S125Abstracts

in all or a particular subset of SSc patients. In contrast, IL-17itself was not detected in the majority of SSc patients.Discussion: The combination of IL-17, IFNγ and TGFβ levels inCD45Ro and CD45Ra cells from SSc patients is useful todistinguish between lSSc, ldSSc or edSSc. Blocking Th17inducing cytokines such as IL-6 and IL-23 may provide a usefultool to intervene in the progression of SSc.

doi:10.1016/j.clim.2009.03.368

F.117. Are HLA-E Restricted CD8+ T CellsFunctionally Altered in Multiple Sclerosis?Kim Pannemans1, An Goris2, Rita Dobosi2, BenedicteDubois2, Bart Van Wijmeersch1, P. Stinissen1, NielsHellings1. 1Hasselt University and TransnationaleUniversiteit Limburg, Diepenbeek, Belgium;2University Hospital Gasthuisberg, Leuven, Belgium

Qa-1 restricted CD8+ regulatory T cells suppress autoreac-tive CD4+ Tcells in the animalmodel formultiple sclerosis (MS).This study aims at elucidating the involvement of CD8+ T cellsrestricted by HLA-E, the human variant of Qa-1, in immunor-egulatory alterations in MS. In a first part, an MS associationstudy of HLA-E polymorphisms was performed. The HLA-E geneharbors two polymorphic sites, codon 107 (HLA-E⁎0101 versusHLA-E⁎0103) and codon 77 (HLA-E⁎010301 versus HLA-E⁎010302). These polymorphisms were studied in 1078 HCand 832 MS patients. Plink option analysis showed a significanthigher presence of HLA-E⁎010301 and lower frequency of HLA-E⁎0101 in MS patients (p = 0.04). Conditioning on DRB1⁎1501revealed that this HLA-E association actually reflected theeffect of DRB1⁎1501 on the development of MS. Although nogenetic association was found, functional alterations in HLA-ErestrictedCD8+ Tcells could still be involved in thedysbalancedautoimmune responses observed in MS patients. Therefore,HLA-E restricted CD8+ Tcells are characterised at the level oftheir phenotype and regulatory potential. First, the phenotypeof both NKG2C+ and NKG2A+CD8+ Tcells was studied using flowcytometry. The frequency of NKG2A+CD8+ T cells was notsignificantly different between HC (n=10, 6.9± 3.2 %) and MSpatients (n=10, 8.7±3.2 %). The same could be concluded forNKG2C+CD8+ T cells (HC: 3.82±2.1 %, MS: 3.32± 1.0 %). Thepercentage of CD28+ T cells within both the NKG2C+ andNKG2A+CD8+ Tcell fraction is declined inMS patients comparedto HC. The functional consequence of this observation iscurrently investigated in coculture experiments with auto-reactive T cells and these CD8+ subsets. Samples will besubdivided according to their HLA-E genotype. This study couldlead to new insights in the pathogenesis of MS.

doi:10.1016/j.clim.2009.03.369

F.118. Association of Impaired NK Cell Activity withImbalance in CD3-CD16+ Bright and Dim SubsetDistribution and Receptor Expression in MetastaticMelanoma PatientsGordana Konjevic2, Katarina Mirjačić Martinović1, AnaVuletic1, Nada Babovic1. 1Institute for Oncology and

Radiology of Serbia, Belgrade, Serbia; 2School of Medicine,University of Belgrade, Beograde, Serbia

As we show that NK cell activity in metastatic melanoma(MM) patients prior to therapy is impaired the aim of this studywas to investigate the expression of a set of NK cell receptorsand activation markers on the two functionally distinct,regulatory CD16dim and cytotoxic CD16bright NK subsets.PBL of MM and controls were analyzed for NK cell activity,percentage of CD3-CD16 dim and bright subsets, expression ofCD107a, NKG2D, CD161, CD158a and CD158b on NK cell andintracellular production of IFN-γ, as well as the expression,after in vitro 4 h co-culture of PBL with tumor cell lines, ofCD161 and CD158a on these subsets by Flow cytometry. Weshow thatMMpatients not only have significantly decreasedNKcell activity, NK cell CD107a expression and IFN-γ production,but also a significantly larger CD16dim and smaller cytotoxicCD16bright NK subset. Furthermore, after co-culture withtarget tumor cell lines, K562and FemX, the percentage of CD3-CD16bright, contrary to CD3-CD16dim NK cells significantlydecreased, aswell as CD161 and CD158a on CD3-CD16bright NKsubset. Also, in support of suppressed NK cells is our novelfinding of decreased NK cell IFN-γ production in MM patients,that can affect their cytotoxicity, as reflected in CD107adecrease. The novel results for NK subsets showing howsecondary NK cell contact with tumor cells changes CD161 andCD158a expression supports the tumor-related nature of thisphenomenon. Therefore, we report an unfavorable tip of thebalance towards the less mature, CD16bright158alow NK cellphenotype in MM patients.

doi:10.1016/j.clim.2009.03.370

F.119. FTY720+Sirolimus Increase Skin AllograftSurvival via Cell Phenotype ChangesCamila Lopes1, Flavia Rosin1, José Cordeiro2, ValquiriaBueno1. 1UNIFESP Federal University of São Paulo, SãoPaulo, Brazil; 2FAMERP São José do Rio Preto MedicalSchool, São José do Rio Preto, Brazil

FTY720(FTY) prevents lymphocyte(Ly) egress from lymphnodes(LN) and thymus whereas Sirolimus(SRL) prevents Tcellactivation. Skin allograft recipient mice were either non-treated(Tx) or treated with SRL or FTYor FTY+SRL(1 mg/Kg/day).FTY+SRL had no rejection vs Tx:9.8±0.8/SRL:15.0±0.5/FTY:14.8±0.9 days,pb0.0005. On day +5, FTY+SRL hadlower counts of splenocytes vs Tx and SRL[Tx:62.0(18.5)/SRL:34.4(10.2)/FTY:30.0(8.7)/FTY+SRL:24.6(6.8)x106,pb0.05];thymocytes (THY) vs Tx and FTY[Tx:43.2(7.8)/SRL:26.3(40.3)/FTY:40.0(6.9)/FTY+SRL:25.2(6.5)x106,pb0.005];CD4+ cells in spleen (SPL) vs Tx and SRL[Tx:15.9(5.2)/SRL:9.0(2.6)/FTY:5.5(3.4)/FTY+SRL:4.3(1.1)x106,pb0.005],in LN vs Tx [Tx:0.6(0.2)/SRL:0.6(0.1)/FTY:0.3(0.2)/FTY+SRL:0.1(0.1)x106,pb0.05], in peripheral blood(PB) vs others[Tx:22.4(13.9)/SRL:25.1(4.4)/FTY:2.2(1.6)/FTY+SRL:1.1(0.4)%,pb0.05;CD8+ cells in SPL vs Tx[Tx:13.0(3.7)/SRL:5.9(1.5)/FTY:4.8(3.6)/FTY+SRL:5.4(1.0)x106,pb0.005],in LN vs others[Tx:0.6(0.5)/SRL:04(0.5)/FTY:0.5(0.2)/FTY+SRL:0.3(0.2)x106,pb0.05] and in PB vs SRL[Tx:15.8(4.0)/SRL:17.9(4.1)/FTY:1.8(0.3)/FTY+SRL:6.5

S126 Abstracts

(3.9)%,pb0.05];CD4+CD8+ THY vs Tx[Tx:39.9(5.6)/SRL:23.4(36.3)/FTY:33.4(10.2)/FTY+SRL:18.3(4.7)x106,pb0.005], Lyin PB vs Tx and SRL[Tx:63.0(27.0)/SRL:50.0(14.8)/FTY:15.0(5.0)/FTY+SRL:17.0(4.3)%,pb0.005];CD11b+ cells in theallografts vs FTY[Tx:8.8(9.2)/SRL:7.2(6.2)/FTY:14.1(22.1)/FTY+SRL:4.6(2.4)%,pb0.05];higher counts of CD11b+ non-Ly inSPL vs Tx[Tx:42.9(18.2)/SRL:22.6(6.0)/FTY:21.7(5.0)/FTY+SRL:20.7(7.1)x106,pb0.005]; CD11b+ lymphocytes vs Tx andSRL in PB[Tx:6.0(3.5)/SRL:7.3(7.4)/FTY:50.7(3.3)/FTY+SRL:43.5(10.6)%,pb0.005];CD11b+ non-Ly vs Tx and SRL in PB[Tx:42.7(28.7)/SRL:56.5(42.4)/FTY:93.5(9.8)/FTY+SRL:93.4(1.5)%,pb0.005].Increased allograft survival in FTY+SRL wasassociated with decreased peripheral and allograft-infiltratingimmune cells.

doi:10.1016/j.clim.2009.03.371

F.120. FTY720 Treatment Impairs Lung TumorDevelopment Due to Decrease of MyeloidSuppressor Cells (CD11b+Gr-1+) and RegulatoryT Cells (CD4+CD25hiFoxp3+)Flavia Rosin1, Camila Lopes1, Patricia Cury2, CelinaOshima1, Valquiria Bueno1. 1Federal University ofSão Paulo - UNIFESP, Sao Paulo, Brazil; 2Sao Jose do Rio PretoMedical School, Sao Jose do Rio Preto, Brazil

Introduction: Tumor growth is associated with the increaseof myeloid suppressor cells (MySC:CD11b+Gr-1+) and regula-tory Tcells (Treg:CD4+CD25highFoxp3+), which inhibit CD8+ Tcell responses to specific tumor-associated antigens. FTY720experimentally reduces tumor growth and metastasis. How-ever, there is no report whether FTY720 interferes with Treg orMySC expression during tumor development. Aim: To investi-gate the role of FTY720 in urethane-induced lung tumor.Methods: Lung tumor was induced in BALB/c mice by twoinjections of urethane (1,5 g/Kg each) with a 48-hour interval.Mice were either non-treated (UR/n=9) or FTY720-treated(5 mg/Kg/day, FTY/n=10) during five consecutive daysstarting 30 days after the last urethane injection. Spleen,thymus and lungs were harvested 120 days later. Using flowcytometry we evaluated CD3+CD4+,CD3+CD8+ and Treg cells inspleen and thymus andMySC in spleen. Nodule and hyperplasiaareas (NHA) were measured in lung sections. Results: FTY720smaller total NHA (UR:2.21 mm2/FTY:2.00 mm2) was asso-ciated with decreased Treg (UR:7.6±2,2%/FTY:6.8±2,3%) andMySC (UR:25.4±8.9%/FTY:23.0±7.%) in spleen and withincreased Treg in thymus (UR:1.0±1.0%/FTY:2.8±3.9%). Also,FTY720 decreased CD3+CD4+ and CD3+CD8+ T lymphocyte inspleen (CD3+CD4+:UR:33.2±10.7%/FTY:29.5±8.2%; CD3+CD8+:UR:16.2±4.2%/FTY: 15.0±4.6%) and thymus (CD3+CD4+:UR:13.3±4.0%/FTY:11.8±3.8%; CD3+CD8+:UR:7.2±3.4%/FTY:6.2±3.1%). Conclusion: In urethane-induced lungtumor FTY720 administration was able to limit tumor devel-opment and the relatedmechanismswere the decrease of Tregand MySC in spleen, which could lead to the non-inhibition ofthe immune response against tumor. Key-words: Lung Cancer,Treg Cell, Suppressor Cell, Flow Cytometry.

doi:10.1016/j.clim.2009.03.372

F.121. B Cell-mediated Autoimmunity inHypomorphic rag1 and lig4 Mouse Mutants asModels for Human Leaky SCIDJolan Walter1, Francesca Rucci1, Laura Patrizi1, Emma-MariaAndersson1, Marton Keszei2, Catherine Yan2, Irina Gavanescu3,Pietro Poliani5, JoAnn Sekiguchi4, Frederick Alt3,Luigi Notarangelo1. 1Children's Hospital Boston, HarvardMedical School, Boston, MA; 2Beth Israel Deaconess MedicalCenter, Harvard Medical School, Boston, MA; 3HarvardMedical School, Boston, MA; 4University of Michigan, AnnArbor, MI; 5University of Brescia, Spedali Civili of Brescia,Brescia, Italy

T-cell mediated immunopathology is a hallmark of humancombined immunodeficiencies (Omenn syndrome, leaky SCID)due to hypomorphic mutations in genes involved in V(D)Jrecombination. These defects are also expected to result inimpaired receptor editing, and hence in B-cell mediatedautoimmunity. However, the contribution of autoantibodyformation to the pathology of Omenn syndrome and leaky SCIDhas not been investigated. We have studied two mouse modelswith hypomorphic mutations in rag1 (rag1S723C/S723C) andlig4 (lig4R278H/R278H) that recapitulate leaky SCID. In bothmodels, V(D)J recombination is impaired, but not abrogated.Both mice show a severe, but incomplete, block in B celldevelopment at the pre to proB cell stage. Both mouse modelshave detectable IgG, IgM, IgA, with significantly higher levelsin case of the rag1 hypomorphicmodel. ELISPOTassays indicatethat antibody-secreting cells are present both in the spleenand bone marrow, thus confirming that the hypomorphicmutations are permissive for residual B cell development andfunction. Importantly, autoantibodies to dsDNA and tochromatin were detected by ELISA, Hep2 cell line stainingand immunohistochemistry in a significant proportion of mice,andweremore frequently observed in rag1mutants.Moreover,the frequency of autoantibody-producing mice increases withage. Overall, these data indicate that B-cell-mediated auto-immunity may be an important component of the immuno-pathology of leaky SCID and Omenn syndrome, presumablybecause of stochastic generation of an autoreactive B cellrepertoire, associated with impaired receptor editing.

doi:10.1016/j.clim.2009.03.373

F.122. Toll Like Receptor 2 Promotes Th2/Th17Responses via TLR4 and TLR8 by Human DendriticCells by Abrogating the Type 1 InterferonAmplification LoopMark Wenink1, Kim Santegoets1, Lenny van Bon1, JasperBroen1, Richard Huijbens1, Erik Lubberts2, Piet van Riel1,Wim van den Berg1, Timothy Radstake1. 1Radboud UniversityNijmegen Medical Center, Nijmegen, Netherlands; 2ErasmusMedical Center, Rotterdam, Netherlands

The precise function of TLR2 in the immune system remainselusive. We delineated the role of TLR2 in the determinationof the fate of human dendritic cells (DCs) and it's interactionwith other TLRs. Monocyte-derived DCswere cultured from 15healthy controls and in vitro experiments were performed.Both P3C (TLR2/1 ligand) and P2C (TLR2/6) dose-dependently