WSC20/6 TLR-4 IS INVOLVED IN TUBULO-INTERSTITIAL INJURY DURING CHRONIC OBSTRUCTIVE NEPHROPATHYW. P. C. Pulskens1, G.J.D. Teske1, L. M. Butter1, I.K. Luirink1, T. van der Poll2, S. Florquin1, J. C. Leemans1

1Academic Medical Centre, University of Amsterdam, Pathology, Amsterdam, Netherlands, 2Academic Medical Centre, University of Amsterdam, CEMM, Amster-dam, Netherlands

Objective: Tubulo-interstitial injury is a common finding in the chronically injured kidney that is characterized by a cascade of events including inflammation.TLRs can initiate an inflammatory response upon activation by endogenous danger ligands released during sterile tissue injury. Interestingly, TLR4 is constitutivelyexpressed in the kidney. However, the role of this organ-specific expression in progressive renal injury is yet unknown.Methods: In order to study the role of TLR4 in a model of chronic kidney disease, wild type C57Bl/6 and TLR4-/- mice (n=7-8/group) were subjected to unilateralurether obstruction (UUO) by a permanent ligation of one urether. Mice were subsequently sacrificed after 1, 3, 7 and 14 days to determine tubular injury, renalinflammation and renal fibrosis. TLR4, biglycan and HMGB1 mRNA levels were measured by quantitative RT-PCR.Results: We found a significant increase in TLR4 mRNA (t=3, 14) following UUO when compared with sham-operated animals, accompanied by enhanced mRNAlevels of the endogenous TLR4 ligands biglycan (t=7, 14) and HMGB1 (t=14).Interestingly, TLR4-/- mice developed more severe tubular damage in the obstructed kidneys than wild type mice 1, 3 and 7 days post-UUO.TLR4 deficiency did not affect renal inflammation upon UUO, as reflected by equal levels of infiltrating macrophages in the renal interstitium at t=1, 7 and 14 daysand equal levels of chemokines KC and MCP1 in renal homogenates at all time points.Remarkably, TLR4-/- mice showed an attenuated total collagen deposition that was significantly lower 7 and 14 days post-UUO when compared with wild typemice. TLR4 deficiency did not affect the accumulation of myofibroblasts in the kidney.Conclusion: TLR4 and its potential ligands are markedly upregulated in the kidney following UUO. TLR4 deficiency was associated with more tubulo-interstitialdamage but less renal fibrosis, whereas TLR4 deficiency does not influence inflammatory responses or myofibroblast accumulation.

WSC22 – Immunology of the Eye

WSC22/1 CORNEAL ANTIGEN PRESENTING CELLS PROMOTE ALLOGRAFT SURVIVALJ. Schwartzkopff1, D. R. Saban2, A. Beiter1, I. T. Gross1, D. Böhringer1, T. Reinhard1

1University Eye Hospital, Freiburg, Germany, 2Schepens Eye Research Institute/ Harvard Medical School, Boston, United States

Objectives: The risk of corneal allograft rejection seen clinically in children and in infants is considerably high. Baby rat recipients have previously been describedas a good model to study this, as they indeed demonstrate accelerated rejection. In this set of experiments a potential difference in the immunogenicity of a cornealtransplant depending on the donor age was analyzed in the baby rat model.Methods: Corneal allografts were harvested from baby or adult Fisher rats and orthotopically placed on baby or adult Lewis rats (3 or 10 weeks old respectively).In addition chimeric corneal allografts consisting of stroma-endothelium from adult Fisher rats covered by corneal epithelium from baby Lewis rats (or reciprocalchimeric grafts) were also placed orthotopically on baby Lewis rats. Furthermore, grafts were depleted of APC prior to keratoplasty by UVB-irradiation and trans-planted. Graft opacity was regularly assessed until rejection and immunohistological analyses of corneal antigen presenting (APC) cells were performed by fluores-cence and confocal microscopy.Results: In contrast to normal adult corneas, the center of normal baby corneas from donor Fisher rats had large numbers of APCs (DC, MHC-II, CD11c, CD163,)in the epithelial layer. Nonetheless, rejection of such baby allografts was markedly decreased in baby recipients (p X 0.01), and similarly observed in adult recipi-ents as well (p X 0.01). Interestingly, chimeric allografts bearing baby corneal epithelium covering adult stroma-endothelium, showed a strong decrease in therejection rate (p X 0.01) compared to reciprocal chimeric allografts. Depletion of corneal APCs reversed the immunological exeptional position of a young graft andlead to significant rejection rate that was comparable to the one obeserved for old corneal allografts.Conclusion: The immunogenicity of baby corneal allografts appears to be markedly reduced relative to adult donors. Furthermore, our data suggest that this is dueto donor corneal APC born by baby corneal epithelium. We therefore demonstrate evidence that donor corneal APC can promote corneal graft survival.

WSC22/2 SYSTEMIC COMPLEMENT ACTIVATION INCREASES THE RISK OF DEVELOPING AMDL.A. Hecker1, A. O. Edwards1, E. Ryu2, K. H. Baratz1, W.L. Brown1, P. C. Issa3, H.P. Scholl3, B. Pollok-Kopp4, K. E. Schmid-Kubista1, M. Oppermann4

1Mayo Clinic, Rochester, Opthalmology, Rochester, United States, 2Mayo Clinic, Rochester, Biomedical Statistics and Informatics, Rochester, United States, 3Univer-sity of Bonn, Opthalmology, Bonn, Germany, 4University of Göttingen, Cellular and Molecular Immunology, Göttingen, Germany

To determine if systemic complement activation increases the risk of having age-related macular degeneration (AMD) and to understand how established geneticvariants for AMD combine with plasma biomarkers of complement activation to predict development of AMD.Proteins in the alternative pathway of complement (substrate protein factor B, pathway regulators factors D and H, and activation products Ba, C3d, and C5a) werequantified in plasma from 125 AMD and 149 control subjects without AMD. Subjects were genotyped for polymorphisms in CFH (rs800292, rs1061170, rs1048663,rs2274700, rs412852, rs11582939), complement factor B (rs4151667), complement component C2 (rs9332739, rs547154), complement component C3(rs2230199), and ARMS2 (rs10490924). The impact of covariates on protein levels in control subjects was determined and correction made for significant effects.Statistical analysis to compare protein medians (mg/ml) between cases and controls employed non-parametric quantile regression. For prediction of AMD status,stepwise logistic regression was performed and AUC values calculated.The chronic activation products Ba (AMD : 1.07 mg/ml, control : 0.78 mg/ml, p= 0.007) and C3d (AMD : 40.73, control : 35.82, p= 0.002) were significantly ele-vated in plasma from AMD subjects demonstrating increased systemic activation of the alternative pathway of complement. Further, the regulatory protein factorD (AMD : 1.50, control : 1.16, p X 0.0001) and factor B substrate (AMD : 1103, control : 984, p =0.01) were significantly elevated in AMD subjects. Risk genotypesin the CFH and C2/BF loci were associated with increased plasma levels of chronic activation products (Ba, C3d), factor B substrate, and factor D. Models usingcombinations of both proteomic and genetic biomarkers predicted having AMD. Plasma complement levels remained as an independent risk for AMD in a combinedproteogenomic model.Systemic activation of the alternative pathway of complement is associated with increased risk of AMD and correlates with genetic variants in complement genespreviously associated with AMD. Both plasma protein levels and genetic markers were predictive of having AMD. Plasma complement activation persisted as anindependent risk, after adjusting for the genetic risks. Thus, non-genetic factors also appear to increase AMD risk through activation of the alternative pathway ofcomplement.

WSC22/3 AUTOIMMUNE RESPONSES IN BACILLUS CALMETTE-GUERIN (BCG)-INDUCED UVEITISM. Diedrichs-Möhring1, A. Garip1, C. Deeg2, S.R. Thurau1, G. Wildner1

1Section of Immunobiology, Dept. of Ophthalmol., Ludwig-Maximilians-University, Munich, Germany, 2Institute for Animal Physiology, Dept. of Veterinary Sci-ences, Ludwig-Maximilians-University, Munich, Germany

Objectives: M. tuberculosis can cause intraocular inflammation (uveitis) by intraocular infection or accompanying noninfectious vasculitis. However, uveitis canalso occur after vaccination or treatment of bladder cancer with BCG, although BCG does not invade the eye. We therefore speculated that BCG-associated uveitisis caused by antigenic mimicry of mycobacterial and retinal antigens. Here we investigated the case of a 72 years old HLA-B27-negative patient who had developedbilateral granulomatous anterior uveitis after repeated intravesicle treatment with BCG for bladder carcinoma (4 cycles with 6 treatments each). We tested thepatient’s peripheral blood mononuclear cells for proliferation and cytokine/chemokine secretion in response to retinal autoantigens and peptides as well as totuberculin.Methods: Proliferation of lymphocytes to purified protein derivative (PPD) from M. tuberculosis, ocular autoantigens S-Ag, IRBP and their peptides as well as toCRALBP and tetanus toxoid (TT) were tested by 3H-thymidine uptake for the last 18h of 4 days culture. Culture supernatants were collected daily and pooled forcytokine/chemokine testing using the multiplex bead array assay.Results: We found strong proliferative responses to PPD and IRBP protein (SI 31.1), as well as slight responses to IRBP peptide R16 and TT. Stimulation with PPDresulted in high secretion of IL-2, IFN-gamma, TNF-alpha, IL-6, IL-8, MCP-1 and MIP-1-alpha in the supernatants, while IL-4, IL-10 or IL-17 were not detected. Reti-nal autoantigens S-Ag, IRBP and CRALBP as well as the peptides SAg 281, R16, R4/R14 and R4T and TT induced the same cytokine pattern, while IL-2 was unde-tectable.Conclusion: In addition to the proliferative responses of peripheral blood lymphocytes to PPD, TT, IRBP and R16 we observed cytokine/chemokine secretion alsoin cultures with other retinal autoantigens, indicating a polyclonal autoimmune response elicited by BCG. Amino acid sequence alignments revealed homologiesbetween proteins from M.tuberculosis and retinal antigens, suggesting antigenic mimicry as a potential cause of uveitis in this patient.Supported by Deutsche Forschungsgemeinschaft, SFB 571

Eur. J. Immunol. 2009: Wednesday, Workshops S 566–S 610 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim S 597

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WSC22/4 INDUCTION OF ACAID AND ORAL TOLERANCE WITH S-ANTIGEN PEPTIDE AND MIMOTOPES IN RAT AUTOIMMUNE UVEITISS. Thurau1, M. Diedrichs-Möhring1, I. Rädler-Angeli1, G. Wildner1

1Section of Immunobiology, Dept. of Ophthalmology, Ludwig-Maximilians-University, München, Germany

Objectives: Anterior chamber associated immune deviation (ACAID) and oral tolerance induction are two potential pathways to prevent autoimmune disease inLewis rats. In our Lewis rat model of experimental autoimmune uveitis (EAU) we have previously described two mimotopes of retinal soluble Antigen (SAg) pep-tide PDSAg, which are derived from environmental antigens: Rota from rotavirus and Cas from bovine milk casein. Both, Rota and Cas, are pathogenic but lack oraltolerogenicity. Here we investigate whether these peptides can induce ACAID for the prevention of EAU.Methods: ACAID was induced by a single injection of saline (control), PDSAg, Rota or Cas into the anterior chambers of both rat eyes. EAU was induced 7 dayslater by subcutaneous immunization with PDSAg or Rota, respectively, in CFA. For oral tolerance induction the same antigens were fed 3 × every other day, withthe last oral treatment 2-3 days prior to immunization. Disease was graded clinically and histologically.Results: PDSAg effectively suppressed EAU induced with PDSAg when applied orally or into the eye, while Rota or Cas did not ameliorate PDSAg-induced EAU.Intraocular injection of PDSAg, but not of Rota, reduced disease severity of both, PDSAg- and Rota-induced EAU.Conclusion: These data indicate that pathogenic antigens or peptides do not necessarily induce tolerance, neither by the oral route (oral tolerance) nor by intraoc-ular injection (ACAID). This suggests that in both pathways of tolerance induction the regulatory T cells are independent of the effector population with respectto antigen recognition.Supported by the Deutsche Forschungsgemeinschaft SFB 571.

WSC22/5 CORRELATION OF ANTIBODY DEPOSITS AND RETINAL GANGLION CELL LOSS IN ANIMALS WITH EXPERIMENTALAUTOIMMUNE GLAUCOMAS.C. Joachim1, O. W. Gramlich1, P. Laspas1, V. Stahl1, P.F. Gottschling1, C. Cuny1, N. Pfeiffer1, F.H. Grus1

1Experimental Ophthalmology, Ophthalmology, Mainz, Germany

Objectives: In the Experimental Autoimmune Glaucoma (EAG) model retinal ganglion cell (RGC) loss is caused by immunization with ocular antigens. The aimof this study was to examine if this RGC loss correlates with accumulation of antibodies in the retina.Methods: Rats were immunized with bovine retinal ganglion cell-layer homogenate (RGH; n=10) or myelin basic protein (MBP, n=10) in combination with per-tussis toxin (PTX) and incomplete Freund’s adjuvant (IFA). Animals received a booster immunization after 4 weeks. A control group (Co, n=10) received only PTXand IFA. All animals were euthanized after 6 weeks. In a 2 week study animals were immunized with MBP (n=5) or bovine optic nerve homogenate (ONH; n=5).The left eye of each animal was used for RGC count after staining with cresyl. In the right eye antibody deposits were detected in cross-sections via anti-rat-IgG orIgM stain. Deposits were counted on one entire cross-section of each retina. Data were analyzed by ANOVA and post hoc test.Results: In both studies significant RGC loss was observed in rats immunized with ONH (P=0.016), RGH (P=0.006), or MBP (P=0.017 after 2 weeks; P=0.018after 6 weeks). IgG-antibody accumulation could be detected in retinae of immunized rats with a mean count of 26.2 for RGH, 15.2 for MBP, and 10.7 for controls(after 6 weeks). The amount of antibody deposits was significantly higher in RGH animals than in controls (P=0.01). IgG-deposits were mainly located in periph-eral areas of the ganglion cell layer. The number of deposits in MBP animals was not significantly different (P=0.77). IgM deposits were observed epiretinally 2weeks after immunization.Discussion: Significant RGC loss as well as specific antibody accumulation patterns could be observed after systemic immunization with ONH, RGH or MBP.These studies demonstrate that neuro-retinal antigens cause an accumulation of antibodies in the retina, which may play an important role in the pathogenesis ofRGC loss in the EAG model. In this model retinal antibodies could capture membrane receptors of ganglion cells which influence important physiological functionssuch as communication and supply deliverance.Support: Boehringer Ingelheim Foundation.

WSC22/6 BLAU SYNDROME-RELATED CARD15 POLYMORPHISMS ARE NOT LINKED TO IDIOPATHIC UVEITIS IN SPANISH PATIENTSN. Rodr ıguez-Perez1, M.B. Gorrono-Echebarr ıa2, A. Aguinaga-Barrilero1, M. Perez-Blas1, J.M. Mart ın-Villa1

1Universidad Complutense de Madrid, Inmunolog ıa, Madrid, Spain, 2Hospital Universitario Pr ı ncipe de Asturias, Oftalmolog ıa, Alcala de Henares, Spain

Background: Uveitis is a clinical feature of the Blau syndrome, a disease linked to CARD15 mutations. However, little is known on the involvement of this genein idiopathic uveitisObjective: To determine the frequency of Blau-related CARD15 mutations in a cohort of Spanish patients with idiopathic uveitis.Settings, Patients and Methods: One hundred and ten patients with idiopathic uveitis, followed at the Department of Ophtalmology of a tertiary hospital (Hospi-tal Universitario Alcala de Henares, Madrid. Spain) were enrolled. As a control population, 104 healthy subjects were used. DNA was extracted form blood samplesand the Blau-related CARD15 polymorphisms were analysed either by PCR-RFLP or direct DNA sequencing.Results: None of the polymorphisms studied was found in any of the individuals tested, whether diseased or healthy.Conclusions: Blau syndrome-related CARD15 mutations are not involved in idiopathic uveitis. This allows us to suggest that the genetic aetiology of the idiopathicuveitis or the Blau-associated uveitis is different.

WSD08 – Tumor Vaccines

WSD08/1 INHIBITION OF PI3K SIGNALLING IN DENDRITIC CELLS REDUCES TLR LIGAND REGULATORY T CELL INDUCTION ANDENHANCES THEIR EFFICACY AS TUMOUR VACCINESN. A. Marshall1, H. Conroy1, A.G. Jarnicki2, K.H.G. Mills1

1Trinity College Dublin, Biochemistry and Immunology, Dublin, Ireland, 2Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Melbourne, Australia

Objectives: Vaccination with antigen pulsed dendritic cells (DC) is an attractive approach for the treatment of tumours. However, hurdles such as the mixed phe-notype of responses (Th1+Treg) induced by such DCs and tumour mediated immune suppression will need to be overcome for clinical success. We tested the suit-ability of PI3K inhibitors for modulating DC vaccines away from regulation and towards anti-tumour effector T cell responses.Methods: Synthetic compounds designed to block each of the four PI3K isoforms are available and allow specific inhibition of signalling by PI3K isoforms. BMDCwere activated with the TLR ligand flagellin (FLG) and/or heat shocked irradiated (hs/ir) tumour cells, and the ability of PI3K inhibitors to modulate cytokine pro-duction (ELISAs for IL-10, IL-12p70, TNF-a, TGF- b) and DC maturation (FACs staining of CD25/40/80/86 and MHCII expression) was examined. PI3K inhibitorswere also tested for the ability to modulate T-cell responses and to prevent tumour growth in vivo, either directly (intra-tumour injection of PI3K inhibitor with FLGand hs/ir tumour cells) or via the transfer of in vitro primed DC into the tumour mass.Results: Maturation of DCs by FLG or tumour antigen resulted in an increase in the production of both IL-12 and IL-10. Co-incubation of DC with specific PI3K sub-type inhibitors demonstrated that PI3K b and d were central for the induction of IL-10 secretion in response to TLR stimulation. PI3K b and d inhibitors were bothable to effectively suppress TLR and tumour cell induced IL-10 secretion, whilst maintaining levels of IL-12 production. In addition, pre incubation of DCs withthese inhibitors prior to TLR and tumour cell stimulation did not prevent or inhibit DC maturation. Immunotherapy with heat shocked-irradiated tumor cells anda TLR agonist in the presence of inhibitor blocking PI3K b and d activation protected against B16 tumors in mice by preventing Treg cell induction and enhancingTh1/CTL responses.Conclusion: The use of PI3K b and d inhibitors to bias DCs away from a regulatory phenotype and towards an anti-tumour response is a promising approach forthe development of effective anti-tumour vaccines.

WSD08/2 DC-SIGN MEDIATED ANTIGEN-TARGETING USING GLYCAN MODIFIED PROTEINS ENHANCES CD8 AND CD4 T CELL RESPONSESW. W. Unger1, S.K. Singh1, J. Stephani2, M. Schaefer2, M. Litjens1, H. Kalay1, J. Garcia-Vallejo1, J. den Haan1, E. Saeland1, T. Sparwasser2, Y. van Kooyk1

1VUMC, Molecular Cell Biology and Immunology, Amsterdam, Netherlands, 2Institute for Infection Immunology, Twincore, Centre of Experimental and ClinicalInfection Research, Hannover, Germany

Dendritic cells (DC) are key antigen presenting cells that have the unique ability to cross present tumor-derived antigens on MHC class I, resulting in effective prim-ing of cytotoxic T lymphocytes (CTL). DC express C-type lectin receptors that bind and subsequently mediate the uptake of carbohydrate structures appended toglycoproteins. Here we have explored the specific expression of the C-type lectin receptor DC-SIGN to target antigens to DC, to improve anti-tumor responses. Oval-bumin (OVA) was used as model antigen and modified with the DC-SIGN specific glycans LewisX or LewisB. The ability of these OVA-glycoconjugates to increasethe efficiency of antigen presentation by murine human DC-SIGN-transgenic (hSIGN Tg) bone marrow-derived DC (BMDC) to OVA-specific CD4 and CD8 T cellswas evaluated. We observed that glycan-modification of OVA targets antigens specifically to BMDC and splenic DC. Furthermore, incubation of hSIGN Tg BMDCwith OVA-glycoconjugates increased CD4 T cell proliferation 10-fold compared to unmodified OVA. Interestingly, we found that incubation of hSIGN Tg BMDC, butnot wt BMDC with OVA- LewisX or -LewisB led to enhanced crosspriming and subsequent CD8 T cell proliferation. Targeting of glycosylated OVA was neither accom-panied with any DC maturation, nor production of inflammatory cytokines. Preliminary data indicate that following OVA-glycoconjugate injection in hSIGN Tgmice a higher percentage of OVA-specific CD8 T cells proliferated and produced IFN-g compared to wt mice.Our results demonstrate that glycan modification of antigen and targeting to DC-SIGN enhanced both CD4 and CD8 T cell responses. Currently, we are evaluatingthe use of glycan-modified liposomes as nanocarriers to target antigens to DC. These vaccination strategies may be valuable for the induction of anti-cancerimmune responses.

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2009: Wednesday, Workshops S 566–S 610S 598