S6 Abstract

translation of adoptive immunotherapy to the clinic willrequire an understanding of the stability and purity of thetransferred Treg population. While several recent animalstudies demonstrate the capacity of Tregs to adopt thelineage-specific transcription program of the cells theysuppress, little is known about plasticity within human Tregs.We previously reported a fraction of FOXP3+ T cells (median4.57%, range 1.1-22.7) were capable of producing the Th1-effector cytokine IFN-γ following in vitroexpansion.Consistentwith this capacity, we demonstrated that CD45RA+ naive Tregscould be induced to produce IFN-γ following Th1-skewingconditions. To characterize the function and stability of thiscell subset, we isolated viable IFN-γ-producing cells using acytokine cell-capture system following in vitro expansion ofhuman CD4+ CD25+ CD127-/lo T cells. Interestingly, isolatedIFN-γ+ cells expressed FOXP3 protein and mRNA at levelscomparable to that observed in IFN-γ- cells and effectivelysuppressed responder T cell proliferation in vitro. Moreover,IFN-γ+ cells expressed elevated levels of the transcriptionfactor Eos compared to effector T cells. However, epigeneticanalysis revealed increased methylation at the FOXP3 Treg-specific demethylation region in IFN-γ+ cells. These data arguethat the IFN-γ+ subset likely originates from bona-fide Tregsrather than from contaminating effectors, yet raises thepotential for lineage instability within this population. Ourresults provide insights into the plasticity and functionaldiversity of human Tregs and suggest novel opportunities formodulating Treg stability for therapeutic applications.

doi:10.1016/j.clim.2010.03.023

OR.7. Characterization of Naturally EmergingPost-EAU Regulatory T CellsDarren Lee, Andrew Taylor. Schepens Eye Research Instituteand Harvard Medical School, Boston, MA

Following resolution of experimental autoimmune uveitis(EAU) TGF-β producing Treg cells emerge in the spleen, butnot in EAU Melanocortin 5 Receptor knock out (MC5r(-/-))mice. MC5r is a receptor for α-MSH, an important mediator ofimmunosuppression within the eye. EAU was induced inC57BL/6J and MC5r(-/-) mice to characterize the IRBP-specific T cell response that emerges in the spleen. Inaddition, the EAU splenocytes were stained for regulatory Tcell markers and analyzed by flow cytometry. Also, the EAU Tcells were assayed for TGF-β, IFN-γ, and IL-17 production,and for regulatory activity. In the course of EAU we foundthat the dominant IRBP-specific T cell types progressed fromTh1, then to Th17, and finally Treg at resolution. Thisprogression did not occur in the MC5r(-/-) EAU mice, rather aTh1 to Th17 and back to Th1 as resolution of EAU wasobserved. After resolution of EAU in wild-type mice thedominant IRBP-specific T cell response were TGF-β producingCD4+CD25+ T cells capable of suppressing EAU. The requirementfor MC5r expression for the generation of these Treg cellsdemonstrates the importance of the melanocortin pathway inthe development of regulatory ocular immunity.

doi:10.1016/j.clim.2010.03.024

OR.8. PHLPP Regulates the Development, Functionand Molecular Signaling Pathways of TRegulatory CellsScott Patterson1, Rosa Garcia1, Audrey O'Neill2, TianyanGao2, Alexandra Newton2, Megan Levings1. 1University ofBritish Columbia, Vancouver, BC, Canada; 2University ofCalifornia-San Diego, San Diego, CA

T regulatory cells (Tregs) have a fundamental role inmaintaining immune homeostasis. There is much interest indefining their unique properties in order to understand theirmechanisms of action and identfy new therapeutic approachesto modulating their activity in vitro or in vivo. We previouslyfound that Tregs have a reduced capacity to activate the PI3K/Akt pathway downstream of the TCR, and that low activity ofAkt was necessary for their suppressive function. In order todefine the molecular basis for their unique molecularphenotype, we investigated Treg expression of phosphatasesthat regulate the PI3K/Akt pathway: PTEN, SHIP and PHLPP.Although ex vivo Tregs and conventional T cells expressedsimilar amounts of the lipid phosphatases PTEN and SHIP, Tregsexpressed significantly higher levels of PHLPP, a novel Ser/Thrprotein phosphatase that negatively regulates Akt. Knock-down of PHLPP using siRNA completely restored TCR-mediatedactivation of Akt in Tregs. Moreover, knock down of PHLPP innaive T cells significantly impaired their capacity to developinto induced Tregs under the influence of TGF-β, indicatingthat PHLPP-mediated suppression of Akt is necessary forperipheral Treg development. To investigate the functionalsignificance of PHLPP in Tregs, we analyzed T cells fromPHLPP1-/- mice. Differentiation of peripheral Tregs wasimpaired in mice genetically deficient for PHLPP1, and thesuppressive capacity of ex vivo Tregs was significantlyreduced. These data define a novel mechanism used by Tcells to control the Akt pathway and provide the first evidencefor a molecular mechanism underlying the functionallyessential reduction of Akt activity in Tregs.

doi:10.1016/j.clim.2010.03.025

Frontiers of AutoimmunityFriday, June 252:45-4:45 pm

OR.9. Insulin Gene VNTR Genotype Associates withFrequency and Phenotype of the AutoimmuneResponse to ProinsulinIvana Durinovic-Belló, Vivian Gersuk, Rebecca Wu, JessicaMatthis, Gerald Nepom. Benaroya Research Institute,Seattle, WA

Immune responses to autoantigens are in part controlledby deletion of autoreactive cells through genetically regu-lated selection mechanisms. The INS-VNTR genetic polymor-phism has been proposed to influence the level of self-tolerance to proinsulin (proINS) by modulating expression ofproINS in the thymus. We have directly analyzed peripheral