1. Volume 36, Issue 5, Pages 782-794 (May 2012)
Adaptor Protein-3 in Dendritic Cells Facilitates Phagosomal Toll-like Receptor Signaling and Antigen Presentation to CD4+ T Cells 
Adriana R. Mantegazza, Susan H. Guttentag, Jamel El-Benna, Miwa Sasai, Akiko Iwasaki, Hao Shen, Terri M. Laufer, Michael S. Marks 
Immunity 
Volume 36, Issue 5, Pages (May 2012)
DOI: /j.immuni
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2. Immunity 2012 36, 782-794DOI: (10.1016/j.immuni.2012.02.018)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 AP-3 Is Not Required for Efficient OVA Cross-Presentation
BMDCs from WT, pearl, or gp91phox-deficient Cybb−/− mice were incubated for 30 min with the indicated concentrations of OVA peptide (A, B), soluble OVA (C, D), or latex beads conjugated to 10 mg/ml of a OVA:BSA mixture in the indicated ratios (E, F), then fixed and cocultured with OT-I cells for 18 hr.
(A, C, E) CD69 expression on OT-I cells was assessed by flow cytometry. Shown are the percentages of vβ5+ T cells that expressed CD69.
(B, D, F) IL-2 production by OT-I cells was measured by ELISA on the culture supernatants.
Data represent mean ± SD of three experiments performed in duplicate. ∗p < 0.05; ∗∗p < See also Figure S1.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
AP-3 Is Selectively Required for Optimal MHC-II Presentation of Phagocytosed Ag
BMDCs from WT or pearl mice were incubated for 30 min with the indicated concentrations of OVA peptide (A, B), soluble OVA (C, D), or OVA:BSA-conjugated latex beads (E, F, G) and then cocultured with OT-II cells for 18 hr.
(A, C, E) CD69 expression by OT-II cells was assessed by flow cytometry. Shown are the percentages of vβ5+ T cells that expressed CD69.
(F) Representative experiment showing CD69 upregulation on OT-II cells after phagocytosis of OVA:BSA beads by BMDCs. Shown are the percentages of vβ5+ cells that expressed CD69.
(B, D, G) IL-2 production by OT-II cells was measured by ELISA on the coculture supernatants.
Data in (A)–(E) and (G) represent mean ± SD of five experiments performed in duplicate. ∗p < See also Figures S1 and S2.
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5. Figure 3
AP-3 Is Required for the Induction of an Optimal Th1 Cell Response to Phagocytosed Ag
BMDCs from WT or pearl mice were incubated with the indicated concentrations of OVA peptide (A, B), soluble OVA (C, D), or OVA:BSA-conjugated latex beads (E–G), then cocultured with OT-II cells for 3 days. T cells were then restimulated with either CD3 and CD28 antibodies or with PMA and ionomycin in the presence of brefeldin A for 5 hr, labeled for intracellular IFN-γ (A, C, E, F) or IL-4 (B, D, G), and analyzed by flow cytometry. Shown are the percentages of vβ5+ T cells expressing each cytokine. Results represent mean ± SD of three experiments performed in duplicate. ∗p < 0.05; ∗∗p < 0.01.
(F) Representative experiment showing intracellular IFN-γ production by OT-II cells. Indicated are the percentages of vβ5+ OT-II cells that are IFN-γ+.
Immunity  , DOI: ( /j.immuni )
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6. Figure 4
AP-3 Is Required for Efficient MHC-II Presentation by Splenic DCs and for Th1 Cell Differentiation of OT-II T Cells during Infection with OVA-Expressing Listeria
(A–D) Splenic DCs were isolated from WT and pearl mice 1 day (A) or 3 days (B) after immunization with rLM, rLM-OVA, or OVA and cocultured in vitro with OT-I or OT-II cells. CD69 expression on T cells was analyzed after 18 hr. Shown are the percentages of vβ5+ T cells that expressed CD69 in representative experiments (A, B) and the mean ± SD from three experiments (C, D).
(E–H) CFSE-labeled CD45.1+ OT-II cells were adoptively transferred into CD45.2+ WT and pearl mice, and mice were infected with rLM or rLM-OVA 24 hr later.
(E and F) CD69 upregulation by CD45.1+CFSE+ OT-II cells from spleen single-cell suspensions was assessed by flow cytometry 18 hr after infection.
(E) Shown are the percentages of CFSE-positive T cells that expressed CD69.
(F) Representative experiment showing percentages of CFSE+-CD69+ OT-II cells after gating on CD45.1+ cells.
(G and H) 8 days after infection, single-cell suspensions of harvested spleens were restimulated for 5 hr with OVA peptide and CD3 antibody in the presence of brefeldin A, labeled for intracellular IFN-γ (G) or IL-4 (H), and analyzed by flow cytometry. Data represent mean ± SD of three experiments with five animals per group.
∗p < 0.05; ∗∗p < See also Figure S4.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5
Defective Presentation of Phagocytosed Ag in Pearl DCs Reflects Inefficient Trafficking of Peptide:MHC-II Complexes to the Cell Surface
BMDCs from WT and pearl mice were pulsed with soluble EαGFP fusion protein (A, B) or EαGFP beads (C–F) for 30 min, washed, and chased for the indicated times.
(A–D) Surface expression of Eα52-68:I-Ab complexes was analyzed by flow cytometry using Yae.
(A and C) Shown are the percentages of CD11c+ BMDCs that were Eα52-68:I-Ab+ over time.
(B and D) Representative experiment showing percentage of CD11c+ cells expressing surface Eα52-68:I-Ab complexes after the pulse. Also shown in (D) is a control in which cells were exposed to OVA beads instead of EαGFP beads and chased for 6 hr.
(E) Surface Eα52-68:I-Ab complexes were blocked with unlabeled Yae, then cells were fixed and permeabilized and intracellular Eα52-68:I-Ab complexes were detected by flow cytometry using biotinylated Yae. Shown are the percentages of CD11c+ BMDCs that were Eα52-68:I-Ab+.
(F) Cells harvested at 4 hr or 18 hr after the pulse were fixed, permeabilized, and analyzed by immunofluorescence microscopy with Yae and secondary antibody (top) and by phase contrast to visualize the beads (bottom).
Data in (A), (C), and (E) represent the mean ± SD from three experiments performed in duplicate. ∗p < 0.05; ∗∗p < See also Figure S5.
Immunity  , DOI: ( /j.immuni )
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8. Figure 6
AP-3 Is Required for Optimal TLR4 and MyD88 Recruitment to Phagosomes
(A–F, H, I) BMDCs from WT, pallid, pearl, or Tlr4−/− mice were pulsed with OVA beads and chased as indicated. Phagosomes were purified, fixed, permeabilized, and stained with FITC-conjugated TLR4 antibody (A, B) or with AP-3 antibody and Dylight 549 anti-mouse Ig (E, F) or with MyD88 or TRAM antibodies and Dylight 549 anti-rabbit Ig (H, I) or negative controls.
(A and H) Shown are histogram plots of representative experiments with the percentages of gated phagosomes that were positive for TLR4, MyD88, or TRAM indicated. Black lines show unstained or secondary antibody-only stained controls.
(B, F, I) Data from three to five independent experiments are represented as mean ± SD.
(E) Histogram plot shows overlaid staining of AP-3 on WT (red) and pearl (green) phagosomes. Individual plots are shown in Figure S6.
(C and D) Purified phagosomes were analyzed by SDS-PAGE and immunoblotting for TLR4 (C), MyD88, TRAM (D), or actin as indicated. Shown are the relevant bands from each blot.
(G) BMDCs from WT and pearl mice were pulsed with LPS-coated beads and chased for 3 hr. Cells were then fixed, permeabilized, labeled with TLR4 (red) and AP3 (green) antibodies and fluorochrome-conjugated secondary antibodies, and analyzed by confocal microscopy. Arrows indicate labeled phagosomes; asterisks denote unlabeled phagosomes. Differential interference contrast (DIC) images (bottom) show phagocytosed beads.
∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S6.
Immunity  , DOI: ( /j.immuni )
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9. Figure 7
AP-3 Is Required for Optimal Inflammatory Cytokine Production Induced by Particulate TLR Ligands
(A) BMDCs from WT, pallid, or pearl mice were incubated for 3 hr with soluble TLR ligands LPS (50 ng/ml), OVA (1 μg/ml), flagellin (Flag.; 10 μg/ml), peptidoglycan (PGN; 10 μg/ml), poly(I:C) (100 μg/ml), or R848 (0.1 μg/ml) or with the indicated ligand-coated or uncoated latex beads, and IL-6 released into the supernatants was measured by ELISA.
(B) BMDCs from WT or pearl mice were incubated in the presence or absence of LPS or LPS-coated beads for 2 hr. cDNA generated from isolated RNA was analyzed by real-time PCR. Data for mRNA levels of IL-12p35, IL-12p40, IL-6, and IFN-β1 from three independent experiments were normalized to the average of five housekeeping genes, and ΔΔCt values were calculated and represented as fold induction of mRNA in stimulated cells relative to unstimulated cells.
(C and D) BMDCs from WT or pearl mice were stimulated with LPS or LPS-coated beads for the indicated times, and cell lysates were analyzed by immunoblotting for phospho-p38, p38, phospho-IRF3, and IRF3.
(C) Shown are the relevant regions of representative blots. Arrows, phospho-IRF3-specific band; asterisks, a nonspecific band.
(D) Quantification of band intensities performed on three independent experiments, showing the fold increase in phospho-p38 (left) and phospho-IRF3 (right) levels relative to unstimulated cells and normalized to total protein levels of p38 or IRF3, respectively.
(A, B, D) Data from at least three independent experiments are represented as mean ± SD.
∗p < See also Figure S7.
Immunity  , DOI: ( /j.immuni )
Copyright © 2012 Elsevier Inc. Terms and Conditions