Article Figures & Data
Figures
- Figure 1. Effect of Arf6 knockdown on cell surface MHC class I expression.
BM-DCs were transduced with lentiviruses carrying shRNA sequences targeting Arf6 (shArf6) or a nontargeting (shNT) control. (A) Arf6 relative gene expression to shNT was measured by qPCR. Data represent mean and SEM of n = 5 independent experiments. (B) Transduced C57BL/6 (Kb) or Balb/c (Ld) BM-DCs were stained with mAb against Kb (B8 and AF6) or Ld (64 and 30), followed by a fluorescently labeled secondary antibody and a Bv421-conjugated antibody against CD11c. The fluorescence was recorded in the CD11c+ cells. Data represent mean and SEM of n ≥ 6 independent experiments. (C) Transduced Balb/c cells were permeabilized and stained with mAbs 64 and 30. Data represent mean and SEM of n = 5 independent experiments. (D) Transduced C57BL/6 BM-DCs were incubated with 5 μg/ml BFA; at each time point, cells were stained with the indicated antibodies and analyzed by flow cytometry. (E) Kb levels at the cell surface of shArf6-transduced BM-DCs during the BFA treatment were plotted as the percentage of Kb levels of shNT-transduced cells. (F) Balb/c BM-DCs were acid-stripped or not and reincubated at 37°C in the presence of 5 μg/ml BFA. Then, acid-treated cells were stained with mAb 64 and untreated cells with mAb 30. (G) The Ld levels at the cell surface of shArf6-transduced BM-DCs during the BFA treatment were plotted as the percentage of Ld levels on shNT-transduced cells. Data represent the means and SEM of n = 3 independent experiments. (H) Transduced C57BL/6 BM-DCs were acid stripped and reincubated at 37°C in the presence or absence of 5 μg/ml BFA before staining with AF6; a representative experiment is shown. (I, H) Data in (H) expressed as a percentage of cell surface values before acid stripping. (J) The Kb levels at the cell surface of acid-stripped shArf6-transduced BM-DCs incubated without BFA are plotted as the percentage of Kb levels of shNT-transduced cells. Data represent the means and SEM of n = 3 independent experiments. Data in (A, B, C, J) were evaluated with a one-sample t test, under the null hypotheses that the column means of the sample are equal to 100%. Data are significantly different if P ≤ 0.05 (*), P ≤ 0.01 (**), or P ≤ 0.001 (***).
- Figure S1. Detection of unfolded Ld molecules by microscopy and flow cytometry.
(A) shNT and shArf6-transduced Balb/c BM-DCs were stained for unfolded and folded Ld molecules at the cell surface. (B, C) shNT and shArf6-transduced Balb/c or C57BL/6 BM-DCs were stained for unfolded and folded Ld molecules intracellularly. (D, E) Balb/c BM-DCs were acid stripped and stained for folded and unfolded Ld molecules using mAbs 30 and 64, respectively, for analysis by immunofluorescence microscopy. (D, E, F) Control C57BL/6 BM-DCs were stained using mAbs 64 and 30 as in (D) and (E) and analyzed by confocal microscopy. Nuclei are stained with DAPI (blue). NT, non-targeting shRNA.
- Figure 2. Intracellular localization of Arf6 in the DC 2.4 cell line.
(A, B, C) Steady state DC 2.4 cells expressing Arf6-mCherry were stained with antibodies against (A) EEA-1, (B) LAMP-1, (C) Rab11. Scale bar: 5 μm. (A, B, C, D) Quantification of (A, B, C). Nuclei are stained with DAPI (grey). Data represent at least 25 cells from n = 3 independent experiments.
- Figure S2. Nocodazole disturbs the intracellular Arf6 compartment.
DC2.4 cells transduced with Arf6-mCherry were treated with 5 μg/ml nocodazole for 30 min at 37°C. (A, B, C, D) Cells were fixed, permeabilized, and stained with antibodies against (A), EEA-1 (B), LAMP-1 (C), Rab11 (D), total MHC class I (Exon 8). Nuclei are stained with DAPI (grey). Scale bar: 5 μm.
- Figure S3. Intracellular localization of Kb after extended internalization.
(A, B), C57BL/6 BM-DCs were acid stripped followed by staining with mAb B8 (B), C57BL/6 BM-DCs were pulsed with mAb AF6 on ice, washed, and analyzed by confocal microscopy after secondary antibody and Rab11 staining either immediately or after a 2 h incubation at 37°C to allow for Kb internalization. Note that visualization of internalized H-2Kb requires overexposure (two bottom row images). Nuclei are stained with DAPI. Scale bars: 20 μm; magnifications: 5 μm. (blue). NT, nontargeting shRNA.
- Figure 3. Folded and unfolded MHC-I enter early endosomes independently of Arf6.
(A, B) Transduced Balb/c BM-DCs were fixed, permeabilized, and stained with a combination of antibodies: EEA-1 (red) and 64 (green, (A)) or 30 (green, (B)). (A, C, D) Cells were preincubated with 64 (after acid stripping) (C) or 30 (D) on ice water 30 min. After washing excess antibodies, cells were incubated for the indicated time points at 37°C, and then treated as in (A). (C, D, E, F) Quantification of (C) and (D). Nuclei are stained with DAPI (blue). Data represent at least 15 cells per condition from two independent experiments. Nuclei are stained with DAPI (blue). Scale bars = 20 μm in upper panels, and 5 μm in lower panels. NT, nontargeting shRNA.
- Figure 4. Delayed disappearance of unfolded MHC-I in Arf6-depleted cells.
(A, B) Transduced Balb/c BM-DCs cells were fixed, permeabilized, and stained with antibodies against LAMP1 (red) and 64 (green, (A)) or 30 (green, (B)). (A, C, D) Cells were preincubated with 64 (after acid stripping) (C) or 30 (D) on ice water 30 min, washed, incubated for the indicated duration at 37°C, and treated as in (A). (E) Cells were preincubated with 20 nM ConB, acid-stripped, pulsed with 64 on ice water 30 min, reincubated for 2 h at 37°C with ConB, fixed, and stained with antibodies against LAMP1 (red), EEA-1 (magenta), and a secondary antibody against 64 (green). (E, F) Cells were treated as in (E) but without acid stripping and stained with mAb 30 (green). (E, G) Quantification of (E). (F, H) Quantification of (F). The area (left panel) and mean grey values (center panel) of each cell were quantified as described in material and methods, and the product area × mean grey value was expressed as the integrated density. Data in (E) and (G) are from at least 62 cells from three independent experiments. Data in (F) and (H) are from at least 42 cells from three independent experiments. Data were evaluated with an unpaired nonparametric Mann–Whitney test, P ≤ 0.05 (*), P ≤ 0.01 (**), or P ≤ 0.001 (***). Nuclei are stained with DAPI (blue). Scale bars = 20 μm in upper panels, and 5 μm in lower panels. NT, nontargeting shRNA.
- Figure 5. Folded MHC-I residing in the ERC are not derived from the cell surface.
(A, B) Transduced Balb/c BM-DCs cells were fixed, permeabilized, and stained with antibodies against Rab11a (red) and with 64 (green, (A)) or 30 (green, (B)). (A, C, D) Cells were pulsed with 64 (after acid stripping, (C)) or 30 (D) on ice water, incubated for the indicated time points at 37°C, and treated as in (A). Nuclei are stained with DAPI (blue). Data represent at least 27 cells per condition from n ≥ 3 independent experiments. Scale bars = 20 μm in upper panels, and 5 μm in lower panels. NT, nontargeting shRNA.
- Figure 6. MHC-I colocalizing with Arf6 is not derived from the cell surface.
(A) DC 2.4 cells expressing Arf6-mCherry were fixed, permeabilized, and stained with the antibody exon 8. Quantification represents 30 cells from three independent experiments. (B, C) DC 2.4 cells expressing Arf6-mCherry were pulsed on ice with AF6, incubated for 20 (B), or 60 min (C), at 37°C to allow for internalization, fixed, permeabilized, and stained for Rab11 and AF6 (secondary antibody only). Nuclei are stained with DAPI (blue). Scale bars 5 = μm.
- Figure 7. MHC-I recycles through a fast Arf6-independent pathway in BM-DCs.
(A) C57BL/6 BM-DCs were pulsed with AF6 for 30 min on ice and shifted to 37°C for 30 min in the presence or absence of primaquine to allow for internalization and stained for AF6 using a secondary antibody. (A, B) BM-DCs were treated as in (A) including the internalization step for 30′, acid stripped, reincubated at 37°C in the absence of primaquine to allow for recycling, and finally stained with a secondary antibody for analysis by flow cytometry. (C) Transduced C57BL/6 BM-DCs were pulsed with AF6 as in (A) and then allowed to internalize AF6-bound H-2Kb for 60 min in the presence of primaquine. (B, D) Recycling of H-2Kb was analyzed as in (B), following an internalization step of 60 min. (A, B, C, D) Data represent means and SEM of n = 3 (A, B) and n = 4 (C, D) independent experiments. NT, nontargeting shRNA.
- Figure S4. Representative recycling assay.
(A, B), Representative histograms of an individual experiment of cells treated as on Fig 7A and B. (A, B, C), Geometric means of all the time points from panels (A) and (B). (A, B, C, D), Zoom into the geometric means of the recycling stage shown in panels (A), (B), and (C). NT, nontargeting shRNA.
- Figure 8. Effect of Arf6 depletion on cross-presentation of various antigen forms.
(A) Transduced C57BL/6 BM-DCs were pulsed with various amounts of SL8 peptide, fixed, and cocultured with OT-I T cells. The next day, the supernatant was recovered and probed for IL-2 by ELISA. (B, C, D, E, F) Cross-presentation to OT-I cells of peptide GS-20 (B), of irradiated yeast cells expressing full length OVA at the cell surface (C), of OVA-anti-OVA IC (D), and of the OVA fusion protein P3UO targeted to CD11c (E), or CD206 (F). (A, B, C, D, E, F) Data represent the means and SD of n = 3 independent experiments (A, B, C, E, F) and n = 4 (D). Data were evaluated with a one-sample t test, under the null hypotheses that the column means of the sample are equal to 100%. Data are significantly different if P ≤ 0.05 (*), P ≤ 0.01 (**), or P ≤ 0.001 (***). NT, nontargeting shRNA.
- Figure S5. Representative IL-2 secretion after cross-presentation experiments.
Cells were treated as on Fig 8. (A, B, C, D, E, F), SL8 peptide (B), GS-20 long peptide (C), OVA-yeast (D), IC anti ovalbumin–ovalbumin (E), fusion protein P3UO targeted to CD11c (F), fusion protein P3UO targeted to CD206. Data represent a single experiment with mean and SD of two duplicate samples. NT, nontargeting shRNA.
- Figure 9. Intracellular routing and degradation of ICs and soluble OVA.
(A) FACS analysis of FcγRI/II cell surface expression in transduced BM-DCs. (B) Arf6-depleted and control BM-DCs were analyzed for IC binding to the surface by pulsing at 4°C with OVA–anti-OVA complexes. Upper panels: focal plane; Lower panels: cumulative z-projection. Scale bar: 20 μm. (C, D) After binding at 4°C, ICs were allowed to internalize at 37°C for 10 or 30 min and cells were stained for EEA-1 (C), and LAMP-1 (D), and a secondary antibody against the OVA antibody. Scale bars, upper panels: 20 μm; lower panels 5 μm. (C, E), Quantification of (C). Data represent at least 15 cells from three independent experiments. Nuclei are stained with DAPI (blue). (F), Transduced BM-DCs were pulsed with soluble OVA-647 on ice and incubated at 37°C for the indicated time points, before recording OVA fluorescence by flow cytometry. Data represent mean and SEM from n = 3 independent experiments. (F, G) As in (F), with the IC formed between an OVA antibody and OVA-647. (H) DC 2.4 cells expressing Arf6-mCherry were fed soluble DQ-OVA at 37°C, and the uptake was followed by live-cell imaging for 1 h. Scale bar: 5 μm. (H, I) As in (H), with IC containing DQ-OVA. Data in (H) and (I) represent n = 2 independent experiments, with 10 cells per condition. NT, nontargeting shRNA.
- Figure S6. Antigen degradation assays.
(A, B) ICs between an OVA antibody and 200 μg/ml soluble OVA-647 were formed at an equimolar ratio for 1 h at 4°C. (A, B) Then, (A), soluble OVA-647 or (B), ICs were added to BM-DCs and incubated on ice for 20 min. After extensive washing, fluorescence was analyzed by flow cytometry. (C), DCs were pulsed with DQ-OVA or IC containing DQ-OVA on ice, washed, and analyzed by FACS. NT, nontargeting shRNA.
Supplementary Materials
- Video 1
DC 2.4 Arf6-mCherry cells were seeded into IBIDI chambers. After equilibrating the chambers at 37°C and 5% CO2 for 5 min, ICs formed between an anti-OVA antibody and DQ-OVA at 200 μg/ml DQ-OVA were added to the cells, and images were taken every 30 s during 60 min.Download video
- Video 2
DC2.4 Arf6-mCherry cells pulsed with IC were monitored for 26 min by live cell imaging.Download video
- Video 3
The cells shown in Videos 2 were monitored for an additional 15 min by live cell imaging.Download video
- Video 4
One individual cell from Videos 2 is shown.Download video
- Video 5
One individual cell from Videos 3 is shown.Download video
- Video 6
DC 2.4 Arf6-mCherry cells were seeded into IBIDI chambers. After equilibrating the chambers at 37°C and 5% CO2 for 5 min, soluble DQ-OVA at 200 μg/ml DQ-OVA was added to the cells, and images were taken every 30 s during 60 min. Recording started 20 min after adding OVA.Download video
- Video 7
An individual cell from Videos 6 is shown.Download video
