TAP-Dependent and -Independent Peptide Import into Dendritic Cell Phagosomes.
Cross-presentation of phagocytosed Ags by MHC class I (MHC-I) molecules is thought to involve transport of cytosolic peptides into dendritic cell phagosomes, mediated by TAP transporters recruited from the endoplasmic reticulum. However, because pure and tightly sealed phagosomes are difficult to obtain, direct evidence for peptide transport into phagosomes has remained limited. Moreover, the parameters determining peptide uptake by, and survival in, phagosomes remain little characterized. In this study, we monitored peptide import into phagosomes by flow cytometry using two types of fluorescent reporter peptides, one of which directly bound to intraphagosomal beads. We observed that a peptide with high TAP affinity is imported into phagosomes in a TAP- and ATP-dependent manner, as expected. However, surprisingly, import of the OVA peptide SIINFEKL, a CD8+T cell epitope frequently used to study cross-presentation, is ATP-dependent but substantially TAP-independent. The half-life of both reporter peptides is shortened by enhanced phagosome maturation triggered by TLR signaling. Conversely, formation of complexes with MHC-I molecules enhances peptide accumulation in phagosomes. Collectively, these results confirm that TAP can import peptides into phagosomes, but they suggest that some peptides, including the popular SIINFEKL, can enter phagosomes also via a second unknown energy-dependent mechanism. Therefore, the frequently reported TAP dependence of cross-presentation of phagocytosed OVA may principally reflect a requirement for recycling MHC-I molecules rather than SIINFEKL import into phagosomes via TAP.
INSERM, Unité 1151, 75015 Paris, France.|CNRS, Unité 8253, 75015 Paris, France.|Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, 75015 Paris, France; and.
Myriam
0000-0002-2757-6195
M
Lawand
INSERM, Unité 1151, 75015 Paris, France.|CNRS, Unité 8253, 75015 Paris, France.|Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, 75015 Paris, France; and.
Anastasia
A
Abramova
INSERM, Unité 1151, 75015 Paris, France.|CNRS, Unité 8253, 75015 Paris, France.|Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, 75015 Paris, France; and.
Valérie
V
Manceau
Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, Germany.
Sebastian
0000-0002-5527-6149
S
Springer
INSERM, Unité 1151, 75015 Paris, France; peter.van-endert@inserm.fr.|CNRS, Unité 8253, 75015 Paris, France.|Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, 75015 Paris, France; and.
Peter
0000-0003-3782-0750
P
van Endert
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000941:Antigens / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D045142:Cross-Priming; D003713:Dendritic Cells / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000941:Antigens; D015395:Histocompatibility Antigens Class I; D010455:Peptides; C486171:Tap1 protein, mouse; D010047:Ovalbumin