Conventional Dendritic Cells Require IRAP-Rab14 Endosomes for Efficient Cross-Presentation

Abstract

Dendritic cells (DCs) use cellular pathways collectively referred to as cross-presentation to stimulate CD8+ T cells with peptide Ags derived from internalized, exogenous Ags. We have recently reported that DCs rely on aminoterminal trimming of cross-presented peptides by insulin-responsive aminopeptidase (IRAP), an enzyme localized in a regulated endosomal storage compartment. Considering a report contending that this role is limited to inflammatory DCs (Segura et al. 2009. Proc. Natl. Acad. Sci. USA 106: 20377–20381), in this study, we examined the role of IRAP in steady-state DC subpopulations. Steady-state conventional DCs (cDCs) and plasmacytoid DCs expressed similar amounts of IRAP. IRAP colocalized with the endosomal markers Rab14 and syntaxin 6, both known to be associated with regulated endosomal storage compartments, in CD8+ and CD8− cDCs—however, to a greater extent in the former population. Likewise, IRAP recruitment to phagosomes was significantly stronger in CD8+ DCs. IRAP deficiency compromised cross-presentation of soluble and particulate Ag by both CD8+ and CD8− cDCs, again with a stronger effect in the former population. Thus, the requirement of IRAP in cross-presentation extends to steady-state cDCs. Moreover, these data suggest that increased recruitment of an IRAP+/Rab14+ compartment to Ag-containing vesicles contributes to the superior cross-presentation efficacy of CD8+ cDCs.

IRAP+ endosomes restrict TLR9 activation and signaling

“…IRAP contains the canonical zinc-binding amino acid motif HELAH, which is essential for the enzymatic activity 26 . A form of IRAP in which the HELAH sequence was changed into HALAH (E465A substitution) co-localized, like the wild-type protein, with syntaxin 6 (Stx6), a SNARE of IRAP + endosomes 16,17 (Fig. 4a).…”

“…IRAP, as well as the small GTPase Rab14 and the Q-SNARE Stx6, two others markers of IRAP + endosomes in DCs 16,17 coincided with VAMP3 endosomes (Supplementary Fig. 3).…”

“…presence of the aminopeptidase IRAP (Insulin Responsive AminoPeptidase), a type II transmembrane protein composed of a catalytic site localized in the endosomal lumen and a cytosolic domain of 110 amino acids. In DCs, IRAP + endosomes are rapidly recruited to DC phagosomes, where the enzymatic activity of IRAP is involved in antigen processing during MHC-I cross-presentation 16,17 .…”

ERAP1–ERAP2 Dimerization Increases Peptide-Trimming Efficiency

“…In the ER, epitope precursor peptides imported by TAP are trimmed by ER aminopeptidases (ERAPs), whereas insulinregulated aminopeptidase, a closely related enzyme residing in endosomes, exerts an equivalent function on cross-presented Ags of extracellular origin (3,4). Although rodents express a single ERAP enzyme, human cells harbor two, ERAP1 and ERAP2 (5).…”

Spatial separation of the processing and MHC class I loading compartments for cross-presentation of the tumor-associated antigen HER2/ neu by human dendritic cells

“…39,40 However, in the case of HER2/neu the lack of co-localization of HLA-A2/HER2/ neu 369-377 complexes with IRAP or Rab14 ruled out these pathways. Also loading of MHC class I molecules with cross-presented epitopes in the MHC class II loading compartment proposed by some authors [41][42][43] seems unlikely because HLA-A2/ HER2/neu 369-377 complexes were not found together with HLA-DR, the chaperon HLA-DM or the invariant chain CD74 in the same compartment. Likewise, loading of the MHC class I molecules with the HER2/neu epitope in early or recycling endosomes proposed by other models 8,9 could be ruled out because of the lack of co-localization of the specific MHC peptide complexes with the respective marker proteins EEA1, CD71 or Rab11.…”