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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 Sepúlveda^*, María López-Bravo, Carlos Ardavín, Sebastian Amigorena, Federica Benvenuti^2,3,* and Oscar R. Burrone^2,3,*

^* International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; U653 Institut National de la Santé et de la Recherche Médicale Institut Curie, Paris, France; and Department of Immunology and Oncology, Centro National de Biotecnologia/Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, Madrid, Spain

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Harnessing 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). Downstream 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⁺ subset using Ab against the C-type lectin receptor DEC205 (4, 5, 6, 7) elicit potent Ag-specific cytotoxic responses in vivo. In contrast, targeting lectins expressed on the CD8^– 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. Collectively, 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 action, 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 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⁺ 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

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 Directive 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 CD11c⁺ 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 CD11c⁺ was higher than 90%.

Phage display screening

For the screening, we used a highly diverse library (>10¹⁰ 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 x 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 µg of anti-SV5tag mAb (20) and 200 µl of 50% protein A-Sepharose CL-4B beads (Amersham Biosciences) for 2 h at 4°C. Biotinylated proteins were immunoprecipitated with scFv-2E5 or scFv-control (ctrl; 20 µg/ml) for 2 h at 4°C, followed by the addition of 5 µg of anti-SV5tag mAb and 50 µl 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-µl aliquot of the peptide solution was mixed with 0.5 µl 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 methionine oxidation and carbamidomethylation was considered. Mass accuracy 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/µg in all of the scFv-OVA proteins used in vivo or in culture.

ScFv^CD36-OVA internalization assay

DCs were incubated at 4°C for 1 h with scFv^CD36-OVA or scFv^ctrl-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 scFv^CD36-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 OVA⁺ cells was determined and used to calculate the percent remaining scFv^CD36-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 µg/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(ab')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 x 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, [³H]thymidine (1µCi; Amersham 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 µM CFSE according to the manufacturer’s instructions. C57BL/6 mice were i.v. injected with 1–2 x 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 x 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 CD45.1 and CD8. Intracellular IFN- production was evaluated using splenocytes or blood cells cultured for 5 h with 1 µM OVA_257–264 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 OVA_257–264 peptide (5 µM) for 1 h at 37°C, washed, and labeled with 5 µM CFSE. Nonpulsed control splenocytes were labeled with low concentration of CFSE (0.5 µM). CFSE^high and CFSE^low cells were mixed at 1:1 ratio (7 x 10⁶ cells each) and injected i.v. into mice. After 15 h, the numbers of CFSE⁺ 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 µg/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 tetramethylbenzidine peroxidase substrate (Sigma-Aldrich).

Tumor rejection experiments

Mice were injected s.c. on days 0 and 7 with 0.5 µg of scFv^CD36-OVA or scFv^Ctrl-OVA. Seven or 30 days after the second immunization, each mouse was challenged s.c. with 2 x 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 = (length x width²)/2. Significance of protection was evaluated using a two-tailed Student t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 (>10¹⁰ independent clones) (19) were performed on CD11c⁺ 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, designated scFv-2E5, which was further analyzed.

View larger version (31K): [in this window] [in a new window]   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. Noninternalized phages were inactivated by subtilisin treatment. DCs were lysed to recover internalized phages by infection of E. coli cells. Rescued phagemids were used for the next round of selection. B, Flow cytometry analysis on DCs to monitor the enrichment of binders in the polyclonal phage preparations 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.

The spleen-derived DC populations used for the phage selection procedure comprises three different subtypes: CD4^–CD8⁺, CD4⁺CD8^–, and double-negative (CD4^–CD8^–) 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 CD11c^high DCs, we found a tight correlation between CD8 expression and binding of scFv-2E5, resulting in two predominant populations of conventional DCs: CD8⁺scFv-2E5⁺ and CD8^–scFv-2E5^–. The same type of correlation was evident on DCs isolated from LNs (data not shown). Further analysis on plasmacytoid and tissue-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⁺ subset of conventional DCs, the main subtype involved in Ag cross-priming.

View larger version (33K): [in this window] [in a new window]   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+/– for conventional DCs and CD11cint and B220+ for plasmacytoid DCs. Langerhans cells and dermal DCs were isolated from the epidermis and identified by gating on CD11c+ cells. Monocytes were defined by gating on Ly-6Chigh, CD11bhigh, and CD11c– cells, macrophages as F4/80+ and Ly-6Cint, B lymphocytes by expression of B220 and T cell by expression of CD3. Filled line, scFv-2E5.

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. 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.

View larger version (61K): [in this window] [in a new window]   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.

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 scFv^CD36-OVA (Fig. 4A). As a control, we constructed a similar recombinant containing an irrelevant scFv (scFv^ctrl-OVA). Both proteins were produced by transient transfection in 293T cells (1–2 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).

View larger version (28K): [in this window] [in a new window]   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.

To investigate the capacity of scFv^CD36-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. CD11c⁺ DCs isolated from the spleen of C57BL/6 mice were incubated with graded concentrations of the recombinant scFv^CD36-OVA or scFv^ctrl-OVA proteins or soluble OVA and then cocultured with OT-I and OT-II cells. T cell proliferation was evaluated 3 days later by [³H]thymidine incorporation. scFv^CD36-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 scFv^ctrl-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.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. In vitro targeting of CD36 on splenic DCs. Proliferation ([3H]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 (•), scFvctrl-OVA (), or OVA alone ().

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 CD11c⁺CD8a⁺ DCs via CD36 would be convenient to induce potent immune responses in vivo.

To investigate the ability of scFv^CD36-OVA to induce proliferation of OVA-specific CD4⁺ T cells, mice were adoptively transferred with CFSE-labeled OT-II cells followed by immunization with graded doses of scFv^CD36-OVA or scFv^ctrl-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 scFv^CD36-OVA (200 ng), whereas priming with a higher amount of scFv^ctrl-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.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. CD36-targeted Ag accesses the exogenous and cross-presentation pathways in vivo. C57BL/6 mice were injected i.v. with 1 x 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.

To evaluate a possible contribution of other APCs to presentation of OVA upon immunization with scFv^CD36-OVA, we compared cross-presentation mediated by DCs and B cells that express high levels of CD36. DCs from spleen and LNs and CD19⁺ B cells were pulsed in vitro with different amounts of scFv^CD36-OVA, scFv^DEC205-OVA, or scFv^ctrl-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. scFv^ctrl-OVA did not cause T cell activation even at the highest Ag dose (1 µg), whereas scFv^DEC205-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.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. Cross-presentation by DCs and B cells. Proliferation of CFSE-labeled OT-I T cells, cocultured for 3 days with 5 x 104 DCs (isolated from spleen or LNs) or B cells (5 x 104 or 5 x 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.

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 proliferated upon immunization with scFv^CD36-OVA and scFv^DEC205-OVA, for comparison. Naive C57BL/6 mice were adoptively transferred with 1.5 x 10⁶ OT-I cells and s.c. injected with 300 ng of scFv^CD36-OVA, scFv scFv^DEC205-OVA, or scFv^ctrl-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 scFv^CD36-OVA without adjuvant, we found a 3- to 4-fold expansion of OT-I cells, relative to mice injected with scFv^ctrl-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 scFv^CD36-OVA did not significantly increase T cell expansion nor the proportion of IFN--secreting cells. In contrast, immunization with scFv^DEC205-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 (Fig. 8, A and B). Thus, according to a previous report (4), Ag targeting to CD8⁺ 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 scFv^CD36-OVA than in mice immunized with scFv^DEC205-OVA plus adjuvant.

View larger version (29K): [in this window] [in a new window]   FIGURE 8. CD36-targeting to steady-state DCs induces long-lasting CTLs. In brief, 1.5 x 106 OT-I (CD45.1+) T cells were adoptively transferred into CD45.2+ C57BL/6 mice followed by immunization with 0.3 µg of scFvCD36-OVA (CD36), scFvDEC205-OVA (DEC-205), or scFvctrl-OVA (ctrl), with or without the addition of anti-CD40 Ab (25 µg). Spleens were harvested at day 12 by flow cytometry: A, the percentage of persistent CD45.1+CD8+ 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.

Targeting a tumor Ag to CD36 elicits a protective immune response

Having demonstrated the ability of scFv^CD36-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 scFv^CD36-OVA or scFv^ctrl-OVA. Seven days after the second immunization, lymphocytes isolated from blood were tested for the ability to produce IFN- upon in vitro restimulation with OVA_257–264 peptide. As shown in Fig. 9A, mice injected with scFv^CD36-OVA, but not with scFv^ctrl-OVA, developed an Ag-specific CTL response as demonstrated by the percentage (4%) of IFN--secreting CD8⁺ 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 scFv^CD36-OVA, but not with scFv^ctrl-OVA (Fig. 9B).

View larger version (27K): [in this window] [in a new window]   FIGURE 9. Activation of endogenous T cells by targeting CD36. A, Induction of OVA-specific CD8+ T cells. Mice were immunized twice at 1-wk intervals with scFvCD36-OVA, scFvctrl-OVA (0.5 µg), 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- secretion profile in the different groups (left panel). Data expressed as means of IFN--secreting CD8+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 = 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 (•) or scFvctrl-OVA ().

View larger version (14K): [in this window] [in a new window]   FIGURE 10. CD36-targeted vaccine induces tumor immunity. C57BL/6 mice were immunized s.c. on days 0 and 7 with 0.5 µg of scFvCD36-OVA (•), scFvctrl-OVA (), or left untreated (). Seven days (A) or 30 days (B) after the second immunization, mice were challenged s.c. with 2 x 105 EG7-OVA cells. Tumor growth was monitored once a week with a caliper. Average tumor size of the six mice (A) or five mice (B) group was expressed in cubic centimeters using the formula V = (length x width2)/2. Significance of protection was evaluated using a two-tailed Student t test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

These results unveil a new function for the CD36 receptor in adaptive immunity. CD36 has been implicated in multiple biological 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 redundant since CD36^–/– 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⁺ 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⁺ DCs to process exogenous Ags for presentation on MHC class I molecules may be regulated at two levels: 1) differential expression of receptors for endophagocytosis 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⁺ specific are cross-presented (5, 12). We provide evidence of another CD8⁺-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). However, 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 CD8⁺ differentiation. It is interesting to note that when comparing the proliferation of Ag-specific CD8⁺ 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 generates 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 coengaging 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 µg) 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.

In summary, we have identified a novel approach to initiate CD8⁺ T cell-mediated immunity by targeting endogenous DCs in vivo and a new tool to specifically study the properties of the unique subset of conventional CD8⁺ DCs.

	Acknowledgments

 
We are grateful to A. Bradbury and D. Sblattero for the phage display library, F. Odreman and M. Hampel for MALDI-TOF analysis, and M. Sturnega for technical assistance.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
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 Engineering 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{at}icgeb.org and burrone{at}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.

Received for publication August 28, 2007. Accepted for publication January 3, 2008.

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