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

Abstract 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-γ 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.