Publications by authors named "Kazutaka Kitaura"

39 Publications

Pharmacological MEK inhibition promotes polyclonal T-cell reconstitution and suppresses xenogeneic GVHD.

Cell Immunol 2021 Sep 13;367:104410. Epub 2021 Jul 13.

Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan.

Rapid immune reconstitution without developing graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation. Here, we analyzed the effects of pharmacological MEK inhibition on human polyclonal T-cell reconstitution in a humanized mouse GVHD model utilizing deep sequencing-based T-cell receptor (TCR) repertoire analysis. GVHD mice exhibited a skewed TCR repertoire with a common clone within target organs. The MEK inhibitor trametinib ameliorated GVHD and enabled engraftment of diverse T-cell clones. Furthermore, trametinib also ameliorated GVHD sparing diverse T cell repertoire, even when it was given from day 15 through 28. Although tacrolimus also reduced development of GVHD, it disturbed diverse T cell reconstitution and resulted in skewed TCR repertoire. Thus, trametinib not only suppresses GVHD-inducing T cells but also promotes human T cell reconstitution in vivo, providing a novel rationale for translational studies targeting human GVHD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellimm.2021.104410DOI Listing
September 2021

T-cell receptor repertoire of cytomegalovirus-specific cytotoxic T-cells after allogeneic stem cell transplantation.

Sci Rep 2020 12 17;10(1):22218. Epub 2020 Dec 17.

Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8677, Japan.

Cytomegalovirus (CMV) infection is a major complication during allogeneic stem cell transplantation (allo-SCT). However, mechanisms of adaptive immunity that drive this remain unclear. To define early immunological responses to CMV after transplantation, we using next-generation sequencing to examine the repertoire of T-cell receptors in CD8/CMV pp65 tetramer cells (CMV-CTLs) in peripheral blood samples obtained from 16 allo-SCT recipients with HLA-A*24:02 at the time of CMV reactivation. In most patients, TCR beta repertoire of CMV-CTLs was highly skewed (median Inverse Simpson's index: 1.595) and, 15 of 16 patients shared at least one TCR-beta clonotype with ≥ 2 patients. The shared TCRs were dominant in 12 patients and, two clonotypes were shared by about half of the patients. Similarity analysis showed that CDR3 sequences of shared TCRs were more similar than unshared TCRs. TCR beta repertoires of CMV-CTLs in 12 patients were also analyzed after 2-4 weeks to characterize the short-term dynamics of TCR repertoires. In ten patients, we observed persistence of prevailing clones. In the other two patients, TCR repertoires became more diverse, major clones declined, and new private clones subsequently emerged. These results provided the substantive clue to understand the immunological behavior against CMV reactivation after allo-SCT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-79363-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747720PMC
December 2020

High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4-28/IGHJ4 gene in primary immune thrombocytopenia.

Sci Rep 2019 06 14;9(1):8645. Epub 2019 Jun 14.

Repertoire Genesis Incorporation, Ibaraki, Japan.

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP)IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4-28 in ITP patients. One hundred eighty-six distinct IGHV4-28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4-28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4-28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4-28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-45264-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570656PMC
June 2019

Characterization of T cell receptors in a novel murine model of nickel-induced intraoral metal contact allergy.

PLoS One 2018 17;13(12):e0209248. Epub 2018 Dec 17.

Department of Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, Sagamihara, Japan.

Nickel is a component of several alloy types that are widely used in our environment, including several dental alloy types that cause intraoral metal contact allergy. However, metal-specific immune responses in the oral mucosa have not been elucidated because a suitable animal model has not been established. In this study, we established a novel murine model of nickel-induced intraoral metal contact allergy and aimed to elucidate the immune response in terms of T-cell receptor repertoire and cytokine profiles in inflamed oral mucosa. The intraoral metal contact allergy model was induced by two sensitizations of nickel plus lipopolysaccharide solution into the postauricular skin followed by a single nickel challenge of the buccal mucosa. Cytokine expression profiles and T-cell phenotypes were determined by quantitative polymerase chain reaction. T cells accumulated in the cervical lymph nodes and inflamed oral mucosa were characterized by analyzing their T-cell receptor α- and β-chain repertoires, and the nucleotide sequences of complementary determining region 3. Significant swelling and pathological features were histologically evident at 1 day after challenge in mice with nickel allergy. At 1 day after the challenge, CD8-positive T cells producing high levels of T helper 1 type cytokines had accumulated in the allergic oral mucosa. At 7 days after the challenge, excessive nickel allergy in the oral mucosa was suppressed by regulatory T cells. Characterization of the T-cell receptor repertoire in nickel allergic mice revealed the presence of natural killer T cells and T cells bearing Trav6-6-Traj57 at 1 day after the challenge. Our murine model of nickel-induced intraoral metal contact allergy showed that natural killer T cells and T cells bearing Trav6-6-Traj57 might be involved in the immune responses of nickel-induced intraoral metal contact allergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209248PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6296741PMC
May 2019

Epstein-Barr virus-related diffuse large B-cell lymphoma in mogamulizumab-treated adult T-cell leukemia with incomplete T-cell reconstitution.

Int J Hematol 2019 Feb 27;109(2):221-227. Epub 2018 Oct 27.

Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan.

Adult T-cell leukemia (ATL) is an aggressive mature T-cell malignancy with a poor prognosis. The anti-C-C motif chemokine receptor 4 (CCR4) antibody mogamulizumab (moga) reduces ATL cells and induces reconstitution of polyclonal T cells; however, ATL cases often remain resistant and moga sometimes causes fatal immunopathology. Epstein-Barr virus (EBV)-related B-cell lymphoma develops in severely immunocompromised subjects, and is particularly associated with impaired T-cell immunity. Here, we report an ATL patient who had received conventional chemotherapy plus moga, and subsequently developed EBV-related diffuse large B-cell lymphoma (DLBCL) of the central nervous system. Next-generation sequencing-based T-cell receptor repertoire analyses identified residual abnormal clones and revealed that reconstitution of polyclonal T cells was incomplete, even after moga treatment. Furthermore, a skin rash that developed after moga treatment was found to contain ATL clones. This case suggests that the limited therapeutic effects of moga and incomplete T-cell reconstitution are associated with severely impaired T-cell immunity and subsequent development of EBV-related DLBCL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12185-018-2552-xDOI Listing
February 2019

Induction of oligoclonal CD8 T cell responses against pulmonary metastatic cancer by a phospholipid-conjugated TLR7 agonist.

Proc Natl Acad Sci U S A 2018 07 2;115(29):E6836-E6844. Epub 2018 Jul 2.

Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093-0695;

Recent advances in cancer immunotherapy have improved patient survival. However, only a minority of patients with pulmonary metastatic disease respond to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T1 breast cancer models, as well as in B16 melanoma, and Lewis lung carcinoma models. In the 4T1 breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist compared with the unconjugated agonist. 1V270 exhibited a wide therapeutic window and minimal off-target receptor binding. The 1V270 therapy inhibited colonization by tumor cells in the lungs in an NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-specific CD8 cell-dependent adaptive immune responses that suppressed late-stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analyses showed that 1V270 therapy induced oligoclonal T cells in the lungs and mediastinal lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy. Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor-specific adaptive immune responses. These results indicate that systemic 1V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers and that TCR repertoire analyses can be used to monitor, and to predict, the response to therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1803281115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055176PMC
July 2018

Three bisabolane-type sesquiterpenes from edible mushroom Pleurotus eryngii.

Fitoterapia 2018 Sep 26;129:108-113. Epub 2018 Jun 26.

Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan.

Three bisabolane-type sesquiterpenes (1-3) were isolated from the fruiting bodies of king trumpet mushrooms (Pleurotus eryngii), together with a known compound (4). All isolated compounds were evaluated for their inhibitory effects on nitric oxide (NO) production. Among these, 2 exhibited a moderate inhibitory effect on NO production with an IC of 90.9 μM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fitote.2018.06.021DOI Listing
September 2018

Next-Generation Immune Repertoire Sequencing as a Clue to Elucidate the Landscape of Immune Modulation by Host-Gut Microbiome Interactions.

Front Immunol 2018 3;9:668. Epub 2018 Apr 3.

Repertoire Genesis Incorporation, Ibaraki, Japan.

The human immune system is a fine network consisted of the innumerable numbers of functional cells that balance the immunity and tolerance against various endogenous and environmental challenges. Although advances in modern immunology have revealed a role of many unique immune cell subsets, technologies that enable us to capture the whole landscape of immune responses against specific antigens have been not available to date. Acquired immunity against various microorganisms including host microbiome is principally founded on T cell and B cell populations, each of which expresses antigen-specific receptors that define a unique clonotype. Over the past several years, high-throughput next-generation sequencing has been developed as a powerful tool to profile T- and B-cell receptor repertoires in a given individual at the single-cell level. Sophisticated immuno-bioinformatic analyses by use of this innovative methodology have been already implemented in clinical development of antibody engineering, vaccine design, and cellular immunotherapy. In this article, we aim to discuss the possible application of high-throughput immune receptor sequencing in the field of nutritional and intestinal immunology. Although there are still unsolved caveats, this emerging technology combined with single-cell transcriptomics/proteomics provides a critical tool to unveil the previously unrecognized principle of host-microbiome immune homeostasis. Accumulation of such knowledge will lead to the development of effective ways for personalized immune modulation through deeper understanding of the mechanisms by which the intestinal environment affects our immune ecosystem.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.00668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891584PMC
June 2019

Quantitative T-cell repertoire analysis of peripheral blood mononuclear cells from lung cancer patients following long-term cancer peptide vaccination.

Cancer Immunol Immunother 2018 06 22;67(6):949-964. Epub 2018 Mar 22.

Department of Chest Surgery, School of Medicine, Fukushima Medical University, Fukushima, 960-1295, Japan.

Therapeutic cancer peptide vaccination is an immunotherapy designed to elicit cytotoxic T-lymphocyte (CTL) responses in patients. A number of therapeutic vaccination trials have been performed, nevertheless there are only a few reports that have analyzed the T-cell receptors (TCRs) expressed on tumor antigen-specific CTLs. Here, we use next-generation sequencing (NGS) to analyze TCRs of vaccine-induced CTL clones and the TCR repertoire of bulk T cells in peripheral blood mononuclear cells (PBMCs) from two lung cancer patients over the course of long-term vaccine therapy. In both patients, vaccination with two epitope peptides derived from cancer/testis antigens (upregulated lung cancer 10 (URLC10) and cell division associated 1 (CDCA1)) induced specific CTLs expressing various TCRs. All URLC10-specific CTL clones tested showed Ca influx, IFN-γ production, and cytotoxicity when co-cultured with URLC10-pulsed tumor cells. Moreover, in CTL clones that were not stained with the URLC10/MHC-multimer, the CD3 ζ chain was not phosphorylated. NGS of the TCR repertoire of bulk PBMCs demonstrated that the frequency of vaccine peptide-specific CTL clones was near the minimum detectable threshold level. These results demonstrate that vaccination induces antigen-specific CTLs expressing various TCRs at different time points in cancer patients, and that some CTL clones are maintained in PBMCs during long-term treatment, including some with TCRs that do not bind peptide/MHC-multimer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00262-018-2152-xDOI Listing
June 2018

Deep sequencing of the T cell receptor visualizes reconstitution of T cell immunity in mogamulizumab-treated adult T cell leukemia.

Oncoimmunology 2018;7(3):e1405204. Epub 2017 Dec 11.

Department of Hematology, Respiratory Medicine and Oncology, Saga University School of Medicine, Saga, Japan.

Although the anti-CCR4 antibody mogamulizumab (moga) shows striking antitumor activity against adult T cell leukemia (ATL), it can also cause fatal immunological pathology such as severe skin rash and graft-versus-host disease, which might be attributed to depletion of CCR4 regulatory T cells. We previously showed that next generation sequencing enables precise analysis of the T cell receptor (TCR) repertoire, and we here used the technique to reveal the immunological dynamics in moga-treated ATL patients. Treatment with moga resulted in remarkable reduction or elimination of clonal cells, and enhanced reconstitution of non-tumor polyclonal CD4 T cells and oligoclonal CD8 T cells. Interestingly, cutaneous T cells infiltrating moga-related skin rashes did not share the same major clones in peripheral blood, which minimizes the possibility of cross-reaction. Thus, deep sequencing of the TCR can reveal the immune reconstitution of moga-treated ATL and provides powerful insights into its mode of action.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/2162402X.2017.1405204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790365PMC
December 2017

Increased diversity with reduced "diversity evenness" of tumor infiltrating T-cells for the successful cancer immunotherapy.

Sci Rep 2018 01 18;8(1):1058. Epub 2018 Jan 18.

Department of Immunotherapeutics, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-Ku, Tokyo, 113-8655, Japan.

To facilitate the optimization of cancer immunotherapy lacking immune-related adverse events, we performed TCR repertoire analysis of tumor-infiltrating CD8 T-cells in B16 melanoma-bearing mice receiving anti-PD-1, anti-CTLA-4, anti-4-1BB, anti-CD4 or a combination of anti-PD-1 and 4-1BB antibodies. Although CD8 T-cells in the tumor were activated and expanded to a greater or lesser extent by these therapies, tumor growth suppression was achieved only by anti-PD-1, anti-PD-1/4-1BB combined, or by anti-CD4 treatment, but not by anti-CTLA-4 or anti-4-1BB monotherapy. Increased CD8 T cell effector function and TCR diversity with enrichment of certain TCR clonotypes in the tumor was associated with anti-tumor effects. In contrast, polyclonal activation of T-cells in the periphery was associated with tissue damage. Thus, optimal combination therapy increases TCR diversity with extended activation of selective CD8 T-cells specifically in the tumor but not in the periphery. Incorporation of the concept of evenness for the TCR diversity is proposed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-19548-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773695PMC
January 2018

Marmosets (Callithrix jacchus) as a non-human primate model for evaluation of candidate dengue vaccines: induction and maintenance of specific protective immunity against challenges with clinical isolates.

J Gen Virol 2017 12 21;98(12):2955-2967. Epub 2017 Nov 21.

National Institute of Infectious Diseases, Tokyo, Japan.

Dengue virus (DENV) is one of the major infectious diseases in tropical regions and approximately half of the world population is at risk of infection. Vaccines would offer an effective control measure against this disease. We previously reported on the utility of marmosets as an animal model for studying primary and secondary dengue infections. Infected marmosets consistently develop viraemia and antibody kinetics that reflect those of patients with dengue. Thus, it is important to determine the utility of marmosets as an animal model for demonstrating vaccine efficacy. In this study, marmosets were inoculated with candidate vaccine and parent strains and challenged with a clinical DENV strain. The viraemia and antibody kinetics in these marmosets were determined. Marmosets consistently develop lower viraemia with an attenuated vaccine strain. During secondary challenge, the IgM response was delayed, whereas the IgG levels rose rapidly, indicating a secondary antibody response. The neutralizing activities against the homotypic serotype were high; all marmosets were protected against viraemia following secondary inoculation. The viraemia markers and antibody responses were consistent with those of human DENV infection and vaccinees. These results demonstrate the utility of marmosets as an animal model for the study of vaccine efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jgv.0.000913DOI Listing
December 2017

Combination immunotherapy with TLR agonists and checkpoint inhibitors suppresses head and neck cancer.

JCI Insight 2017 09 21;2(18). Epub 2017 Sep 21.

Moores Cancer Center, UCSD, La Jolla, California, USA.

Checkpoint inhibitors have demonstrated efficacy in patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, the majority of patients do not benefit from these agents. To improve the efficacy of checkpoint inhibitors, intratumoral (i.t.) injection with innate immune activators, TLR7 and TLR9 agonists, were tested along with programmed death-1 receptor (PD-1) blockade. The combination therapy suppressed tumor growth at the primary injected and distant sites in human papillomavirus-negative (HPV-negative) SCC7 and MOC1, and HPV-positive MEER syngeneic mouse models. Abscopal effects and suppression of secondary challenged tumor suggest that local treatment with TLR agonists in combination with anti-PD-1 provided systemic adaptive immunity. I.t. treatment with a TLR7 agonist increased the ratio of M1 to M2 tumor-associated macrophages (TAMs) and promoted the infiltration of tumor-specific IFNγ-producing CD8+ T cells. Anti-PD-1 treatment increased T cell receptor (TCR) clonality of CD8+ T cells in tumors and spleens of treated mice. Collectively, these experiments demonstrate that combination therapy with i.t. delivery of TLR agonists and PD-1 blockade activates TAMs and induces tumor-specific adaptive immune responses, leading to suppression of primary tumor growth and prevention of metastasis in HNSCC models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.93397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621908PMC
September 2017

Fexofenadine Suppresses Delayed-Type Hypersensitivity in the Murine Model of Palladium Allergy.

Int J Mol Sci 2017 Jun 25;18(7). Epub 2017 Jun 25.

Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, 18-1 Sakuradai, Minami-ku, Sagamihara 252-0392, Japan.

Palladium is frequently used in dental materials, and sometimes causes metal allergy. It has been suggested that the immune response by palladium-specific T cells may be responsible for the pathogenesis of delayed-type hypersensitivity in study of palladium allergic model mice. In the clinical setting, glucocorticoids and antihistamine drugs are commonly used for treatment of contact dermatitis. However, the precise mechanism of immune suppression in palladium allergy remains unknown. We investigated inhibition of the immune response in palladium allergic mice by administration of prednisolone as a glucocorticoid and fexofenadine hydrochloride as an antihistamine. Compared with glucocorticoids, fexofenadine hydrochloride significantly suppressed the number of T cells by interfering with the development of antigen-presenting cells from the sensitization phase. Our results suggest that antihistamine has a beneficial effect on the treatment of palladium allergy compared to glucocorticoids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms18071357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535850PMC
June 2017

Highly functional T-cell receptor repertoires are abundant in stem memory T cells and highly shared among individuals.

Sci Rep 2017 06 16;7(1):3663. Epub 2017 Jun 16.

Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine (RIRBM), Hiroshima University, Hiroshima, Japan.

To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals. Using an HLA-A*02-restricted cytomegalovirus (CMV) pp65-derived immunogenic peptide, we found that the more dominant pp65-specific TCR clonotypes in the blood of healthy donors have higher binding affinities for the CMV peptide and arise from clonotypes that are highly shared among individuals. Interestingly, these highly shared HLA-A*02-restricted CMV-specific TCRs were detected in a CMV-seronegative individual as well as in HLA-A*02-negative donors albeit at lower frequency. More intriguingly, these shared TCR clonotypes were abundant in the stem memory T-cell subset, and TCR diversity of the stem memory T-cell repertoire was significantly lower than in the central memory and effector memory T-cell repertoires. These results suggest that the stem memory T-cell subset may serve as a reservoir of highly shared and highly functional memory T-cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-03855-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473819PMC
June 2017

Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis.

Front Immunol 2017 3;8:389. Epub 2017 May 3.

Repertoire Genesis Incorporation, Ibaraki, Japan.

A diverse antibody repertoire is primarily generated by the rearrangement of V, D, and J genes and subsequent somatic hypermutation (SHM). Class-switch recombination (CSR) produces various isotypes and subclasses with different functional properties. Although antibody isotypes and subclasses are considered to be produced by both direct and sequential CSR, it is still not fully understood how SHMs accumulate during the process in which antibody subclasses are generated. Here, we developed a new next-generation sequencing (NGS)-based antibody repertoire analysis capable of identifying all antibody isotype and subclass genes and used it to examine the peripheral blood mononuclear cells of 12 healthy individuals. Using a total of 5,480,040 sequences, we compared percentage frequency of variable (V), junctional (J) sequence, and a combination of V and J, diversity, length, and amino acid compositions of CDR3, SHM, and shared clones in the IgM, IgD, IgG3, IgG1, IgG2, IgG4, IgA1, IgE, and IgA2 genes. The usage and diversity were similar among the immunoglobulin (Ig) subclasses. Clonally related sequences sharing identical V, D, J, and CDR3 amino acid sequences were frequently found within multiple Ig subclasses, especially between IgG1 and IgG2 or IgA1 and IgA2. SHM occurred most frequently in IgG4, while IgG3 genes were the least mutated among all IgG subclasses. The shared clones had almost the same SHM levels among Ig subclasses, while subclass-specific clones had different levels of SHM dependent on the genomic location. Given the sequential CSR, these results suggest that CSR occurs sequentially over multiple subclasses in the order corresponding to the genomic location of IGHCs, but CSR is likely to occur more quickly than SHMs accumulate within Ig genes under physiological conditions. NGS-based antibody repertoire analysis should provide critical information on how various antibodies are generated in the immune system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.00389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413556PMC
May 2017

A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains.

BMC Immunol 2016 10 11;17(1):38. Epub 2016 Oct 11.

Repertoire Genesis Incorporation, 104 Saito-Bioincubator, 7-7-15, Saito-asagi, Ibaraki, Osaka, 567-0085, Japan.

Background: High-throughput sequencing of T cell receptor (TCR) genes is a powerful tool for analyses of antigen specificity, clonality and diversity of T lymphocytes. Here, we developed a new TCR repertoire analysis method using 454 DNA sequencing technology in combination with an adaptor-ligation mediated polymerase chain reaction (PCR). This method allows the amplification of all TCR genes without PCR bias. To compare gene usage, diversity and similarity of expressed TCR repertoires among individuals, we conducted next-generation sequencing (NGS) of TRA and TRB genes in peripheral blood mononuclear cells from 20 healthy human individuals.

Results: From a total of 267,037 sequence reads from 20 individuals, 149,216 unique sequence reads were identified. Preferential usage of several V and J genes were observed while some recombinations of TRAV with TRAJ appeared to be restricted. The extent of TCR diversity was not significantly different between TRA and TRB, while TRA repertoires were more similar between individuals than TRB repertoires were. The interindividual similarity of TRA depended largely on the frequent presence of shared TCRs among two or more individuals. A publicly available TRA had a near-germline TCR with a shorter CDR3. Notably, shared TRA sequences, especially those shared among a large number of individuals', often contained TCRα related with invariant TCRα derived from invariant natural killer T cells and mucosal-associated invariant T cells.

Conclusion: These results suggest that retrieval of shared TCRs by NGS would be useful for the identification of potential new invariant TCRα chains. This NGS method will enable the comprehensive quantitative analysis of TCR repertoires at a clonal level.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12865-016-0177-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059964PMC
October 2016

Overexpression of ErbB4 is an independent marker for lymph node metastasis in Japanese patients with oral squamous cell carcinoma.

Oral Surg Oral Med Oral Pathol Oral Radiol 2016 Sep 11;122(3):313-21. Epub 2016 May 11.

Department of Clinical Immunology, Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, National Hospital Organization, Kanagawa, Japan. Electronic address:

Objective: Overexpression of the epidermal growth factor receptor (EGFR) family is common in oral squamous cell carcinoma (OSCC). Therefore, we analyzed the expression profiles of the four EGFR family members (ErbB1, ErbB2, ErbB3, and ErbB4) in OSCC of Japanese patients.

Study Design: Sixty-eight primary tumors and 18 normal oral mucosal tissue specimens were evaluated in this study. We analyzed EGFR family members using quantitative polymerase chain reaction and immunohistochemistry, as well as their relationships with clinical factors.

Results: The expression level of ErbB1 messenger RNA (mRNA) was markedly increased in OSCC. By comparing the gene expression levels of EGFR family members in OSCC tissues that had lymph node metastasis with those in the absence of lymph node metastasis, we found that ErbB4 mRNA expression was increased significantly. There was also a significant correlation between the mRNA expression level of ErbB4 and those of ErbB2 and ErbB3 in cases with lymph node metastasis. Moreover, we confirmed protein expression of ErbB4 in the cytoplasm and membrane of tumor cells, which was stronger in cases with lymph node metastasis.

Conclusions: ErbB4 is an independent marker for lymph node metastasis in OSCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.oooo.2016.04.017DOI Listing
September 2016

Effector Regulatory T Cells Reflect the Equilibrium between Antitumor Immunity and Autoimmunity in Adult T-cell Leukemia.

Cancer Immunol Res 2016 08 23;4(8):644-9. Epub 2016 May 23.

Department of Hematology, Respiratory Medicine and Oncology, Saga University School of Medicine, Saga, Japan.

The regulatory T cells (Treg) with the most potent immunosuppressive activity are the effector Tregs (eTreg) with a CD45RA(-)Foxp3(++)CCR4(+) phenotype. Adult T-cell leukemia (ATL) cells often share the Treg phenotype and also express CCR4. Although mogamulizumab, a monoclonal antibody to CCR4, shows marked antitumor effects against ATL and peripheral T-cell lymphoma, concerns have been raised that it may induce severe autoimmune immunopathology by depleting eTregs. Here, we present case reports for two patients with ATL who responded to mogamulizumab but developed a severe skin rash and autoimmune brainstem encephalitis. Deep sequencing of the T-cell receptor revealed that ATL cells and naturally occurring Tregs within the cell population with a Treg phenotype can be clearly distinguished according to CADM1 expression. The onset of skin rash and brainstem encephalitis was coincident with eTreg depletion from the peripheral blood, whereas ATL relapses were coincident with eTreg recovery. These results imply that eTreg numbers in the peripheral blood sensitively reflect the equilibrium between antitumor immunity and autoimmunity, and that mogamulizumab might suppress ATL until the eTreg population recovers. Close monitoring of eTreg numbers is crucial if we are to provide immunomodulatory treatments that target malignancy without severe adverse events. Cancer Immunol Res; 4(8); 644-9. ©2016 AACR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2326-6066.CIR-15-0303DOI Listing
August 2016

Downregulation of epidermal growth factor receptor family receptors and ligands in a mutant K-ras group of patients with colorectal cancer.

Mol Med Rep 2016 Apr 1;13(4):3514-20. Epub 2016 Mar 1.

Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, Sagamihara, Kanagawa 252‑0392, Japan.

The present study investigated the expression profiles of the epidermal growth factor receptor (EGFR) family, which consists of four transmembrane tyrosine kinase receptors and their eight ligands, in 122 patients with colorectal cancer (CRC) using reverse transcription-quantitative polymerase chain reaction analysis. On comparison of the CRC primary tumor and matched adjacent normal mucosa (ANM) tissue samples, the mRNA expression levels of ErbB3, but not ErbB1, were significantly increased in CRC tissue samples, compared with those in the ANM tissues. The expression levels of the ligands exhibited opposing trends to their corresponding receptors, including EGF, BTC, AREG, EREG and HB‑EGF, which were increased in the CRC tissues, whereas NRG1 and NGR2 were decreased in thee CRC tissues, compared with those in the AMN tissues. Subsequently, the present study investigated the frequency of K-ras mutations in the patients with CRC. The K‑ras mutations were found to be present in 36.8% (45/122) of the cases, however, no correlation was observed between K‑ras mutations and clinicopathological characteristics. In the CRC tissues, the expression levels of the EGFR family receptors and their ligands were determined in wild-type and mutant K-ras CRC cases. The expression levels of ErbB1, ErbB2, ErbB3, BTC, AREG, EREG, NRG1 and NRG2 were significantly decreased in the mutant K‑ras cases, compared with those in the wild‑type K‑ras cases. These results suggested that the tumorigenesis of CRC with wild‑type K‑ras was mediated through, not only ErbB1, but also through the ErbB2 and ErbB3 pathways. Notably, although ErbB2 does not bind any ErbB ligands, ErbB2 may activate tumorigenesis via a heterodimer, rather than a homodimer. Therefore, the results of the present study suggest that the most effective strategy to target not only ErbB1, but also ErbB2 and ErbB3, is the use of monoclonal antibody treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/mmr.2016.4951DOI Listing
April 2016

Possible Immune Regulation of Natural Killer T Cells in a Murine Model of Metal Ion-Induced Allergic Contact Dermatitis.

Int J Mol Sci 2016 Jan 12;17(1). Epub 2016 Jan 12.

Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, 18-1 Sakuradai, Minami-ku, Sagamihara 252-0392, Japan.

Metal often causes delayed-type hypersensitivity reactions, which are possibly mediated by accumulating T cells in the inflamed skin, called irritant or allergic contact dermatitis. However, accumulating T cells during development of a metal allergy are poorly characterized because a suitable animal model is unavailable. We have previously established novel murine models of metal allergy and found accumulation of both metal-specific T cells and natural killer (NK) T cells in the inflamed skin. In our novel models of metal allergy, skin hypersensitivity responses were induced through repeated sensitizations by administration of metal chloride and lipopolysaccharide into the mouse groin followed by metal chloride challenge in the footpad. These models enabled us to investigate the precise mechanisms of the immune responses of metal allergy in the inflamed skin. In this review, we summarize the immune responses in several murine models of metal allergy and describe which antigen-specific responses occur in the inflamed skin during allergic contact dermatitis in terms of the T cell receptor. In addition, we consider the immune regulation of accumulated NK T cells in metal ion-induced allergic contact dermatitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms17010087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730330PMC
January 2016

Clinicopathological significance and prognostic value of Wilms' tumor gene expression in colorectal cancer.

Cancer Biomark 2015 ;15(6):789-97

Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, Sagamihara, Japan.

Background: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, and novel effective treatments and diagnostic tools are urgently required.

Objective: The selection of appropriate targeting tumor-associated antigens (TAAs) is critical for immunotherapy. Therefore, we analyzed TAA expression levels and investigated their relationship with clinical factors in adjacent normal mucosa (ANM) and CRC tissue.

Methods: We obtained specimens of CRC primary tumors and matched ANM from 137 patients with CRC who underwent surgical resection. The mRNA levels of seven TAAs, Wilms' tumor gene (WT1), kinetochore associated-2 (KNTC2), cell division cycle associated-1 (CDCA1), M phase phosphoprotein-1 (MPHOSPH1), DEP domain-containing 1 (DEPDC1), 34-kDa translocase of the outer mitochondrial membrane (TOMM34) and ring finger protein-43 (RNF43), were analyzed using quantitative real-time reverse transcription-polymerase chain reaction, and their relationships with clinicopathological factors and the cell cycle were analyzed.

Results: The expression levels of all seven TAAs were significantly higher in CRC tissues than in ANM. Expression levels of WT1 in CRC tissues did not correlate with the cell cycle. Furthermore, WT1 expression in CRC tissues was significantly related to tumor progression, lymph node metastasis, distant metastasis and clinical stage.

Conclusions: WT1 is a potential marker for prognosis and tumor progression in CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/CBM-150521DOI Listing
October 2016

Qualitative differences in brain-infiltrating T cells are associated with a fatal outcome in mice infected with Japanese encephalitis virus.

Arch Virol 2015 Mar 22;160(3):765-75. Epub 2015 Jan 22.

Department of Virology 1, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan.

Japanese encephalitis (JE) is the most important form of viral encephalitis in Asia. The critical factors determining mortality and severity of JE virus (JEV) infection remain unclear. We identified brain-infiltrating T cells associated with a fatal outcome of JEV infection in mice. Dying mice were defined as those that lost more than 25 % of their body weight by day 13 and died by day 21, while surviving mice were defined as those that lost less than 10 % by day 13, based on the result of the survival time course study. Two groups of five mice that demonstrated brain virus titers of >1 × 10(6) pfu/g were randomly selected from the dying and surviving groups and used in the analyses. Cytokine patterns in brains were first examined, revealing a higher ratio of Th1-related cytokine genes in dying mice. The expression levels of CD3, CD8, CD25, and CD69 increased in JEV-infected mice relative to mock-infected mice. However, expression levels of these cell-surface markers did not differ between the two groups. T-cell receptor (TCR) usage and complementary determining region 3 (CDR3) sequences were analyzed in the brain-infiltrating T cells. T cells expressing VA8-1, VA10-1, and VB2-1 increased in both groups. However, the dominant T-cell clones as defined by CDR3 amino acid sequence differed between the two groups. The results indicate that the outcome of JEV infection, death or survival, was determined by qualitative differences in infiltrating T-cell clones with unique CDR3 amino acid sequences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00705-014-2154-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336650PMC
March 2015

Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus.

Am J Trop Med Hyg 2015 Feb 29;92(2):370-6. Epub 2014 Dec 29.

Department of Virology 1, National Institute of Infectious Diseases, Tokyo, Japan; Division of Experimental Animal Research, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan; Department of Rheumatology and Clinical Immunology, Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, National Hospital Organization, Kanagawa, Japan; College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan; National Institute of Infectious Diseases, Tokyo, Japan

Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4269/ajtmh.14-0455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347344PMC
February 2015

Genomic sequence analysis of the MHC class I G/F segment in common marmoset (Callithrix jacchus).

J Immunol 2014 Apr 5;192(7):3239-46. Epub 2014 Mar 5.

Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa 259-1143, Japan;

The common marmoset (Callithrix jacchus) is a New World monkey that is used frequently as a model for various human diseases. However, detailed knowledge about the MHC is still lacking. In this study, we sequenced and annotated a total of 854 kb of the common marmoset MHC region that corresponds to the HLA-A/G/F segment (Caja-G/F) between the Caja-G1 and RNF39 genes. The sequenced region contains 19 MHC class I genes, of which 14 are of the MHC-G (Caja-G) type, and 5 are of the MHC-F (Caja-F) type. Six putatively functional Caja-G and Caja-F genes (Caja-G1, Caja-G3, Caja-G7, Caja-G12, Caja-G13, and Caja-F4), 13 pseudogenes related either to Caja-G or Caja-F, three non-MHC genes (ZNRD1, PPPIR11, and RNF39), two miscRNA genes (ZNRD1-AS1 and HCG8), and one non-MHC pseudogene (ETF1P1) were identified. Phylogenetic analysis suggests segmental duplications of units consisting of basically five (four Caja-G and one Caja-F) MHC class I genes, with subsequent expansion/deletion of genes. A similar genomic organization of the Caja-G/F segment has not been observed in catarrhine primates, indicating that this genomic segment was formed in New World monkeys after the split of New World and Old World monkeys.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1302745DOI Listing
April 2014

Accumulation of metal-specific T cells in inflamed skin in a novel murine model of chromium-induced allergic contact dermatitis.

PLoS One 2014 20;9(1):e85983. Epub 2014 Jan 20.

Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, Sagamihara, Japan.

Chromium (Cr) causes delayed-type hypersensitivity reactions possibly mediated by accumulating T cells into allergic inflamed skin, which are called irritants or allergic contact dermatitis. However, accumulating T cells during development of metal allergy are poorly characterized because a suitable animal model is not available. This study aimed to elucidate the skewing of T-cell receptor (TCR) repertoire and cytokine profiles in accumulated T cells in inflamed skin during elucidation of Cr allergy. A novel model of Cr allergy was induced by two sensitizations of Cr plus lipopolysaccharide solution into mouse groin followed by single Cr challenge into the footpad. TCR repertoires and nucleotide sequences of complementary determining region 3 were assessed in accumulated T cells from inflamed skin. Cytokine expression profiles and T-cell phenotypes were determined by qPCR. CD3+CD4+ T cells accumulated in allergic footpads and produced increased T helper 1 (Th1) type cytokines, Fas, and Fas ligand in the footpads after challenge, suggesting CD4+ Th1 cells locally expanded in response to Cr. Accumulated T cells included natural killer (NK) T cells and Cr-specific T cells with VA11-1/VB14-1 usage, suggesting metal-specific T cells driven by invariant NKT cells might contribute to the pathogenesis of Cr allergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085983PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896422PMC
January 2015

Characterization of T cell receptors of Th1 cells infiltrating inflamed skin of a novel murine model of palladium-induced metal allergy.

PLoS One 2013 3;8(10):e76385. Epub 2013 Oct 3.

Department of Oral and Maxillofacial Surgery, School of Dental Medicine, Tsurumi University, Yokohama, Japan ; Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, Sagamihara, Japan ; Department of Oral and Maxillofacial Surgery, Nagano Matsushiro General Hospital, Nagano, Japan.

Metal allergy is categorized as a delayed-type hypersensitivity reaction, and is characterized by the recruitment of lymphocytes into sites of allergic inflammation. Because of the unavailability of suitable animal models for metal allergy, the role of T cells in the pathogenesis of metal allergy has not been explored. Thus, we developed a novel mouse model for metal allergy associated with infiltration of T cells by multiple injections of palladium (Pd) plus lipopolysaccharide into the footpad. Using this model, we characterized footpad-infiltrating T cells in terms of phenotypic markers, T cell receptor (TCR) repertoires and cytokine expression. CD3+ CD4+ T cells accumulated in the allergic footpads 7 days after Pd challenge. The expression levels of CD25, interleukin-2, interferon-γ and tumor necrosis factor, but not interleukin-4 and interleukin-5, increased in the footpads after challenge, suggesting CD4+ T helper 1 (Th1) cells locally expanded in response to Pd. Infiltrated T cells in the footpads frequently expressed AV18-1 and BV8-2 T cell receptor (TCR) chains compared with T cells in the lymph nodes and exhibited oligoclonality. T-cell clones identified from Pd-allergic mouse footpads shared identical CDR3 sequences containing AV18-1 and BV8-2. These results suggest that TCR AV18-1 and BV8-2 play dominant and critical parts in the antigen specificity of Pd-specific Th1 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076385PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789730PMC
July 2014

Accumulation of invariant NKT cells into inflamed skin in a novel murine model of nickel allergy.

Cell Immunol 2013 Jul-Aug;284(1-2):163-71. Epub 2013 Aug 2.

Department of Oral and Maxillofacial Surgery, School of Dental Medicine, Tsurumi University, 2-1-3, Tsrumi, Tsurumi-ku, Yokohama, Kanagawa 230-8501, Japan; Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization, 18-1, Sakura-dai, Minami-ku, Sagamihara, Kanagawa 252-0392, Japan; (c)Department of Oral and Maxillofacial Surgery, Toshiba Rinkan Hospital, 7-9-1, Kamitsuruma, Sagamihara, Kanagawa 252-0385, Japan.

Nickel (Ni) can cause delayed-type hypersensitivity reactions, which are thought to be mediated by the accumulation of T cells into inflamed skin. Accumulated T cells at the developmental stages in metal allergy are poorly characterized because a suitable animal model has not been established. To investigate the accumulated T cells in allergic inflamed skin, we generated a novel murine model of Ni-induced allergy. The murine model of Ni allergy was induced by two sensitizations of Ni plus lipopolysaccharide solution into the groin followed by three challenges with Ni solution into the footpad. Here we show that a specific TCR repertoire bearing Vα14Jα18, called natural killer (NK) T cells, was expanded monoclonally in BALB/c or C57BL/6 mice. Accumulation of NKT cells was characterized as CD4(+) or CD4(-)CD8(-) T cells. These results suggested that NKT cells are major pathogenic T cells at the elicitation phase of Ni allergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellimm.2013.07.010DOI Listing
March 2014

TNF-α acts as an immunoregulator in the mouse brain by reducing the incidence of severe disease following Japanese encephalitis virus infection.

PLoS One 2013 5;8(8):e71643. Epub 2013 Aug 5.

Department of Virology, Institute of Tropical Medicine, GCOE program, Leading Graduate School Program, Nagasaki University, Nagasaki, Nagasaki, Japan.

Japanese encephalitis virus (JEV) causes acute central nervous system (CNS) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. However, the mechanism of severe encephalitis has not been fully elucidated. In this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal JEV infection. Following extraneural infection with the JaOArS982 strain of JEV, infected mice exhibited clinical signs ranging from mild to fatal outcome. Comparison of the pathogenetic response between severe and mild cases of JaOArS982-infected mice revealed increased levels of TNF-α in the brains of severe cases. However, unexpectedly, the mortality rate of TNF-α KO mice was significantly increased compared with that of WT mice, indicating that TNF-α plays a protective role against fatal infection. Interestingly, there were no significant differences of viral load in the CNS between WT and TNF-α KO mice. However, exaggerated inflammatory responses were observed in the CNS of TNF-α KO mice. Although these observations were also obtained in IL-10 KO mice, the mortality and enhanced inflammatory responses were more pronounced in TNF-α KO mice. Our findings therefore provide the first evidence that TNF-α has an immunoregulatory effect on pro-inflammatory cytokines in the CNS during JEV infection and consequently protects the animals from fatal disease. Thus, we propose that the increased level of TNF-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. In future, further elucidation of the immunoregulatory mechanism of TNF-α will be an important priority to enable the development of effective treatment strategies for Japanese encephalitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071643PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733918PMC
April 2014

Immune-related gene expression profile in laboratory common marmosets assessed by an accurate quantitative real-time PCR using selected reference genes.

PLoS One 2013 25;8(2):e56296. Epub 2013 Feb 25.

Department of Rheumatology and Clinical Immunology, Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, National Hospital Organization, Kanagawa, Japan.

The common marmoset (Callithrix jacchus) is considered a novel experimental animal model of non-human primates. However, due to antibody unavailability, immunological and pathological studies have not been adequately conducted in various disease models of common marmoset. Quantitative real-time PCR (qPCR) is a powerful tool to examine gene expression levels. Recent reports have shown that selection of internal reference housekeeping genes are required for accurate normalization of gene expression. To develop a reliable qPCR method in common marmoset, we used geNorm applets to evaluate the expression stability of eight candidate reference genes (GAPDH, ACTB, rRNA, B2M, UBC, HPRT, SDHA and TBP) in various tissues from laboratory common marmosets. geNorm analysis showed that GAPDH, ACTB, SDHA and TBP were generally ranked high in stability followed by UBC. In contrast, HPRT, rRNA and B2M exhibited lower expression stability than other genes in most tissues analyzed. Furthermore, by using the improved qPCR with selected reference genes, we analyzed the expression levels of CD antigens (CD3ε, CD4, CD8α and CD20) and cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12β, IL-13, IFN-γ and TNF-α) in peripheral blood leukocytes and compared them between common marmosets and humans. The expression levels of CD4 and IL-4 were lower in common marmosets than in humans whereas those of IL-10, IL-12β and IFN-γ were higher in the common marmoset. The ratio of Th1-related gene expression level to that of Th2-related genes was inverted in common marmosets. We confirmed the inverted ratio of CD4 to CD8 in common marmosets by flow cytometric analysis. Therefore, the difference in Th1/Th2 balance between common marmosets and humans may affect host defense and/or disease susceptibility, which should be carefully considered when using common marmoset as an experimental model for biomedical research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056296PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581525PMC
September 2013
-->