Pam3Cys4 internalization by naïve CD4+T cells accelerates t-bet promoter epigenetic modification and restrains TGF–β1 expressing tumor growth (P2153)

Pam3Cys4 internalization by naïve CD4+T cells accelerates t-bet promoter epigenetic modification and restrains TGF–β1 expressing tumor growth (P2153) Mohsen Ibrahim,1,3Kelsey Toth,1Donatella Ponti,4Antonella Calogero,4Claudio Andreetti,3Howard Huang,2Daniel Kreisel,1Sasha Kreisel,1Erino Rendina,3 and Andrew Gelman1,3 1Surgery, Washington Univ. in St. Louis, St. Louis, MO 2Medicine, Washington Univ. in St. Louis, St. Louis, MO 3Thoracic Surgery, Sapienza University of Rome, Rome, Italy 4Pathology, Sapienza University of Rome, Rome, Italy To promote Th1 responses TLR2 engagement is conducted on ex vivo-activated T cells but this hinders Ag-specific clonal expansion in vivo. We asked if naive CD4+T cells could internalize the TLR2 agonist Pam3Cys4 to promote Th1 responses in vivo. Methods: WT, TLR2-/- or OT2 CD69-CD25-CD44-CD62L+CD4+T cells were incubated with rhodamine-labeled or unlabeled Pam3Cys4 for 3 hrs and extensively washed to remove all soluble agonist. CD4+T(iPam) activation ex vivo was conducted under Th1 conditions +/- TGF-β1 and bisulfite conversion t-bet promoter sequence analysis was performed. 4x10(5) OT2 or OT2(iPam) cells were adoptively transferred into B6 mice with established EG-7 OVA tumors. Results: Intracellular FACS revealed WT or TLR2-/- CD4+T cells readily internalize Pam3Cys4. As compared to untreated CD4+T cells CD4+T(iPam) displayed > than 50% t-bet promoter CpG hypomethylation at -134 and -75 at 36 hrs post-activation leading to sharply higher t-bet and IFN-{gamma} expression in a TLR2 dependent manner. OT2 (iPam) halted EG-7 OVA growth with high numbers of OVA-specific CD4+ and CD8+ T cells in the DLN and tumor. In contrast, untreated OT2 induced little Th1 accumulation and accelerated tumor growth when compared to mice that did not receive T cells but this rapid growth pattern could be reversed with TGF-β1 Abs. Also, unlike with untreated OT2 cells TGF-β1 could not block naïve OT2(iPam) differentiation into Th1 cells. These data suggest a novel approach to tumor immunotherapy.