Cross-presentation of synthetic long peptides by human dendritic cells: a process dependent on ERAD component p97/VCP but Not sec61 and/or Derlin-1

Abstract

Antitumor vaccination using synthetic long peptides (SLP) is an additional therapeutic strategy currently under development. It aims to activate tumor-specific CD8(+) CTL by professional APCs such as DCs. DCs can activate T lymphocytes by MHC class I presentation of exogenous antigens - a process referred to as "cross-presentation". Until recently, the intracellular mechanisms involved in cross-presentation of soluble antigens have been unclear. Here, we characterize the cross-presentation pathway of SLP Melan-A16-40 containing the HLA-A2-restricted epitope26-35 (A27L) in human DCs. Using confocal microscopy and specific inhibitors, we show that SLP16-40 is rapidly taken up by DC and follows a classical TAP- and proteasome-dependent cross-presentation pathway. Our data support a role for the ER-associated degradation machinery (ERAD)-related protein p97/VCP in the transport of SLP16-40 from early endosomes to the cytoplasm but formally exclude both sec61 and Derlin-1 as possible retro-translocation channels for cross-presentation. In addition, we show that generation of the Melan-A26-35 peptide from the SLP16-40 was absolutely not influenced by the proteasome subunit composition in DC. Altogether, our findings propose a model for cross-presentation of SLP which tends to enlarge the repertoire of potential candidates for retro-translocation of exogenous antigens to the cytosol.

Figure 1. SLP_{16 –40}-FITC is cross-presented efficiently by the DC.

Flow cytometry of DC activation of the 10C10 CD8+ T-cell clone 10C10. DC were pulsed for 3 h in the presence of short peptide MelanA_26–35, SLP_16–40, or SLP_16–40-FITC before co-culture with the 10C10 clone at a 1∶1 cell ratio. The histogram represents the fluorescence emitted by the 10C10 clone stained with anti-IFN-γ.

Figure 2. SLP_{16 –40}-FITC fluorescence monitored by confocal microscopy.

Immunofluorescence microscopy. Kinetics of internalization of SLP_16–40-FITC (green) by DCs. DCs were stained at the cell membrane with antibody w6–32 (red) and nuclei were counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Data are representative of two independent experiments. Scale bar represent a distance of 5 µm.

Figure 3. Study of SLP_{16 –40}-FITC colocalization in DCs : colocalization in early endosomes and lysosomes.

(A) Immunofluorescence microscopy. Kinetics of internalization of DCs incubated with SLP_16–40-FITC (green), 15, 30, 60 or 120 minutes after pulse. DCs are stained at the early endosomes with antibody anti-EEA-1 (red) and the nuclei are counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Single scans are representative for multiple cells analysed in at least 2 experiments. (B) Immunofluorescence microscopy. Kinetics of internalization of DCs incubated with SLP_16–40-FITC (green), 15, 30, 60 or 120 minutes after pulse. DCs are stained at the lysosomes with antibody LAMP-1 (red) and the nuclei are counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Single scans are representative for multiple cells analysed in at least 2 experiments. Scale bar represent a distance of 5 µm.

Figure 4. Study of SLP_{16 –40}-FITC colocalization in DCs: no colocalization in Golgi and ER.

(A) Immunofluorescence microscopy. Kinetics of internalization of DCs incubated with SLP_16–40-FITC (green), 30, 60 or 120 minutes after pulse. DCs are stained at the Golgi with antibody anti-GM130 (red) and the nuclei are counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Data are representative of two independent experiments. (B) Immunofluorescence microscopy. Kinetics of internalization of DCs incubated with SLP_16–40-FITC (green), 30, 60 or 120 minutes after pulse. DCs are stained at the ER with antibody anti-calreticulin (red) and the nuclei are counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Data are representative of two independent experiments. Scale bar represent a distance of 5 µm.

Figure 5. Kinetics of internalization of SLP_{16 –40} in DCs.

(A) Volume measurement of green fluorescence (SLP_16–40-FITC) with red fluorescence (ER) colocalization at each time point. For each point, average volume was determined on five different cells of two independent experiments. (B) Volume measurement of green fluorescence (SLP_16–40-FITC) with red fluorescence colocalization (early endosomes or lysosomes) at each time point. Representations of colocalization in early endosomes are in white bars and colocalization in lysosomes are in black bars. For each point and each compartiment, average volume was determined in five different cells from two independent experiments. (C) The colocalization of SLP_16–40-FITC immunofluorescence with intracellular compartments (early endosomes, lysosomes, ER and Golgi apparatus) was quantified by measuring the Pearson correlation coefficient (Rr) with Velocity software. A Pearson correlation of 1 indicates complete colocalization, a value of 0 indicates no specific colocalization and a value of −1 indicates a perfect but inverse correlation (exclusion). Measurements of the Pearson correlation coefficient indicate a reasonable degree of partial colocalization of SLP_16–40 with early endosomes between 5 and 60 minutes and with lysosomes between 45 and 120 minutes. The Pearson correlation coefficient of SLP_16–40 with ER and Golgi apparatus lysosomes indicates no specific colocalization. The Pearson correlation coefficient was measured with n = 5 cells. Statistical significance of colocalization was compared to the null hypothesis of no specific colocalization (Pearson correlation coefficient value of 0).

Figure 6. Immature and mature DC express mixed-type proteasomes.

(A) Untreated day 5-immature DC and DC treated with LPS (1 µg/ml) for 24 hours were analysed for their proteasome content by western-blotting using antibodies against β1, β2, β5, β1i, β2i, β5i, as indicated. Purified 26 proteasomes (250 ng) from erythrocytes (standard proteasome) and spleen (mixture of standard and immunoproteasome) were used as sources to ensure antibody specificity. To control for equal loading, proteins were subjected to western blotting using the anti-β-actin antibody. (B) Flow cytometry of DC recognition by CD8⁺ T-cell clone 10C10. DCs were treated with Epoxomicin (1 and 5 µM) for 30 min, then DCs were pulsed for 3 h in the presence of short peptide MelanA_26–35, or SLP_16–40, and inhibitor before co-culture with the 10C10 clone at a 1∶1 cell ratio. Data are representative of at least three independent experiments. Statistical analysis was performed using non-parametric Mann-Whitney test and values in the presence of inhibitor were significantly different (p<0,04).

Figure 7. Effect of siRNA depletion of each of the inducible proteasome subunit β1i, β2i, β5i on the DC-mediated cross-presentation of the SLP 16–40.

(A) DC were transfected with 1 µM of control siRNA or β1i, β2i, β5i -targeting siRNA for 72 hours. The knockdown of the above-stated inducible subunits as well as its impact on the steady-state level of each of the standard proteasome subunits (β1, β2, β5) was analysed by western-blotting using specific antibodies, as indicated. Antibody against b-actin was used to ensure an equal protein loading. (B) IFN-g production of the LT CD8+10C10 responded to β1i, β2i, β5i -depleted DC pulsed with either SLP16–40 or Melan- A26–35. All data are shown as means +/− SD and are representative of three independent experiments.

Figure 8. Effects of different inhibitors on SLP_{16 –40} cross-presentation.

(A) TAP transport: Flow cytometry of DC recognition by LT CD8⁺10C10. DCs were treated with inhibitor, ICP47 (50 and 100 µM) for 30 min, then DCs were pulsed for 3 h with the short peptide Melan-A_26–35, or SLP_16–40, and in the presence of the inhibitor before co-culture with the LT CD8⁺10C10 at a 1∶1 cell ratio. Data are representative of at least three independent experiments. Statistical analysis was performed using non-parametric Mann-Whitney’s test and values in the presence of inhibitor were significantly different (p<0.04). The molecular mechanisms involved in cross-presentation of SLP_16–40. Flow cytometry of DC recognition by LT CD8⁺10C10. DCs were treated with inhibitor, (B) NH₄Cl (20 mM and 50 mM) or (C) ExoA (10 and 20 µg/mL) for 30 min, then DCs were pulsed for 3 h in the presence of short peptide Melan-A_26–35, or SLP_16–40, and inhibitor before co-culture with the LT CD8⁺10C10 at a 1∶1 cell ratio. Data are representative of at least three independent experiments. Statistical analysis was performed using non-parametric Mann-Whitney’s test and values in the presence of inhibitor were significantly different (p<0,04).

Figure 9. Gene silencing of the ERAD-related component p97/VCP substantially impairs cross-presentation of the SLP16–40.

(A) Day5-immature HLA-A2+ DC were electroporated with 1 µM of either control siRNA or siRNA against p97/VCP, Derlin-1 or Sec61a1, as indicated. The steady-state protein level of each of the targeted gene was determined by western-blot analysis 72 hours later. To control for equal loading, samples were subjected to western blotting using the anti-b-actin antibody. (B) The Melan-A26–35 CTL response against siRNAtreated DC loaded with either 10 µM of SLP16–40 or 1 µM of Melan-A26–35 (as a positive control) was examined using an IFN-γ ELISA. All data are shown as means +/− SD and are representative of three independent experiments. **p<0.01 (t-Test).

Figure 10. Putative cross-presentation mechanisms of SLP Melan-A_{16 –40}.