The isotype of epidermal growth factor receptor variant III (EGFRvIII) is often identified in glioblastomas. Previously, we created a mouse monoclonal antibody, 3C10 (IgG2b), that specifically recognized EGFRvIII, and a recombinant single-chain variable fragment of 3C10. The aim of the current study was to develop genetically engineered T cells, termed T-bodies, that express a chimeric receptor consisting of the 3C10 single-chain variable fragment coupled to signaling modules such as the CD3zeta (f) chain, for the treatment of tumors expressing mutant EGFR. After successful construction of the chimeric 3C10 ⁄ CD3f T-cell receptor, its expression on the T-body was observed using western blotting and flow cytometry. The specificity of the T-body for EGFRvIII was evaluated using an interferon-gamma Elispot assay and a standard 51 Cr-release cytotoxicity assay. Furthermore, we demonstrated that the systemically delivered T-body infiltrated the intrabrain tumor and significantly delayed tumor growth. These results indicate that the T-body expressing the chimeric 3C10 ⁄ CD3f T-cell receptor specifically recognized glioma cells expressing EGFRvIII. In conclusion, T-body-based immunotherapy appears to be a promising approach for the treatment of glioma. (Cancer Sci 2010; 101: 2518-2524 T he expression of epidermal growth factor receptor (EGFR) is amplified in approximately 50% of glioblastomas (GBM).(1) The binding of a ligand to EGFR leads to receptor dimerization, autophosphorylation and activation of several downstream signaling pathways such as the Ras ⁄ Raf ⁄ MEK ⁄ ERK pathway, the PI3K ⁄ Akt pathway and the PLC-gamma (c) ⁄ PKC pathway, resulting in cell proliferation, motility and survival.(2) Approximately 40-70% of brain tumors with EGFR amplification express mutant EGFR variant III (EGFRvIII); EGFRvIII has a deletion of exons 2-7 that causes a defect in the extracellular ligand-binding domain and induces constitutive activation in a ligand-independent manner.(3-6) Notably, EGFRvIII is characterized by an 801-base pair (bp) in-frame deletion, which results in a unique sequence with a glycine residue at the fusion junction between amino acid residues 5 and 274. Epidermal growth factor receptor variant III is an attractive target antigen for cancer immunotherapy because it is not expressed in normal tissue and is associated with survival, invasion and angiogenesis in cancers.(4,7) Previously, we generated the monoclonal antibody (mAb) 3C10 and a recombinant singlechain variable fragment (scFv) antibody (Ab) that specifically recognizes EGFRvIII.

Behçet's disease (BD) is widely known to be strongly associated with human leukocyte antigen (HLA) B51 in many different ethnic groups.Recently, HLA-B51 allele typing of Greek BD patients was performed to study the distribution of B*5101-B*5107 alleles in this Greek population, the B51 antigen strongly associated with BD was found to be predominantly encoded by allele B*5101. As it is now known that the B51 antigen can be encoded by 21 alleles, B*5101-B*5121, we performed HLA-B*51 allele genotyping among 58 Greek patients with BD. After serological HLA typing, typing of HLA-B*51 alleles was performed using the polymerase chain reaction-sequencing-based typing (PCR-SBT) method. The frequency of the B51 antigen was found to be significantly higher in the patient group as compared with the control group (75.9% of patients vs 22.0% of controls. In the genotyping of B51 alleles, 34 out of 44 B51-positive patients possessed B*5101, 13 out of the 44 carried B*5108. In contrast, all of the 9 B51-positive normal controls carried B*5101. This study revealed a strong association between Greeks with BD, both B*5101, B*5108, provided important insights into the molecular mechanism underlying the association between HLA status, this disease.

We recently advocated in favour of naming a novel H2-haplotype consisting of Kd, D/Ldm7, I-Ak and I-Ek in the atopic dermatitis (AD) mouse model NC/Nga as “H-2nc.” The role of the H2-haplotype in AD development was investigated in H2b-congenic NC/Nga mice (NC.h2b/b and NC.h2b/nc) established by backcrossing. A severe 2,4-dinitrofluorobenzene (DNFB)-induced dermatitis in NC/Nga was alleviated partially in NC.h2b/nc and significantly in NC.h2b/b. The AD phenotype was correlated with thymic stromal lymphopoietin (TSLP)-epidermal expression levels and serum levels of total IgE and IL-18/IL-33. Histologically, allergic contact dermatitis (ACD) was accompanied by lymphocytes and plasma cells-infiltrating perivasculitis in NC.h2b/nc and NC.h2b/b and clearly differed from AD accompanied by neutrophils, eosinophils and macrophages-infiltrating diffuse suppurative dermatitis in NC/Nga. Interestingly, IFN-γ/IL-17 production from autoreactive CD4+ T-cells remarkably increased in DNFB-sensitised NC.h2b/b but not in NC/Nga. Our findings suggest that AD or ACD may depend on haplotype H-2nc or H-2b, respectively, in addition to the NC/Nga genetic background.

Background Th9 cells have been implicated in the development of allergic inflammation, though its contribution to allergic rhinitis and the effect of steroid on Th9 cell-mediated nasal responses are unclear. Objective In this study, allergen-induced nasal inflammatory responses and their steroid responsiveness were investigated in ovalbumin (OVA)-specific Th9 cell-transferred mice. Methods BALB/c mice were transferred with in vitro -differentiated Th9 cells and challenged by intranasal injection of OVA with or without subcutaneous administration of dexamethasone (Dex). Then, the infiltration of inflammatory cells in the nasal mucosa and nasal hyperresponsiveness (NHR) was assessed. Results The significant NHR accompanied by nasal infiltration of eosinophils as well as allergen-specific T cells was induced in Th9 cell-transferred mice upon allergen challenge. These responses were strongly suppressed by the treatment with Dex. Conclusion The participation of Th9 cells in the pathogenesis of allergic rhinitis was suggested.

Background: Although immunosuppressants for therapy of atopic dermatitis (AD) are still being sought, proteasome inhibitors are also potential candidates for the treatment of AD. Proteasome inhibitors exert various effects by blocking proteasomal degradation and help regulate processes such as apoptosis induction via caspase-9, cell cycle progression via cyclins, NF-κB inactivation via IκB, and downregulation of antigen cross-presentation. The cells targeted by proteasome inhibitors are therefore activated cells undergoing proliferation or differentiation, and antigen-presenting cells carrying out protein degradation. Objectives: This study investigated the therapeutic effects and side effects of a proteasome inhibitor, MG132, on the treatment of AD. Methods: AD-like disease in NC/Nga mice housed under specific pathogen-free conditions was induced by repeated application of 2,4-dinitrofluorobenzene (DNFB). Disease progression was evaluated by inflammation score, histopathology, and serum IgE level, and the effects of systemic MG132 administration were investigated. The percentages and absolute numbers for each population of Th1, Th2, and Th17 cells in the axillary lymph nodes were analyzed by flow cytometry. Results: DNFB application increased the expression of a unique major histocompatibility complex class I mutant molecule D/L^dm7 in dendritic cells (DCs), and increased Th1 and Th17 cells in NC/Nga mice. In vivo MG132 administration to NC/Nga mice with DNFB-induced dermatitis reduced Th17 cells but maintained the level of Th1 cells, resulting in the alleviation of dermatitis lesions by decreasing both serum IgE hyperproduction and mast cell migration. To understand the mechanisms maintaining Th1 cell levels following in vivo MG132-administration, we focused on the role of proteasomes regulating D/L^dm7 expression. Interestingly, 20S proteasome activity was higher in NC/Nga DCs than in BALB/c DCs. In vitro MG132 administration partially increased D/L^dm7 expression in a dose-dependent manner during DC maturation, and induced IFN-γ production from autoreactive CD8+ T cells but not from CD4+ T cells following coculturing with D/L^dm7-upregulated DCs. Conclusion: Although MG132 administration temporarily alleviated AD pathogenesis in NC/Nga mice, prolonged MG132 treatment may result in immunopathogenesis leading to chronic AD due to its side effect of maintaining Th1 levels via autoreactive CD8+ T cells.

Background The activation of Th2 cells that play a pivotal role in the development of allergic eosinophilic inflammation is regulated by an L-type amino acid transporter (LAT) 1. However, the contribution of LAT1 to the pathogenesis of Th2 cell-mediated airway inflammation has not been investigated. Objective In this study, we investigated the effect of a LAT1 inhibitor, JPH203, on Th2 cell-mediated airway eosinophilic inflammation. Methods BALB/c mice were transferred with ovalbumin (OVA)-specific Th2 cell and challenged by corresponding allergen with or without administration of JPH203. Then, the infiltration of inflammatory cells including eosinophils and allergen-specific Th2 cells in the lungs and bronchial hyperresponsiveness (BHR) was assessed. Results Inflammatory responses in the lungs with massive accumulation of eosinophils and BHR were induced in Th2 cell-transferred mice upon challenge with OVA. The treatment with JPH203 significantly suppressed the allergen-induced BHR but not eosinophil infiltration. The infused Th2 cells were also accumulated in the lungs upon allergen challenge, though the response was not affected by JPH203 treatment. Conclusion JPH203 suppressed Th2 cell-mediated BHR through the mechanisms independently of the lung accumulation of eosinophils and Th2 cells.

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