Autonomous phagosomal degradation and antigen presentation in dendritic cells

Abstract

Phagocytosis plays a critical role in both innate and adaptive immunity. Phagosomal fusion with late endosomes and lysosomes enhances proteolysis, causing degradation of the phagocytic content. Increased degradation participates in both innate protection against pathogens and the production of antigenic peptides for presentation to T lymphocytes during adaptive immune responses. Specific ligands present in the phagosomal cargo influence the rate of phagosome fusion with lysosomes, thereby modulating both antigen degradation and presentation. Using a combination of cell sorting techniques and single phagosome flow cytometry-based analysis, we found that opsonization with IgG accelerates antigen degradation within individual IgG-containing phagosomes, but not in other phagosomes present in the same cell and devoid of IgG. Likewise, IgG opsonization enhances antigen presentation to CD4(+) T lymphocytes only when antigen and IgG are present within the same phagosome, whereas cells containing phagosomes with either antigen or IgG alone failed to present antigen efficiently. Therefore, individual phagosomes behave autonomously, in terms of both cargo degradation and antigen presentation to CD4(+) T cells. Phagosomal autonomy could serve as a basis for the intracellular discrimination between self and nonself antigens, resulting in the preferential presentation of peptides derived from opsonized, nonself antigens.

Fig. 1.

Phagosomal degradation of OVA is dependent on phagosome maturation kinetics. (A) Latex beads conjugated to OVA were internalized by resting bone marrow-derived DCs and MOs and analyzed by high-resolution fluorescence microscopy after labeling of OVA (green), LAMP-1 (red), and F-actin. Shown are maximum projections of five focal planes with a step width of 0.3 μm from representative cells. (Scale bar: 10 μm.) (B) DCs were allowed to internalize beads conjugated to OVA or IgG-OVA, and phagosomes were isolated from these cells after different chase times. After simultaneous labeling of OVA and LAMP-1, 5,000 phagosomes of each sample were analyzed by flow cytometry. Results are representative of five independent experiments.

Fig. 2.

Phagosomes have differing capabilities for degrading OVA and acquiring LAMP-1 depending on the particle ligand. DCs (A) and MOs (B) were allowed to internalize beads conjugated to OVA, LPS-OVA, and IgG-OVA. Phagosomes were isolated from these cells after different chase times, labeled with antibodies against OVA and LAMP-1, and analyzed by flow cytometry. Results shown are representative of 4,500 analyzed phagosomes labeled for OVA or LAMP-1, as well as isotype controls (gray). The graphs to the right show quantitative data from five independent experiments with DCs (A) and three independent experiments with MOs (B). The mean fluorescence intensity (MFI) of all samples was normalized to the initial chase time point (OVA degradation) or the final chase time point (LAMP-1 acquisition) of control phagosomes containing OVA beads.

Fig. 3.

Phagosome maturation occurs autonomously in DCs. Different mixtures of beads coupled to OVA (blue), IgG-OVA (red), or IgG alone (black) were applied to DCs. The cells were then sorted into different populations, and their phagosomes containing OVA beads (Upper) or IgG-OVA beads (Lower) were analyzed for phagosomal degradation of OVA (A) and phagosomal acquisition of LAMP-1 (B) by phagoFACS. Shown are MFI profiles of 3,000 analyzed phagosomes per condition representative of at least five independent experiments.

Fig. 4.

IgG increases MHC class II-restricted antigen presentation in DCs. (A) Beads conjugated to OVA or IgG-OVA were added to DCs or MOs at two different concentrations and cocultured with OT-II lymphocytes to assess MHC II antigen presentation. T-cell activation was examined by FACS analysis of their CD69 surface expression. (B) As controls, both cell types were incubated with the synthetic peptide and cocultured with OT-II cells. (C–H) Comparable experiments were performed with DCs after phagocytosis of beads conjugated to different ligands and sorted into populations containing only one bead. (C) CD69 expression of OT-II cells after coincubation with 100,000 sorted DCs. (D) Quantification of OT-II CD69 expression after coincubation with different numbers of sorted DCs and background subtraction. (E) As a control, the minimal peptide was added to 50,000 sorted DCs containing one BSA bead or one IgG-BSA bead before OT-II coincubation. (F–H) T-cell activation was also assessed using CFSE-labeled OT-II cells. (F) Proliferation of OT-II cells after coincubation with 5,000 sorted DCs. (G) Quantification of OT-II proliferation after coincubation with 5,000 or 7,500 sorted DCs and background subtraction. (H) As a control, the minimal peptide was added to 5,000 sorted DCs containing one BSA bead or one IgG-BSA bead before OT-II coincubation. Representative histograms as well as average data of at least two independent experiments are shown.

Fig. 5.

MHC II-restricted presentation of OVA occurs autonomously in DCs. (A) Schematic representation of the sorting strategy used for antigen presentation experiments. After simultaneous phagocytosis of different bead types, cells were sorted according to their content into populations containing one of each type or both together within the same cell. (B) After sorting and antigen processing, cells were coincubated with OT-II cells and analyzed for expression of CD69. (C) The same cells were also coincubated with CFSE-labeled OT-II cells and analyzed for proliferation. Average data as well as representative histograms of three independent experiments are shown.