1A) consistent with a naïve state. Upon culture with OVA323–339, CD4+ T cells rapidly upregulated the expression of the early activation marker CD69 and, as indicated by their FSC profile, began to enlarge and undergo blastogenesis (Fig. 1A). CD69 expression remained high on CD4+ T cells in OVA peptide-containing

cultures until day 5 and during this time a CD44hi phenotype was acquired. This indicated ongoing activation of CD4+ T cells in the presence of antigen which was confirmed by continued CFSE dilution until peptide removal (data not shown). CD62L expression Lumacaftor in vitro was transiently reduced upon culture but after 3 days, even in the presence of OVA peptide, returned to the levels equivalent to that on naïve OT-II cells (Fig. 1A). Upon washing and reculture in the absence of OVA peptide, but in the presence of IL-7, CD69 expression rapidly declined to baseline levels and OT-II T cells developed a CD44hi CD62Lhi phenotype as displayed by central memory T cells 12. Restimulation of OT-II cells recovered from culture demonstrated that a large proportion of the “central memory-like” T cells were capable of rapidly producing the effector cytokines IFN-γ

and IL-2 (Fig. 1B) unlike naïve T cells (data not shown), HSP inhibitor indicating substantial acquisition of rapid effector function. Notably, post-activated OT-II T cells produced little IL-4, IL-17 or IL-10, indicating that these conditions promoted Th1-like differentiation. No Foxp3 expression was detected in CD4+ T cells recovered from these cultures (data not shown). Therefore, we conclude that these culture conditions generate a population of OT-II T cells phenotypically similar to central memory T cells and skewed toward a Th1 phenotype. Using a model in which OVA expression is targeted to DC by the CD11c promoter, we have shown that steady-state presentation of OVA by OVA-expressing DC induces peripheral tolerance in naïve CD4+ and CD8+ T cells 13, 14 and memory and effector CD8+ T cells 4, 15. As the CD11c promoter appears to drive low-level transgene expression in CD11cint cells Sorafenib chemical structure 16, which includes plasmacytoid DC, some activated macrophages,

subsets of intraepithelial lymphocytes and NK1.1+ cells, we previously showed that the presentation of immunogenic MHC/OVA peptide complexes was restricted to CD11chi conventional DC 13. Additionally, Taqman qPCR studies have shown OVA message is restricted to DC and not expressed in B and T cells of 11c.OVA mice 15. Therefore, we used this model to test the susceptibility of activated CD4+ T cells to DC-induced peripheral tolerance. To determine whether CD4+ memory T cells were activated by OVA peptides presented by steady-state OVA-expressing DC, cultured OT-II cells were CFSE labeled and transferred to 11c.OVA and nontransgenic controls. Three days after transfer, little CFSE dilution was observed in the spleens or LN of nontransgenic recipients, although a small number of cells appeared to have undergone one or two divisions (Fig. 2A). In 11c.