Cross-talk between TGF-β and IL-6 has been shown to direct the differentiation of CD4⁺ cells into special IL-17-secreting cells, which are termed Th17 cells. In this study, we demonstrated that TGF-β and IL-6 could stimulate CD8⁺ cells to differentiate into noncytotoxic, IL-17-producing cells in MLC. These IL-17-producing CD8⁺ cells exhibit a unique granzyme B⁻IFN-γ⁻IL-10⁻ phenotype. The mRNA level of Th2/T cytotoxic 2 (Tc2) transcription factors GATA3 and Th1/Tc1 transcription factors T-box expressed in T cell (T-bet) as well as its target H2·O-like homeobox (Hlx) is decreased in CD8⁺ cells from TGF-β- and IL-6-treated MLC. In addition, these CD8⁺ cells display a marked up-regulation of retinoic acid-related orphan receptor-γt, a key IL-17 transcription factor. These results demonstrate that the existence of an IL-17-producing CD8⁺ subset belongs to neither the Tc1 nor the Tc2 subset and can be categorized as a T noncytotoxic 17 (Tnc17) subset.

We have previously shown that a plasmid (pE) encoding the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection against a lethal viral challenge. In the present study, we used adoptive transfer experiments and gene knockout mice to demonstrate that the DNA-induced E-specific antibody alone can confer protection in the absence of cytotoxic T-lymphocyte (CTL) functions. Plasmid pE administered by either intramuscular or gene gun injection produced significant E-specific antibodies, helper T (Th)-cell proliferative responses, and CTL activities. Animals receiving suboptimal DNA vaccination produced low titers of anti-E antibodies and were only partially or not protected from viral challenge, indicating a strong correlation between anti-E antibodies and the protective capacity. This observation was confirmed by adoptive transfer experiments. Intravenous transfer of E-specific antisera but not crude or T-cell-enriched immune splenocytes to sublethally irradiated hosts conferred protection against a lethal JEV challenge. Furthermore, experiments with gene knockout mice showed that DNA vaccination did not induce anti-E titers and protective immunity in Igμ^−/−and I-Aβ^−/−mice, whereas in CD8α^−/−mice the pE-induced antibody titers and protective rate were comparable to those produced in the wild-type mice. Taken together, these results demonstrate that the anti-E antibody is the most critical protective component in this JEV challenge model and that production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4⁺T cells but independent of CD8⁺T cells.

Cross-presentation is a key function of dendritic cells (DCs), which present exogenous Ags on MHC class I molecules to prime CTL responses. The effects of TLR triggering on the cross-presentation of exogenous Ags by DCs remain unclear. In this study, we used synthetic dipalmitoylated peptides and TLR2 agonist–conjugated peptides as models to elucidate the mechanisms of TLR2-mediated cross-presentation. We observed that the internalization of dipalmitoylated peptides by bone marrow–derived DCs was facilitated by TLR2 via clathrin-mediated endocytosis. The administration of these dipalmitoylated peptide-pulsed bone marrow–derived DCs eliminated established tumors through TLR2 signaling. We further demonstrated that the induction of Ag-specific CTL responses and tumor regression by dipalmitoylated peptides was TAP independent. In addition, presentation of dipalmitoylated peptides by MHC class I molecules was blocked in the presence of an endosomal acidification inhibitor (chloroquine) or a lysosomal degradation inhibitor (Z-FL-COCHO). The endocytosed dipalmitoylated peptide also passed rapidly from early endosome Ag-1–positive endosomes to RAS-related GTP-binding protein 7 (Rab7)–associated late endosomes compared with their nonlipidated counterparts. Furthermore, we found that dipalmitoylated peptide–upregulated Rab7 expression correlated with Ag presentation via the TLR2/MyD88 pathway. Both JNK and ERK signaling pathways are required for upregulation of Rab7. In summary, our data suggest that TLR2-mediated cross-presentation occurs through the upregulation of Rab7 and a TAP-independent pathway that prime CTL responses.