Lek2, Tracy Tabib1, Robert Lafyatis1, Creg Workman, PhD1, Dario Vignali, PhD1 1 University of Pittsburgh,

Lek2, Tracy Tabib1, Robert Lafyatis1, Creg Workman, PhD1, Dario Vignali, PhD1 1 University of Pittsburgh, Pittsburgh, PA, USA; 2Children’s Hopsital of Pittsburgh, Pittsburgh, PA, USA Correspondence: Dario Vignali ([email protected]) Journal for ImmunoTherapy of Cancer 2018, 6(Suppl 1):P481 Background Regulatory T cells (Tregs) are a suppressive cell population that limit the anti-tumor response. Even so, systemic ablation of Tregs can not be utilized as a therapy due to enormous autoimmune defects. Our lab has demonstrated that Treg-restricted deletion of cell surface protein Neuropilin (Nrp1, CD304) outcomes in substantially decreased tumor growth with no autoimmune defects [1]. We’ve shown that Tregrestricted deletion of Nrp1 inside the TME will not lead to loss of Foxp3 expression and “PKA Storage & Stability ex-Treg” generation but rather causes them to exhibit an effector-like phenotype such as loss of suppressive function and production of interferon gamma (IFN), which we refer to as Treg fragility [2]. Techniques We sought to understand the epigenetic underpinnings in between Nrp1-sufficient and -deficient Tregs from the tumor microenvironment that could lead to this `fragile’ state. To perform so we performed bisulfite treatment from ZymoEZ Direct Kit followed by Sanger Sequencing to determine differences in DNA methylation. We utilized ATAC sequencing to recognize discrepancies in chromatin accessibility following the Greenleaf protocol [3]. We also utilized TCR sequencing from Adaptive Biotechnologies per the manufacturer’s protocol. For single cell RNAseq, we loaded 3500 cells/sample utilizing ChromiumTM Single Cell 3′ Gel Bead Kit and Chromium Single Cell 3’v2 Library Kit. Samples were sequenced on a NextSeq500. Finally, Cut RUN ChIPseq was carry out following the Henikoff protocol [4]. Results We located that Tregs lacking Nrp1 Enterovirus drug within the TME have a differential methylation signature at the Conserved Non-coding Sequence two (CNS2) locus in the Foxp3 gene, albeit no difference within the chromatin accessibility at this locus, no adjust in single cell RNAseq, and maintenance of Foxp3 protein expression. We also discovered that Nrp1-deficient Tregs are not peripherally-induced Tregs but rather are thymically-derived.Journal for ImmunoTherapy of Cancer 2018, six(Suppl 1):Page 251 ofConclusions We’ve got identified an intriguing change in the DNA methylation status of your CNS2 locus of Foxp3 in the Nrp1-deficient Tregs in the tumor microenvironment but no loss in Foxp3 expression. This finding conflicts with present data suggesting that CNS2 hypermethylation shuts off Foxp3 expression. Extra experiments will probably be necessary to understand how this locus maintains Foxp3 protein in spite of DNA methylation. Future studies may also examine the epigenetic mediators that could possibly bring about this differential methylation or if extrinsic factors in the TME promote differential methylation.References 1. Delgoffe GM, Woo SR, Tunis ME, Gravano DM, Guy C, Overacre AE, Bettini ML, Vogel P, Finkelstein D, Bonnevier J, Workman CJ, Vignali DA. Stability and function of regulatory T cells is maintained by a neuropilin-1semaphorin-4a axis. Nature. 2013; 7466, 252-6 two. Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, Horne W, Moskovitz JM, Kolls JK, Sander C, Shuai Y, Normolle DP, Kirkwood JM, Ferris RL, Delgoffe GM, Bruno TC, Workman CJ, Vignali DAA. Interferon- derives treg fragility to market anti-tumor immunity. Cell. 2017; 169, 1130-41 3. Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenle.