S35Abstracts

pathways involved in autoimmune diseases. However,increasing new evidence points to the limitations of thissimplistic paradigm as well as to the role of the IL-17/IL-23axis in certain autoimmune diseases. In our studies in the AAmodel, we observed that arthritic Lewis rats showedcytokine profiles during the course of disease that werecontrary to the expected patterns. These rats showedhighest Th1 cytokine response in the recovery phase insteadof the acute phase of the disease, whereas the AA-resistantWistar Kyoto rats revealed highest Th1 response early after apotentially disease-inducing antigenic challenge. Similarly, aparadoxical increase in the Th1 cytokines along with anergyinduction in T cells was observed in Lewis rats given atolerogenic dose of soluble Hsp65, which led to suppressionof arthritis. No change in Th2 cytokines was observed. Theseresults apparently are in contradiction with the Th1-Th2paradigm. Further exploration into cytokine interactionsrevealed an increased Th1 response coupled with suppres-sion of the Th17 response. Our results unravel a novelimmunoregulatory attribute of pro-inflammatory cytokinesthat could be exploited for immunomodulation of IL-17-mediated autoimmune inflammation.

doi:10.1016/j.clim.2009.03.097

OR.83. TSLP Modulates the Balance between Th1and Th2 Responses in the SkinRyan Larson1, Steven Ziegler2. 1University of Washington,Seattle, WA; 2Benaroya Research Institute, Seattle, WA

TSLP is an IL-7 related cytokine produced by epithelialcells in response to tissue damage, inflammation, or TLRligation. Overexpression of TSLP in mouse skin or lung leadsto the development of atopic dermatitis and asthma,respectively; however, these models fail to elucidate whichcell types are acted on by TSLP. TSLP has also beenhypothesized to inhibit aberrant Th1 responses by inhibitingthe production of IL-12 family members by DCs. TSLPreceptor deficiency significantly inhibits the developmentof Th2-type contact hypersensitivity (CHS) response inducedby the irritant dibutyl phthalate in combination with thehapten fluorescein isothiocyanate (FITC). Specifically, FITC-sensitized TSLPR-/-mice do not experience swelling oreosinophilia at the site of challenge; nor do they experienceincreased serum IgE, all hallmarks of Th2-type CHS observedin WT mice. The reduced CHS response is due in part to thesignificant reduction in frequency and number of skin-derived antigen-bearing FITC+CD11c+DC in skin-draininglymph nodes 24 hours post FITC sensitization of TSLPR-/-mice. Furthermore, skin-derived TSLPR-/-FITC+CD11c+DCsare reduced in their ability to drive proliferation of naïveantigen specific CD4+Tcells compared to WTcounterparts. Inaddition, we have found that there is no requirement forCD4+T cells to respond to TSLP for the development of FITC-induced CHS. Interestingly, TSLPR-/-mice experience anenhanced Th1-type CHS response to the Th1-inducingcontact sensitizing agent 2,4-dinitrofluorobenzene, com-pared to WT. These results support a role for TSLP actingon DC in vivo resulting in the development of Th2 allergic

response and potentially playing a regulatory role byinhibiting Th1 CHS responses.

doi:10.1016/j.clim.2009.03.098

Tuning Adaptive ImmunitySunday, June 142:45 pm–4:45 pm

OR.84. The Role of Aire in Mediating AutoimmunePeripheral NeuropathyMaureen Su, Jason Devoss, Dan Davini, Una Fan, CedricLouvet, Helene Bour-Jordan, Jeffrey Bluestone, MarkAnderson. University of California, San Francisco, SanFrancisco, CA

The Autoimmune Regulator (Aire) gene plays an importantrole in central tolerance by driving the expression ofperipheral self-antigens in the thymus. The presentation ofthese antigens within the thymus drives the negative selectionof T cells that recognize these self-antigens, thus preventingautoimmunity.We have recently described amousemodel, theG228W knockin mouse, with a dominant negative pointmutation in Aire. Unexpectedly, this mouse develops aspontaneous, fulminant autoimmune peripheral neuropathyin the NOD background. The autoimmune nature of thisneuropathy is supported by the appearance of lymphocyticinfiltration in the sciatic nerves on histology and the ability ofsplenocytes from affected mice to transfer disease into animmunodeficient host. In people, the pathophysiology ofautoimmune peripheral neuropathy is not well understood,and, importantly, the pathogenic antigen important in causingautoimmune peripheral neuropathy is not known. We haveidentified Myelin Protein Zero (MPZ) as the antigen predomi-nantly recognized by sera fromG228Wmice. Interestingly, thisantigen has also been described as the pathogenic antigen inthe CD86-deficient mouse model of autoimmune peripheralneuropathy. We show here that G228W mice that are alsodeficient in CD86 have accelerated neuropathy, demonstratingthat these two alterations affect two separate tolerancepathways. G228W mice treated with anti-CD86 blockingantibody also develop accelerated neuropathy. Thus, twoseparate tolerance pathways appear to utilize the sameantigen to protect against autoimmuneperipheral neuropathy.

doi:10.1016/j.clim.2009.03.099

OR.85. Regulation of GRAIL Expression by mTORControls Naive CD4 T Cell ProliferationJack Lin, Neil Lineberry, Michael Kattah, Leon Su, Paul Utz,C. Garrison Fathman. Stanford University School ofMedicine, Stanford, CA

In this study of mouse and human naïve CD4 T cells wedemonstrate that the E3 ubiquitin ligase Gene Related toAnergy in Lymphocytes (GRAIL) is expressed in quiescentcells and functionally inhibits cell proliferation. Upon T cellactivation with CD28 co-stimulation, IL-2 signals through its

S36 Abstracts

receptor and activates the Akt-mTOR (Mammalian Target ofRapamycin) pathway. The activation of mTOR results inselective mRNA translation, including the epistatic regulatorof GRAIL, Otubain-1 (Otub1), whose expression results in thedegradation of GRAIL, allowing T cell proliferation. Thisactivation of mTOR appears to be the crucial pathway of IL-2receptor (IL-2R) signaling that regulates GRAIL expression.Use of CTLA4-Ig treatment blocks CD28 co-stimulation andresultant IL-2 expression, whereas anti-IL-2 or Rapamycintreatment block mTOR activation downstream of IL-2Rsignaling. We show that all three of these biotherapeuticsinhibit mTOR-dependent translation of mRNA transcripts,resulting in blockade of Otub1 expression, maintenance ofGRAIL, and inhibition of CD4 T cell proliferation. Theseresults provide a mechanistic pathway involving CD28 co-stimulation, IL-2R signaling, and mTOR activation as impor-tant checkpoints for naïve CD4 T cell proliferation via Otub1and GRAIL regulation. Our findings also reveal a role forGRAIL apart from anergy, suggesting that endogenous GRAILregulates general cell cycle and proliferation of primarynaïve CD4 T cells.

doi:10.1016/j.clim.2009.03.100

OR.86. TIM-4 Negatively Regulates ImmuneResponses by Reducing Antigen SpecificT Cell NumbersLee Albacker1, Piia Karisola1, Sarah Umetsu2, Omid Akbari1,Gordon Freeman3, Dale Umetsu1, Rosemarie DeKruyff1.1Children's Hospital Boston and Harvard Medical School,Boston, MA; 2Feinberg School of Medicine, NorthwesternUniversity, Chicago, IL; 3Dana-Farber Cancer Institute andHarvard Medical School, Boston, MA

TIM-4, a member of the TIM gene family, is a cellsurface molecule expressed by macrophages and dendriticcells that serves as a receptor for phosphatidyl-serine(PtdSer), an important marker of apoptotic cells. We nowshow that TIM-4 regulates adaptive immunity by controllingthe engulfment, and therefore regulating the number ofantigen-specific T cells. In vitro and in vivo blockade ofTIM-4 prevented the phagocytosis of apoptotic T cells byTIM-4 expressing cells, and enhanced the proliferation andcytokine secretion of antigen-primed T cells. In contrast,transgenic over-expression of TIM-4 on MHC class II positivecells reduced T cell responses by reducing the number ofantigen specific T cells. The diminished response ofantigen-specific T cells was not due to altered T cellactivation, or the development of regulatory T cells. Theseresults suggested that TIM-4 regulates immune responsesby mediating the phagocytosis and removal of antigenspecific T cells, an idea supported by an in vitro systemutilizing CFSE labeled T cells, plate bound anti-CD3, and3T3 cells expressing TIM-4 or empty vector. The number ofactivated T cells increased much more rapidly in culturescontaining 3T3-vector control cells than in cultures con-taining TIM-4 expressing 3T3 cells. The rate of T cellproliferation and the number of divisions were not changedby the presence of 3T3-TIM-4 cells. We conclude that TIM-4expressing cells remove apoptotic and activated T cells

expressing PtdSer, thereby regulating the number ofantigen-specific T cells.

doi:10.1016/j.clim.2009.03.101

OR.87. Aire-deficient Mice Develop SpontaneousAutoimmunity to the Central NervousSystem AntigensAsako Tagawa1, Toshimasa Aranami1, Mitsuru Matsumoto2,Takashi Yamamura1. 1National Institute of Neurosicence,NCNP, Kodaira, Japan; 2Institute for Enzyme Research,University of Tokushima, Tokushima, Japan

Autoimmune regulator (Aire) gene is expressed inmTECs and plays a pivotal role for negative selection ofautoreactive thymocytes. Aire-deficiency causes sponta-neous organ-specific autoimmune inflammations in bothhuman and animals. In Aire-deficient mice, infiltration of Tcells and B cells are observed in the affected tissues.However, whether Aire-deficiency results in spontaneouscentral nervous system (CNS) autoimmunity reactive tomyelin antigens is not known. Here we addressed whetherAire-deficient (Aire−/−) C57BL/6J mice might developspontaneous CNS autoimmunity. A substantial number oflymphocytes could be isolated from the CNS of naive agedC57BL/6J mice (2.5±0.4×105/head). The number of thespontaneous infiltrating lymphocytes was increased in agedAire−/− mice (4.8±1.1×105/head) compared with controlAire+/+mice, although this difference was not significant.Interestingly, the number of CD4+and CD8+T cells withinthe CNS were significantly increased in Aire−/− mice.Strikingly, after in vitro culture of the Aire−/− CNSinfiltrating cells in the presence with CNS myelin-derivedpeptides (MOG35-55 or PLP178-191), certain amounts of IL-17(100-300 pg/ml) and IFN-γ(200-250 pg/ml) were producedin the culture supernatant. By contrast, Aire+/+CNS cellsdid not produce any of these cytokines, indicating thatthey are not responsive to self. Furthermore, PLP178-191-specific IgG titers were elevated in the aged Aire−/− mice,compared with the Aire+/+mice. These results imply thataged Aire-deficient mice spontaneously develop autoim-mune Th1 and Th17 responses to encephalitogenic myelinpeptides. Aire−/− mice provide a opportunity to investigatethe mechanism on the spontaneous CNS autoimmunity,which could significantly differ from that for the inducedexperimental autoimmune encephalomyelitis.

doi:10.1016/j.clim.2009.03.102

OR.88. Identification of a Subset of AIRE ExpressingDendritic Cell in Human Peripheral LymphoidTissues: Evidences for a Role in the Induction ofPeripheral TolerancePietro Poliani1, Kai Kisand2, Veronica Marrella3, MariaRavanini1, Luigi Notarangelo4, Anna Villa3, Pärt Peterson2,Fabio Facchetti1. 1University of Brescia, Brescia, Italy;2University of Tartu, Tartu, Estonia; 3Humanitas, Rozzano,Italy; 4Children's Hospital, Harvard Medical School,Boston, MA