Molecular mechanisms of TLR2-mediated antigen cross-presentation in dendritic cells

Abstract

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.

FIGURE 1.

TLR2 was required for the endocytosis and cross-presentation of Pam2IDG in BMDCs. (A) WT or TLR2KO BMDCs were pulsed with 1 μM FITC-conjugated Pam2IDG and incubated at 4°C or 37°C for 30 min. BMDCs that were not pulsed were used as controls. The results are shown as MFI value at 37°C, obtained by subtracting MFI at 4°C. Three independent experiments were performed. BMDCs were treated with 50 μg/ml anti-TLR2 Ab or an isotype control Ab (B), 50 μM chlorpromazine (CM) (C), or 2.5 μM filipin (D) for 30 min and then coincubated with 1 μM FITC-conjugated Pam2IDG for another 30 min. After free peptides were washed away, the cells were stained with anti-CD11c Ab. Data were collected by gating on CD11c⁺ cells. Naive C57BL/6 mice (five per group) were inoculated with 2 × 10⁵ TC-1 tumor cells via s.c. injection before immunization. To examine the presentation of IDG and Pam2IDG, BMDCs were pulsed with 1 μM peptides for 3 h. TC-1 tumor-bearing mice were immunized with PBS as a control group, 2 × 10⁵ IDG- or Pam2IDG-pulsed WT BMDCs (E), or 2 × 10⁵ Pam2IDG-pulsed WT or TLR2KO BMDCs (F) via i.v. injection at 7 d after tumor inoculation. Average tumor sizes are shown (cm³). The data are expressed as the mean ± SD. *p < 0.05, **p < 0.01.

FIGURE 2.

TLR2 agonist–conjugated peptides could be cross-presented by BMDCs via a TAP-independent pathway. BMDCs cultured from C57BL/6 WT or TAPKO mice were incubated with 1 μM Pam2IDG for 3 h, and free peptides were then washed away. (A) Seven days after C57BL/6 mice were immunized with BMDCs via i.v. injection, the cytotoxic ability of Ag-specific T cells was assessed via an in vivo killing assay. The specific lysis percentages of the target cells were determined by flow cytometry. The data represent the average of two independent experiments. (B) TC-1 tumor-bearing mice (six per group) were immunized with 2 × 10⁵ peptide-pulsed BMDCs via i.v. injection. Average tumor sizes are shown (cm³). The data are expressed as the mean ± SD. WT BMDCs were pretreated with 25 μM chloroquine (CQ) or 5 μM cathepsin S (CS) inhibitor for 30 min and then pulsed with 1 μM Pam2IDG (C) or RAH (D) for 2.5 h at 37°C. After free peptides were washed away, 2 × 10⁴ BMDCs were cocultured for 48 h with 2 × 10⁵ CD8⁺ T cells that were purified from the splenocytes of RAH/IFA-immunized mice. IFN-γ–secreting cells were then detected by an ELISPOT assay. WT (E) or TAPKO (F) BMDCs were pretreated as in (C) but pulsed with 1 μM Pam2EQL for another 2.5 h at 37°C. After free peptides were washed away, 2 × 10⁴ BMDCs were cocultured for 48 h with 2 × 10⁵ CD8⁺ T cells that were purified from OT-I splenocytes. Cell proliferation was determined using a [³H]thymidine incorporation assay. The data shown are representative of two independent experiments. **p < 0.01.

FIGURE 3.

TLR2 agonist–conjugated peptides were internalized by Rab7⁺ late endosomes and generated the epitope in endolysosomes. BMDCs were incubated with 1 μM FITC-conjugated IDG or Pam2IDG (green) for 10, 20, or 30 min at 37°C. To detect endosomes, BMDCs were stained with anti-EEA1 (A, C) or anti-Rab7 (B, D) to visualize EEA1⁺ and Rab7⁺ endosomes (red), respectively. (E and F) The colocalization rates of IDG-FITC and Pam2IDG-FITC colocalized with EEA1⁺ and Rab7⁺ endosomes. WT (G) or TAPKO (H) BMDCs were incubated with 1 μM Pam2EQL for 30 or 60 min. After incubation, the cells were double stained with 25.D1-16 Ab (red) to detect H2-K^b/OVA_257–264 and with anti-Rab7 or anti-LAMP1 (green). Colocalization is depicted in yellow. The data shown are representative of two independent experiments. **p < 0.01.

FIGURE 4.

TLR2 agonist–conjugated peptides upregulated Rab7 expression via the TLR2–MyD88 pathway to enhance cross-presentation. (A) Lysates were collected from IDG- or Pam2IDG-treated WT BMDCs at the indicated time points. Lysates were collected from Pam2IDG-treated TLR2KOor TLR6KO (B), MyD88KO (C), JNK inhibitor– (D), SB203580- (E), or PD98059-treated (F) BMDCs at the indicated time points. Rab7 and GAPDH expression were then determined by Western blotting.

FIGURE 5.

Rab7 overexpression could promote Pam2EQL cross-presentation by BMDCs. WT (A) or TAPKO (B) BMDCs were transduced with Lenti-vector, Lenti-Rab7, or Lenti-Rab7T22N for 2 d before being peptide pulsed. The transduced BMDCs were pulsed with 1 μM Pam2IDG or Pam2EQL for 2.5 h at 37°C. After free peptides were washed away, 2 × 10⁴ BMDCs were cocultured for 48 h with 2 × 10⁵ CD8⁺ T cells that were purified from the splenocytes of OT-I–transgenic mice. Cell proliferation was determined using a [³H]thymidine incorporation assay. The data shown are representative of two independent experiments. **p < 0.01.