J Immunol-2008-Tagliani-3201-9

1. of August 4, 2016. This information is current as Dendritic Cells+αCD36 on Steady-State CD8 Pathway to Induce Immunity by Targeting Selection of an Antibody Library Identifies a Federica Benvenuti and Oscar R. Burrone López-Bravo, Carlos Ardavín, Sebastian Amigorena, Elisa Tagliani, Pierre Guermonprez, Jorge Sepúlveda, María http://www.jimmunol.org/content/180/5/3201 doi: 10.4049/jimmunol.180.5.3201 2008; 180:3201-3209; ;J Immunol References http://www.jimmunol.org/content/180/5/3201.full#ref-list-1 , 25 of which you can access for free at:cites 36 articlesThis article Subscriptions http://jimmunol.org/subscriptions is online at:The Journal of ImmunologyInformation about subscribing to Permissions http://www.aai.org/ji/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/cgi/alerts/etoc Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved. Copyright © 2008 by The American Association of 9650 Rockville Pike, Bethesda, MD 20814-3994. The American Association of Immunologists, Inc., is published twice each month byThe Journal of Immunology atICGEBonAugust4,2016http://www.jimmunol.org/DownloadedfromatICGEBonAugust4,2016http://www.jimmunol.org/Downloadedfrom

2. Selection of an Antibody Library Identifies a Pathway to Induce Immunity by Targeting CD36 on Steady-State CD8␣؉ Dendritic Cells1 Elisa Tagliani,* Pierre Guermonprez,† Jorge Sepu´lveda,* Marıá Lo´pez-Bravo,‡ Carlos Ardavıń,‡ Sebastian Amigorena,† Federica Benvenuti,2,3 * and Oscar R. Burrone2,3 * Improvement of the strategy to target tumor Ags to dendritic cells (DCs) for immunotherapy requires the identification of the most appropriate ligand/receptor pairing. We screened a library of Ab fragments on mouse DCs to isolate new potential Abs capable of inducing protective immune responses. The screening identified a high-affinity Ab against CD36, a multi-ligand scavenger receptor primarily expressed by the CD8␣؉ subset of conventional DCs. The Ab variable regions were genetically linked to the model Ag OVA and tested in Ag presentation assays in vitro and in vivo. Anti-CD36-OVA was capable of delivering exogenous Ags to the MHC class I and MHC class II processing pathways. In vivo, immunization with anti-CD36-OVA induced robust activation of naive CD4؉ and CD8؉ Ag-specific T lymphocytes and the differentiation of primed CD8؉ T cells into long-term effector CTLs. Vaccination with anti-CD36-OVA elicited humoral and cell-mediated protection from the growth of an Ag-specific tumor. Notably, the relative efficacy of targeting CD11c/CD8␣؉ via CD36 or DEC205 was qualitatively different. Anti-DEC205- OVA was more efficient than anti-CD36-OVA in inducing early events of naive CD8؉ T cell activation. In contrast, long-term persistence of effector CTLs was stronger following immunization with anti-CD36-OVA and did not require the addition of exogenous maturation stimuli. The results identify CD36 as a novel potential target for immunotherapy and indicate that the outcome of the immune responses vary by targeting different receptors on CD8␣؉ DCs. The Journal of Immunology, 2008, 180: 3201–3209. H arnessing the immunostimulatory properties of dendritic cells (DCs)4 to induce tumor rejection is a major goal in immunotherapy. The outcome of the immune response induced by targeting Ags to DCs depends on multiple parameters. DCs are phenotypically heterogeneous, with different populations playing distinct roles in immunity. The first level of specialization is given by the differential expression of membrane receptors. Fc receptors, scavenger receptors, and C-type lectin receptors confer the ability to recognize and internalize different forms of Ags. In addition, the expression of different sets of receptors for pathogen- associated motifs tunes the type of immune response (1). Down- stream of Ag capture, a second level of specialization is given by the intrinsic capacity to handle Ags for presentation of peptide on their MHC class I and class II molecules. Internalized Ags are presented by default onto MHC class II molecules by all APCs. DCs, in addition, have the capacity to perform cross-presentation, a process that permits the presentation of peptide-MHC class I complexes from exogenous Ags. This property is essential for the priming of antiviral and antitumor immunity. Data accumulated in the last few years have clearly established that cross-presentation of soluble and cell-associated Ags in vivo is mainly accomplished by a subset of conventional DCs that express the CD8␣ marker (2, 3). Indeed, targeting Ags to the CD8␣ϩ subset using Ab against the C-type lectin receptor DEC205 (4–7) elicit potent Ag-specific cytotoxic responses in vivo. In contrast, targeting lectins expressed on the CD8␣Ϫ subset of conventional DCs induce Ab responses but poor cellular-mediated responses (8, 9). Whether the relative ability to display peptides on class I or II products is mainly cell type or receptor regulated, however, requires further studies (10). Several other molecules like the scavenger receptor Lox-1 (11), the mannose receptor (12), some chemokine receptors (13, 14), and TLRs (15, 16) have been tested for their potential in immunotherapy. A critical lesson from all of these studies is that some receptors (4, 5, 11, 12), but not others (16, 17), require the addition of a DC maturation signal to induce protective immunity. Collec- tively, it is difficult to predict which strategy among the proposed ones is the most efficient on a comparative basis. Thus, there is the necessity to identify new receptors, characterize their mode of ac- tion, and develop technologies that facilitate comparative studies. With the aim to improve current existing strategies, we set up a screening of phage-displayed libraries of Ab fragments on DCs. This technique is powerful in that it provides new Ab candidates and an easy procedure to test them functionally by genetically *International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; † U653 Institut National de la Sante´ et de la Recherche Me´dicale Institut Curie, Paris, France; and ‡ Department of Immunology and Oncology, Centro National de Biotec- nologia/Consejo Superior de Investigaciones Cientificas, Universidad Autońoma de Madrid, Madrid, Spain Received for publication August 28, 2007. Accepted for publication January 3, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by “Associazione Italiana per la Ricerca sul Cancro.” E.T. was supported by an International Centre for Genetic Engineering and Biotechnology Predoctoral Fellowship from the Corso di Perfezionamento of the Scuola Normale Superiore di Pisa. F.B. was supported by an International Centre for Genetic Engi- neering and Biotechnology Postdoctoral Fellowship. 2 These authors contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Federica Benvenuti and Dr. Oscar R. Burrone, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy. E-mail addresses: benvenut@icgeb.org and burrone@icgeb.org 4 Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived DC; LN, lymph node; scFv, single chain fragments; SIP, small immunoprotein; RT, room temperature; PFA, paraformaldehyde; ctrl, control. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org atICGEBonAugust4,2016http://www.jimmunol.org/Downloadedfrom

3. linking the isolated specificities to the Ag of interest. In this study, we provide the proof of principle of such methodology. We focus on the characterization of a selected high-affinity Ab directed against the class B scavenger receptor CD36, expressed exclusively on the CD8␣ϩ subset of blood-derived conventional DCs. Our results show that a fusion protein that contains the variable regions of the Ab against CD36 linked to the model Ag OVA is internalized and delivered to a processing pathway able to generate both MHC class II and class I peptide complexes and to induce a long-term protective response in vivo in the absence of any added DC maturation stimuli. Materials and Methods Mice Female C57BL/6 (B6) mice (6–7 wk old) were purchased from Harlan Breeders. OVA-specific, TCR-transgenic OT-I and OT-II mice were purchased from The Jackson Laboratory. CD45.1-congenic C57BL/6 (a gift from P. Guermonprez, Institut Curie Paris, Paris, France) were bred to OT-I mice to obtain OT-I/CD45.1. Animal care and treatment were conducted in conformity with institutional guidelines in compliance with national and international laws and policies (European Economic Community Council Di- rective 86/609; OJL 358; December 12, 1987) and housed at the International Centre for Genetic Engineering and Biotechnology animal house. Abs and reagents All mAb, if not specified otherwise, were purchased from BD Biosciences. Anti-mouse CD40 (clone HM40-3) was obtained from eBioscience. Rabbit polyclonal anti-CD36 Ab was purchased from Abcam. All reagents, if not specified otherwise, were obtained from Sigma-Aldrich. Cells DCs were isolated from single-cell suspensions of lymph nodes or spleen treated with 400 U/ml collagenase D (Roche) for 30 min followed by enrichment (depletion of B, T, NK) or positive selection by CD11cϩ beads (Miltenyi Biotec). OT-II and OT-I cells were isolated from total lymph node (LN) suspensions by negative selection using a MACS isolation kit (Miltenyi Biotec). Langerhans cells and dermal DCs were isolated from the epidermis as described previously (18). Bone marrow-derived DCs (BMDCs) were generated using Fms-like tyrosine kinase 3 ligand as described previously (18). DCs were used at day 10 and purity of CD11cϩ was higher than 90%. Phage display screening For the screening, we used a highly diverse library (Ͼ1010 independent clones) of single-chain Ab fragments (scFv) derived from samples of human PBLs (19). Phages were prepared following standard protocols. Phage selection was performed on DCs isolated from spleen of C57BL/6 mice. Briefly, 1013 CFU of phage were blocked in PBS/5% milk and allowed to bind to 6 ϫ 106 DCs for 2 h at 4°C. After PBS washings, cells were resuspended in IMDM and incubated at 37°C for 30 min. Extracellular phages were inactivated with subtilisin (3 mg/ml) in buffer B (HBSS), 20 mM Tris, and 2 mM EDTA, pH 8) for 30 min. Cells were washed and lysed in 1 ml of triethylamine (100 mM) for 8 min at room temperature (RT). The lysate was neutralized with 0.5 ml of 1 M Tris-HCl (pH 7.4). Internalized phages were recovered by infecting Escherichia coli (TG1) cells. Samples were processed for quantitation of phage titer (19). FACS and immunofluorescence analysis using phage Phages (1012 CFU) were blocked in PBS/5% milk and allowed to bind to BMDCs for 1 h at 4°C. For FACS analysis, phage binding was detected by anti-M13 IgG mAb (Amersham Pharmacia) and FITC-conjugated anti- mouse IgG (Kirkegaard &Perry Laboratories). For confocal analysis, cells were plated on poly-L-lysine-treated coverslips and incubated either at 4 or 37°C for 30 min. After PBS washings, cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with PBS/0.1% Triton X-100. The coverslips were saturated with PBS/1% BSA (30 min at RT). Phage particles were detected with rabbit anti-fD Bacteriophage Ab (Sigma-Aldrich) and FITC-conjugated swine anti-rabbit IgG (DakoCytomation). Samples were analyzed by confocal microscopy (Axiovert; Zeiss). Immunoprecipitation/Western blotting In brief, 107 BMDCs or BO9 T cells were surface biotinylated (EZ-Link Sulfo-NHS-biotinylation kit) and lysed. Cell debris were spun out (12,000 rpm, 15 min) and supernatants were precleared with 10 ␮g of anti-SV5tag mAb (20) and 200 ␮l of 50% protein A-Sepharose CL-4B beads (Amer- sham Biosciences) for 2 h at 4°C. Biotinylated proteins were immunoprecipitated with scFv-2E5 or scFv-control (ctrl; 20 ␮g/ml) for 2 h at 4°C, followed by the addition of 5 ␮g of anti-SV5tag mAb and 50 ␮l of 50% protein A-Sepharose CL-4B. Beads were washed three times in ice-cold lysis buffer before SDS-PAGE analysis. Upon transfer on polyvinylidene difluoride membranes (Millipore), the presence of biotinylated protein was revealed by HRP-conjugated streptavidin (Amersham Biosciences). In-gel digestion and mass spectrometry In-gel digestion was performed as described elsewhere (21). Briefly, protein gel pieces were excised and tryptically digested with porcine trypsin. After incubation overnight, gel pieces were centrifuged at 14,000 rpm for 5 min, then extracted once with 5% formic acid/50% acetonitrile and dried by vacuum centrifugation. Mass spectrometry data were acquired working in reflectron mode with a 4800 MALDI TOF/TOF. A 0.5-␮l aliquot of the peptide solution was mixed with 0.5 ␮l of a-cyano-4-hydroxycinnamic acid matrix and subjected to MALDI analysis. Mass spectrometry data were subjected to database searching using Mascot (Matrix Science) against SwissProt database. Up to one missed tryptic cleavage and optional me- thionine oxidation and carbamidomethylation was considered. Mass accu- racy was limited to 80 ppm. Production of scFv-OVA proteins The scFv isolated from the library and an irrelevant scFv (scFv-ctrl) were engineered into a small immunoprotein (SIP) format by cloning them between the secretory signal sequence from a mouse Ig H chain and the third constant domain of human IgG1 (␥1-CH3) (22). The sequence encoding for full-length OVA was cloned in-frame downstream of the ␥1-CH3 domain, followed by a His6 tag. For comparison, the DNA fragment encoding for V regions of anti-DEC-205/CD205 rat mAb (clone NLDC-145) were amplified and cloned into a similar SIP-OVA format. SIP-OVA-H6 proteins were transiently expressed in 293T cells after calcium-phosphate transfection. Proteins were purified by affinity chromatography using a poly-H6 tag purification system (Ni-NTA His Bind Resin; Novagen) and quantified by Coomassie staining and Western blot using a rabbit anti- OVA Ab (Abcam). The recombinant scFv-OVA proteins were tested for the presence of endotoxin by the chromogenic Limulus amebocyte lysate assay (Charles River ENDOSAFE). The levels of endotoxin were below 0.05 endotoxin units/␮g in all of the scFv-OVA proteins used in vivo or in culture. ScFvCD36 -OVA internalization assay DCs were incubated at 4°C for 1 h with scFvCD36 -OVA or scFvctrl -OVA, washed and then either kept at 4°C or incubated at 37°C for 15, 30, or 60 min. For FACS analysis, the level of scFvCD36 -OVA remaining at the cell surface was detected using rabbit anti-OVA and FITC-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories). The median fluorescent values of OVAϩ cells was determined and used to calculate the percent remaining scFvCD36 -OVA at the surface, with 1 h at 4°C incubated cells taken as 100%. For confocal analysis, after binding at 4°C for 1 h, cells were washed, plated on poly-L-lysine-treated coverslips, and incubated either at 4 or 37°C for 30 min. Membranes were counterstained with 5 ␮g/ml cholera toxin subunit B-FITC, washed, fixed in 4% PFA, and permeabilized with 0.02% saponin. ScFvCD36-OVA was detected with Alexa Fluor 594-F(abЈ)2 goat anti-human IgG (Molecular Probes). In vitro T cell proliferation assay DCs were pulsed with graded doses of scFv-OVA proteins or soluble OVA (Worthington Biochemical) for 3 h at 37°C. After washing, 1 ϫ 105 OT-I or OT-II cells were cocultured with Ag-pulsed DCs in round-bottom 96- well plates (1 DC:5 T cell ratio). After 48 h, [3 H]thymidine (1␮Ci; Am- ersham Biosciences) was added for 18 h, and incorporation was measured by liquid scintillation counting after collection of cells on a glass fiber filter with and automatic cell harvester (Tomtec). Adoptive transfer and T cell proliferation responses OT-I or OT-II cells were labeled with 7 ␮M CFSE according to the man- ufacturer’s instructions. C57BL/6 mice were i.v. injected with 1–2 ϫ 106 OT-I or OT-II cells followed by injection in the footpad of scFv-OVA proteins. Three days later, lymph node cell suspensions were stained for CD8 or CD4, respectively, and the CFSE dilution was evaluated by FACS. To evaluate long-term OT-I persistence, 1.5 ϫ 106 OT-I/CD45.1 were injected i.v. into recipient CD45.2 hosts immunized with scFv-OVA proteins. Twelve days later, spleen cell suspensions was stained for surface 3202 TARGETING CD36 INDUCES CROSS-PRESENTATION BY DCs atICGEBonAugust4,2016http://www.jimmunol.org/Downloadedfrom

4. CD45.1 and CD8. Intracellular IFN-␥ production was evaluated using splenocytes or blood cells cultured for 5 h with 1 ␮M OVA257–264 peptide in the presence of brefeldin A (BD Biosciences). Cells were stained with anti-mouse CD8 and CD3 Abs for 25 min at 4°C. After fixation with 2% PFA, cells were stained for intracellular IFN-␥ in PermWash solution (BD Biosciences) for 30 min at 4°C. In vivo cytotoxicity assay Naive syngenic splenocytes were pulsed with OVA257–264 peptide (5 ␮M) for 1 h at 37°C, washed, and labeled with 5 ␮M CFSE. Nonpulsed control splenocytes were labeled with low concentration of CFSE (0.5 ␮M). CFSEhigh and CFSElow cells were mixed at 1:1 ratio (7 ϫ 106 cells each) and injected i.v. into mice. After 15 h, the numbers of CFSEϩ cells in spleen and lymph nodes were determined by FACS. Anti-OVA Ab ELISA Ninety-six-well Maxisorp ELISA plates (Nunc) were coated overnight with 3 ␮g/ml OVA. Plates were washed and blocked in PBS/1% BSA/ 0.1% Tween 20. Sera collected from the clotted blood of immunized mice were serially diluted in blocking solution and incubated for 1 h at RT. Plates were washed and Ab binding was detected using HRP-conjugated anti-mouse IgG Ab (Kirkegaard & Perry Laboratories) followed by tetra- methylbenzidine peroxidase substrate (Sigma-Aldrich). Tumor rejection experiments Mice were injected s.c. on days 0 and 7 with 0.5 ␮g of scFvCD36 -OVA or scFvCtrl -OVA. Seven or 30 days after the second immunization, each mouse was challenged s.c. with 2 ϫ 105 EG7-OVA cells (ATCC CRL- 2113). Tumor size was measured with a caliper ruler at different time points after the challenge. Average size is expressed in cubic centimeters using the formula V ϭ (length ϫ width2 )/2. Significance of protection was evaluated using a two-tailed Student t test. Results Selection of internalizing phages by panning on DCs We used a phage display library of single-chain Ab fragments (scFv) to isolate Abs specific for surface molecules of mouse DCs. We set up a whole cell panning procedure to specifically select Abs able to trigger the internalization of the bound DC receptors. Multiple rounds of positive selection using a highly diverse scFv- phage library (Ͼ1010 independent clones) (19) were performed on CD11cϩ cells purified from the spleen of C57BL/6 mice. To selectively recover and propagate only the internalized phages, we used subtilisin, a protease that inactivates extracellular phages by cleavage of the phage protein pIII, thus rendering the phage particles noninfectious (23) (Fig. 1A). To evaluate enrichment for specific binders, we analyzed by flow cytometry the pool of clones recovered after each round of selection. The polyclonal scFv- phage population from the third round of panning, compared with the pools of the first and second round, showed significantly increased binding on DCs (Fig. 1B) but not on an irrelevant cell type (A20) used as control (data not shown). We determined by immunofluorescence that the polyclonal scFv-phage population from the third round of panning was internalized by DCs upon incubation at 37°C, suggesting that most of the isolated ligands target endocytic receptors (Fig. 1C). Randomly picked individual phage clones from the third round of panning were analyzed by DNA fingerprinting and DNA sequencing. Sixty percent of the 40 clones analyzed showed the same fingerprinting pattern (data not shown). DNA sequencing confirmed the presence of a dominant clone, des- ignated scFv-2E5, which was further analyzed. Characterization of the selected scFv The spleen-derived DC populations used for the phage selection procedure comprises three different subtypes: CD4Ϫ CD8ϩ , CD4ϩ CD8Ϫ , and double-negative (CD4Ϫ CD8Ϫ ) DCs. Therefore, we asked whether the Ag recognized by scFv-2E5 was expressed on a specific DC subset. We evaluated by flow cytometry the binding of scFv-2E5 on splenic DCs. When we analyzed CD11chigh DCs, we found a tight correlation between CD8␣ expression and binding of scFv-2E5, resulting in two predominant populations of conventional DCs: CD8␣ϩ scFv-2E5ϩ and CD8␣Ϫ scFv-2E5Ϫ . The same type of correlation was evident on DCs isolated from LNs (data not shown). Further analysis on plasmacytoid and tis- sue-derived DCs (Langerhans cells and dermal DCs) did not show any specific staining (Fig. 2). We therefore concluded that the Ag recognized by scFv-2E5 is uniquely expressed by the CD8␣ϩ subset of conventional DCs, the main subtype involved in Ag cross-priming. When binding specificity was investigated for other hemopoietic cell types present in the spleen, only macrophages and a percentage (30%) of B cells were positive, whereas both monocytes and T lymphocytes were negative (Fig. 2). These data indicate that scFv-2E5 recognizes a cell surface marker specifically expressed by APCs. To characterize the molecule targeted by scFv-2E5, we conducted immunoprecipitation experiments using extracts of surface biotinylated BMDCs or T cells (BO9 T lymphocytes) as a negative control. These experiments identified a DC-specific protein with an apparent molecular mass of 90 kDa (Fig. 3A). By MALDI mass spectrometry, we identified the immunoprecipitated band as the mouse class B scavenger receptor CD36. The band immunoprecipitated by scFv-2E5 reacted in Western blot with a commercially available anti-CD36 Ab (Fig. 3B). Moreover, the full-length cDNA sequence of CD36 was amplified and transfected in 293T cells. FIGURE 1. Selection of internalizing phages by panning on DCs. A, Schematic representation of the panning procedure. Phages were allowed to bind to DCs and to be internalized by incubation at 37°C. Noninternal- ized phages were inactivated by subtilisin treatment. DCs were lysed to recover internalized phages by infection of E. coli cells. Rescued phage- mids were used for the next round of selection. B, Flow cytometry analysis on DCs to monitor the enrichment of binders in the polyclonal phage prep- arations from each round of selection. C, Internalization of phage particles by DCs incubated with the original library or the pool of phages from the third round of panning (Sel III) using the anti-pIII Ab and a FITC-conjugated secondary Ab. 3203The Journal of Immunology atICGEBonAugust4,2016http://www.jimmunol.org/Downloadedfrom

5. Flow cytometry analysis showed that scFv-2E5 specifically stained CD36-transfected but not mock-transfected 293T cells (Fig. 3C). These results demonstrate that scFv-2E5 recognizes an epitope in the extracellular portion of CD36. Targeting Ags to CD36 induces presentation of MHC class I and MHC class II epitopes in vitro CD36 belongs to the class B scavenger receptors family. It is a multi-ligand receptor involved in cellular adhesion and lipid metabolism. In vitro studies with human DCs have suggested a possible role of CD36 in cross-presentation of apoptotic material (24). However, this molecule is not essential for either cross-presentation or cross-tolerance of cell-associated Ags in mice (25, 26). Nevertheless, a role for CD36 in other pathways of cross-presentation cannot be excluded. To investigate whether CD36 could be used as a targeting molecule for the induction of T cell immune response by DCs, the scFv-2E5 was engineered into the SIP format, a molecule that contains, downstream of the scFv, the third constant domain of human IgG1 (␥1-CH3), thus producing a dimeric scFv-2E5 (22). The C terminus of the SIP-2E5 was fused in-frame to the full- length sequence of OVA, followed by a His6 tag, to generate the dimeric recombinant protein that we named scFvCD36 -OVA (Fig. 4A). As a control, we constructed a similar recombinant containing an irrelevant scFv (scFvctrl -OVA). Both proteins were produced by transient transfection in 293T cells (ϳ1–2 mg/L) and purified from culture supernatants by anti-His6 tag affinity chromatography. Analysis by Western blot of the purified proteins revealed, for each of them, a single band of the expected molecular mass (90 kDa; Fig. 4B). To study the endocytosis of scFvCD36 -OVA, we performed a flow cytometry-based internalization assay. scFvCD36 -OVA was rapidly internalized with ϳ70% of the protein removed from the cell surface within 30 min at 37°C (Fig. 4C). These data were confirmed by immunofluorescence: scFvCD36 -OVA was efficiently internalized at 37°C while it remained associated to the plasma membrane upon incubation at 4°C (Fig. 4D). To investigate the capacity of scFvCD36 -OVA to deliver OVA- derived peptides to the MHC class I and MHC class II pathways of Ag presentation, we performed in vitro proliferation assays using OT-I and OT-II cells. CD11cϩ DCs isolated from the spleen of FIGURE 3. ScFv-2E5 recognizes an epitope in the extracellular portion of the scavenger receptor CD36. Western blot of extracts from cell surface- biotinylated DCs or BO9 T cells, immunoprecipitated with scFv-2E5 (2E5) or an irrelevant scFv (ctrl) and revealed with streptavidin-HRP (A) or with an anti-CD36 Ab (B) as indicated. C, Binding of scFv-2E5 or scFv-ctrl to 293T cells transiently transfected with the cDNA-encoding mouse CD36. FIGURE 4. Internalization of the recombinant anti-CD36-Ag fusion. A, Schematic representation of the scFv-OVA recombinant molecule. B, Western blot (anti-OVA) of recombinant Ag molecules: soluble OVA (lane 1), scFvCD36 - OVA (lane 2), and scFvctrl -OVA (lane 3). C, Time course of scFvCD36 -OVA internalization. After binding at 4°C, DCs were incubated either at 37 or at 4°C for the indicated time points. The plot shows the ratio of remaining surface-bound scFvCD36 -OVA at the two temperatures, measured with an anti-OVA Ab. Data show means of duplicate measurements. D, Confocal microscopy of scFvCD36 - OVA internalization by DCs. After binding at 4°C, cells were washed and incubated for 30 min at 4 or 37°C as indicated. Fixed and permeabilized DCs were stained with Alexa 594-conjugated anti-human Ab and FITC-conjugated cholera toxin subunit B to counterstain plasma membranes. Images from the central Z section of representative cells are shown. FIGURE 2. Binding profile of scFv-2E5 on splenic DCs and hemopoietic cells. DCs from the spleen of C57BL/6 mice were labeled with scFv- 2E5 and a panel of different markers gating on: CD11chigh and CD8␣ϩ/Ϫ for conventional DCs and CD11cint and B220ϩ for plasmacytoid DCs. Langerhans cells and dermal DCs were isolated from the epidermis and identified by gating on CD11cϩ cells. Monocytes were defined by gating on Ly-6Chigh , CD11bhigh , and CD11cϪ cells, macrophages as F4/80ϩ and Ly-6Cint , B lymphocytes by expression of B220 and T cell by expression of CD3. Filled line, scFv-2E5. 3204 TARGETING CD36 INDUCES CROSS-PRESENTATION BY DCs atICGEBonAugust4,2016http://www.jimmunol.org/Downloadedfrom

6. C57BL/6 mice were incubated with graded concentrations of the recombinant scFvCD36 -OVA or scFvctrl -OVA proteins or soluble OVA and then cocultured with OT-I and OT-II cells. T cell proliferation was evaluated 3 days later by [3 H]thymidine incorporation. scFvCD36 -OVA induced OT-I and OT-II proliferation at a concentration as low as 20 ng/ml, whereas no proliferation was detected even at the highest concentration (1600 ng/ml) of scFvctrl - OVA. Relative to soluble OVA, uptake via CD36 increased the efficiency of presentation by at least 400-fold for the MHC class I epitope (Fig. 5A) and by 300-fold for the MHC class II epitope (Fig. 5B). These results demonstrate that targeting CD36 on splenic DCs increases the efficiency of presentation of protein Ags on MHC class II and, more importantly, is able to deliver OVA to the MHC class I pathway of Ag cross-presentation. CD36 mediates CD4ϩ and CD8ϩ naive T cells activation in vivo A major drawback of the current DC-based clinical approaches derives from the necessity of their ex vivo manipulation. Few studies have addressed the possibility to use Abs to specifically target and activate DCs in vivo (4, 11, 12). We asked whether targeting OVA Ag to CD11cϩ CD8aϩ DCs via CD36 would be convenient to induce potent immune responses in vivo. To investigate the ability of scFvCD36 -OVA to induce proliferation of OVA-specific CD4ϩ T cells, mice were adoptively transferred with CFSE-labeled OT-II cells followed by immunization with graded doses of scFvCD36 -OVA or scFvctrl -OVA. T cell proliferation was evaluated in the draining LNs at day 3 upon injection. We detected OT-II proliferation only in LNs of mice immunized with scFvCD36 -OVA (200 ng), whereas priming with a higher amount of scFvctrl -OVA (500 ng) did not elicit OT-II activation (Fig. 6A). These data indicate that targeting of CD36 enhances the efficiency of MHC class II peptide Ag presentation in vivo. We next focused on the ability to trigger CTLs since this is particularly challenging and would be valuable for the design of effective vaccines. To evaluate the relative ability of DCs to cross- present OVA in vivo, we performed a comparative study with a well-characterized system of Ag delivery to DCs, the DEC-205 receptor (4, 5). The V regions of the anti-DEC-205 mAb (NLDC- 145) were amplified and engineered in the same format as scFvCD36 -OVA. The recombinant scFvDEC205 -OVA protein main- tained the same binding specificity as the original anti-DEC mAb (data not shown). Mice were adoptively transferred with CFSE- labeled OT-I cells and s.c. injected with increasing amounts of scFvCD36 -OVA, scFvDEC205 -OVA, or scFvctrl -OVA. Ag-specific T cell proliferation was determined in the draining LNs at day 3 upon transfer. Practically all of the OT-I cells in LNs of scFvCD36 - OVA-immunized mice entered cell cycle and underwent up to six divisions after a dose of just 100 ng of OVA protein, whereas immunization with the highest dose of scFvctrl -OVA (300 ng) did not induce naive OT-I cells to divide. Immunization with scFvDEC205 -OVA, however, was more efficient in inducing entry in the cycle since as little as 10 ng of protein induced extensive T cell proliferation (Fig. 6B). Altogether these data indicate that delivery of Ags through CD36 in vivo promotes uptake and processing for peptides presentation on MHC class II and class I molecules. To evaluate a possible contribution of other APCs to presentation of OVA upon immunization with scFvCD36 -OVA, we compared cross-presentation mediated by DCs and B cells that express high levels of CD36. DCs from spleen and LNs and CD19ϩ B cells were pulsed in vitro with different amounts of scFvCD36 -OVA, FIGURE 5. In vitro targeting of CD36 on splenic DCs. Proliferation ([3 H]thymidine uptake) of OT-I cells (A) or OT-II cells (B) cocultured for 72 h with spleen-derived DCs pulsed with different concentrations of scFvCD36 -OVA (F), scFvctrl -OVA (ࡗ), or OVA alone (Œ). FIGURE 6. CD36-targeted Ag accesses the exogenous and cross-presentation pathways in vivo. C57BL/6 mice were injected i.v. with 1 ϫ 106 CFSE-labeled OT-II (A) or OT-I (B) T cells and subsequently injected s.c. with graded doses of scFvCD36 -OVA (CD36), scFvctrl -OVA (ctrl), or scFvDEC205 -OVA (DEC205) as indicated. T cell proliferation was measured at day 3 in draining LNs. Histograms represent the CFSE dilution profile of OT-II cells (A) and OT-I cells (B) at day 3. 3205The Journal of Immunology atICGEBonAugust4,2016http://www.jimmunol.org/Downloadedfrom

7. scFvDEC205 -OVA, or scFvctrl -OVA and cocultured with CFSE-labeled OT-I cells. At day 3, we evaluated T cell proliferation as well as IL-2 production. Results show that only DCs are able to efficiently take up and present OVA to OT-I cells. B cells did not stimulate T cell proliferation above the background levels, even at Ag and cell doses 10-fold higher than those required to observe proliferation by DCs. scFvctrl -OVA did not cause T cell activation even at the highest Ag dose (1 ␮g), whereas scFvDEC205 -OVA was at least twice as efficient as CD36 in inducing T cell proliferation (Fig. 7). Only T cells primed by DCs secreted IL-2 (data not shown). These results indicate that targeting the CD36 receptor induces efficient cross-presentation exclusively in DCs. ScFvCD36 -OVA immunization induces differentiation of naive OT-I cells into effector CTLs in the absence of adjuvancy Procedures for targeting Ags to DCs in vivo should, in addition, induce the activation of DCs, since targeting Ags to immature DCs may lead to tolerance (4, 5). To evaluate the long-term effects of targeting Ags to the CD36 receptor on DCs, we followed the fate of the OT-I cells that pro- liferated upon immunization with scFvCD36 -OVA and scFvDEC205 - OVA, for comparison. Naive C57BL/6 mice were adoptively transferred with 1.5 ϫ 106 OT-I cells and s.c. injected with 300 ng of scFvCD36 -OVA, scFv scFvDEC205 -OVA, or scFvctrl -OVA either in the presence or absence of adjuvant (anti-CD40 mAb). At day 12 upon transfer, we evaluated the expansion of Ag-specific effector T cells in the spleen. In mice immunized with scFvCD36 - OVA without adjuvant, we found a 3- to 4-fold expansion of OT-I cells, relative to mice injected with scFvctrl -OVA, or PBS (Fig. 8A) and a high proportion of cells secreted IFN-␥ upon in vitro restimulation, indicating acquisition of effector functions (Fig. 8B). Coadministration of anti-CD40 mAb with scFvCD36 -OVA did not significantly increase T cell expansion nor the proportion of IFN- ␥-secreting cells. In contrast, immunization with scFvDEC205 - OVA, despite initial T cell proliferation, did not induce detectable expansion of Ag-specific effector cells unless codelivered with the anti-CD40 mAb. The few OT-I cells that persisted were unable to secrete IFN-␥, but were rescued by coadministration of adjuvant FIGURE 7. Cross-presentation by DCs and B cells. Proliferation of CFSE-labeled OT-I T cells, cocultured for 3 days with 5 ϫ 104 DCs (isolated from spleen or LNs) or B cells (5 ϫ 104 or 5 ϫ 105 ), preincubated with scFvCD36 -OVA (CD36), scFvctrl -OVA (ctrl), or scFvDEC205 -OVA (DEC-205). Plots represent the percentage of OT-I cells that underwent proliferation. FIGURE 8. CD36-targeting to steady-state DCs induces long-lasting CTLs. In brief, 1.5 ϫ 106 OT-I (CD45.1ϩ ) T cells were adoptively transferred into CD45.2ϩ C57BL/6 mice followed by immunization with 0.3 ␮g of scFvCD36 -OVA (CD36), scFvDEC205 -OVA (DEC-205), or scFvctrl -OVA (ctrl), with or without the addition of anti-CD40 Ab (25 ␮g). Spleens were harvested at day 12 by flow cytometry: A, the percentage of persistent CD45.1ϩ CD8ϩ cells (OT-I) and B, their ability to produce IFN-␥ following restimulation in vitro. C, In vivo cytotoxicity assay. Mice were treated as above. The ability of primed OT-I cells to kill an Ag-specific target cell was evaluated at day 12 by injecting mice with a mix of CFSE-labeled syngeneic splenocytes pulsed (CFSEhigh ) or not pulsed (CFSElow ) with the OVA257–264 class I peptide. Fifteen hours postinjection, the ratio between CFSEhigh and CFSElow (r) was evaluated as a measure of specific CTL activity. 3206 TARGETING CD36 INDUCES CROSS-PRESENTATION BY DCs atICGEBonAugust4,2016http://www.jimmunol.org/Downloadedfrom

8. (Fig. 8, A and B). Thus, according to a previous report (4), Ag targeting to CD8ϩ DCs via the DEC205 receptor requires a maturation stimulus to induce immunity. Instead, targeting the same DC subsets via CD36 induces T cell expansion per se. It should be noticed that T cell expansion was higher in mice primed with scFvCD36 -OVA than in mice immunized with scFvDEC205 -OVA plus adjuvant. To directly evaluate the effector functions of OT-I cells generated upon targeting Ags to CD36, we performed an in vivo cytotoxicity assay. Mice were adoptively transferred with 1.5 ϫ 106 OT-I cells and then s.c. injected with 200 ng of scFvCD36 -OVA or scFvctrl -OVA either with or without anti-CD40 Ab. At day 12 after immunization, we injected a mixture of OVA peptide-pulsed and nonpulsed syngeneic splenocytes to assess the cytolytic activity of in vivo-primed OT-I cells. Effective cytotoxicity was observed only in LNs and spleen of mice immunized with scFvCD36 -OVA regardless of the coadministration of anti-CD40. In mice primed with scFvCD36 -OVA, ϳ90% of the peptide-pulsed targets were eradicated from LNs, while a small proportion of target cells could still be detected in the spleen (Fig. 8C). Together, these data demonstrate that a single immunization with scFvCD36 -OVA is sufficient to induce a durable formation of effector T cells even in the absence of any additional maturation stimulus. Targeting a tumor Ag to CD36 elicits a protective immune response Having demonstrated the ability of scFvCD36 -OVA to induce priming and differentiation of adoptively transferred OT-I cells into effector T cells, we next sought to determine whether targeting OVA to CD36 could activate, as well, an endogenous Ag-specific T cell response. To this purpose, naive C57BL/6 mice were immunized twice in 14 days with 500 ng of scFvCD36 -OVA or scFvctrl -OVA. Seven days after the second immunization, lymphocytes isolated from blood were tested for the ability to produce IFN-␥ upon in vitro restimulation with OVA257–264 peptide. As shown in Fig. 9A, mice injected with scFvCD36 -OVA, but not with scFvctrl -OVA, devel- oped an Ag-specific CTL response as demonstrated by the percentage (4%) of IFN-␥-secreting CD8ϩ T cells. In parallel, we examined the humoral immune response by measuring the titers of OVA-specific IgG Abs by ELISA. High titers of anti-OVA- specific Abs were found in mice primed with scFvCD36 -OVA, but not with scFvctrl -OVA (Fig. 9B). We next determined whether the activation of the endogenous OVA-specific T cell response confers protection against the graft of EG7-OVA tumor cells. Mice were immunized at days 0 and 7 FIGURE 9. Activation of endogenous T cells by targeting CD36. A, Induction of OVA-specific CD8ϩ T cells. Mice were immunized twice at 1-wk intervals with scFvCD36 -OVA, scFvctrl -OVA (0.5 ␮g), or left untreated. Seven days later, lymphocytes isolated from blood were restimulated in vitro with the OVA257–264 peptide. Dot plots show one representative IFN- ␥ se- cretion profile in the different groups (left panel). Data expressed as means of IFN-␥-secreting CD8ϩ T cells (right panel). B, OVA-specific IgG Ab titers from sera of animals immunized as in A. Seven days after the second immunization, sera of mice (n ϭ 5) were collected and anti-OVA IgG Ab titer was determined by ELISA. Data are expressed as means of OVA-specific IgG Ab from serially diluted sera of mice immunized with scFvCD36 -OVA (F) or scFvctrl -OVA (f). FIGURE 10. CD36-targeted vaccine induces tumor immunity. C57BL/6 mice were immunized s.c. on days 0 and 7 with 0.5 ␮g of scFvCD36 -OVA (F), scFvctrl - OVA (f), or left untreated (). Seven days (A) or 30 days (B) after the second immunization, mice were challenged s.c. with 2 ϫ 105 EG7-OVA cells. Tumor growth was monitored once a week with a caliper. Av- erage tumor size of the six mice (A) or five mice (B) group was expressed in cubic centimeters using the formula V ϭ (length ϫ width2 )/2. Significance of protection was evaluated using a two-tailed Student t test. 3207The Journal of Immunology atICGEBonAugust4,2016http://www.jimmunol.org/Downloadedfrom

9. with 500 ng of scFvCD36-OVA or scFvctrl-OVA and challenged 1 wk after the last immunization, a time point that corresponds to the peak of effector T cell expansion. To assess vaccine memory, we performed a parallel experiment by challenging mice 1 mo after the last boost. Results in Fig. 10 show that mice immunized with scFvCD36-OVA exhibited a significant tumor growth inhi- bition compared to mice vaccinated with scFvctrl-OVA, both when the challenge was performed at early (7 days; pϭ0.0005) or later (30 days) time points ( pϭ0.008). Altogether these results demonstrate that targeting DCs via CD36 with low doses of Ag and without adjuvant is capable of inducing endogenous T cell responses that delay the growth of an Ag-specific tumor. Discussion The selection of an appropriate Ab is crucial to improve Ag targeting to DCs for vaccination purposes. In this study, we report the successful application of a procedure to select, from a filamentous phage display library, scFv Ab fragments internalized by DCs. We isolated a panel of scFvs able to bind to DC surface molecules and capable of triggering internalization of the bound receptor. We focused on one binder specific for the CD8␣ϩ subset of conventional DCs and whose target was characterized as the class B scavenger receptor CD36. To create an Ab-based vaccine, we rescued the sequence of the Ab V regions isolated from the screening and we fused them to an antigenic unit. Targeting Ag to DCs via CD36 greatly enhanced the efficiency of Ag presentation to T cells on both MHC class I and class II products. In vivo, immunization with the anti-CD36 recombinant vaccine induced long-term T cell expansion and tumor protection. These results unveil a new function for the CD36 receptor in adaptive immunity. CD36 has been implicated in multiple biolog- ical processes that define it as a multi-ligand scavenger receptor, mainly involved in cellular adhesion and lipid metabolism (27). Few reports have addressed the role of CD36 in APCs. At first, CD36 was shown to be involved in phagocytosis of apoptotic bodies (24). Subsequent studies indicated that CD36 function is re- dundant since CD36Ϫ/Ϫ APC were fully competent to carry out cross-presentation of Ags derived form apoptotic cells in vivo (25, 26). Most recent data suggest that CD36 is selectively implicated in the presentation of Ags derived from apoptotic bodies on MHC class II molecules (28). In this study, we show that CD36-mediated endocytosis of soluble Ags increases at least 300-fold the delivery of peptides to the classical MHC class II and the cross-presentation pathways (Fig. 5). This property is unique to DCs since B cells, that express the receptor and bind the recombinant Ab, were unable to cross-present the OVA epitope to T cells (Fig. 7). This new mechanism for Ag cross-presentation is particularly relevant to understand the biology of blood-derived CD8␣ϩ DCs. Among lymphoid organ resident DCs, those expressing the CD8␣ marker are clearly the most efficient at cross-presenting cellular (3), soluble (2, 10), and pathogen-associated Ags (29, 30). Current data suggest that the ability of CD8␣ϩ DCs to process exogenous Ags for presentation on MHC class I molecules may be regulated at two levels: 1) differential expression of receptors for endoph- agocytosis and 2) expression of a specialized pathway to promote the generation of MHC class I peptides (31). Ags captured via the C-type lectin DEC205 and the mannose receptors that are both CD8␣ϩ specific are cross-presented (5, 12). We provide evidence of another CD8␣ϩ -specific receptor that induces cross-presentation of endocytosed Ags. Thus, it is tempting to speculate that targeting any endocytic receptors on CD8␣ would lead to cross- presentation, although with different efficiencies. For vaccination purposes, the targeting molecule should be able to generate both classes of MHC peptide complexes at the cell surface and provide additional signals to achieve full activation of naive T lymphocytes. Indeed, a clear comparison of the long-term effects triggered by different receptors remains an open issue. The C-type lectin DEC205 has been the prototype DC target receptor to induce immune responses against various Ags (7, 32, 33). How- ever, targeting DEC205 leads to tolerance unless a second maturation signal such as CD40L is provided (5). We demonstrate that when targeting CD36, such an additional signal is dispensable for effector CD8ϩ differentiation. It is interesting to note that when comparing the proliferation of Ag-specific CD8ϩ T cells induced by targeting DEC205 or CD36 via identical recombinant molecules, the early response was stronger in the case of DEC205. The dichotomy arises only at day 12 when almost no expanded T cells persisted in animals immunized with anti-DEC205, whereas efficient expansion is obtained in the case of CD36. How can these divergent outcomes be explained? We can exclude that long-term accumulation of cytotoxic T cells by immunization through CD36 reflects higher levels of TCR engagement. Rather the T cell proliferation profile at day 3 indicates that targeting DEC205 gener- ates higher amounts of MHC class I peptide complexes at the cell surface. A second possibility is that CD36 but not DEC205 provides per se a DC maturation signal. Although we did not investigate the direct effect of the scFv anti-CD36 on the maturation state of DCs in vivo, several reports have shown that engagement of CD36 can modulate DC maturation and functions (28, 34, 35). It would be interesting to investigate whether engagement of CD36 in our model may activate proinflammatory responses by coengag- ing TLRs at the cell surface, as shown to occur during the uptake of Staphylococcus aureus (36). We tested the potential of immunization via CD36 in a tumor rejection model. Mice immunized with low doses (0.5 ␮g) of anti- CD36-OVA in the absence of adjuvancy mounted an endogenous T cell response that was sufficient to protect them from the growth of an Ag-specific tumor. Although further studies are needed to fully evaluate the efficiency of CD36 immunization against other tumor-associated Ags, these results indicate that targeting CD36 may be a valuable strategy to induce immunity without the need to coadminister immunostimulatory molecules. 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