Tomonori Ishii

CD26-mediated signaling for T cell activation occurs in lipid rafts through its association with CD45RO

CD26 is a T cell activation antigen that contains dipeptidyl peptidase IV activity and is known to bind adenosine deaminase. The mechanism by which CD26 costimulation potentiates T cell receptor-mediated T cell activation, leading to subsequent exertion of T cell effector function, is still not clearly defined. In this article, we demonstrate that CD26 localizes into lipid rafts, and targeting of CD26 to rafts is necessary for signaling events through CD26. Importantly, aggregation of CD26 by anti-CD26 mAb crosslinking also causes coaggregation of CD45 into rafts. Moreover, we show that CD26 directly binds to the cytoplasmic domain of CD45. Our results therefore indicate a mechanism whereby CD26 engagement promotes aggregation of lipid rafts and facilitates colocalization of CD45 to T cell receptor signaling molecules p56Lck, ZAP-70, and TCRζ, thereby enhancing protein tyrosine phosphorylation of various signaling molecules and subsequent interleukin-2 production.

A Genome-Wide Association Study Identified AFF1 as a Susceptibility Locus for Systemic Lupus Eyrthematosus in Japanese

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Although recent genome-wide association studies (GWAS) have contributed to discovery of SLE susceptibility genes, few studies has been performed in Asian populations. Here, we report a GWAS for SLE examining 891 SLE cases and 3,384 controls and multi-stage replication studies examining 1,387 SLE cases and 28,564 controls in Japanese subjects. Considering that expression quantitative trait loci (eQTLs) have been implicated in genetic risks for autoimmune diseases, we integrated an eQTL study into the results of the GWAS. We observed enrichments of cis-eQTL positive loci among the known SLE susceptibility loci (30.8%) compared to the genome-wide SNPs (6.9%). In addition, we identified a novel association of a variant in the AF4/FMR2 family, member 1 (AFF1) gene at 4q21 with SLE susceptibility (rs340630; P = 8.3×10−9, odds ratio = 1.21). The risk A allele of rs340630 demonstrated a cis-eQTL effect on the AFF1 transcript with enhanced expression levels (P<0.05). As AFF1 transcripts were prominently expressed in CD4+ and CD19+ peripheral blood lymphocytes, up-regulation of AFF1 may cause the abnormality in these lymphocytes, leading to disease onset.

Soluble CD26/Dipeptidyl Peptidase IV Induces T Cell Proliferation Through CD86 Up-Regulation on APCs

CD26 is a T cell costimulatory molecule with dipeptidyl peptidase IV enzyme activity in its extracellular region. We have previously reported that the addition of soluble CD26 (sCD26) resulted in enhanced proliferation of peripheral blood T lymphocytes induced by the recall Ag, tetanus toxoid (TT). However, the mechanism involved in this immune enhancement has not yet been elucidated. In this paper, we demonstrate that the enhancing effect of sCD26 on TT-induced T cell proliferation occurred in the early stages of immune response. The cells directly affected by exogenously added sCD26 are the CD14-positive monocytes in the peripheral blood. Mannose-6 phosphate interfered with the uptake of sCD26 into monocytes, suggesting that mannose-6 phosphate/insulin-like growth factor II receptor plays a role in the transportation of sCD26 into monocytes. When sCD26 was added after Ag presentation had taken place, enhancement in TT-induced T cell proliferation was not observed. In addition, enhancement of TT-mediated T cell proliferation by sCD26 does not result from trimming of the MHC-bound peptide on the surface of monocytes. Importantly, we also showed that exogenously added sCD26 up-regulated the expression of the costimulatory molecule CD86 on monocytes through its dipeptidyl peptidase IV activity, and that this increased expression of CD86 was observed at both protein and mRNA level. Therefore, our findings suggest that sCD26 enhances T cell immune response to recall Ag via its direct effect on APCs.

Rheumatoid arthritis after human parvovirus B19 infection

Polyarthropathy associated with human parvovirus B19 (B19) is usually transient, but often resembles rheumatoid arthritis (RA).1-3 However, B19 DNA screened by polymerase chain reaction (PCR) may be positive not only in autoimmune diseases, but also in non-arthropathy.4 To study whether or not RA occurs after acute B19 infection, we conducted a prospective study of patients with acute onset polyarthritis. Sixty seven cases showing acute inflammatory polyarthritis from January 1990 to June 1990 were examined and followed up for six years. Among them, 12 (9 female and 3 male) exhibited IgM anti-B19 antibodies.5 PCR, followed by a Southern blot analysis6 also showed the presence of B19 DNA in all 12 cases with IgM anti-B19 antibodies, but did not in the remainder. Initially, rheumatoid factor (RF) was negative in all B19 positive cases except one, but became positive in four, two to four months after the infection (table 1). Among RF …

Human Parvovirus B19 Transgenic Mice Become Susceptible to Polyarthritis

Human parvovirus B19 (B19) often causes acute polyarthritis in adults. In this paper, we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructual protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type II collagen (CII)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-α increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CII increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2b haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CII-induced arthritis resembling RA.

An association analysis of HLA-DRB1 with systemic lupus erythematosus and rheumatoid arthritis in a Japanese population: effects of *09:01 allele on disease phenotypes.

Objective. To re-evaluate the roles of HLA-DRB1 alleles in susceptibility to SLE and RA and their effects on autoantibody status in large-scale Japanese cohorts. Methods. A total of 656 SLE, 2410 RA and 911 control subjects, who were all Japanese, were genotyped for HLA-DRB1 alleles using sequence-specific oligonucleotide probes. The association of alleles with disease susceptibility was tested by logistic regression analysis and by the relative predispositional effect method. The association with autoantibody status was examined by the standard χ(2) test. Results. HLA-DRB1*15:01, *09:01, *08:02 and *04:01 were significantly associated with SLE susceptibility, while shared epitope (SE) alleles and DRB1*09:01 were associated with RA susceptibility. The compound heterozygote of DRB1*09:01/*15:01 conferred an increased risk for SLE compared with the homozygotes for DRB1*09:01 and *15:01 and was associated with earlier onset of disease, whereas the compound effect of DRB1-SE/*09:01 was not clear in RA. DRB1*09:01 was significantly associated with the appearance of anti-Sm antibody in SLE as well as ACPA in RA, while protectively associated with anti-dsDNA antibody in SLE. No significant interaction was observed between DRB1*09:01 and smoking status for the appearance of ACPA, unlike that observed in SE alleles in RA. Conclusion. We identified HLA-DRB1 alleles associated with SLE and RA in a Japanese population and demonstrated a shared susceptibility of DRB1*09:01 between the diseases as well as its effect on autoantibody production.

G1/S cell cycle arrest provoked in human T cells by antibody to CD26.

CD26 is T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity located in its extracellular region. The expression of CD26 is enhanced after activation of T cells, while it is preferentially expressed on a subset of CD4+ memory T cells in the resting state. In this paper, we demonstrate that binding of the soluble anti‐CD26 monoclonal antibody (mAb) 1F7 inhibits human T‐cell growth and proliferation in both CD26‐transfected Jurkat T‐cell lines and human T‐cell clones by inducing G1/S arrest, which is associated with enhancement of p21Cip1 expression. This effect depends on the DPPIV enzyme activity of the CD26 molecule. Moreover, we show that expression of p21Cip1 after treatment with the anti‐CD26 mAb 1F7 appears to be induced through activation of extracellular signal‐regulated kinase (ERK) pathway. These data thus suggest that anti‐CD26 treatment may have potential use in the clinical setting involving activated T cell dysregulation, including autoimmune disorders and graft‐vs.‐host disease.

Efficacy and safety of tocilizumab in patients with refractory Takayasu arteritis: results from a randomised, double-blind, placebo-controlled, phase 3 trial in Japan (the TAKT study)

Objective To investigate the efficacy and safety of the interleukin-6 receptor antibody tocilizumab in patients with Takayasu arteritis (TAK). Methods Patients with TAK who had relapsed within the previous 12 weeks were induced into remission with oral glucocorticoid therapy. In this double-blind, placebo-controlled trial, patients were randomly assigned 1:1 to receive weekly tocilizumab 162 mg or placebo subcutaneously, and oral glucocorticoids were tapered 10 %/week from week 4 to a minimum of 0.1 mg/kg/day until 19 patients relapsed. The primary endpoint was time to relapse of TAK, defined as ≥2 of the following: objective systemic symptoms, subjective systemic symptoms, elevated inflammation markers, vascular signs and symptoms or ischaemic symptoms. Results The intent-to-treat and safety populations included 18 tocilizumab-treated and 18 placebo-treated patients. The per-protocol set (PPS) included 16 tocilizumab-treated and 17 placebo-treated patients. HRs for time to relapse of TAK were 0.41 (95.41% CI 0.15 to 1.10; p=0.0596) in the intent-to-treat population (primary endpoint) based on relapse in eight tocilizumab-treated and 11 placebo-treated patients and 0.34 (95.41% CI 0.11 to 1.00; p=0.0345) in the PPS. The secondary endpoints, time to relapse assessed by Kerr’s definition and clinical symptoms only, were consistent with the primary endpoint. Serious adverse events were reported in one tocilizumab-treated and two placebo-treated patients. There were no serious infections and no deaths. Conclusion Although the primary endpoint was not met, the results suggest favour for tocilizumab over placebo for time to relapse of TAK without new safety concerns. Further investigation is warranted to confirm the efficacy of tocilizumab in patients with refractory TAK. Trial registration number JapicCTI-142616.

Fratricide of natural killer cells dressed with tumor-derived NKG2D ligand

The natural killer group 2 membrane D (NKG2D) activating receptor plays crucial roles not only in host defense against tumors and viral infections, but also in autoimmune diseases. After NKG2D-mediated activation, Natural killer (NK) cells must be regulated to avoid potentially harmful reactivity. However, the negative regulation of these activated NK cells is poorly understood. Here, we reveal that the engagement of NKG2D by its ligand elicits not only target cell lysis, but also NK cell fratricide. Conventional mouse NK cells underwent cell death when cocultured with RMA cells expressing the NKG2D ligand retinoic acid early-inducible protein 1 (Rae-1), but not with RMA cells lacking MHC class I. NK cells from mice deficient for DAP10 and DAP12 or perforin did not undergo death, highlighting the importance of the NKG2D pathway for NK cell death. However, NKG2D does not transmit direct death signals in NK cells. Rather, the interaction between NKG2D and Rae-1 allowed NK cells to acquire tumor-derived Rae-1 by a membrane transfer process known as ”trogocytosis,” which was associated with clathrin-dependent NKG2D endocytosis. NK cells dressed with Rae-1 were lysed by neighboring NK cells through the NKG2D-induced perforin pathway in vitro and in vivo. These results provide the unique NKG2D function in negative regulation of activated NK cells.

Anti-K-ras ribozyme induces growth inhibition and increased chemosensitivity in human colon cancer cells

Colon cancer is one of the carcinomas that is resistant to a variety of therapies. To develop a new therapy by regulating the activated K-ras gene in colon cancers, we prepared synthetic DNA encoding the ribozyme (catalytic RNA), and inserted it into an expression vector (LNCX) originated from a retrovirus. Transfection of the vector into SW620 human colon cancer cells brought about significant suppression of K-ras mRNA synthesis and inhibition of the cell growth. Studies in athymic mice, in which K-ras ribozyme-introduced SW620 cells were transplanted, also revealed a marked reduction of tumor growth in vivo. Furthermore, the ribozyme-introduced tumors became more sensitive to treatment with anti-cancer drugs such as cisplatin, adriamycin, 5-fluorouracil, vincristine, and etoposide. These data suggest that the possible efficacy of anti-K-ras ribozyme increases the chemosensitivity of human colon cancers as well as the inhibitory effect on the growth of human colon cancers.