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[Cancer Research 66, 6843-6850, July 1, 2006]
© 2006 American Association for Cancer Research

Immunology

CD1d-Restricted T Cells License B Cells to Generate Long-Lasting Cytotoxic Antitumor Immunity In vivo

Yeonseok Chung, Byung-Seok Kim, Yeon-Jeong Kim, Hyun-Jeong Ko, Sung-Youl Ko, Dong-Hyeon Kim and Chang-Yuil Kang

Laboratory of Immunology, Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Kwanakgu, Seoul, Korea

Requests for reprints: Chang-Yuil Kang, Laboratory of Immunology, College of Pharmacy, Seoul National University, Shillim-9-dong, Kwanakgu, Seoul 151-742, Korea. Phone: 82-2-880-7860; Fax: 82-2-885-1373; E-mail: cykang{at}snu.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although resting B cells are known for being poorly immunogenic and for inducing T-cell tolerance, we have here attempted to test whether their immunogenicity could be enhanced by CD1d-restricted invariant T cells (iNKT) to a point where they could be used in cellular vaccines. We found that the addition of the iNKT ligand {alpha}-galactosylceramide ({alpha}GalCer) to peptide-loaded B cells overcame peptide-specific T-cell unresponsiveness and allowed for the generation of peptide-specific memory CTL immunity. This CTL was induced independently of CD4 T and natural killer cells but required iNKT and CD8 T cells. B cells directly primed CTL, and the {alpha}GalCer and the peptide must be presented on the same cell. Importantly, our B-cell–based vaccine is comparable in efficiency with dendritic cell–based vaccines, inducing similar CTL responses as well as providing an effective regimen for preventing and suppressing s.c. and metastatic tumors. Therefore, with the help of iNKT, peptide-pulsed B cells can establish long-lasting antitumor immunity and so show promise as the basis for an alternative cell-based vaccine. (Cancer Res 2006; 66(13): 6843-50)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Of the available vaccine approaches, cellular vaccines using antigen-presenting cells (APC), such as dendritic cells (DC), are reliable at generating effective T-cell immunity (1). DCs are ideal APCs for immunotherapy because they can capture antigen and then migrate into lymphoid organs where they present the antigen to the relevant T cell. More importantly, they provide strong costimulation to the T cells (2, 3). The DC-based vaccine approach is well established for both experimental and clinical studies. However, DCs are relatively scarce in blood and lymphoid tissues, and it is difficult to increase their numbers ex vivo from blood monocytes, both of which present major drawbacks to their widespread use in vaccines (4).

B cells offer an attractive alternate source for cellular vaccines in that they are abundant in lymphoid tissues and blood (4), easily expanded ex vivo (5, 6), and home to lymphoid organs after parenteral administration. Despite these advantages, B cells have been ignored as a vaccinating APC because they are poorly immunogenic. In fact, accumulating evidence shows that they induce T-cell tolerance in both CD4 and CD8 T cells directly, probably due to the lack of costimulation (7–9). However, ‘activated’ B cells can prime both CD4 and CD8 T cells (5, 6, 10–13), suggesting that, when activated by the appropriate stimuli, B cells can act as immunogenic APCs capable of inducing antigen-specific T-cell immunity.

It is well established that iNKT cells play a crucial role in a variety of immune responses and in immunopathology as a whole (14, 15). They act as regulators of immunity in tumor (16), diabetes (17, 18), and at immune-privileged site (19). In sharp contrast, ligand-activated iNKT cells lead to the activation of T, B, and natural killer (NK) cells as well as DCs. Injection of {alpha}-galactosylceramide ({alpha}GalCer), an iNKT ligand, generates antitumor immunity via the mediation of NK and T cells (20). Mice, to which protein antigen and {alpha}GalCer have been cogiven, develop humoral and cell-mediated immunity, including CTL responses (21, 22). Interestingly, Crowe et al. (23) has recently reported that CD4– iNKT cells in liver could induce better antitumor immunity than CD4+ iNKT in liver or iNKT from other organs. Furthermore, Terabe et al. (24) showed that type II NKT population negatively regulates antitumor immunity. Thus, it is likely that respective NK T-cell (NKT) subsets in different lymphoid organs possess their own novel function in vivo. Moreover, a recent study has shown that {alpha}GalCer-loaded DCs generate longer lasting iNKT cell responses than does free form of {alpha}GalCer (25). Based on these findings, we hypothesized that presentation of the iNKT ligand on B cells could convert them from tolerogenic to immunogenic, thereby generating strong immunity against antigen displayed on MHC molecules of the B cells. To test this hypothesis, we determined the efficiency of {alpha}GalCer-loaded, peptide-pulsed B cells in generating cytotoxic immunity and antitumor activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and antibodies for depletion. Female C57BL/6 and BALB/c mice (Charles River, Seoul, Korea) were used at 6 to 10 weeks of age. The OT-I and C57BL/6bm1 (bm-1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME), whereas J{alpha}281–/– and MHC II–/– mice were kindly provided by Dr. Doo-Hyun Chung (Seoul National University, Seoul, Korea) and Dr. Se-Ho Park (Korea University, Seoul, Korea), respectively. All mice were kept under specific pathogen-free conditions in the Animal Center for Pharmaceutical Research (Seoul National University). Antibodies from hybridomas [i.e., GK1.5 (anti-CD4), 2.43 (anti-CD8), and PK136 (anti-NK1.1)] were obtained and injected i.p. (150 µg/mouse) to deplete the respective lymphocyte subsets in vivo. All animal studies were approved by the Institutional Animal Care and Use Committee of Seoul National University.

Loading {alpha}GalCer and peptide on B cell or DC. B220+ cells were purified from splenocytes using microbeads once CD11c+ cells had been depleted by anti-CD11c microbeads. These cells were >99% CD19 positive. DCs were purified from spleen by treatment with collagenase IV and DNase I and by density gradation followed by sorting with CD11c microbeads as described previously (26).

Purified cells (B cells or DCs) were cocultured with {alpha}GalCer (1 µg/mL), which had been prepared as described previously (27), or the vehicle (0.5% polysorbate) for 14 hours in a CO2 incubator. In some experiments, these cells were also additionally cocultured with ovalbumin257-264 peptide or HER-2/neu63-71 for 1 hour. After extensive washing, these cells were i.v. injected into their syngenic mice or cocultured with a NKT hybridoma (DN32.D3).

Carboxylfluorescein diacetate succinimidyl ester–labeled OT-I adoptive transfer study. Ovalbumin-specific CD8 T cells (>90% of which were V{alpha}2 positive) were isolated from OT-I mice using magnetic beads. These cells were labeled with 10 µmol/L carboxylfluorescein diacetate succinimidyl ester (CFSE) and i.v. transferred into their syngenic mice (26). On the following day, mice were i.v. injected with B cells manipulated in vitro with indicated conditions. Forty-eight hours later, lymphoid cells from the lymph nodes or spleen of the recipient mice were stained with phycoerythrin (PE)-conjugated anti-V{alpha}2 antibody and then analyzed by flow cytometry.

Intracellular cytokines staining. For detection of intracellular cytokines in OT-I T cells, lymphoid cells were stimulated for 6 hours in RPMI 1640 supplemented with 10% FCS and Golgistop in the presence or absence of 1 µmol/L ovalbumin257-264. Cells were permeabilized with Cytofix/Cytoperm reagents in accordance with the manufacturer's recommendations before being stained with PE-conjugated anti-IFN-{gamma} monoclonal antibody (mAb) or PE-conjugated anti-CD25 antibody (BD PharMingen, San Diego, CA). For intracellular staining of IFN-{gamma} in iNKT cells, splenocytes from mice injected with vehicle-pulsed B cells (B/veh) or {alpha}GalCer-loaded B cells (B/{alpha}GalCer) were stained with {alpha}GalCer/CD1d-multimer together with PE-Cy5-conjugated anti-T-cell receptor ß (TCRß) antibody. These cells were permeabilized and stained with APC-conjugated anti-IFN-{gamma} antibody.

In vivo and in vitro cytotoxicity assay. The in vivo ovalbumin-specific cytolytic activity of CD8 T-cell responses was measured using flow cytometry. Syngenic lymphocytes were either loaded with 1 µmol/L peptides or left untouched before being labeled with CFSE at different concentrations (20 and 2.5 µmol/L, respectively). Equal numbers of the two populations were mixed and injected i.v. into mice. Eighteen to 24 hours later, lymphoid cells from spleen and lymph nodes were analyzed to assess peptide-specific killing. The ovalbumin-specific lysis was calculated as follows: r = % CFSElow / % CFSEhigh and % lysis = [1 – (runprimed / rprimed)] x 100, where r is the ratio. In vitro cytolytic activity against EG-7 target cells was measured using a standard 51Cr release assay as described previously (28).

Detection of a peptide-specific CTL population. The population of peptide-specific CTL was calculated based on IFN-{gamma}-producing CD8 T cells induced in response to ovalbumin257-264 (26). Briefly, cells from spleen were stimulated with 1 µmol/L ovalbumin257-264 for 4 hours in the presence of 1 µg/mL GolgiPlug. Cells were fixed, permeabilized, stained with antibodies to mouse IFN-{gamma}-PE and CD8-FITC, and then analyzed by flow cytometer.

Preventive and therapeutic tumor model. Ovalbumin-transfected B16 melanoma (MO-5, kindly provided by Dr. Kenneth Rock, University of Massachusetts, Worcester, MA) and HER-2/neu-transfected CT26 colon carcinoma (CT26-HER-2/neu; ref. 29) were used as model tumors. To test preventive effects, C57BL/6 mice were given various cellular vaccines at day 0 and then 2 x 105 MO-5 were s.c. injected into their left flank on day 7. To test therapeutic effects, mice received 2 x 105 MO-5 on day 0 and were then given cellular vaccines on days 1 or 9. In the HER-2/neu tumor model, BALB/c mice were challenged i.v. or s.c. with 2 x 105 CT26-HER-2/neu, causing them to develop tumors. The tumor-bearing mice were then vaccinated with B-cell-based or DC-based cellular vaccines, and rates of survival or tumor growth were measured. In some experiments, tumor-free mice were further inoculated s.c. with the same tumor and tumor growth was monitored.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bidirectional activation of B/{alpha}GalCer and iNKT cells in vivo. Because it was already well established that {alpha}GalCer-loaded DCs activate iNKT cells (25), we first examined whether B/{alpha}GalCer would do likewise. After depleting CD11c+ cells from the splenocytes to remove DC contamination, we isolated pure B cells using anti-B220 microbeads. The sorted cells were CD19+, CD1d+, but CD11c– (>99%; Fig. 1A ). Primary DCs were isolated from spleen (26). These purified B cells or DCs were pulsed with various concentrations of {alpha}GalCer and then cocultured with a NKT hybridoma, DN32.D3 (30). B/{alpha}GalCer efficiently stimulated DN32.D3 cells to produce interleukin (IL)-2 (Supplementary Fig. S1A), equaling the rate of the DC group of IL-2 production when at higher ratios to the hybridoma but falling short when at lower ratios (Supplementary Fig. S1B).


Figure 1
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  		Figure 1. In vivo reciprocal activation between iNKT cells and B/{alpha}GalCer. A, B cells were purified from spleen using anti-B220 beads after depletion of CD11c+ cells. The expression of CD19, CD11c, and CD1d was measured by flow cytometry. Shaded histograms, isotype controls. B, C57BL/6 mice were injected i.v. with B/veh or B/{alpha}GalCer (2 x 106 per mouse). Six hours later, TCRß+CD1d-dimer+ cells among total splenocytes were analyzed for the expression of IFN-{gamma} by intracellular cytokine staining and flow cytometric analysis. Shaded histogram, isotype control. C, B/veh or B/{alpha}GalCer were stained with 10 µmol/L CFSE and i.v. injected into C57BL/6 mice. After 24 hours, CFSE+ cells from splenocytes were analyzed. B and C, numbers, mean fluorescence intensity. Data are representative of two separate experiments.

 
To examine if B/{alpha}GalCer could directly induce iNKT activation in vivo, we injected B/{alpha}GalCer or B/veh i.v. into syngenic mice and measured the level of intracellular IFN-{gamma} in iNKT cells. Indeed, injection of B/{alpha}GalCer stimulated iNKT to produce IFN-{gamma} whereas B/veh did not (Fig. 1B). Consistent with this finding, we observed the induction of IL-4 and IFN-{gamma}-producing cells on B/{alpha}GalCer injection in wild-type (WT) mice but not in J{alpha}281–/– mice, showing the activation of iNKT cells by B/{alpha}GalCer in vivo (Supplementary Fig. S1C).

To determine what, if any, changes were induced in B cells after injection, we next i.v. injected CFSE-labeled B/{alpha}GalCer into syngenic mice and then analyzed the costimulatory molecules on the CFSE+ cells. Flow cytometric analysis of B cells revealed that {alpha}GalCer minimally affected the level of CD40, CD80, and CD86 on B cells during the in vitro pulsing period (Supplementary Fig. S1D). Interestingly, high levels of CD86 but not CD80 expression were induced within 24 hours (Fig. 1C). CD40 and MHC II were also slightly up-regulated. Even 48 hours after injection, no up-regulation of CD80 was observed, whereas all other results remained largely consistent with the 24-hour level (data not shown). Therefore, both DC/{alpha}GalCer and B/{alpha}GalCer are capable of activating iNKT cells both in vitro and in vivo.

Peptide-pulsed B/{alpha}GalCer promotes the activation of peptide-specific CD8 T cells. Next, we addressed whether copulsing of {alpha}GalCer and MHC I–restricted peptide on B cells could prime peptide-specific CD8 T cells. To this end, we first adoptively transferred CFSE-labeled ovalbumin-specific CD8 T cells (OT-I) into C57BL/6 mice and then i.v. injected B/veh (B alone), B/{alpha}GalCer, vehicle plus peptide-pulsed B cells (B/pep), or {alpha}GalCer plus peptide-pulsed B cells (B/{alpha}GalCer/pep), respectively.

As expected, little division of OT-I cells was induced in mice receiving B alone or B/{alpha}GalCer (Fig. 2 ). Injection of B/pep induced a substantial division of OT-I, but very few of these cells produced IL-2 (<4%) and some population (38%) produced IFN-{gamma} after restimulation ex vivo. By contrast, mice given B/{alpha}GalCer/pep showed an enhanced division of OT-I, with >40% of the resultant cells producing IL-2 and, most surprisingly, >90% producing IFN-{gamma} at much higher levels than the B/pep group. These results suggest that a far higher rate of CD8 T-cell activation could be achieved by the loading of {alpha}GalCer onto B/pep.


Figure 2
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  		Figure 2. B cells copulsed with {alpha}GalCer and ovalbumin257-264 generate long-lasting cytotoxic T-cell immunity. A, CFSE-labeled OT-I T cells were transferred into C57BL/6 mice. On the following day, the recipient mice were injected i.v. with B cells (1 x 106 per mouse) after culture with the indicated condition. Top, 48 hours later, lymphoid cells were obtained from the spleen and CFSE dilution. Numbers, number of cell divisions. Lymphoid cells were further stimulated with ovalbumin257-264, and intracellular IL-2 (middle) or IFN-{gamma} (bottom) was measured. B, C57BL/6 mice were vaccinated with the indicated form of B cells. One, 3, or 5 weeks later, in vivo CTL assays were done by injecting CFSE-labeled syngenic targets (see Materials and Methods). CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. C, C57BL/6 mice (four per group) were vaccinated as in (B), and the response of IFN-{gamma}-producing CD8 T cells to ovalbumin257-264 was calculated 7 days after vaccination (left) or 7 days after additional CTL priming with ovalbumin-loaded syngenic splenocytes (right). Points, mean; bars, SE. *, P < 0.05, compared with B alone control. Data are representative of three separate experiments.

 
B/{alpha}GalCer/pep induces long-lasting cytotoxic T-cell responses. In the next study, we asked if our B-cell-based vaccine approach could induce cytotoxic immunity. To answer this question, we injected i.v. groups of C57BL/6 mice with B alone, B/{alpha}GalCer, B/pep, or B/{alpha}GalCer/pep and then determined in vivo CTL activity. As shown in Fig. 2B, only B/{alpha}GalCer/pep completely lysed peptide-pulsed targets and, to our surprise, it also maintained complete cytotoxicity even 5 weeks after a single vaccination. Echoing this finding, only the B/{alpha}GalCer/pep-treated group showed a significant increase in the number of IFN-{gamma}-producing CD8 T cells against the peptide (Fig. 2C, left).

In another experiment, we additionally primed the B-cell-vaccinated mice with ovalbumin-coated syngenic splenocytes to delineate the CTL responsiveness against subsequent immunization with the same antigen. Mice given B alone or B/{alpha}GalCer responded normally toward the priming and generated substantial peptide-specific IFN-{gamma}-producing CD8 T cells (Fig. 2C, right). However, mice given B/pep showed no increase in the number of IFN-{gamma}-producing CD8 T cells, suggesting that these mice were tolerant of the peptide. By contrast, mice vaccinated with B/{alpha}GalCer/pep displayed far greater peptide-reactive CD8 T cells than did either the group receiving B/{alpha}GalCer or B alone, suggesting that this is a recall response. Based on these collective findings, we concluded that the loading of {alpha}GalCer on B/pep generated long-lasting memory cytotoxic immunity.

B/{alpha}GalCer/pep is as efficient a generator of CTL as DC/{alpha}GalCer/pep or DC/pep. We next sought to compare the efficacy of our B-cell-based vaccine strategy at generating cytotoxicity with that of DC-based vaccine. To this end, we determined the minimum cell number required for achieving complete target lysis in vivo. Serial dilutions of B/{alpha}GalCer/pep or DC/{alpha}GalCer/pep were i.v. injected into syngenic mice, and an in vivo CTL assay was done. As depicted in Fig. 3A , mice injected with DC/{alpha}GalCer/pep showed complete target cell lysis with as few as 16,000 cells. Of interest, a single vaccination with 80,000 B/{alpha}GalCer/pep cells was enough to establish a complete peptide-specific lysis, whereas vaccination with 16,000 cells generated a moderate cytotoxicity. However, given that the surface area of DCs is far larger than that of B cells, B/{alpha}GalCer/pep may be as efficient as DC/{alpha}GalCer/pep in generating cytotoxicity.


Figure 3
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  		Figure 3. Comparative efficiency of B-cell-based and DC-based cellular vaccine. B cells and DCs were isolated from the spleens of C57BL/6 mice and then pulsed with ovalbumin257-264 plus {alpha}GalCer ({alpha}GC; A) or peptide alone (pep; B). Left, serial dilutions were i.v. given into syngenic mice. Seven days later, in vivo cytotoxicity against ovalbumin257-264 was measured. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. Nil, no treatment before receiving targets. Data are representative of three separate experiments.

 
DC/pep efficiently generated ovalbumin-specific cytotoxicity, whereas B/pep treatment, regardless of the number of cells tested, did not (Fig. 3B). Of note, the pattern of in vivo cytotoxicity of the DC/pep-treated group was very similar to that of the DC/{alpha}GalCer/pep-treated group, indicating that the loading of {alpha}GalCer onto DCs did not further enhance the vaccine efficacy of DC/pep in the system.

Generation of CTL by B/{alpha}GalCer/pep does not require CD4 T or NK cells but requires CD8 T and iNKT cells. We next examined which types of immune cell were involved in the generation of the CTL response. We injected mice with anti-CD4, anti-CD8, or anti-NK1.1 antibodies 4 days before or 4 days after vaccination with B/{alpha}GalCer/pep. Flow cytometric analysis showed that these antibodies efficiently depleted their respective populations in vivo (Supplementary Fig. S2). As it turned out, the timing of depletion made no difference, as the generation of CTL activity was not hampered in either case by the depletion of CD4+ or NK1.1+ cells (Fig. 4A ). It is noteworthy that, although the injection of anti-NK1.1 antibody depleted NK1.1+ cells, {alpha}GalCer/CD1d-dimer-positive cells were still detectable (Supplementary Fig. S2). As expected, CD8 depletion completely blocked the killing of target cells. Consistent with these results, MHC II–/– mice (lacking CD4 T cells) developed normal CTL responses, whereas J{alpha}281–/– mice (lacking iNKT cells) failed to do so (Fig. 4B). In short, the generation of the CTL immunity required both CD8 T and iNKT cells but not CD4 T nor NK cells.


Figure 4
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  		Figure 4. Mechanism for the generation of cytotoxicity by B-cell-based vaccine. A, B cells copulsed with {alpha}GalCer and ovalbumin257-264 (1 x 106 per mouse) were i.v. injected into C57BL/6 mice at day 0. The recipient mice received anti-CD4, anti-CD8, anti-NK1.1 depleting mAbs, or rat IgG as a control (con IgG) on days –4 (before depletion) or 4 (after depletion). These mice were tested for in vivo cytotoxicity against ovalbumin257-264. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. B, WT, J{alpha}281–/–, or MHC II–/– mice were i.v. injected with B cells copulsed with {alpha}GalCer and peptide. Splenocytes were restimulated with EG-7 for 5 days, and cytotoxicity against ovalbumin257-264 was measured using a standard 51Cr release assay. C, B cells from WT or bm-1 mice were copulsed with {alpha}GalCer and ovalbumin257-264 before being i.v. injected into WT mice. A week later, an in vivo CTL assay was done. D, C57BL/6 mice were vaccinated with B cells copulsed with {alpha}GalCer and ovalbumin257-264 (1 x 106 per mouse) or ‘a combination of B cells pulsed with ovalbumin257-264 and of B cells pulsed with {alpha}GalCer’ (1 x 106 each). A week later, an in vivo CTL assay was done. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. Data are representative of two separate experiments.

 
B cells act as real APCs rather than a peptide reservoir, and loading of {alpha}GalCer and peptide on the same B cell is required for CTL generation. It could be argued that peptide-pulsed B cells in our model act not as APC but as reservoirs of peptide from which the host DCs withdraw peptides to induce CTL responses. To explore this possibility, we used bm-1 mice model. The cells of these mice can load ovalbumin257-264 peptide onto their MHC I, but the resulting complex is not recognized by the cognate CD8 T cells due to a mutation in the H-2K region (31). As shown in Fig. 4C, mice vaccinated with B/{alpha}GalCer/pep derived from B cells of bm-1 mice failed to develop ovalbumin-specific cytolytic activity, suggesting that DC or other professional APC in the recipient mice was not responsible for the CTL generation in this system.

We next asked if it were possible to generate CTL when {alpha}GalCer and peptide were pulsed separately and then injected together. To this end, C57BL/6 mice were i.v. injected with ‘B/{alpha}GalCer plus B/pep’ or B/{alpha}GalCer/pep alone. As shown in Fig. 4D, mice vaccinated with B/{alpha}GalCer plus B/pep failed to generate in vivo cytotoxicity, showing that peptide and {alpha}GalCer must be presented on the same B cell to generate the cytotoxicity.

B/{alpha}GalCer/pep establishes antitumor immunity. Finally, we asked whether vaccination with B/{alpha}GalCer/pep would generate antitumor immunity. To test prophylactic antitumor activity, groups of mice were vaccinated once with B alone, B/{alpha}GalCer, B/pep, B/{alpha}GalCer/pep, DC/pep, or DC/{alpha}GalCer/pep before an ovalbumin-transfected B16 melanoma (MO-5) was transplanted s.c. into them. We observed a slightly delayed pattern of tumor growth in mice vaccinated with B/{alpha}GalCer, although all mice finally developed tumors (Fig. 5A ). In contrast, no mice receiving B/{alpha}GalCer/pep, DC/pep, or DC/{alpha}GalCer/pep developed tumor growth. To examine whether these mice established long-term antitumor activity, we rechallenged the surviving mice with s.c. MO-5 tumors s.c. 70 days after the first tumor inoculation. As depicted in Fig. 5B, we observed no tumor growth in those mice, showing that vaccination with B/{alpha}GalCer/pep established memory immunity against the tumor.


Figure 5
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  		Figure 5. B-cell-based vaccine can offer both preventive and therapeutic antitumor immunity against ovalbumin-transfected B16 melanoma. A and C to D, groups of C57BL/6 mice (five-six per group) were vaccinated with the indicated cellular vaccine at day 0. MO-5 cells (2 x 105) were s.c. injected into the mice 7 days after (A), 1 day before (C), or 9 days before (D) the vaccination. Tumor mass was measured thrice weekly. Points, mean; bars, SE. B, 70 days after the first tumor inoculation, tumor-free mice were rechallenged s.c. with MO-5 cells (2 x 105) and tumor mass was measured. *, P < 0.05, compared with B alone control group (A and C-D) or with age-matched naive mice (B). Data are representative of two or three separate experiments.

 
We next asked whether vaccination with B/{alpha}GalCer/pep would eradicate a preexisting tumor. To this end, we established two therapeutic models: mice were vaccinated (a) 1 or (b) 9 days after s.c. transplant when tumors had become palpable. In the 1-day model, vaccination with either DC/pep or DC/{alpha}GalCer/pep almost completely suppressed tumor growth (Fig. 5C). Interestingly, tumor growth was also completely diminished in mice vaccinated with B/{alpha}GalCer/pep. In the 9-day model, none of these vaccinations completely destroyed the growing tumor due to the aggressive nature of the B16 melanoma. However, in mice vaccinated with B/{alpha}GalCer/pep, tumor growth was less pronounced than in ‘B alone’ group of mice and resembled that observed in the DC/pep-vaccinated or DC/{alpha}GalCer/pep-vaccinated groups (Fig. 5D).

To determine whether this B-cell-based vaccine regimen can be applied to real tumor antigen, we chose the HER-2/neu model because this tumor antigen is well characterized and its CTL epitope is known (32). Again, we observed a significant level of HER-2/neu-specific cytotoxicity in vivo in mice given {alpha}GalCer-loaded HER-2/neu63-71-pulsed B cells (Fig. 6A ). To examine antitumor activity in this model, we injected HER-2/neu-expressing colon carcinoma (CT26-HER-2/neu; ref. 29) i.v. or s.c. into BALB/c mice before vaccinating them with HER-2/neu63-71-pulsed B/{alpha}GalCer. After i.v. tumor inoculation, survival rates were slightly better for those mice vaccinated with B/{alpha}GalCer or B/pep than those vaccinated with B alone (Fig. 6B). In sharp contrast, all mice vaccinated with B/{alpha}GalCer/pep survived the duration of the experiment. We observed very similar results in the s.c. tumor growth model (Fig. 6C). Collectively, our B-cell-based vaccine regimen proved to be as effective as DC-based vaccines in generating both prophylactic and therapeutic antitumor immunity.


Figure 6
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  		Figure 6. B-cell-based vaccine triggers therapeutic antitumor immunity against HER-2/neu-expressing tumor. A, BALB/c mice were vaccinated with B cells or DCs after coculture with {alpha}GalCer or vehicle plus HER-2/neu63-71 as indicated. One week later, in vivo CTL assays were done. Numbers, percentage of specific lysis. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. B and C, BALB/c mice were challenged i.v. (B) or s.c. (C) with CT26-HER-2/neu. The next day, these tumor-bearing mice were vaccinated with B cells or DCs as described in (A) and the survival rates (B) or tumor growth (C) of these mice was measured. Data are representative of two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although several studies have shown that iNKT cell–based vaccines induce antitumor immunity through DC-dependent mechanism (21, 22, 25, 33), we attempted to test whether B cells could be used as a source of cellular vaccine for generating antitumor T-cell immunity in this study. Our findings suggest that iNKT cells can convert tolerogenic B cells into immunogenic APCs that can generate long-lasting cytotoxic immunity. To our surprise, the CTL responses induced by our B-cell-based vaccine were comparable with those induced by DC-based vaccine in efficiency as well as prevention and suppression of tumor growth in several different tumor models. These findings suggest that B-cell-based cellular vaccines would provide an additional resource in the fight against tumors and infectious pathogens.

B cells are known as tolerogenic APCs for CD8 T cells (7). Furthermore, a recent study has shown that B cells suppress iNKT activation when {alpha}GalCer is given as free form (34), suggesting that B cells are also poorly immunogenic for iNKT cells. In the current study, however, we observed that cell-associated form of {alpha}GalCer on B cells directly activates iNKT cells in vivo as well as in vitro. This discrepancy in findings could be attributed to the different forms of {alpha}GalCer (free form versus cell-associated form). It is also possible that the density of {alpha}GalCer on B cells may affect the level of iNKT activation. B cells are activated when iNKT cells recognize the {alpha}GalCer they harbor. These ‘licensed’ B cells, probably together with other cytokines from iNKT cells, are then able to fully activate cytotoxic T cells whose TCRs are specific for the peptide presented on the surface of the same B cells.

Essential to the design of any vaccine is an antigen-specific long-term memory response. Strikingly, our B-cell-based vaccine approach triggered long-lasting memory cytotoxic immunity, whereby vaccinated mice successfully resisted rechallenge with the same tumor long after the initial tumor had been eradicated. It is generally accepted that CD4 T help is required to elicit efficient CD8 T-cell recall response. However, in the study reported here, primary CTL responses did not require CD4 T cells. Based on these findings, we tentatively propose that iNKT cells acted in place of CD4 T help producing and triggering enough signals to generate memory CTL responses.

It is important to note that cytotoxic activity in this study did not depend on NK1.1+ cells. However, we believe that NK cells weakly contributed to the suppression of tumor growth in our tumor challenge model because B cell pulsed with {alpha}GalCer alone delayed tumor growth (35). NK1.1 depletion does not mean that the entire iNKT population has been eradicated; despite their name, a segment of the iNKT population does not express NK1.1, and NK1.1+ iNKT cells down-regulate NK1.1 after activation (36–38). Thus, the activation of the NK1.1– iNKT population alone would be enough to generate equally effective CTL responses in vivo in NK-depleted mice.

Although several groups have investigated the use of CD40 agonists as adjuvants (4, 6, 10, 11), we instead tested {alpha}GalCer because (a) {alpha}GalCer activates B cells to express costimulatory molecules with the help of iNKT cells (39), (b) in our previous study, {alpha}GalCer but not CD40 agonists were shown to reverse the induction of peripheral tolerance (27, 28, 40, 41), and (c) iNKT can complement the costimulation (signal 2) provided by activated B cells by promptly producing various cytokines and chemokines (signal 3) that enhance T-cell immunity (25). When we used anti-CD40 antibody–activated, peptide-loaded B cells, we failed to observe impressive CTL activity in our system (Supplementary Fig. S3). Interestingly, we observed an impressive up-regulation of CD86 but not CD80. A recent study has shown that CD86 preferentially interacts with CD28, whereas CD80 shows a preference for CTLA-4 (42). Thus, the early expression of CD86 without CD80 on peptide-presenting B cells could contribute to the successful induction of CTL in the current model. Further studies are needed to elucidate which factors are involved in generating CTL by this B-cell-based vaccine.

Theoretically, however, DCs have unique advantages as a source for cellular vaccine because they can capture and process both particulate and soluble antigens and cross-present peptides from those antigens efficiently. Thus, our ‘B-cell-based vaccine’ approach would be useful for the defined CTL epitopes. In fact, several CD8 T-cell epitopes for tumor-associated antigen have been identified and used in clinical trials (43). Moreover, innovative techniques, such as protein transduction by TAT-fusion protein and antigen-expressing viral vector, could offset and compensate for the less efficient capture and cross-presentation of antigen by B cells, allowing them to act as CTL-stimulating APCs against antigens whose CTL epitopes are not defined (11, 12, 44). Although B cells are not a major population in blood, they can be increased exponentially ex vivo by CD40 ligation and maintained for a long period (4–6), obvious clinical assets.

To our knowledge, the current study is the first to show that, with the help of iNKT cells, B cells can induce cytotoxic immunity as well as preventive and therapeutic antitumor immunity as effectively as DCs. Because they also express an abundance of MHC II, B cell could plausibly be made to induce effector CD4 T cells using the procedure outlined above. Recent studies have shown that tumors of blood origin, such as B-cell malignancy and myeloid leukemia, expressed functional CD1d (45, 46). It will be interesting to address whether loading of NKT ligand on these tumors would lead to cytotoxic immunity against their respective natural tumor antigens. For these reasons, we believe that a cellular vaccine strategy using B cell as APC would offer a promising tool for immunotherapy against tumors and infectious pathogens.


   Acknowledgments
 
Grant support: National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea grant 0420090-1 (C-Y. Kang).

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.

We thank Dr. Albert Bendelac (University of Chicago, Chicago, IL) for DN32.D3 hybridoma, Dr. Se-Ho Park for MHC II–deficient mice, Dr. Doo-Hyun Chung for J{alpha}281-deficient mice, and Dr. Chen Dong (M.D. Anderson Cancer Center, Houston, TX) for critical review and discussion of this article.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/ 8/06. Revised 4/13/06. Accepted 5/ 1/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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