Naomi Takahashi

Impaired CD4 and CD8 Effector Function and Decreased Memory T Cell Populations in ICOS-Deficient Patients

Interaction of ICOS with its ligand is essential for germinal center formation, T cell immune responses, and development of autoimmune diseases. Human ICOS deficiency has been identified worldwide in nine patients with identical ICOS mutations. In vitro studies of the patients to date have shown only mild T cell defect. In this study, we report an in-depth analysis of T cell function in two siblings with novel ICOS deficiency. The brother displayed mild skin infections and impaired Ig class switching, whereas the sister had more severe symptoms, including immunodeficiency, rheumatoid arthritis, inflammatory bowel disease, interstitial pneumonitis, and psoriasis. Despite normal CD3/CD28-induced proliferation and IL-2 production in vitro, peripheral blood T cells in both patients showed a decreased percentage of CD4 central and effector memory T cells and impaired production of Th1, Th2, and Th17 cytokines upon CD3/CD28 costimulation or PMA/ionophore stimulation. The defective polarization into effector cells was associated with impaired induction of T-bet, GATA3, MAF, and retinoic acid-related orphan nuclear hormone receptor (RORC). Reduced CTLA-4+CD45RO+FoxP3+ regulatory T cells and diminished induction of inhibitory cell surface molecules, including CTLA-4, were also observed in the patients. T cell defect was not restricted to CD4 T cells because reduced memory T cells and impaired IFN-γ production were also noted in CD8 T cells. Further analysis of the patients demonstrated increased induction of receptor activator of NF-κB ligand (RANKL), lack of IFN-γ response, and loss of Itch expression upon activation in the female patient, who had autoimmunity. Our study suggests that extensive T cell dysfunction, decreased memory T cell compartment, and imbalance between effector and regulatory cells in ICOS-deficient patients may underlie their immunodeficiency and/or autoimmunity.

Variations in the C3, C3a receptor, and C5 genes affect susceptibility to bronchial asthma

Bronchial asthma (BA) is a common chronic inflammatory disease characterized by hyperresponsive airways, excess mucus production, eosinophil activation, and the production of IgE. The complement system plays an immunoregulatory role at the interface of innate and acquired immunities. Recent studies have provided evidence that C3, C3a receptor, and C5 are linked to airway hyperresponsiveness. To determine whether genetic variations in the genes of the complement system affect susceptibility to BA, we screened single nucleotide polymorphisms (SNPs) in C3, C5, the C3a receptor gene (C3AR1), and the C5a receptor gene (C5R1) and performed association studies in the Japanese population. The results of this SNP case-control study suggested an association between 4896C/T in the C3 gene and atopic childhood BA (P=0.0078) as well as adult BA (P=0.010). When patient data were stratified according to elevated total IgE levels, 4896C/T was more closely associated with adult BA (P=0.0016). A patient-only association study suggested that severity of childhood BA was associated with 1526G/A of the C3AR1 gene (P=0.0057). We identified a high-risk haplotype of the C3 gene for childhood (P=0.0021) and adult BA (P=0.0058) and a low-risk haplotype for adult BA (P=0.00011). We also identified a haplotype of the C5 gene that was protective against childhood BA (P=1.4×10−6) and adult BA (P=0.00063). These results suggest that the C3 and C5 pathways of the complement system play important roles in the pathogenesis of BA and that polymorphisms of these genes affect susceptibility to BA.

Functional promoter polymorphism in the TBX21 gene associated with aspirin-induced asthma

Asthma is a phenotypically heterogeneous disorder with many etiologic factors and clinical characteristics. T-bet, a Th1-specific transcription factor of T-box family, has been found to control interferon-γ (IFN-γ) expression in T cells. Mice lacking the T-bet gene (tbx21) demonstrate multiple physiological and inflammatory features reminiscent of human asthma. In order to examine whether polymorphisms in the candidate gene, TBX21, located on chromosome 17q21.32, are related to the risk of human asthma phenotypes, we have searched for genetic variations in the human TBX21 gene and identified 24 single nucleotide polymorphisms (SNPs), including five novel SNPs, by direct sequencing in Japanese subjects. Among asthma phenotypes, a promoter −1993T→C SNP, which is in linkage disequilibrium with a synonymous coding 390A→G SNP in exon 1, is significantly associated with a risk of aspirin-induced asthma (AIA; P=0.004, Pc=0.016). This association has also been confirmed in additional independent samples of asthma with nasal polyposis (P=0.008), regardless of aspirin hypersensitivity. Furthermore, our data indicate that the −1993T→C substitution increases the affinity of a particular nuclear protein to the binding site of TBX21 covering the −1993 position, resulting in increased transcriptional activity of the TBX21 gene. Thus, in addition to the antigen-driven excess Th2 response, increased T-bet (and subsequent IFN-γ) production in human airways of individuals with the −1993T→C polymorphism could contribute to the development of certain asthma-related phenotypes, such as AIA.

Two Brothers with Ataxia-Telangiectasia-like Disorder with Lung Adenocarcinoma

We report on 2 brothers with ataxia-telangiectasia-like disorder with lung adenocarcinoma. They both had ataxia with cerebellar atrophy and mental retardation. They had the same mutation of the MRE11 gene, which has not been reported previously (c.727T>C and g.24994G>A).

Polymorphisms in ADAM33 are associated with allergic rhinitis due to Japanese cedar pollen

Background A recent report provided evidence that a disintegrin and metalloprotease domain 33 (ADAM33), a member of the ADAM family, is a novel susceptibility gene in asthma linked to bronchial hyper‐responsiveness. However, there has been no investigation of the genetic role of ADAM33 variants in nasal allergy.

Substrate Recognition by Ca2+/Calmodulin-dependent Protein Kinase Kinase ROLE OF THE ARG-PRO-RICH INSERT DOMAIN

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584–587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK α and β shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathioneS-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.

Ca2+/Calmodulin-dependent Protein Kinase Cascade in Caenorhabditis elegans IMPLICATION IN TRANSCRIPTIONAL ACTIVATION

We have recently demonstrated thatCaenorhabditis elegansCa2+/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803–15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI.CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr179 in a Ca2+/CaM-dependent manner, resulting in a 30-fold decrease in the K m of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI,CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH2-terminal six residues (2PLFKRR7) contain a functional nuclear localization signal. We have also demonstrated thatCeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca2+-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved inC. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca2+-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

Phenotypic variations between affected siblings with ataxia-telangiectasia: ataxia-telangiectasia in Japan.

A nationwide survey was conducted for identifying ataxia-telangiectasia (AT) patients in Japan. Eighty-nine patients were diagnosed between 1971 and 2006. Detailed clinical and laboratory data of 64 patients including affected siblings were collected. Analyses focused on malignancy, therapy-related toxicity, infection, and hematological/immunological parameters. The phenotypic variability of AT was assessed by comparing 26 affected siblings from 13 families. Malignancy developed in 22% of the cases and was associated with a high rate of severe therapy-related complications: chemotherapy-related cardiac toxicity in 2 children, and severe hemorrhagic cystitis requiring surgery in 2 patients. The frequency of serious viral infections correlated with the T cell count. Hypogammaglobulinemia with hyper-IgM (HIGM) was recorded in 5 patients, and 3 patients developed panhypogammaglobulinemia. Differences in immunological parameters were noted in siblings. Four patients showed an HIGM phenotype, in contrast to their siblings with normal IgG and IgM levels. The patients with HIGM phenotype showed reduced levels of TRECs and CD27+CD20+ memory B cells. The findings suggest that hitherto unidentified modifier genes or exogenous environmental factors can influence the overall immune responses. Our data along with future prospective study will lead to better understanding of the hematological/immunological phenotypes and to better care of the patients.

Skin Barrier Dysfunction and Low Antimicrobial Peptide Expression in Cutaneous T-cell Lymphoma

Purpose: Atopic dermatitis is characterized by decreased expression of filaggrin and loricrin. Patients with atopic dermatitis often suffer from skin infections, which are also frequently seen in patients with cutaneous T-cell lymphoma (CTCL). In this study, we aimed to investigate the skin barrier in CTCL. Experimental Design: We assessed skin moisture and transepidermal water loss (TEWL) in patients with CTCL. We next examined mRNA expression levels of filaggrin, loricrin, and antimicrobial peptides (AMP) in skin samples of CTCL, using skin from healthy volunteers and patients with atopic dermatitis or psoriasis as controls. Immunostainings for filaggrin, loricrin, and S100 proteins were also performed. Results: Lower levels of skin moisture accompanied by higher levels of TEWL were seen in lesional skin of CTCL than in normal skin. CTCL lesional skin contained lower levels of filaggrin and loricrin mRNA than normal skin, which was also true with atopic dermatitis and psoriatic skin. mRNA expression levels of filaggrin in CTCL skin negatively correlated with disease severity markers. Expression levels of AMPs in lesional skin of CTCL and atopic dermatitis were significantly lower than in psoriatic skin. Immunohistochemistry confirmed decreased expression of filaggrin and loricrin in CTCL, atopic dermatitis, and psoriatic skin and enhanced expression of S100 proteins in psoriatic skin. Conclusions: Our results show that there is barrier dysfunction in CTCL skin, similar to what is seen with atopic dermatitis skin. In addition, low AMP expression in CTCL skin was documented when compared with psoriatic skin, which may explain frequent infections that can occur in patients with CTCL. Clin Cancer Res; 20(16); 4339–48. ©2014 AACR.

Association between genetic variation in the gene for death-associated protein-3 (DAP3) and adult asthma.

AbstractLung epithelium plays a central role in modulation of the lung inflammatory response, and lung repair and airway epithelial cells are targets in asthma and viral infection. Activated T lymphocytes release cytokines such as interferon-gamma (IFN-γ) that induce apoptosis, or programmed cell death, of damaged epithelial cells. Death-associated protein-3 (DAP3) is involved in mediating IFN-γ-induced cell death. To assess the possible involvement of genetic variants of DAP3 with asthma, we searched for single-nucleotide polymorphisms (SNPs) in the gene and conducted a case-control study with 1,341 subjects. We found a strong association between bronchial asthma (BA) in adults (P=0.0051, odds ratio=1.87, 95% CI=1.20-2.92), whereas no association was found with childhood asthma. The tendency was more prominent in patients with higher serum total immunoglobulin E (IgE) (>250 IU/ml) (P=0.00061, odds ratio=2.40, 95% CI=1.44-4.00). DAP3 was expressed in normal bronchial epithelial cells, and the expression was induced by IFN-γ. These results indicated that specific variants of the DAP3 gene might be associated with the mechanisms responsible for adult BA and contribute to airway inflammation and remodeling.