-extraction with DCM and MeOH, followed by water and alkali extraction and ethanol precipitation gave two water extracts and two alkali extracts. Neutral and acidic polysaccharide fractions were obtained after anion-exchange chromatography of the water extracts. The neutral polysaccharides (60-73 kDa) were heterogeneous and branched and consisted of a (1 → 3)-linked ß-Glc backbone with (1 → 6)-linked kinks in the chain at approximately every fifth residue, with branches of (1 → 6)-linked ß-Glc in addition to substantial amounts of (1 → 6)-linked α-Gal with 3-O-methylation at about every third Gal residue. The acidic polysaccharide fractions (10-31 kDa) showed similar structural motifs as the neutral fractions differing mainly by the presence of (1 → 4)-linked α-GalA and α-GlcA. ß-Xyl, α-Man and α-Rha were also present in varying amounts in all fractions. No major structural differences between the IOI and IOE fractions were observed. An alkaline polysaccharide fraction (>450 kDa) was obtained from the IOI alkali extract, and consisted mainly of (1 → 3)- and (1 → 6)-linked ß-Glc and (1 → 4)-linked ß-Xyl. Several of the fractions showed in vitro immunomodulatory effect by increasing NO production in the murine macrophage and dendritic cell lines J774.A1 and D2SC/1. Most fractions managed to increase NO production only at the highest concentration tested (100 µg/ml), while the neutral fraction IOE-WN activated potent NO production at 10 µg/ml and was considered the most promising immunomodulating fraction in this study.

Immune Cell Composition in Human Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the world. Immunological analysis of the tumor microenvironment (immunoscore) shows great promise for improved prognosis and prediction of response to immunotherapy. However, the exact immune cell composition in NSCLC remains unclear. Here, we used flow cytometry to characterize the immune infiltrate in NSCLC tumors, non-cancerous lung tissue, regional lymph node, and blood. The cellular identity of >95% of all CD45+ immune cells was determined. Thirteen distinct immune cell types were identified in NSCLC tumors. T cells dominated the lung cancer landscape (on average 47% of all CD45+ immune cells). CD4+ T cells were the most abundant T cell population (26%), closely followed by CD8+ T cells (22%). Double negative CD4-CD8- T cells represented a small fraction (1.4%). CD19+ B cells were the second most common immune cell type in NSCLC tumors (16%), and four different B cell sub-populations were identified. Macrophages and natural killer (NK) cells composed 4.7 and 4.5% of the immune cell infiltrate, respectively. Three types of dendritic cells (DCs) were identified (plasmacytoid DCs, CD1c+ DCs, and CD141+ DCs) which together represented 2.1% of all immune cells. Among granulocytes, neutrophils were frequent (8.6%) with a high patient-to-patient variability, while mast cells (1.4%), basophils (0.4%), and eosinophils (0.3%) were less common. Across the cohort of patients, only B cells showed a significantly higher representation in NSCLC tumors compared to the distal lung. In contrast, the percentages of macrophages and NK cells were lower in tumors than in non-cancerous lung tissue. Furthermore, the fraction of macrophages with high HLA-DR expression levels was higher in NSCLC tumors relative to distal lung tissue. To make the method readily accessible, antibody panels and flow cytometry gating strategy used to identify the various immune cells are described in detail. This work should represent a useful resource for the immunomonitoring of patients with NSCLC.

Toll-Like Receptor Ligands and Interferon-γ Synergize for Induction of Antitumor M1 Macrophages.

Tumor-associated macrophages may either promote or suppress tumor growth depending on their activation status. Interferon-γ (IFN-γ) has been identified as a key factor for inducing tumoricidal M1 phenotype in macrophages. However, it remains unclear whether IFN-γ is sufficient or if additional stimuli are required. Here, we tested IFN-γ and a panel of toll-like receptor (TLR) agonists for the ability to activate murine macrophages toward a tumoricidal M1 phenotype. The following TLR ligands were used: TLR1/TLR2 agonist Pam3CSK4, TLR2/TLR6 agonist lipotechoic acid, TLR3 agonist poly(I:C), TLR4 agonist lipopolysaccharide (LPS), TLR5 agonist flagellin, TLR7 agonist CL264, and TLR9 agonist CpG. We used an in vitro growth inhibition assay to measure both cytotoxic and cytostatic activity of mouse macrophages against Lewis lung carcinoma (LLC) and MOPC315 plasmacytoma tumor cells. Production of nitric oxide (NO) and cytokines by activated macrophages was quantified. We found that IFN-γ alone was not able to render macrophages tumoricidal. Similarly, macrophage activation with single TLR agonists was inefficient. In sharp contrast, IFN-γ was shown to synergize with TLR agonists for induction of macrophage tumoricidal activity and production of both NO and pro-inflammatory cytokines (TNF-α, IL-12p40, and IL-12p70). Furthermore, IFN-γ was shown to suppress macrophage IL-10 secretion induced by TLR agonists. NO production was necessary for macrophage tumoricidal activity. We conclude that two signals from the microenvironment are required for optimal induction of antitumor M1 macrophage phenotype. Combination treatment with IFN-γ and TLR agonists may offer new avenues for macrophage-based cancer immunotherapy.

Generation and Functional In Vitro Analysis of Semliki Forest Virus Vectors Encoding TNF-α and IFN-γ.

Cytokine gene delivery by viral vectors is a promising novel strategy for cancer immunotherapy. Semliki Forest virus (SFV) has many advantages as a delivery vector, including the ability to (i) induce p53-independent killing of tumor cells via apoptosis, (ii) elicit a type-I interferon (IFN) response, and (iii) express high levels of the transgene. SFV vectors encoding cytokines such as interleukin (IL)-12 have shown promising therapeutic responses in experimental tumor models. Here, we developed two new recombinant SFV vectors encoding either murine tumor necrosis factor-α (TNF-α) or murine interferon-γ (IFN-γ), two cytokines with documented immunostimulatory and antitumor activity. The SFV vector showed high infection rate and cytotoxicity in mouse and human lung carcinoma cells in vitro. By contrast, mouse and human macrophages were resistant to infection with SFV. The recombinant SFV vectors directly inhibited mouse lung carcinoma cell growth in vitro, while exploiting the cancer cells for production of SFV vector-encoded cytokines. The functionality of SFV vector-derived TNF-α was confirmed through successful induction of cell death in TNF-α-sensitive fibroblasts in a concentration-dependent manner. SFV vector-derived IFN-γ activated macrophages toward a tumoricidal phenotype leading to suppressed Lewis lung carcinoma cell growth in vitro in a concentration-dependent manner. The ability of SFV to provide functional cytokines and infect tumor cells but not macrophages suggests that SFV may be very useful for cancer immunotherapy employing tumor-infiltrating macrophages.