The role of MHC class I recycling and Arf6 in cross-presentation by murine dendritic cells

(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.

(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.

(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.

(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.

(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.

(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-2K^b for 60 min in the presence of primaquine. (B, D) Recycling of H-2K^b 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.

(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.

(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.