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Exosome-Driven Antigen Transfer for MHC Class II Presentation Facilitated by the Receptor Binding Activity of Influenza Hemagglutinin This information is current as of September 25, 2021. James S. Testa, Geraud S. Apcher, Joseph D. Comber and Laurence C. Eisenlohr J Immunol 2010; 185:6608-6616; Prepublished online 3 November 2010; doi: 10.4049/jimmunol.1001768 Downloaded from http://www.jimmunol.org/content/185/11/6608

Supplementary http://www.jimmunol.org/content/suppl/2011/05/31/jimmunol.100176 http://www.jimmunol.org/ Material 8.DC1 References This article cites 79 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/185/11/6608.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Exosome-Driven Antigen Transfer for MHC Class II Presentation Facilitated by the Receptor Binding Activity of Inﬂuenza Hemagglutinin

James S. Testa,1 Geraud S. Apcher,2 Joseph D. Comber, and Laurence C. Eisenlohr

The mechanisms underlying MHC class I-restricted cross-presentation, the transfer of Ag from an infected cell to a professional APC, have been studied in great detail. Much less is known about the equivalent process for MHC class II-restricted presentation. After infection or transfection of class II-negative donor cells, we observed minimal transfer of a proteasome-dependent “class I- like” epitope within the inﬂuenza neuraminidase glycoprotein but potent transfer of a classical, H-2M–dependent epitope within the hemagglutinin (HA) glycoprotein. Additional experiments determined transfer to be exosome-mediated and substantially enhanced by the receptor binding activity of incorporated HA. Furthermore, a carrier effect was observed in that incorporated Downloaded from HA improved exosome-mediated transfer of a second membrane protein. This route of Ag presentation should be relevant to other enveloped viruses, may skew CD4+ responses toward exosome-incorporated glycoproteins, and points toward novel vaccine strategies. The Journal of Immunology, 2010, 185: 6608–6616.

D4+ T lymphocytes are activated through the recogni- The dichotomy of exogenous Ag for MHC II and endogenous

tion of Ag-derived peptides complexed to MHC class II Ag for MHC I (4) is becoming increasingly indistinct. For ex- http://www.jimmunol.org/ C (MHC II) molecules. Conventionally, Ag is acquired ample, there are now numerous examples of MHC II-restricted through uptake of exogenous material in the form of whole or peptides that are generated from endogenous sources of Ag (5–7), partial pathogens, soluble proteins, or cellular fragments from and several processing mechanisms have been reported including necrotic or apoptotic cells (1). Internalized Ags are then unfolded macroautophagy (8), chaperone-mediated autophagy (9), and a class and digested within the endosomal network by resident reductases I-like proteasome/TAP-dependent pathway (10). The NA79 epi- and proteases. Once sufficiently disordered, epitopes are loaded tope (aa 79–93), derived from the influenza neuraminidase (NA) onto MHC II molecules in a late endosomal compartment with the andalsoH-2–IEd restricted, is generated via this last pathway assistance of the chaperone H-2M. The H-2–IEd-restricted site 1 (10). A major exception to the conventional class I pathway is the (S1) epitope aa 107–119 of the A/Puerto Rico/8/34 (PR8) in- phenomenon of cross-presentation in which Ag is transferred from by guest on September 25, 2021 fluenza hemagglutinin (HA) is generated in this manner (2, 3). infected cells to professional APCs, primarily dendritic cells (DCs). The conventional pathway for generation of MHC class I (MHC This route appears to be critical for naive T cell activation in cases I)-restricted epitopes is distinctly different in requiring delivery where the professional APC cannot directly acquire Ag (11). of Ag to the cytosol, digestion by the proteasome, and transport of Additionally, it allows for escape from immunosuppressive effects resultant peptides to the endoplasmic reticulum via TAP for load- imposed by the pathogen in the infected cell (12). Conventionally, ing onto nascent MHC I molecules. cross-presented material is delivered to the cytosol for proteasome/ TAP-dependent processing (13, 14), but there are also reports in which cross-presented Ag is confined to a phagolysosomal compartment where it is processed and loaded onto resident MHC I Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107 molecules, a decidedly MHC II-like scenario (13, 15). 1Current address: Immunotope Inc., Pennsylvania Biotechnology Center, Doyles- A priori, there is no reason why Ag transfer would be restricted town, PA. to MHC I. Limited study of class II-restricted transfer may be due 2Current address: Target Therapeutics, INSERM U940, Institute of Molecular Ge- to the general view that class II processing is limited to the en- netics, University of Paris 7, Hospital St. Louis, Paris, France. dosomal compartment after uptake of exogenous Ag. Thus, Ag Received for publication May 27, 2010. Accepted for publication September 30, transfer would not be much different from direct acquisition. How- 2010. ever, given the aforementioned endogenous MHC II presentation This work was supported by National Institutes of Health Grant AI39501. pathways, there is ample reason for exploring cross-presentation Address correspondence and reprint requests to Dr. Laurence C. Eisenlohr, Thomas within the class II system. Whereas several studies have sug- Jefferson University, BLSB Room 730, 233 South 10th Street, Philadelphia, PA 19107. E-mail address: [email protected] gested that class II-restricted Ag transfer is limited (16–22), others The online version of this article contains supplemental material. have described transfer of soluble proteins, apoptotic bodies, pre- processed peptides, and exosomes as being viable mechanisms (1, Abbreviations used in this paper: CQ, chloroquine; DC, dendritic cell; HA, hemagglutinin; HAU, hemagglutinating unit; iPR8, infectious PR8; MHC I, MHC class I; 23–30). With the goal of better understanding the parameters of MHC II, MHC class II; MUG, b-galactosidase substrate methyl-umbelliferyl-b-D- class II-restricted Ag transfer, we tested the widely disparate S1 and galactoside; MVB, multivesicular body; NA, neuraminidase; NA79, neuraminidase aa 79–93 epitope; PR8, influenza A/Puerto Rico/8/34; Rv6, Rv6 variant of PR8 HA; NA79 epitopes in this setting. Given the relative efficiency of class S1, site 1; S1–Tac, S1 appended on the N terminus of Tac; SS, signal sequence; TEM, I-restricted cross-presentation, our expectation was that NA79 transmission electron microscopy; TM, transmembrane domain; UVPR8, UV- would be transferred whereas the classically presented S1 epitope inactivated PR8; WT, wild-type. would not. Instead, results were the opposite, revealing a mecha- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 nism for efficient Ag transfer of classically presented epitopes. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001768 The Journal of Immunology 6609

Materials and Methods 200 mU NA (Sigma) to remove terminal sialic acid residues 2 h before the Mice addition of B6-HA donor cells. To evaluate the degree of anti-HA–mediated neutralization and NA-mediated de-sialation, treated B6IEd cells were All mice used were purchased from The Jackson Laboratory (Bar Harbor, infected with PR8, and NA79 presentation was determined. ME) and maintained by the Thomas Jefferson University Office of Labo- ratory Animal Services (Philadelphia, PA). All experimental protocols were Exosome purification preapproved by the Thomas Jefferson University Institutional Animal Care Exosomes were purified from 4-d- or 16-h-old supernatants of appropriate and Use Committee. cells according to standard protocol (34). Briefly, supernatant was first Synthetic peptides centrifuged at 10,000 3 g for 30 min to clear cells and cell debris. Sub- sequently, it was ultracentrifuged at 100,000 3 g to generate an exosome Synthetic S1 (HA 107–119), NA79 (NA 79–93), and SIINFEKL (OVA257– pellet, which was then washed with PBS and re-pelleted at 100,000 3 g. 264) peptides were purchased from Invitrogen (Carlsbad, CA). Peptides Pellets were then floated on a continuous 5–65% sucrose gradient by ultra- were pulsed onto APCs at 1025 M. centrifugation at 24,000 rpm in a Beckman-Coulter (Brea, CA) SW41TI rotor (∼76,000 3 g) overnight. The following day, the gradient fractions were Cell lines, transfection, and recombinant retroviral analyzed for exosome content, and exosome-containing fractions were di- transduction alyzed against PBS prior to storage at 4˚C. Cell lines described in this study were primary skin fibroblasts derived from Coupling of exosomes to latex beads C57B6 or H-2M2/2 C57B6, which were then transduced with appropriate retroviruses. All lines were maintained in DMEM supplemented with 10% Exosomes were prepared for analysis by flow cytometry as previously de- FCS and 0.05 mM 2-ME. scribed (35). Briefly, purified exosomes were adsorbed to 0.4-mm-diameter The retrovirus construct used was MSCV–CMVor MSCV–CMV–IRES– aldehyde/sulfate latex beads (Invitrogen) for 15 min at room temperature. GFP with pCL–Ampho or pCL–Eco as the helper plasmid (generous gifts Next, the bead/exosome mixture was diluted with 1 ml PBS and incubated Downloaded from from Dr. Jianke Zhang, Thomas Jefferson University). Retrovirus was pro- another 2 h. After this incubation, beads were washed and stained for flow duced as previously described (10). Briefly, 293T cells were transfected with cytometric analysis using anti-HA. the appropriate MSCV construct along with the helper plasmid. Constructs used include IEda,IEdb, human CIITA, HA [H1 and H1-Rv6 (31)], NA, and Electron microscopy S1–Tac. Supernatant collected was then either used or stored at 280˚C for For transmission electron microscopy analysis, purified exosomes were later use. For transductions, supernatant plus polybrene (10 mg/ml) was adsorbed onto carbon-coated grids (Ladd Research Laboratories, Williston, overlaid onto cells for 24 h and replaced with complete medium. Once http://www.jimmunol.org/ VT) for 3 min. After adsorption, the samples were fixed for 1 min with 1% confluent, cells were expanded and sorted based on surface IEd or IRES–GFP glutaraldehyde. The grids were then washed with a drop of ddH Oand expression using a MoFlo (Dako Cytomation, Fort Collins, CO) cell sorter. 2 transferred to a drop of 1% uranyl acetate for 1 min for negative staining. For all transfections of fibroblasts, Genejuice (Novagen, Madison, WI) Excess uranyl acetate was absorbed with Whatman paper no. 1, and grids was used according to the manufacturer’s instructions. Transfected cells were dried on filter paper for 2–3 h. Grids were then visualized on an Tecnai were used in Ag presentation assays 48 h posttransfection. (FEI, Hillsboro, OR) 12 transmission electron microscope at 80 kV. Primary DCs Immunoblotting For experiments involving the use of primary DCs, splenocytes from CB6F1 3 We followed standard techniques for immunoblotting. Specifically, we used mice (H-2b d) were enriched for DCs as described previously (32). Briefly, exosomal fractions generated by sucrose gradient separation. Dialyzed 1E7 B16 melanoma cells, stably transfected with flt3L (generous gift from by guest on September 25, 2021 fractions were concentrated using Vivaspin 6 columns (Sartorious, Goettin- Christopher Norbury), were injected s.c. at the left flank. After 10–14 d, the gen, Germany), stained using anti-S1 or anti-flotillin (BD Pharmingen), and mice were sacrificed, and whole splenocytes were used for the negative analyzed using SDS-PAGE. isolation DC (Invitrogen, Carlsbad, CA). T cell hybridomas Statistics All experiments were analyzed using Prism (GraphPad, La Jolla, CA) soft- S1- and NA79-specific T cell hybridomas, which express b-galactosidase ware. Statistical significance was calculated using a paired Student t test with upon recognition of peptide-MHC class II complexes, and the SIINFEKL: one- and two-tailed distributions. Differences were determined to be sig- Kb–specific T hybridoma (B3Z), which does so upon recognition of peptide- nificant if the p value was ,0.05. MHC class I complexes, have been described previously (10, 33). T cell hybridomas were maintained in RPMI 1640 plus 10% FCS and 0.05 mM 2-ME. Activation was measured by detection of fluorometric b-galactosidase substrate methyl-umbelliferyl-b-D-galactoside as previously described (2). Results Cells expressing influenza HA can transfer Ag for S1 Ag presentation assays presentation For experiments where cells were infected with influenza, the strain PR8, As previously reported (5, 10), S1 is presented from both infectious subtype H1N1, was used. Cells were infected with 50 hemagglutinating units PR8 (iPR8) and UV-inactivated PR8 by B6IEd, B6 skin fibroblasts (HAU) per million cells for 30 min in PBS with 0.1% BSA. Postinfection, da b cells were washed three times with serum-containing medium to remove any stably expressing IE , , and human CIITA (Fig. 1A). This re- unbound virus. flected the ability of this epitope to be generated in the endosomal In some experiments, we separated B6-HA donor cells from B6IEd compartment. In contrast, NA79 is presented from only iPR8, re- m acceptor cells using Transwell inserts with 0.4- m pores (Corning, Corning, flecting the requirement for cytosolic delivery and proteasome- NY). For drug treatments of donor cells (all from Sigma-Aldrich, St. Louis, dependent processing, properties that we speculated would allow MO), working concentrations were PMA at 0.1 mg/ml, LPS at 1 mg/ml, it to be cross-presented as an MHC I-restricted epitope. chloroquine at 0.4 mM, and NH4Cl at 200 mM. Cells were co-incubated Biosynthesized HA does not traffic efficiently to the endosomal with the compounds for 16 h followed by three washes with PBS before compartment (36). Thus, because transduction of APCs with an co-incubation with acceptor cells and T cell hybridomas. In some cases, NA-expressing retrovirus, just like infection with iPR8, should re- supernatants were collected for exosome harvest. Purified exosomes were quantified by protein concentration analysis using a Nanodrop spectro- sult in presentation of NA79, we expected that transduction with an photometer (Thermo-Fisher, Waltham, MA) at an absorbance of 280 nm. HA-expressing retrovirus would result in minimal or absent pre- ToassessreceptorbindingactivityofHA, blocking Abswereused. H28E23 sentation of S1. When this experiment was performed in B6IEd (anti-HA), CMI-1.1 (anti-S1), and NA21C1 (anti-NA) were purified from cells, both epitopes were presented efficiently (Fig. 1A). This led us hybridoma supernatant using recombinant immobilized protein A beads to consider the possibility that some form of Ag transfer is possible (Thermo-Fisher). Purifiedand unconjugatedanti-TfR waspurchasedfromBD d Pharmingen (Franklin Lakes, NJ). Working concentrations of all Abs were in both cases. To test this, we infected H-2–IE -negative B6 fibro- 100 ng/ml to 10 ng/ml. In some cases, B6IEd acceptor cells were treated with blasts with iPR8 and co-incubated these “donor cells” with 6610 MHC II EXOSOME TRANSFER ENHANCED BY HEMAGGLUTININ Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. MHC II presentation via Ag transfer. A (top panel), B6IEd cells were treated with the indicated amounts of iPR8 or UV-inactivated PR8 (UVPR8). As positive controls, B6IEd cells were pulsed with 1025 M synthetic S1 and NA peptides. Upon treatment, APCs were serially diluted, and equal number of S1 and NA79 T cell hybridomas were then added to assess presentation. A (bottom panel), B6IEd cells were transduced with PR8 HA or NA (B6- HA-IEd or B6-NA-IEd). As positive controls, B6-IEd were pulsed with S1 or NA79 peptides. S1 and NA79 T cell hybridomas were used to assess Ag presentation. B, Primary B6 skin ﬁbroblasts (not expressing IEd) were infected with 50 HAU iPR8 and used as donor cells in an Ag transfer assay. Prior to coculture with B6IEd cells as recipients, donor cells were washed. After serial dilution of donors and acceptors, S1 and NA79 T cell hybridomas were added and used to read out Ag presentation. C, Primary B6 ﬁbroblasts stably transduced with HA and NA (B6-HA and B6-NA) were cocultured overnight with B6IEd cells or primary DCs and with S1 T cell hybridomas. D, 293T cells were transfected with genes encoding OVA, SIINFEKL-cytosolic-NP, or HA and incubated with B6IEd. Presentation of NA79, S1, and SIINFEKL were assessed with the appropriate T cell hybridomas. All experiments are representative of three independent experiments. *p , 0.05. MUG, b-galactosidase substrate methyl-umbelliferyl-b-D-galactoside.

“acceptor” B6IEd cells or primary DCs extracted from H-2b3d mice. apoptosis might have facilitated the presentation of the S1 epitope Despite its class I-like nature, NA79 was poorly presented under and/or inhibited the presentation of the NA79 epitope (38, 39). To these conditions, whereas the classically presented S1 epitope was investigate this, we stably transduced donor B6 skin fibroblasts with potently presented (Fig. 1B). The remainder of the experiments the HA or NA genes, a procedure that produced no evidence of cell reported in this study focused on the latter finding. death (data not shown). Upon coculturing these donors with primary A caveat to using influenza-infected cells as donors is the pos- DCs or B6IEd cells as recipients, we observed that S1 was efficiently sibility that nascent virions bud from the surface and infect neigh- presented (Fig. 1C), whereas NA79 was once again poorly presented boring acceptor cells, thereby giving the false impression of Ag (Supplemental Fig. 2). Thus, the disruptive effects of viral infection transfer. This seemed unlikely because the NA79 epitope would are not essential for S1 presentation via Ag transfer, nor can they then be presentable via the direct, endogenous route. Nevertheless, explain the near lack of NA79 presentation. to rule this out, donor cells were infected, allowed sufficient time for We also considered the possibility that NA79 was not efficiently de novo protein synthesis of viral proteins, andthen UV-irradiated be- transferred because our system is unable to facilitate true MHC I fore coculture with acceptor cells (Supplemental Fig. 1). UV irradi- cross-presentation. Thus, we transfected donor H-2Kb–negative ation of donor cells had no impact on S1 presentation by the acceptor 293T cells with genes encoding HA, NA, OVA, and a version of b cells indicating that reinfection was not the basis for Ag transfer. influenza nucleoprotein that contains the OVA257–264 H-2K –re- In addition, because transfer of apoptotic bodies has been reported stricted epitope. Upon coculture of these transfectants with B6IEd to mediate class II-restricted Ag transfer (1), and influenza infection cells, we observed robust cross-presentation of OVA257–264 and can lead to apoptosis (37), we considered the possibility that presentation of S1 but not NA79 (Fig. 1D). The Journal of Immunology 6611

The transferred material is a soluble factor dependent upon electron microscopy (TEM) of the active fractions (Fig. 3A, inset) processing by the acceptor cell revealed structures typical of exosomes imaged by TEM (∼100 nm). In exploring the basis for Ag transfer, we first eliminated the pos- An exosome preparation from influenza-infected B6 donor cells sibility that the transferred material was simply S1 peptide released was similarly stimulatory (Fig. 3B). from the donor cells and loaded onto acceptor cell IEd at the plasma To verify exosome-mediated transfer, donor cells were treated with PMA and LPS, enhancers of exocytosis (45, 46), and cocultured membrane as previously described (27–30). Ultracentrifugation of d the medium from PR8-infected B6 cells cleared nearly all of the with B6IE cells after extensive washing (Fig. 3C). In both cases, stimulatory activity, and the effect was complete for medium from the amount of material recovered from the exosome fraction was the B6-HA transfectants (Fig. 2A). Furthermore, loading of syn- increased (Supplemental Fig. 4), and enhanced presentation was thetic peptide has been reported to be primarily H-2M independent observed (Fig. 3C). These compounds did not alter the amount of (40), and, as shown in Fig. 2B, absence of H-2M in the acceptor but surface HA or cell viability (Supplemental Fig. 5), indicating that not the donor cells completely abrogates presentation. We also ob- the effect is not due to increased Ag expression. The experiment served that fixation of acceptor cells by paraformaldehyde signifi- was repeated with chloroquine and NH4Cl, inhibitors of exocytosis cantly impairs their ability to present S1 from transferred Ag but not (47, 48). Both compounds reduced exosome production (Supple- synthetic peptide (data not shown). mental Fig. 4) and similarly affected presentation of S1 (Fig. 3C) Pulsing B6IEd cells with conditioned media from B6-HA and while having no effect on HA expression or viability (Supplemental B6-NA revealed that the transferred material is not cell associated Fig. 5). and transmitted without direct contact between donor and acceptor Another reported transfer mechanism is the release of peptide– (Supplemental Fig. 3A). To solidify this conclusion, B6IEd acceptor MHC II complex-containing exosomes by DCs (49), allowing for Downloaded from the acquisition of “prepresented” Ag. However, exosomes from cells were separated from B6-HA or B6-NA donor cells by an 0.4- d mm membrane (Supplemental Fig. 3B). This had no impact on the B6IE -HA cells pulsed onto B6 cells did not result in S1 presentation potency of S1 presentation. (Fig. 3D), nor did they directly activate the S1-specific T hybridoma (data not shown). This form of Ag transfer may be limited to exo- Ag is transferred in the context of secreted exosomes from the somes secreted from and taken up by professional APCs.

donor cells http://www.jimmunol.org/ Based on these results, we hypothesized that Ag transfer is medi- HA incorporated into exosomes is intact and in an ated by exosomes secreted from donor cells and internalized by outward-facing orientation recipient cells, a mechanism previously described for tumor cell- We next examined the possibility that the S1 epitope is transferred derived Ags (41, 42). Accordingly, we purified exosomes from B6- as part of soluble HA fragments within the exosome lumen, as would HA supernatant over a sucrose gradient and pulsed fractions onto occur with cytosolic proteins (50). We first immunoblotted exosomal B6IEd cells. As shown in Fig. 3A, S1 presentation was only observed lysate with a monoclonal anti-S1 Ab and detected a single band at 64 in fractions whose density corresponded with that of typical exo- kDa (Fig. 4A), the expected size of full-length HA. Because mem- somes (1.13–1.19 g/cm3) (43). Furthermore, immunoblotting of brane proteins can be incorporated into exosomes in either orien- by guest on September 25, 2021 fractionated supernatants revealed co-migration with the exosomal tation (50), we next determined the directionality of HA by flow marker flotillin (44) (Fig. 3A, lower panel). Finally, transmission cytometry. Latex beads adsorbed with purified exosomes from B6-

FIGURE 2. Ag transfer is dependent on pel- letable material and processing by the acceptor cell. A, Supernatants from PR8-infected B6 cells (left panel) or B6-HA cells (right panel) were subjected to ultracentrifugation. The resuspended pellets and the supernatants were then added to B6IEd cells and S1-T cells. Whole supernatant and cells were added as positive controls, and data are depicted with uninfected B6 subtracted. As an additional control, media were “spiked” with synthetic S1 peptide prior to ultracentrifugation and observed to be fully stimulatory (data not shown). After ultracentrifugation, supernatant from infected B6 cells was confirmed to be clear of virions using a flow cytometry-based infectivity assay (data not shown). B, Primary skin fibroblasts from B6 mice or H2M2/2 mice were transduced for stable expression of HA or NA. These cells were used as donors with B6IEd or H2M2/2 B6IEd as acceptor cells in an S1-specific presentation assay. All experiments are representative of three independent experiments. MUG, b-galactosidase substrate methyl-umbelliferyl-b-D-galactoside. 6612 MHC II EXOSOME TRANSFER ENHANCED BY HEMAGGLUTININ

FIGURE 3. Ag is presented via secreted exosomes produced by donor cells. A, Exosomes were purified from B6-HA supernatant by sucrose gradient fraction- ation. Fractions were analyzed for their sucrose density by refractometry. Dialyzed fractions were then pulsed onto B6IEd cells and cocultured with S1-T cells. Fraction number is graphed against T cell activation (solid line) and sucrose density (dotted line). The accepted density range of exosomes (43) is highlighted. These fractions were also blotted for S1 and flotillin-1, an exosomal marker (44). Inset, Purified exosomes from B6-HA were negatively stained using uranyl ac- Downloaded from etate and imaged by TEM. Original magnification 326,000. This image is representative of a typical field for three independent experiments. B, Exosomes were purified from supernatant of infected B6 cells that were infected with 50 HAU iPR8 and pulsed onto B6IEd cells. S1 presentation was assessed with the S1-specific

T hybridoma. C, B6-HA cells were treated with PMA, http://www.jimmunol.org/

LPS, NH4Cl, or chloroquine (CQ) for 16 h. Subse- quently, exosomes were puriﬁed from condition supernatant, or extensively washed cells were co-incubated with B6IEd and S1-T hybridomas. D, Exosomes puriﬁed from B6IEd-HA cells were pulsed onto B6 cells and incubated with S1-T cells. All experiments are representative of three independent experiments. *p , 0.05. MUG, b-galactosidase substrate methyl-umbelliferyl- b-D-galactoside. by guest on September 25, 2021

HA cells demonstrated HA-speciﬁc staining (Fig. 4B). Together neutralizing Ab because sialated glycoproteins and glycolipids these results indicate that exosome-incorporated HA is full length will reappear at the plasma membrane during NA-free coculture with its extracellular domain facing outward. with donor cells and T hybridomas.

Sialic acid binding function of HA enhances transfer of HA can enhance cross-presentation of a “bystander” exosomal Ag exosome-associated Ag HA mediates influenza cellular attachment by binding to terminal Finally, with an eye toward practical application, we investigated sialic acid residues at the plasma membrane. Thus, we speculated whether HA enhances transfer and presentation of other Ags in- that incorporation of HA rendered the exosome capable of receptor- corporatedintotheexosome.Tothis end,wegeneratedstableB6 cells mediated attachment, increasing efficiency of Ag transfer. This was that express a modified version of the human IL-2 receptor a-chain tested in two ways. First, a transfer assay was performed in the (Tac), in which S1 was appended at the N terminus (S1–Tac) (Fig. presence of an amount of neutralizing anti-HA Abs that was 6A). First, cells transfected with the S1–Tac gene were tested as confirmed to inhibit virus infectivity (Supplemental Fig. 6A). In- donors and observed to facilitate a low level of H-2M–dependent hibition was substantial, though incomplete (Fig. 5A). A similar presentation (Fig. 6B). We then transfected stably expressing S1– effect was observed when donor cells were replaced with purified Tac cells with a gene encoding the Rv6 variant of PR8 HA (H1- exosomes (Fig. 5B). Next, acceptor cells were pretreated with NA Rv6), which lacks the S1 epitope while retaining sialic acid binding to remove surface sialic acid residues, with efficacy of this pro- activity (31). When HA-Rv6 was coexpressed with the S1-bearing cedure also assessed via infectivity assay (Supplemental Fig. 6B). Tac construct, presentation of S1 was markedly enhanced (Fig. 6C). NA-treated acceptor cells pulsed with exosomes from B6-HA or As was the case with PR8 HA, HA-Rv6 expression alone did not B6-NA cells presented S1 less efficiently than did untreated cells enhance exocytosis (Supplemental Fig. 7). Taken together, our (Fig. 5C). As anticipated, this treatment was less effective than the results demonstrate that the receptor binding activity of HA The Journal of Immunology 6613 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. S1 is transferred in the context of full-length, native HA on exosomes from the donor cells. A , Exosomes were purified from B6-HA FIGURE 5. Receptor binding activity of HA enhances exosome-medi- cells and subjected to Western blot analysis using anti-S1 Ab (top panel)or ated Ag transfer. A, B6-HA cells were cocultured with B6IEd cells and pulsed onto B6IEd cells in the presence of S1-T hybridomas (bottom T cell hybridomas with or without purified anti-HA Ab (H28E23) or pu- panel). B, Purified exosomes from B6-HA or B6-NA cells were adsorbed rified anti-TfR Ab. Peptide controls consisted of B6IEd cells cocultured with to aldehyde/sulfate latex beads and stained for HA or NA by flow cyto- 2 B6-NA and S1-T hybridomas in addition to 10 5 M synthetic S1 peptide. metry. All experiments are representative of three independent experi- B, Exosomes were purified from B6-HA and then pulsed onto B6IEd cells ments. MUG, b-galactosidase substrate methyl-umbelliferyl-b-D-galacto- and T cell hybridomas with or without equivalent amounts of anti-HA Ab, side. anti-NA Ab, or anti-S1 Ab. C, B6-HA cells were co-incubated with B6IEd cells pretreated with NA and then co-incubated with S1-T cells. All experiments are representative of three independent experiments. *p , 0.05. enhances exosome-mediated Ag transfer, not just of HA-associated MUG, b-galactosidase substrate methyl-umbelliferyl-b-D-galactoside. epitopes but also of exosome-incorporated proteins in general.

Discussion as part of the pathogen and uptake of the same Ag released from an The concept of MHC II-restricted Ag transfer is ostensibly of infected cell. However, there is now convincing evidence for many limited interest because, as exempliﬁed by the S1 epitope, classical alternative processing pathways including the loading of Ag onto MHC II processing entails internalization of exogenous Ag, un- “recycling” class II molecules in early endosomes (2, 52–54), de- folding and/or digestion in the endocytic compartment, and H-2M– livery of Ag from cytosol to the endolysosomal compartment via dependent loading onto nascent class II molecules (51). In essence, autophagy (8, 9), and a cytosolic/proteasome-dependent pathway there would be minimal difference between direct uptake of an Ag (10). Because this last route, which produces the NA79 epitope, is 6614 MHC II EXOSOME TRANSFER ENHANCED BY HEMAGGLUTININ

FIGURE 6. HA can enhance presentation of another exosome-associated Ag. A, Schematic of the S1–Tac construct. The S1 epitope was appended to the N terminus of wild-type (WT) Tac just after the signal sequence (SS). TM, transmembrane domain. B, B6-S1-Tac cells were co-incubated with either B6IEd cells or H2M2/2 B6IEd cells in addition to S1-T hybridomas. Peptide controls consist of acceptor cells pulsed with 1025 M synthetic S1 peptide in the absence of donor cells. C, B6-S1-Tac cells were transduced with HA-Rv6 and incubated for 48 h. After incubation, these cells and empty vector-transduced cells were cocultured with B6IEd cells and T cell hybridomas. All experiments are representative of three independent experiments. MUG, b-galactosidase substrate methyl-umbelliferyl-b-D-galactoside. Downloaded from

similar to conventional class I processing, we investigated whether have speculated for some time that the NA79 epitope is destroyed a scheme similar to MHC I-restricted cross-presentation exists on immediately after virus or exosome internalization, precluding the class II side. This was possible because NA79 is produced via binding to class II. Such susceptibility has been noted for several

only the cytosolic route (and not the classical route). Despite ob- other epitopes (63–65). In our own Ag system, presentation of the http://www.jimmunol.org/ serving substantial direct presentation of NA79 from endogenous H-2–IEd-restricted and HA-derived S3 epitope is considerably NA, presentation by Ag transfer was quite limited. However, we increased by addition of leupeptin, a serine and cysteine protease observed robust presentation of the classical S1 epitope driven by inhibitor (2, 66). We stress that proteolytic susceptibility is likely the incorporation of HA into exosomes. Because HA-bearing exo- a property of the epitope itself and not the parent protein. Thus, somes are similar to influenza virions in having the extracellular other epitopes within NA may be presentable from exogenous domain of HA facing outward, we investigated whether the sources. An intriguing possibility is that some MHC I epitopes are receptor-binding function of HA enhances the efficiency of transfer. similarly susceptible to endosomal proteases such that they are Blocking Abs and NA treatment of the acceptor cells confirmed that presentable mainly or exclusively from endogenous sources. Ef- this was the case. fective responses to such epitopes might depend upon direct in- by guest on September 25, 2021 Exosomes originate as intralumenal vesicles in the multivesicular fection of the professional APCs. Indeed, endosomal susceptibility body (MVB), and protein incorporation is selective. One basis for may be an explanation for MHC I epitopes that are inefficiently localization to this compartment is monoubiquitination (55). Mon- cross-presented, a property that has been correlated with subdomi- oubiquitinated proteins interact with a heterooligomeric complex nance, and low immunogenicity (67, 68) termed the endosomal sorting complex required for transport, which We have refrained from describing exosome-mediated trans- mediates targeting to the MVB. This is an unlikely basis for in- fer of S1 as “cross-presentation.” Cross-presentation of MHC I- corporation of HA, however, because it is not known to contain restricted epitopes is generally understood to involve transfer of a ubiquitination motif. Another partitioning mechanism, exempli- biosynthesized Ag in complex form (apoptotic bodies, heat shock fied by TfR, involves the rerouting of internalized proteins aggre- protein associated, etc.) from an infected cell to the cytosol of gated into lipid rafts (56, 57) that are preferentially sorted into a DC for proteasome and TAP-dependent processing and pre- MVBs rather than recycling compartments (55). Because HA is well sentation (11, 14, 69). In the case of S1 transfer, biosynthesis known to aggregate into raft domains for the budding of new virions, of HA is required for exosome incorporation, but cytosolic de- we favor this mechanism as the basis for sorting into exosomes (58, livery is not. Additional studies may reveal MHC II-restricted Ag 59). In addition, the high levels of expression associated with in- transfer that features both biosynthesis and cytosolic delivery and fection and the transfection conditions used might cause “overflow” could rightfully be termed cross-presentation. In fact, this may be into certain cellular compartments. This phenomenon has been de- the case for the small amount of NA79 presentation by the re- scribed for protein disulfide isomerase; when superexpressed, it cipient cell (Fig. 1). In preliminary experiments, anti-HA blocking traffics beyond the endoplasmic reticulum into the endosomal com- Ab does not reduce presentation of NA (data not shown), sug- partments by saturating the relevant endoplasmic reticulum-reten- gesting a transfer mechanism that is not exosome mediated. Fur- tion machinery (60). ther, if transfer of NA79 is limited by susceptibility to endosomal The near absence of NA79 presentation via Ag transfer is not proteases, less susceptible epitopes might be “cross-presented.” explained by failure to localize into exosomes because NA is also Further complicating the terminology is the existence of an alter- incorporated into lipid rafts. Although NA levels are ∼5-fold less native TAP-independent MHC I cross-presentation pathway that than those of HA during a viral infection (61, 62), expression level involves delivery of Ag to MHC I-containing phagolysosomes (11, does not appear to be the reason. NA is readily detected on the 70, 71). In outline, this scheme is very similar to the Ag transfer surface of exosomes from infected cells (data not shown), and mechanism that we have described for S1. titration of exosomes in S1 stimulation assays indicate that the MHC I cross-presentation facilitates immune responses in at amount of NA should not be limiting. Furthermore, marginal Ag least two ways. First, it effectively separates Ag from pathogen- transfer was also observed from NA- but not HA-transfected cells, directed mechanisms that specifically interfere with processing despite comparable levels of expression (data not shown). We and presentation within the infected cell. Although most examples The Journal of Immunology 6615 of interference are in conjunction with MHC I (12, 72), several 3. Sinnathamby, G., M. Maric, P. Cresswell, and L. C. Eisenlohr. 2004. 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