An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine

Abstract

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell–tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c⁺ expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-010-0921-2) contains supplementary material, which is available to authorized users.

Introduction

Spontaneous mammary cancer is among the most common malignancies afflicting unspayed female dogs with most cases resulting in death from widespread metastatic disease [1]. Canine mammary tumors (CMTs) have been characterized extensively for both genetic defects and phenotypes, and share many characteristics with human breast cancer including hormone dependence [2–6], frequent oncogene Her-2/neu activation [7] and defects in expression of cyclin dependent kinases (CDKs) and their inhibitors (CKIs) such as p16/INK4A and p21/Cip1 [8–10]. CMTs represent one of the best homologous models of human breast cancer known consisting of the same malignant tissue types, highly similar pathology and natural history, and involve no viral etiology [2, 3, 11].

Tumors express specific antigens that are recognized by the immune system [12, 13] and in some cases promote natural remission [14–16]. A therapeutic strategy that could reliably stimulate immune recognition could promote better management strategies in treatment of neoplastic disease particularly in metastatic cases. However, naturally occurring immune management is, at best, unreliable and, at worst, ineffective in eliminating tumor cells.

One approach, designed to improve immune recognition, is based on creation of hybrid-cell vaccines through fusion of antigen presenting cells (APCs) with tumor cells [17–20]. Hybrid-cell fusion constructs express both tumor-specific antigens and necessary machinery for antigen presentation and, if they are autologous (MHC I matched), T cell activation is possible. Among APCs, dendritic cells (DCs) are considered most effective in antigen presentation. They express MHC class I and II molecules as well as costimulatory and adhesion molecules [17–20]. Because DCs can transport presented antigens to activate effector T cells in lymph nodes they have been suggested as promising candidates for the APC component of hybrid-cell vaccines [19].

Our work and others, have demonstrated that immune recognition can be enhanced by vaccination with cellular constructs composed of allogeneic hybrid-cell fusions of APCs and tumor cells [17, 19–23]. Uptake, processing and presentation of tumor cell antigens by APCs, including antigen loading of DCs, is thought to be promoted by cell fusion stimulating antigen recognition. Such hybrid-cell fusions express both tumor-specific antigens and the necessary machinery needed for antigen presentation but have been thought to require MHC matching for T cell activation. We have previously demonstrated that MHC-matching was not required and that measurable cytotoxic T cell responses could be induced using only allogeneic cell lines that could be explained by cross-presentation between allogeneic and autologous DCs [21, 24]. Although some success has been reported using cellular cancer vaccines, suggesting that these strategies possess the potential to improve immune recognition of tumors, and they remain experimental [12, 25–28].

Canine mammary tumor cell lines have been developed and represent stable lines derived from malignant canine mammary cancers with well characterized morphology and genetic changes [2, 3, 7, 9–11, 21, 29]. We have previously published a strategy for creating allogeneic (unmatched) canine hybrid-cell fusion vaccines against canine mammary cancer utilizing CMT cell lines and their fusion to a DC-like cell line [21]. The resulting fusions were of very high quality typically fusing at rates of 40–70% of all cells and vaccination demonstrated a clear, statistically significant enhancement of cytotoxic T lymphocyte (CTL) activity in normal healthy dogs. However, because no component cell in the allogeneic hybrid-cell fusions was MHC matched, there was uncertainty as to the mechanism of immune presentation. We have now developed the technology for high-speed sorting of relatively rare canine DCs isolated from peripheral blood to provide a population of APCs suitable for making hybrid-DC fusions that are autologous (MHC matched) to each dog vaccinated. We now report the fusion of these well described CMT cell models and primary DC-enriched populations sorted from fresh canine peripheral blood to create autologous DC hybrid-cell fusions for use as cancer vaccines.

Materials and methods

Cell culture

CMT12, CMT27, and CMT28 canine mammary tumor-derived cells were grown under standard conditions in Leibovitz’s L-15-medium, as previously described, in preparation for hybrid-cell fusion [8, 10, 21]. Primary CMT cells were librated from fresh canine mammary tumor biopsy materials as previously described [2].

Primary autologous dendritic cell sorting

Flow cytometry and high-speed cell sorting were used to isolate DC populations labeled with specific antibodies to CD antigens. Peripheral blood mononuclear cell (PBMC) populations were prepared from whole canine blood (40–60 ml) collected from normal outbred Beagles into EDTA tubes (Becton, Dickinson), gently mixed and centrifuged 30 min at 1,000g at room temperature. The buffy coat containing the PBMCs was isolated in 1–2 ml and combined with 8 ml HBS (HEPES-buffered saline, pH 7.2, no Mg/Ca, Gibco). The cell suspension was layered over 5 ml Histopaque 1077 (Sigma-Aldrich) and centrifuged at 1,000g at room temperature for 30 min. The PBMC containing band was extracted in 1–2 ml and diluted with 2 ml HBS, mixed, and centrifuged. The supernatant was removed and cells gently resuspend in 5 ml HBS. Cells were centrifuged once and resuspended in 4 ml flow wash buffer (FWB: HBS, 10% fetal bovine serum) and incubated at room temperature for 40 min.

PBMC populations (1–3 × 10⁹ cells in 1 ml) were labeled with antibodies against canine CD4 (FITC-conjugated polyclonal rat anti-canine, Serotec), CD8 (RPE-conjugated polyclonal rat anti-canine, Serotec), and CD11c (monoclonal mouse anti-canine, Serotec) as previously described [21] in combinations of up to three antibodies (100 μl of each antibody solution per 1 ml reaction volume). Anti-CD11c antibodies were labeled with secondary anti-mouse monoclonal antibody conjugated to Alexa-Fluor 660 (70 μl/reaction) as previously described [21]. Cells were analyzed and sorted in a MoFlo flow cytometer with 488 and 635 nm lasers (Beckman). Dot-plots and histograms were analyzed using Summit 4.3 (Beckman). The entire cell suspension was sorted for each experiment and sorted cells were collected under sterile conditions into tubes containing 1 ml of fetal bovine serum. Both sample and sorted cell populations were maintained at 4°C. Following sorting, cells were gently mixed, collected by centrifugation, as described above, and cultured in RPMI-1640 (Gibco/BRL) containing 10% fetal bovine serum (Hyclone) and penicillin/streptomycin/fungizone (Gibco/BRL) as described above.

Hybrid-DC fusion and vaccine preparation

Populations of CMT28 and autologous DCs were fused by incubation with sterile solutions of 50% w/v PEG (~3,350 MW) in Improved MEM as previously described [21]. Parallel fusions of stained and unstained cells provided cells to be analyzed by flow cytometry and unstained cells for injection as previously described except CellTracker Orange CMTMR (Invitrogen) was used to stain DC populations (1:2 dilution of CellTracker Orange CMTMR stock to sterile clear 1× Hanks and then 1 μl of the diluted dye added per 1 × 10⁶ cells for 30 min at 37°C) [21]. Cell suspensions were collected by centrifugation, resuspended in Improved MEM and incubated for 15 min followed by centrifugation and washed three times with Hanks and 5 × 10⁴ cells analyzed by flow cytometry on a MoFlo flow cytometer (green 530 ± 20 nm and orange 580 ± 15 nm) to determine percentages of single or dual-stained populations following fusion. Populations of CMT28/DC fusions (5 × 10⁶ DCs fused to 1 × 10⁷ CMT28 cells, ratio of 1:2 in 0.5 ml PBS containing CpG oligonucleotides, adjusted for DC yield) were prepared as previously described [21, 30].

Polyclonal canine IgG assay

Polyclonal antibody-containing sera from vaccinated dogs were assayed for CMT-specific cIgG (canine IgG) by collecting clotted blood samples at 37°C from each dog, rimming each clot followed by clot contraction at 4°C for 1 h and then centrifugation and extraction of the serum and storage in aliquots at −85°C. For the standard assay, sera were thawed on ice and aliquots (100 μl) of live 5 × 10⁵ CMT28 cells in growth medium were reacted with 5 μl of polyclonal dog serum (or HBS for no-primary antibody controls) for 60 min at room temperature, washed three times in 1 ml HBS and resuspended in 100 μl FWB. Secondary antibody (4 μl/assay, sheep pan-anti-dog IgG-FITC, Serotec) was added and incubated 60 min at room temperature in the dark, washed two times in 1 ml HBS, resuspended in 700 μl HBS with 1% w/v BSA (fraction V), and filtered to 53 μm [21]. Live labeled cells were analyzed by flow cytometry on a MoFlo flow cytometer (Beckman). Volumes of primary and secondary antibodies were altered in those experiments designed to optimize the standard assay. In some experiments FITC-conjugated cIgG class 1 (goat anti-dog IgG1, Serotec) or cIgG class 2 (sheep anti-dog IgG1, Serotec) antibodies were substituted or an anti-MHC I antibody known to react with canine MHC I (mouse monoclonal anti-MHC I/poly-mammalian, H58A, VMRD, Inc.) was reacted with target cells prior to assay. This is the only type of commercially anti-MHC I antibody available that has been proven to bind canine MHC I. In other cases the order of antibody addition was reversed by applying polyclonal cIgG first and then the anti-MHC class I antibody followed by secondary Alexa660-conjugated anti-mouse IgG monoclonal antibody (Invitrogen). All cytometry analysis ensured collection of at least 10,000 events in the positive gates.

MHC I typing of dogs and canine cell lines

Each vaccinated dog and the CMT27 cell line were partially typed by direct DNA sequencing of exon 2 of the major MHC I locus DLA88 in the canine genome as previously described [21].

Vaccine injection and blood collection

All experiments were conducted under oversight of the Auburn University IACUC committee in AAALAC approved animal care facilities. Healthy adult, reproductively intact (unspayed) female Beagles (1–2 years old) were acclimated for 2 weeks and randomly assigned (three dogs/group: vaccinated and vehicle injected control). Vaccines were prepared and injected into popliteal lymph nodes, guided by ultrasound, of mildly sedated dogs given local anesthesia at the site of injection as previously described at weeks 1, 5, and 9 [21]. Blood was collected prior to, and one week after, each vaccination from each animal by venipuncture. Both sera and PBMC populations were isolated and frozen for storage at each time point. Each dog was examined daily for physical signs of tumor growth at the injection site and any signs of physical distress. Regular examinations for acute cytotoxicity and long-term effects were also conducted [21].

Cell-mediated immune assays

Cell-mediated immune (CMI) assays were assessed by ⁵¹Cr-release using vaccine strain CMT28 cells as targets as well as other unmatched CMT cell lines and primary CMT cells freshly isolated from tumor biopsy specimens as previously described [21]. Target cells were incubated with dilutions (25:1 or 50:1) of effector lymphocytes:targets from vaccinated or unvaccinated dogs, or medium alone and supernatants counted for released radioactivity by liquid scintillation counting in triplicate. Corrected percent lysis was calculated to determine relative CTL activity as previously described [21].

Results

Construction of autologous DC hybrid-cell fusion vaccines

We previously published a strategy for creating allogeneic canine hybrid-DC fusion vaccines against canine cancer [21]. Although fusions were of high quality (typically fusing at rates of 40–70%) and elicited an enhancement of cytotoxic T lymphocyte (CTL) activity in normal healthy dogs no component of this vaccine was MHC matched as determined by individual DLA88 (HLA equivalent) gene sequencing. To provide MHC-matched DCs, we have developed technology to isolate relatively rare peripheral blood subsets of autologous canine DCs from individual dogs based on combinations of CD4/CD8/CD11c staining of PBMC populations and sorting the CD11c⁺ cells under live and aseptic conditions. Canine PBMCs labeled for CD4, CD8, and CD11c were analyzed by flow cytometry and sorted into CD11c⁺/CD4⁻/CD8⁺ (blue), CD11c⁺/CD4⁺/CD8⁻ (green), CD11c⁺/CD4⁻/CD8⁻ (red), and CD11c⁺⁺/CD4 +/CD8 + (light blue) antigen expressing populations representing DC-enriched populations from peripheral blood (Fig. 1). We routinely sort 1–3 × 10⁹ PBMCs recovering approximately 1–5 × 10⁶ CD11c⁺ cells sufficient to prepare a single hybrid-DC fusion vaccine or approximately 0.1–0.5% of total PBMCs.

Fig. 1.

Cytometry and cell sorting of primary canine DC populations from PBMCs. Canine PBMCs (60 ml fresh EDTA-treated blood) were labeled for CD4, CD8, and CD11c, analyzed by flow cytometry and sorted into CD11c⁺/CD4⁻/CD8⁺ (b lower right gate), CD11c⁺/CD4⁺/CD8⁻ (a lower right gate), CD11c⁺/CD4⁻/CD8⁻ (b lower left gate excluding CD11c⁺/CD4⁺/CD8⁻ cells in the a lower right gate), and CD11c⁺⁺/CD4⁺/CD8⁺ (b upper left gate) populations enriched in peripheral blood-derived DCs. DC-enriched/labeled populations are indicated by color back-gates. Sorted populations were gated first on whole cells in forward/side scatter dot plots and then analyzed for fluorescence. Typical sorts involved labeling approximately 1–3 × 10⁹ PBMCs recovering 1–5 × 10⁶ CD11c⁺ cells sufficient to prepare a single hybrid-DC fusion vaccine

Blood was collected from six normal healthy female Beagles and DC-enriched populations were sorted from three of them for preparation of autologous DC hybrid-cell fusion vaccines. Fusions were injected three times at 4-week intervals into popliteal lymph nodes in formulations composed of cell fusions in pyrogen-free PBS with CpG-oligonucleotide adjuvant (Fig. 2) as previously described [21]. Three control dogs were bled identically but injected with vehicle with CpG alone. No acute or longer-term effects of vaccination were detected and blood was collected on 1 and 2 weeks following each vaccination for immune assessment (Fig. 2).

Fig. 2.

Autologous DC hybrid-cell fusion vaccine and blood collection strategy. Six normal healthy reproductively intact female adult laboratory Beagles were randomly divided among two groups: vaccinated (animals Di, M, and L) and control vehicle injected (animals Da, B, G). Vaccines were composed of LPS-free PBS, CpG oligonucleotides, and hybrid-DC fusions while controls were injected with the identical formulation minus the cell fusions both delivered into the popliteal lymph node. Autologous DC hybrid-cell fusion vaccines (V×) were administered at weeks 1, 5, and 9 and blood (B) was collected (40 ml/dog) 1 and 2 weeks after each vaccination. Acute effects were monitored daily for 1 week and then twice weekly for 12 weeks

Autologous DC hybrid-cell fusion vaccination induces a robust IgG response

To overcome barriers with fixed CMT cell targets in ELISA for cIgGs [21], a live CMT cell target assay was developed using flow cytometry to provide greater sensitivity and quantitation by single cell fluorescence. Amount of primary cIgG-containing sera from dogs and secondary anti-cIgG antibody was varied to ensure that primary antibodies were applied at less than saturating levels and secondary antibodies were applied at saturating levels to allow quantitation of amount of the CMT-specific cIgG present in each canine serum sample (Fig. 3). Levels of 5 μl of primary serum and 4 μl of secondary serum (for 100 μl reactions containing 5 × 10⁵ CMT targets) were optimal providing sufficient dynamic range for assay of varying levels of CMT-specific cIgG.

Fig. 3.

Anti-CMT cIgG assay optimization. Flow cytometry assays of cIgG recognizing CMT antigens were developed such that primary cIgG was delivered below saturation of live CMT28 cell targets and secondary sheep anti-cIgG-FITC was applied at saturating concentrations so that mean channel fluorescence (MCF) was proportional to strength of the primary cIgG reaction to CMT28 antigens. Histograms show fluorescence (FITC-channel) for cells treated with serum containing cIgG from naïve control dogs (Control), for cells reacted with primary cIgG containing serum from vehicle-injected dogs (Vehicle), and for cells reacted with cIgG containing serum from vaccinated dogs (V×). Primary antibodies (1^oAb) were applied at 5 or 10 μl serum per 100 μl cells (5 × 10⁵) and secondary antibodies (2^oAb) were applied at 2 or 4 μl serum per 100 μl cells (5 × 10⁵) as noted. Optimal fluorescence protocol (arrow) was judged as lowest level of primary serum used and maximum level of secondary antibody used without background staining. Data shown is the last summary experiment of several trials adjusting serum levels for optimization assay conditions as 10 μl primary antibody, 4 μl secondary antibody to 5 × 10⁵ cells in 100 μl reaction volume

A time course of cIgG activities binding CMT28 cells for each dog was prepared from sera collected on 1 and 2 weeks following each of three vaccinations (Fig. 4a). Activities were expressed relative to an average for cells labeled with secondary antibody only (no primary antibody). Control injected animals ranged near the mean without significant response while hybrid-DC fusion vaccinated dogs reacted strongly but to varying degrees. All vaccinated dogs demonstrated a statistically significant anamnestic response to subsequent vaccinations after week 3 with vaccine matched CMT28 cell targets.

Fig. 4.

Anti-CMT cIgG assay time course and cross-reactivity. Flow cytometry assays of cIgG from vaccinated dogs recognizing CMT antigens were delivered at below saturation of live CMT28 cell targets and using secondary sheep anti-cIgG-FITC was applied at saturating concentrations were performed. Mean channel fluorescence (MCF) was proportional to strength of the primary cIgG reaction to CMT28 antigens for each dog. Titers were calculated as fold difference relative to an average for cells labeled with secondary antibody only. Relative levels from 2 naïve (never injected) Beagles are shown for comparison. a Time course of autologous DC vaccinated (V × 1, 2, and 3) and control (Control 1, 2, and 3) dog antibody titers against CMT28 target cells for each of six blood collections (1 and 2 weeks after each of three vaccinations weeks 2, 3, 6, 7, 10, and 11 for each dog). Inset: examples of polyclonal CMT-specific cIgG binding from naïve dog (black), Dog V × 3/L 1 week post vaccination 1 (light gray) and 1 week post vaccination 3 (medium gray). Statistical analysis employed a mixed model for repeated measures analysis of variance. Statistical significance (asterisk) was achieved by the first blood collection after the second vaccination (week 6) for all vaccinated animals (week 6 p = 0.0274, week 7 p = 0.013, week 10 p = 0.0004, week 11 p = 0.0029). b Autologous DC vaccinated (Vaccine) and control (Control) dog IgG titers (1 week following vaccination 3) against vaccine matched CMT28 target cells and allogeneic target CMT27 and CMT12 cells. For each cell target, sera from 3 control (left) and 3 vaccinated (right) dogs from 1 week post vaccine 3 are shown. Relative MCF levels were calculated as described above. Inset: examples of polyclonal CMT-specific cIgG binding allogeneic CMT12 (black) and CMT27 (light gray) cells as well as vaccine-matched CMT28 (medium gray) cell targets. Histograms are plotted as frequency/cell number against log fluorescence. Statistical significance (asterisk) was achieved for all sera from vaccinated animals against all three cell lines (unpaired t test: CMT12 p = 0.0105, CMT27 p = 0.0068, CMT28 p = 0.0134). c Anti-CMT cIgG assay cross-reactivity against primary CMT cells. Flow cytometry assays of cIgG recognizing CMT antigens in primary biopsy-derived canine CMT cells were performed to evaluate autologous DC vaccinated (Vaccine) and control (Control) dog IgG titers (1 week following vaccination 3). Left: Percent-labeled population (hatched) was calculated compared to controls (gray) analyzed without primary antibody (representative example shown). Right: Percent-labeled populations compared for 2 control (Da and B) and 2 vaccinated (M and L) dog sera. Sufficient primary cells were recovered to evaluate sera from 2 control and 2 vaccinated dogs. T test (1 tailed) analysis revealed statistical significance between vaccinated and unvaccinated sera against primary biopsy-derived CMT cells at p = 0.0866

Antibody immunity in sera, derived 1 week after vaccination 3 against vaccine matched CMT28 cells, was compared to reactions using MHC-unmatched CMT12 and CMT27 cells. In vaccinated dogs, antibody recognition of shared CMT cell antigens was evident in all CMT cell lines (Fig. 4b). Reactions in all CMT cell targets (matched and unmatched) suggested expression of shared antigens although reactions to CMT12 were approximately half that observed for CMT27 and CMT28 cells. No antibody recognition was detected with any cell targets reacted with sera from control animals. Differences in reactions to CMT28, CMT27, and CMT12 cells were all statistically significant from control sera.

To ensure that such immune reactions were not recognizing common antigens expressed as a consequence of culture in vitro, primary CMT cells freshly recovered from a canine mammary tumor biopsy were also evaluated as targets of cIgG binding. Although limited numbers of cells reduced the number of assays possible and the binding levels were lower, there was a clear statistically significant binding of primary CMT cells by the vaccinated dog sera compared to control sera (Fig. 4c). This data reinforce the conclusion that the sera from vaccinated dogs recognized shared antigens among distinct canine mammary tumor-derived cell lines that were also shared by primary tumors. No significant reaction of these antisera with unrelated tumor-derived cell lines from canine melanoma or lymphoma was detected (data not shown).

Each of the dogs and cell lines express unique MHC I nucleotide sequences (data not shown) and, with one exception (CMT27 cells and Dog Di/6), also express unique MHC I alleles as demonstrated by direct DNA sequencing of exon 2 of the canine DLA88 locus (Fig. S1) encoding most of the canine DLA88 locus variable region of MHC I [21]. In an attempt to assess the contribution of MHC class I mismatch to cIgG recognition assays, CMT cells were pre-reacted with anti-MHC class I antibody, known to react with canine MHC I on CMT cells [21], prior to addition of vaccinated dog sera (Fig. S2). Pre-reaction to block MHC class I neither results in diminished reactions against CMT28 vaccine cells nor did such attempts to block reaction affect binding to unmatched CMT12 or CMT27 cells employing sera from any of the vaccinated dogs (Di, M, L). This data could suggest that MHC I mismatch made little contribution to cIgG reactions although, because the anti-cMHC I antibody reacts to an epitope conserved in multiple species, it is also possible that this antibody does not provide sufficient steric hindrance to interfere with polyclonal cIgG binding to MHC I determinants on target cells. These results were confirmed by reversing the order of reaction applying the cIgG first and then the anti-MHC class I antibody followed by Alexa 660-conjugated anti-mouse IgG antibody. This experiment produced essentially the same results where cIgG pre-reaction was unable to block antibodies directed against MHC I (Fig. S3). Because the only commercially available anti-MHC I antibodies proven to bind canine MHC I react with multiple species no further conclusions were possible.

To determine if IgG class switching occurred during vaccination, IgG class 1- and 2-specific anti-cIgG antibodies were substituted as secondary antibodies staining matched CMT28 and unmatched CMT27 targets assaying sera from vaccinated dogs 1 week following vaccination 1 and 3 (Fig. 5). In all vaccinated dogs there were strong balanced IgG1/2 reactions to matched CMT28 cells after vaccination 1. However, statistically significant bias toward stronger IgG2 reactions to CMT28 cells following the third vaccination was detected. Although not significant, this was also observed with unmatched CMT27 targets except in dog Di where a weaker near background level IgG2 reaction was observed.

Fig. 5.

Anti-CMT cIgG assay of isotype switching in vaccinated dogs. Flow cytometry analysis of cIgG isotype (1 or 2) binding to matched CMT28 and unmatched CMT27 cell targets was performed. Sera from the three vaccinated dogs (Di, M, and L) from 1 week following vaccination 1 and 3 (V× 1 and 3 for each isotype and each dog) were reacted with target cells as noted (Target). FITC-conjugated secondary antibody specific for canine IgG class 1 or class 2 (IgG1 or IgG2) were used for secondary labeling. Negative controls included primary sera omitted (N1, N2) and no antibody (N) for both target cells. Inset: Representative examples of polyclonal CMT cIgG-specific histograms reacted against CMT28 cells for IgG1 (gray) and IgG2 (hatched) isotypes for dogs Di and L at 1 week following vaccination 3. Statistical significance (asterisk) was achieved for all vaccinated animals for CMT28 targets detecting cIgG2 after vaccination 3 (paired t test: p = 0.0363). Mean channel fluorescence (MCF) was expressed as a proportion of the cIgG reaction (in fold) to CMT28 antigens as described above

Autologous DC hybrid-cell fusion vaccination results in a weak CTL response

CMT-specific CTL assays were performed by ⁵¹Cr-release from vaccine matched CMT28 or unmatched CMT12 cell targets by control and hybrid-DC fusion injected dog PBMCs at dilution ratios of 50:1 and 25:1 (PBMCs to ⁵¹Cr-loaded CMT targets in triplicate) and percent-specific lysis calculated as a percentage of total releasable isotope available [21]. Some significant increase in CTL activity was detected at ratios of 25:1 against CMT28 targets but no consistent pattern of CTL activity was evident suggesting that, despite the presence of CpG oligonucleotides, immune reactions were biased toward an IgG response (Fig. 6).

Fig. 6.

Cell-mediated immunity of hybrid-DC fusion immunized dogs against matched and unmatched CMT cells. CMT-specific cytotoxic T cell (CTL) assays were performed by standard ⁵¹Cr-release assays from vaccine matched CMT28 or unmatched CMT27 cell targets in the presence of PBMCs recovered from immunized or control vehicle injected animals. CTL activity in PBMC populations was measured independently in triplicate and relative CTL response calculated as percent-specific lysis (% CMI) for each of 2 PBMC:CMT target cell ratios (50:1—50× or 25:1—25×) calculated as the percentage of total releasable isotope available due to specific lysis [21]. PBMCs were obtained from dogs 2 weeks after vaccine 1 (1.2 samples) and 1 week after vaccine 3 (3.1 samples). CMI assays for hybrid-DC fusion vaccine and control groups were performed on labeled vaccine-matched CMT28 cell targets (right) and unmatched CMT12 target cells (left). The entire experiment was repeated twice and representative data is shown. Some statistical significance was detected after vaccine 1 at a ratio of 25:1 but no consistent pattern of CTL responses was evident (mixed model for repeated measures analysis of variance)

Discussion

Mammary neoplasms express surface antigens that are recognized by the patient’s immune system but fail to promote an effective killing response [13]. We have previously demonstrated the potential of inducing such immune responses to mammary cancer in vaccine development through fusion of CMT cells with DC-like cells utilizing all-allogeneic cell line sources [21]. This strategy appears to potentiate uptake and promote processing and presentation of CMT antigens by DCs enhancing antigen recognition [13, 21, 24]. The success of CMT cell-DC fusions as vaccines suggests that they can overcome the rate limiting step, which is thought to be the transfer of antigens to the DC [13]. Cell fusion strategies for DC loading also avoid introduction of antigen selection bias although a theoretical risk of induction of autoimmunity has been postulated. There appears to be no evidence of this potential [13, 21–24]. We have previously reported only mild and rapidly resolving side-effects from lymph node vaccination of these hybrid-DC fusions and our current data also supports previous reports suggesting that injection of live tumor cells does not result in establishment of cell colonies [21, 31]. To date we have vaccinated approximately 30 dogs in which more than 100 hybrid-cell fusion injections have been made revealing no long-term adverse effects [21]. Adverse affects were confined to mild and rapidly resolving lameness (within 2 days) in the injected leg and transient and mild elevation of body temperature. Previous investigations also revealed a significant CTL response in vaccinated dogs as a consequence of vaccination with an allogeneic cell construct [21].

We have further developed this strategy to incorporate an autologous DC population isolated directly from laboratory Beagles to provide an individualized MHC-matched component to these vaccine constructs. Preparative isolation of primary canine DCs from individual dogs was possible due to their size and blood volume which allowed recovery of sufficient PBMCs to isolate approximately 1–5 × 10⁶ DCs from each animal for each vaccine construct. Such volumes bode well for the translation of such strategies to humans from this intermediate canine model as body size and blood volume are comparable. Previously published procedures for cell fusion were also successfully adapted to autologous DC hybrid-cell fusions [21].

Immune responses to antigens common to several CMT cell lines were assessed by comparing IgG reactions to CMT cells matched or unmatched for MHC to the vaccine construct based on CMT28 cells [21]. The assay measured variation in level of CMT-specific cIgGs present in serum able to stably bind CMT cells from unrelated dogs. The data demonstrate that a specific antibody response could be reliably detected and recognized native CMT antigens on live cells when healthy dogs were vaccinated with autologous DC hybrid-cell fusions composed of autologous DCs and CMT28 cells. Further, the response was also detected in unmatched CMT12 and CMT27 cells and even freshly isolated primary CMT cells from a tumor biopsy. Such comparisons strongly suggested the presence and detection of shared CMT cell antigens common to these CMT cells since the CMT cell lines used here have been previously shown to encode unique canine MHC class I DLA88 (HLA-equivalent) alleles [21]. Thus, a positive immune reaction with any of the CMT target cells, unmatched for MHC I, would strongly suggest that shared CMT antigens were recognized. This was true even though the reaction was less intense than that detected against vaccine-matched CMT28 cells. The data also suggest that the antigens recognized were not the result of culture in vitro as primary CMT cells were also recognized by these polyclonal sera. We have presumed that these antigens are located on the cell surface, as antibodies effectively labeled live cells, although at present we do not have any direct indication as to the identity of these apparently shared antigens. We have evidence that the important breast cancer antigen mucin 1 (MUC1) is expressed in CMT cells although not at the unusually high levels observed in human breast cancer lines (Fig. S4) [32]. Canine mammary tumors have previously been shown to express MUC1 protein by immunohistochemistry and this protein could account for some of this shared antigen [33].

There was also a distinct class shift detected in the cIgG response following the third vaccination from IgG1 to a stronger IgG2 response. This was true for all vaccinated animals when vaccine-matched CMT28 cell targets were used and in two of three vaccinated animals when non-matched CMT27 cells were employed as targets. A clear class shift biased toward the IgG2 from the IgG1 isotype was detected. Such observations in canine systems cannot be directly associated with specific T-helper type responses [34] although it has been suggested that a class shift to an IgG2-biased profile can be associated with a Th1-type response with a cytokine profile characterized as similar to human immune responses [35–38]. The enhanced cIgG2/Th1 induction, with characteristic cytokine expression, is suppressed in many cancers and is reduced in dogs with spontaneous metastatic disease [39]. The shift to a cIgG2 immune response may indicate potential for a more effective anti-tumor response even though direct correlation between cIgG isotype and Th1/Th2 responses is not yet possible in canine models. That recognition of MHC I miss-match likely accounted for part of the IgG response was suggested by the lower reaction with unmatched CMT cell lines. Limitations in the specificity of available antibodies recognizing canine MHC I precluded further conclusions regarding dependence of this reaction on MHC I recognition.

Subsequent assessment of CTL responses were surprisingly low in vaccinated dogs in these experiments compared to allogeneic hybrid-DC fusion vaccinated dogs [21]. This could be due to a difference in the autologous nature of the DCs employed and the mechanisms of antigen presentation now available. It is also possible that regulatory T cells (T_reg or suppressor T cells) were induced by this improved vaccine strategy thus suppressing a CTL response. Further investigation will be needed to explore these responses but evidence concerning cIgG responses clearly demonstrated that vaccination stimulated immune recognition of canine mammary tumor antigens and that reaction was biased toward an antibody response that in most cases peaked after two vaccinations. We have previously determined that enhanced immune recognition of shared CMT cell antigens using allogeneic vaccines were the result of enhanced CTL recognition most likely of shared CMT antigens. Development of the autologous DC vaccine component has caused most of the reaction to be directed through an antibody response. The possible consequences of an antibody bias and any enhanced inflammatory response remain to be elucidated.

We have established that vaccination with autologous DC hybrid-cell fusions constructed using freshly isolated autologous DC-enriched cell populations can be accomplished and such cellular vaccines have the potential to develop enhanced individualized immune responses in dogs. This demonstrates the potential of the hybrid-DC fusion strategy and the practicality of this technology for promoting immune recognition of mammary tumors. This strategy will serve as a platform for development of an immunotherapeutic vaccine treatment of canine mammary cancer.

Electronic supplementary material

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