DEC-205 is a cell surface receptor for CpG oligonucleotides

Abstract

Synthetic CpG oligonucleotides (ODN) have potent immunostimulatory properties exploited in clinical vaccine trials. How CpG ODN are captured and delivered to the intracellular receptor TLR9, however, has been elusive. Here we show that DEC-205, a multilectin receptor expressed by a variety of cells, is a receptor for CpG ODN. When CpG ODN are used as an adjuvant, mice deficient in DEC-205 have impaired dendritic cell (DC) and B-cell maturation, are unable to make some cytokines such as IL-12, and display suboptimal cytotoxic T-cell responses. We reveal that DEC-205 directly binds class B CpG ODN and enhances their uptake. The CpG-ODN binding function of DEC-205 is conserved between mouse and man, although human DEC-205 preferentially binds a specific class B CpG ODN that has been selected for human clinical trials. Our findings identify an important receptor for class B CpG ODN and reveal a unique function for DEC-205.

Fig. 1.

DEC-205 directly binds CpG ODN. (A) CHO-K1 cells or CHO-K1 cells expressing DEC-205 or Clec12A were incubated on ice with 125 nM of FITC-conjugated 1668 CpG ODN (1 h). Binding was analyzed by flow cytometry. Data are representative of three experiments. ELISA plates were coated with 10 μg/mL of mAb against (B) DEC-205, Clec9A, Clec12A, or mouse DC-SIGN or (C) the Flag peptide. Graded doses of soluble flag-tagged DEC-205, Clec9A, Clec12A, or mouse DC-SIGN were then captured onto ELISA plates. Captured proteins were incubated with 1668 CpG ODN-biotin (10 μg/mL), and this binding was detected using SA-HRP and visualized with ABTS. (D and E) Sensorgrams showing binding of increasing concentrations of (D) DEC-205 protein or (E) control protein (Clec9A) injected over immobilized 1668 CpG ODN-biotin and (F) comparison of maximum response of DEC-205 and Clec9A binding as a percentage of maximal response as a function of concentration. Circles represent the mean values of two experiments ± SEM. The binding of CpG ODN by DEC-205 is not dependent on the CpG motif. (G–I) ELISA plates were coated with 10 μg/mL of 9B4 mAb against the Flag peptide and then incubated with mouse DEC-205 (5 μg/mL). Bound DEC-205 was interrogated with graded doses of biotinylated (G) 1668 CpG ODN, (H) 1826 CpG ODN, or (I) 2006 CpG ODN that either contained the CpG motif or not (No CpG). Representative data of three experiments are shown. (J) ELISA plates were coated with 10 μg/mL of mAb against the Flag peptide. Graded doses of either human or mouse flag-tagged DEC-205 were captured by the anti-Flag mAb and then exposed to various biotinylated CpG ODN (1 μg/mL). Alternatively, captured (K) mouse or (L) human DEC-205 (5 μg/mL) were exposed to graded doses of A- (2216), B- (1668 and 2006), or C- (2395) ODN. Bound CpG ODN were detected using SA-HRP and visualized using ABTS. This analysis was performed twice with similar findings, and one experiment is presented.

Fig. 2.

DEC-205 facilitates the uptake of CpG ODN. B6 or DEC-205^–/– mice were injected i.v. with 20 μg 1668 CpG ODN-Cy3. Spleens were extracted 30 min later, DC purified, and examined by flow cytometry (A) for their uptake of CpG ODN. CD8⁺ DC were sorted by flow cytometry and then analyzed by confocal microscopy (B) for their internalization of CpG ODN. Nuclei were stained with DAPI (blue), MHC class II (green), and CpG ODN-Cy3 (red). Pictures depict one slice in the middle of the cell. Experiment was conducted four times, with this particular staining combination performed twice. Representative data are presented.

Fig. 3.

DEC-205 facilitates DC maturation induced by CpG ODN. B6 or DEC-205^–/– mice were injected i.v. with 5 or 20 nmols of 1668 CpG ODN, and then 5 h later spleens were extracted, DC isolated, and CD8⁺ DC examined by flow cytometry for expression of CD40, CD80, and MHC class II. Representative histograms of (A) CD40, (B) CD80, and (C) MHC class II expression of steady-state or 1668 CpG ODN-activated CD8⁺ DC. Percent increase of mean fluorescence intensity of (D) CD40, (E) CD80, and (F) MHC class II in CD8⁺ DC and CD8^– DC in response to CpG. Cumulative data of five experiments ± SEM. Statistical significance was determined by a one-tailed unpaired Student t test.

Fig. 4.

DEC-205 facilitates B-cell activation induced by CpG ODN. (A) B6 or DEC-205^–/– mice were injected i.v. with 20 nmols of 1668 CpG ODN, and then 24 h later spleens were extracted and B cells analyzed for their expression of CD40, CD80, and MHC class II by flow cytometry. Representative histograms of three experiments are presented. (B) B cells (CD19⁺CD3^–) from B6 and DEC-205^–/– mice were purified by flow cytometry, incubated with 31.25 nM of 1668 CpG ODN for 16–18 h, and then assessed for their expression of CD40, CD86, and MHC class II. This experiment was performed three times, and representative histograms are shown. (C) The supernatants from the cultured B cells from (B) were collected, and IL-6 concentrations were measured by ELISA. Pooled data from two experiments are presented. Samples were run in duplicates or triplicates.

Fig. 5.

DEC-205 facilitates production of cytokines and the induction of CTL in response to CpG. B6 or DEC-205^–/– mice were injected i.v. with (A and B) 5 or 20 nmols of 1668 CpG ODN or (C) graded doses of LPS. Plasma samples were taken 3 h later, and levels of (A) IL-12p70 and (B and C) IL-6 were measured by Bio-Plex Pro cytokine bead arrays. For CpG-treatment, the cumulative data of four experiments are presented (n = 8) as mean ± SEM. For LPS-treated mice, the cumulative data of two experiments are presented (n = 4) as mean ± SEM. Statistical significance was determined by a one-tailed unpaired Student t test. n/s, not significant. (D) B6 or DEC-205^–/– mice were injected i.v. with 1 μg of anti-Clec12A-OVA mAb in the presence or absence of 5 nmols of CpG (1668). Six days later, mice were injected with OVA-peptide-coated target cells, and killing was measured 18 h later. The experiment was performed twice, and each symbol represents the lytic activity of an individual mouse.