Antigenic Targeting of the Human Mannose Receptor Induces Tumor Immunity

Generation of hMR Tg mice

The hMR Tg mice were made in the C57BL/6 strain using standard microinjection techniques. Based on genomic sequence information on the 30-exon hMR gene (38), basic local alignment sequence tool sequence searches were conducted on National Center for Biotechnology Information (NCBI) and several human bacterial artificial chromosome (BAC) clones corresponding to this region were identified. One in particular, 211-kb BAC clone RP11-457D2, containing the 5′ and 3′ extremes of the exon sequence previously identified by Kim et al. (38), was obtained from Research Genetics, and characterized further. Sequence verification of exons 1 and 30 were conducted; in addition, sequence identity of a region −26 kb upstream of exon 1 was verified. Full-length NotI-digested BAC insert DNA was prepared as per Chrast et al. (39). This DNA was used for microinjection into fertilized C57BL/6 oocytes to generate hMR Tg mice.

Routine PCR genotyping of mouse tail genomic DNA prepared by the Roche Magnapure method (Roche Diagnostics) used PCR Supermix (Invitrogen Life Technologies) with primers located in the 5′ and 3′ end of the BAC insert DNA (732bp amplicon from −26 kb upstream of exon 1 and 547 bp corresponding to exon 30). The hMR Tg mice were expanded and housed under pathogen-free conditions. WT mice were littermates of Tg or age- and gender-matched C57BL/6 mice purchased from Taconic Farms. CD11c-DTR ((Itgax-DTR/GFP)57Lan/J) mice (C57BL/6 background) were obtained from The Jackson Laboratory and crossed with hMR Tg mice.

MR Abs

The B11 mAb (a human IgG1) were purified from supernatants of transfected Chinese hamster ovary cells as previously described (36, 37). B11-OVA fusion proteins were made as an in-frame fusion of the entire OVA cDNA at the C terminus of the B11 Ig H chain. Mouse anti-hMR Ab clones 19.2 and 15-2 were purchased from BD Biosciences/BD Pharmingen and Serotec, respectively. Anti-mouse MR (muMR) MR5D3 was obtained from BD Biosciences/BD Pharmingen. All reagents/Abs that were used in vivo or in cultures were endotoxin-free (<10 EU/mg, tested by Limulus amebocyte lysate assay), either purchased as functional grade or run through the endotoxin removal gel column (Pierce Biotechnology).

Preparation of peritoneal and bone marrow (BM)-derived cells

Peritoneal exudates cells (PEC) were elicited by i.p. injection with 1 ml of 3% thioglycolate 4 days before harvesting. BMDCs cells were generated from BM harvested from femurs and tibias flushed with RPMI 1640 medium. After depletion of the lineage-positive cells, BM precursor-enriched cells were cultured with 10% FBS, 50 μM 2-ME, 10 ng/ml GM-CSF in RPMI 1640 medium at 3 × 10⁶ cells/well in 6-well plate. The cultures were fed at days 2 and 4 by gently swirling the plates, aspirating 75% of medium and adding back fresh complete medium containing GM-CSF (PeproTech). On day 6, nonadherent cells were removed by gently swirling plates and aspirating cultured medium. Adherent cells were harvested by incubation with Versene (Invitrogen Life Technologies). The majority of these cells were positive for the expression of CD11c as determined by flow cytometry (data not shown). hMR expression was evaluated flow cytometrically with anti-hMR mAbs clone 19.2 and by Western blot analysis with anti-hMR mAb clone15-2.

In vivo Ag targeting and immunohistochemical analysis

Mice were injected s.c. in the two front paws with 5 μg of B11 mAb in 5 μl of PBS to each side. The same amount of irrelevant human IgG1 (Sigma-Aldrich) was administered s.c. as a control for FcR interactions. The skin near and distal to the injection site, proximal (axillary) and distal (inguinal) lymph nodes, spleen, liver, and lung were harvested 24 h later. These organs were snap-frozen and sectioned for immunohistochemical staining with Abs against human IgG. To assess the expression profiles of hMR in the Tg mice, cryostat sections from a panel of 14 different tissues were stained with B11 or 19.2 following the methods published previously (36).

Immunization for humoral responses

For humoral responses, mice of 8–12 wk of age were immunized i.p. with 0.1or 10 μg of B11 in 100 μl of PBS or in 100 μl of Ribi adjuvant per mouse at 2-wk intervals, for a total of three doses. The anti-hMR 19.2 mAb (mouse IgG1) in combination with 100 μl of Ribi was administered to evaluate the generation of anti-Id Abs. Each group consisted of a minimum of five hMR Tg mice or age and gender-matched wild-type (WT) mice. Blood specimens were collected from orbital sinus under anesthetics on day 0 for baseline and 1 wk after each injection and sera were isolated and stored at −20°C for ELISA.

ELISA

Anti-B11 titers were measured by standard ELISA using immobilized B11 on microtiter plates and AP-conjugated goat anti-mouse IgG Fcγ Ab (Jackson ImmunoResearch Laboratories) for detection. Plasma specimens were diluted serially and titers were determined by the highest dilution of each specimen giving an absorbance at OD₄₀₅ that was greater than double the value of control mouse serum. Ab subclass determination was done by means of ELISA with SBA clonotyping system/AP (Southern Biotechnology Associates). For anti-Id responses microtiter plates were coated with 19.2 F(ab)₂ or irrelevant isotype-matched F(ab)₂ and blocked with 5% BSA. Anti-Id IgG specific for 19.2 were detected by a goat anti-mouse IgG, Fc-specific probe. Pooled plasmas from immunized mice were tested for blocking of 19.2 mAb binding to hMR by flow cytometry. Human monocyte-derived DC were incubated with 19.2-PE alone or in the presence of the plasma pool at various ratio at 4°C for 1 h.

In vivo T cell proliferation assays

CD45.1⁺ OT-I and OT-II cells were labeled with CFSE (Molecular Probes) and 5 × 10⁶ cells were injected i.v. into recipient mice before immunization. On day 3 after transfer flow cytometric analysis identified OT-I and OT-II populations among splenocytes using the following mAbs (BD Pharmingen): anti-CD45.1 PE (A20) and either anti-CD4-PerCP-Cy5.5 (GK1.5) or anti-CD8α-PerCP-Cy5.5 (53-6-7). The percentage of divided OT-I or OT-II T cells was calculated as the CFSE⁺, CD45.1⁺, CD4⁺ or 8⁺ population outside the unimmunized single peak/total CD45.1⁺, CD4⁺ or 8⁺ population. For systemic DC depletion, RIP-mOVA/CD11c-DTR mice were treated i.p. with 4 ng/g body weight of diphtheria toxin (DT; Sigma-Aldrich) 18 h before transfer of T cells. One dose of DT leads to 48–72 h of CD11c⁺ cell depletion (40).

IFN-γ intracellular staining

Effector responses were assessed 5 days after transfer of CD45.1⁺ OT-I cells in mice immunized with B11-OVA with or without CpG (InvivoGen). A total of 2 × 10⁶ splenocytes/500 μl of complete medium were incubated with 0.1 μM SIINFEKL peptide and GolgiStop solution (BD Biosciences) for 4 h at 37°C. Cell fixation and permeabilization was done using the Cytofix/Cytoperm Plus kit (BD Biosciences) before intracellular staining with anti-IFN-γ-Alexa Fluor-647 (XMG1.2). Dot plots show the percentage of IFN-γ cells among CD45.1⁺, CD8⁺ gated cells.

ELISPOT

Mice were immunized on days 0 and 14 with 10 μg of B11-OVA with or without 50 μg of CpG (InvivoGen) both given i.p. Splenocytes were harvested on day 21 and plated at 3 × 10⁵ cells/well on a Millipore 96-well membrane plate coated overnight with anti-IFN-γ capture Ab (AN18) and blocked with 10% FBS in RPMI 1640 (Mabtech). Cells were incubated overnight with either no Ag or OVA (300 μg/ml). Wells were washed and incubated with biotinylated anti-IFN-γ (R4-6A2-biotin) and streptavidin-alkaline phosphatase and developed with 5-bromo-4-chloro-3-indolyl phosphate/NBT substrate. A CTL Immunospot Series 3A Analyzer was used for imaging and counting with Immunospot software.

Tumor prevention model

Mice were immunized on days 0 and 14 with 10 μg of B11-OVA with or without 50 μg of CpG both given i.p. On day 21 mice were injected s.c. with 2 × 10⁵ M04 tumor cells (57). Mice were monitored for tumors until sacrifice at day 18 and three-dimensional tumor sizes measured with calipers. A Student t test was used to evaluate significance of growth inhibition of immunization vs no treatment.

Generation of hMR Tg mice

To establish an animal model enabling antigenic targeting of the hMR in vivo hMR Tg mice were generated. A 211-kb BAC clone containing the entire hMR gene and its 5′ and 3′ flanking regions was integrated into the genome of C57BL/6 mice (Fig. 1⇓A), inclusive of 56 kb of 5′ flanking regulatory sequences upstream of exon 1, allowing for transgene expression under the control of its own promoter. Multiple PCR primer pairs established that the transgene was integrated intact and real-time PCR analysis was consistent with a single copy insertion (data not shown) in the founder line. The expression of the transgene was confirmed using RT-PCR analysis and hMR-specific Abs in several immunoassays (Fig. 1⇓B). Immunoblotting analysis on lysates derived from activated PEC revealed a ∼180 kb band only in the hMR Tg mice, which corresponded to the size of hMR in lysates from human DC (Fig. 1⇓C). The endogenous muMR, detected with species specific reagents, was expressed at similar levels in hMR Tg mice and WT mice in PEC-Mφ and BM-derived DC, indicating that bearing the transgene did not interrupt the endogenous MR expression (Fig. 1⇓D). Immunostaining with the human anti-hMR mAb B11 in a panel of 14 tissues, including skin, lymph node, spleen, thymus, liver, lung, brain, heart, kidney, colon, ileum, ovary, oviduct, and testis, demonstrated a distribution pattern of hMR (Fig. 2⇓A and data not shown) consistent with that previously described in human tissues (5, 15) and with the reported expression of muMR (5, 41, 42). These cells were distributed in capsular, trabecular, and interstitial connective tissues, as well as around vessels. In particular, the specific immunoreactivity with mAb B11 was observed in sinusoidal endothelium in the spleen and liver, as well as tissue-specific Mφ, such as alveolar Mφ in the lung, and Kupffer cells in the liver. Immunofluorescence studies demonstrated colocalization of B11 in MHC class II⁺ APCs of the skin which likely represent dermal Mφ and DCs (Fig. 2⇓B).

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FIGURE 1.

hMR transgene and its expression in mice. A, Schematic map of the 211-kb BAC clone corresponding to the hMR. Exons are indicated by vertical lines. The arrowheads beneath the map indicate positions of PCR primers used for sequence confirmation and PCR genotyping. Black arrowheads are specific to exon sequences; gray arrowheads correspond to intronic or nontranscribed regions. B, PCR genotyping and RT-PCR amplification of mRNA. Left panel, Primer combinations as defined in Fig. 1⇓A were used on genomic tail DNA from F₁ mouse 44 and are indicated over the lanes. M, phiX 174 + HaeIII markers; (−), no input primers. Middle and right panels: 1, no input RNA; 2, human placental poly(A)⁺ RNA; 3, F₁ mouse 44 BM total RNA; 4, F₁ mouse 44 spleen total RNA; 5 and 6, non-Tg spleen total RNA; 7, F₁ mouse 43 BM total RNA; 8, F₁ mouse 43 spleen total RNA; M, phiX 174 + HaeIII markers. C, Western blot analysis with Abs specific for hMR (15-2) or muMR (MR5D3) on cell lysates from PEC-Mφ derived from hMR Tg and WT mice, and human DCs. Precision plus protein standards were included as molecular mass markers. The band corresponding to hMR and muMR are indicated with arrowheads. D, Cell surface expression of MR. Cells from hMR Tg and WT mice were stained with hMR-specific (19.2-PE) or muMR-specific (MR5D3-PE) Abs (bold line for PEC-Mφ and shaded histograms for BM-derived DC). Mouse IgG1-PE and rat IgG2a-PE were used as isotype controls. PEC-Mφ were the gated population that were negative in lineage markers Ter-119, Gr-1, CD3ε, and B220, while BM-derived DC were gated on all viable cells.

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FIGURE 2.

In vivo targeting of hMR. A, Immunohistochemical localization of hMR expression in humans and hMR Tg and WT mice. Comparable expression patterns in hMR Tg mice, but not WT mice, are seen in human skin and lymph node. B, hMR is expressed by MHC class II⁺ cells in the skin. Acetone-fixed cryostat sections were stained with FITC-conjugated anti-hMR and biotinylated anti-MHC class II mAb, and subsequently incubated with Alexa 488 goat anti-FITC Ab or Cy3-strepavidin. C, Draining lymph node and the skin of injection site were harvested from hMR Tg mice 24 h after intradermal administration of anti-hMR mAb B11 (5 μg). Staining with rabbit anti-human IgG demonstrates prominent B11 uptake in the epidermis and in the lymph node, specifically the capsular sinus and in medullary structures.

APC targeting with the anti-hMR mAb B11

To accomplish specific targeting of the hMR in vivo the anti-hMR mAb, B11 was injected s.c. into WT and hMR Tg mice. In the draining lymph node of Tg mice, hMR-targeted cells were mainly localized to medullary sinusoids and cords, and particularly in a subset of scattered clusters of cells in paracortical regions (Fig. 2⇑C). In the splenic red pulp, hMR targeted cells were readily apparent in hMR Tg mice but not in WT mice (data not shown).

Induction of humoral responses to hMR-targeted Ag

hMR Tg mice and their WT littermates were immunized with B11 to determine the effect of MR targeting on humoral immunity (Fig. 3⇓A). Administration of 10 μg of B11 i.p. with a single immunization, even in the absence of adjuvant, was sufficient to generate high-titered anti-B11 IgG responses in hMR Tg mice that were further inducible with boosting with second and third rounds of immunizations. Immunological memory in hMR Tg mice was apparent since high titered responses were induced upon recurrent immunization, some 3 mo after the final boost. Under the same stimulations, anti-B11 human responses were undetectable in WT mice demonstrating hMR targeting.

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FIGURE 3.

Antigenic targeting to MR enhances humoral responses. A, B11 mAb 10 μg without adjuvant was administered i.p. on days indicated by arrows. Serum anti-B11 titers were measured by ELISA at the indicated time points. Solid and dashed lines represent individual hMR Tg and WT mice (10 mice/group), respectively. B, Induction of anti-Id responses in hMR Tg mice. A total of 10 μg of mAb 19.2 mixed with 100 μl of Ribi was administered i.p. on days indicated by arrows. The anti-19.2 F(ab′)₂ titers were analyzed by ELISA using a murine IgG-Fc-specific secondary Ab. Solid and dashed lines represent individual hMR Tg and WT mice (six mice per group), respectively.

Enhancement of humoral responses was also investigated using the murine anti-hMR mAb 19.2. The Id of murine mAb can serve as a model of a poorly immunogenic Ag in mice, as the induction of anti-Id immune responses generally requires conjugation to an immunogenic carrier protein such as keyhole limpet hemocyanin or genetic fusion with GM-CSF (43, 44). Humoral responses to 19.2 mAb administered with Ribi adjuvant were readily generated in hMR-Tg but not WT mice (Fig. 3⇑B). The anti-19.2 IgG titers were predominantly Id-specific, based on specificity for 19.2 and the ability to block 19.2 binding to human monocyte-derived DC (data not shown). Importantly, the anti-Id titers were boosted by subsequent immunizations, despite the presence of potentially neutralizing Abs. Taken together, these data confirm that targeting to hMR with a human or a murine mAb can enhance humoral immunity to the targeted Ag, presumably by enhancing the efficiency of Ag uptake, processing, and presentation.

hMR-targeted Ag accesses the exogenous and cross-presentation pathway in vivo

To directly assess the ability of hMR targeting to promote T cell priming in vivo, a B11-OVA fusion protein was genetically engineered containing an in-frame fusion of the entire OVA cDNA at the C terminus of the B11 Ig H chain. This produced a fusion protein of the predicted 232 kDa, containing two OVA molecules per B11 IgG1 molecule. Generation of the B11-OVA fusion molecule enabled the use of immunization models using naive Tg OT-I and II cells, two well-characterized Tg TCR systems that, respectively, allow evaluation in vivo of MHC class I- and II-restricted OVA-specific T cell priming.

C57BL.6 recipients of either CFSE-labeled naive TCR Tg OT-I CD8 or OT-II CD4 cells were immunized i.p. with either 1 or 10 μg of B11-OVA. In WT mice, OT-I cells failed to proliferate with either dose of B11-OVA (Fig. 4⇓). In contrast, B11-OVA immunization induced robust and dose-dependent OT-I proliferative responses in hMR Tg mice; 14 and 91% of OT-I cells had divided in hMR Tg mice injected with either 1 or 10 μg of B11-OVA, respectively. CD4 OT-II proliferative responses were not detected with 1 μg of B11-OVA in either WT or hMR Tg mice but when mice were immunized with 10 μg of B11-OVA, OT-II proliferative responses were strongly induced in hMR Tg mice (81% divided) but not in WT mice. Thus, hMR targeting accesses both the exogenous and cross-presentation pathway in vivo allowing priming of both MHC class I- and MHC class II-restricted T cell responses.

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FIGURE 4.

hMR-targeted Ags access the exogenous and cross-presentation pathway in vivo. WT (gray) or hMR Tg (black) mice were injected with either CFSE-labeled OT-I or OT-II T cells and subsequently injected i.p. with PBS, 1 or 10 μg of B11-OVA. Splenocytes were obtained 72 h later for flow cytometric analysis. Percentages of divided OT-I or II cells are shown. Data representative of three mice/group are shown. Immunization with B11-OVA-induced OT-I and OT-II brisk proliferative responses occurred in hMR Tg mice and were absent in WT mice.

hMR targeting induces adjuvant-dependent effector T cell priming

Targeting of APCs in vivo using another Ag uptake receptor, DEC-205, resulted in T cell proliferation without concomitant effector differentiation (34). To assess whether OT-I cell proliferative responses were accompanied by effector CD8 differentiation in hMR-targeted mice, WT or hMR Tg mice OT-I recipients were immunized i.p. with 10 μg of B11-OVA either in the presence or absence of the TLR 9 ligand CpG as adjuvant. After 5 days, splenocytes were restimulated with OT-I peptide, and IFN-γ production evaluated by intracellular staining to assess OT-I effector differentiation (Fig. 5⇓A). In WT mice, there was no evidence of increased OT-I effector differentiation after immunization with B11-OVA in either the presence or absence of CpG. In contrast, there was robust induction of IFN-γ production by OT-I cells in hMR Tg mice immunized with B11-OVA in the presence of CpG. Notably, immunization of hMR Tg mice with B11-OVA alone failed to induce OT-I IFN-γ production. Thus, despite the robust proliferative response seen after immunization of hMR Tg mice with B11-OVA alone (Fig. 4⇑) there was no concomitant enhancement of IFN-γ effector differentiation, which instead required addition of the TLR 9 agonist CpG.

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FIGURE 5.

Adjuvants enhance hMR-targeted immune responses. A, CpG enhances OT-I effector differentiation. OT-I recipients were immunized i.p. with 10 μg of B11-OVA with or without 50 μg of CpG. Splenocytes were harvested 5 days later, restimulated for 4 h with OT-I (257–264) peptide, and stained intracellularly for IFN-γ production. Data are expressed as the percent of IFN-γ⁺ producers among CD45.1⁺, CD8⁺ gated input cells. B, Ribi adjuvant enhances Ab responses upon low dose of targeting Ag. B11 mAb (0.1 μg) with or without Ribi was administered i.p. at 2-wk intervals in hMR Tg and WT mice. Anti-B11 titers were measured by a murine total IgG-specific ELISA on sera harvested 1 wk after the third immunization. Each dot denotes an individual mouse and the experiment is representative of three separate studies. C, Ribi adjuvant promotes Th1-type humoral responses. The identical sera of immunized mice were analyzed by ELISA using IgG1 and IgG2a subclass-specific secondaries. Although anti-B11 responses of the IgG1 subclass were produced upon B11 immunization in the presence or absence of Ribi adjuvant, IgG2a responses were only induced in Ribi recipients.

To evaluate whether the addition of a strong adjuvant could enhance the hMR-targeted humoral response, WT and hMR Tg mice were immunized once with a low dose of B11 (0.1 μg). In WT mice no detectable humoral responses were detectable even in the presence of a strong adjuvant (Fig. 5⇑B). Robust humoral responses occurred in hMR Tg mice with IgG1 being the predominant isotype in the absence of adjuvant. However, in the presence of adjuvant (Ribi) humoral responses were quantitatively and qualitatively enhanced (Fig. 5⇑C). Importantly, induction of significant IgG2a titers required immunization together with adjuvant. Thus, hMR targeting induces strong humoral responses, however, Th1-type isotype switched responses requires addition of adjuvant.

T cell priming in vivo by hMR targeting requires DCs

DCs have been implicated as the relevant cross-presenting cell for effector CD8 T cell priming. Multiple potential APCs express MR in vivo including DC. To determine the contribution of DCs to hMR-targeted priming, hMR Tg mice were mated with CD11c-DT receptor (DTR) Tg mice that express a human high-affinity DTR on the surface of CD11c⁺ cells, making them specifically sensitive to DT-mediated apoptosis (40). Using this DC depletion model, DCs have been previously shown to be required for the cross-presentation of cell-associated (40) and soluble exogenous Ag (45), as well as for the presentation of bacterial (40) and viral Ags (46, 47). Preliminary experiments in DT-treated WT and CD11c-DTR mice, showed that transferred OT-I cells accumulated and localized to the T cell zones of the lymph nodes and spleen in comparable numbers regardless of the presence or absence of CD11c⁺ cells (data not shown). Therefore, DT-treated hMR Tg mice and hMR Tg/CD11c-DTR mice were transferred with 5 × 10⁶ CFSE-labeled OT-I cells, injected with B11-OVA and proliferation was assessed after 3 days. Strikingly, no proliferation of the OT-I cells was seen in DC-depleted recipient mice (Fig. 6⇓). Although roles for subpopulations of marginal zone splenic macrophages cannot be ruled out (48), these data are consistent with a major contributing role for DCs in hMR-mediated T cell priming in vivo. We confirmed the ability of hMR targeted Ag to mediate MHC class I-restricted Ag presentation by BMDCs in vitro, although the induction of OT-I proliferative responses in vitro by B11-OVA Ab was not seen without the addition of CpG (data not shown).

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FIGURE 6.

hMR-targeted cross-presentation requires DCs in vivo. A, Flow cytometric analysis of splenocytes isolated from DT-treated or untreated hMR Tg/CD11c-DTR mice 3 days posttransfer of CFSE-labeled OT-I cells alone or together with 10 μg of B11-OVA (right panels). Histograms are gated on CD8⁺CFSE⁺ cells and represent one of three independent experiments. OT-I cell proliferative responses were abolished in the absence of DCs. B, hMR Tg BMDCs mediate cross-presentation by B11-OVA. BMDCs cultured from BM precursors for 7 days were treated with 10 μg/ml CpG and cultured with OT-I CD8 Tg T cells (DC:T ratio of 1:20) and graded doses of soluble OVA or B11-OVA mAb. Thymidine incorporation was measured after 36 h. In the absence of CpG, hMR Tg BMDCs failed to induce detectable B11-OVA-mediated OT-I proliferation (data not shown).

hMR-targeted Ag can induce protective tumor immunity

The capacity of B11-OVA vaccines to target hMR-dependent Ag-specific CD8 responses was further evaluated in tumor immunity models. WT and hMR Tg mice were immunized twice with 10 μg of B11-OVA in the presence or absence of CpG. One week following the secondary immunization, OVA-specific T cell responses were evaluated with IFN-γ ELISPOTs. Consistent with the results seen using the OT-I system, IFN-γ responses only occurred in hMR Tg mice immunized with both B11-OVA and CpG (Fig. 7⇓A). Humoral responses of the IgG1 subclass were induced in all hMR Tg mice immunized with B11-OVA, however, as was the case with Ribi adjuvant (Fig. 5⇑), IgG2a responses required concomitant immunization with CpG (data not shown). Thus, B11-OVA and CpG immunization induced cellular effector responses and this immunization protocol was further evaluated in a tumor prevention model. Mice were immunized twice with B11-OVA and CpG and challenged s.c. with 2 × 10⁵ MO-4 cells, a B16F10 melanoma OVA transfectant. In WT mice, tumors grew rapidly in all mice regardless of immunization strategy. However, in hMR Tg mice significant tumor growth inhibition was exhibited in mice immunized with B11-OVA and CpG adjuvant (p = 0.009 vs no treatment) (Fig. 7⇓B). Thus, immunization strategies incorporating both adjuvant and hMR targeting of tumor Ag can induce protective tumor immunity.

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FIGURE 7.

hMR-targeted vaccines induce tumor immunity. A, IFN-γ effector responses after B11-OVA immunization requires addition of adjuvant. Mice were immunized twice with 10 μg of B11-OVA, 50 μg of CpG, or both on days −21 and −7; splenocytes were harvested on day 0. Splenocytes were stimulated with either no Ag or 300 μg/ml OVA overnight and IFN-γ production was assessed by ELISPOT. Data are expressed as the mean of three wells. B, Mice were immunized as above and challenged s.c. with B16-OVA tumor cells. Mean tumor sizes of five mice per group are shown. Tumors in mice immunized with B11-OVA in combination with CpG were significantly smaller on day 18 (p = 0.009, Student’s t test (one-sided)).