Yohko Nakagawa

Predominant Role of T Cell Receptor (TCR)-α Chain in Forming Preimmune TCR Repertoire Revealed by Clonal TCR Reconstitution System

The CDR3 regions of T cell receptor (TCR)-α and -β chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-α and -β to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-α or -β chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-β or -α chains. Surprisingly, one-third of such TCR-β containing random CDR3β from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-α. A similar dominant function of TCR-α in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus–specific TCR. Subsequently, we generated TCR-α and/or -β transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-α Tg generated CTLs with heterogeneous TCR-β, whereas TCR-β Tg-induced CTLs bearing a single TCR-α. These findings of Ag recognition with minimum involvement of CDR3β expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-α chain in determining the preimmune repertoire of Ag-specific TCR.

Identification of an antigenic epitope in Helicobacter pylori urease that induces neutralizing antibody production

ABSTRACT We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pyloriinfection.

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes.

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.

Rapid Induction of Apoptosis in CD8+ HIV-1 Envelope-Specific Murine CTLs by Short Exposure to Antigenic Peptide

During primary viral infection, in vivo exposure to high doses of virus causes a loss of Ag-specific CD8+ T cells. This phenomenon, termed clonal exhaustion, and other mechanisms by which CTLs are deleted are poorly understood. Here we show evidence for a novel form of cell death in which recently stimulated CD8+ HIV-1 envelope gp160-specific murine CTLs become apoptotic in vitro after brief exposure to free antigenic peptide (P18-I10). Peak apoptosis occurred within 3 h of treatment with peptide, and the level of apoptosis was dependent on both the time after initial stimulation with target cells and the number of targets. Using T cell-specific H-2Dd/P18-I10 tetramers, we observed that the apoptosis was induced by such complexes. Induction of apoptosis was blocked by cyclosporin A, a caspase 3 inhibitor, and a mitogen-activated protein kinase inhibitor, but not by Abs to either Fas ligand or to TNF-α. Thus, these observations suggest the existence of a Fas- or TNF-α-independent pathway initiated by TCR signaling that is involved in the rapid induction of CTL apoptosis. Such a pathway may prove important in the mechanism by which virus-specific CTLs are deleted in the presence of high viral burdens.

Purification of antigenic peptide from murine hepatoma cells recognized by Class-I major histocompatibility complex molecule-restricted cytotoxic T-lymphocytes induced with B7-1-gene-transfected hepatoma cells

BACKGROUND/AIM It has been reported that expression of costimulatory molecules, such as B7, on tumors is essential for priming tumor-specific cytotoxic T-lymphocytes (CTLs). Here, we have attempted to induce murine hepatoma-specific CTLs by immunizing with the B7-1-gene-expressing hepatoma cells, and to identify the epitope(s) presented on the hepatoma cells. METHODS The B7-1-gene encoding plasmid was transferred into the murine hepatoma cell line, Hepa1-6. Syngeneic C57BL/6 mice were immunized with the B7-1-transfected cells via various routes to prime CTLs. The mild acid elution method was used to isolate antigenic fractions from the class-I major histocompatibility complex (MHC) molecules on the Hepa1-6 cells. Cytotoxicity was measured by standard 51Cr-releasing assay. The effect of the CTLs on hepatoma growth was evaluated in hepatoma-bearing SCID mice to which the cells were preadministered. RESULTS A clone, termed L1, highly expressing the B7-1-gene, has been established. Killer cells generated from mice immunized intraperitoneally with L1 cells eliminated both L1 and Hepa1-6 cells, and also another syngeneic hepatoma cell line, Hepa1-clc7. The killer cells were CD8+ and the class-I MHC molecule-restricted CTLs which might recognize hepatoma-specific antigenic peptide(s) in association with the D(b)class-I MHC molecules. A functional peptide fraction was obtained from eluted fluid of the Hepa1-6 cells. In addition, intravenous preadministration of the CTLs inhibited the hepatoma growth in SCID mice. CONCLUSIONS The hepatoma epitope-specific CTLs which suppressed hepatoma growth in vivo could be generated with the B7-1-gene-transfected hepatoma cells. These results will be useful in establishing immunotherapy against hepatocellular carcinoma.

Effects of extracellular pH and hypoxia on the function and development of antigen-specific cytotoxic T lymphocytes.

The major effector cells for cellular adaptive immunity are CD8(+) cytotoxic T lymphocytes (CTLs), which can recognize and kill virus-infected cells and tumor cells. Although CTLs exhibit strong cytolytic activity against target cells in vitro, a number of studies have demonstrated that their function is often impaired within tumors. Nevertheless, CTLs can regain their cytotoxic ability after escaping from the tumor environment, suggesting that the milieu created by tumors may affect the function of CTLs. As for the tumor environment, the patho-physiological situation present in vivo has been shown to differ from in vitro experimental conditions. In particular, low pH and hypoxia are the most important microenvironmental factors within growing tumors. In the present study, to determine the effect of these factors on CTL function in vivo, we examined the cytolytic activity of CTLs against their targets using murine CTL lines and the induction of these cells from memory cells under low pH or hypoxic conditions using antigen-primed spleen cells. The results indicated that both cytotoxic activity and the induction of functional CTLs were markedly inhibited under low pH. In contrast, in hypoxic conditions, although cytotoxic activity was almost unchanged, the induction of CTLs in vitro showed a slight enhancement, which was completely abrogated in low pH conditions. Therefore, antigen-specific CTL functions may be more vulnerable to low pH than to the oxygen concentration in vivo. The findings shown here provide new therapeutic approaches for controlling tumor growth by retaining CTL cytotoxicity through the maintenance of higher pH conditions.

Suppression of an Already Established Tumor Growing through Activated Mucosal CTLs Induced by Oral Administration of Tumor Antigen with Cholera Toxin

Priming of CTLs at mucosal sites, where various tumors are originated, seems critical for controlling tumors. In the present study, the effect of the oral administration of OVA plus adjuvant cholera toxin (CT) on the induction of Ag-specific mucosal CTLs as well as their effect on tumor regression was investigated. Although OVA-specific TCRs expressing lymphocytes requiring in vitro restimulation to gain specific cytotoxicity could be detected by OVA peptide-bearing tetramers in both freshly isolated intraepithelial lymphocytes and spleen cells when OVA was orally administered CT, those showing direct cytotoxic activity without requiring in vitro restimulation were dominantly observed in intraepithelial lymphocytes. The magnitude of such direct cytotoxicity at mucosal sites was drastically enhanced after the second oral administration of OVA with intact whole CT but not with its subcomponent, an A subunit (CTA) or a B subunit (CTB). When OVA plus CT were orally administrated to C57BL/6 mice bearing OVA-expressing syngeneic tumor cells, E.G7-OVA, in either gastric tissue or the dermis, tumor growth was significantly suppressed after the second oral treatment; however, s.c. or i.p. injection of OVA plus CT did not show any remarkable suppression. Those mucosal OVA-specific CTLs having direct cytotoxicity expressed CD8αβ but not CD8αα, suggesting that they originated from thymus-educated cells. Moreover, the infiltration of such OVA-specific CD8+ CTLs was observed in suppressed tumor tissues. These results indicate that the growth of ongoing tumor cells can be suppressed by activated CD8αβ CTLs with tumor-specific cytotoxicity via an orally administered tumor Ag with a suitable mucosal adjuvant.

Production of Autoantibodies by Murine B-1a Cells Stimulated with Helicobacter pylori Urease through Toll-Like Receptor 2 Signaling

ABSTRACT Helicobacter pylori infection is associated with several autoimmune diseases, in which autoantibody-producing B cells must be activated. Among these B cells, CD5-positive B-1a cells from BALB/c mice were confirmed to secrete autoantibodies when cocultured with purified H. pylori urease in the absence of T cells. To determine the mechanisms for autoantibody production, CD5-positive B-1a cells were sorted from murine spleen cells and stimulated with either purified H. pylori urease or H. pylori coated onto plates (referred to hereafter as plate-coated H. pylori), and autoantibody production was measured by enzyme-linked immunosorbent assay (ELISA). Complete urease was not secreted from H. pylori but was visually expressed over the bacterium-like endotoxin. Urease-positive plated-coated H. pylori stimulated B-1a cells to produce autoantibodies, although urease-deficient isotype-matched H. pylori did not. Autoantibody secretion by B-1a cells was inhibited when bacteria were pretreated with anti-H. pylori urease-specific antibody having neutralizing ability against urease enzymatic activity but not with anti-H. pylori urease-specific antibody without neutralizing capacity. The B-1a cells externally express various Toll-like receptors (TLRs): TLR1, TLR2, TLR4, and TLR6. Among the TLRs, blocking of TLR2 on B-1a cells with a specific monoclonal antibody (MAb), T2.5, inhibited autoantibody secretion when B-1a cells were stimulated with plate-coated H. pylori or H. pylori urease. Moreover, B-1a cells from TLR2-knockout mice did not produce those autoantibodies. The present study provides evidence that functional urease expressed on the surface of H. pylori will directly stimulate B-1a cells via innate TLR2 to produce various autoantibodies and may induce autoimmune disorders.

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances.

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8+ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both Db class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4–CD8– and CD8+ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4−CD8−, but not CD8+ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.

Effects of Dendritic Cell Subset Manipulation on Airway Allergy in a Mouse Model.

Background: Two major distinct subsets of dendritic cells (DCs) are arranged to regulate immune responses: DEC-205+ DCs drive Th1 polarization and 33D1+ DCs establish Th2 dominancy. Th1 polarization can be achieved either by depletion of 33D1+ DCs with a 33D1-specific monoclonal antibody (mAb) or by activation of DEC-205+ DCs via intraperitoneal injection of α-galactosylceramide (α-GalCer). We studied the effect of 33D1+ DC depletion or DEC-205+ DC activation in vivo using an established mouse model of allergic rhinitis (AR). Methods: Mice were injected intraperitoneally with OVA plus alum and challenged 4 times with daily intranasal administration of OVA. Immediately after the last challenge, allergic symptoms such as sneezing and nasal rubbing as well as the number of cells in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NALF) were counted. The levels of serum OVA-specific IgG1, IgG2a, and IgE were also determined by ELISA. Results: The allergic symptom scores were significantly decreased in 33D1+ DC-depleted or DEC-205+ DC-activated AR mice. The levels of OVA-specific IgG1, IgG2a, and IgE, and the number of NALF cells, but not BALF cells, were reduced in 33D1+ DC-depleted but not in DEC-205+ DC-activated AR mice. Moreover, the activated DEC-205+ DCs suppressed histamine release from IgE-sensitized mast cells, probably through IL-12 secretion. Conclusions: The manipulation of innate DC subsets may provide a new therapeutic strategy for controlling various allergic diseases by reducing histamine release from IgE-sensitized mast cells by driving the immune response towards Th1 dominancy via activation of DEC-205+ DCs in vivo.