Dendritic cells pulsed or fused with AML cellular antigen provide comparable in vivo antitumor protective responses

Experimental Hematology 34 (2006) 1403–1412

Dendritic cells pulsed or fused with AML cellular antigen provide comparable in vivo antitumor protective responses Brenda J. Weigela, Angela Panoskaltsis-Mortaria, Miechaleen Diersa, a Melissa Garcia , Chris Leesa, Arthur M. Kriegb, Wei Chena, and Bruce R. Blazara a University of Minnesota Cancer Center and Department of Pediatrics, Division of Pediatric Hematology/Oncology and Blood & Marrow Transplant, Minneapolis, Minn., USA; bColey Pharmaceutical Group, Wellesley, Mass., USA

(Received 3 March 2006; revised 28 April 2006; accepted 16 May 2006)

Objective. To investigate whether syngeneic BM-derived DCs generated in vitro and fused with syngeneic C1498 tumor cells (murine AML line) could induce a better antitumor protective effect compared to similarly generated DCs pulsed with C1498 lysate with or without co-injection of a class B CpG oligodeoxynucleotide (CpG 7909) in vivo. Methods. DCs were pulsed with C1498 lysate prior to intravenous administration 14 and 7 days prior to tumor challenge. Separate cohorts received DCs electrically fused to irradiated C1498 cells. Cohorts were administered DCs that were lysate-pulsed or fused with tumor cells on days 14 and 7 prior to tumor injection. Some cohorts were co-injected with CpG 7909 at the time of DC administration. Results. All DC vaccines significantly improved survival (p ! 0.01) vs nonvaccinated controls. There was no difference in the antitumor protective response between mice that received pulsed vs fused DCs (47% vs 45% survival). Both DC vaccines generated a fivefold increase in splenic tumor-reactive cytotoxic T-lymphocyte precursor cells and significantly (p ! 0.05) higher mean frequencies of IFN-g-producing splenocytes compared to controls. CpG 7909 improved the survival of mice receiving the fused DCs (p ! 0.05) but not the pulsed DCs. Surviving mice were rechallenged and found to be resistant to lethal tumor injection. Conclusions. DC vaccine strategies may be effective in generating anti-AML responses. No advantage was observed between lysate-pulsed and tumor cell–fused DCs. CpGs may provide an adjuvant effect depending on the type of DC vaccine administered. Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) and have been used to present tumorspecific antigens as tumor vaccines [1–7]. DC vaccines are being tested as a means of inducing antitumor immune responses in patients [4–7]. Multiple sources of antigen and different methods of loading antigen on DCs have been used in an attempt to optimize antitumor responses. These have included using irradiated whole-cell suspensions, necrotic cell lysates, cellular DNA, or RNA as

Offprint requests to: Brenda J. Weigel, M.D., University of Minnesota Cancer Center, Department of Pediatrics, Division of Pediatric Hematology/Oncology and Blood & Marrow Transplant, MMC 366, 420 Delaware St. SE., Minneapolis, MN 55455; E-mail: [email protected]

well as chemical or electrical fusion of whole cells and DCs [1,3,4,8]. CpG oligodeoxynucleotides (CpG) are synthetic oligodeoxynucleotides that mimic immunostimulatory bacterial DNA [9,10]. They are unmethylated DNA sequences containing characteristic CpG dinucleotides in particular base contexts [9–11]. These CpG motifs are thought to be responsible for initiating a potent immune response in mice, nonhuman primates, and humans [12–14]. CpGs have been shown to activate APCs (particularly tissue DCs), leading to upregulation of costimulatory molecules necessary for T-cell activation and conversion to effector cytotoxic T lymphocytes (CTLs) and stimulate DC secretion of proinflammatory cytokines, particularly interferon g (IFN-g), IL-12, IL-6, TNF-a, and type I interferons

0301-472X/06 $–see front matter. Copyright Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2006.05.011

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that are necessary for effective immune responses [9,15– 17]. CpGs have been shown to have antitumor effects both when used as an adjuvant to idiotypic vaccines or as a single agent. Recently, we have shown that CpGs have potent antileukemia effects when administered as a single agent in naı¨ve and bone marrow (BM)-transplanted mice [18]. CpGs are currently in clinical trials for the treatment of lymphoma [19] and lung carcinoma [20] as well as adjuvant therapy for vaccines [21–24]. Despite major advances in the treatment of acute myelogenous leukemia (AML), the overall survival rate still is only 40 to 50%. Immunotherapy offers the potential to improve survival for patients with AML. Immunization using DCs pulsed with tumor cell lysate has been shown to be a powerful method of priming tumor-reactive T cells and inducing host protective and therapeutic antitumor immunity in mice and, more recently, in humans [2,3]. Previously, we have shown that tumor cell lysate–pulsed DCs provide significant antileukemia responses in a murine model [25,26]. An alternative approach to tumor cell lysate loading of DCs has been to fuse tumor cells with DCs, thereby providing an intracellular source of tumor antigen, avoiding the need for tumor antigen uptake and loading onto MHC class I and II molecules. Fused DCs have the cell-surface characteristics of both the tumor cell and DC. Fusion of whole tumor cells to DCs has provided an efficient and effective vaccine strategy for many tumor models [27–29]. Shimizu et al. [28], using a murine melanoma model, compared fused whole tumor:DC vaccines to lysate-pulsed vaccines administered intranodally to tumor-bearing mice. They showed that the fused cell vaccine provided the greatest antitumor response and highest level of IFN-g in the tumor-draining lymph node when IL-12 was used as adjuvant. It has recently been shown that autologous DCs fused with AML cells had a superior antitumor response in vitro compared to lysate-pulsed DCs [30,31]. Given our previous data suggesting that lysate-pulsed DCs provide significant anti-AML tumor protection and the suggestion that fusion of AML tumor cells to DCs may generate a more potent response, we sought to determine if DC:tumor cell fusions would further improve survival in a murine model of AML. Our studies are the first to evaluate systemic administration of fused DC vaccines in a leukemic model of widespread disease. The studies were done in a prophylactic leukemia model to more accurately assess an immune response by eliminating any confounding immunosuppressive effects that tumor may have on the system. We also sought to determine whether the co-administration of CpGs as adjuvant with either tumor lysate–pulsed or tumor cell–fused DCs could potentiate in vivo antitumor immunity. These results have implications for the design of clinical trials of DC vaccines and the concurrent use of CpG as a vaccine adjuvant.

Materials and methods Mice C57BL/6, female mice, aged 5 to 6 weeks, were obtained from The National Institutes of Health (Bethesda, MD, USA), housed in a specific pathogen-free environment and fed ad libitum. C57BL/6 mice were used for in vitro DC generation and in vivo antitumor immune response evaluation between 8 and 10 weeks of age. All experiments using mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota and cared for according to institutional guidelines. Cells and cell culture reagents The AML cell line, C1498 (MHC Iþ, II
) was maintained in RPMI-1640 culture medium supplemented with 1 mM penicillin/streptomycin, 2 mM L-glutamine, and 10% FBS (HyClone, Ogden, UT, USA). The cells were maintained at 37 C and 5% C02. C1498 cells grown in AIM-V serum-free medium (Life Technologies, Carlsbad, CA, USA) were used to generate tumor cell lysate by subjecting AML cells suspended in phosphate-buffered saline (PBS) through 4 freeze/thaw cycles in liquid nitrogen and a 37 C water bath. The cell viability was evaluated by trypan blue exclusion and no viable cells were present at the completion of 4 cycles. Lysates were frozen at
80 C until use. Prior to use, lysates were thawed, centrifuged at 1200 rpm, and the supernatant used as the source of tumor antigen. For fusion documentation studies, C1498 cells stably transfected to express the fluorescent Discosoma coral-derived protein DsRed2 were utilized as previously described [32]. Generation of DCs from BM progenitors BM was harvested from the long bones of the femurs, tibiae, and fibulae of mice as previously described [33]. Red cells were lysed by ammonium chloride incubation and the single cell suspension depleted of mature T cells, B cells, granulocytes, and IA (MHC II)þ cells using an antibody cocktail followed by complement lysis. The antibody cocktail contained the following antibodies: antiThy 1.2 (30-H-12), anti-B220 (RA3-6B2), anti-Gr-1 (RA-8C5), and anti-IAb (AF6-120.1.2) (American Type Culture Collection [ATCC], Manassas, VA, USA). The DC progenitors were then incubated at 1  106 cells/mL of DMEM-complete media with cytokine for 5 to 7 days at 37 C and 10% C02 in 6-well plates with 3 mL per well. To generate mature DCs, murine GM-CSF (R&D Systems, Minneapolis, MN, USA) 150 U/mL and murine IL-4 (Schering-Plough, Kenilworth, NJ, USA) 75 U/mL were added for 7 days followed by 2 further days in culture with CpG oligodeoxynucleotide (CpG 7909, 2 mg/mL) (Coley Pharmaceutical Group, Wellesley, MA, USA) [26]. Cytokines were replenished on day 3 to 4 by removing 2/3 of the media and replenishing with fresh media supplemented with cytokine. Nonadherent and loosely adherent cells were removed by pipetting on day 4 to 5 and replated with fresh cytokine-containing media in new 6-well plates. Cells were again replated with fresh cytokine-containing media when CpG was added on day 7 to 9. Phenotypic evaluation of DCs The cell-surface antigen expression of the DCs was confirmed prior to in vivo administration and after DCs were pulsed or fused with tumor antigen. There was no phenotypic difference detected between DCs evaluated prior to pulsing with tumor lysate and

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after an 18-hour incubation with lysate (data not shown). Cells were washed and incubated with a-FCR (CD16/CD32) (2.4G2) (Pharmingen, San Diego, CA, USA) at 4 C for 10 minutes to block nonspecific binding of fluorochromes. The following directly conjugated antibodies (Pharmingen) were incubated with DCs at 4 C for 30 minutes: CD8a-FITC, CD4-FITC, NK1.1-PE, B220-PE, CD11b-PE, CD80-FITC, CD86-FITC, CD40-FITC, H2Kb-PE, IAb-FITC, and CD11c-PE. Cells were washed and analyzed using the FACS Calibur (Becton-Dickinson, San Diego, CA, USA), gated and analyzed using forward- and side-scatter plots on 10,000 live events. Fusion of AML cells and dendritic cells DCs were generated as described above. C1498 tumor cells grown in serum-free medium (Aim V) were irradiated with 100 Gy of radiation (Cs source) prior to electrical fusion to DCs. DCs and tumor cells were mixed 1:1, washed, and resuspended in fusion buffer (10 mM MgCl2, 10 mM CaCl2, 5% D-mannitol adjusted to a pH of 7.2–7.4). Cells were loaded (a maximum of 20  106 cells per fusion) into sterile fusion chambers and electrically fused using AC voltage currents (53 seconds) generated from a BTXTM instrument (ECM2001). Fused cells were immediately resuspended in media with fresh cytokines and plated at 1.25  106 cells/mL for overnight incubation at 37 C and 10% C02 in 6-well plates with 3 mL per well. Fusion efficiency and confirmation of fused cells Fusion was documented using flow cytometry, electron microscopy, and FISH (fluorescence in situ hybridization). For confirmation of fusion by flow cytometry, tumor cells were labeled with the fluorescent antibody SNARF and DCs were labeled with IAb-PE as described. The double-positive cells were evaluated using the FACS Calibur (Becton-Dickinson, San Diego, CA, USA), gated using forward- and side-scatter plots on 10,000 live events. To further document fusion, the fused cell population was labeled with CD11c-FITC (a DC marker) and DRAQ5 (nuclear stain) and evaluated using multispectral imaging flow cytometry [34]. Fused cells were compared to single cell suspensions of DCs and tumor cells. Fusion was confirmed by the presence of dual nuclei staining by DRAQ5 and unilateral staining of CD11c. For confocal microscopy, DCs and DsRed2-expressing tumor cells were fused, stained with anti-CD11c-FITC, and compared to single cell suspensions of DCs and tumor cells. Fusion was documented by the coexistence of two nuclei in a red and green expressing cell. As a final confirmation of fusion, fused cells were evaluated using FISH for the X and Y chromosomes. DCs were generated as described from male C57BL/6 mice (XY) and fused to tumor cells known to be female (XX) in origin. To make the X probe, mouse chromosome X bacterial artificial chromosome (BAC) probes for FISH were identified using the National Center for Biotechnology Information (NCBI) website and obtained from the RP23 mouse library at Children’s Hospital Oakland Research Institute (CHORI). After purification of BAC DNA (Roche, Basil, Switzerland), probes were labeled by nick translation (Invitrogen, Carlsbad, CA, USA) using digoxigenin11-dUTP (Roche Diagnostics, Basil, Switzerland). The fragments were checked for size by electrophoresis on a 1% TBE gel. Labeled DNA was precipitated in COT-1 DNA, sodium acetate and 95% ethanol, and resuspended in 50% formamide hybridization buffer. For the Y probe, mouse chromosome Y, whole chromo-

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some paint probe (FITC labeled) was obtained from Open Biosystems (Huntsville, AL, USA). FISH hybridizations were performed according to published protocols [35]. Interphase analysis was performed on 500 interphase cells and scored from each of the single cell suspension controls as well as the fusion samples. Cells and probe signals were visualized under an Olympus BX51microscope outfitted with a 175 W Zenon lamp and DAPI, FITC, and Texas Red filters. The red signal from the Dsred2 tumor cells was quenched prior to FISH to allow visualization of the X probe. FISH images were captured and analyzed using an interferometerbased cooled coupled device (CCD) camera and FISH view software (Applied Spectral Imaging, Vista, GA, USA). In vivo AML protection Bone marrow–derived DCs were generated as described above and then pulsed with C1498 lysate at a ratio of 3:1 (lysate cell equivalents:DC) on day 7 to 9 of culture and incubated in 6-well plates for 18 hours or fused with irradiated C1498 cells as described above. DCs were washed three times in PBS and resuspended in sterile PBS for injection into mice. Mice (n 5 5–10/group) were vaccinated 14 and 7 days prior to tumor challenge. A total of 0.5  106 DCs, consisting of DCs subjected to lysate pulsing or fusion, was given so that the same total number of DCs were administered intravenously regardless of the technique used (fusion vs lysate pulsing) or the efficiency of either technique in providing a source of AML antigens to DC cells used for vaccination. DCs had to be greater then 75% viable to be administered. Seven days after the second DC vaccine, control nonvaccinated mice and DC vaccinated mice were challenged with C1498 cells (106) given intravenously. The DC dose and schedule and C1498 dose and schedule used was based upon previous data with lysate-pulsed DCs, which had been shown to provide a significant level of protection against a subsequent challenge with a uniformly lethal dose of C1498 (1–2  106 cells) given 7 days after the second weekly DC vaccination [26]. To further enhance the potential for in vivo antitumor response, some cohorts of mice received CpG 7909 100 mg per mouse per injection [18,26] as an adjuvant given intraperitoneally at the time of DC vaccination on days 14 and 7 prior to tumor injection. No CpGs were given at the time of C1498 challenge. To determine if surviving mice were able to generate a memory response, mice surviving greater than 120 days were rechallenged with 1  106 C1498 cells/mouse. In vivo CTLp generation The cytotoxic T-lymphocyte precursor (CTLp) frequency was analyzed by limiting dilution as previously described [36]. Spleens from mice (n 5 3 per group) that received DCs and nonvaccinated controls were harvested on day 0 and a single cell suspension was obtained by passing the cells through a wire mesh and red cells were lysed using ammonium chloride. Graded concentrations of splenocytes (1  105
3  102) (30 replicates per dilution) were stimulated with 1  104 irradiated (100 Gy, Cs source) C1498 tumor cells per well and IL-2 (20 U/mL, Amgen, Thousand Oaks, CA, USA) in 96-well plates for 7 days. Tritiated thymidine–labeled C1498 tumor cells (1  104 per well) were then added to the cultures and incubated for 4 hours at 37 C. Cytotoxicity was assessed by the JAM assay [37], which measures the level of tritium retained by cells that have not undergone lysis. The CTLp frequency was calculated as previously described by means of Poisson distribution statistics [38]. Results are expressed as the

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absolute numbers of CTLp per spleen, which takes into account the variability in spleen size with vaccination. Data are depicted graphically as the mean values of absolute numbers of CTLp per spleen of cohorts of mice studied concurrently. IFN-g ELISPOT (enzyme-linked ImmunoSPOT) assay The immune response generated on a per-cell basis was measured using the ELISPOT assay for IFN-g. The assay was modified from that previously published [39]. Briefly, ELISPOT plates (Millipore, Billerica, MA, USA) were coated with purified rat antimouse IFN-g (Pharmingen, San Diego, CA, USA) and incubated overnight at 4 C. Plates were washed with sterile PBS and unbound sites were blocked with PBS–5% bovine serum albumin (BSA) during a 1-hour incubation. Plates were washed with sterile PBS. Single cell suspensions of splenocytes from each group were placed in the wells in graded concentrations from 0 to 106 cells/ mL. Stimulators of irradiated C1498 tumor cells (100 Gy, Cs source) at 105 cells/mL were added to splenocytes at each concentration. ConA (5 mL/well) and PBS were used as positive and negative controls, respectively, at all cell concentrations. Plates were incubated at 37 C, 5% CO2 for 24 hours. After 24 hours, plates were washed 5 times with PBS–0.05% Tween and the second antibody, biotinylated rat anti-mouse IFN-g, was added (Pharmingen, San Diego, CA, USA). Plates were wrapped in plastic wrap and incubated overnight at 4 C. The next day the plates were again washed 5 times with PBS–0.05% Tween and streptavidin alkaline phosphatase (Jackson Immunoresearch, West Grove, PA, USA) was added and plates allowed to incubate for 90 minutes. Plates were washed 5 times in PBS–0.05% Tween and substrate detected by the addition of NBT-BCIP (Sigma, St. Louis, MO, USA). IFN-g-producing cells were detected and quantitated using the Zeiss ELISPOT reader. All wells were compared to background controls. Data are presented as the mean number of positive spots per well from individually analyzed mice with wells containing a concentration of splenocytes at 105 cells/mL. Statistics The Kaplan-Meier product-limit method was used to calculate survival rates. Differences between groups were determined using the Generalized Wilcoxon test.

Results Electrical fusion of AML cells and DCs produces a hybrid cell population To determine whether hybrid cellular vaccines provide antitumor protection, we generated cellular vaccines by electrically fusing irradiated tumor cells to BM-derived DCs. The whole fusion product was evaluated and used in vivo as the fused cells are large and do not tolerate sorting procedures well. We utilized flow cytometry to quantitate fusion as it is the most readily available method and used most often in studies evaluating fused cell products. Fusion was confirmed by flow cytometry for all cells used in vivo. Identification of cells that were positive for both SNARF (tumor cells) and IAb or CD80 (DCs), as determined by flow cytometry, indicated that 11 to 12% of the cells

were double-positive (Fig. 1A). For each experiment, cellular fusion was confirmed using flow cytometry with fusion efficiency ranging from 10 to 20%. This is consistent with the level of fusion found in other studies using electrical fusion [40]. We were able to clearly document fusion using multiple methods to evaluate the fused cell population. Confocal microscopy identified cells with dual red and green signals and two nuclei consistent with DCs (CD11c FITC: green) fused to tumor cells (DsRed2: red) (Fig. 1B). Fused cells were imaged using multispectral flow cytometry. Staining with DRAQ5 allowed for a dual nuclear signal to be confirmed in the fused population (Fig. 1C) that had unilateral expression of CD11c (the tumor cells are CD11c
). Finally, FISH identified tumor cells as XO and DCs as XY. The tumor cells had lost an X possibly due to spontaneous mutation. Fused cells were documented as XXY (Fig. 1D). Phenotypic evaluation of DCs pulsed with tumor lysate or fused with whole tumor cells did not show any differences in expression (frequency and mean fluorescence intensity) of CD11c, CD11b, B220, H2Kb, IAb, or the costimulatory molecules CD80, CD86, and CD40 (data not shown). The cells were pulsed or fused after in vitro maturation with CpG, possibly accounting for uniform expression of surface markers on the DCs. Due to the large size and possibly the fragility of recently fused cells, high-speed cell sorting was not successful in isolating a pure population of viable fused DCs that retained function (data not shown). Moreover, such purification of fused DCs by high-speed cell sorting would not be practical for most centers that lack GMP cell sorters and therefore the current experiments simulate conditions that would be amenable to clinical testing of either fused DCs or lysate-pulsed DC preparations. DCs pulsed with AML lysate or electrically fused with tumor cells provide similar antitumor protection Previous work from our laboratory has demonstrated that DCs pulsed with tumor lysate provide significant protection from AML challenge [25,26]. To determine if DCs fused with tumor cells also generated an antitumor response, mice were given the DC/tumor hybrid cells intravenously 14 and 7 days prior to lethal tumor injection. In aggregate data of two replicate experiments with similar results, we observed that mice receiving DC fusion or lysate-pulsed DCs had similar survival (45% vs 47% survival, respectively), which was significantly higher than the 5% survival rates in nonvaccinated control mice (Fig. 2). Augmentation of antitumor protective effects by the coadministration of CpG 7909 with tumor:DC hybrid but not with pulsed DCs We have previously shown that CpG 7909 induces an antitumor response as a single agent in this model of AML via immune system activation in vivo and not via a direct apoptotic mechanism [18,26]. Both T cells and NK cells with

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Figure 1. Multiple methods document cell fusion. (A) The fused cell population was identified using a double-positive population of SNARF-positive (tumor cells) (y-axis, labeled FL2) and IAb- (left x-axis) or CD80 (B7.1)- (right x-axis) positive (DCs) as determined by flow cytometry. Cells were analyzed on a live gate of 10,000 events. (B) Confocal microscopy identified cells with dual red and green signals and two nuclei consistent with fused DCs (CD11c FITC: green) and tumor cells (DsRed2: red). (C) Multispectral flow cytometry with DRAQ5 identified a dual nuclear signal in the fused population. (D) FISH identified tumor cells as XO (single red signal) and DCs as XY (red and green signal). Fused cells were documented as XXY. Experiments were replicated and representative data are presented.

anti-AML effector cell properties are activated after CpG 7909 administration. Since CpGs are known vaccine adjuvants, we compared the survival rates of mice that were vaccinated with pulsed or fused DC vaccines intravenously in the presence or absence of CpG 7909 given intraperitoneally at the same time as DC vaccination. Mice vaccinated with tumor lysate–pulsed DCs in the presence or absence of CpGs 7909 had a significantly higher survival rate than nonvaccinated controls (40% vs 41% vs 4%, respectively) (Fig. 3A). Mice administered intravenous DC:tumor cell hybrids and CpG 7909 intraperitoneally had a significantly 1.0

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days Figure 2. Comparable survival in mice vaccinated with lysate-pulsed or tumor cell fused DCs prior to challenge with AML cells. Cohorts of mice were not vaccinated or were vaccinated with either lysate-pulsed DCs or DC:tumor fusion vaccines (0.5  106 DC cells/mouse, iv) 14 and 7 days prior to lethal tumor injection (1  106 C1498 tumor cells/ mouse, iv). On the y-axis is the proportion of mice surviving and on the x-axis are the days from tumor challenge. Ten mice per group were analyzed and replicate data from two experiments were pooled for analysis. Mice receiving DCs pulsed with tumor lysate or fused with tumor cells had a significantly higher survival rate than non-DC-vaccinated controls (45% vs 47% vs 5% survival, respectively).

improved survival compared to mice receiving the hybrid cells without adjuvant (60% vs 32% survival respectively, p ! 0.05) or the nonvaccinated controls (4% survival) (Fig. 3B). As no significant differences in survival were found between groups that received lysate-pulsed or fused DC vaccines without adjuvant (Fig. 2), this suggests that the CpG may be providing an immune-activating effect that favors antigen presentation by the fused vaccine preparation. DC:tumor cell hybrids and DCs pulsed with tumor lysate generate tumor-reactive responses in vitro To determine if the in vivo antitumor responses correlated with in vitro tumor-reactive immune responses, we evaluated cohorts of mice (n 5 3 mice/group/experiment) at the time of initial tumor challenge for the total number of splenic CTLps (CTLs generated after repriming to irradiated C1498 cells in vitro) and the frequency of splenocytes capable of producing interferon-g in response to overnight in vitro repriming with irradiated C1498 cells. A representative experiment quantifying CTLp (Fig. 4) and interferon-g production (Fig. 5) is shown. DC vaccines pulsed with tumor lysate or fused with tumor cells generated at least a fivefold increase in the mean numbers of anti-AML reactive CTLps per spleen when compared to naı¨ve mice and no significant differences were seen between the types of DC vaccines given (Fig. 4). The addition of CpG as an in vivo adjuvant modestly, albeit not significantly, increased mean numbers of tumor-reactive CTLps per spleen in mice that received either DC vaccine. Similarly, splenocyte tumor-responsive interferon-g production reflected the CTLp data and was significantly (p ! 0.05) increased in DC vaccinated cohorts compared to controls but was not significantly different between the type of DC vaccine given (Fig. 5). As seen in Figure 4, CpG administration increased the mean numbers

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Figure 4. Increased splenic antitumor-reactive CTLp generation in mice administered DC vaccines. Mice were left unvaccinated or were given two weekly DC vaccines (0.5  106 cells, iv) using tumor lysate–pulsed or DC:tumor hybrids on days 14 and 7 prior to analysis. On the y-axis are the mean values and error bars are 1 standard deviation of the mean for the absolute number of antitumor-reactive CTLp per spleen. The xaxis lists the cohort of mice analyzed. Data are derived from 3 mice per cohort and one representative experiment is shown. DC vaccines pulsed with tumor lysate or fused with tumor cells generated at least a fivefold increase in the mean values for tumor-reactive CTLps per spleen when compared to naı¨ve mice. Although the addition of CpG as adjuvant modestly increased the generation of tumor-reactive CTLps, there was no statistical difference between DC-vaccinated groups.

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Figure 3. CpG adjuvant improves survival in mice receiving tumor cell– fused but not lysate-pulsed DCs. CpG 7909 (100 mg/mouse, ip) was administered as an adjuvant along with DC vaccines (0.5  106 cells/mouse, iv) on days 14 and 7 prior to tumor injection (1  106 C1498 tumor cells/ mouse, iv). On the y-axis is the proportion of mice surviving and on the x-axis are the days from tumor challenge. Data are pooled from two replicate experiments with similar results that were comprised of 8–10 mice per group per experiment. (A) As compared to nonvaccinated controls, mice receiving tumor lysate–pulsed DCs had a significantly higher survival rate which was not increased by CpG 7909 administration (4% vs 41% vs 40%, respectively). (B) As compared to nonvaccinated controls, mice administered DC:tumor cell hybrids with CpG as adjuvant had significantly higher survival compared to mice receiving the hybrid cells without adjuvant (4% vs 60% vs 32% survival, respectively).

of splenocytes capable of producing interferon-g in response to C1498 cells, although as observed for CTLp per spleen, the increase was modest and no significant differences were observed in recipients of either type of DC vaccination cohort. Thus, at least as of the time of live tumor challenge, we conclude that both types of DC vaccines increased antitumor-reactive T-cell responses to a similar extent, which was associated with increased resistance to tumor challenge (Fig. 2). The addition of CpG 7909 at the time of DC vaccination only modestly increased these antitumor immune responses. These latter data are consistent with the fact that CpG 7090 did not increase the survival outcome of mice vaccinated with lysate-pulsed DCs but do not provide an explanation for the improved survival seen in mice receiving

CpG 7909 and DC:tumor hybrids compared to DC:tumor hybrids alone. DCs pulsed with tumor lysate or fused DC:tumor cell hybrids generate an anti-tumor memory response As vaccinated mice generated a sufficient in vivo antitumor response to allow some mice to resist initial tumor challenge, we sought to determine if the surviving mice had memory for tumor by rechallenging the mice. Indeed, mice that received either pulsed or fused DCs with or without CpG as adjuvant all had significantly improved survival, following rechallenge at 120 days after initial AML challenge, compared to controls (p ! 0.05). There were no differences between the groups of mice receiving DC pulsed or DC:tumor hybrid cell vaccines with or without CpG as adjuvant (Fig. 6). These data indicate that the DC vaccines are capable of providing long-lasting protection against AML. Discussion Our data represent the first study of direct in vivo comparison of pulsed and fused DC vaccines delivered systemically in a disseminated leukemia model and support two important conclusions. Firstly, in our model system using electrical fusion of tumor cells to DCs, we found protective antitumor responses generated with DC fusion vaccines to be comparable to tumor lysate–pulsed DC vaccines when given prior to AML challenge. Second, the intraperitoneal

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Days Post Rechallenge Figure 5. DC vaccines increase the frequency of interferon g–producing splenocytes as assessed by an ImmunoSPOT assay. Mice were left unvaccinated or were given two weekly DC vaccines (0.5  106 cells, iv) using tumor lysate–pulsed or DC:tumor hybrids on days 14 and 7 prior to analysis. Data are derived from 3 mice per cohort and one representative experiment is shown. On the y-axis are the mean values and error bars are 1 standard deviation of the mean for the frequency of interferon g–producing splenocytes responding to overnight in vitro repriming with irradiated tumor cells. The x-axis lists the cohort of mice analyzed. DC vaccines pulsed with tumor lysate or fused with tumor cells generated at least a twofold increase in the frequency of tumor-reactive interferon g–producing splenocytes when compared to naı¨ve mice. The inclusion of CpG 7909 along with DC vaccination did not significantly increase the frequency of spots per well, although modest increases in the mean values were observed with each type of DC vaccine. )p ! 0.05 for each group compared to control.

administration of CpG 7909 at the time of intravenous injection of DC:tumor fusion vaccines but not of tumor lysate–pulsed DC vaccines enhanced the vaccine efficacy. In contrast to the similar survival and anti-AML immune responses we observed using AML lysate–pulsed DC and AML:DC fusion vaccines, Galea-Lauri et al. [31] found that chemical fusion of irradiated human tumor cells (myeloid leukemia cell lines U937 and K562) to DCs stimulated a more potent antileukemia CTL response than DCs pulsed with apoptotic tumor cell fragments or whole-cell lysates when used to prime T cells in vitro. Similar to our in vivo anti-AML CTLp data, Klammer et al. [41] reported that using autologous irradiated human myeloid blasts and mature DCs to prime autologous T cells in vitro, it was found that both DC:fusion heterokaryons (mean fusion efficiency 26%) and lysate-pulsed DCs could induce antiAML CTLs or interferon-g-producing T cells assessed by ImmunoSPOT assay using samples from some AML patients, with neither product proving to be uniformly superior to the other. Shimizu et al. [28] compared the in vivo administration of B16 melanoma lysate-pulsed DC and DC:tumor cell fusion vaccines administered by intranodal injection to 3-day melanoma tumor–bearing mice along with four days of IL-12 injections given intraperitoneally. They found that the fused cells lead to greater antitumor

Figure 6. DC vaccines provide a memory response. Mice surviving greater than 120 days post initial tumor challenge were rechallenged with iv injection of 1  106 C1498 tumor cells/mouse. Six to eight mice per group were concurrently challenged with live tumor. Data are representative of replicate experiments. On the y-axis is the proportion surviving and on the x-axis are the days after tumor challenge (naı¨ve mice) or rechallenge (mice previously vaccinated with DCs, challenged with live tumor and then rechallenged $ 4 months later). Mice that received either pulsed or fused DCs with or without CpG as adjuvant all had significantly improved survival compared to naı¨ve controls that were concurrently challenged (p ! 0.05). There were no differences between the groups of mice receiving pulsed or hybrid cell vaccines with or without CpG as adjuvant.

responses in this tumor system. These two studies (AML vs melanoma, respectively) differ for a number of potentially important variables including but not limited to differences in the homing, migration, (widely metastatic vs pulmonary and skin) and immune properties of AML vs b galactosidase–expressing melanoma cells (MHC class I high vs low/negative and moderately vs poorly immunogenic), maturation of the DCs used (more mature vs less mature DCs), fusion efficiency (10–20% vs 40–60% of adherent cells), use of multiple vs one DC vaccine, timing of DC vaccinations related to tumor administration (pre-AML vs post-melanoma cell challenge), and use of no adjuvant vs IL-12 adjuvant. Regarding fusion efficiency, we cannot exclude the possibility that a higher fusion efficiency with AML cells and DC to levels comparable to Shimizu et al. [28] would have demonstrated superiority of electrofusion. Our tumor:DC fusion efficiency obtained by electrofusion of mature DCs with murine AML cells is similar to that reported by others for human myeloid leukemia cells [31,41] and higher than that routinely observed with human DCs and tumor lines representing a range of solid tumor types using electrofusion techniques [42]. Many other studies evaluating hybrid DC vaccines have utilized chemical fusion with polyethylene glycol (PEG) [30,31,43–45]. Using PEG to induce fusion, Gong et al. report success in all 16 AML patients from which AML blasts were obtained, with an illustrated

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flow cytometry example showing 34% fusion efficiency in one patient [30]. Thus, it is possible that for AML cells, our fusion efficiencies of 10 to 20% are within the range likely achievable by clinical electrofusion procedures. However, very few comparisons exist of the two methods of cellular fusion for the generation of DC vaccines. Gottfried et al. [44] compared the characteristics of DC:tumor cell hybrids using melanoma cell lines and human peripheral blood mononuclear cell–derived DCs. They found that PEG fusion generated fourfold to eightfold higher numbers of dual fluorescent cells (as detected by flow cytometry) compared to electrically fused cells. Contrary to this, Scott-Taylor et al. [45] have shown comparable results between methods when using carcinoma cells derived from prostate or breast cancer fused to DCs generated from CD34þ cells. In a murine model of mammary carcinoma, Lindner and Schirrmacher found similar fusion efficiency and similar in vivo antitumor protective effects between DCs fused to tumor cells using PEG or electrofusion methods [46]. As the number of fused cells required for optimal in vivo antitumor response is not known, it is possible that by using electrical fusion instead of PEG we may have eliminated the advantage of the PEG-fused DCs as observed by Galea-Lauri et al. [31]. Alternatively, it is possible that the testing of the two types of DCs (fused vs lysate-pulsed) at different DC or tumor cell doses could uncover more subtle differences in antitumor protective effects. The electrical fusion method was chosen for our studies as it is easier, faster, and more readily translated than PEG fusion strategies. Effective induction of antitumor CTL response requires DC priming of T cells within lymphoid organs. As compared to pulsed DCs, the larger size of heterokaryons and the coexpression of cell-surface antigens from DC:tumor fusions likely lead to migration and homing differences between these two types of DCs. Morse et al. indium-111 oxyguinolone labeled GM-CSF- and IL-4-propagated peripheral blood DCs pulsed with tumor RNA and compared sites of migration after subcutaneous, intravenous, and intradermal administration [47]. No DCs were found in the draining lymph node after subcutaneous injection and few were found after intradermal injection, while intravenous injection resulted in initial pulmonary localization followed by redistribution to the liver, spleen, and BM, potential sites of AML disease. Our previous studies have shown a high degree of protection against murine AML challenge with intravenous injection of AML lysate–pulsed DCs [25,26]. Since it also would be anticipated that the migration of mature DC:tumor heterokaryons from the site of injection might be suboptimal to induce an immune response of sufficient magnitude to generate CTLs to permit resistance of systemically administered AML cells, we chose to use an intravenous route of DC administration for comparing these two types of DC vaccines although other studies have indicated that the heterokaryons may be most effective when

administered directly into the lymphatic system (intranodal or intrasplenic) [27,31,48]. Thus, it is possible that our studies with fused DCs would have shown superiority over AML lysate–pulsed DCs if administered into the lymphatics. Because the optimal route of administration for DC vaccines has not been established in humans and many studies have been performed with intravenous DC vaccines [49], our studies using the intravenous route are relevant to clinical trials. Regardless as to whether there are differences in the homing, migration, or survival of tumor lysate–pulsed vs electrically fused DCs, the net result in survival was comparable in the absence of CpG 7909 administration. It is possible that the improved outcome using electrically fused but not lysate-pulsed DC vaccines with CpG 7909 was due to the differential biological effects of CpG 7909 on homing, migration, or survival of these two DC preparations. Another important difference between our comparative study of fusion vs lysate-pulsed DCs and that of Shimizu et al. [28] could be the immune responses required for a positive therapeutic response in the model tested, systemic AML, as compared to a 3-day B16 melanoma lung metastasis model. Like Galea-Lauri et al. [31], we used mature DCs to fuse or pulse with antigen and we did not detect any difference in phenotype between the groups. This may favor an advantage to fused cells as the cells do not need to uptake or process antigen, a function that is greater in immature DCs rather than mature DCs [40,50], which may mitigate the potential adverse consequences of electrofusion in terms of DC:tumor heterokaryon fragility, clearance in the lung and reticuloendothelial system after intravenous administration, or potential adverse alterations in the homing and migration into lymph nodes after intravenous injection. Several strategies have been utilized to enhance DC vaccine responses in vivo. One of those strategies has been to use an adjuvant to enhance the priming response of tumor vaccines. We have previously shown that CpGs have an antitumor effect in our murine model of AML [18]. We have also shown that CpGs are effective in maturing murine DCs in vitro and in enhancing an in vivo anti-AML response that is T cell dependent when compared to other methods of in vitro DC maturation [26]. To potentiate the antitumor effects of the DCs pulsed with tumor lysate or fused with tumor cells, we administered CpG intraperitoneally at the time of the intravenous DC administration. These routes of administration were chosen to be consistent with what we had previously found to be optimal for DC and CpG administration in this model system [18,26]. Interestingly, only the survival response to the DC:tumor cell fusion and not the tumor lysate–pulsed DC vaccine was enhanced by the use of CpG as adjuvant. Merard et al. have used an approach to recruit DCs in the host to the tumor site and when combined with intratumoral CpG, a more potent antitumor immune response could be generated with either intratumoral CpG or

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recruitment of DCs into the tumor [51]. In those studies, CpG overcame the tumor-mediated inhibition of DC activation in vivo at the tumor site. In other studies, Hiraoka et al., using DC:tumor fusions coadministered intradermally with CpG adjuvant, showed a greater antitumor cytokine response and CTL generation as well as improved survival compared to fused cells alone or DCs mixed with tumor cells [52]. CpG failed to provide or enhance survival or antitumor immune activation as a single agent or as an adjuvant to the DC:tumor cell mixture in that study, similar to the conclusions we reached with lysate-pulsed DCs and CpG coinjection in our study. The reason for the superior efficacy in both studies using fused DCs and CpG coadministration are unknown. However, it is possible that the fused cell preparation is less stable in vivo and releases tumor antigen to endogenous DCs that are matured by CpG administration, whereas tumor lysate–pulsed DCs, which are smaller and not exposed to electrochemical currents, are more stable in vivo. CpG 7909 administration results in a burst of proinflammatory cytokines that peak at 24 to 48 hours as we have previously reported [18], and these cytokines would be included amongst those that are known to activate DCs. As the DCs used for lysate pulsing and fusion were already matured using CpG 7909 in vitro, the additive effect of CpG 7909 with fused DCs more likely relates to effects on host cell populations, including APCs, T cells, or NK cells. Since the second CpG dose is given 1 week before tumor challenge, the burst of cytokine response will have concluded and the effects on stimulating NK cell responses [18] most likely would have subsided as well. While we cannot exclude a long-lasting effect of fused DCs and CpG on NK cells with anti-AML reactivity, there is no a priori reason to speculate that electrically fused DCs would be preferable to lysate-pulsed DCs in activating long-lived NK cells. Instead, we favor the explanation that CpG 7909 bolstered the antitumor protective effects of fused DCs via activation of endogenous DCs that had taken up antigen released from the DC vaccine. While our data indicate that total splenic anti-AML CTLp and frequency of interferon-g-producing splenocytes in response to AML cells is increased by CpG 7909, albeit only modestly, and there was no significant difference between fused and lysate-pulsed DCs given in the context of CpG 7909, it is possible that antitumor reactive CTLs present in lymph nodes or as memory cells in nonlymphoid organs would have been higher in the cohort of mice receiving fused DC:tumor vs tumor lysate–pulsed DC vaccines and CpG. Alternatively, it is possible that the height or longevity of these responses at some time after tumor challenge would be different in cohorts of recipients given either DC preparations with CpG 7909. In conclusion, our data suggest that DC vaccine strategies may be effective in developing strategies to treat AML. Our data fail to show a clear advantage of DC fused with tumor cells compared to DC pulsed with tumor antigen under the conditions tested. We also show that adjuvant strategies using CpGs may enhance DC vaccine responses

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but this may depend on the method of antigen loading. These data have potential clinical implications for the use of fused vs lysate-pulsed DCs to treat AML. Acknowledgments The authors would like to thank Greg Veltri of the University of Minnesota Cancer Center’s Flow Cytometry Core Laboratory for the multispectral flow analysis and LeAnn Oseth from the University of Minnesota Cytogenetics Laboratory for the FISH analysis. The authors would also like to thank Stacy Bohl for her technical assistance. Supported in part by grants from the Children’s Cancer Research Fund, Viking Children’s Fund, Coley Pharmaceutical Group, and NIH grants R01 CA-72669 (BRB) and R01 CA-85922 (WC).

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