Immunological kidney diseases (WS-098): Chairpersons: Yasuhiko Tomino, Jan Novak

International Immunology, Aug 2010

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Immunological kidney diseases (WS-098): Chairpersons: Yasuhiko Tomino, Jan Novak

Chairpersons: Yasuhiko Tomino 2 3 4 5 6 Jan Novak 2 3 4 5 6 0 Berlin-Brandenburg Center for Regenerative Therapies (BCRT) , Berlin , Germany 1 Department of Nephrology,Charite University Medicine Berlin , Berlin , Germany 2 H. Trydzenskaya 3 M. Masuda , S. Morimoto, H. Y. Shi, T. Morita, N. Nishimura, N. Takahashi, H. Masaki, K. Kouno, T. Yokoi, M. Yoshika, Y. Komiyama, T. Iwasaka , H. Takahashi. Kansai Medical University , Osaka , Japan 4 J. D. Ooi, J. Chang, S. R. Holdsworth, A. R. Kitching. Dept. of Medicine, Monash Medical Centre, Monash University , Clayton , Australia 5 V. Bueno , J. F. Pedregosa, G. N. Gomes, M. F. Franco. UNIFESP - Federal University of Sa ? o Paulo , Sa ? o Paulo , Brazil 6 S y m p o s i a WS/PP-098-01 Measurement of soluble FcgRIIIa and soluble FcgRIIIaMw in urine from patients with proteinuria Conclusion: Our results show that pathogenesis of FSGS involves down regulation of Treg cells. Identification of steroid resistant FSGS prior to initiation of therapy may help in avoiding steroids and hence its side effects in these patients. A larger patient population with longer follow-up is required to establish the clinical significance of these results. - M eL a i s o p m y S item res cLunh tecLu ss pp oo hh ss kk r r oo WW Autoimmunity directed against the neutrophil enzyme, myeloperoxidase (MPO), leads to glomerulonephritis. MPO specific anti-neutrophil cytoplasmic anti- H. Suzuki1, M. Raska2,3, B. A. Julian3, R. J. Wyatt4, J. Mestecky3, bodies are pathogenic while MPO specific T cells also play a role. This study A. Gharavi5, Y. Tomino1, J. Novak3. 1Juntendo University Faculty of sought to identify the immunogenic T cell epitopes of MPO in C57BL/6 mice and demonstrate that an MPO peptide can induce glomerulonephritis. The Medicine, Tokyo, Japan, 2Palacky University at Olomouc, Olomouc, entire MPO molecule was screened by immunizing mice with pools of overlap- Czech Republic, 3University of Alabama at Birmingham, Birmingham, AL, ping 20-mers (n ? 4/group). The 5 strongest responding peptides: peptides 10, United States, 4University of Tennessee Health Science Center, 24, 52, 57 and 61, measured by IFNg ELISPOT, were then used to immunize Memphis, TN, United States, 5Columbia University, New York, NY, mice (n ? 4/group). Cellular responses to the immunizing peptide and to recom- United States binant mouse MPO were measured in vitro by [3H]-thymidine proliferation and IFNg and IL-17A ELISPOTs. All 5 peptides made responses to themselves Galactose (Gal)-deficient IgA1 (Gd-IgA1) plays a key role in the pathogenesis of and to recombinant mouse MPO with peptide 52 making the strongest IgA nephropathy (IgAN). Glycans on Gd-IgA1 constitute neoantigens that are responses both to itself and to recombinant mouse MPO. Immunized mice did recognized by anti-glycan antibodies, leading to formation of nephritogenic not develop glomerulonephritis, but planting MPO in glomeruli using low dose immune complexes. IgA1-producing cells from circulation and tonsils of IgAN anti-GBM antibodies allows effector CD4+ cells to recognize MPO as a planted patients secrete, Gd-IgA1 with sialylated or terminal N-acetylgalactosamine autoantigen. Mice were immunized with either peptide 52 (n ? 6) or with whole (GalNAc)-containing O-glycans, due to a decreased activity of MPO (n ? 4) and disease triggered using low dose sheep anti-GBM antibodies. ?1,3-galactosyltransferase (C1GALT1 gene) and elevated activity of Mice sensitized to OVA323-339 (n ? 5) were controls. MPO peptide and whole a2,6-GalNAc-sialyltransferase (ST6GALNACII gene). To confirm the roles of MPO immunized mice developed comparable levels of glomerulonephritis, these glycosyltransferases, we knocked-down expression of C1GALT1 and measured by histological glomerular injury, urinary protein:creatinine ratios, ST6GALNACII genes in immortalized IgA1-producing cells from circulation of and glomerular infiltration of cellular effectors. Peptide 52 immunized mice IgAN patients (IgAN cells) and healthy controls using siRNA. C1GALT1 also developed anti-MPO antibodies. These results define an immunopatho- expression decreased 50% after siRNA knock-down and increased genic T cell epitope of MPO. This work supported by the Australian NHMRC. Gal-deficiency of IgA1 (from 31% to 44% in IgAN cells, P , 0.0001, and from 12% to 16% in cells from controls, P ? 0.0035). Conversely, galactosylation of IgA1 O-glycans increased by 15% (P ? 0.0033) after 50% knock-down of ST6GALNACII gene in IgAN cells. Next, we measured the levels of IgA1 galacWS/PP-098-03 Down regulation of regulatory T cells (Treg cells) tosylation with or without prior enzymatic sialylation. The results showed that in patients with focal segmental glomerulosclerosis- A pilot study enzymatic sialylation of terminal GalNAc blocked its subsequent galactosylaS. Anis, E. Ahmed, R. Muzaffar. Sindh Institute of Urology and tion. Furthermore, we showed that knock-down of the over-expressed ST6GALNACII gene in IgAN cells reduced Gal deficiency. Detailed analysis of Transplantation, Karachi, Pakistan the pathways leading to the production of the nephritogenic Gd-IgA1 may offer leads for new therapeutic strategies for IgAN. WS/PP-098-05 Premature sialylation contributes to galactose deficiency of IgA1 in patients with IgA nephropathy Background: Focal segmental glomerulosclerosis (FSGS) comprises of a group of glomerular lesions with various clinical manifestations and is a common cause of renal failure both in children and adults. Pathogenesis of FSGS may involve imbalance in Treg cells or autoreactive B cells. We have analyzed and compared the frequencies of Treg cells (CD4+25+ and CD8+122+) and CD5+B cells in blood and urine samples of 50 individuals of which 29 were diagnosed with FSGS and 21 belonged to non-FSGS-group. Data was analyzed using spss software and t-test was applied to determine significance. Results: The percentage of CD4+25+ cells was low in FSGS-group compared to non-FSGS-group (3.8 + 3.3 vs. 4.5 + 4.4) with a significantly low ratio of these cells to CD4+122+ cells in FSGS patients (9.5 + 13.6 vs. 23.15 + 38.3; p ? 0.024). CD8+122+ cells were significantly low in steroid-resistant FSGS patients compared to steroid-dependent-group (1.1+ 1.2 vs. 6.0 +7.0; p ? 0.042). This is also reflected in patients who did not show remission and developed renal dysfunction (p ? 0.039). CD25+122+ B cells were higher in the urine of steroiddependent and resistant-FSGS compared to steroid-responsive patients (p ? 0.023 and 0.022 respectively). WS/PP-098-06 New protocol of BKV-specific T cell analysis allows for improved assessment of phenotypic/functional characteristics of BKV-specific immunity BKV-associated nephropathy (BKVAN) represents a serious complication of the post-transplant period in kidney recipients leading to organ loss in 50 % of all cases. Monitoring of BKV-specific immunity is very important in management of patients with BKV infection/BKVAN. Our previous data demonstrated all five BKV-specific antigens inducing CD4+ T-cell responses with BKV-antigenic immunodominance varying within affected patients. Therefore, BKV-specific T-cell response directed against all five BKV proteins should be analyzed in order to assess the entire BKV-specific immunity. The aim of the present study was to establish a fast/sensitive method for the analysis of BKV-specific CD4/CD8-T-cells and apply it for phenotypic/functional T cell analysis in patients with post-BKV infection. Using mixture of overlapping peptide pools encompassing all 5 BKV antigens (VP1, VP2, VP3, LTAg and stAg) and multiparameter flow cytometry we evaluated BKV-specific CD4/CD8 T cells in renal transplant patients with resolved BKV infection. We found 0.3 + 0.093% of IFNg + CD4+ T cells; 1.5 + 0.368% of IL-2 + CD4+ T cells; 0.031 + 0.0126% of IFNg + CD8+ T-cells. The frequencies of BKV-specific T-cells demonstrated with the new protocol were significantly higher as compared to previously used methods. In addition, polyfunctional IFNg + IL-2 + TNFa+ BKV-specific CD4+ T cells were found in all patients with resolved BKV infection/BKVAN. Interestingly, neither polyfunctional CD8+ T cells nor single IL-17+ T cells were detected. Here, we demonstrated, that, using mixture of overlapping peptides pools spanning all five BKV proteins enabled us to identify CD4- and CD8-T-cell responses and its prenotypic/functional characteristics that were not consistently detectable with the previously applied protocol with a single protein stimulation. which is a cryptic binding site to integrins within OPN created by thrombin cleavage, or a small interfering RNA (siRNA) targeted on OPN coding sequence. Both treatments with either M5 antibody or siRNA could prevent EAU development with suppression of TNF-a, IFN-g, and IL-17 production. Our results suggest that OPN represents a novel therapeutic target to control uveoretinitis. However, both blockades have been applied at the time of immunization and application after the onset was not as effective as the preventive regimen. Thus, we are further seeking the therapeutic application of OPN blockade for actual amelioration of EAU. WS/PP-099-02 Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a retina-specific T cell receptor Experimental autoimmune uveitis (EAU) is induced by immunization with retinal antigens and mimics human uveitis. We generated transgenic (Tg) mice that express a T cell receptor (TCR) specific to the uveitogenic retinal protein IRBP WS/PP-098-07 Treatments for MPO-ANCA-associated vasculitis cloned from an autopathogenic (wild type) T cell line. in SCG/Kj mouse baTchkegrpoeurnipdhecroanltTaicneeldlre1p5e- 3rt0o%ire, oafnIdRBoPn TthCeR RTAgGm2ic-deeofinciethnet cboancvkegnrtoiounnadl R. Kusunoki1, T. Nagao1, C. Iwamura2, S. Kobayashi3, W. Yumura4, 90-95%, IRBP-specific CD4+ T cells, as judged by binding of Ag-specific T. Nakayama2, K. Suzuki1. 1Inflammation Program, Department of MHC class II-Ig dimers. The cells proliferated to IRBP161-180 peptide and Immunology, Chiba University Graduate School of Medicine, Chiba, transferred EAU to na??ve recipients. The proportion of memory T cells in the Japan, 2Department of Immunology, Chiba University Graduate School IRBP-specific population was substantially lower than in the polyclonal population, consistent with the notion of immune sequestration/privilege of the retinal of Medicine, Chiba, Japan, 3Juntendo University Koshigaya Hospital, antigens. Nevertheless, IRBP TCR Tg mice developed early spontaneous uveitis Koshigaya-Saitama, Japan, 4Jichi Medical University, with most of the eye-infiltrating T cells displaying a memory phenotype. The Shimotsuke-Tochigi, Japan ocular inflammatory infiltrate included Th1, Th17 and T regulatory cells Myeloperoxidase-antineutrophil cytoplasmic autoantibody (MPO-ANCA) associ- (Tregs), detected by intracellular staining for IFN-g, IL-17A, and Foxp3. ates with rapidly progressive glomerulonephritis (RPGN) and glomerular cres- IRBP-specific Tregs were enriched in the eye compared to the periphery. cent formation. We analyzed the pathogenetic factor of RPGN and the Interestingly, IRBP-specific Foxp3+ T cells did not seem to be positively effective therapy against RPGN in SCG/Kj mice, spontaneously developing selected in the thymus, suggesting that Tregs in the eye had been peripherally MPO-ANCA-associated RPGN. We revealed a significant correlation between or locally converted from conventional T cells. We are currently studying where the rate of crescent formation and IL-6/MCP-1. Then, IL-6 was selected as a priming to ?sequestered? retinal antigens occurs in this model. candidate factor of RPGN because anti-IL-6 receptor (IL-6R) antibody was The IRBP TCR Tg mice provide a new valuable model of spontaneous uveitis recently proven to be effective against rheumatoid arthritis. So, we conducted to study basic mechanisms involved in maintenance and breakdown of immune administration of anti-IL-6R antibody (Chugai Pharmaceutical Co.). In addition, homeostasis affecting privileged sites such as the eye. we also conducted administration of mizoribine (Asahi Kasei Pharmaceutical Co.) and 15-deoxyspergualin (DSG, Nippon Kayaku Co.), reported its therapeutic effect against RPGN of SCG/Kj mice. As a result, MPO-ANCA titer WS/PP-099-03 Common and different gene loci susceptible to decreased after treatment with anti-IL-6R antibody. It was also found that mizor- sialoadenitis and dacryoadenitis in a Sjo? gren?s syndrome mouse ibine had tendency to bring relief and there was significant therapeutic effect in model urynalysis, histopathology, MPO-ANCA level and the rate of CD3+ B220+ cells after treatment with DSG. Furthermore, significant reduction of IL-2, IL-12(p40), T. Kamao, T. Miyazaki, Y. Soga, H. Komori, M. Terada, M. Nose. Ehime and MIP-1b was observed by DSG treatment. As a consequence, it is indicated University Graduate School of Medicine, Toon, Japan that IL-6 involves the production of autoantibody. Additionally, IL-2, IL-12(p40), and MIP-1b could be new candidates of pathogenetic factor. Moreover, it was considered that DSG is effective against RPGN as a drug for remission induction. And it might be more effective therapy using mizoribine as a maintenance therapy after remission by DSG. Sjo?gren?s syndrome (SS) is clinically manifested by sicca syndrome, associated with autoimmune sialoadenitis and dacryoadenitis. These lesions are commonly characterized by destruction of the ductules in the onset, associated with the accumulation of mononuclear cells, followed by destruction of the acinar glands. However, it still remains unclear whether these lesions develop coincidentally and in a common mechanism. An MRL/lpr strain of mice is well known as a SS mouse model, which spontaneously develops sialoadenitis and dacryoadenitis coincidentally. However, in the F2 generation of the interWS-099 Immunological diseases of the eye crosses of MRL/lpr mice and non-SS-prone mice, C3H/lpr, these lesions were genetically dissociated. Thus, we graded the severity of both lesions in the F2 Chairpersons: Koh-hei Sonoda, Nobuyuki Ohguro mice under histopathological scoring and then performed a quantitative trait locus analysis with polymorphic microsatellite markers. We identified three sigWS/PP-099-01 Amelioration of experimental autoimmune nificant susceptibility loci to the severity of sialoadenitis on chromosomes 1 and uveoretinitis by blockade of osteopontin with antibody or small 2, designated autoimmune sialoadenitis in MRL mice (Asm)3, Asm4 and Asm5. interfering RNA To those of dacryoadenitis, three significant susceptibility loci were determined on chromosomes 1, 2 and 5, designated autoimmune dacryoadenitis in MRL K. Iwabuchi1,2, D. Iwata3, M. Kitamura3, N. Kitaichi3, S. Kon4, H. Kitamei3, mice (Adacm)1, Adacm2 and Adacm3. Interestingly, Asm3 and Adacm1, and K. Namba3, K. Yoshida3, S. Ishida3, S. Ohno2, S. R. Rittling5, Asm5 and Adacm2 located in the common chromosomal regions each other, D. T. Denhardt6, T. Uede4, K. Onoe1. 1Div Immunobiol, Inst Genetic Med, while Asm4 and Adacm3 were independent. These results indicate that sialoaHokkaido University, Sapporo, Japan, 2Dept Ocular Inflamm Immunol, denitis and dacryoadenitis may develop under the control of susceptibility Grad Sch Med, Hokkaido University, Sapporo, Japan, 3Dept Ophthalmol, genes with a different allelic combination to lead a regular variation of clinical phenotypes of SS. Positional candidate genes for the Adacm3 were discussed. Grad Sch Med, Hokkaido University, Sapporo, Japan, 4Div Mol Immunol, Inst Genetic Med, Hokkaido University, Sapporo, Japan, 5The Forsyth Institute, Boston, MA, United States, 6Dept Cell Biol Neurosci, Rutgers University, Piscataway, NJ, United States WS/PP-099-04 Accumulation of regulatory T cells in the aqueous humour of uveitis patients sEtxupdei eridmeanstaal mauotdoeimlomfuhnuemuavneoernedtiongiteisno(EuAsUu)veinitims,icwehhicahs ibseceonmemxotennssiigvehlty- S. H. Kottoor1,2, K. Morsley1,2, R. Khanfer1,2, A. K. O. Denniston1,2, threatening intraocular inflammatory disease. We have examined an aggravat- I. J. Khan2, K. S. Oswall2, M. Salmon1, K. Piper3, P. I. Murray2, ing role of osteopontin, a protein of pleiotropic functions and contributing to S. J. Curnow1,2. 1Institute of Biomedical Research, School of Immunity the development of Th1/Th17 cell-mediated immunity. Wild type (WT; C57BL/ and Infection, University of Birmingham, Birmingham, United Kingdom, 6) and OPN2/2 mice were immunized with human interphotoreceptor 2Academic Unit of Ophthalmology, School of Immunity and Infection, retionoid-binding protein (hIRBP) peptide sequence 1-20 to induce EAU. University of Birmingham, Birmingham, United Kingdom, 3School of OPN2/2 mice showed milder disease progression compared with WT in clini- Cancer Sciences, University of Birmingham, Birmingham, United cal and histopathological EAU scores with reduced Ag-specific cell proliferation Kingdom and pro-inflammatory cytokine production. OPN was elevated in WT mice along with the progression of EAU. Thus, OPN blockade was performed in WT mice with administration of either M5 antibody specific to SLAYGLR sequence, eL M trcesu trsae tecL cnuL resu itehm Downloaded from of Immunology by guest on 21 April 2018

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Immunological kidney diseases (WS-098): Chairpersons: Yasuhiko Tomino, Jan Novak, International Immunology, 2010, v20-v21, DOI: 10.1093/intimm/dxq357