The aim of the current study was to explore immunisation protocols to enhance protective responses against DFTD. These included immunisation with DFTD cells expressing MHC-I to increase their antigenicity, in combination with ISCOMATRIX™, Poly I:C and CpG, adjuvants that enhance both innate and adaptive immune responses. As access to Tasmanian devils for research purposes is limited, we were restricted to the use of nine healthy devils, some of which had reached an advanced age. These devils were used over a five-year period. In an endeavour to improve the immune response, four immunisation protocols were tested sequentially. Seven devils were immunised with a variety of cell preparations and adjuvants to determine if they could be protected against the development of DFTD following challenge with live DFTD cells. One devil was used as an adjuvant-alone control and one devil as a non-immunised control. Devils were monitored for adverse reactions to the immunisations and none were identified. In the absence of a reliable cytotoxicity assay we used DFTD-specific IgG antibody levels in devil serum to evaluate the immune responses after the immunisations in each protocol. As devils were immunised with either MHC-I+ or MHC-I− DFTD cells, antibody reactivity was evaluated against cytokine treated (MHC-I+) and untreated (MHC-I−) DFTD cells. As IgG production requires T cell help, this also provided an indicator of T cell involvement. Six of the immunised devils and the non-immunised control devil were later challenged with live DFTD cells. If tumours developed, immunotherapy was commenced and tumour rejection and anti-tumour antibody responses were measured. Tumour biopsies were taken before and after immunotherapy, and evaluated for immune cell infiltration by immunohistochemistry. An outline of the immunisations, challenges and tumour development is presented in Supplementary Table S1.

A comparison of the four protocols can be seen in Figs 1–4. The responses to immunisation are shown in Fig. 1 and highlight that immunisation with MHC-I+ DFTD cells consistently produced antibody responses. Figure 2 shows the histology of the engrafted DFTD tumours, which occurred in six of the seven devils challenged with live tumour cells. Immunohistochemistry did not reveal evidence of immune cell infiltration into the tumours. Figure 3 outlines the growth of the engrafted tumours and their response to immunotherapy. Immunotherapy with live MHC-I+ DFTD cells was associated with tumour regression in three devils and the regression correlated with antibody responses against DFTD cells. The histology in Fig. 4 supports the immune-mediated regression of the tumours as indicated by the large numbers of MHC-II and CD3 positive cells infiltrating the tumour.

Figure 1: Antibody responses to immunisation in protocols A to D and adjuvant control. Immunisation with MHC-I+ DFTD cells consistently induced antibodies responses. The left column shows a brief description of the immunisation protocols and the histograms in the right column show the corresponding antibody responses against both MHC-I+ and MHC-I− DFTD cells assessed by flow cytometry. Each colour in the histogram relates to a particular protocol as indicated. (a) Protocol A (TD1-My), immunisation with DFTD cell protein extracts did not induce antibody responses. (b) Protocol B, immunisation with MHC-I+ DFTD cells elicited antibodies responses particularly against MHC-I+ cells in both TD2-Ga and TD3-Ty devils. Immunisation with a combination of firstly sonicated and then irradiated MHC-I+ DFTD cells in Protocol C (c) or firstly irradiated and then sonicated in Protocol D (d) consistently induced antibody responses in the four devils. (e) Immunisation of the adjuvant control devil TD9-Pl followed the same regime as Protocol D but received only the adjuvant component of the vaccine. This devil did not produce detectable antibodies against DFTD cells. Full size image

Figure 4: Tumour histology and immunohistochemistry of DFTD tumours following immunotherapy showing tumour regression and immune cell infiltration. (a) Protocol A. Biopsy sections of the tumour site of TD1-My taken one week after completion of immunotherapy. Tumour regression correlated with strong immune cell infiltration of MHC-II+ cells and T cells, mainly CD8+ cells. (b) Protocol B. A tumour biopsy taken one week after the last therapy in TD2-Ga shows no evidence of immune cell infiltration. Biopsies of the scar tissue 3 weeks after dislodgment of the tumour in TD3-Ty show the persistence of few DFTD (PRX+) cells and a large number of MHC-II+ cells but very few CD3+ cells. (c) Protocol D. Tumour regression in TD6-Tp and TD7-Sy correlated with strong immune cell infiltration of MHC-II+ cells and CD3+ cells with CD8+ cells more abundant than CD4+ cells as evidenced by biopsies taken 4 weeks after therapy. (d) No-immunisation control (TD8-Mk), a biopsy of the LHS tumour taken 4 weeks after therapy shows well established DFTD tumours with virtually no immune cell infiltration. Standard haematoxylin and eosin (H&E) staining and immunohistochemical labelling using anti-periaxin (PRX) antibody and anti-MHC-II, CD3, CD8 and CD4 antibodies. Scale bar, 50 μm. Full size image

Figure 2: Histology and immunohistochemistry of DFTD tumours following challenge, showing lack of immune cell infiltration. (a) Sections of a biopsy of the induced tumour in TD1-My (Immunisation Protocol A) taken 10 weeks after challenge. There is no evidence of immune cell infiltration. (b) Development of grafted DFTD tumours in devils immunised with Protocol B. The biopsy sections of the tumour in TD2-Ga taken 12 weeks after challenge show scattered MHC-II+ cells but virtually no infiltrating CD3+ cells. Similarly, no immune cell infiltration was detected in a biopsy of the DFTD tumour in TD3-Ty taken 10 weeks after the challenge. (c) TD4-Mm (Protocol C) did not develop tumours after the challenge with live DFTD cells. (d) Development of grafted DFTD tumours in devils immunised with Protocol D. TD6-Tp developed DFTD tumours after challenge at both sides of injection (left hand side - LHS, and right hand side - RHS of the rump). The images are representative histology of a biopsy from the LHS tumour taken 14 weeks after challenge showing very poor immune cell infiltration. TD7-Sy also developed DFTD tumours at both sides of injection. The images show no evidence of immune cell infiltration in a biopsy of the LHS tumour taken 20 weeks after challenge. (e) The adjuvant control (TD8-Mk) developed tumours at both sides of the challenge. A biopsy of the LHS tumour taken 10 weeks after challenge shows scattered MHC-II+ cells within the tumour and very occasional CD3+ cells. All panels (a to e) standard haematoxylin and eosin (H&E) staining and immunohistochemical labelling using anti-periaxin (PRX) antibody to detect DFTD tumour cells and anti-MHC-II and anti-CD3 antibodies to detect an immune response. Scale bar, 50 μm Full size image

Figure 3: Tumour growth and antibody responses following immunotherapy. Tumour regression associated with antibody responses was observed in three devils after therapy with live MHC-I+. Graphs on the left show a time-line of the growth of the induced tumours following challenge with live DFTD cells (day 0) for each protocol (a to d) and the no-immunisation control (e). Immunotherapy protocols are indicated in coloured vertical dashed lines. Histograms on the right show the antibody responses after the therapy assessed by flow cytometry against both MHC-I− and MHC-I+ DFTD cells. (a) Protocol A, immunotherapy in TD1-My induced complete tumour regression and was associated with high levels of antibody, particularly against MHC-I+ DFTD cells. (b) Protocol B, immunotherapy in TD2-Ga was ineffective and the tumour grew. Antibodies were detected against both MHC-I− and MHC-I+ DFTD cells. Immunotherapy in TD3-Ty was ineffective in controlling tumour growth. The tumour ulcerated and dislodged from the skin. A few weeks later the tumour re-established and continued to increase in size. Antibodies were not detected in the serum. (c) Protocol C. TD4-Mm did not develop tumours after challenge. This devil died 189 days after challenge. A post-mortem examination did not detect DFTD tumours or metastases. (d) Protocol D. Immunotherapy in TD6-Tp consisting of a single injection of live MHC-I+ DFTD cells given in the interscapular region induced complete regression of both tumours. The same immunotherapy in TD7-Sy also induced complete regression of both tumours. A small third tumour developed in the site of the immunotherapy injection. This tumour did not increase in volume and immune cell infiltration was observed in histology (see Supplementary Table S2). Both devils TD6-Tp and TD7-Sy had elevated serum antibodies against DFTD cells. (e) The therapy administered to the no-immunisation control (TD8-Mk) was ineffective and both tumours grew. A third tumour developed in the site of the immunotherapy and this tumour also continued to increase in size. Antibodies were not detected after therapy. Full size image

The following sections describe the assessment of the responses to immunisation and immunotherapy in each protocol.

Protocol A

Devil TD1-My was immunised subcutaneously three times with protein extracted from heat-treated DFTD cells and boosted six months after the third immunisation. Each immunisation and the booster used the ISCOMATRIX™ adjuvant. This devil did not show any evidence for an antibody response against the DFTD cells (Fig. 1a). While there was no DFTD-specific antibody response detectable in the in vitro assay, an in vivo immune response may have occurred. To test this, the devil was challenged with 25,000 live DFTD cells one month after the booster immunisation in one site in the rump. A tumour was detected 37 days after this challenge. DFTD was confirmed on biopsy by periaxin expression and there were no signs of immune cell infiltration in the tumour (Fig. 2a).

The lack of an antibody response indicated that the DFTD cells were poorly recognised by this devil’s immune system. When the tumour in this animal reached approximately 30 cm3 and with no signs of regression, the devil was injected subcutaneously with live interferon-γ (IFN-γ) treated MHC-I+ DFTD cells between the shoulder blades. As DFTD cells cultured with IFN-γ upregulate MHC-I expression9, they have the potential to become targets for an allogeneic immune response. The original tumour continued to increase in size, but after one week it began to regress (Fig. 3a). To maintain the response, a cytokine rich conditioned medium (supernatant obtained from mitogen stimulated devil lymphocytes) was injected intra-tumourally each week for three weeks. This was followed by an additional injection of live MHC-I+ DFTD cells near the tumour. The tumour continued to regress until it was no longer palpable four weeks after the last immunotherapy (Fig. 3a).

One week after all treatments were completed, the serum contained elevated levels of antibodies against MHC-I+ DFTD cells, almost 30 times the median fluorescence intensity (MFI) of the pre-immune serum. Antibodies against MHC-I− DFTD cells were also detected, but at lower levels (Fig. 3a). A tumour biopsy, taken a week after regression was first detected, showed sparse DFTD cells, with a strong infiltration of MHC-II+ cells and CD3+ cells (predominantly CD8+) into the tumour (Fig. 4a, Supplementary Table S2). Table 1 describes the immunotherapy and summarises the immune response to the therapy.

Table 1 Summary of antibody and cellular responses to immunotherapy. Full size table

Due to an age-related health problem, this devil was euthanised 40 weeks after the last treatment. There were no signs of tumour recurrence or metastases during post-mortem examination. The remarkable T cell infiltration into the tumour and the strong antibody response provided the first evidence that immunotherapy can stimulate the devil’s immune system to recognise and target an established DFTD tumour. One concern was that immunotherapy with live MHC-I+ DFTD cells could pose a risk of tumour engraftment at the tumour immunotherapy site. Therefore, when immunisation protocols failed to protect against experimentally induced DFTD the subsequent immunotherapy was inoculation with irradiated IFN-γ treated MHC-I+ DFTD cells (to mimic intact live MHC-I+ DFTD cells) and IFN-γ therapy (protocol B).

Protocol B

Two devils, TD2-GA and TD3-Ty were immunised with frozen/thawed DFTD cells that had been treated with either Trichostatin A (TSA), a histone deacetylase inhibitor (TD2-Ga) or cytokine rich conditioned medium (TD3-Ty) to upregulate MHC-I expression. The adjuvant ISCOMATRIX™ was used in all immunisations.

TD2-Ga developed low to medium antibody responses against IFN-γ treated MHC-I+ DFTD cells and untreated DFTD cells (Fig. 1b). The devil was then challenged with 25,000 live DFTD cells and a DFTD tumour was first identified at the inoculation site 67 days after challenge. Immunohistochemistry at this time showed few MHC-II+ cells and occasional CD3+ cells, mostly located at the periphery of the tumours or in proximity to blood vessels. (Fig. 2b). Cells with dendritic morphology, presumably dendritic cells, were seen in the epidermis, dermis and subcutaneous tissue, but not associated with the tumours (Supplementary Table S2).

When the tumour reached approximately 20 cm3 in volume the devil was subcutaneously injected, on the rump near the tumour, with irradiated MHC-I+ DFTD cells followed one week later by an intra-tumoural injection of devil recombinant IFN-γ, which became available for the first time. The tumour continued to grow (Fig. 3b). For the duration of the immunotherapy, devil TD2-Ga maintained medium levels of antibodies against IFN-γ treated MHC-I+ and untreated DFTD cells (Fig. 3b). Tumour biopsies showed very few MHC-II+ cells and occasional T cells were present, but mostly in the surrounding connective tissue (Fig. 4b). This devil died naturally of an unrelated cause. A post-mortem showed an encapsulated DFTD tumour with strong evidence of tumour vascularisation including large blood vessels within the tumour. Few MHC-II+ and T cells were identified within the tumour. MHC-II+ and T cells were present in the granulation tissue and connective tissue away from the tumour. (Supplementary Table S2). Table 1 provides a summary of the immune responses to therapy.

TD3-Ty developed a low antibody response post-immunisation, but only against IFN-γ treated MHC-I+ DFTD cells (Fig. 1b). A DFTD tumour was first identified at the inoculation site 67 days after challenge with 25,000 live DFTD cells. Immunohistochemistry revealed almost no immune cell infiltration within the tumour (Fig. 2b). MHC-II+ and CD3+ cells were scarce, and when present were located at the periphery of the tumours or in proximity to blood vessels (Supplementary Table S2).

When the tumour reached approximately 20 cm3 in volume the devil received an intra-tumoural injection of IFN-γ each week for three weeks. The tumour continued to grow rapidly. Two weeks after the last dose of IFN-γ this devil was injected subcutaneously, on the rump near the tumour, with irradiated IFN-γ treated MHC-I+ DFTD cells (Fig. 3b). At this time the tumour appeared to ulcerate and it dislodged from the skin. A biopsy of the tumour site revealed granulation tissue, fibroblasts with an activated-like appearance and a small amount of necrotic tumour. Large dendritic MHC-II+ cells were identified in the periphery of the necrotic tumour cells associated with CD3+/CD8+ cells. Biopsies taken one, three and four weeks later showed the re-establishment of nests of DFTD tumours suggesting that dislodgment of the tumour may have been mechanical, and not due to an immune process (Supplementary Table S2). Serum antibodies against DFTD cells were not detected after treatment (Fig. 3b). MHC-II+ and CD3+ cells were present around the periphery of the tumour clusters but very few infiltrated the tumour (Fig. 4b). The tumour continued to grow and this devil was euthanised. A summary of the immunotherapy and the immune response to the therapy is described in Table 1.

From the immunisation of TD2-Ga and TD3-Ty, it was concluded that MHC-I+ DFTD cells induced better DFTD-specific IgG antibody responses. However, the immunisation protocol alone was not enough to induce an effective anti-tumour immune responses. Therefore, additional adjuvants were used in subsequent immunisation protocols.

Protocol C

Two devils (TD4-Mm and TD5-Br) were immunised with sonicated DFTD cells that had been treated with IFN-γ to upregulate MHC-I (immunisation 1), followed by irradiated, IFN-γ treated DFTD cells (immunisation 2) and boosters with irradiated MHC-I+ DFTD cells. In all immunisations/boosters the adjuvants ISCOMATRIX™, Poly I:C and CpG were used.

TD4-Mm produced medium antibody responses against IFN-γ treated MHC-I+ and untreated DFTD cells (Fig. 1c). Two booster injections of irradiated MHC-I+ DFTD cells six and eleven months after the last immunisation did not appear to increase the antibody levels (Fig. 1c). This devil remained free of DFTD tumours for 189 days after challenge with live DFTD cells but was euthanized for age-related health reasons. Medium to high antibody responses against IFN-γ treated MHC-I+ DFTD cells and untreated DFTD cells were detected after the live tumour cell challenge and similar levels identified in the post-mortem sample (Table 1).

TD5-Br produced medium antibody responses against IFN-γ treated MHC-I+ and untreated DFTD cells (Fig. 1c). This devil was euthanized for age-related health reasons before it was challenged with live DFTD cells.

Immunisation Protocol C showed the first preliminary evidence that the combination of adjuvants led to a stronger anti-tumour antibody response. It is feasible that this strong immune response also prevented experimental DFTD tumour engraftment.

Protocol D

Two devils (TD6-Tp and TD7-Sy) were immunised with irradiated DFTD cells that had been treated with IFN-γ to upregulate MHC-I (immunisation 1), followed by sonicated, IFN-γ treated MHC-I+ DFTD cells (immunisation 2) and a booster with irradiated MHC-I+ DFTD cells. All immunisations/boosters were in combination with ISCOMATRIX™, Poly I:C and CpG adjuvants.

TD6-Tp produced medium antibody responses against IFN-γ treated MHC-I+ DFTD cells and untreated DFTD cells, but only after receiving the second immunisation (Fig. 1d). 80 days following challenge with 25,000 live DFTD cells at the right hand side (RHS) of the rump and 100,000 live DFTD cells at the left hand side (LHS) of the rump, DFTD tumours developed at both sites. The higher dose was used to confirm that if a tumour did not develop, it was not due to insufficient cells being inoculated. Immunohistochemistry of the LHS tumour revealed only a few MHC-II+ and CD3+ cells mainly at the periphery of the tumour cell nests (Fig. 2d). When this tumour reached approximately 20 cm3 in volume the devil was subcutaneously injected in the interscapular region (between the shoulders blades), with live IFN-γ treated MHC-I+ DFTD cells. One week later the original tumours began to regress (Fig. 3d). Low to medium antibody responses against IFN-γ treated MHC-I+ and untreated DFTD cells were evident after immunotherapy (Fig. 3d). Biopsies of the LHS tumour, four weeks after immunotherapy, revealed moderate numbers of MHC-II+ cells towards the tumour periphery and a few within the tumour. A large number of CD3+ cells showed a similar distribution to the MHC-II+ cells, with CD8+ cells more abundant than CD4+ cells (Fig. 4c). Neither tumour (LHS and RHS) was palpable 70 days after immunotherapy commenced. Biopsies at the LHS tumour site showed scar tissue composed of hyalinised and relatively acellular dense connective tissue. Evidence of immune infiltration was indicated by moderate to large numbers of MHC-II+ cells, individually or in clusters, adjacent to the connective tissue. Similarly, a moderate to high number of CD3+ T cells paralleled the MHC-II+ cells (Supplementary Table S2).

TD7-Sy produced medium/high antibody responses against IFN-γ treated MHC-I+ DFTD cells and untreated DFTD cells after receiving immunisation 1. Responses increased after immunisation 2 (Fig. 1c). This devil was challenged with live DFTD cells at the RHS and LHS of the rump as described above and DFTD tumours developed at both inoculation sites 110 days later. Immunohistochemistry of the LHS tumour revealed limited immune cell infiltration (Fig. 2d). When the LHS tumour reached approximately 30 cm3 in volume the devil was subcutaneously injected, in the interscapular region, with live IFN-γ treated MHC-I+ DFTD cells. The tumours increased in size, but one week later both tumours began to regress (Fig. 3d). Medium to high antibody responses against IFN-γ treated MHC-I+ DFTD cells and untreated DFTD cells were evident after treatment (Fig. 3d). Biopsies of the LHS and RHS tumours, four weeks after immunotherapy, revealed moderate numbers of MHC-II+ cells towards the periphery of the tumour and some cells within the tumour. A large number of CD3+ cells showed a similar distribution to the MHC-II+ cells, with CD8+ cells more abundant than CD4+ cells (Fig. 4c). The LHS and RHS tumours were not palpable 70 days after treatment commenced.

With devil TD7-Sy, the live MHC+ DFTD cells that were used for immunotherapy developed into a small tumour (Fig. 3d). This tumour did not increase in volume after it had reached 10 cm3. MHC-II+ and CD3+ cells could be found throughout the tumour (Supplementary Table S2). A summary of the immunotherapy and the immune response to the therapy is described in Table 1.

Immunisation protocol D produced antibody responses to DFTD cells. Although this did not protect from tumour development, immunotherapy with a single injection of live MHC-I+ DFTD cells was followed by immune cell infiltration and tumour regression.

Non-immunised control devil

TD8-Mk was not immunised and did not receive adjuvant. 40 days after challenge with live DFTD cells, tumours were palpable at both the 25,000 and 100,000 cell inoculation sites. The tumours continued to grow, with no indication of immune cell infiltration (Fig. 2e). When the tumour reached approximately 10 cm3 in volume the devil was subcutaneously injected, in the interscapular region, with live IFN-γ treated MHC-I+ DFTD cells. For this devil, the immunotherapy via injection of live MHC-I+ DFTD tumour cells produced a small tumour (Fig. 3e). There was no evidence for antibody production (Fig. 3e). Biopsies taken 28 days after the immunotherapy showed well established and encapsulated tumours. Average to large sized intra-tumoural blood vessels and extensive necrosis between the pockets of tumours were observed (Supplementary Table S2). A few foci of MHC-II+ cells appeared in the periphery of the tumours with very few within the tumour. Very few CD3+ (either CD4 and CD8) T cells were found within or surrounding the tumour (Fig. 4d). Due to the progression of tumour size, this devil was euthanised. Table 1 describes the immunotherapy and summarises the immune responses.

Adjuvant control devil

TD9-Pl received five injections of the adjuvant components only (i.e. excluding DFTD cells) and did not produce detectable anti-DFTD antibodies (Fig. 1e). This devil was not challenged with live DFTD cells.

MHC-I genotypes within the test cohort

Devils have three classical MHC-I genes, Saha-UA, -UB and -UC, with Saha-UA converted to a pseudogene in certain MHC haplotypes due to a 1.6-kb-long deletion in the genomic region14. All nine devils (seven immunised and two controls) and the DFTD tumour cell line were genotyped at these three loci for the peptide-binding domains (α1 and α2; Supplementary Table S3).

A total of 18 MHC-I variants was identified across all samples; 12 have been previously reported and assigned to MHC loci based on BAC contig assembly and sequencing and genotype analysis14,15, whereas six are novel variants (SahaI*35:02, 88:02, 99, 100, 101, and 102; GenBank accession numbers KY194692- KY194697). These were assigned to gene UA, UB or UC based on their phylogenetic relationship with previously reported variants (Supplementary Fig. S1). Four MHC-I variants, SahaI*35, 46, 90 and 28, were identified in the DFTD cells used in the immunisation and immunotherapy procedures. This result differs from the previously reported MHC genotype of the tumour (Pye et al. 2015), which is likely due to the use of different starting material. The previous study used tumour biopsies (Pye et al. 2015), which usually contain host connective tissue (Siddle et al.10 PNAS), whereas in this study tumour DNA was extracted from cell culture and therefore was unlikely to contain host DNA contamination. The other 14 MHC-I variants showed high sequence similarities (94.2% average amino acid sequence identity; Supplementary Table S4) with the four variants in the tumour, consistent with previous findings of low sequence variability in devil MHC genes14. Two control devils each shared one MHC-I antigen with DFTD cells (SahaI*35 in TD9-Pl and SahaI*28 in TD8-Mk), while five of the seven immunised devils, TD1-My (Protocol A), TD2-Ga and TD3-Ty (Protocol B), TD6-Tp and TD7-Sy (Protocol D), did not share any MHC-I molecules with the tumour. TD1-My (protocol A) had one MHC-I variant (SahaI*101) with relatively lower sequence similarity (89.2%) with one of the tumour antigen (SahaI*90). This animal produced lower antibody responses against MHC-I+ DFTD cells post-immunisation and post-booster than the other devils (Fig. 1). Of the two devils in protocol B, TD2-Ga appeared to produce stronger antibody responses than TD3-Ty. This may be attributed to TD2-Ga possessing MHC-I variant SahaI*87, which shows the lowest sequence identity among all variants to SahaI*46 (89.2%) and SahaI*28 (89.7%) of the tumour. The two devils in protocol C produced good immune responses even though they shared one and two MHC-I molecules with tumour cells. The two devils in protocol D, TD6-Tp and TD7-Sy, had similar MHC-I genotypes, both possessing variants SahaI*29, 36, 37 and 27. Both produced responses against DFTD cells.