Route of antigen uptake differentially impacts presentation by dendritic cells and activated monocytes

Abstract

Dendritic cells (DCs), which maintain tolerance and orchestrate T cell immune responses, comprise a heterogeneous group of cells. For example, in the steady state, murine spleen contains pre-DC-derived CD8(+) and CD8(-) conventional DCs. During inflammation, monocytes become activated and acquire some DC-like features, such as expression of CD11c and MHC class II. Although each of these cell types can present Ag, the relative efficiency of processing and presentation after Ag capture by different routes has not yet been systematically compared. To this end, we administered OVA to various conventional DCs and activated monocytes by receptor-mediated endocytosis, pinocytosis, or phagocytosis and measured internalization and presentation to MHC class I- and MHC class II-restricted T cells. We find that CD8(-) DCs are more efficient than any other type of APC tested in terms of presenting Ag to MHC class II-restricted T cells, irrespective of the route of Ag capture. In contrast, both subsets of splenic DCs are highly effective in cross-presenting Ags to CD8(+) T cells. DCs and activated monocytes cross-presented Ags delivered by DEC205-mediated endocytosis and pinocytosis. However, DCs differ from activated monocytes in that the latter are several orders of magnitude less efficient in presenting Ags captured by phagocytosis to CD8(+) or CD4(+) T cells. We conclude that DCs derived from pre-DCs differ from monocyte-derived cells in that DCs process and present Ags efficiently irrespective of the route of Ag capture. Our observations have significant implications for understanding initiation of immune responses and vaccination strategies targeting DCs and activated monocytes.

FIGURE 1

hDEC205-mediated endocytosis in vivo promotes cross-presentation by both CD8⁻ and CD8⁺ DC subsets. A, Representative histograms show proliferation as measured by CFSE dye dilution of OTI (upper panels) and OTII (lower panels) T cells upon culture with 5×10³ CD8⁻ or CD8⁺ DCs isolated from CD11c-hDEC transgenic mice injected with αhDEC-OVA, αmDEC-OVA or αDCIR2-OVA, as indicated. B, Summary of 3 independent experiments as in A. Panels show the percentage of divided, CFSE low OTI (upper panel) and OTII (lower panel) after incubation with 5 × 10³ (open symbols) or 20 × 10³ (solid symbols) CD8⁻ or CD8⁺ DCs that were targeted with OVA, as indicated. Each symbol indicates independent experiments and represents the average of duplicate measurements. Spleens of 3-10 injected mice were pooled in each experiment. Data was analyzed by repeated measures ANOVA and Tukey’s test was used to compare groups: * p < 0.05; **p < 0.01; ***p<0.001.

FIGURE 2

Antigen presentation after DEC205-mediated endocytosis in vitro. A-C, APCs from CD11c-hDEC mice were isolated from naive (solid lines) or mBSA-CFA immunized (dashed lines) mice (left panels), or cultured from bone marrow (right panels). A, The Y-axis shows relative cell-associated OVA, as measured by rabbit anti-OVA and developed with anti-rabbit-HRP, after targeting with the indicated concentrations of αhDEC-OVA on the X-axis. B, Activation and proliferation of OTI (upper panels) and OTII (lower panels) T cells in response to OVA containing APCs. The Y-axis shows the percentage of divided T cells. C, Graphs show OTI (upper panels) and OTII (lower panels) T cell proliferation in response to peptide pulsed APCs. D-F, APCs were isolated or cultured from spleen or bone marrow of WT mice. D and E, as in A and B, but targeting was with αmDEC-OVA. F, As in C. A-F, graphs represent pooled data from 3-7 independent experiments.

FIGURE 3

DEC205-mediated endocytosis promotes cross-presentation in B cells. NP-specific B cells were isolated from B1-8^hi transgenic mice and stimulated with LPS and IL-4 for 50-60 hours. A, The Y-axis shows relative cell-associated OVA, after targeting with the indicated concentrations of αmDEC-OVA, or αmDEC-OVA and BSA-NP, or OVA-NP on the X-axis. Maximum cell-associated OVA was normalized to 100 in each experiment. B, Activation and proliferation of OTI (upper panel) and OTII (lower panel) T cells in response to OVA containing B cells. The Y-axis shows percentage of divided T cells. A and B, represent pooled data from 3 independent experiments.

FIGURE 4

Antigen presentation after pinocytosis. APCs were isolated from spleen or cultured from bone marrow or spleen of WT mice. A, The Y-axis shows relative cell-associated OVA, developed with streptavidin-HRP, after incubation of APCs with the indicated concentrations of OVA-biotin on the X-axis. B, Activation and proliferation of OTI (upper panels) and OTII (lower panels) T cells in response to OVA containing APCs. The Y-axis shows percentage of divided T cells. C, Graphs show OTI (upper panels) and OTII (lower panels) T cell proliferation in response to peptide pulsed APCs. A-C represent pooled data from 3-5 independent experiments.

FIGURE 5

Antigen presentation after phagocytosis. A, APCs were isolated from spleen of naive or mBSA-CFA immunized mice. GM-DCs were derived from bone marrow. Enriched populations were incubated with OVA-beads before sorting. Panels show activation and proliferation of OTI (left panels) and OTII (right panels) T cells after incubation with the indicated number of APCs in the X-axis, that were sorted to contain a single OVA-bead. The Y-axis shows percentage of divided T cells. In lower panels each symbol indicates independent experiments and represents the average of duplicate measurements where 2,500 sorted APCs were incubated with T cells. Data was analyzed by ANOVA and Tukey’s test was used to compare groups: * p < 0.05; ***p<0.001. B, as in A, but DCs from naive mice were incubated with beads adsorbed with 25% OVA and 75% keyhole limpet hemocyanin protein. A and B represent pooled data from 2-7 independent experiments.