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The Journal of Immunology, 2007, 178, 7587-7597
Copyright © 2007 by The American Association of Immunologists, Inc.
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Uptake of Human Papillomavirus Virus-Like Particles by Dendritic Cells Is Mediated by Fc{gamma} Receptors and Contributes to Acquisition of T Cell Immunity1

Diane M. Da Silva*, Steven C. Fausch*, J. Sjef Verbeek{dagger} and W. Martin Kast2,*

* Department of Molecular Microbiology and Immunology and Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033; and {dagger} Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chimeric human papillomavirus virus-like particles (HPV cVLP) are immunogens able to elicit potent CTL responses in mice against HPV16-transformed tumors; however, the mechanism of T cell priming has remained elusive. HPV VLP bind to human MHC class II-positive APCs through interaction with Fc{gamma}RIII, and immature dendritic cells (DC) become activated after incubation with HPV VLP; however, it is unclear whether Fc{gamma}R on DC are involved. In mice, Fc{gamma}RII and Fc{gamma}RIII are homologous and bind similar ligands. In this study, we show that binding and uptake of VLP by DC from Fc{gamma}RII, Fc{gamma}RIII, and Fc{gamma}RII/III-deficient mice are reduced by up to 50% compared with wild-type mice. Additionally, maturation of murine DC from Fc{gamma}RII/III-deficient mice by VLP is also reduced, indicating that DC maturation, and thus Ag presentation, is diminished in the absence of expression of Fc{gamma}R. To investigate the in vivo contribution of Fc{gamma}R in the induction of cellular immunity, Fc{gamma}R single- and double-knockout mice were immunized with HPV16 L1/L2-E7 cVLP, and the frequency of E7-specific T cells was analyzed by tetramer binding, IFN-{gamma} ELISPOT, and cytotoxicity assays. All readouts indicated that the frequency of E7-specific CD4+ and CD8+ T cells induced in all Fc{gamma}R-deficient mice after immunization with cVLP was significantly diminished. Based on these results, we propose that the low-affinity Fc{gamma}R contribute to the high immunogenicity of HPV VLP during T cell priming by targeting VLP to DC and inducing a maturation state of the DC that facilitates Ag presentation to and activation of naive T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human papillomavirus (HPV)3 infection in humans is linked to both cervical dysplasia and cervical cancer, with high-risk HPVs being detected in virtually all cases of cancer of the cervix (1). A promising prophylactic vaccine candidate for prevention of HPV infection is papillomavirus virus-like particles (VLP). VLP are highly immunogenic, nonreplicative structures that mimic their virus counterparts in morphology and immunogenicity (2, 3, 4, 5). The L1 major capsid protein and the L2 minor capsid protein self-assemble into VLP that induce high titers of virus-neutralizing Abs and provide protective immunity in animals against viral infection and development of papillomas (5, 6, 7, 8). HPV VLP are immunogenic in humans (9, 10), and recent data show that they prevent high-risk HPV infection in vaccinated women (11, 12, 13). For individuals with existing HPV-induced lesions, therapeutic strategies must be used. The HPV E6 and E7 proteins are selectively expressed in cervical cancer cells (14), making them unique targets for specific T cell-mediated immunotherapy of HPV-associated disease.

HPV16, chimeric VLP (cVLP) comprising HPV16 L1 and an L2-E7 fusion protein (HPV16 L1/L2-E7 cVLP), have been shown to deliver the HPV16 E7 tumor Ag to the immune system, prime E7-specific CD8+ T cells, and protect mice against challenge with E7-transformed tumor cells in an experimental murine tumor challenge model (15). The mechanism(s) of how cVLP induce cell-mediated immunity (CMI), however, was unknown. A study of VLP binding to cells of the immune system showed that VLP bind to MHC class II+ APCs and that Fc{gamma}RIII (CD16) is part of a complex that mediates binding (16). Previously, we and others have shown that direct VLP binding to dendritic cells (DC) results in DC activation and up-regulation of costimulatory molecules, secretion of Th1 cell-stimulating cytokines, and priming of naive T cells against cVLP-derived peptides in vitro (17, 18, 19).

DC are professional APCs that play a key role in the induction of adaptive immune responses by capturing Ags and processing them for efficient presentation to naive T cells on MHC class I and II molecules, together with expression of costimulatory molecules such as CD80, CD86, and CD40 (20, 21, 22). Fc{gamma}R are expressed on most cells of the hemopoietic lineages, including DC, and link the humoral and cellular arms of the immune system through internalization of Ag-Ab immune complexes (IC). Recent observations suggest that uptake of IC through Fc{gamma}R leads to maturation of immature DC, cross-presentation of Ags, and induction of Ag-specific CD8+ responses in vivo, resulting in tumor immunity (23, 24, 25).

In light of these previous studies (16, 17, 18), we hypothesized that Fc{gamma}RIII-VLP interactions are involved in targeting VLP to DC and result in the induction of CMI against cVLP-derived Ags. Therefore, in this study, the contribution of Fc{gamma}R in direct uptake of VLP and activation of immature DC was investigated. Additionally, the in vivo importance of Fc{gamma}R expression on DC for the priming of CD8+ T cell and CD4+ T cell responses against cVLP-derived peptides was determined. Fc{gamma}R studies in the murine system are complicated by the fact that in mice, Fc{gamma}RIII is 95% homologous to Fc{gamma}RII in the extracellular domain and binds similar ligands (26). Therefore, because of the extremely high homology, Fc{gamma}RII as well as Fc{gamma}RIII were investigated as possible receptors for VLP on DC. Our results indicate that both Fc{gamma}RII and Fc{gamma}RIII are involved in the uptake of VLP by murine DC, and internalization of VLP through Fc{gamma}R leads to DC maturation, as indicated by an increase in MHC and costimulatory molecules. More importantly, in the absence of Fc{gamma}RII/III expression on APCs in vivo, both CD4+ and CD8+ cellular immune responses against E7 were diminished, indicating that Fc{gamma}R play an important role in the induction of both CD4+ and CD8+ T cell immunity against HPV cVLP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 (H-2b) female mice 5–6 wk of age were purchased from Taconic Farms. Fc{gamma}RIIb-deficient (B6.129S4; H-2b) female mice 4–6 wk of age were purchased from Taconic Farms. Fc{gamma}RIII-deficient mice (B6; H-2b) (27) and Fc{gamma}RII/III double-knockout mice (B6.129/OLA; H-2b) were obtained from Leiden University Medical Center. Fc{gamma}RII/III-deficient mice were obtained by crossing Fc{gamma}RII–/– mice (B6.129 background) with Fc{gamma}RIII–/– mice (B6 background). Homozygous Fc{gamma}RII/III double-knockout mice were backcrossed to C57BL/6 mice once, and heterozygotes were intercrossed to obtain a homozygote line. Fc{gamma}RIII–/– and Fc{gamma}RII/III–/– mice were maintained as a colony at our facility under specific pathogen-free conditions. Mice were 5–8 wk of age at the time of vaccination. All procedures were performed in accordance with institutional guidelines and approved by the institutional animal care and use committee.

Abs and reagents

The following Abs and reagents were purchased from BD Biosciences: anti-I-Ab FITC, anti-Db FITC, anti-mouse CD8a FITC, anti-mouse CD4 PE, anti-mouse CD11b PE, anti-mouse CD11c PE, anti-mouse CD19 FITC, anti-mouse CD40 FITC, anti-mouse CD80 FITC, anti-mouse CD86 FITC, anti-mouse CD16/CD32 (2.4G2) FITC, anti-mouse NK1.1 PE, anti-mouse Ly6 (GR-1) PE, goat anti-mouse IgG FITC, mouse IgG2a FITC, mouse IgG2b FITC, mouse IgG2b, rat IgG2b FITC, purified anti-mouse CD4 (GK1.5), purified anti-mouse CD8a (5H10-1), purified anti-mouse I-A/I-E (M5/114.15.2), purified anti-mouse CD45RA (14.8), purified anti-mouse Ter119, purified anti-mouse Ly6 (Gr-1), and streptavidin FITC. Peptides HPV16 E7(49–57) (28), HPV16 L1(165–173) (29), and HPV16 E7(30–67) containing Th epitopes (30, 31) were synthesized at the University of Chicago and purified by reverse-phase HPLC. Purity was assessed by analytical HPLC and was determined to be >99% pure. FITC-labeled dextran and FITC-labeled BSA were purchased from Sigma-Aldrich.

Virus-like particles

HPV16 L1/L2 VLP, HPV16 L1/L2-E7 cVLP, JC VP1 VLP, and BK VP1 VLP were produced in Trichoplusia ni (High Five) cells by infection with recombinant baculovirus. JC VP1 and BK VP1 recombinant baculoviruses were a gift from P. Jensen (National Institutes of Health, Bethesda, MD). VLP were purified from cells by sucrose and CsCl gradient centrifugation, quantified, and examined by electron microscopy, Western blot, and ELISA, as previously described (15, 32, 33). Endotoxin levels of VLP preparations were detected and semiquantitated against an endotoxin standard (Sigma-Aldrich) in a Limulus assay (E-toxate; Sigma-Aldrich) following manufacturer’s instructions. The level of endotoxin was determined to be <0.12 endotoxin U/mg VLP (equivalent to <24 pg/mg VLP), a concentration that does not activate DC in our experimental system (17, 34).

Generation of bone marrow-derived DC (BMDC)

Hind extremities of mice were collected, soft tissues were removed, and bones were rinsed in 70% ethanol. Ends of femurs and tibias were cut and bone marrow was flushed out with RPMI 1640 (BioWhittaker). DC precursors were enriched by negative selection of lineage marker-positive cells using Abs (anti-CD4, anti-CD8, anti-I-A/I-E, anti-CD45, anti-Gr-1, and anti-Ter119) and Lowtox rabbit complement (Cedarlane Laboratories). Hemopoietic precursors were isolated over a Lympholyte-M cushion (Cedarlane Laboratories); cells at the interface were collected and washed thoroughly. Remaining cells were plated in 24-well plates at 2 x 105 cells/well in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 50 µg/ml kanamycin, 50 µM 2-ME, 1 mM sodium pyruvate, and 2 mM nonessential amino acids (i.e., complete medium), and supplemented with murine rGM-CSF (500 U/ml) and murine rIL-4 (500 U/ml) (both from PeproTech). Cells were incubated at 37°C, 5% CO2 for 6–8 days. Every other day, cultures were fed by aspirating 50% of the medium and adding back fresh medium supplemented with GM-CSF and IL-4. At day 6–8, cells were harvested for RNA isolation or were used for binding, uptake, or activation studies. Approximately 2 x 106 cells were recovered from each well using this method. Cultures at day 6 contained 80–85% DC, as assessed by CD11c expression. Cultures at day 8 contained >90% CD11c+ DC. For DC activation, day 6 immature DC were activated directly in wells without replating as follows: 100% of medium was aspirated and replaced with complete medium containing 500 U/ml GM-CSF alone or in combination with 10 µg/ml HPV16 L1/L2 VLP or 5 µg/ml LPS (LPS from Escherichia coli 026:B6; Sigma-Aldrich).

RNA isolation and RT-PCR

RNA from immature DC or splenocytes was isolated using the Qiagen RNeasy Mini Kit for total RNA isolation, followed by treatment with RQ1 RNase-free DNase (Promega) for 1 h at 37°C. RT-PCR was performed on freshly isolated RNA using the Access RT-PCR System (Promega), according to manufacturer’s instructions. Primers for murine CD16 were as follows: forward, 5'-ATGTTTCAGAATGCACACTCTGG-3' and reverse, 5'-TCACTTGTCTTGAGGAGCCTGG-3'. Primers for murine CD32 were as follows: forward, 5'-ATGGGAATCCTGCCGTTCCTAC-3' and reverse, 5'-CTAAATGTGGTTCTGGTAATCA-3'. Primers for murine GAPDH were as follows: forward, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and reverse, 5'-CATGTAGGCCATGAGG TCCACCAC-3'.

Flow cytometry

Spleens were isolated from mice, and single-cell suspensions were made. RBC were lysed using red cell lysis buffer (Sigma-Aldrich). DC were collected from plates after activation and washed with FACS buffer (PBS/0.5% FBS/0.1% sodium azide). Single or double staining was performed on ice using directly labeled Abs against mouse surface Ags or the appropriate isotype control at a concentration of 1 µg/106 cells for 45 min. Cells were washed three times, and fluorescence was determined on a FACSCalibur using CellQuest software (BD Biosciences). Propidium iodide (Sigma-Aldrich) was used to gate on viable cells.

VLP binding and uptake

HPV VLP, BK VLP, and JC VLP were biotinylated using sulfo-NHS-SS-biotin (Pierce), following the manufacturer’s instructions, and were dialyzed against four changes of PBS/0.5 M NaCl overnight at 4°C. Binding experiments were performed by incubating DC (106) with 0.1 µg up to 4 µg of biotinylated HPV16 L1/L2 VLP, BK VLP, or JC VLP for 1 h on ice. After extensive washing, cells were incubated with streptavidin FITC for 30 min on ice. Cells were counterstained with CD11c PE. Fluorescence was analyzed by two-color flow cytometry. Propidium iodide (Sigma-Aldrich) was used to gate on viable cells. VLP uptake experiments were performed, as previously described (34, 35). Briefly, HPV16 L1/L2 VLP, BK VLP, or JC VLP was labeled with CFSE (Vybrant CFDA SE Cell Tracer Kit; Molecular Probes) for 4 h at room temperature. Excess CFSE was removed by dialysis against 4 L of PBS/0.5 M NaCl overnight at 4°C. One million DC were incubated with 1 µg/ml FITC-labeled dextran or 1 µg/ml FITC-labeled BSA for 90 min or 1 µg/ml CFSE-labeled VLP for 4 h at 37°C. For experiments in which 5 times more VLP was used for uptake, DC were incubated with 5 µg/ml CFSE-labeled VLP. At various time points, fractions were collected and fixed with 1% paraformaldehyde. Intracellular fluorescence resulting from VLP uptake was measured by flow cytometry. The percentage of VLP uptake by DC at each time point was calculated by the following: ((mean fluorescence intensity (MFI) experimental) – (MFI time zero experimental)/(MFI experimental maximum) – (MFI time zero experimental)) x 100.

IL-12 ELISA

Ninety-six-well Maxisorp ELISA plates were coated with anti-mouse IL-12 p70 capture Ab (Pierce) in 100 µl of PBS overnight at 4°C, following manufacturer’s guidelines. Plates were blocked with 3% BSA/PBS for 2 h at room temperature, then washed three times with PBST. One hundred microliters of 24-h culture supernatant from DC-VLP cultures was added in triplicate and incubated overnight at 4°C. Murine IL-12 standards were added ranging from 3000 to 1.5 pg/ml. Biotinylated anti-mouse IL-12 p70 detection Ab (Pierce) was added at 0.5 µg/ml and incubated at room temperature for 2 h. After washing three times, peroxidase-labeled streptavidin (Sigma-Aldrich) was added for 1 h at room temperature. Plates were washed and developed with o-phenylenediamine substrate, and OD values were read in an ELISA plate reader at 490 nm.

MLR and proliferation assay

Irradiated LPS-matured BMDC (104) from wild-type (wt) or FcR knockout mice were cultured with BALB/c CD4+ and CD8+ T cells (0.25–2 x 105) purified by magnetic positive selection (MACS system; Miltenyi Biotec) in triplicate in a 96-well U-bottom plate. Control T cells were incubated with medium alone or with 10 µg/ml Staphylococcus enterotoxin B (SEB; Sigma-Aldrich). Cells were incubated for 96 h (37°C/5% CO2); 100 µl of medium was removed and was replaced with 50 µl of medium containing 1 µCi of [3H]thymidine. After an additional 18-h incubation, cells were harvested onto 96-well filtermate plates and incorporated radioactivity was measured in a TopCount Microplate Scintillation and Luminescence Counter (Packard Instrument). For proliferation of T cells from wt or Fc{gamma}R knockout mice, 2 x 105 MACS-purified CD90+ (Thy-1.2) T cells were cultured with 8 x 105 irradiated BALB/c splenocytes for 96 h before addition of [3H]thymidine. For Ag-specific proliferation of T cells from cVLP-immunized wt or Fc{gamma}R knockout mice, untouched CD4+ T cells from vaccinated mice were purified by negative selection MACS sorting (Miltenyi Biotec) and were cultured in triplicate with medium alone, 5 µg/ml PHA (Sigma-Aldrich), wt C57BL/6 BMDC alone, or DC pulsed with 10 µg/ml HPV16 L1/L2-E7 cVLP. Cultures were incubated for 5 days before addition of [3H]thymidine.

Immunization with VLPs

Mice were immunized s.c. with 100 µg of chimeric HPV16 L1/L2-E7 cVLP in PBS. For control immunizations, mice were immunized with E7(49–57) peptide. Briefly, 50 µg of E7 peptide per mouse was dissolved in PBS and emulsified in IFA (Difco) at a 1:1 ratio and injected s.c. Ten days after immunization, mice were sacrificed and spleens were harvested.

ELISPOT assay

Spleens from vaccinated mice (n = 3 per experiment) were harvested and made into single-cell suspensions by filtering through a 70-µm mesh filter (BD Biosciences). Splenocytes were cultured at 5 x 106 cells/well in 24-well tissue culture dishes in IMDM (BioWhittaker) containing 2 IU/ml IL-2 and 2.5 µg/ml HPV16 E7(49–57) peptide or no peptide for 18 h. M200 ELISPOT plates (Cellular Technologies) were coated with 10 µg/ml anti-mouse IFN-{gamma} capture Ab (BD Biosciences) in PBS at 4°C overnight. Plates were washed with PBS/0.5% Tween 20 and blocked with culture medium containing 10% FBS. Splenocytes were harvested from wells and counted. The number of viable CD8+ T cells from each spleen sample recovered after in vitro culture was enumerated by flow cytometry by staining cells with FITC-labeled anti-mouse CD8 and counterstaining with propidium iodide. Splenocytes were plated in the precoated ELISPOT plates at 1 x 105 to 1.25 x 104 CD8+ cells/well in triplicate. After 24-h incubation at 37°C and 5% CO2, plates were washed with PBS-0.5% Tween 20 and incubated with 1 µg/ml biotinylated anti-mouse IFN-{gamma} Ab (BD Biosciences), followed by 1 µg/ml avidin-HRP (Sigma-Aldrich). Spots were developed with 3-amino-9-ethyl-carbazole substrate (Sigma-Aldrich) for 5 min. Individual spots were counted using an ImmunoSpot analyzer with ImmunoSpot software (Cellular Technologies). The number of background spots from medium-only wells was subtracted from spots obtained after stimulation with specific peptide. For studies in which CD4+ T cells or CD8+ T cells were depleted, splenocytes were harvested from HPV16 L1/L2-E7 cVLP-immunized mice. CD4+ or CD8+ cells were removed using anti-mouse CD4 or anti-mouse CD8a positive selection MACS microbeads (Miltenyi Biotec), respectively. The remaining untouched cells containing CD8+ T cells were cultured for 18 h with medium alone or with E7(49–57) peptide. The remaining untouched cells containing CD4+ T cells were cultured 18 h with medium alone or an extended E7 peptide, E7(30–67), containing a Th cell epitope. Cells were harvested and plated into 96-well ELISPOT plates coated with IFN-{gamma} capture Ab, as described above.

Cytotoxicity assay

T cells from groups of mice (n = 3) were enriched from splenocytes by nylon wool adherence. T cells were cocultured at a 10:1 ratio with RMA-S (36) cells that had been cultured at 26°C for 40 h, loaded with a H2-Db-binding peptide derived from the L1 protein of HPV16 (29), and treated with mitomycin C (Sigma-Aldrich). Cocultures were supplemented with 2% T cell supplement without Con A (BD Biosciences) and incubated for 5 days at 37°C, 5% CO2 in a humidified incubator. Cells were collected by gentle pipetting, and live cells were isolated over a Lympholyte-M (Cedarlane Laboratories) cushion. T cells were added to 96-well V-bottom plates in triplicate wells containing 2 x 103 51Cr-labeled EL4 target cells loaded with HPV16 L1 peptide (aa 165–173) or control Db-binding peptide at E:T ratios of 90:1, 30:1, 10:1, and 3:1. Maximum release was determined by culturing target cells with 1% Triton X-100. Spontaneous release was determined by culturing target cells with medium alone. Plates were incubated for 5 h at 37°C, and supernatant was collected and placed onto LumaPlate-96 (PerkinElmer), dried overnight, and then counted using a TopCount Microplate Scintillation and Luminescence Counter (PerkinElmer). Percent lysis was calculated as ((experimental release – spontaneous release)/(maximum release – spontaneous release)) x 100.

Tetramer analysis

H2-Db tetramers labeled with PE and containing the HPV 16E7(49–57) peptide RAHYNIVTF were obtained from the National Institute of Allergy and Infectious Diseases Tetramer Facility. CD8+ T cells from 5 x 107 freshly isolated splenocytes from immunized mice were enriched using positive selection with CD8a MACS microbeads through a LS separation column (Miltenyi Biotec). One million CD8+-enriched cells were incubated for 1 h on ice with 20 µl of 1/100 diluted PE-labeled E7 tetramer and 2 µg of anti-mouse CD8 FITC in FACS buffer. Cells were washed four times and analyzed by flow cytometry for the percentage of double-positive cells.

Mouse Ab isotyping

Mice (n = 3) were bled on days 0 (before VLP vaccination), 3, 7 (before boost), 14 (before boost), 21, 28, and 52. Serum was isolated using serum separator tubes (BD Biosciences) and frozen at –20°C until analysis. Mouse serum isotypes were quantified using the Beadlyte Mouse Ig Isotyping Kit (Upstate Biotechnology) following the manufacturer’s instructions. Data were collected and analyzed on the Bio-Plex 200 system and suspension array technology using Bio-Plex Manager Software (Bio-Rad). Specific anti-VLP IgG titers were determined by ELISA, as previously described (33). Briefly, purified HPV16 L1/L2 VLPs (500 ng/well) were used to coat 96-well Maxisorp ELISA plates (Nunc). Mice sera were added to wells at dilutions from 1/100 to 1/100,000. After washing, bound Ab was detected by the addition of peroxidase-labeled goat anti-mouse IgG Ab (BioSource International). Concentration of anti-VLP IgG was determined from a standard curve using a conformational anti-HPV16 VLP mAb (H16.V5 provided by N. Christensen, Penn State University, Hershey, PA).

Statistical analysis

Mean values were analyzed by one-way ANOVA using GraphPad Prism software 4.0. A p value <0.05 is considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Fc{gamma}RII/III expression on wt and knockout murine DC

To investigate the role of Fc{gamma}RII and Fc{gamma}RIII on DC in VLP binding, internalization, and DC activation, we obtained homozygous mice deficient in expression of Fc{gamma}RIIb (CD32–/– mice) or Fc{gamma}RIII (CD16–/– mice) or both Fc{gamma}RII and III (CD16/32–/– mice). RT-PCR and flow cytometry were performed to determine which, if any, Fc{gamma}R were expressed on DC from wt and Fc{gamma}R knockout mice. BMDC were generated by culturing hemopoietic precursors with GM-CSF and IL-4 in vitro. RT-PCR analysis on day 6 immature DC revealed the presence of RNA transcripts for both CD16 and CD32 in wt C57BL/6 (wt B6) mice (Fig. 1A). Splenocytes isolated from B6 mice, which contain a mixture of cell types that express Fc{gamma}R, were also positive for CD16 and CD32. DC from CD16–/– mice lacked CD16 RNA, but maintained expression of CD32 RNA transcript. Likewise, DC from CD32–/– mice did not express the full-length CD32 RNA transcript, but maintained expression of CD16 RNA. In DC from double-knockout CD16/32–/– mice, both CD16 and CD32 RNA transcripts were absent (Fig. 1A). In all cases, GAPDH RNA was amplifiable and the omission of reverse transcriptase did not lead to any PCR product, indicating that we were indeed analyzing RNA and not genomic DNA. Surface expression of CD16 and CD32 was detected on DC using the mAb 2.4G2 (anti-mouse Fc{gamma}RII/III), which recognizes both CD16 and CD32 due to their homology. Positive staining on DC from CD16–/– mice would therefore be attributable to CD32 surface expression, and vice versa for CD32–/– mice. Confirming the RT-PCR analysis, the 2.4G2 Ab stained DC from B6, CD16–/–, and CD32–/– mice, but not DC from CD16/32–/– mice (Fig. 1B). The level of Ab staining on the DC from the single-knockout mice also indicated that CD32 is expressed more predominantly than CD16 based on the MFI (MFI = 319 for CD16–/– DC; MFI = 195 for CD32–/– DC) (Fig. 1B). Together, these data indicate that both low-affinity Fc{gamma}R are expressed normally on wt B6 DC, although at different levels, and the knockout mice give us tools to study each contribution of CD16 and CD32 in the induction of CMI by VLP.


Figure 1
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  		FIGURE 1. Characterization of Fc{gamma}R expression on DC from wt and Fc{gamma}R knockout mice. DC were generated from mouse bone marrow hemopoietic precursors in the presence of murine GM-CSF and murine IL-4 for 7 days in 24-well tissue culture dishes. A, RT-PCR for mouse CD16, mouse CD32, or mouse GAPDH. C57BL/6 splenocytes were used as a positive control for amplification of CD16 and CD32 transcripts. B, Cell surface CD16 and CD32 expression on DC as detected by flow cytometry. Cells were stained with FITC-labeled anti-2.4G2 (Fc{gamma}RII/III) Ab or with an isotype control Ab. One of two independent experiments is shown.

 
Binding and uptake of VLP by Fc{gamma}R-deficient DC

Previous data showed that the cells of the immune system with the highest capability of binding VLP were APCs such as DC, B cells, macrophages, and monocytes (16), cells that incidentally express CD16, CD32, or both. Because DC are the most efficient APCs for inducing CMI, we focused on this cell type to analyze HPV16 VLP binding and internalization. Analysis of HPV16 VLP binding to DC from B6 or knockout mice showed a partial dose-dependent decrease in binding to CD16–/– and CD32–/– DC and an even more pronounced decrease in CD16/32–/– DC (Fig. 2A). This decrease in binding was specific for HPV VLP, because there was no comparable decrease seen when VLP from either JC or BK viruses were tested, which also have nonenveloped capsids similar in size and shape to HPV (Fig. 2, B and C). However, even in the absence of both CD16 and CD32, binding to DC was not abrogated completely, indicating that although CD16 and CD32 do contribute to the DC-VLP interaction, these molecules are not solely responsible for mediating binding.


Figure 2
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  		FIGURE 2. Analysis of VLP binding to wt and Fc{gamma}R-deficient DC. VLP binding was analyzed by flow cytometry. Immature DC were incubated with increasing concentrations of biotinylated HPV16 L1/L2 VLP (A), BK VP1 VLP (B), or JC VP1 VLP (C) on ice, washed, then incubated with streptavidin FITC to detect VLP binding. Shown is the MFI of binding at concentrations tested. Shown in one of four independent experiments.

 
Some viruses have the ability to use multiple receptors, some for attachment and others for entry into the target cell. To determine whether CD16, CD32, or both are involved in entry of HPV VLP into DC, we analyzed uptake of fluorescently labeled VLP by wt and Fc{gamma}R-deficient DC. CFSE-labeled HPV16 VLP were incubated with BMDC from B6 or Fc{gamma}R knockout mice for 4 h at 37°C. At various time points, aliquots were removed, fixed, and analyzed by flow cytometry to determine whether there was a difference in the kinetics of VLP uptake. VLP taken up by DC results in time-dependent quantitative intracellular fluorescence due to cleavage of the CFSE by intracellular esterases (34). Uptake of HPV VLP by wt DC occurred within 30 min and was maximal between 60 and 120 min (Fig. 3A). Intracellular fluorescence did not increase substantially after 90 min when DC were incubated with labeled VLP for longer periods. DC from CD16–/–, CD32–/–, and CD16/32–/– mice took up half the amount of VLP compared with wt DC within the first 60 min (Fig. 3A). Extended incubation for up to 4 h with Fc{gamma}R-deficient DC did not result in any further significant VLP uptake (B6 DC vs single-knockout DC, p ≤ 0.001; B6 DC vs double-knockout DC, p ≤ 0.001). As controls, DC were assessed for uptake of FITC-labeled dextran, which uses the mannose receptor for endocytosis into cells, and FITC-labeled BSA, which is taken up by cells by nonspecific pinocytosis. The wt DC and DC from all Fc{gamma}R-deficient mice took up an equivalent amount of FITC dextran within 90 min with no significant difference (p > 0.05) in the kinetics of uptake, indicating that no differences exist in basic receptor-mediated endocytosis mechanisms between wt and Fc{gamma}R-deficient mice (Fig. 3B). The wt DC and DC from Fc{gamma}R-deficient mice also took up equivalent amounts of BSA (Fig. 3C), indicating that there are no obvious differences in plasma membrane integrity of the DC or differences in internalization of nominal proteins. Because protein uptake by DC appears to plateau after 90 min, extended time points were not analyzed for dextran and BSA. Uptake of JC VLP and BK VLP was additionally tested to exclude the possibility that receptor-mediated uptake of other large virus particles was not affected in DC that lack Fc{gamma}R. No differences in uptake of either JC or BK VLPs were observed by any of the Fc{gamma}R knockout mice (Fig. 3, D and E). To analyze whether oversaturating amounts of VLP lead to a greater increase in either receptor-mediated or nonspecific VLP uptake, DC from wt or Fc{gamma}R-deficient mice were incubated with 5 times more fluorescent labeled VLP as used in Fig. 3A. As expected for receptor-mediated uptake, addition of more VLP did not lead to equivalent amounts of uptake in the Fc{gamma}R-deficient mice, with uptake still being significantly decreased in the knockout mice (B6 DC vs single-knockout DC, p ≤ 0.05; B6 DC vs double-knockout DC, p ≤ 0.01) (Fig. 3F). Together, these data indicate that over a period of 4 h, DC that lack CD16, CD32, or both take up quantitatively less VLP than DC that express the Fc{gamma}R, and this effect is specific for HPV VLP. Collectively, these data indicate that CD16 and CD32 can indeed function for both binding and as HPV16 VLP uptake receptors.


Figure 3
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  		FIGURE 3. Quantification of VLP uptake by wt and Fc{gamma}R-deficient DC. Intracellular uptake of HPV VLP was assessed by flow cytometry. A, CFSE-labeled HPV16 L1/L2 VLP were added to immature DC and incubated at 37°C for 240 min. Fractions were taken at 0, 15, 30, 60, 120, and 240 min; fixed; and assessed for intracellular VLP fluorescence. The percentage of VLP uptake by DC at each time point was calculated and is shown for wt and Fc{gamma}R knockout DC. Shown is the average uptake (±SEM) of four independent experiments; ***, p ≤ 0.001 compared with C57BL/6 mice. B, FITC-labeled dextran uptake by DC. Percentage of dextran uptake was determined, as described for VLP. C, FITC-labeled BSA uptake by DC. Percentage of BSA uptake was determined, as described for VLP. D, BK VLP uptake by DC was performed as for HPV VLP uptake. One of two experiments is shown. E, JC VLP uptake by DC was performed as for HPV VLP uptake. One of two experiments is shown. F, CFSE-labeled VLP uptake using five times more VLP than was used in A. Shown is the average uptake (±SEM) of four independent experiments; **, p ≤ 0.01; *, p ≤ 0.05 compared with C57BL/6 mice.

 
Activation of wt and Fc{gamma}R-deficient DC by VLP

Previous data demonstrated that DC become activated after direct incubation with VLP (17, 18). To determine whether VLP-induced activation is Fc{gamma}R dependent, we incubated HPV16 VLP with immature BMDC from wt or Fc{gamma}R-deficient mice, then harvested and analyzed the cells for an increase in cell surface markers associated with maturation and activation by flow cytometry. DC from wt B6 mice up-regulated MHC class I and class II molecules and the costimulatory molecules CD40, CD80, and CD86 by 2-fold or more after incubation with HPV VLP (Fig. 4A). DC from CD16–/–, CD32–/–, and CD16/32–/– mice responded similarly to LPS as wt DC, indicating that LPS-mediated activation pathways are intact in Fc{gamma}R-deficient mice (Fig. 4B). LPS-matured DC from Fc{gamma}R-deficient mice also produced similar amounts of the immunostimulatory cytokine IL-12 compared with wt mice (Fig. 4C). However, when incubated with VLP, DC from both CD16–/– and CD32–/– mice and CD16/32–/– mice displayed a less mature phenotype indicated by lower expression of MHC and costimulatory molecules (Fig. 4A). VLP-mediated activation of DC as measured by marker up-regulation was not completely dependent on the presence of Fc{gamma}R, indicating that, like binding and uptake, signaling through Fc{gamma}R only partially accounts for the VLP-induced activation and maturation of DC. BMDC grown and activated with VLP in serum-free medium displayed the same up-regulation of activation markers as BMDC grown in the presence of serum, indicating that VLP-induced DC activation is not dependent on the presence of Abs in the culture medium that can bind simultaneously to VLP and Fc{gamma}R (data not shown). Therefore, our results suggest that direct VLP binding to CD16 and/or CD32 contributes to, but is not required for, DC activation.


Figure 4
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  		FIGURE 4. Activation of DC from wt or Fc{gamma}R knockout mice by HPV16 VLP. Day 6 immature DC from wt C57BL/6 mice, CD16–/– mice, CD32–/– mice, and CD16/32–/– mice were cultured with medium alone, LPS, or HPV16 L1/L2 VLP for 48 h. Cells were harvested from wells and analyzed for MHC class I, MHC class II, CD40, CD80, and CD86 cell surface marker expression by flow cytometry. Fold change in marker expression was calculated vs the MFI of marker expression on unactivated cells. Shown is the average fold change (±SEM) of four independent experiments using HPV VLP (A) or LPS (B). C, IL-12 p70 secretion from unactivated or LPS-activated DC. Error bars, SD of triplicate wells. Data are representative of four independent experiments.

 
Cellular immune response against HPV cVLP in Fc{gamma}R-deficient mice

To determine whether Fc{gamma}R on DC are involved in the in vivo priming of CD8+ T cell responses against cVLP-derived peptides, wt and Fc{gamma}R-deficient mice were immunized with HPV16 L1/L2-E7 cVLP, and the frequency of E7 peptide-specific CD8+ T cells induced after immunization was analyzed by E7(49–57)-MHC tetramer binding, IFN-{gamma} ELISPOT, and a 51Cr release cytotoxicity assay. Enriched CD8+ T cells from the spleens of immunized mice were stained with fluorescently labeled MHC tetramers containing the HPV16 E7(49–57) peptide and counterstained with a CD8 Ab. The percentage of CD8+tetramer+ cells from cVLP-immunized single- and double-Fc{gamma}R knockout mice was significantly reduced compared with wt B6 mice (Fig. 5A) (p < 0.05). The number of IFN-{gamma}-secreting E7-specific CD8+ T cells from cVLP-immunized CD16–/– mice was also reduced by 40–50%, and the responses in CD32–/– and CD16/32–/– mice were almost completely absent (p < 0.05 compared with wt mice) (Fig. 5B). The absence of Fc{gamma}R on APCs did not have any effect on the induction of E7-specific CD8+ T cells by peptide immunization, because both wt and Fc{gamma}R-deficient mice all had equal or greater numbers of tetramer+CD8+ cells (Fig. 5A) and equivalent numbers of IFN-{gamma}-secreting cells (Fig. 5B) (p > 0.05). Cytotoxicity against L1 peptide-loaded target cells showed that CTL from wt mice immunized with cVLP had an enhanced ability to kill target cells compared with CTL from CD16–/–, CD32–/–, and CD16/32–/– mice, with the double-knockout mice having 2- to 3-fold less ability to kill across the E:T ratios (p < 0.05, wt vs CD16–/–; p < 0.01, wt vs CD32–/–; and p < 0.001, wt vs CD16/32–/– at the 90:1 ratio; Fig. 5C). The absence of Fc{gamma}R did not decrease CTL killing after L1 peptide immunization, because all mice were able to kill target cells in a similar manner (p > 0.05; Fig. 5D).


Figure 5
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  		FIGURE 5. CMI analysis of wt and Fc{gamma}R-deficient mice immunized with HPV cVLP. A, C57BL/6, CD16–/–, CD32–/–, and CD16/32–/– mice (n = 3/group) were immunized once s.c. with PBS, HPV16 L1/L2-E7 cVLP, or HPV16 E7(49–57) peptide control in IFA. Splenocytes were harvested at day 10 postimmunization and tested for binding of H2-Db tetramers loaded with HPV16 E7(49–57) peptide. CD8+ cells from spleen were enriched by MACS before tetramer staining. Shown are the mean number of tetramer-binding CD8+ T cells (±SEM) for each group of mice. B, HPV16 E7-specific IFN-{gamma} production after 24-h stimulation with E7(49–57) peptide. Shown are the mean number of spot-forming cells per 105 CD8+ T cells (±SEM). *, p < 0.05 compared with cVLP-immunized C57BL/6 mice. Groups of naive mice or peptide-immunized mice were not statistically different. C and D, Chromium release cytotoxicity assay after 5 days of in vitro stimulation against the HPV16 L1(165–173) H2-Db-binding peptide. Shown is the mean percent lysis (±SD) (n = 3 pooled splenocytes) from HPV16 VLP-immunized mice (C) or L1 peptide-immunized mice (D). Nonspecific lysis against unpulsed target cells was subtracted from specific lysis. All naive mice exhibited <5% lysis against peptide-pulsed target cells. One of three independent experiments is shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with cVLP-immunized C57BL/6 mice at the 90:1 ratio. E, CD8+ T cell IFN-{gamma} secretion in CD4-depleted cultures stimulated with E7(49–57) peptide. Shown is the mean number of spot-forming cells per 106 splenocytes (±SD) (n = 3 pooled splenocytes) representative of two experiments. ***, p < 0.001 compared with C57BL/6 mice. F, CD4+ T cell IFN-{gamma} secretion in CD8-depleted cultures stimulated with E7(30–67) peptide. Shown is the mean number of spot-forming cells per 106 splenocytes (±SD) (n = 3 pooled splenocytes) representative of two experiments. ***, p < 0.001 compared with C57BL/6 mice. G, Proliferative response of purified CD4+ cells from VLP-immunized mice in response to VLP-pulsed wt DC. Radioactive thymidine uptake from T cells stimulated with DC alone was subtracted from DC-VLP stimulation. Shown is the mean thymidine uptake (±SD) (n = 3 pooled splenocytes) representative of two experiments.

 
To determine whether CMI responses in Fc{gamma}R-deficient mice involved only the CD8+ T cell subset or both the CD8+ and CD4+ T cell subsets, CD8+ and CD4+ T cell responses were analyzed separately after cVLP vaccination by IFN-{gamma} ELISPOT. In cultures in which CD4+ cells were depleted from splenocytes before E7(49–57) peptide restimulation, the number of IFN-{gamma}-secreting CD8+ T cells from all three Fc{gamma}R-deficient mice was significantly reduced (p < 0.001, wt vs CD16–/–, CD32–/–, or CD16/32–/–; Fig. 5E). Similarly, in cultures in which CD8+ cells were depleted, the number of IFN-{gamma}-secreting CD4+ T cells responding to the E7(30–67) peptide, containing a Th cell epitope, also was significantly reduced in all three Fc{gamma}R-deficient mice (p < 0.001, wt vs CD16–/–, CD32–/–, or CD16/32–/–; Fig. 5F). In addition, untouched purified CD4+ T cells from cVLP-vaccinated Fc{gamma}R-deficient mice all proliferated less than T cells from wt B6 mice when cultured with wt B6 DC pulsed with cVLP (Fig. 5G). Collectively, these data indicate that E7-specific T cells can be induced in Fc{gamma}R-deficient mice; however, they are not induced optimally after cVLP immunization. To exclude intrinsic defects in either T cells or DC from Fc{gamma}R-deficient mice that could lead to these observations, DC from wt and Fc{gamma}R-deficient mice were used to stimulate purified BALB/c T cells or T cells from wt and Fc{gamma}R-deficient mice were stimulated by BALB/c DC in an allogeneic MLR. DC from wt and Fc{gamma}R-deficient mice were equally capable of stimulating proliferation of BALB/c T cells (Fig. 6A), and likewise, T cells isolated from wt and Fc{gamma}R-deficient mice were equally capable of proliferating in response to stimulation by BALB/c DC, but not to autologous DC (Fig. 6B). In addition, no significant changes in the percentage of cell types in the spleens of Fc{gamma}R-deficient mice were evident aside from the loss of expression of CD16 and CD32 in CD32–/– and CD16–/– mice, respectively, and a small, but noticeable decrease in the percentage of NK cells in CD16/32–/– mice (Fig. 6C). Altogether, these results suggest that expression of both CD16 and CD32 on murine DC contributes to binding and uptake of cVLP, maturation of DC, and subsequently for the full activation of CD8+ and CD4+ T cells against HPV cVLP-derived Ags.


Figure 6
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  		FIGURE 6. DC Ag-presenting function and T cell function from wt and Fc{gamma}R-deficient mice. A, LPS-matured BMDC from wt or knockout mice were used as stimulator cells and cultured with purified allogeneic T cells (2.5 x 104 to 2 x 105) from BALB/c mice in an MLR. [3H]Thymidine incorporation was determined as a measure of T cell proliferation. For controls, BALB/c T cells were cultured alone or with SEB. B, Purified T cells (1 x 105) from wt or knockout mice were cultured with allogeneic T cell-depleted BALB/c splenocytes as APCs, autologous APCs, or SEB in a MLR. Responder T cell proliferation was detected by incorporation of [3H]thymidine. Error bars, SD of triplicate values. One of two independent experiments is shown. C, Characterization of splenocyte subpopulations in 12- to 16-wk-old wt and Fc{gamma}R-deficient mice. Splenocytes (n = 5 mice/group) were analyzed for expression of various cell surface markers by flow cytometry. CD4 and CD8 were used as markers for T lymphocytes; 2.4G2 for cells expressing either Fc{gamma}RII or Fc{gamma}RIII; CD19 for B lymphocytes; NK1.1 for NK cells; Gr-1 for granulocytes; CD11b/MAC-1 for monocytes/macrophages; and CD11c for DC. Shown is the mean percentage of cells (±SEM).

 
Humoral immune response against HPV VLP in Fc{gamma}R-deficient mice

Activating and inhibitory Ig-binding molecules on the surface of B cells help set thresholds for BCR signaling (37). VLP-directed Abs induced by VLP Ag priming would form IC with the VLP upon secondary exposure. VLP-IC would then be available for binding to FcRs and could result in secondary VLP internalization and signaling on B cells and other cells that express Fc{gamma}R. To analyze Ab production in Fc{gamma}R-deficient mice in response to VLP, mice were immunized with HPV16 VLP and boosted twice. Sera were collected and analyzed for total levels of serum IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 isotypes, and also specific levels of total anti-HPV VLP IgG (Fig. 7). Notably, in CD16–/– mice the early Ab responses were significantly elevated in IgM and IgG2a isotypes (Fig. 7, C and F) and slightly elevated in IgG3 (Fig. 7B). In all other mice, the majority of isotype-specific responses were comparable, indicating there was a negligible effect of the loss of CD16, CD32, or the combination of CD16/32. Importantly, in CD32–/– mice, there did not appear to be an increase in the levels of Abs that would be expected if a negative regulator of B cell signaling were missing. VLP-specific IgG persisted longer in CD32–/– and CD16/32–/– mice compared with wt and CD16–/– mice, although not statistically significant (Fig. 7G). Importantly, no VLP-specific IgG was detected until 7 days after vaccination, suggesting that it is unlikely that VLP-Ab complexes would form during the priming event, thus contributing to binding to FcRs.


Figure 7
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  		FIGURE 7. Ab production in Fc{gamma}R-deficient mice in response to VLP. Mice (n = 3) were immunized with HPV16 VLP and boosted twice. A–F, Sera were collected and analyzed for total levels of serum IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 isotypes using murine isotype-specific suspension bead array technology. G, Specific levels of total anti-HPV VLP IgG were determined by Ag-specific ELISA. Shown are mean Ab concentrations (±SEM) over time.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In DC, but not in other cell types, Fc{gamma}R-mediated internalization targets extracellular Ags to a unique DC-specific Ag transport pathway. This targeting results in delivery of the Ags to the cytosol, TAP-dependent translocation of peptides into the lumen of the endoplasmic reticulum, and loading onto MHC class I molecules (38, 39, 40). This specialized process can result in the activation of Ag-specific CD8+ T cells that can differentiate into cytokine-secreting cytotoxic effectors in response to extracellular Ags (23, 24, 39, 41, 42).

We previously reported that mAbs to human CD16, but not CD32, were capable of inhibiting binding of HPV16 VLP to human APCs (16). Unlike in the mouse in which the extracellular domains of CD16 and CD32 are 95% homologous, human CD16 and CD32 isoforms are only ~50% homologous. Therefore, CD16 on human cells of the immune system may function as the predominant receptor for HPV VLP, whereas in mice we found both low-affinity Fc{gamma}R to function as receptors for HPV VLP on immunocytes. A new low-affinity IgG FcR with homology to CD16, Fc{gamma}RIV, has recently been identified in mice and is also expressed on BMDC (43, 44). Our data do not rule out a function for this novel Fc{gamma}R in VLP binding and uptake. Indeed, expression of Fc{gamma}RIV in the absence of Fc{gamma}RII/III expression may account for part of the remaining VLP binding and uptake, DC maturation, and T cell priming by DC in our experimental system. Further studies analyzing the contribution of this receptor are warranted.

Although not likely to be relevant to the infectious life cycle of HPV, DC expressing CD16 are highly relevant for induction of immune responses after immunization with VLP and cVLP, as evidenced by this study. Targeting of certain Ags to Fc{gamma}R through formation of IC dramatically increases the efficiency of cross-presentation of exogenous Ags by mouse BMDC to CD8+ T cells by several orders of magnitude (23, 24, 25). Fc{epsilon}RII is also able to enhance internalization of IC in B cells, resulting in efficient focusing of Ag into the degradative pathway and presentation to Th cells (45). Because VLP are multivalent Ags, they too may be able to induce cross-linking of Fc{gamma}R and cellular signaling leading to DC maturation. Although signaling was not analyzed in this study, we found that when BMDC lacked expression of CD16, CD32, or both molecules, up-regulation of both MHC and costimulatory molecules was suppressed. However, it is not conclusive from the data presented in this work whether VLP uptake mediated by Fc{gamma}R is the same or distinct from Ab-Ag IC uptake.

The reduced intracellular uptake of VLP by DC from CD16/32–/– mice that was observed could also be attributable to the reduced binding of VLP to the DC. We have attempted to separate binding from uptake by incubating DC with 5 times more VLP per DC, a concentration that results in near equivalent VLP binding between wt and Fc{gamma}R knockout DC. Even in the presence of excess VLP, Fc{gamma}R-deficient DC still do not endocytose similar amounts of VLP as wt DC, indicating that virus binding and virus uptake can indeed be separated. Alternatively, the use of 5 times more VLP per DC in uptake studies might have also increased nonspecific receptor-mediated uptake of VLP. Inhibition of binding, uptake, and DC activation in DC from Fc{gamma}R-deficient mice was not complete, indicating that even in the absence of Fc{gamma}R, DC are still able to take up some VLP and become activated. In addition to receptor-mediated clathrin-dependent endocytosis as a mechanism for uptake of VLP, DC are also capable of endocytosing VLP through macropinocytosis (46). Both mechanisms of uptake could account for uptake that occurs in the absence of Fc{gamma}R, and most likely still occurs even in the presence of these Ig-binding receptors. The possibility also remains that there is yet another receptor on the DC that is responsible for binding and/or uptake of VLP into the cell. Furthermore, other molecules that have been characterized as HPV receptors may also contribute to binding and/or uptake to DC (47, 48, 49) and possibly signaling DC activation, although this has not been studied to date (50, 51). However, it is highly likely that a VLP binding to Fc{gamma}R will be endocytosed into the cell for processing, as IC are under normal conditions. The reduced fluorescence resulting from VLP uptake could also have been due to differences in processing of the CFSE label due to differential uptake mechanisms; however, we view this possibility as unlikely, because different cell types with different endocytosis mechanisms have been shown to take up CFSE-labeled VLP (35, 46).

Our in vivo immunization data with cVLP indicate that in the absence of CD16 or CD32 expression, mice do not mount a quantitatively equivalent E7-specific CD8+ T cell and CD4+ T cell response compared with mice that do express these Fc{gamma}R. The reduction of mature Fc{gamma}R-deficient DC after in vitro activation by VLP does seem to correlate with the reduced in vivo cellular immune response seen after VLP immunization. Because some DC activation does occur in the absence of FcR expression, these DC may be involved in initiating the observed T cell responses in Fc{gamma}R-deficient mice. L1 was used as a readout for CTL in part because L1 protein is more abundant than the E7 protein in cVLP and cytotoxic responses have been observed against an H-2Db-binding epitope after vaccination with L1 capsomeres (29). The decrease in CTL also correlated with the reduced number of E7-specific T cells detected by IFN-{gamma} ELISPOT and tetramer staining. We did note a discrepancy between the number of IFN-{gamma}-secreting T cells detected in the CD16/32–/– double-knockout mice after cVLP immunization and the frequency of T cells detected by E7-tetramer binding. This observation could be due to T cells that bind the MHC class I tetramers, but do not secrete IFN-{gamma} upon stimulation because they are not fully activated. Although tetramer analysis can only indicate that T cells are present, it does not indicate T cells are functional. Moreover, both CD8+ and CD4+ CMI were reduced in cVLP-immunized Fc{gamma}R-deficient mice, indicating that both MHC class I and class II peptides are presented less well or without the proper costimulation compared with wt mice. Overall, all of our analyses indicated that the most severe phenotype was observed when both CD16 and CD32 were absent.

In the CD16 and CD32 single-knockout and CD16/32 double-knockout mice, all cells that normally express CD16, CD32, or both are deficient for these molecules. However, our in vitro data together with the in vivo data suggest that it is specifically CD16/32 expression on DC that is critical for the initiation of class I-restricted CMI by cVLP. Fc{gamma}R are not thought to participate directly in Ag presentation to T cells. Rather, Fc{gamma}R expression on DC functions to capture IC for Ag processing and signal the cell for maturation (23). Although our immunization regimen with cVLP is not conducive to formation of VLP-IC, IC may still play an important role in targeting VLP to DC under some circumstances. We have previously shown that human DC are activated by HPV VLP-IC and can induce T cells specific for VLP-derived epitopes in vitro (52). However, we have also shown that in vivo immunization of wt mice with VLP-IC does not result in sufficient CMI to reject murine HPV-transformed tumors (33). In the latter circumstance, however, VLP-IC can target Fc{gamma}R on many cell types, not just DC, which may affect overall T cell priming.

The lack of CMI induced by cVLP in CD16/32–/– mice cannot be due to intrinsic defects of APCs because DC from Fc{gamma}R-deficient mice retain the ability to stimulate allogeneic T cells in an MLR and do up-regulate costimulatory molecules and secrete IL-12 in response to LPS stimulation. DC from Fc{gamma}R-deficient mice were also equally capable of binding and taking up VLP from unrelated viruses. Additionally, because T cells themselves do not express Fc{gamma}R, the lack of these molecules should not have an effect on T cell function. In FcR{gamma} chain-deficient mice, the lack of MHC class II-restricted CD4+ T cell responses has been found to lie in defective Ag presentation and not in T cell function (53). Our data similarly showed intact T cell function from Fc{gamma}R-deficient mice in that HPV16 E7 peptide immunization resulted in comparable MHC class I-restricted CD8+ T cell responses to wt mice and T cells from Fc{gamma}R-deficient mice respond equally to alloantigens. Our data support the conclusion that the defective T cell responses in the Fc{gamma}R-deficient mice in response to cVLP are due to impaired Ag presentation by DC during in vivo priming and not to a defect in T cells.

In the natural course of HPV infection, virions first encounter Langerhans cells, DC-like APCs that reside in the skin and mucosal epidermal layers. Human Langerhans cells, unlike human DC, are not activated by HPV VLP and are not capable of inducing HPV-specific immunity after incubation with cVLP (34). Although expression of CD16 is detected on endocervical and transformation zone epithelial cells, that may contribute to tissue-specific homing by HPV, very few Langerhans cells in the vaginal and cervical epithelium express CD16 (54). Therefore, the lack of CD16 expression on human Langerhans cells may account for the generally poor immune responses observed against HPV infection in addition to the differences in how DC and Langerhans cells respond to the HPV virus capsid (46).

Although protective neutralizing Ab responses to HPV VLP are critical for preventing initial virus infection, they most likely do not play a role in the initial encounter of HPV VLP with APCs. Rather, Abs against the surface structure of VLP would become important upon secondary exposure to Ag, at which time IC can bind to FcRs. Because Fc{gamma}R are expressed also on B cells as well as APCs, we analyzed Ab production in our Fc{gamma}R-deficient mice after VLP vaccination. Although IgG1 and IgG2b are the predominant isotypes made in response to s.c. VLP vaccination in wt mice (D. Da Silva and W. Kast, unpublished data), there were significant increases in IgM, IgG2a, and to a lesser extent IgG3 in CD16–/– mice. Although it is not immediately clear why these responses would be higher in CD16–/– mice, one possibility may be indirectly through the role of T cell help during isotype switching. Human monocyte-derived CD16+ DC stimulate Th2 cells to secrete more IL-4 than CD16– DC (55). Preferential isotype switch to IgG2a and IgG3 could be the result of altering the balance of IL-4 to IFN-{gamma} secreted by Th1 cells. Further studies extending beyond the scope of the work presented in this study would help answer these questions. Moreover, whereas the total amount of anti-VLP-specific IgG did not significantly vary in the early response, our data do suggest that in mice lacking CD32, a negative regulator in B cells, Ab levels did not decline compared with wt and CD16–/– mice. However, further time points would be needed to confirm these data.

Overall, the work presented in this study defines a key contribution for the low-affinity Fc{gamma}R in targeting HPV VLP to DC and elucidates a mechanism for VLP-induced maturation of DC that facilitates cVLP-derived peptide presentation to naive Ag-specific CD4+ and CD8+ T cells. The interaction of VLP with human CD16 and both murine CD16 and CD32, combined with the possibility of Fc{gamma}R signaling-induced DC maturation, may account for the high intrinsic immunogenicity of HPV VLP.


   Acknowledgment
 
We thank deMauri Mackie from the Beckman Center for Immune Monitoring 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 National Institutes of Health Grants R01 CA74397 and P01 CA97296, and the V and Whittier Foundations (to W.M.K.). D.M.D.S. was supported by National Institutes of Health Postdoctoral Fellowship T32 CA09659 and the University of Southern California Norris Cancer Center Postdoctoral Scholarship Program. W.M.K. holds the Walter A. Richter Cancer Research Chair. Back

2 Address correspondence and reprint requests to Dr. W. Martin Kast, Norris Comprehensive Cancer Center, Zilkha Institute Building, Room 245, University of Southern California, 1501 San Pablo Street, MC 2821, Los Angeles, CA 90033. E-mail address: mkast{at}usc.edu Back

3 Abbreviations used in this paper: HPV, human papillomavirus; BMDC, bone marrow-derived dendritic cell; CMI, cell-mediated immunity; VLP, virus-like particle; cVLP, chimeric VLP; DC, dendritic cell; IC, immune complex; MFI, mean fluorescence intensity; SEB, Staphylococcus enterotoxin B; wt, wild type. Back

Received for publication June 13, 2006. Accepted for publication April 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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