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Keywords:

  • Cross presentation;
  • Dendritic cells;
  • Protein trafficking

Adaptive immunity is induced when DCs internalize antigens for processing and presentation on MHC molecules. While antigens targeted toward lysosomes are degraded by lysosomal proteases and loaded onto MHC class II (MHC II), antigens internalized by the mannose receptor (MR) are routed toward a distinct endosome subpopulation, which retains characteristics of early endosomes [1]. From these endosomes, MR internalized antigens are processed for loading onto MHC class I (MHC I) [1], a process termed as cross-presentation. Such processing includes antigen transport into the cytoplasm for proteasomal degradation [2, 3]. Despite intensive investigations, the key proteins regulating the export of antigen into the cytoplasm or the endosomal loading of MHC I molecules remain unknown. Hence, the protein content of such antigen-bearing endosomes is of high interest. While purification and analysis of phagosomes have been established quite extensively [4], the analysis of endosomes containing soluble antigens remains difficult. In this comment, we describe the establishment of a novel method to analyze the protein content of such endosomes by flow cytometry. Importantly, particular care was taken to avoid unwanted clustering of individual endosomes, which can occur during centrifugation, as this severely affects the reliability of the flow cytometric analysis.

To analyze endosomes specifically supplied by the MR, we generated MR expressing HEK293 T cells (HEK-MR), which internalize high amounts of OVA (Fig. 1A). We then disrupted HEK-MR cells, which had been incubated with fluorochrome-labeled OVA, and isolated endosome preparations. Flow cytometric analysis of these preparations revealed OVA-containing particles in the endosomal preparations of HEK-MR cells but not of control cells (Fig. 1B, Supporting Information Fig. 1 and 2). This indicated that these structures might indeed be endosomes. To test this hypothesis, we compared them with calibrated size beads, which revealed that the OVA-containing structures have a size of around 500 nm (Fig. 1C), which corresponds to the size of endosomes supplied by the MR [5]. To ensure that the observed structures are indeed intact organelles, we treated them with different detergents before analysis (Supporting Information Fig. 3), which resulted in a release of OVA, demonstrating that these structures are indeed membrane-bound vesicles.

image

Figure 1. Analysis of endosome preparations from HEK-MR cells. (A) OVA uptake by HEK-MR (black line histogram) or HEK293 T (dotted line histogram) cells. Gray-filled histogram: without OVA. (B) Flow cytometric analysis of enriched endosomes from OVA-treated HEK-MR or HEK293 T cells (C) Flow cytometric analysis of enriched endosomes from OVA-treated cells (gray) and comparison to calibrated size beads. Endosomes from OVA-treated cells were isolated, stained for (D) Rab5 or (E) the cytoplasmic tail of the MR and analyzed by flow cytometry. (F) Endosome preparations from OVA-treated cells were fixed, permeabilized, and stained against the intraendosomal part of the MR. In all experiments, endosomal preparations were gated as shown in Supporting Information Fig. 2. Data are representative of at least three independent experiments.

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Subsequently, we tested if such analysis could be used to analyze the protein content of individual endosomes. To this end, we mixed HEK-MR cells that were pretreated with either Alexa Fluor 488- or Alexa Fluor 647-conjugated OVA, isolated endosomes from this cell mixture, and analyzed them by flow cytometry (Supporting Information Fig. 4A). Importantly, a clear population of endosomes, which were positive for both Alexa Fluor 488 and Alexa Fluor 647, was detected, ind-icating undesired clustering or fusion of endosomes during the preparation. Such clustering was not observed directly after cell disruption, showing that it might have been caused by the centrifugation of the endosome preparation. Similar observations were observed after isolation of endosomes from OVA-treated wild-type BM-DCs or from untreated MHC II-GFP expressing BM-DCs (Supporting Fig. 4B). If these endosomes were mixed before centrifugation, a clear population positive for both GFP and OVA was observed. To test whether the presence of double positive endosomes was caused by centrifugation, we centrifuged our preparations at different velocities, which revealed a close correlation between the presence of double positive endosomes and centrifugation speed (Supporting Information Fig. 5). Since the occurrence of such double positive endosomes is an unwanted artifact, which falsifies a flow cytometric analysis, we aimed to avoid such clustering by fixing the endosomes before centrifugation. Subsequent flow cytometric analysis revealed indeed that after fixation, no double positive endosomes were detected (Supporting Information Fig. 5), indicating that the inclusion of a fixation step allows reliable analysis of individual endosomes.

Next, we stained the endosomal prep-arations with an antibody against Rab5, which colocalizes with MR-containing endosomes as shown by immune fluorescence microscopy [1], and against the cytoplasmic region of the MR. Importantly, we fixed the endosomes before centrifugation to prevent endosome clustering as shown in Supporting Information Fig. 5. Flow cytometric analysis showed that nearly all OVA-positive structures were positive for Rab5 and the MR (Fig. 1D and E), demonstrating that these structures indeed are endosomes and that this method can be used to detect proteins at the surface of endosomes. The Rab5- and MR-specific antibodies used here both recognize their epitope at the cytoplasmic site of the endosome. To investigate whether it is also possible to perform antibody stainings against epitopes within the endosome, we developed an intraendosomal staining protocol including fixation and permeabilization. Importantly, we used a different MR-specific antibody that recognizes the carbohydrate recognition domains 4–7, which are in the luminal portion of the MR. Figure 1F shows specific intraendosomal staining of the MR in nearly all OVA-positive endosomes of HEK-MR cells. Importantly, no staining was observed in endosomes derived from OVA-treated control cells, further demonstrating the specificity of the staining.

Subsequently, we analyzed whether we could extend this approach to the characterization of endosomes from murine BM-DCs. Such DCs express high levels of the MR, which they use to internalize large quantities of OVA [1]. Also in these cells, MR internalized antigens colocalized with the early endosome marker Rab5 and the MR itself, but not with the late endosome marker proteins Rab7 and Lamp1 as shown by immune fluorescence microscopy (Supporting Information Fig. 6 and [1]). Similar to the experiments with the HEK-MR cells, we enriched endosomes from OVA-treated wild-type or MR-deficient BM-DCs and stained them with antibodies against Rab5 or the cytoplasmic tail of the MR. Flow cytometric analysis revealed that nearly all OVA-containing endosomes expressed Rab5 and the MR (Fig. 2A and B), confirming previous observations by immune fluorescence microscopy (Supporting Information Fig. 6 and [1]). As a negative control, we stained the endosomes with an antibody against the late endosomal marker Rab7, which is not present in OVA-containing endosomes (Supporting Information Fig. 6 and [1]). Figure 2C shows that Rab7 was only detected in OVA-negative organelles, confirming the specificity of the endosomal flow cytometric analysis.

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Figure 2. Staining of endosomal preparations from BM-DCs. (A)–(C) Endosomes from OVA-treated DCs were isolated and stained for (A) Rab5, (B) the cytoplasmic tail of the MR, or (C) Rab7. (D) Endosomes from OVA-treated cells were fixed, permeabilized, and stained against the intraendosomal part of the MR. MR-deficient BM-MΦs were incubated with OVA for 10 min and chased with medium for the indicated time points. Histograms depict (E) Rab5 or (F) LAMP1 expression on OVA-positive endosomes that were gated as indicated. In all experiments, endosomal preparations were gated as shown in Supporting Information Fig. 2. Data are representative of at least three independent experiments.

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Subsequently, we stained the endosomes with an antibody against the intraendosomal part of the MR. Figure 2D shows that nearly all OVA-containing endosomes from wild-type cells can be stained specifically for the MR, demonstrating that also proteins within endosomes from BM-DCs can be analyzed by flow cytometry.

In contrast to BM-DCs, which internalize high amounts of OVA only via the MR [1], BM-MΦs additionally internalize OVA via the scavenger receptor (SR) [1]. While antigens internalized by the MR are not transported into lysosomes [1], antigens internalized by the SR are targeted into endosomes that undergo normal lysosomal maturation [1]. During such maturation, these endosomes lose the expression of Rab5 and accumulate LAMP1. To further validate our technique, we performed time course experiments with endosomes from MR-deficient BM-MΦs. Immediately after SR-mediated OVA uptake, Rab5 could be detected in nearly all OVA-positive endosomes (Fig. 2E, Supporting Information Fig. 7) and decreased steadily over time. In contrast, expression of LAMP1 was not detected immediately after OVA uptake, but accumulated in OVA-positive endosomes over time (Fig. 2F). Such downregulation of Rab5 and upregulation of LAMP1 was not observed in endosomes derived from OVA-treated BM-DCs (Supporting Information Fig. 8). In these cells, the MR targets OVA toward a distinct endosomal compartment, which retains all characteristics of early endosomes as analyzed previously by immune fluorescence microscopy [1].

A previous study described a flow cyto-metric analysis of isolated early endosomes [6]. In that study, however, endo-somes were isolated using high-speed centrifugation steps. Here, we demonstrated that high-speed centrifugation causes clustering of individual endosomes and developed a reliable protocol, in which such clustering is prevented by fixation of the endosomes. Previous studies also report of flow cytometric analysis of isolated phagosomes that arose after invagination of antigen-coated latex beads [7-9]. Due to the characteristics of these beads, pure preparations of such phagosomes can be obtained relatively easily. In this study, we used a technically more demanding procedure to analyze the protein composition of endosomes containing soluble antigens. Classical approaches to determine the protein content of such endosomes generally include time consuming density gradient centrifugation procedures followed by Western blot analysis. An important restriction of such approaches is that the endosomal fractions with a similar density cannot be analyzed separately. Flow cytometric analysis overcomes this restriction and allows investigating the presence of specific proteins also in a subpopulation of endosomes, like the OVA-containing endosomes reported here. Importantly, the endosomal fraction does not need to be purified extensively before analysis, which displays another major advantage over classical purification methods.

Finally, specific staining of enriched endosomes may allow FACS sorting to isolate endosomal subpopulations. Ongoing experiments of our group point out that FACS sorting of pure OVA-containing endosomes indeed yields sufficient material for subsequent analysis by mass spectrometry, which could provide a detailed description of the endosomal protein composition allowing the identification of unknown proteins involved in cross-presentation.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. Conflict of interest
  4. References
  5. Supporting Information

We thank the House for Experimental Therapy and the Flow Cytometry Core Facility at the Institute of Molecular Medicine for technical support. This work was supported by the collaborative research center SFB645 and grant number BU2441/1–1, both funded by the German Research Foundation.

Conflict of interest

  1. Top of page
  2. Acknowledgements
  3. Conflict of interest
  4. References
  5. Supporting Information

The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Acknowledgements
  3. Conflict of interest
  4. References
  5. Supporting Information
Abbreviations
ERAD

ER-associated degradation machinery

HEK-MR

MR-expressing HEK293 T cells

MR

Mannose receptor

SR

Scavenger receptor

Supporting Information

  1. Top of page
  2. Acknowledgements
  3. Conflict of interest
  4. References
  5. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

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eji2324-sup-0001-s1.pdf794K

Figure S1. Electron microscopy of enriched endosome preparation.Figure S2. Gating scheme of endosome preparations for flow cytometrical analysis.

Figure S3. Influence of saponin and Triton X-100 on isolated OVA containing endosomes.Figure S4: Influence of centrifugation on endosome clustering.Figure S5. Influence of centrifugation on endosome clustering.

Figure S6. Intracellular localization of MR-internalized OVA.

Figure S7. Time course experiments of endosomal maturation MR-deficient BM-M were incubated with fluorochrome-labeled OVA for 10 min and chased with medium for the indicated time points.Figure S8. Expression of Rab5 and LAMP1 on endosomes containing MR endocytosed OVA.

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