S49Abstracts

proteins of the src family (Lyn, Fyn) that are activated earlyin the pathway. Studies in mice performed by our group havepointed out that CSK expression levels are associated withthe development of autoantibodies. Furthermore, prelimi-nary data generated from the UCSF Lupus Genetics Projectcollection suggested an association (odds ratio (OR)=1.20)between several CSK variants and systemic lupus erythema-tosus (SLE) in subjects with European background. All thevariants associated with SLE were located on a commonhaplotype. Therefore, we sequenced the 14 exons of the CSKgene, as well as five intronic regions of high homologybetween mice and humans in 24 lupus patients homozygousfor the associated haplotype. The results of the sequencingshowed the A variant of rs34933034 (G/A), located within aphylogenetically conserved intronic region, was present in 23of the 24 subjects. We then carried out a case–control studyin 480 SLE samples and 538 controls. The results show an ORof 1.32 (Fisher test, P=0.026) for the A variant. Genotypingfor rs34933034 in an expanded SLE and control cohort is inprogress. Given that signaling through the BCR regulates notonly the response of the B cells towards an antigenicchallenge but also the selection of clones for maturationand the survival of cells once in the periphery, the study ofthe physiological consequences of this variation for Cskexpression is being addressed.

doi:10.1016/j.clim.2010.03.148

T.34. Systemic Activation of Macrophages byInterferon-gamma Rapidly Induces Severe Anemiaand HemophagocytosisErin Zoller, Catherine Terrell, Julio Aliberti, AlexandraFilipovich, Michael Jordan. Cincinnati Children's HospitalMedical Center, Cincinnati, OH

Hemophagocytosis, or ‘blood eating’, by macrophages is aphenomenon seen in hemophagocytic lymphohistiocytosis(HLH) and other severe inflammatory settings. The mechan-isms of this uncharacterized physiologic phenomenon andwhether or not it is a cause of cytopenias in these settings arelargely unknown. It is speculated to be caused by hypercy-tokinemia, including high levels of interferon gamma (IFN-γ), as IFN-γ is linked to HLH disease severity. We have shownpreviously that IFN-γ is necessary for development of severecytopenias in a mutant mouse model of HLH. We report herethe necessity of IFN-γ in the development of anemia in wild-type mice infected with Toxoplasma gondii. We alsodetermined that systemic in vivo infusion of IFN-γ outsideof the context of an infection is sufficient to cause rapid andsevere consumptive anemia in a dose-dependent manner aswell as cause hemophagocytosis of red blood cells bymacrophages. This anemia is dependent on STAT1 and IRF-1, molecules directly downstream of the IFN-γ receptor.Importantly, we see that transgenic mice expressing adominant negative mutant IFN-γ receptor under a macro-phage-specific promoter (CD68) are protected from bothanemia and hemophagocytosis. This, along with our otherdata, 1) indicates that high but physiologically relevant dosesof IFN-γ alone can act directly on macrophages to cause

anemia development and hemophagocytosis and 2) high-lights a novel common mechanism of the two processes.

doi:10.1016/j.clim.2010.03.149

T.35. Decreased Soluble TNF-α in SpondyloarthritisVersus Rheumatoid Arthritis SynovitisCarmen Ambarus, Nathaliya Yeremenko, Huriatul Masdar,Paul-Peter Tak, Dominique Baeten. Academic MedicalCenter-University of Amsterdam, Amsterdam, Netherlands

Background: Tumor necrosis factor-α (TNF) is a pivotalmediator of synovial tissue (ST) inflammation. However, thelevels of soluble TNF (sTNF) are strongly reduced in SpAversus RA synovial fluid (SF). Therefore, we aimed to unravelpotential differences in TNF signalling between SpA and RA.Methods: ST and SF were obtained from actively inflamedknee joints of RA and SpA patients. Soluble mediators in SFwere analyzed by ELISA. ST expression was assessed by qPCRand immunohistochemistry. Results: sTNF was significantlylower in SpA than in RA SF (Pb0.001), while mRNA levels ofTNF were comparable. This discrepancy could be explainedby increased soluble TNF (decoy) receptors, by relativelyhigher tmTNF versus sTNF or by increased intracellulardegradation of TNF in SpA. Expression of TNF-R1 and TNF-R2was moderately decreased at mRNA level in SpA versus RA,but similar when assessed by immunohistochemistry. sTNF-R1 and sTNF-R2 in SF were substantially lower in SpA than RA(Pb0.001). TACE (TNF-cleaving enzyme) mRNA levels in ST,as well as sIL-6R (also cleaved by TACE) and TIMP-3 (inhibitorof TACE) levels in SF were similar in SpA and RA. Westernblotting on SpA and RA ST is currently being performed.Conclusion: The differences in SF sTNF levels between SpAand RA cannot be explained by increased decoy receptors orTACE activity in SpA. Our data suggest that maintainedmembrane expression of both TNF receptors in combinationwith decreased decoy receptors might potentiate TNFsignaling in SpA. It remains to be assessed whether thissignaling is dependent on tmTNF rather than sTNF.

doi:10.1016/j.clim.2010.03.150

T.36. Constitutive Expression of CD25 on theSurface of Neonatal (invariant) Natural Killer T CellsPrimes Them to Proliferate with Lower AntigenicStimulationAshish Sharma1, Mihoko Whalen1, Qing Huang1, AdeleWang2, Lixin Xu1, Indira Genowati1, Meghan Levings2,Pascal Lavoie1. 1Child and Family Research Institute,Vancouver, BC, Canada; 2University of British Columbia,Vancouver, BC, Canada

Invariant natural killer T (iNKT) cells in neonates areknown to constitutively express the IL-2 receptor alpha chain(CD25). We explored the functional consequence of theexpression of CD25 on the surface of neonatal iNKT cells by

S50 Abstracts

comparing their functional properties to other common CD25-expressing T cell subsets. Unlike CD25-expressing activated Tcells, iNKT cells seem to express high levels of Kruppel-LikeFactor-2, a transcription factor highly expressed in quiescent Tcells. Furthermore, neonatal iNKT cells differ from CD25+CD4+

T regulatory cells as they do not suppress conventional T-cellproliferation. Upon stimulationwith CD1d-restricted antigens,neonatal iNKT cells display a dramatically lower proliferationthreshold compared to adult iNKT cells. Further experimentsusing a competitive blocking IL-2 antibody suggest that themarkedly reduced proliferation threshold of neonatal iNKTcells is, at least in part, due to the constitutive expression ofCD25. The functional consequence of CD25 expression onneonatal iNKT cells appears to be distinct from that ofactivated T cells, in that the former do not proliferate whenexposed to IL-2 in the absence of T-cell receptor (TCR)/CD28costimulation. In light of the limited structural diversity ofiNKT cells' TCR, we speculate that neonatal iNKT cells' uniqueand distinct phenotype plays a role in the maintenance of adiverse repertoire with repeated low avidity antigenicchallenge early in life.

doi:10.1016/j.clim.2010.03.151

T.37. Ethylenecarbodiimide-treated SplenocytesCarrying Male CD4 Epitopes Confer Hya TransplantTolerance by Interfering with CD40/CD154 SignalsAaron Martin, Derrick McCarthy, Stephen Miller.Northwestern University, Chicago, IL

In humans and certain strains of laboratory mice, maletissue is recognized as non-self and destroyed by the immunesystem of females. Destruction of male tissue grafts isaccomplished by CTLs in a helper-dependent fashion.Drawing on our laboratory's experience in inducing antigen-specific tolerance in autoimmune diseases, we attachedpeptides representing the immunodominant CD4 T cellepitope in male antigen (Dby) to donor leukocytes with thechemical cross-linker ethylene–carbodiimide (ECDI). Admin-istration of the antigen-coupled leukocytes (Dby-SP) torecipient B6 females prior to male skin engraftment resultedin a significant increase in survival of male skin graftscompared to nontolerized recipients while treatment withleukocytes coupled to the CD8 epitopes (Uty and Smcy) failedto prolong graft survival. In accord with previous autoimmu-nity studies from our laboratory, we demonstrate that CD4cells tolerized in this manner fail to proliferate or secretecytokines in response to male antigen. Here we alsodemonstrate that tolerized CD4s fail to effectively prime aCD8 response. In Dby-tolerized recipients, male antigen-specific CD8 cells retain a naïve surface phenotype, fail tosecrete interferon-γ, and fail to lyse labeled targets in vivo.Two lines of evidence suggest that CD40-CD40L interactionscontribute to these observations: (1) tolerized CD4 cellsexhibit a defect in CD40L upregulation following subsequentantigen challenge in vitro and (2) treatment with agonisticanti-CD40 at the time of transplantation abrogates toler-ance. Interestingly, anti-CD40 treatment completely re-

stored the function of Dby-specific CD4 cells, but not of CD8cells specific for Uty or Smcy.

doi:10.1016/j.clim.2010.03.152

T.39. Serum Neopterin Levels as a Diagnostic Markerof Hemophagocytic Lymphohistiocytosis SyndromeMaria Ibarra, Marisa Klein-Gitelman, Elaine Morgan, MariaProytcheva, Christine Sullivan, Gabrielle Morgan, LaurenPachman, Maurice O'Gorman. Children's Memorial Hospital,Chicago, IL

Hemophagocytic lymphohistiocytosis (HLH) is a potentiallylethal condition characterized by exaggerated macrophageactivation leading to hyper cytokines production. Early diagnosiscan be difficult because complete diagnostic criteria are notfulfilled at initial presentation in substantial proportion ofpatients. Neopterin (activated macrophages byproduct) is aninflammationmarker associatedwith cell mediated immunity invarious diseases. In this study, we evaluated serum neopterin asHLH diagnostic marker. Patients with HLH (primary andsecondary) between January 2000 andMay 2009were identifiedand retrospective chart reviewwas performed. Neopterin levelsand clinical/laboratory information related to HLH criteriawererecorded. Neopterin results from HLH group were compared toresults from control group [50 active juvenile dermatomyositis(JDM) patients]. Neopterin diagnostic accuracy was measuredby area under curve and tradeoffs in sensitivity and specificityobtained by receiver operating characteristic analysis. Logisticregression modeling measured association between serumneopterin and HLH-related laboratory data. Serum neopterinlevels were measured by competitive immunoassay (normalrangeb10 nmol/L). Results: Serum neopterin results wereavailable from 16 patients with secondary and 5 patients withprimary HLH. Mean neopterin level for HLH was 84.9 (SD: 83.4)and for JDM was 21.5 (SD: 10.13). Cutoff value (38.9 nmol/L)was 70% sensitive and 95% specific for HLH.Within HLH patients,neopterin levelswere associatedwith ferritin (P=0.0007) andD-dimer (P=0.018) and inversely with platelet count (P=0.034)and fibrinogen (P=0.035). Conclusion: HLH patients hadneopterin levels three times greater than normal cutoff.Compared to JDM patients, elevated serum neopterin was asensitive and specific HLH marker.

doi:10.1016/j.clim.2010.03.153

T.40. Myeloid-derived Regulatory Cells in theAirways of Asthmatic and COPD SubjectsJessy Deshane, Meiqin Zeng, Jaroslaw Zmijewski, JohnAnderson, Jack Lancaster, Marion Spell, Naomi Fineberg,David Redden, Edward Abraham, Amit Gaggar, MarkDransfield, David Chaplin. University of Alabama atBirmingham, Birmingham, AL

Free radicals such as nitric oxide (NO) and superoxide(O2.−) and infiltrating myeloid cells contribute to airway