Takaji Matsutani

Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice

Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is a chronic systemic inflammatory disease of unknown aetiology that primarily affects the synovial membranes of multiple joints. Although CD4+ T cells seem to be the prime mediators of RA, it remains unclear how arthritogenic CD4+ T cells are generated and activated. Given that highly self-reactive T-cell clones are deleted during normal T-cell development in the thymus, abnormality in T-cell selection has been suspected as one cause of autoimmune disease. Here we show that a spontaneous point mutation of the gene encoding an SH2 domain of ZAP-70, a key signal transduction molecule in T cells, causes chronic autoimmune arthritis in mice that resembles human RA in many aspects. Altered signal transduction from T-cell antigen receptor through the aberrant ZAP-70 changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells. Thymic production of arthritogenic T cells due to a genetically determined selection shift of the T-cell repertoire towards high self-reactivity might also be crucial to the development of disease in a subset of patients with RA.

Polyclonal expansion of TCRBV2- and TCRBV6-bearing T cells in patients with Kawasaki disease

We examined T‐cell receptor (TCR) usage, cytokine production and antibody responses to superantigens in patients with Kawasaki disease (KD) to facilitate a better understanding of the immunopathogenesis of KD. The mean percentage of VB2‐ or VB6.5‐bearing T cells in peripheral blood mononuclear cells (PBMC) of patients with acute‐phase KD was significantly higher than that of patients in the convalescent phase of KD or in healthy donors. Expansion of VB2‐ or VB6.5‐bearing T cells was polyclonal because DNA sequences in the complementarity determining region 3 of VB2‐ and VB6.5‐positive cDNA clones were all different from each other. The plasma levels of interleukin (IL)‐1β, IL‐2, IL‐6, IL‐8, IL‐10, interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and granulocyte colony‐stimulating factor (G‐CSF) were elevated in the acute phase of KD. We previously reported that streptococcal pyrogenic exotoxin C (SPEC) was a potent stimulator of VB2‐ and VB6.5‐positive T cells and, furthermore, serum levels of anti‐SPEC antibodies were significantly higher in patients with acute and convalescent KD than in age‐matched controls. The results of the present study, together with those of our previous report, suggest that SPEC induces activation and polyclonal expansion of VB2‐ and VB6.5‐positive T cells, and that SPEC‐induced activation of T cells may lead to the pathogenesis of KD.

Relation of streptococcal pyrogenic exotoxin C as a causative superantigen for Kawasaki disease

We previously reported that the frequency of TCRBV2 and TCRBV6S5-bearing T-cells was high in patients in the acute phase of Kawasaki disease (KD) and that streptococcal pyrogenic exotoxin C (SPE-C) was a potent stimulator of these TCRBV-bearing T-cells. To further elucidate the pathogenesis of KD, we examined the T-cell receptor (TCR) repertoire, human leukocyte antigen (HLA)-DRB1 genotype, and antibody responses to recombinant(r) SPE-C in patients with KD. We also performed in vitro stimulation with rSPE-A and rSPE-C of peripheral blood mononuclear cells from healthy donors and characterized the reacting T-cells. The percentage of T-cells bearing TCRBV2 and TCRBV6S5 was high in patients in the acute stage of KD. rSPE-C stimulation of PBMC from healthy donors induced expansion of TCRBV2 and TCRBV6S5-bearing T-cells. Furthermore, serum levels of anti-SPEC antibodies, which did not display antimitogenic activity, were higher in patients with acute KD than in age-matched controls. The frequencies of the DRB1*04051, 0406, and 0901 were high, whereas that of the DRB1*1101 was low among patients with KD as compared with the healthy adults.

Serotonin-like immunoreactivity in the central nervous system and gonad of the scallop, Patinopecten yessoensis

SummaryThe localization of neurons containing serotonin in the central nervous system and the gonad of the scallop, Patinopecten yessoensis, was examined immunohistochemically. In the central nervous system a large number of immunoreactive perikarya were observed in the following regions: a part of the anterior lobe of the cerebral ganglion; the posterior lobe of the cerebral ganglion; the pedal ganglion; and the accessory ganglion. No immunoreactive perikarya were found in the visceral ganglion. Numerous immunoreactive fibers were revealed in the neuropil of all central ganglia. In the gonadal region immunoreactive fibers were distributed around the gonoduct and along the germinal epithelium.

Restricted usage of T-cell receptor α-chain variable region (TCRAV) and T-cell receptor β-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation

We analysed T‐cell receptor α‐chain variable region (TCRAV) and T‐cell receptor β‐chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo‐BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6–7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1‐4 (26%, 11 of 41 recipients), VA3‐1 (32%) and VB24‐1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity‐determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0·05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T‐cell regeneration via a thymic‐dependent pathway in children.

Determination of T-cell receptors of clonal CD8-positive T-cells in myelodysplastic syndrome with erythroid hypoplasia

We determined T-cell receptor alpha-chain variable (TCRAV) and T-cell receptor beta-chain variable (TCRBV) region repertoires in peripheral bloods from patients with myelodysplastic syndrome (MDS) with erythroid hypoplasia. T-cells bearing TCR ADV14S1/BV5S2, AV21S1/BV21S4, and AV2S2/BV7S2 segments were markedly increased in three of four MDS patients, respectively. In addition, there was a positive relationship between the increase in the number of CD8-positive T-cells and the expression levels of these TCR transcripts. These findings suggest that CD8-positive T-cells monoclonally or oligoclonally expanded in the peripheral blood. We also determined the nucleotide and amino acid sequences of the complementarity-determining region 3 (CDR3) of TCR alpha- and beta-chains of the expanded T-cells. Unique sequences were detected in a high percentage of the respective CDR3 clones. The gene segment of the variable and joining regions, however, varied among the patients. The deduced amino acid sequences of CDR3 were heterogeneous among the patients, and there was no common motif. These results indicate there is monoclonal or oligoclonal proliferation of CD8-positive T-cells in MDS patients with erythroid hypoplasia, and suggest that these proliferating T-cells are responsible for the pathogenesis of the MDS entity.

Quantitative analysis of the usage of human T cell receptor α and β chain variable regions by reverse dot blot hybridization

Abstract We previously developed an adaptor ligation-mediated PCR method to amplify the T cell receptor (TCR) cDNA pools. In the present study we applied reverse dot blot hybridization to PCR-amplified specimens for quantitative analysis of the usage of TCR α and β chain variable (V) region. 44 VA sequence-specific oligonucleotide probes (SSOPs) and 38 VB SSOPs were synthesized corresponding to unique sequences of VA and VB subfamilies. Peripheral blood lymphocytes of ten healthy donors and five T cell clones established from bone marrow cells were examined for VA and VB usage using this method. The results were consistent with those obtained by a colony hybridization method and those by immunofluorescence staining using monoclonal antibodies to VA and VB. Thus, reverse dot blot hybridization for TCR Vα and Vβ is a new, easy and dependable technique useful for analysis of VA and VB usage by human T cells.

Spontaneous scratching behaviour in DS-Nh mice as a possible model for pruritus in atopic dermatitis.

Itching is one of the major clinical symptoms in atopic dermatitis (AD) and complicates the management of this pathological condition. An animal model of AD‐like pruritus would contribute to a better understanding of AD and could lead to the development of safe and effective antipruritic agents. DS non‐hair (DS‐Nh) mice raised under conventional conditions spontaneously develop pruritus, which is associated with a dermatitis similar to human AD. There is a significant positive correlation between disease severity and the period of scratching behaviour in DS‐Nh mice. In the present study, we found that levels of histamine and nerve growth factor (NGF) in serum and/or skin tissue were higher in DS‐Nh mice with AD‐like dermatitis than in age‐matched mice without dermatitis. The histopathological data indicated that nerve fibres extend into and mast cells infiltrate the surrounding area of the skin lesion. NGF production by XB‐2 cells, which was derived from mouse keratinocytes, was enhanced by histamine via the H1 receptor. We also found that prolonged treatment with an H1‐antagonist was effective against pruritus through depression of the production of NGF, which is thought to be generated by keratinocytes. We conclude that DS‐Nh mice can serve as a suitable model for gaining a better understanding of pruritus in AD, and that prolonged treatment with an H1‐antagonist may be beneficial in patients with AD‐associated pruritus.

Inflammation-induced IgA + cells dismantle anti-liver cancer immunity

The role of adaptive immunity in early cancer development is controversial. Here we show that chronic inflammation and fibrosis in humans and mice with non-alcoholic fatty liver disease is accompanied by accumulation of liver-resident immunoglobulin-A-producing (IgA+) cells. These cells also express programmed death ligand 1 (PD-L1) and interleukin-10, and directly suppress liver cytotoxic CD8+ T lymphocytes, which prevent emergence of hepatocellular carcinoma and express a limited repertoire of T-cell receptors against tumour-associated antigens. Whereas CD8+ T-cell ablation accelerates hepatocellular carcinoma, genetic or pharmacological interference with IgA+ cell generation attenuates liver carcinogenesis and induces cytotoxic T-lymphocyte-mediated regression of established hepatocellular carcinoma. These findings establish the importance of inflammation-induced suppression of cytotoxic CD8+ T-lymphocyte activation as a tumour-promoting mechanism.

DS-Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C.

DS–Nh mice raised under conventional conditions spontaneously develop dermatitis similar to human atopic dermatitis (AD), which is associated with staphylococcal infection. In the present study, we show that Staphylococcus aureus producing staphylococcus exotoxin C (SEC) was recovered from the culture of the skin lesions of DS–Nh mice with AD‐like dermatitis and that the serum levels of anti‐SEC antibodies from these mice were elevated. We describe here how to promote experimental AD by epicutaneous injection with SEC‐producing S. aureus to DS–Nh mice. In order to assess the role of SEC in the pathogenesis of AD, the mitogenic activity, TCRBV repertoire analysis and the production of IL‐4 and IFN‐γ from spleen mononuclear cells (MNC) from DS–Nh stimulated by SEC were compared with those due to SEA, SEB and TSST. The weakest was the mitogenic activity of SEC, and higher IL‐4 responses and lower IFN‐γ responses to SEC showed correlation with TCRBV8S2‐positive T cells, which were selectively stimulated by SEC. We also demonstrate that SEC‐producing S. aureus was able to survive in DS–Nh after intradermal injection. These results suggest a possible role for SEC in the pathogenesis of AD through host–S. aureus relationships.