All mice were obtained from the University of Tasmania Central Animal House and housed in a pathogen free environment and provided with food and water ad libitum. The two strains used were C57BL/6 and BALB/c. The mice were at least 5 weeks old at commencement of experimental treatments.

All animal experiments were performed with approval of the Animal Ethics Committee of University of Tasmania (UTAS), approval numbers A0010231 and A0010888.

Devil Facial Tumour Disease cell line, C5065, was obtained from stocks stored in liquid nitrogen at the UTAS. The cell line was established from primary tumor biopsy samples taken under the approval of the Animal Ethics Committee of Tasmania’s Park and Wildlife Services (permit numbers 33/2004–5 and 32/2005–6) and provided by A.-M. Pearse and K. Swift, Tasmanian Department of Primary Industries, Parks, Wildlife and Environment (DPIPWE). Cells were grown in RPMI-10FCS, which consisted of RPMI-1640 medium (GIBCO, New York, USA) supplemented with 10% fetal calf serum (FCS) (Bovogen Biological, VIC, Australia), 5 mM l-glutamine (Sigma-Aldrich, St Louis, MO, USA), and 40 mg/ml of gentamicin (Pfizer, Bentley Australia). The C5065 cell line culture was maintained in a fully humidified 5% CO₂ incubator at 35°C. Assays required co-culturing of mouse splenocytes with C5065 were maintained at 37°C.

Blood was collected from mice post mortem via cardiac puncture or via mandible bleed with living mice, allowed to clot and the serum was separated by centrifuging. Serum was stored at −20 or −80°C until required. One microliter of each serum sample was incubated with 10⁵ DFTD cells in 50 μl of staining buffer [phosphate-buffered saline (PBS) with 1% (w/v) bovine serum albumin (BSA) (Roche Diagnostics, Germany) and 0.01% NaN₃], on ice for 30 min. Following two washes with PBS, the cells were incubated with 2 μg of the relevant anti-mouse immunoglobulin Alexa-fluor conjugated antibody (Life Technologies, USA, catalog numbers IgG A-31553, IgG₁ A-21121, IgG_2a A-21136, IgG_2b A-21146, IgG₃ A-21151, IgM A-21042) in 100 μl of staining buffer on ice for 30 min. Negative controls included DFTD cells with either no serum or pooled naïve serum. Following three washes in PBS, the samples were resuspended in 100 μl of PBS for prompt reading on the flow cytometer (BD Canto II, Becton Dickinson, NJ, USA).

The forward scatter, side scatter, thresholds, and PMT voltages were optimized to place the negative mean fluorescence intensity (MFI) for the negative controls at approximately the second decade of the log scale. FCS 4 Express Flow Cytometry software (De nova Software, USA) was used to perform histogram overlays to compare indirect immunofluorescence levels of samples with naïve serum control. The geometric mean of the MFI for immune serum was divided by the geometric mean of the MFI for naïve serum control to give the relative antibody levels.

Devil Facial Tumour Disease cells in log growth phase were harvested from culture and viability was ascertained by trypan blue exclusion. Cell viability ranged from 50 to 80%. Cells were washed, resuspended in PBS, and 2 × 10⁶ viable cells were injected via the subcutaneous (SC) route or intraperitoneal (IP) route into C57BL/6 and BALB/c mice. With multiple sites SC injections, the cells were divided between multiple sites as stated. No adjuvants were used.

Spleens were removed from naïve or IP immunized mice. Lymph nodes were obtained from SC immunized mice. Mononuclear cells (MNC) were obtained by pressing spleens or lymph nodes through 40 μm cell strainers (BD Falcon, USA) and then separating them by density-gradient centrifugation on Histopaque-1083 gradient (Sigma-Aldrich, USA) following the manufacturer’s protocol. The MNC were washed twice with PBS and resuspended in RPMI-10FCS for use in in vitro cytotoxicity and cytokine assays.

Mononuclear cells were prepared as described above and resuspended in RPMI-10FCS at 10⁷ cells/ml. DFTD cells were harvested and resuspended at 10⁵ cells/ml in RPMI-10FCS. One hundred microliters each of DFTD and lymphocyte suspensions were combined in V-bottomed 96 well plates (Greiner Bio-one, Frickenhausen, Germany). Control wells were also prepared in the same plate by combining 100 μl RPMI with 100 μl of either DFTD or MNC as indicated. The plates were covered by lids and incubated for 72 h at 37°C with 5% CO₂. The plates were centrifuged to pellet the cells and the supernatant collected and stored at −20°C until required. Supernatant samples were assayed individually for cytokine activity using BD Biosciences CBA T_H1, T_H2, T_H17 micro-bead array kit and FACS array software as per manufacturer’s instruction (BD Biosciences, Cat # 560485). In brief, 50 μl of each supernatant sample and cytokine standards were incubated with mixed capture beads and PE detection reagent for 3 h, washed, resuspended, and run on the BD Canto II, flow cytometer. Cytokine data were analyzed using FCAP Array software (BD Bioscience, San Jose, CA, USA).

DFTD (10⁷; target cells) were labeled with 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) by incubating with 2 μl of 5 mM CFSE for 30 min at 23°C on shaker plate protected from light. Cells were washed twice and resuspended at 10⁵ cells/ml in RPMI-10FCS.

Mononuclear cells were resuspended in RPMI-10FCS at 10⁷ cells/ml. A V-bottomed 96 well plate (Greiner Bio-one, Frickenhausen, Germany) was prepared with 100 μl serial dilutions of the MNC to provide effector ratios of 100:1, 50:1, 25:1, 12:1, 6:1, and 3:1 with 10⁴ target cells.

The cytotoxicity assays were performed using four replicate samples and incubating for 18 h at 37°C in a fully humidified 5% CO₂ incubator. The plates were centrifuged for 5 min at 500 g, supernatant removed, pellet resuspended in 100 μl PBS + 1 μl propidium iodide (PI), and read on a BD Canto II flow cytometer with the High Throughput Sampler unit directly recovering samples from the wells.

Flow cytometry data analysis was performed using Flowing Software (Turku Centre for Biotechnology, Finland), or FCS 4 Express Flow Cytometry (De Nova Software, USA), with further analysis and graphs produced in Microsoft Excel 2007 and GraphPad Prism 5.

Cells positive for CFSE and PI were identified as dead target cells whereas cells positive for CFSE, but negative for PI, were identified as viable target cells. Cytotoxicity per well was calculated as dead target cells divided by the sum of dead and viable target cells and expressed as a percentage. The average cytotoxicity for each effector target ratio was calculated and the standard error of the mean (SEM) values was used for the error bars.

Quantitative data comparing two groups are expressed as the mean ± SEM and P-values calculated using Student’s unpaired two tailed t-test. Quantitative data comparing more than two groups are expressed as the mean with the probability calculated by one-way ANOVA followed by Dunnett’s test.

There was no evidence for substantial levels of preformed antibodies as serum from naïve mice showed minimal binding to DFTD cells. This was the case for both IgG and IgM. As shown in Figure 1, flow cytometry profiles of serum from three representative naïve BALB/c and C57/BL6 mice show barely detectable levels of preformed antibodies. When immunized intraperitoneally, both strains of mice had considerable increases in levels of specific antibodies to DFTD cells. As future experiments were aimed at evaluating specific immunity following DFTD cell immunization, the results were summarized as histograms rather than displaying multiple flow cytometry plots. The geometric mean of the MFI of the serum from immunized mice was divided by the geometric mean of the MFI of the serum from naïve mice.

BALB/c mice were twice injected subcutaneously with viable DFTD cells, which were obtained from cell culture. The objective was to determine if living DFTD cells could elicit an antibody response. Flow cytometry was used to analyze serum for IgG and IgM antibodies specific to DFTD surface antigens. All the IgG isotypes tested for were detected, but IgM was not detected (Figure 2), thus indicating that DFTD cells can induce an antibody response. The responses to single site SC injections were compared with multiple site SC injections to determine if multiple site injections with the same number of total cells produced a greater response. Multiple site SC injections produced greater total IgG (P < 0.05; unpaired Student’s t-test) and IgG1 (P < 0.01; unpaired Student’s t-test) responses than injecting the same number of DFTD cells into a single site (Figure 2).

C57BL/6 mice were intraperitoneally or subcutaneously injected twice with viable DFTD cells. The objective was to determine, which route produced a better antibody response. Results in Figure 3 show that the IP route produced both IgM and IgG antibodies. The responses following the IP injections were stronger than the SC route for IgM (P < 0.01; unpaired Student’s t-test), total IgG, IgG1, IgG2a (P < 0.001; unpaired Student’s t-tests), and IgG3 (P < 0.05; unpaired Student’s t-test).

Having established that immunization with DFTD cells induces strong IgG responses following two injections, we next determined if IgG and IgM responses could be detected after a single injection. Results in Figure 4 show 4 days was insufficient to generate optimum levels of antibody in the serum with IgM detected in only two of the four mice and IgG detected in only one of these two mice. By 7 days all mice produced both IgM and IgG antibodies with the predominant isotype being IgG (P < 0.05; Student’s unpaired t-test) (Figure 4).

Responses to secondary immunizations following 95 days showed enhanced responses in IgG1, IgG2b, and IgG3 antibodies but not IgM (Figures 4 and 5). The enhanced response is consistent with a primed immune system memory response because it was evident 95 days after the primary immunization.

As IP injections of DFTD cells produced the best antibody response, we used this route to determine if an enhanced cytotoxic response could be induced against DFTD cells. Splenocytes from naïve C57BL/6 mice demonstrated dose response cytotoxicity against DFTD cells following 18 h incubation (Figure 6). This was not improved following two IP injections with DFTD cells, indicating that unprimed cells of the immune system mediated the cytotoxicity.

Since tumor cells can weaken anti-tumor immune responses by skewing them toward less effective T_H2 responses, we examined the antibody and cytokine profiles for such a bias. Two strains of mice were compared because of their reportedly opposing T_H1 (C57BL/6) or T_H2 (BALB/c) dominated immune responses (10, 11).

Mice were injected intraperitoneally twice with DFTD cells and serum was collected 7–8 days following the second immunization. Levels of IgM, the T_H2 antibody, IgG1, and the T_H1 antibody IgG3 were not significantly different between the two strains (Figure 7). Of the other T_H1 antibodies, BALB/c mice expressed higher levels of IgG2a and lower levels of IgG2b compared to C57BL/6 (P < 0.05; unpaired Student’s t-test).

As up-regulation of certain cytokines in the tumor microenvironment can enhance or suppress tumor rejection, we next assessed the cytokine production of splenocytes from naïve mice, mice given a single IP injection of DFTD and mice given two IP injections of DFTD cells. Splenocytes were obtained from the mice and incubated in vitro for 72 h with DFTD cells and the supernatant analyzed for cytokines. Cytokine levels expressed into the growth media were then measured.

Splenocytes from non-immunized mice did not produce detectable cytokines. Evidence for IFN-γ, TNF, IL-6, and IL-10 production was apparent for splenocytes obtained at 4 and 21 days following a single immunization (Figure 8). These same cytokines were also detected when splenocytes were obtained 5 days after a secondary DFTD immunization.

Of the T_H1 cytokines, peak levels of IFN-γ and TNF-α were detected from splenocytes obtained at day 21 after a single immunization. Peak levels of the T_H2 cytokine IL-10 were detected following a secondary immunization. The pro-inflammatory cytokine IL-6 followed a similar pattern to IL-10 with peak values occurring after a secondary immunization. IL-4 and IL-12 were not produced at detectable levels and are therefore not shown. The antibody and cytokine profiles did not suggest that immunization with DFTD cells polarized the immune response toward a T_H1 or T_H2 profile.

Inactivated DFTD cells that maintain their immunogenicity are required for vaccine and immunotherapy trials. To determine the best means of achieving this, BALB/c mice were injected twice IP with DFTD cells. The cells were either viable or inactivated by irradiation, sonication, or rapid freeze/thawing. Serum was collected for analysis of anti-DFTD antibodies by flow cytometry. Splenocytes from DFTD immunized mice were co-cultured with DFTD cells and the supernatant analyzed for cytokine expression.

Mice immunized with irradiated cells produced higher levels of DFTD specific antibodies compared to sonicated and freeze/thaw lysates. When cultured in vitro with DFTD cells, splenocytes from mice immunized with sonicated or freeze/thaw lysates produced lower levels of IFN-γ and TNF-α compared to splenocytes from mice immunized with irradiated cells (Figure 9). Irradiation of cells is the better method for inactivation because immunogenicity is maintained.