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. 2010 Mar 2;107(9):4287-92.
doi: 10.1073/pnas.0910609107. Epub 2010 Feb 8.

Mature dendritic cells use endocytic receptors to capture and present antigens

Craig D Platt  1 , Jessica K Ma, Cécile Chalouni, Melanie Ebersold, Hani Bou-Reslan, Richard A D Carano, Ira Mellman, Lélia Delamarre
Affiliations

Mature dendritic cells use endocytic receptors to capture and present antigens

Craig D Platt et al. Proc Natl Acad Sci U S A. .

Abstract

In response to inflammatory stimuli, dendritic cells (DCs) trigger the process of maturation, a terminal differentiation program required to initiate T-lymphocyte responses. A hallmark of maturation is down-regulation of endocytosis, which is widely assumed to restrict the ability of mature DCs to capture and present antigens encountered after the initial stimulus. We found that mature DCs continue to accumulate antigens, especially by receptor-mediated endocytosis and phagocytosis. Internalized antigens are transported normally to late endosomes and lysosomes, loaded onto MHC class II molecules (MHCII), and then presented efficiently to T cells. This occurs despite the fact that maturation results in the general depletion of MHCII from late endocytic compartments, with MHCII enrichment being typically thought to be a required feature of antigen processing and peptide loading compartments. Internalized antigens can also be cross-presented on MHC class I molecules, without any reduction in efficiency relative to immature DCs. Thus, although mature DCs markedly down-regulate their capacity for macropinocytosis, they continue to capture, process, and present antigens internalized via endocytic receptors, suggesting that they may continuously initiate responses to newly encountered antigens during the course of an infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Antigens bound to FcγR and anti-DEC-205 are internalized and trafficked to late endosomal compartments in fully mature DCs. (A) Immature or LPS-matured BMDCs were incubated with Lucifer Yellow (1 mg/mL) for 15 min at 37 °C, washed, incubated with HRP-Cy5/rabbit anti-HRP ICs (10 μg/mL) or no IC as a control for 20 min at 4 °C, washed, and further incubated at 4 °C or 37 °C for the indicated time. The remaining surface ICs were detected with anti-rabbit IgG-phycoerythrin (PE). Cells were then stained for CD86 and CD11c. Mature DCs are gated on the CD11c+, CD86-high cells, Lucifer Yellow-low (poorly macropinocytic) cells, and immature DCs are gated on the CD11c+, CD86-low, Lucifer Yellow-high (macropinocytic) cells. Average values of two experiments are displayed. (B) As in A, but BMDCs were incubated with anti-DEC-205-PE or isotype control (5 μg/mL) and remaining surface antibodies were detected using anti-rat IgG-Alexa 647. (C) LPS-matured BMDCs were incubated with soluble OVA-Alexa 647 (0.5 μg/mL) for 10 min, stained for CD86 and CD11c, and sorted on CD11c+, CD86-high, OVA-Alexa 647-low (poorly macropinocytic) cells, ensuring a pure population of mature DCs that did not internalize soluble antigens. DCs were then incubated with FITC and 10 nm of gold HRP ICs (10 μg/mL) or with Alexa 488 and 10 nm of gold anti-DEC-205 (5 μg/mL) as in A before fixation, staining for the indicated markers, and analysis by confocal or electron microscopy.
Fig. 2.
Fig. 2.
Fully mature DCs efficiently phagocytose IgG-coated latex beads. (A) Immature and LPS-matured BMDCs were sorted according to CD86 surface expression and macropinocytic activity as in Fig. 1C. Cells were incubated with IgG or BSA-coated fluorescent beads for 30 min at 37 °C, washed, and chased for 15 min at 37 °C. After pulse chase, the cells were fixed, stained for the indicated markers, and analyzed by confocal microscopy. (B) At least 90 DCs from each condition were selected at random and examined for the presence of beads within H-2M+ compartments, thus confirming complete internalization.
Fig. 3.
Fig. 3.
Fully mature DCs efficiently present antigen internalized by FcγR and DEC-205. (A) Untreated and LPS-matured BMDCs were incubated with OVA-Alexa 647 (0.5 μg/mL) for 10 min at 37 °C, stained for CD86 and CD11c, and sorted into highly pure populations of mature and immature cells. Mature DCs were defined as CD86-high OVA-low LPS-treated cells, and immature DCs were defined as CD86-low OVA-intermediate/high untreated cells. Sorted cells were fed with OVA-rabbit anti-OVA ICs or with soluble OVA and LPS for 30 min at 37 °C. DCs were cultured with 105 OT.2 CD4+ T cells for 24 h on anti-CD28-coated plates. T-cell activation was assessed by measuring IL-2 secretion by ELISA. Averages of triplicates are displayed. (B) Performed as in A but with anti-DEC-205-OVA and isotype control-OVA fusion proteins. (C) Untreated and LPS-matured BMDCs were stained for CD86 and then incubated with Eα aggregates or Eα-aggregate ICs (30 μg/mL) and LPS for 7 h or with LPS alone as a control. Cells were then stained with YAe antibody and anti-CD11c and analyzed by flow cytometry. Mature DCs are gated on the CD11c+ CD86-high cells, and immature DCs are gated on the CD11c+ CD86-low cells. Values are normalized to those of DCs receiving ICs that were immature when first exposed to antigen. Bars indicate SE. Results are averages of two independent experiments. (D) Performed as in A and B but DCs were cultured with CD8+ OT.1 T cells specific for MHCI-OVA peptide complexes to assess presentation on MHCI.
Fig. 4.
Fig. 4.
Fully mature DCs load peptide onto MHCII in the lysosomal compartment. (A) Immature or LPS-matured BMDCs were sorted as in Fig. 3A and incubated at 37 °C for 90 min with Eα ICs (30 μg/mL) and LPS. Cells were washed and then fixed or incubated at 37 °C for an additional 18.5 h before fixation. Cells were stained with YAe, anti-H2M, and anti-MHCII antibodies and then analyzed by confocal microscopy. (B) Immature or LPS-matured BMDCs were stained for CD86 and then incubated with CHX (1 μg/mL) for 30 min. Eα ICs (30 μg/mL) were added to the DCs for 5 h at 37 °C in presence of CHX. Cells were then stained with YAe and anti-CD11c antibodies and analyzed by flow cytometry. Mature DCs are gated on the CD11c+ CD86-high cells, and immature DCs are gated on the CD11c+ CD86-low cells.
Fig. 5.
Fig. 5.
Mature splenic DCs efficiently present antigen in vitro but have reduced access to antigen in the setting of systemic inflammation. (A) Reduced presentation of antigen by mature splenic DCs in vivo. Twenty-four hours after adoptive transfer of 106 carboxyfluorescein succinimidylester (CFSE)-labeled OT.2 CD4+ T cells, B6/C57J mice were injected i.p. with 3 μg of LPS or PBS control. After an additional 5 h, OVA ICs, soluble OVA, SIINFEKL-OVA peptide, or rabbit anti-OVA antibody as a control was i.v. injected. Spleens were harvested 4 h later, and T-cell activation was assessed by CD69 up-regulation. (B) DCs were isolated from the spleens of mice injected i.p. with 3 μg of LPS or PBS 9 h before harvest. DCs were sorted on CD11c+ CD86-high cells; incubated with anti-DEC-205-OVA, OVA ICs, or soluble OVA for 2 h; and cultured with CFSE-labeled CD4+ OT.2 T cells. Sixty hours later, the extent of cell division was determined by flow cytometry. Average values of duplicates are displayed. (C) Three micrograms of anti-DEC-205-FITC or isotype control antibody was injected i.v. into mice pretreated for 9 h with 3 μg of LPS injected i.p. or with PBS control. Spleens were harvested 2 h later, and DCs were analyzed by flow cytometry. Histograms of CD11c+ cells are displayed. (D) Mice were injected i.p. with 3 μg of LPS or with PBS control. Nine hours later, Eα aggregates (30 μg), Eα-aggregate ICs (30 μg), or PBS control was injected s.c. into the hind leg hocks (each injection contained 1 μg of LPS). Draining popliteal LNs were harvested 4 h later, and cells were stained with the YAe antibody. Histograms are of CD11c+ cells.
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