S132 Abstracts

but also can be a driving force of chronic inflammation. Th1and Th17 cells are distinct lineages of effector/memory cells,imprinted for reexpression of interferon-γ (IFN-γ) andinterleukin-17 (IL-17), by upregulated expression of T-betand RORγt, respectively. Apparently, Th1 and Th17 cells sharetasks in the control of inflammatory immune responses. Thcells coexpressing IFN-γ and IL-17 have been observed in vivo,but it remained elusive, how these cells had been generatedand whether they represent a distinct lineage of Th differen-tiation. Ex vivo isolated IL-17 secreting Th17 cells, generatedin vivo, have a stablememory for reexpression of IL-17 in vitro,upon restimulation by antigen. They do not respond to IL-12 orIL-4, the Th1 and Th2 polarizing signals (Lexberg et al., Eur. J.Immunol. (2008) 38(10):2654-64). Here we show that ex vivoisolated Th17 cells can be converted into Th1/17 cells bycombined IFN-γ and IL-12 signaling. IFN-γ is required toupregulate expression of the IL12Rβ2 chain, and IL-12 for Th1polarization. These Th1/17 cells stably coexpress RORγt andT-bet on the single cell level, and are imprinted for reexpressionof both IFN-γ and IL-17. Th1/17 cells thus represent a distinctlineage of Th cell differentiation, combining the pro-inflam-matory potential of Th1 and Th17 cells.

doi:10.1016/j.clim.2010.03.397

S.76. Increased Fraction of IL-17 Secreting Cells inType 1 DiabetesAshish Marwaha, Megan Levings, Sarah Crome, ConstadinaPanagiotopoulos, John Priatel, Rusung Tan. University ofBritish Columbia, Vancouver, BC, Canada

Type 1 diabetes (T1D) results from the autoimmune self-destruction of insulin producing pancreatic beta-cells. Theestablished role of regulatory T cells (Tregs) in suppressingautoimmunity has lead to the hypothesis that aberrations intheir numbers may underlie T1D pathogenesis in humans.Previous studies have enumerated Treg numbers based onexpression of the transcription factor FOXP3 for their identifi-cation. However, recent evidence has emerged that human CD4+ FOXP3 expressing cells include subsets that are non-suppressive and secrete the proinflammatory cytokine interleu-kin-17 (IL-17). IL-17 is produced by a distinct T helper celllineage (Th17) that have also been implicated in a number ofautoimmune diseases butwhose role in human T1D has not beendefinitively established. We observed an increase in theproportion FOXP3-expressing CD4+ T cells in new onset T1Dpatients as compared to age-matched healthy controls, which isnot representativeof an increase in suppressiveTreg cell subsetsbut is confined to a subset of cells that express FOXP3 at anintermediate level, lack thememorymaker CD45RA and secreteIL-17. Furthermore, we found that there is an increase in theproportion of other IL-17 secreting cell subsets, both Th17 andTc17 cells, in T1D subjects as compared to healthy controls. Thisstudy confirms that the sole use of FOXP3 as a marker for Tregcells will inadvertently include IL-17 secreting cells. We alsoestablish, for the first time, an increase in the proportion ofmultiple IL-17 secreting cell subsets in new onset T1D patients.

doi:10.1016/j.clim.2010.03.398

S.77. Blockade of CD40 Signaling on DiabetogenicCD4 T Cells Inhibits Effector FunctionRocky Baker1, Thierry Mallevaey1, Laurent Gapin2, KathrinHaskins1. 1University of Colorado Health Sciences Center,Denver, CO; 2National Jewish Health, Denver, CO

IFN-γ-secreting Th1 T cells play a pivotal role in thedevelopment of type 1 diabetes. We previously reported thatdiabetogenic Th1 T cell clones express the costimulatorymolecule CD40 and that treatment of these cells with anantibody to CD40 abrogates their capacity to transfer disease,indicating that engagement of CD40 on T cells contributes totheir diabetogenicity. To further investigate the effect ofCD40blockade on effector function of Th1 cells,weproducedavariant of a diabetogenic T cell clone BDC-5.2.9 expressing adominant-negative (DN) form of the CD40 molecule. WhenBDC-5.2.9 was transduced with CD40DN, the T cells producedonly very low levels of IFN-γ when challenged with antigen invitro, suggesting that signaling through the CD40 molecule isnecessary for effector function. BDC-5.2.9 is highly diabeto-genic in young NOD or NOD.scid recipient mice and inducesfull-blown disease within a two-week period followinginjection. When the CD40DN variant of BDC-5.2.9 was testedin vivo, none of the recipients developed diabetes. Thedemonstration that effector function of pathogenic T cells isinhibited through expression of a non-functional CD40 mole-cule on the T cells suggests a signaling requirement throughCD40. Since diabetogenic T cells can co-express both CD40 andits ligand CD154 upon activation, it is possible that theirpathogenic properties are enhanced through T-T interactionsvia CD40-CD154. We conclude from these experiments thatblocking CD40 on autoreactive T cells alters their effectorfunction and hypothesize that engagement of CD40 on Th1 Tcells promotes diabetogenicity.

doi:10.1016/j.clim.2010.03.399

S.78. IL-12 Induces HumanCD4+CD45RA-CD25hiCD127low/neg RegulatoryT Cells to Secrete IFNγ and IL-10 and Acquire aNon-regulatory Effector PhenotypeMargarita Dominguez-Villar1, David Hafler2, ClareBaecher-Allan1. 1Brigham and Women′s Hospital, Boston,MA; 2Yale School of Medicine, New Haven, CT

Regulatory T cells (Treg) play a pivotal role inmaintaining abalance between the immune response and peripheraltolerance. Recent evidence indicates that regulatory T cellsare more functionally plastic than originally thought. Not onlydoes the cytokine milieu modulate their capacity to suppress,but it can also induceTregs to secrete cytokines (IL-17). The IL-12 family of cytokines (IL-12, IL-23, IL-27) are keyplayers in theregulation of T cell responses. Despite their similarity, each ofthem has a distinct role in regulating the immune response. IL-12 is required for the induction of IFN-γ-producing Th1 cellsfrom naïve T cells. IL-27 plays a role in the differentiation ofnaïve CD4 T cells into IL-10 producing Tr1 cells. In contrast, IL-23 plays a key role in the induction of Th17 cell differentiation.We have studied the distinct effects that these cytokines (IL-12, IL-23 and IL-27) exert on human Treg function and