Neonatal Fc Receptor for Igg (Fcrn) Regulates Cross-Presentation of Igg Immune Complexes by CD8 Cd11b Dendritic Cells

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

Kristi Bakera,b, Shuo-Wang Qiaoc,d, Timothy T. Kuoa,b, Victoria G. Avesona,b, Barbara Platzere,f, Jan-Terje Andersenc,d, Inger Sandliec,d, Zhangguo Chena,b, Colin de Haarg, Wayne I. Lencere,f,h, Edda Fiebigere,f,h,1, and Richard S. Blumberga,b,h,1,2

aDivision of Gastroenterology, Brigham and Women’s Hospital, Boston, MA 02115; bDepartment of Medicine, Harvard Medical School, Boston, MA 02115; cDepartment of Immunology, Oslo University Hospital Rikshospitalet, 0027 Oslo, Norway; dInstitute of Immunology, University of Oslo, 0027 Oslo, Norway; eDivision of Gastroenterology and Nutrition, Children’s Hospital Boston, Boston, MA 02115; fDepartment of Pediatrics, Harvard Medical School, Boston, MA 02115; gDepartment of Gastroenterology and Hepatology, Erasmus Medical Centre, 3000 CA, Rotterdam, The Netherlands; and hHarvard Digestive Diseases Center, Boston, MA 02115

Edited by Emil R. Unanue, Washington University, St. Louis, MO, and approved May 10, 2011 (received for review December 17, 2010) Cross-presentation of IgG-containing immune complexes (ICs) is an ported back to the endoplasmic reticulum (ER) for loading onto important means by which dendritic cells (DCs) activate CD8+ T cells, MHC-I or reimported into the phagosomal compartment con- yet it proceeds by an incompletely understood mechanism. We taining components of the ER retrotranslocation and protein show that monocyte-derived CD8−CD11b+ DCs require the neona- loading machinery (8, 9). All pathways of cross-presentation are tal Fc receptor for IgG (FcRn) to conduct cross-presentation of IgG characterized by carefully controlled acidic degradation, which is ICs. Consequently, in the absence of FcRn, Fcγ receptor (FcγR)- achieved by balancing acidification and oxidation within the rele- mediated antigen uptake fails to initiate cross-presentation. FcRn vant intracellular compartments (6). is shown to regulate the intracellular sorting of IgG ICs to the To date, the majority of studies examining cross-presentation proper destination for such cross-presentation to occur. We dem- mechanisms have focused on the processing of soluble anti- onstrate that FcRn traps antigen and protects it from degradation gens. However, extracellular antigens can also be directed to in- within an acidic loading compartment in association with the rapid tracellular cross-presentation pathways by specific receptor- recruitment of key components of the phagosome-to-cytosol cross- mediated internalization. An important example of the latter is presentation machinery. This unique mechanism thus enables uptake of IgG-containing immune complexes (IgG ICs) by acti- cross-presentation to evolve from an atypically acidic loading com- vating Fcγ receptors (FcγR; refs. 10–14). It was therefore inter- partment. FcRn-driven cross-presentation is further shown to con- esting to consider the role played by the neonatal Fc receptor for trol cross-priming of CD8+ T-cell responses in vivo such that during IgG (FcRn) in cross-presentation and its relationship to FcγR- chronic inflammation, FcRn deficiency results in inadequate induc- mediated internalization. FcRn is a nonclassical Fc receptor tion of CD8+ T cells. These studies thus demonstrate that cross- encoded by the Fcgrt gene that binds its ligand, IgG, exclusively at presentation in CD8−CD11b+ DCs requires a two-step mechanism acidic pH ≤ 6.5 and resides predominantly intracellularly (15). IMMUNOLOGY that involves FcγR-mediated internalization and FcRn-directed in- Despite the insinuation of its name, FcRn is expressed throughout tracellular sorting of IgG ICs. Given the centrality of FcRn in controlling life in a variety of cell types. These include mouse and human cross-presentation, these studies lay the foundation for a unique DCs (15, 16), where it has been shown to play a role in MHC-II- means to therapeutically manipulate CD8+ T-cell responses. restricted presentation of IgG IC-derived antigens (17, 18). Given these characteristics, we sought to determine whether endritic cells (DCs) function as central orchestrators of im- FcRn is necessary for directing antigen as an IgG IC toward γ Dmunity by providing necessary innate signals and adaptive a cross-presented fate subsequent to Fc R-mediated entry of functions through the processing and presentation of antigens antigen. Our results demonstrate that FcRn indeed plays an γ to which T cells respond. DCs are highly specialized for the indispensible role downstream of Fc R in the intracellular sorting processing of extracellular antigens in the context of major histo- of IgG IC-delivered antigens to the appropriate destination for cross-presentation to occur. This pathway is especially important compatibility complex class II (MHC-II) molecules for presenta- − + + in monocyte-derived CD8 CD11b DCs wherein FcRn expres- tion to CD4 T cells and MHC class I (MHC-I) molecules for + + sion is required for the in vivo induction of CD8 T cells. Col- presentation to CD8 T cells (1). This latter process, known as lectively, our data identify a previously undescribed role for FcRn “cross-presentation,” is important for the induction of immune and as such delineate a unique intracellular mechanism by which responses against a variety of pathogens, including viruses and FcγR-initiated cross-presentation proceeds. bacteria, that do not primarily infect DCs (2, 3). Not all DCs are equal in their ability to cross-present antigens because a very Results specific intracellular environment is required for this process. In + − FcRn Enables Cross-Presentation of IgG-Complexed Antigens in particular, CD8 CD11b DCs in mice and their recently identi- CD8−CD11b+ DCs. To investigate the role of FcRn in cross- fi + + ed human counterparts, CD141 (BCDA-3) DCs, are major presentation of IgG ICs, we used a series of previously described mediators of cross-presentation (4, 5). These DC subsets uniquely process antigen in a minimally degradative manner that relies

upon maintaining a near-neutral compartmental pH and pre- Author contributions: K.B., E.F., and R.S.B. designed research; K.B., B.P., and J.-T.A. performed serving antigenicity to generate peptides compatible with MHC-I research; T.T.K. and V.G.A. contributed new reagents/analytic tools; K.B., S.-W.Q., I.S., Z.C., − loading (6, 7). CD8+CD11b DCs have been shown to use two C.d.H., W.I.L., E.F., and R.S.B. analyzed data; and K.B., E.F., and R.S.B. wrote the paper. predominant mechanisms to achieve this result. In the vacuolar The authors declare no conﬂict of interest. pathway, antigens are degraded by proteases, such as cathepsins, This article is a PNAS Direct Submission. within phagosomes where loading of peptides directly onto MHC- 1E.F. and R.S.B. contributed equally to this work. I occurs. In the phagosome-to-cytosol pathway, antigens are pro- 2To whom correspondence should be addressed. E-mail: [email protected]. tected from local degradation and exported to the cytosol for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proteasomal degradation. Antigenic peptides are either trans- 1073/pnas.1019037108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1019037108 PNAS | June 14, 2011 | vol. 108 | no. 24 | 9927–9932 Downloaded by guest on September 25, 2021 chimeric anti-4-hydroxy-3-iodo-5-nitrophenylacetic acid (anti- as an IC with IgG versus an FcRn-disabled (IHH-IgG) ICs were NIP) antibodies, one of which, IHH-IgG, harbors three muta- not due to unequal FcRn expression (Fig. S1E), the binding of the tions that disable binding to FcRn [I253A, H435A, and H436A IgG containing IC to FcγR on the cell surface (Fig. S1 F–H), the (IHH); refs. 18 and 19; Fig. S1A]. quantities of internalized antigen (Fig. S1I), the ability of adop- − − We compared CD8 CD11b+ DCs and CD8+CD11b DCs for tively transferred DCs to reach the draining LN, or the viability of their ability to elicit cross-priming of a CD8+ T-cell response homed DCs (Fig. 1C and Fig. S1 J and K). Therefore, whereas − in vivo. In the first series of experiments, the desired subsets of CD8+CD11b DCs are superior in the cross-presentation of − DCs were obtained from the spleens of mice s.c. inoculated with soluble antigen, CD8 CD11b+ DCs are more effective in cross- either GM-CSF or Flt-3L secreting melanoma cells to selectively presenting antigen as an IgG IC in a pathway that is dependent − − enrich for CD8 CD11b+ or CD8+CD11b DCs, respectively, as upon FcRn. described in SI Methods (18, 20, 21). The different DC subsets − were isolated to purity by flow cytometric sorting and character- FcRn-Mediated Cross-Presentation by CD8 CD11b+ DCs Is Highly ized for expression of cell surface markers (Fig. S1B). The sorted Efficient at Inducing T-Cell Responses to Low Doses of Complexed DCs were pulsed with IgG- or IHH-IgG–complexed NIP-ovalbumin and Particulate Antigen. To investigate the efficiency of FcRn-me- (OVA) and adoptively transferred into the opposite hind foot- diated cross-presentation, we studied the ability of these cells to pads of recipient mice that had received carboxyfluorescein activate OT-I cells over a broad dose range of IgG ICs and par- diacetate succinimidyl ester (CFSE)-labeled OT-I T cells (Fig. ticulate antigens in vitro using CD11c-microbead isolated DCs S1C). Ipsilateral popliteal lymph nodes (PLN) were harvested from mice inoculated with GM-CSF or Flt3L secreting melanoma − after 72 h, and antigen-specific T-cell proliferation was assessed. cells (SI Methods and Fig. S2A). We observed that CD8 CD11b+ − Strikingly, CD8 CD11b+ DCs loaded with IgG-complexed DCs were able to cross-present OVA at doses as low as 0.05 μg/mL OVA (0.5 μg/mL) induced robust proliferation of adoptively in a dose-dependent and entirely FcRn-restricted fashion when transferred OT-I T cells and did so to a much greater extent provided as an IgG IC (Fig. 2 A and B). FcRn dependence for cross- − than CD8+CD11b DCs and in an entirely FcRn-dependent presentation extended to opsonized particulate antigens because − manner (Fig. 1A and Fig. S1D). In contrast, CD8+CD11b DCs only OVA-coated beads opsonized with IgG, but not IHH-IgG, − were more potent than CD8 CD11b+ DCs in inducing pro- efficiently activated CD8+ T cells (Fig. 2 C). Furthermore, the liferation to 5 μg/mL soluble antigen (Fig. 1B), consistent with ability of FcRn to promote cross-presentation was not limited to − previous observations (4, 22). These differences in the ability of CD8 CD11b+ DCs generated under inflammatory conditions be- − − − CD8+CD11b and CD8 CD11b+ DCs to cross-present antigen cause spleen CD8 CD11b+ DCs from untreated mice loaded with IgG ICs were also capable of activating CD8+ T cells in an FcRn- − dependent manner (Fig. S2 B and C). Thus, CD8 CD11b+ DCs are extremely potent in the cross-presentation of low doses of antigen in various forms when delivered in an IgG-complexed form. We next examined the relationship between FcγR and FcRn in the cross-presentation of antigen as IC with IgG. As shown in Fig. − 2D, cross-presentation by CD8 CD11b+ DCs of small ICs was highly dependent upon the ability of ICs to bind to both FcRn and FcγR since CD8+ T-cell activation was greatly reduced when ICs were formed with either IHH-IgG or N297A-IgG, the latter of which cannot bind FcγRs but retains the ability to bind FcRn (23). The requirement for FcγR was similarly observed for opsonized particulate antigen (Fig. 2E). Given that FcRn characteristically binds IgG at acidic pH not typical of the extracellular milieu where FcγRs function, we considered whether pH might be a primary factor in determining at which stage each of the IgG binding receptors is engaged while shuttling antigen into a cross- presentation pathway. As shown in Fig. 2F, IgG was observed to immunoprecipitate CD16/CD32 between pH levels of 8.0 and 6.5, consistent with what would be observed in an extracellular or early endosomal environment. In contrast, FcRn was only precipitated by IgG between pH levels 6.5 and 5.0, a pH more likely to be found within deeper endocytic compartments. Our data are consistent with a model in which IgG ICs interact sequentially with FcγRs and FcRn, binding first to FcγRs at the neutral pH of the cell surface and subsequently, following internalization, to FcRn within acidic intracellular endolysosomal compartments.

FcRn Mediates Entry of IgG ICs into a Rab27a, Vacuolar ATPase, and − Fig. 1. CD8 CD11b+ DCs use FcRn-dependent cross-presentation in vivo to gp91phox Containing Acidic Phagosome That Allows for Cross- induce primary CD8+ T-cell responses to low-dose antigen in IgG ICs. (A) Only Presentation. Given our proposed pH-dependent mechanism, CD8−CD11b+ DCs efficiently cross-prime when loaded with IgG ICs contain- we next sought to elucidate the characteristics of the intracel- ing low doses of antigen (0.5 μg/mL) and do so in an FcRn-dependent lular loading compartments involved in FcRn-mediated cross- manner. IC-loaded DCs were injected into the opposite hind footpads of − + fi − presentation in CD8 CD11b DCs. We rst examined the pH recipient mice (see Fig. S1C). (B) CD8+CD11b DCs efficiently cross-prime + reached by phagosomal compartments after IgG IC internalization CD8 T cells when loaded with a standard dose (5 μg/mL) of soluble antigen. − + 647 − + within CD8 CD11b DCs using FITC- and Alexa -conjugated (C) Viable CD8 CD11b DCs loaded with IgG ICs or IHH-IgG ICs migrate μ equally to the draining PLN. DCs were labeled with CFSE before IC loading 3- m beads. As shown in Fig. 3A, phagosomes that had in- and footpad injection and assessed 12 h later. Percentages indicate the ternalized FcRn-binding IgG-opsonized beads maintained a pH of fraction of MHC-II+CD11c+CFSE+ cells. Annexin V and 7-AAD staining con- ∼5.5, which was consistent with 1-log lower acidification of the firmed DCs viability. phagosomes than that observed within DCs that had internalized

9928 | www.pnas.org/cgi/doi/10.1073/pnas.1019037108 Baker et al. Downloaded by guest on September 25, 2021 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.

IHH-IgG-opsonized beads (pH ∼ 6.5). To understand the mecha- cross-presentation by proteasome (lactacystin and MG132) and nism for this increased phagosomal acidification in the presence of Sec61α (Exotoxin A; Fig. 4D and Fig. S3 C and D) inhibitors in- IC binding to FcRn, we performed biochemical studies on phag- dicated that this process proceeds via a phagosome-to-cytosol − osomes purified from CD8 CD11b+ DCs having internalized cross-presentation pathway. A contribution for the alternative magnetic particles opsonized with IgG or IHH-IgG (Fig. 3B). vacuolar pathway was ruled out by the lack of insulin-regulated Phagosomes from DCs that received IgG-opsonized particles rap- amino peptidase (IRAP) enrichment within phagosomes from idly acquired greater quantities of vacuolar ATPase (V-ATPase), IgG-opsonized beads (Fig. 4C) and the inability of cathepsin gp91phox, and Rab27a than those from DCs treated with IHH- inhibitors to abrogate IgG IC-mediated cross-presentation (Fig. IgG-opsonized particles. Consistent with the functional importance 4D). FcRn is thus necessary for both the movement of IgG ICs to of the recruited phagosomal proteins, blockade of V-ATPase ac- an acidified endosome equipped for phagosome-to-cytosol medi- tivity with concanamycin A or gp91phox-mediated oxidation with ated cross-presentation as well as protection of the complexed DPI completely abrogated the contribution of FcRn to cross- antigen from rapid degradation in this harsh environment. presentation (Fig. 3C). We therefore next examined the oxidation + state of internalized IgG-opsonized beads conjugated to the oxi- FcRn-Mediated Cross-Presentation Induces Cross-Priming of CD8 IMMUNOLOGY dation-sensitive probe dihydrorhodamine (DHR). Interestingly, T-Cell Responses in Vivo During Intestinal Inflammation. To demon- despite the presence of increased gp91phox within the phagosomes strate the physiological relevance of our findings, we investigated of IgG-containing beads, beads opsonized by IgG underwent less oxidation than beads opsonized by IHH-IgG (Fig. 3D).

FcRn Retains IgG ICs in an Intracellular Compartment Equipped with Components of the Cross-Presentation Machinery. In light of these findings, we examined the stability and distribution of ICs using − confocal microscopy on CD8 CD11b+ DCs obtained from bone marrow (BMDCs), as described in SI Methods (Fig. S3A). In cells pulsed with IgG ICs, the antigenic component of the ICs remained visible within intracellular compartments 60 min after pulsing and remained localized with FcRn (Fig. 4A). In comparison, despite containing comparable quantities of internalized antigen at 15 min after the pulse, BMDCs that received IHH-IgG containing ICs exhibited little evidence of the internalized antigen after 60 min and loss of FcRn coalescence into punctate structures (Fig. 4A). Consistent with the morphologic evidence for persistence of antigen within phagosomes when IgG ICs bind to FcRn, quantita- tively more OVA was detected after 60 min within the membrane − + fractions of CD8 CD11b DCs pulsed with IgG ICs than in DCs Fig. 3. FcRn-dependent cross-presentation of IgG ICs relies on acidification pulsed with IHH-IgG ICs (Fig. 4B and Fig. S3B). We therefore and oxidation of the loading compartment. (A) Phagosomes containing IgG- conclude that FcRn protects IgG ICs from rapid degradation in opsonized beads acidify more efficiently than do those containing IHH-IgG- − + the acidic phagosomes of CD8 CD11b DCs. opsonized beads. DCs were pulsed with opsonized 3-μm beads labeled with Because of the prolonged sequestration of FcRn-bound IgG ICs FITC and Alexa647 dyes and chased as indicated. (B) Phagosomes containing within phagosomal compartments, we sought to determine IgG-opsonized beads were enriched in Rab27a, V-ATPase, and gp91phox. whether these compartments contained known components of Phagosomes were purified from ICs pulsed DCs by using magnetic separa- cross-presentation machinery. Indeed, only the phagosomes con- tion. (C) FcRn-mediated cross-presentation of IgG-complexed antigens is blocked by inhibitors of acidification (concanamycin A, 50 nM) and oxidation taining beads capable of FcRn ligation were enriched in TAP1, μ α (DPI, 5 M). (D) IgG-complexed antigens that bind FcRn are protected from Sec61 , and MHC-I molecules (Fig. 4C). These data suggest that oxidation. DCs were pulsed with opsonized beads conjugated to DHR and FcRn facilitates IC trafficking into a compartment equipped for Alexa647 and chased as indicated. Results are representative of at least three cross-presentation. Furthermore, inhibition of FcRn-mediated independent experiments. Data are mean ± SEM of triplicates. **P < 0.01.

Baker et al. PNAS | June 14, 2011 | vol. 108 | no. 24 | 9929 Downloaded by guest on September 25, 2021 Fig. 4. FcRn ligation by IgG ICs traps the complexed antigen in a membrane- bound compartment optimized for cross-presentation. (A) IgG ICs are re- tained for prolonged periods within endosomal compartments where they colocalize with FcRn. BMDCs were pulsed with ICs containing OVA- Alexa488,chasedasindicated,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 cyto- plasmic 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.

− whether CD8 CD11b+ DCs were capable of inducing FcRn- consistent with infiltrating monocyte-derived inflammatory DCs, − mediated cross-presentation during the course of an active in- and CD8 CD11b+CD11c++ cells, consistent with expanded − flammatory response in vivo. To do so, we chose a chronic model resident DCs, but not CD8+CD11b CD11c+ DCs (Fig. 5A). of intestinal inflammation induced by sequential rounds of dex- These infiltrating DC subsets were present in equivalent pro- − − tran sodium sulfate (DSS) administration that is associated with portions in both wild-type and FcRn / mice, did not display the generation of high levels of IgG anti-bacterial antibodies differences in the level of activation, and exhibited similar levels (Fig. S4A; refs. 17 and 24). Both the mesenteric lymph nodes of MHC-I, MHC-II, and FcγR(Fig. S4B) as well as FcRn ex- (MLN) and the colonic lamina propria (LP) of DSS-treated mice pression (Fig. S4C). When these three DC subsets were sorted − contained a marked increase in CD8 CD11b++CD11c+ cells, by flow cytometry and loaded ex vivo with IgG- or IHH-IgG–

− 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.

9930 | www.pnas.org/cgi/doi/10.1073/pnas.1019037108 Baker et al. Downloaded by guest on September 25, 2021 − complexed OVA, only the CD8 CD11b++CD11c+ monocyte- well as phagosome-to-cytosol associated cross-presentation. The − derived DC subset efficiently cross-presented the IC-derived net effect of FcRn ligation in CD8 CD11b+ DCs may thus antigen and did so in an FcRn-dependent fashion (Fig. 5B). be to emulate for the immune-complexed antigen the mildly Furthermore, only FcRn-mediated cross-presentation by the degradative conditions experienced by soluble antigens within − − CD8 CD11b++CD11c+ DCs was associated with strong in- CD8+CD11b DCs. duction of IFNγ secretion by primary OT-I cells (Fig. 5B). These A notable aspect of our study is that a role for FcRn in IgG IC- − − findings were confirmed by DSS treatment of FcRn / mice whose mediated cross-presentation is mainly demonstrable, especially at − CD8 CD11b++CD11c+ DCs failed to elicit strong cross-priming low antigen concentrations, within a subset of monocyte-derived − under a variety of different conditions (Fig. 5C). Moreover, when CD8 CD11b+ DCs. Prior studies that have identified a role for total CD8+ T cells were isolated from the MLNs, but not spleens, these DCs in cross-presentation have examined instances of in vivo of the colitic mice, it was observed that the T cells from wild-type inflammation in the context of parasite- or adjuvant-induced dis- mice exhibited significantly higher levels of IFNγ secretion upon ex ease (26–28). Under such circumstances, although not directly − − vivo restimulation than did the CD8+ T cells from FcRn / mice shown, it is highly likely that the observed effects were at least (Fig. 5D). These CD8+ T cells also expressed significantly greater partially mediated by IgG-complexed antigen rather than strictly quantities of transcripts for Granzyme-B and T-bet relative to the by soluble antigen, thereby implicating a role for FcRn. Most − − amounts observed in those obtained from FcRn / mice (Fig. 5E studies on cross-presentation to date have focused on the pre- − − and Fig. S4D). Given the monocyte-derived origin of CD8 sentation of soluble antigen by CD8+CD11b DCs. However, CD11b++CD11c+ DCs, we ruled out a possible contribution of several of the very elements that enable this subset of DCs to ef- monocytic cells by demonstrating that peritoneal macrophages ficiently cross-present such antigens may also limit FcRn from entirely lack the ability to induce FcRn-dependent cross-pre- optimally participating in the cross-presentation of IgG IC-derived sentation (Fig. S4 E and F). Thus, FcRn-dependent cross-pre- antigens in these DCs. Specifically, whereas the phagosomes of − sentation of IgG ICs is carried out by monocyte-derived CD8 CD11b+ DCs are buffered at a highly acidic pH (≤5.5), − CD8 CD11b++CD11c+ DCs and leads to greater levels of cyto- which is optimal for FcRn function, the phagosomes within − toxic CD8+ T-cell activation in vivo during the course of colitis. CD8+CD11b DCs are buffered at near-neutral pH (6.5–7.0), a range at which FcRn exhibits minimal IgG binding (7). This − Discussion finding may explain the relatively limited ability of CD8+CD11b Despite existing literature documenting that antigens within IgG DCs to exhibit cross-presentation of antigen as an IC relative to ICs are efficiently cross-presented after FcγR-mediated inter- that observed with soluble antigen. This result is in stark contrast − nalization (10–14), the intracellular mechanisms responsible to the very potent ability of CD8 CD11b+ DCs, which can achieve for directing IgG-complexed antigen into cross-presentation- a high degree of acidification amenable to IgG–FcRn binding, enabling compartments is not known. In the present study, we to exhibit cross-presentation of antigen as a complex with IgG. We show that intracellular routing by FcRn after FcγR-mediated up- believe these data complement a previous study demonstrating − take is necessary for the cross-presentation of extremely low cross-presentation of IgG ICs by CD8 CD11b+ DCs (11). The ICs − concentrations of IgG-complexed antigens by CD8 CD11b+ DCs. used in the study by den Haan & Bevan (11) contained very high FcRn is perfectly suited to facilitate cross-presentation be- concentrations of antigen (250 μg/mL), whereas the antigen con- cause of several unique characteristics. These characteristics in- centrations in our ICs were >2 orders of magnitude lower (0.5 μg/ IMMUNOLOGY clude the fact that the pH range in which FcRn binds its ligand, mL). Thus, it is likely that our findings reflect the specific behaviors − − pH 5.0–6.5, is complementary to the relatively neutral pH of CD8+CD11b and CD8 CD11b+ DCs at different points along binding range of FcγR on the cell surface. Together with the a spectrum of IC antigen concentrations. Although this concept predominantly intracellular distribution of FcRn in DCs, this undoubtedly requires further study, the present data indicate that − bias toward binding IgG at acidic pH means that FcRn is per- CD8 CD11b+ DCs are primarily involved in initiating immune fectly poised to bind IgG ICs as soon as they are released by the responses as a consequence of cross-presentation under con- − internalizing FcγR within acidic endosomes. Consistent with this ditions of low-dose antigen, whereas CD8+CD11b DCs are more hypothesis, our data demonstrate that the intracellular com- potent in controlling such immune responses once they are un- − partment within which FcRn functions in CD8 CD11b+ DCs is derway and abundant antigen is present. Even so, the demon- atypically acidic for a cross-presenting compartment but is one strable level of proliferation detected with IgG ICs, but not − that would be predicted to permit IC interactions with FcRn. IHH-IgG ICs, by the CD8+CD11b DCs in our footpad assay Our results therefore favor a pH-dependent relay model in which supports a role for FcRn-dependent T-cell activation via cross- FcγR and FcRn act consecutively to shuttle IgG ICs from the cell presentation at low antigen concentrations in this DC subset. surface into a cross-presentation compartment. Therefore, it is likely that cross-presentation of IgG ICs by both We have also shown that the acidic microenvironment of the subsets of DCs is nearly entirely dependent upon the expression phagosome not only stabilizes the IC–FcRn interaction but also of FcRn, only with varying degrees of efficiency. slows the rate of antigen degradation and enables retention in Importantly, we show that FcRn-mediated cross-presentation − a compartment equipped for cross-presentation. These latter by CD8 CD11b+ DCs is critical for cross-priming of CD8+ T points are a key feature of cross-presentation because antigenic cells in vivo when antigen is provided as an IC with IgG and that epitopes require conservation for efficient loading onto MHC-I during inflammation, the generation of CD8+ cytotoxic T cells is (7, 25). The intracellular compartment to which FcRn directs dependent upon FcRn expression by these DCs. Recently, FcRn in IgG ICs is enriched in Sec61α, TAP1, and MHC-I (9). The hematopoietic cells was shown to play a pathogenic role in colitis presence of these membrane-associated proteins within FcRn- in the presence of flagellin containing IgG ICs (17, 18). Further- expressing phagosomes containing multimeric IgG ICs, together more, FcRn has been implicated in the pathogenesis of several with our data demonstrating their functional importance, indi- CD8+ T cell-dependent inflammatory diseases, including glo- cate that FcRn-enabled cross-presentation occurs by reuptake of merular disease, arthritis, and myasthenia gravis (29–34). Given proteasomally generated peptides into a cross-presentation that FcRn-mediated cross-presentation by infiltrating monocyte- − equipped vesicle for direct loading onto MHC-I molecules. To- derived CD8 CD11b+ DCs likely operates at extremely low-dose gether, these studies show that FcRn not only functions to ranges of antigen and that pathogen-, autoimmune-, and tumor- relay FcγR internalized IgG ICs to the appropriate intracellular associated immune responses are typically associated with a hu- locale but also, potentially, directly controls the recruitment moral IgG response (35), our results indicate that these DCs have of components necessary for optimized acidic degradation as evolved the ability to use FcRn to be highly sensitive in the de-

Baker et al. PNAS | June 14, 2011 | vol. 108 | no. 24 | 9931 Downloaded by guest on September 25, 2021 tection of antigen and to convert this stimulus into a functional beads for 2–3 h and cocultured with primary CD8+ T cells isolated from naïve CD8+ T-cell response. Thus, the knowledge that FcRn plays OT-I mice. a critical role in cross-presentation signiﬁcantly extends its importance to immunity beyond its previously identiﬁed roles in Isolation of Intracellular Compartments. Phagosomes were prepared as de- MHC-II-restricted antigen presentation, antibody protection, and scribed (SI Methods).

transcellular transport of IgG (18, 19, 30). − + + − In summary, our data reveal an important role for FcRn in the In Vivo T-Cell Proliferation. Splenic CD8 CD11b and CD8 CD11b DCs were proper intracellular trafficking of IgG-derived ICs during cross- sorted from spleens of donor mice, loaded with IgG or IHH-IgG ICs for 3 h, and injected into opposite hind footpads of Wt mice adoptively transferred with presentation. Our studies show that, in the absence of FcRn γ CFSE-labeled OT-I T cells, as outlined in Fig. S1C. For assessing DC migration function, Fc R-dependent internalization of ICs is not able to and viability, DCs were stained with CFSE before transfer and for Annexin V elicit cross-presentation, indicating that these two molecules and 7-AAD after isolation of the draining LN, as described in SI Methods. function in tandem and are cooperative. This role for FcRn is − + especially notable in CD8 CD11b DCs that are recruited to Chronic DSS Colitis. Chronic DSS colitis was induced by using a 2% DSS solution fl sites of in ammation, such that in the absence of FcRn function, (36). On day 52, cells were isolated from the colonic LP and MLN as described + the induction of CD8 T-cell responses are disabled. Thus, al- (37). CD8−CD11b++CD11c+, CD8−CD11b+CD11c++, and CD8+CD11b−CD11c++ though it is well-established that cross-presentation of antigen in DC subsets were sorted, loaded with ICs, and cocultured with primary OT-I T the context of IgG ICs can be initiated by FcγR-mediated in- cells. In separate experiments, CD8+ T cells were isolated and restimulated ternalization, our data not only elucidate the intracellular for 24 h with anti-CD3/anti-CD28. Supernatants were analyzed by using a mechanism by which this process proceeds but also demonstrate Cytokine Bead Array (BD Pharmingen). RNA was analyzed by using a Nano- the necessity of FcRn for the induction of CD8+ T-cell responses string assay (NanoString Technologies). during the course of inflammation. Statistical Analysis. Statistical significance was assessed by two-tailed un- Methods paired Student’s t test. − − Mice. OT-I mice were obtained from The Jackson Laboratory. FcRn / mice on a C57BL/6 background, which are deficient in the Fcgrt gene, have been ACKNOWLEDGMENTS. We thank Dr. Sherie Morrison (University of California described (18). In all experiments, wild-type (Wt) mice and FcRn−/− mice were Los Angeles) for the J558L cells, Dr. Siew Yeen Chai (University of Melbourne, – matched littermates. All procedures were approved by the Harvard Medical Australia) for the anti-IRAP antibody, and Dr. Glenn Dranoff (Dana Farber Cancer Institute) for the Flt3L- and GM-CSF-secreting melanoma cell lines. This Area Standing Committee on Animals. work was supported by the Canadian Institutes of Health Research (K.B.); National Institutes of Health Grants DK071798 (to T.T.K.), AI075037 (to E.F.), Antigen Presentation Assays. ICs were preformed as indicated in SI Methods. and DK53056 (to W.I.L. and R.S.B.); and the Harvard Digestive Diseases Center DCs isolated as described in SI Methods were loaded with ICs or opsonized (National Institutes of Health Grant DK34854 to W.I.L. and R.S.B.).

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