Abstract Book - Pathology and Laboratory Medicine - University of ...

ABSTRACT #36BASIC SCIENCESGRADUATE STUDENTPOSTER presentationThe role of hypoxia induced factor 1α and notch signaling in anergic T cellmaintenance during homeostatic expansionKevin Tsai 1 , John Priatel 1 , and RuSung Tan 11Department of Pathology and Laboratory Medicine48BacgroundThe process of central tolerance, which weeds out autoreactive CD8+ T cells, is important in preventing autoimmunediseases. The process however, is imperfect, resulting in the release of some of these auto reactive T cells intocirculation. It is widely believed that through the tolerizing cytokines secreted by the T regulatory cells and or thecontinual exposure to the self-antigen without the presence of co-stimulatory signals, these auto-reactive T cellsbecome antigen unresponsive (anergized). This state of anergy was shown to be reversible under certain conditionssuch as homeostatic expansion (HE), or under the influence of certain cytokines such as IL-7 and or IL-15. In arecent publication by Greenburg et al. these anergized autoreactive T cells were shown to be activated during HE,but revert back to anergy after lymphocyte population was restored independent of cytokines or antigens. Epigeneticregulation was strongly implicated in the study. We hypothesize that: the epigenetically programmed CD8+ T cellanergy is driven by Notch signaling and hypoxia induced factor 1α signaling, thus modulating the actions of Notchsignaling, and Hif-1a signaling, will affect the establishment/maintenance of their epigenetic memory.ObjectivesTo determine whether or not modulating Notch and or Hif-1α signaling could result in the impairment of anergyestablishment/maintenance in the autoreactive CD8+ T cells during homeostatic expansion using a rat insulinpromoter OVA (RIP-OVA), OT1 TCR transgenic mouse model.MethodsWe will first generate a transgenic mouse strain carrying a specific T cell receptor recognizing a specific self-antigenby crossing OT1 mice with RIP-OVA mice. The anergized OT1 T cells will be harvested from the OT1-RIP-OVAmice using H2Kb-SIINFEKL tetramer and FACS. They will then be treated with HIF-1α activator Desferrioxamine(DFO) before being reintroduced into age-matched, syngenic partially lymphodepleted recipients treated with orwithout anti-notch ligand antibodies. The autoreactivity of the CD8+ OT1 cells will be monitored in several ways:We will assess the autoreactivity in vivo by monitoring onset of autoimmune diabetes (AD) in the recipient miceafter treatment. We will assess the autoreactivity in vitro using ELISA, and chromium 51 assay. The expression levelsof the components of the epigenetic machinery and microRNAs will be measured using qPCR, and the epigeneticconfiguration on the loci of important CD8 T cells effector molecules such as granzyme will be assessed usingchromatin immunoprecipitation.ResultsModulation of Hif-1α and or Notch ligand during homeostatic expansion caused differential expression of theepigenetic machinery. As the result, reactivated OTI CD8+ T cells were no longer anergized after the homeostaticexpansion was completed. This defect is manifested in vivo as AD and in vitro as enhanced cytotoxicity andcellularity.ConclusionsThe elucidation of the molecular mechanisms of T cell anergy maintenance allows us to device new strategies inmodulating T cell anergy in order to treat diseases such as autoimmunity and cancer.2012 * Oral/Poster Presentations