Yuki Orito

A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell.

Plasmodium parasites are fertilized in the mosquito midgut and develop into motile zygotes, called ookinetes, which invade the midgut epithelium. Here we show that a calcium‐dependent protein kinase, CDPK3, of the rodent malarial parasite (Plasmodium berghei) is produced in the ookinete stage and has a critical role in parasite transmission to the mosquito vector. Targeted disruption of the CDPK3 gene decreased ookinete ability to infect the mosquito midgut by nearly two orders of magnitude. Electron microscopic analyses demonstrated that the disruptant ookinetes could not access midgut epithelial cells by traversing the layer covering the cell surface. An in vitro migration assay showed that these ookinetes lack the ability to migrate through an artificial gel, suggesting that this defect caused their failure to access the epithelium. In vitro migration assays also suggested that this motility is induced in the wild type by mobilization of intracellular stored calcium. These results indicate that a signalling pathway involving calcium and CDPK3 regulates ookinete penetration of the layer covering the midgut epithelium. Because humans do not possess CDPK family proteins, CDPK3 is a good target for blocking malarial transmission to the mosquito vector.

A mosquito salivary protein inhibits activation of the plasma contact System by binding to factor XII and high molecular weight Kininogen

The salivary glands of female mosquitoes contain a variety of bioactive substances that assist their blood-feeding behavior. Here, we report a salivary protein of the malarial vector mosquito, Anopheles stephensi, that inhibits activation of the plasma contact system. This factor, named hamadarin, is a 16-kDa protein and a major component of the saliva of this mosquito. Assays using human plasma showed that hamadarin dose-dependently inhibits activation of the plasma contact system and subsequent release of bradykinin, a primary mediator of inflammatory reactions. Reconstitution experiments showed that hamadarin inhibits activation of the plasma contact system by inhibition of the reciprocal activation of factor XII and kallikrein. Direct binding assays demonstrated that this inhibitory effect is due to hamadarin binding to both factor XII and high molecular weight kininogen and interference in their association with the activating surface. The assays also showed that hamadarin binding to these proteins depends on Zn2+ ions, suggesting that hamadarin binds to these contact factors by recognizing their conformational change induced by Zn2+ binding. We propose that hamadarin may attenuate the hosts acute inflammatory responses to the mosquitos bites by inhibition of bradykinin release and thus enable mosquitoes to take a blood meal efficiently and safely.

Regulatory T Cell–Resistant CD8+ T Cells Induced by Glucocorticoid-Induced Tumor Necrosis Factor Receptor Signaling

We previously found that a Salmonella typhimurium vector engineered to secrete soluble tumor antigen induces CD4(+) T cells resistant to CD4(+)CD25(+) regulatory T cells (Treg) and that glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) signal is involved in the development of this resistance. In this study, we address the potential of incorporating GITR ligand (GITRL) as a way to augment the immunogenicity of cancer vaccines. BALB/c mice were immunized by gene gun with plasmids encoding the mutated extracellular signal-regulated kinase 2 (mERK) with or without plasmids encoding mouse GITRL. Coadministration with GITRL during primary and secondary immunization enhanced the induction of mERK-specific CD8(+) T cells. Antibody depletion and minigene analysis suggested that GITRL directly activated CTL epitope-specific CD8(+) T cells independently of CD4(+) T cells. Immunization with plasmids encoding a CTL epitope and GITRL resulted in strong tumor inhibition in a CD8(+) T cell-dependent manner. Furthermore, CTL epitope-specific CD8(+) T cells induced by immunization with plasmids encoding CTL epitope coadministered with GITRL were refractory to suppression by CD4(+)CD25(+) Tregs compared with CD8(+) T cells induced without GITR signaling. We propose that coadministration of GITR signaling agents with tumor antigens constitutes a promising novel strategy for cancer vaccine development.

LISP1 is important for the egress of Plasmodium berghei parasites from liver cells.

Most Apicomplexa are obligatory intracellular parasites that multiply inside a so‐called parasitophorous vacuole (PV) formed upon parasite entry into the host cell. Plasmodium, the agent of malaria and the Apicomplexa most deadly to humans, multiplies in both hepatocytes and erythrocytes in the mammalian host. Although much has been learned on how Apicomplexa parasites invade host cells inside a PV, little is known of how they rupture the PV membrane and egress host cells. Here, we characterize a Plasmodium protein, called LISP1 (liver‐specific protein 1), which is specifically involved in parasite egress from hepatocytes. LISP1 is expressed late during parasite development inside hepatocytes and locates at the PV membrane. Intracellular parasites deficient in LISP1 develop into hepatic merozoites, which display normal infectivity to erythrocytes. However, LISP1‐deficient liver‐stage parasites do not rupture the membrane of the PV and remain trapped inside hepatocytes. LISP1 is the first Plasmodium protein shown by gene targeting to be involved in the lysis of the PV membrane.

Liver-specific protein 2: a Plasmodium protein exported to the hepatocyte cytoplasm and required for merozoite formation.

The liver stage is the first stage of the malaria parasite that replicates in the vertebrate host. However, little is known about the interplay between the parasite liver stage and its host cell, the hepatocyte. In this study, we identified an exported protein that has a critical role in parasite development in host hepatocytes. Expressed sequence tag analysis of Plasmodium berghei liver‐stage parasites indicated that transcripts encoding a protein with an N‐terminal signal peptide, designated liver‐specific protein 2 (LISP2), are highly expressed in this stage. Expression of LISP2 was first observed 24 h after infection and rapidly increased during the liver‐stage schizogony. Immunofluorescent staining with anti‐LSP2 antibodies showed that LISP2 was carried to the parasitophorous vacuole and subsequently transported to the cytoplasm and nucleus of host hepatocytes. Gene targeting experiments demonstrated that majority of the LISP2‐mutant liver‐stage parasites arrested their development during formation of merozoites. These results indicate that exported LISP2 is involved in parasite–host interactions required for the development of liver‐stage parasites inside hepatocytes. This study demonstrated that mid‐to‐late liver‐stage malarial parasites have a system for exporting proteins to the host cell as intraerythrocytic stages do and presumably to use the proteins to modify the host cell and improve the environment.

Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans

To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen‐induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin‐1 and ‐2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen‐related peptide, a specific agonist of the major collagen‐signaling receptor glycoprotein (GP)VI. Moreover, triplatin‐1 inhibited Fc receptor γ‐chain phosphorylation induced by collagen, which is the first step of GPVI‐mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen‐induced platelet aggregation inhibitors from hematophagus invertebrates.

Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans.

Two plasma kallikrein–kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin‐1 and triafestin‐2, have been identified and characterized. Reconstitution experiments showed that triafestin‐1 and triafestin‐2 inhibit the activation of the kallikrein–kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin‐1 and triafestin‐2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+‐dependent manner, suggesting that they specifically recognize Zn2+‐induced conformational changes in factor XII and high molecular weight kininogen. Triafestin‐1 and triafestin‐2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N‐terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin‐1 and triafestin‐2 inhibit activation of the kallikrein–kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood‐feeding.

Stimulation through very late antigen-4 and -5 improves the multifunctionality and memory formation of CD8⁺ T cells.

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T‐cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH‐296, we demonstrated that stimulation via very late Ag (VLA)‐4 and VLA‐5 in human and BALB/c mouse CD8+ T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR‐transgenic mouse‐derived CD8+ T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma‐derived tumor Ag, we showed that stimulation by CH‐296 improved the ability of tumor‐specific CD8+ T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3+CD4+ Treg cells in tumors. These results suggest that stimulation via VLA‐4 and VLA‐5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.

193. Efficacy and Safety of Immunotherapy with Chimeric Antigen Receptor Targeting WT1 and HLA-A24:02 pMHC Complex

Recent success in the treatment of patients with B cell malignancy by CD19-CAR encourages the development of successive CAR therapy targeting broad range of tumor-associated antigens. However, the search for the appropriate target molecule for CAR, other than B cell markers, has not met the needs. The molecules recognized by CAR is generally limited to the cellular surface molecules, making difficult the search for the tumor-specific targets. Inspired by the physiological recognition of epitope peptide and MHC molecule (pMHC) by T cells, we have generated a series of antibodies that recognize the pMHC complexes with peptides derived from tumor antigens expressed intracellularly. Screening a phage display library of human antibody scFv, we isolated an scFv antibody clone “WT#213” that can specifically recognize WT1 p235-243 peptide (CMTWNQMNL) complexed with HLA-A24:02 molecule. We have constructed a retroviral vector that encodes the CAR consists of WT#213 and intracellular signal transduction domains of CD3ζ and GITR (WT#213 CAR). We confirmed the specific recognition of endogenous WT1-expressing cells by the CAR-T cells with the intracellular cytokine staining and the 51Cr release cytotoxic assay. Moreover, we demonstrated the effectiveness of adoptive cell therapy with WT#213 CAR against the WT1 expressing HLA-A24:02 positive human leukemia cells, utilizing NOG immunodeficient mice. To evaluate the safety of the WT#213 CAR, we predicted the potential property of WT#213 CAR to cross-react to normal tissues in humans. We conducted alanine scan analysis of WT1p235-243 peptide that was recognized by WT#213 CAR as well as the TCR derived from CTL clone TAK-1 which recognizes same epitope peptide in association with HLA-A24:02 to define the amino acids that were critically important in the recognition by the WT#213 CAR or TAK-1-derived TCR. After BLAST search, we synthesized the normal protein-derived peptides with potential risk of cross-reactivity, and tested the recognition of these peptides by WT#213 CAR or TAK-1-derived TCR. Although the critical peptides, and therefore the peptides with potential risk, were quite different between the WT#213 CAR and TAK-1-derived TCR, none of these peptides showed the stimulation of WT#213 CAR or TAK-1-derived TCR. The results here suggest that the immunotherapy with WT#213 CAR will be effective for the treatment of the leukemia patients without the predicted risk in the evaluation we performed.View Large Image | Download PowerPoint Slide