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. 2011 Jun 14;108(24):9927-32.
doi: 10.1073/pnas.1019037108. Epub 2011 May 31.

Neonatal Fc receptor for IgG (FcRn) regulates cross-presentation of IgG immune complexes by CD8-CD11b+ dendritic cells

Kristi Baker  1 , Shuo-Wang Qiao, Timothy T Kuo, Victoria G Aveson, Barbara Platzer, Jan-Terje Andersen, Inger Sandlie, Zhangguo Chen, Colin de Haar, Wayne I Lencer, Edda Fiebiger, Richard S Blumberg
Affiliations

Neonatal Fc receptor for IgG (FcRn) regulates cross-presentation of IgG immune complexes by CD8-CD11b+ dendritic cells

Kristi Baker et al. Proc Natl Acad Sci U S A. .

Abstract

Cross-presentation of IgG-containing immune complexes (ICs) is an important means by which dendritic cells (DCs) activate CD8(+) T cells, yet it proceeds by an incompletely understood mechanism. We show that monocyte-derived CD8(-)CD11b(+) DCs require the neonatal Fc receptor for IgG (FcRn) to conduct cross-presentation of IgG ICs. Consequently, in the absence of FcRn, Fcγ receptor (FcγR)-mediated antigen uptake fails to initiate cross-presentation. FcRn is shown to regulate the intracellular sorting of IgG ICs to the proper destination for such cross-presentation to occur. We demonstrate that FcRn traps antigen and protects it from degradation within an acidic loading compartment in association with the rapid recruitment of key components of the phagosome-to-cytosol cross-presentation machinery. This unique mechanism thus enables cross-presentation to evolve from an atypically acidic loading compartment. FcRn-driven cross-presentation is further shown to control cross-priming of CD8(+) T-cell responses in vivo such that during chronic inflammation, FcRn deficiency results in inadequate induction of CD8(+) T cells. These studies thus demonstrate that cross-presentation in CD8(-)CD11b(+) DCs requires a two-step mechanism that involves FcγR-mediated internalization and FcRn-directed intracellular sorting of IgG ICs. Given the centrality of FcRn in controlling cross-presentation, these studies lay the foundation for a unique means to therapeutically manipulate CD8(+) T-cell responses.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CD8−CD11b+ DCs use FcRn-dependent cross-presentation in vivo to induce primary CD8+ T-cell responses to low-dose antigen in IgG ICs. (A) Only CD8−CD11b+ DCs efficiently cross-prime when loaded with IgG ICs containing low doses of antigen (0.5 μg/mL) and do so in an FcRn-dependent manner. IC-loaded DCs were injected into the opposite hind footpads of recipient mice (see Fig. S1C). (B) CD8+CD11b− DCs efficiently cross-prime CD8+ T cells when loaded with a standard dose (5 μg/mL) of soluble antigen. (C) Viable CD8−CD11b+ DCs loaded with IgG ICs or IHH-IgG ICs migrate equally to the draining PLN. DCs were labeled with CFSE before IC loading and footpad injection and assessed 12 h later. Percentages indicate the fraction of MHC-II+CD11c+CFSE+ cells. Annexin V and 7-AAD staining confirmed DCs viability.
Fig. 2.
Fig. 2.
FcRn-mediated cross-presentation by CD8−CD11b+ DCs is particularly efficient at inducing T-cell responses over a low-dose range of complexed and particulate antigen. (A) FcRn enables cross-presentation of very low antigen concentrations contained within IgG ICs. DCs were loaded with ICs or soluble NIP-OVA and cocultured with primary OT-I cells. (B) CD8−CD11b+ DCs from FcRn−/− mice fail to cross-present IgG ICs. (C) FcRn enables cross-presentation of antigens derived from large opsonized particles. DCs were loaded with opsonized 3-μm beads at the indicated ratios and cocultured with OT-I cells. (D) FcRn-mediated cross-presentation of ICs is abrogated in the absence of FcγR binding. (E) Internalization via FcγR is required for FcRn-mediated cross-presentation of antigen from IgG-opsonized particulate antigen. (F) FcγR binds IgG at neutral pH, whereas FcRn binds IgG at acidic pH. CD8−CD11b+ DCs were lysed in buffers of the indicated pH and immunoprecipitated with IgG. All results are representative of at least three independent experiments with n = 3 per group. All data are mean ± SEM of triplicates. *P < 0.05; **P < 0.01.
Fig. 3.
Fig. 3.
FcRn-dependent cross-presentation of IgG ICs relies on acidification and oxidation of the loading compartment. (A) Phagosomes containing IgG-opsonized beads acidify more efficiently than do those containing IHH-IgG-opsonized beads. DCs were pulsed with opsonized 3-μm beads labeled with FITC and Alexa647 dyes and chased as indicated. (B) Phagosomes containing IgG-opsonized beads were enriched in Rab27a, V-ATPase, and gp91phox. Phagosomes were purified from ICs pulsed DCs by using magnetic separation. (C) FcRn-mediated cross-presentation of IgG-complexed antigens is blocked by inhibitors of acidification (concanamycin A, 50 nM) and oxidation (DPI, 5 μM). (D) IgG-complexed antigens that bind FcRn are protected from oxidation. DCs were pulsed with opsonized beads conjugated to DHR and Alexa647 and chased as indicated. Results are representative of at least three independent experiments. Data are mean ± SEM of triplicates. **P < 0.01.
Fig. 4.
Fig. 4.
FcRn ligation by IgG ICs traps the complexed antigen in a membrane-bound compartment optimized for cross-presentation. (A) IgG ICs are retained for prolonged periods within endosomal compartments where they colocalize with FcRn. BMDCs were pulsed with ICs containing OVA-Alexa488, chased as indicated, and stained for FcRn. (Scale bar: 10 μm.) (B) IgG-complexed antigen persists within a membrane-bound compartment when bound to FcRn. DCs were pulsed with ICs and chased as indicated before isolation of cytoplasmic and membrane fractions. The density of each major band at 60 min was quantified for each of three independent experiments. *P < 0.05. (C) Key proteins of the cross-presentation machinery, including TAP1, Sec61α, and MHC-I, are enriched in phagosomes containing FcRn-binding IgG-opsonized beads. (D) Generation of peptides for FcRn-mediated cross-presentation is dependent on proteasomal processing rather than vacuolar protease digestion and requires Sec61α endosomal export. DCs were pretreated with solvent, Lactacystin (10 μM), MG132 (4 μM), Cathepsin S inhibitor (0.1 μM), Cathepsin B inhibitor (10 μM), or Exotoxin A (5 μg/mL) before ICs or OVA257–264 (100 pg/mL) loading. All results are representative of at least two independent experiments. All data are mean ± SEM of triplicates. **P < 0.01.
Fig. 5.
Fig. 5.
FcRn-dependent cross-presentation of IgG ICs by CD8−CD11b++CD11c+ inflammatory DCs induces a proinflammatory T-cell response during chronic DSS colitis. (A) The MLN and LP of mice chronically treated with DSS contain a population of CD8−CD11b++CD11c+ inflammatory DCs. R1, CD8−CD11b++CD11c+; R2, CD8−CD11b+CD11c++; R3, CD8+CD11b−CD11c++. (B) Only FcRn-mediated cross-presentation by CD8−CD11b++CD11c+ DCs induces the production of proinflammatory IFNγ by CD8+ T cells. Sorted DCs were loaded with ICs and cocultured with OT-I T cells. (C) CD8−CD11b++CD11c+ DCs from DSS-treated FcRn−/− mice are deficient in their ability to induce proinflammatory cytokine production by CD8+ T cells in response to IgG ICs. (D and E) CD8+ T cells from the MLNs of chronically DSS-treated WT mice, but not those from FcRn−/− mice, secrete large quantities of IFNγ (D) and produce large quantities of Granzyme B and T-bet (E) upon anti-CD3/CD28 restimulation. Data are representative of at least two independent experiments with n = 5 per group. All data are mean ± SEM of triplicates. *P < 0.05; **P < 0.01.
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