Distinct antigen uptake receptors route to the same storage compartments for cross-presentation in dendritic cells

Abstract

An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcγ receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation.

Keywords: C-type lectin receptors; Fc receptors; antigen cross-presentation; cathepsin; dendritic cells.

FIGURE 1

Antigen storage in dendritic cells. DCs were pulsed with OVA IC for 2 h, washed and chased for 2, 24, 48 or 72 h. CFSE‐labelled CD8⁺ OTI T cells were added, and T‐cell proliferation was analysed by flow cytometry (a). DCs were pulsed with OVA IC (Alexa Fluor 488) for 15 min (b, left panel) or pulsed with OVA IC for 2 h and chased for 48 h (b, right panel). OVA IC uptake and presence in DCs were imaged by confocal microscopy, and differential interference contrast (DIC) was additionally used to image cell contrast. Immuno‐electron microscopy images of DCs after 15‐min OVA IC (Alexa Fluor 488) pulse (c, left) and DCs pulsed with OVA IC for 2 h and chased for 48 h (c, right). Sections were labelled with immunogold for Alexa488 with 10‐nm gold particle size. EE = early endosome, M = mitochondria, AS = antigen storage compartment, NC = nucleus

FIGURE 2

Characterization of the antigen storage compartments in DCs upon FcγR targeting. DCs were pulsed with OVA IC (Alexa Fluor 488, green) for 15 min or pulsed with OVA IC for 2 h and chased for 48 h. OVA IC presence in DCs was imaged by confocal microscopy, and DIC was used to image cell contrast. Specific antibodies against EEA1, Rab5, Rab7, LAMP1, MHCI, MHCII, TAP1 and PA28β (red) were used and analysed for co‐localization with OVA IC. Co‐localization between OVA IC and the antibodies is summarized in a table, ‘+’ indicates co‐localization, ‘‐’ indicates no co‐localization

FIGURE 3

Characterization of the antigen storage compartments in DCs with imaging flow cytometry. DCs were pulsed with OVA IC (Alexa Fluor 647 labelled OVA) for 1, 2, 5, 10, 15 min or 1 h (different for each antibody), and pulsed for 2 h and chased for 24 and 48 h. Co‐localization between OVA IC and EEA1, Rab5, Rab7, LAMP1, MHCI, MHCII, TAP1 and PA28β was analysed by imaging flow cytometry

FIGURE 4

The presence of cathepsins in the antigen storage compartments in DCs. Naïve DCs or DCs pulsed with OVA IC for 2 h and chased for 24 h were run on a 15% PAGE gel. Quenched activity‐based probe BMV109 was used to stain active cathepsin X, B, S and L, indicated by arrows (a). Co‐localization between BMV109 (red) and specific antibody against cathepsin S (blue) was analysed in naïve DCs with confocal microscopy. DCs were pulsed with OVA IC (Alexa Fluor 488) for 30 min or pulsed for 2 h and chased for 24 h. Co‐localization between OVA IC (green), cathepsin S (blue) and BMV109 (red) was analysed by confocal microscopy (b). Naïve DCs, DCs pulsed with OVA IC (Alexa Fluor 488) for 30 min, 1, 2, 3 h or DCs pulsed for 2 h and chased for 24 h were stained with cathepsin X antibody and BMV109. Co‐localization between OVA IC (green), cathepsin X (blue) and BMV109 (red) was analysed by confocal microscopy. Co‐localization between OVA IC, BMV109, cathepsin X and cathepsin S is summarized in a table, ‘+’ indicates co‐localization, ‘+/−’ indicates partial co‐localization, and ‘‐’ indicates no co‐localization (c)

FIGURE 5

Characterization of the antigen storage compartments in DCs upon MGL1 targeting. DCs were pulsed with OVA‐Le^X for 2 h, washed and chased for 2, 24, 48 or 72 h. CFSE‐labelled OTI CD8⁺ T cells were added, and T‐cell proliferation read‐out was analysed by flow cytometry (a). DCs were pulsed with OVA‐Le^X (DyLight 488) for 15 min or pulsed with OVA‐Le^X for 2 h and chased for 48 h. OVA‐Le^X presence in DCs was imaged by confocal microscopy and specific antibodies against EEA1, Rab5, Rab7, LAMP1, MHCI, MHCII, TAP1 and PA28β (red) were used and analysed for co‐localization with OVA‐Le^X (green). Co‐localization between OVA‐Le^X and the antibodies is summarized in a table, ‘+’ indicates co‐localization, and ‘−’ indicates no co‐localization (b)

FIGURE 6

Antigens targeted to FcγRs and MGL1 on DCs end up in the same compartments. DCs were pulsed with OVA IC (Alexa Fluor 647) or OVA‐Le^X (DyLight 488) for 15 min and 1 h, or pulsed for 2 h and chased for 24 or 48 h. Co‐localization between OVA IC (red) and OVA‐Le^X (green) was visualized by confocal microscopy and DIC was used to image cell contrast. Histograms for each fluorophore were created for a selected area (indicated by a line on the image) and overlays were made with the ImageJ software. Arrows indicate co‐localization between OVA IC (red) and OVA‐Le^X (green)