S39Abstracts

potential pathogenic mechanism for RA associated auto-antibodies in the initiation and propagation of synovialinflammation.

doi:10.1016/j.clim.2010.03.117

T.3. Effects of Diacylglycerol Kinase-ζ Deficiency onCD8+ T Cell FunctionMatthew Riese, Gary Koretzky. University of Pennsylvania,Philadelphia, PA

The mechanism underlying tumor-mediated impairmentof CD8+ T-cell function is poorly understood. We sought todetermine if CD8+ T cells with enhanced signaling down-stream of the T-cell receptor (TCR) could overcome tumor-mediated inhibitory effects. To that end, we evaluated micewith targeted deletion of diacylglycerol kinase ζ (dgkζ), aprotein partially responsible for terminating DAG-mediatedsignal transduction after TCR activation. We found that CD8+

T cells from dgkζ-deficient mice demonstrated enhancedproliferation and cytokine production after TCR stimulation.Moreover, we observed that mice inoculated with EL4-ovacells grew smaller tumors and developed increased numbersof activated CD8+ T cells. In vitro, we found that dgkζ-deficient CD8+ T cells were less susceptible to inhibition byTGFβ or regulatory T cells. We conclude that deficiency ofdgkζ results in CD8+ T cells with enhanced functionalcapacity and hypothesize that targeting dgkζ could representa novel strategy for enhancing antitumor immune responses.Future work will evaluate CD8+ T-cell responses in a model ofspontaneous mouse tumor development. Supported by NIHAI-058019 and GM-053256.

doi:10.1016/j.clim.2010.03.118

T.4. Complex Modulation of De Novo Foxp3 Inductionand CD4 T Cell Tolerance by Inflammatory StimuliLucas Thompson, Steven Ziegler. Benaroya Research Institute,Seattle, WA

Foxp3+ regulatory T cells (Treg) are important media-tors of self-tolerance in the periphery. Treg can differen-tiate from thymic precursors or naïve CD4 T cells in theperiphery. We sought to address the role of de novoperipheral induction of Foxp3+ Treg in a model of CD4 Tcell tolerance to a tissue antigen. Upon adoptive transferof monoclonal DO11.10 CD4 T cells into lymphopenic miceexpressing membrane-bound OVA in the pancreatic islets(RIP-mOVA/RAG), a low frequency of the chimeric micebecome diabetic, with most remaining healthy for morethan 80 days posttransfer. If Foxp3-deficient monoclonalDO11.10 cells are transferred into RIP-mOVA/RAG mice,100% of these mice become diabetic by 40 days post-transfer, and this can be prevented by cotransfer of Foxp3+ Treg. Despite an absence of Foxp3 expression amongmonoclonal DO11.10 cells at the time of transfer, at

20 days posttransfer, healthy mice have a substantialfraction (10%–25%) of donor CD4 T cells that haveupregulated de novo Foxp3. Transfer of monoclonalDO11.10 cells into RIP-mOVA/RAG mice followed byinjection of polyIC, a strong inducer of Th1 inflammatorycytokines, resulted in rapid diabetes in 100% of treatedmice by day 11 posttransfer and reduced expression ofFoxp3. Conversely, pretreatment of the T cells withpolyIC-mediated inflammation results in enhanced expres-sion of Foxp3 upon later encounter with antigen, andmaintenance of tolerance. These data suggest thattemporal coordination of inflammatory and antigen signalscan result in divergent outcomes for effector or Tregdifferentiation.

doi:10.1016/j.clim.2010.03.119

T.5. Identification of Asthma and Human SpecificEnhancers in the Th2 Cytokine LocusGregory Seumois1, Maria Vedanayagam2, Nada Omran2,Lukas Kalinke2, Anjana Rao3, Mark Ansel1, PanduranganVijayanand1. 1University of California-San Francisco, SanFrancisco, CA; 2University of Southampton, Southampton,United Kingdom; 3Harvard Medical School, Boston, MA

Asthma is a pulmonary disorder characterized byairways inflammation initiated and maintained by Th2lymphocytes. Upon activation, Th2 cells release Th2-cytokines IL4, IL5, and IL13. Th2-cytokine genes arelocated in a common locus. In mouse T cells, differentcis-regulatory regions (enhancers) across this locus havebeen identified and functionally investigated. Since theseregions are not fully conserved across mammalian species,we aimed to study the epigenetic status of T cells fromasthmatic patients to identify human and asthma specificenhancers. Blood, bronchial biopsy, and BAL samples wereobtained from patients categorized in relation to diseaseseverity. T cells were sorted by flow cytometry. Blood Tcells were further subsorted into CD4+-naïve, memory Th1-enriched (CCR4−) and Th2-enriched (CCR4+) cells. Toidentify potential enhancers, histone H3 K4me2 andK27me3 modification across the locus was analyzed bychromatin immunoprecipitation (ChIP). As the number ofsorted cells ranged between 1×103 and 1×106, we havedeveloped a microscaled ChIP-on-chip assay. We showedthat our microscaled ChIP-on-chip assay result performedwith 1×105 cells is comparable to standard ChIP assayrequiring 2–10×106 cells. By histone profiling the Th2-cytokine locus, we identified 9 asthma and human specificcis-regulatory regions (4 conserved and 5 nonconserved),which may play an important role in Th2-cytokine generegulation in asthma. We demonstrated the feasibility toperform epigenetic analysis on primary T cells obtainedfrom blood and diseased tissue of asthma patients. Thisinformation is likely to provide valuable insights into roleof epigenetic mechanisms in asthma pathogenesis.

doi:10.1016/j.clim.2010.03.120