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.
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
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
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,
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