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. 2005 Jun;3(6):e185.
doi: 10.1371/journal.pbio.0030185. Epub 2005 Apr 26.

Apoptotic cells deliver processed antigen to dendritic cells for cross-presentation

Nathalie E Blachère  1 , Robert B Darnell, Matthew L Albert
Affiliations

Apoptotic cells deliver processed antigen to dendritic cells for cross-presentation

Nathalie E Blachère et al. PLoS Biol. 2005 Jun.

Abstract

Antigen derived from engulfed apoptotic cells can be cross-presented by dendritic cells (DCs) for the generation of major histocompatibility class I/peptide complexes. While the early events of recognition and internalization of the dying cell have been characterized, the antigen-processing pathway or pathways remain unknown. We established a mouse model assaying for the activation of polyclonal T cells reactive to antigen derived from apoptotic cells, and demonstrated two distinct pathways for the trafficking of exogenous epitopes. In the first, exogenous antigen is dependent on the DC's expression of a functional transporter associated with antigen processing (TAP). Surprisingly, we found evidence that a second pathway exists in which transfer of processed antigen from the dying cell allows formation of major histocompatibility class I/peptide complexes in TAP-deficient DCs. In vivo data suggest that in situations of stress (e.g., viral infection), this latter pathway may be more efficient, illustrating that dying cells may preselect immunologically important antigenic determinants.

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Figures

Figure 1
Figure 1. TAP−/− DCs Cross-Present Antigen Derived from Apoptotic Cells
(A and B) Antigen presentation via the endogenous pathway was evaluated in WT DCs and TAP−/− DCs by directly infecting cells with influenza virus and assaying for T cell activation. IFN-γ production and T cell precursor frequency were determined using an ELISPOT assay. (C) To evaluate transfer of TAP activity from the dying cells to DCs, WT or TAP−/− DCs after capture of apoptotic cells were directly infected and tested for their respective ability to activate CD8+ T cells via the endogenous pathway. (D and E) WT DCs (D) or TAP−/− DCs (E) were co-cultured for 36–48 h with influenza-infected or uninfected allogeneic cells in the presence of TNF-α. As above, mature DCs were harvested and assayed for their ability to stimulate influenza-reactive CD8+ T cells. To bypass the requirement for CD4+ T cell help in the activation of CD8+ T cells via the exogenous pathway, IL-12 was added to the cultures. Spot-forming cells (SFCs) per 106 T cells are reported. Data are representative of three experiments. Values are averages of triplicate wells with error bars indicating standard deviation.
Figure 2
Figure 2. Processed Antigen from the Dying Cell Is Required for MHC I Presentation in TAP−/− DCs
To generate apoptotic cells lacking processed antigen, lactacystin pretreatment of influenza-infected H-2d 3T3 cells was performed. Expression of influenza antigen was evaluated by intracellular FACS analysis using influenza NP mAbs followed by PE-conjugated goat anti-mouse mAb (A). Expression of MHC I/pep complexes in the lactacystin-treated 3T3 cells was evaluated by monitoring the activation of H-2d-restricted influenza hemagglutanin-reactive T cells. The Kd-restricted immunodominant peptide (HA210–219) derived from hemagglutanin was pulsed onto 3T3 cells and served as a positive control (B). The influenza-infected H-2d 3T3 cells were then induced to undergo apoptosis, and co-cultures were generated using C57BL/6 WT DCs (C) or TAP−/− DCs (D). To evaluate T cell activation and expansion, DCs cross-presenting antigen were cultured with CD8+ T cells in the presence of IL-12 for 7–8 d. T cells were then harvested and tested for influenza reactivity in a 20-h IFN-γ ELISPOT. H-2b EL4 cells with or without influenza infection served as the stimulators in the ELISPOT assay as above. Data are representative of two experiments. Values are averages of triplicate wells with error bars indicating standard deviation.
Figure 3
Figure 3. Transporter Activity Is Required in Either the Apoptotic Cell or the DC for Efficient Cross-Presentation of Antigen
RMA/S cells were infected with influenza and irradiated with UVB to allow for antigen loading and the induction of apoptotic death. Co-cultures were generated as described in the Materials and Methods using WT DCs (A) or TAP−/− DCs (B). DCs were harvested and tested for their ability to cross-present antigen and activate influenza-reactive CD8+ T cells as measured in a 40-h ELISPOT assay. To evaluate loading of MHC II, WT DCs and TAP−/−DCs that had captured apoptotic antigen were tested for their ability to activate influenza-reactive CD4+ T cells. Data are representative of four experiments. Values are averages of triplicate wells with error bars indicating standard deviation.
Figure 4
Figure 4. Immunization with Apoptotic Cells Results in the Selective Priming of T Cells Reactive to Processed Antigen
(A and B) C57BL/6 mice were immunized intraperitoneally with 300 HAU of influenza (A), or 5 × 106 infected apoptotic 3T3 cells (B). After 14 d, splenocytes were harvested, and CD8+ T cells were purified. To assay for the specificity of these cells, an IFN-γ ELISPOT was performed using the following stimulators: DCs alone or DCs pulsed with either 1 μM NP366–374 or 1 μM PA224–233 peptide. (C) C57BL/6 mice were immunized intraperitoneally with 5 × 106 untreated versus lactacystin-treated influenza-infected apoptotic 3T3 cells. As above, 14 d after priming, splenocytes were harvested, and CD8+ T cells were purified and assayed for their reactivity to NP366–374 versus PA224–233. In this experiment, peptide-pulsed EL4 cells were employed as the stimulators. Data are representative of two experiments. Values are averages of triplicate wells with error bars indicating standard deviation.
Figure 5
Figure 5. Processed Antigen within the Dying Cell Is Required for Efficient In Vivo Priming
C57BL/6 mice were immunized intraperitoneally with 300 HAU of influenza (A), or 2 × 106 infected apoptotic kidney epithelial cells derived from β2m-deficient (B) or TAP-deficient mice (C). After 14 d, splenocytes were harvested, and CD8+ T cells were purified. To assay for the specificity of these cells, an IFN-γ ELISPOT was performed using the following stimulators: EL4 cells alone or EL4 cells pulsed with either 1 μM NP366–374 or 1 μM PA224–233 peptide. Values are averages of triplicate wells with error bars indicating standard deviation.
Figure 6
Figure 6. An Active Role for Apoptotic Cells in the Transfer of Antigen to DCs
We propose that apoptotic cells play an active role through the transfer of processed antigen to DCs for the generation of MHC I/pep complexes. This pathway may be dominant in the presentation of infectious antigen as the virus may co-opt cellular translational machinery, resulting in high levels of viral protein, and the upregulation of stress proteins, as well as inducing apoptotic cell death. Defective ribosomal initiation products chaperoned by HSPs offer a potential source of antigen. Within the DC, HSPs derived from the internalized apoptotic cell may traffic via a retrograde transport pathway, shuttled to the trans-Golgi and then the ER via binding to KDEL receptors (A). Alternatively, the evidence for phagosome–ER (PHAGO-ER) fusion and/or the recycling of MHC I from the plasma membrane offers the possibility that processed antigen may interact directly with the DC's MHC I (B). As ER chaperones within the phagocytosed cell would be bound to the pool of peptides derived from newly synthesized proteins, these pathways offer the DC an accurate representation of what occurred immediately prior to death (A and B). At high concentrations of protein, we also find evidence for the DC processing the cross-presented antigen. This likely occurs via a phago–ER-to-cytosol pathway as has been previously described (C).
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