IL9 Polarizes Macrophages to M1 and Induces the Infiltration of Antitumor Immune Cells via MIP-1 and CXCR3 Chemokines.

Tumor-associated macrophages (TAM) are involved in tumor progression, metastasis, and immunosuppression. Because TAMs are highly plastic and could alter their phenotypes to proinflammatory M1 in response to environmental stimuli, reeducating TAMs has emerged as a promising approach to overcoming the challenges of solid cancer treatment. This study investigated the effect of IL9 on macrophage M1 polarization and verified its antitumor potential to retrain TAMs and promote chemokine secretion. We demonstrated that IL9 stimulated macrophage proliferation and polarized them toward the proinflammatory M1 phenotype in an IFNγ-dependent manner. Tumor-localized IL9 also polarized TAMs toward M1 in vivo and made them release CCL3/4 and CXCL9/10 to recruit antitumor immune cells, including T and natural killer cells, into the tumor microenvironment. Furthermore, peritoneal treatment with recombinant IL9 delayed the growth of macrophage-enriched B16F10 melanoma and 4T1 breast cancer in syngeneic mice, although IL9 treatment did not reduce tumor growth in the absence of macrophage enrichment. These results demonstrate the efficacy of IL9 in macrophage polarization to trigger antitumor immunity. Significance: These findings clarified the effect of IL9 on macrophage M1 polarization and verified its antitumor potential through retraining TAMs and chemokine secretion.

Protective and Therapeutic Effects of an IL-15:IL-15Rα-Secreting Cell-Based Cancer Vaccine Using a Baculovirus System.

This study reports the use of the BacMam system to deliver and express self-assembling IL-15 and IL-15Rα genes to murine B16F10 melanoma and CT26 colon cancer cells. BacMam-based IL-15 and IL-15Rα were well-expressed and assembled to form the biologically functional IL-15:IL-15Rα complex. Immunization with this IL-15:IL-15Rα cancer vaccine delayed tumor growth in mice by inducing effector memory CD4+ and CD8+ cells and effector NK cells which are tumor-infiltrating. It caused strong antitumor immune responses of CD8+ effector cells in a tumor-antigen specific manner both in vitro and in vivo and significantly attenuated Treg cells which a control virus-infected cancer vaccine could induce. Post-treatment with this cancer vaccine after a live cancer cell injection also prominently delayed the growth of the tumor. Collectively, we demonstrate a vaccine platform consisting of BacMam virus-infected B16F10 or CT26 cancer cells that secrete IL-15:IL-15Rα. This study is the first demonstration of a functionally competent soluble IL-15:IL-15Rα complex-related cancer vaccine using a baculovirus system and advocates that the BacMam system can be used as a secure and rapid method of producing a protective and therapeutic cancer vaccine.

Interleukin-9 Inhibits Lung Metastasis of Melanoma through Stimulating Anti-Tumor M1 Macrophages.

Interleukin-9 (IL-9) is well known for its role in allergic inflammation. For cancer, both pro- and anti-tumor effects of IL-9 were controversially reported, but the impact of IL-9 on tumor metastasis has not yet been clarified. In this study, IL-9 was expressed as a secretory form (sIL-9) and a membrane-bound form (mbIL-9) on B16F10 melanoma cells. The mbIL-9 was engineered as a chimeric protein with the transmembrane and cytoplasmic region of TNF-α. The effect of either mbIL-9 or sIL-9 expressing cells were analyzed on the metastasis capability of the cancer cells. After three weeks of tumor implantation into C57BL/6 mice through the tail vein, the number of tumor modules in lungs injected with IL-9 expressing B16F10 was 5-fold less than that of control groups. The percentages of CD4+ T cells, CD8+ T cells, NK cells, and M1 macrophages considerably increased in the lungs of the mice injected with IL-9 expressing cells. Among them, the M1 macrophage subset was the most significantly enhanced. Furthermore, peritoneal macrophages, which were stimulated with either sIL-9 or mbIL-9 expressing transfectant, exerted higher anti-tumor cytotoxicity compared with that of the mock control. The IL-9-stimulated peritoneal macrophages were highly polarized to M1 phenotype. Stimulation of RAW264.7 macrophages with sIL-9 or mbIL-9 expressing cells also significantly increased the cytotoxicity of those macrophages against wild-type B16F10 cells. These results clearly demonstrate that IL-9 can induce an anti-metastasis effect by enhancing the polarization and proliferation of M1 macrophages.

Crosstalk between the Producers and Immune Targets of IL-9.

IL-9 has been reported to play dual roles in the pathogenesis of autoimmune disorders and cancers. The collaboration of IL-9 with microenvironmental factors including the broader cytokine milieu and other cellular components may provide important keys to explain its conflicting effects in chronic conditions. In this review, we summarize recent findings on the cellular sources of, and immunological responders to IL-9, in order to interpret the role of IL-9 in the regulation of immune responses. This knowledge will provide new perspectives to improve clinical benefits and limit adverse effects of IL-9 when treating pathologic conditions.

Cell-Based IL-15:IL-15Rα Secreting Vaccine as an Effective Therapy for CT26 Colon Cancer in Mice.

Interleukin (IL)-15 is an essential immune-modulator with high potential for use in cancer treatment. Natural IL-15 has a low biological potency because of its short half-life and difficulties in mass-production. IL-15Rα, a member of the IL-15 receptor complex, is famous for its high affinity to IL-15 and its ability to lengthen the half-life of IL-15. We have double-transfected IL-15 and its truncated receptor IL-15Rα into CT26 colon cancer cells to target them for intracellular assembly. The secreted IL-15:IL-15Rα complexes were confirmed in ELISA and Co-IP experiments. IL-15:IL15Rα secreting clones showed a higher anti-tumor effect than IL-15 secreting clones. Furthermore, we also evaluated the vaccine and therapeutic efficacy of the whole cancercell vaccine using mitomycin C (MMC)-treated IL-15:IL15Rα secreting CT26 clones. Three sets of experiments were evaluated; (1) therapeutics, (2) vaccination, and (3) longterm protection. Wild-type CT26-bearing mice treated with a single dose of MMC-inactivated secreted IL-15:IL-15Rα clones prolonged survival compared to the control group. Survival of MMC-inactivated IL-15:IL-15Rα clone-vaccinated mice (without any further adjuvant) exceeded up to 100%. This protection effect even lasted for at least three months after the immunization. Secreted IL-15:IL-15Rα clones challenging trigger anti-tumor response via CD4+ T, CD8+ T, and natural killer (NK) cell-dependent cytotoxicity. Our result suggested that cell-based vaccine secreting IL-15:IL-15Rα, may offer the new tools for immunotherapy to treat cancer.

Application of antihelix antibodies in protein structure determination.

Antibodies are indispensable tools in protein engineering and structural biology. Antibodies suitable for structural studies should recognize the 3-dimensional (3D) conformations of target proteins. Generating such antibodies and characterizing their complexes with antigens take a significant amount of time and effort. Here, we show that we can expand the application of well-characterized antibodies by "transplanting" the epitopes that they recognize to proteins with completely different structures and sequences. Previously, several antibodies have been shown to recognize the alpha-helical conformation of antigenic peptides. We demonstrate that these antibodies can be made to bind to a variety of unrelated "off-target" proteins by modifying amino acids in the preexisting alpha helices of such proteins. Using X-ray crystallography, we determined the structures of the engineered protein-antibody complexes. All of the antibodies bound to the epitope-transplanted proteins, forming accurately predictable structures. Furthermore, we showed that binding of these antihelix antibodies to the engineered target proteins can modulate their catalytic activities by trapping them in selected functional states. Our method is simple and efficient, and it will have applications in protein X-ray crystallography, electron microscopy, and nanotechnology.

Higd-1a regulates the proliferation of pancreatic cancer cells through a pERK/p27KIP1/pRB pathway.

Higd-1a/HIMP1-a/HIG1, a mitochondrial inner membrane protein, promotes cell survival under low glucose and hypoxic conditions. We previously reported that it interacts with Opa1, a factor involved in mitochondrial fusion, to regulate mitochondrial homeostasis. In the present study, we found that depletion of Higd-1a inhibited the proliferation of pancreatic cancer cells in vitro and in mice xenografts. Higd-1a knockdown did not itself lead to cell death but it caused cell cycle arrest through induction of p27KIP1 and hypo-phosphorylation of RB protein. Knockdown of Higd-1a also induced cellular senescence as shown by increased granularity and SA-ß-galactosidase activity. We further showed that the mitochondrial stress induced by Higd-1a led to reduced ERK phosphorylation. Inhibition of the ERK pathway with U0126 induced p27KIP1 expression in the pancreatic cancer cells, confirming that the cell cycle retardation was the result of inhibition of the ERK pathway. Array analysis of human pancreatic cancers revealed that expression of Higd-1a was significantly elevated in pancreatic cancer tissues compared to normal tissue. Collectively, our results demonstrate that Higd-1a plays an important role in the proliferation of pancreatic cancer cells by regulating the pERK/p27KIP1/pRB signaling pathway.

Attachment of flagellin enhances the immunostimulatory activity of a hemagglutinin-ferritin nano-cage.

Hemagglutinin (HA) displayed on a ferritin nano-cage has been shown to be effective in generating a potent immune response against a broad range of influenza infections. Here, we showed that conjugation of flagellin together with HA to the exterior surface of the ferritin cage greatly enhanced not only the humoral immune response in mice but also antigen-specific T cell responses that include Th1 cytokine secretion. The effect of flagellin remained essentially unchanged when the molar ratio of flagellin to HA was reduced from 1:1 to 1:3. Injection of the ferritin-HA-flagellin cage provided protection against lethal virus challenge in mice. We used a small immunoglobulin fragment VL12.3 as a convenient method for attaching HA and flagellin to the ferritin cage. This attachment method can be used for rapid screening of a variety of protein cages and nano-assemblies to identify the most suitable carrier and adjuvant proteins for the target antigen.

Ectopically Expressed Membrane-bound Form of IL-9 Exerts Immune-stimulatory Effect on CT26 Colon Carcinoma Cells.

IL-9 is a known T cell growth factor with pleiotropic immunological functions, especially in parasite infection and colitis. However, its role in tumor growth is controversial. In this study, we generated tumor clones expressing the membrane-bound form of IL-9 (MB-IL-9) and investigated their influences on immune system. MB-IL-9 tumor clones showed reduced tumorigenicity but shortened survival accompanied with severe body weight loss in mice. MB-IL-9 expression on tumor cells had no effect on cell proliferation or major histocompatibility complex class I expression in vitro. MB-IL-9 tumor clones were effective in amplifying CD4+ and CD8+ T cells and increasing cytotoxic activity against CT26 cells in vivo. We also observed a prominent reduction in body weights and survival period of mice injected intraperitoneally with MB-IL-9 clones compared with control groups. Ratios of IL-17 to interferon (IFN)-γ in serum level and tumor mass were higher in mice implanted with MB-IL-9 tumor clones than those observed in mice implanted with control cells. These results indicate that the ectopic expression of the MB-IL-9 on tumor cells exerts an immune-stimulatory effect with toxicity. To exploit its benefits as a tumor vaccine, a strategy to control the toxicity of MB-IL-9 tumor clones should be developed.

Construction of novel repeat proteins with rigid and predictable structures using a shared helix method.

Generating artificial protein assemblies with complex shapes requires a method for connecting protein components with stable and predictable structures. Currently available methods for creating rigid protein assemblies rely on either complicated calculations or extensive trial and error. We describe a simple and efficient method for connecting two proteins via a fused alpha helix that is formed by joining two preexisting helices into a single extended helix. Because the end-to-end ligation of helices does not guarantee the formation of a continuous helix, we superimposed 1-2 turns of pairs of connecting helices by using a molecular graphics program. Then, we chose amino acids from the two natural sequences that would stabilize the connecting helix. This "shared helix method" is highly efficient. All the designed proteins that could be produced in Escherichia coli were readily crystallized and had the expected fusion structures. To prove the usefulness of this method, we produced two novel repeat proteins by assembling several copies of natural or artificial proteins with alpha helices at both termini. Their crystal structures demonstrated the successful assembly of the repeating units with the intended curved shapes. We propose that this method could dramatically expand the available repertoire of natural repeat proteins.

Membrane-bound p35 Subunit of IL-12 on Tumor Cells is Functionally Equivalent to Membrane-bound Heterodimeric Single Chain IL-12 for Induction of Anti-tumor Immunity.

In this study, we compared two different tumor cell vaccines for their induction of anti-tumor immunity; one was a tumor cell clone expressing a membrane-bound form of IL-12 p35 subunit (mbIL-12 p35 tumor clone), and the other was a tumor clone expressing heterodimeric IL-12 as a single chain (mb-scIL-12 tumor clone). The stimulatory effect of mb-scIL-12 on the proliferation of ConA-activated splenocytes was higher than that of mbIL-12 p35 in vitro. However, the stimulatory effect of mbIL-12 p35 was equivalent to that of recombinant soluble IL-12 (3 ng/ml). Interestingly, both tumor clones (mbIL-12 p35 and mb-scIL-12) showed similar tumorigenicity and induction of systemic anti-tumor immunity in vivo, suggesting that tumor cell expression of the membrane-bound p35 subunit is sufficient to induce anti-tumor immunity in our tumor vaccine model.

Crystal structures of mono- and bi-specific diabodies and reduction of their structural flexibility by introduction of disulfide bridges at the Fv interface.

Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Diabodies are engineered antibody fragments that are composed of two Fv domains connected by short peptide linkers. They are attractive candidates for mediators in assembling protein nano-structures because they can simultaneously bind to two different proteins and are rigid enough to be crystallized. However, comparison of previous crystal structures demonstrates that there is substantial structural diversity in the Fv interface region of diabodies and, therefore, reliable prediction of its structure is not trivial. Here, we present the crystal structures of ten mono- and bi-specific diabodies. We found that changing an arginine residue in the Fv interface to threonine greatly reduced the structural diversity of diabodies. We also found that one of the bispecific diabodies underwent an unexpected process of chain swapping yielding a non-functional monospecific diabody. In order to further reduce structural flexibility and prevent chain shuffling, we introduced disulfide bridges in the Fv interface regions. The disulfide-bridged diabodies have rigid and predictable structures and may have applications in crystallizing proteins, analyzing cryo-electron microscopic images and building protein nano-assemblies.

The Membrane-Bound Form of IL-17A Promotes the Growth and Tumorigenicity of Colon Cancer Cells.

Interleukin-17A is a member of the IL-17 family, and is known as CTLA8 in the mouse. It is produced by T lymphocytes and NK cells and has proinflammatory roles, inducing cytokine and chemokine production. However, its role in tumor biology remains controversial. We investigated the effects of locally produced IL-17A by transferring the gene encoding it into CT26 colon cancer cells, either in a secretory or a membrane-bound form. Expression of the membrane-bound form on CT26 cells dramatically enhanced their proliferation in vitro. The enhanced growth was shown to be due to an increased rate of cell cycle progression: after synchronizing cells by adding and withdrawing colcemid, the rate of cell cycle progression in the cells expressing the membrane-bound form of IL-17A was much faster than that of the control cells. Both secretory and membrane-bound IL-17A induced the expression of Sca-1 in the cancer cells. When tumor clones were grafted into syngeneic BALB/c mice, the tumor clones expressing the membrane-bound form IL-17A grew rapidly; those expressing the secretory form also grew faster than the wild type CT26 cells, but slower than the clones expressing the membrane-bound form. These results indicate that IL-17A promotes tumorigenicity by enhancing cell cycle progression. This finding should be considered in treating tumors and immune-related diseases.

Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker.

Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a 'fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.

Higd-1a interacts with Opa1 and is required for the morphological and functional integrity of mitochondria.

The activity and morphology of mitochondria are maintained by dynamic fusion and fission processes regulated by a group of proteins residing in, or attached to, their inner and outer membranes. Hypoxia-induced gene domain protein-1a (Higd-1a)/HIMP1-a/HIG1, a mitochondrial inner membrane protein, plays a role in cell survival under hypoxic conditions. In the present study, we showed that Higd-1a depletion resulted in mitochondrial fission, depletion of mtDNA, disorganization of cristae, and growth retardation. We demonstrated that Higd-1a functions by specifically binding to Optic atrophy 1 (Opa1), a key element in fusion of the inner membrane. In the absence of Higd-1a, Opa1 was cleaved, resulting in the loss of its long isoforms and accumulation of small soluble forms. The small forms of Opa1 do not interact with Higd-1a, suggesting that a part of Opa1 in or proximal to the membrane is required for that interaction. Opa1 cleavage, mitochondrial fission, and cell death induced by dissipation of the mitochondrial membrane potential were significantly inhibited by ectopic expression of Higd-1a. Furthermore, growth inhibition due to Higd-1a depletion could be overcome by overexpression of a noncleavable form of Opa1. Collectively, our observations demonstrate that Higd-1a inhibits Opa1 cleavage and is required for mitochondrial fusion by virtue of its interaction with Opa1.

The survival effect of mitochondrial Higd-1a is associated with suppression of cytochrome C release and prevention of caspase activation.

Higd-1a (hypoxia induced gene domain family-1a) is a mitochondrial inner membrane protein with a conformation of N-terminal outside-C-terminal outside and loop inside. There are four Higd genes, Higd-1a, -1b, -1c and -2a, in the mouse. Higd-1a and -2a are expressed primarily in the brain, heart, kidney and leukocytes. HIF (hypoxia-inducible factor) overexpression induced the endogenous expression and promoter activity of Higd-1a. Mutation of the HRE (hypoxia-response element) site at -32bp in the Higd-1a promoter reduced the promoter activity, suggesting that transcription of Higd-1a is regulated by binding of the transcription factor HIF to the HRE. Higd-1a promoted cell survival under hypoxia. RAW264.7 cells stably transfected with Higd-1a underwent less apoptosis than control cells in a hypoxic condition, and hypoxia-induced apoptosis was strongly enhanced when endogenous Higd-1a was silenced by siRNA. The survival effect of Higd-1a was completely abolished by deletion of the 26 N-terminal amino acids, and we showed that Higd-1a increased survival by inhibiting cytochrome C release and reducing the activities of caspases. However, expression of Bcl-2, Bax, Bad, and BNIP3 and translocation of AIF were unaffected under the same conditions. Higd-2a also enhanced cell survival under hypoxia. Cells transfected with Higd-2a underwent less apoptosis than control cells in hypoxic conditions, and hypoxia-induced apoptosis increased when endogenous Higd-2a was depleted. Together these observations indicate that Higd-1a is induced by hypoxia in a HIF-dependent manner and its anti-apoptotic effect results from inhibiting cytochrome C release and reducing caspase activities.

Silencing of BNIP3 results from promoter methylation by DNA methyltransferase 1 induced by the mitogen-activated protein kinase pathway.

We have previously shown that Ras mediates NO-induced BNIP3 expression via the MEK-E RK-HIF-1 pathway i n mouse macrophages, and that NO-induced death results at least in part from the induction of BNIP3. In the present study, we describe another aspect of Ras regulation of BNIP3 expression in pancreatic cancer cells. Human BNIP3 promoter-driven luciferase activity was efficiently induced by activated Ras in AsPC-1, Miapaca-2, PK-1 and PANC-1 cells. However, expression of endogenous BNIP3 was not induced, and BNIP3 up-regulation by hypoxia was also inhibited. Treatment of the cells with the DNMT inhibitor, 5-aza-2-deoxycytidine, restored BNIP3 induction, indicating that DNA methylation of the BNIP3 promoter was responsible for the inhibition of BNIP3 induction. Furthermore, inhibition of the MEK pathway with U0126 reduced DNMT1 expression, but not that of DNMT3a and 3b, and restored the hypoxia-inducibility of BNIP3, suggesting that the DNA methylation of the BNIP3 promoter was mediated by DNMT1 via the MEK pathway.

Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation.

CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser(132) loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38- and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di- or trimerization is necessary but not sufficient for complete activation of CD40.

Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer.

Toll-like receptor 2 (TLR2) initiates potent immune responses by recognizing diacylated and triacylated lipopeptides. Its ligand specificity is controlled by whether it heterodimerizes with TLR1 or TLR6. We have determined the crystal structures of TLR2-TLR6-diacylated lipopeptide, TLR2-lipoteichoic acid, and TLR2-PE-DTPA complexes. PE-DTPA, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, is a synthetic phospholipid derivative. Two major factors contribute to the ligand specificity of TLR2-TLR1 or TLR2-TLR6 heterodimers. First, the lipid channel of TLR6 is blocked by two phenylalanines. Simultaneous mutation of these phenylalanines made TLR2-TLR6 fully responsive not only to diacylated but also to triacylated lipopeptides. Second, the hydrophobic dimerization interface of TLR2-TLR6 is increased by 80%, which compensates for the lack of amide lipid interaction between the lipopeptide and TLR2-TLR6. The structures of the TLR2-lipoteichoic acid and the TLR2-PE-DTPA complexes demonstrate that a precise interaction pattern of the head group is essential for a robust immune response by TLR2 heterodimers.

The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex.

The lipopolysaccharide (LPS) of Gram negative bacteria is a well-known inducer of the innate immune response. Toll-like receptor (TLR) 4 and myeloid differentiation factor 2 (MD-2) form a heterodimer that recognizes a common 'pattern' in structurally diverse LPS molecules. To understand the ligand specificity and receptor activation mechanism of the TLR4-MD-2-LPS complex we determined its crystal structure. LPS binding induced the formation of an m-shaped receptor multimer composed of two copies of the TLR4-MD-2-LPS complex arranged symmetrically. LPS interacts with a large hydrophobic pocket in MD-2 and directly bridges the two components of the multimer. Five of the six lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD-2, forming a hydrophobic interaction with the conserved phenylalanines of TLR4. The F126 loop of MD-2 undergoes localized structural change and supports this core hydrophobic interface by making hydrophilic interactions with TLR4. Comparison with the structures of tetra-acylated antagonists bound to MD-2 indicates that two other lipid chains in LPS displace the phosphorylated glucosamine backbone by approximately 5 A towards the solvent area. This structural shift allows phosphate groups of LPS to contribute to receptor multimerization by forming ionic interactions with a cluster of positively charged residues in TLR4 and MD-2. The TLR4-MD-2-LPS structure illustrates the remarkable versatility of the ligand recognition mechanisms employed by the TLR family, which is essential for defence against diverse microbial infection.