The newly-arisen Devil facial tumour disease 2 (DFT2) reveals a mechanism for the emergence of a contagious cancer
Contagious cancers have emerged and circulate in two species of mammals ( dogs and Tasmanian devils ) and four species of molluscs ( Metzger et al . , 2015 , 2016; Novinski , 1876; Pearse and Swift , 2006 ) . The Tasmanian devil is the only mammalian species in which two independent contagious cancers exist , Devil Facial Tumour 1 ( DFT1 ) and Devil Facial Tumour 2 ( DFT2 ) ( Pye et al . , 2016b ) . Although both cancers have similar gross morphology , causing tumours on the face , neck and oral cavity , genetic analysis shows that they emerged in different individuals ( Pearse and Swift , 2006; Pye et al . , 2016b; Stammnitz et al . , 2018 ) . DFT1 was first identified in 1996 in the northeast of Tasmania ( Hawkins et al . , 2006 ) , but has since spread widely , causing close to 100% mortality and drastic decline of affected populations ( Hamede et al . , 2015 ) . In contrast , DFT2 is a more recent contagious cancer , it was first characterised in 2014 and has been formally identified in eleven devils from the Channel region of Southwest Tasmania ( Pye et al . , 2016b; Kwon et al . , 2018 ) . Recent immunohistochemical and drug sensitivity analyses of DFT2 indicate it arose from a similar tissue to DFT1 ( Stammnitz et al . , 2018 ) . As allografts , DFT1 and DFT2 cells should be rejected by the host devil through T cell recognition of non-self MHC class I molecules encoded by the major histocompatibility complex ( MHC ) ( Sherman and Chattopadhyay , 1993 ) . The mature MHC class I molecule is a trimer that acts as a ligand for the T cell receptor ( TCR ) and is composed of a light chain , β2-microglobulin ( β2m ) , in non-covalent association with a heavy chain , which provides a binding cleft for self and non-self peptides . Classical MHC class I molecules are important transplantation antigens that are highly polymorphic , ubiquitously expressed and involved in peptide-antigen presentation . In contrast , non-classical MHC class I molecules have little polymorphism and more diverse functions , including regulation of the immune response particularly by inhibiting natural killer ( NK ) cell function ( Braud et al . , 1999 ) . Allografts can be rejected within 7–14 days when host CD8+ T cells are exposed to non-self MHC class I molecules and their bound peptides on the surface of donor cells ( Rosenberg , 1993 ) . Vertebrates have multiple MHC class I genes that are generally not orthologous between species due to rapid evolution ( Adams and Parham , 2001 ) . Five MHC class I genes have been identified in the genomic region encoding the Tasmanian devil MHC ( Cheng et al . , 2012 ) . Saha-UA , -UB and –UC , are likely classical , with moderate levels of polymorphism and ubiquitous expression , while Saha-UD , –UK and -UM , are non-classical , with limited polymorphism and restricted expression ( Cheng et al . , 2012; Cheng and Belov , 2014 ) . We have previously demonstrated that DFT1 cells down-regulate MHC class I and class II molecules from their cell surface , thereby removing the targets for a T cell response ( Siddle et al . , 2013 ) . This is the first indication that DFT1 cells have similar immune evasion mechanisms to those found in single organism tumours ( Hicklin et al . , 1999; McGranahan et al . , 2017 ) . Loss of MHC class I is due to epigenetic down-regulation of β2m , and the peptide transporter encoded by TAP1 and TAP2 ( Siddle et al . , 2013 ) but MHC class I can be restored in DFT1 cells when treated with IFNγ ( Siddle et al . , 2013 ) . MHC class I positive DFT1 cells have since been used with some success in initiating immune responses to DFT1 cells ( Tovar et al . , 2017 ) . Down-regulation of MHC is also observed in the canine transmissible venereal tumour ( CTVT ) , where MHC molecules are down-regulated during transmission and growth ( Hsiao et al . , 2002; Pérez et al . , 1998 ) . However , subsequent cytokine signalling leads to lymphocyte infiltration of tumours , MHC up-regulation and either tumour regression or stasis of growth ( Yang et al . , 1987 ) . These findings have suggested that loss of MHC class I expression is necessary for the emergence and subsequent transmission of contagious cancers . Why DFT2 cells can pass between individuals without a protective immune response from the host is not known , nor are the conditions that have facilitated the emergence of two contagious cancers in this species . Here we show that in contrast to DFT1 , DFT2 cells express MHC class I molecules . However , the dominantly expressed MHC class I alleles are either non-classical or shared with host devils and common in the wider population . Finally , DFT2 tumours were identified with evidence of MHC class I loss , indicating the emergence of MHC class I down-regulation in this tumour .
Flow cytometry using a monoclonal antibody against Tasmanian devil β2m was used to assess the level of MHC class I expression on the surface of a representative DFT1 cell line ( DFT1_4906 ) ( Figure 1A ) , DFT1 cells treated with IFNγ ( DFT1_4906 + IFNγ ) ( Figure 1B ) , three DFT2 cell lines ( DFT2_RV , DFT2_SN and DFT2_TD549 ) ( Figure 1C , D and E ) and devil fibroblast cells ( Fibroblasts_Salem ) ( Figure 1F ) . As has been previously reported ( Siddle et al . , 2013 ) , DFT1 cells do not express β2m , but DFT1 cells treated with IFNγ upregulate β2m to the same level as devil fibroblast cells . In contrast , all three DFT2 cell lines express cell surface β2m , but the level of expression varies , DFT2_TD549 cells have the highest level of expression ( Mean Fluorescence Intensity ( MFI ) 42 . 1 ) followed by DFT2_RV ( MFI 37 . 6 ) and DFT2_SN ( MFI 12 . 1 ) . Although all three DFT2 cell lines express β2m , they have lower levels of β2m than DFT1 cells that have been treated with IFNγ ( MFI 92 . 5 ) and devil fibroblasts ( MFI 93 . 3 ) . These results indicate that DFT2 cells grown in culture express surface MHC class I molecules in contrast to DFT1 , which is MHC class I negative . RT-qPCR was used to quantify the relative level of MHC class I gene expression for β2m , classical MHC class I and non-classical MHC class I genes in vitro ( Figure 2A–D ) . Due to high nucleotide similarity between the classical MHC class I genes in the Tasmanian devil , Saha-UA , -UB and –UC , and their respective alleles , a single primer set was used to amplify transcripts from these genes as a group . Gene specific primers were used to amplify the non-classical MHC class I genes , Saha-UK ( Figure 2C ) and Saha-UD , which was found only at trace levels ( Figure 2—figure supplement 1 ) . RT-qPCR shows that DFT2 cells express significantly higher levels of β2m ( Figure 2A ) , classical MHC class I heavy chain genes ( Saha-UA , -UB and -UC ) ( Figure 2B ) and non-classical MHC class I ( Saha-UK ) ( Figure 2C ) than DFT1_4906 . However , expression of β2m and Saha-UA , -UB and -UC by DFT2 cells is lower than that of DFT1_4906 + IFNγ and Fibroblasts , which is consistent with the levels of β2m expression observed on the DFT2 cell lines . Interestingly , while the levels of Saha-UA , -UB and -UC in the three DFT2 cell lines is lower than DFT1_4906 + IFNγ ( Figure 2B and D ) , the levels of Saha-UK are not significantly different ( Figure 2C and D ) . This is despite the fact that the expression level of the classical MHC class I genes reflects the amplification of three different MHC class I loci compared to a single locus , Saha-UK . Thus , DFT2 cells in vitro express both classical and non-classical MHC class I heavy chain genes . To extend this analysis to primary DFT2 tumours we used RT-PCR to amplify β2m and MHC class I heavy chain transcripts from the DFT2 cell lines and six primary DFT2 tumours collected between March 2014 and January 2016 ( Figure 2E and Supplementary file 1 ) . The primary tumours include DFT2_RVT1 and DFT2_SNT2 from which the cell lines DFT2_RV and DFT2_SN were derived . RT-PCR shows evidence of expression for β2m in the cell lines and in primary tumour samples ( Figure 2E ) , reflecting the expression of cell surface β2m protein in the cell lines . Saha-UK is expressed in all cell lines and primary biopsies . The cell lines and primary tumours express classical MHC class I , but the expression levels appear to be variable between the primary tumours . While this analysis is not quantitative , as the amount of stroma in each sample varies between tumours , these results show that DFT2 cells express both classical and non-classical MHC class I transcripts alongside β2m . To further investigate the expression of MHC class I molecules between DFT2 tumours in vivo , a shared peptide immunogen was used to raise a pan-classical MHC I antibody against the classical MHC class I heavy chains ( Saha-UA , -UB and -UC ) . A second peptide , specific in sequence to Saha-UK , was used to raise an antibody against the non-classical MHC class I , Saha-UK . Monoclonal antibodies were initially screened by western blot using protein from devil fibroblast cells . Positive clones were re-screened for molecule specificity against recombinant Saha-UK and recombinant Saha-UC protein ( Figure 3—figure supplement 1 ) . Clones specific for Saha-UK ( clone - α-UK_15-29-1 ) and Saha-UA –UB and -UC ( clone - α-UA/UB/UC_15-25-18 ) were identified . Staining of DFT2 serial sections from six primary DFT2 tumours ( Supplementary file 1 ) with these antibodies demonstrates expression of both classical ( Saha-UA , -UB and –UC ) and non-classical ( Saha-UK ) MHC class I molecules in vivo ( Figure 3 and Figure 3—figure supplement 2 ) . However , this analysis also demonstrates that MHC class I expression is variable in DFT2 tumours . Three of the tumours , DFT2_RVT1 , DFT2_SNT2 and DFT2_818T1 ( Figure 3 ) , retain strong expression of classical class I molecules , with localisation to the cell membrane . This result is consistent with the cell surface expression of β2m observed on the DFT2_RV and DFT2_SN cell lines , derived from two of these primary tumours ( Figure 1C and D ) . However , expression of classical MHC class I in DFT2_547 and DFT2_523 is weaker , appears mostly cytoplasmic and shows some variation in staining intensity , with some cells in DFT2_547 showing very low levels of expression . Strikingly , DFT2_812 is negative for classical MHC class I ( Figure 3 ) . The expression of Saha-UK also varies between and within DFT2 biopsies ( Figure 3 ) . DFT2_RVT1 cells are uniformly positive for Saha-UK , whereas DFT2_SNT2 and DFT2_547 have variable expression , with some areas staining more weakly . Interestingly , there is some evidence for Saha-UK staining in DFT2_812 , a tumour negative for classical MHC class I , but DFT2_523 is largely Saha-UK negative ( Figure 3 ) . While the DFT2 tumours with strong classical class I expression show localisation to the cell membrane ( i . e . cell surface expression ) , staining for Saha-UK shows a cytoplasmic distribution in some of the DFT2 tumours . Notably , DFT2 tumours also show morphological heterogeneity ( Figure 3—figure supplement 3 ) . DFT2 tumour cells are sometimes arranged as nests separated by a fibroblastic stroma , for example DFT2_547T1 , but can also be arranged in diffuse sheets separated by a fibrous component , for example DFT2_818T1 and DFT2_523T1 . The degree of individual tumour cell nuclear pleomorphism can also vary within the same tumour ( DFT2_SNT2 ) . The expression of MHC molecules by DFT2 cells may mean that this tumour is more immunogenic than DFT1 . As infiltration of lymphocytes has been correlated with a positive prognostic outcome for some cancers ( Galon et al . , 2006; Chee et al . , 2017 ) we stained DFT2 tumours for the lymphocyte marker , CD3 . Staining of the primary tumours shows that lymphocyte infiltration does occur in some individuals ( Figure 4 ) . No CD3 positive cells were observed in DFT2_RV or DFT2_547 ( Figure 4 ) , but a more significant number of CD3 positive cells is evident in DFT2_818 , DFT2_523 and DFT2_812 ( Figure 4 ) , where the lymphocytes are clustered at the stroma and some cells can be seen infiltrating the tumour mass . A small number of CD3 positive cells are visible in DFT2_SN ( Figure 4 ) . The classical MHC class I alleles expressed by DFT2 cells should play a role in any host immune response against the tumour . To assess the MHC class I alleles expressed by DFT2 cells we amplified , cloned and sequenced exon 2 ( the region of the devil MHC class I where the majority of variation is located ) of MHC class I transcripts from DFT2 cell lines . Previous analysis of DFT2 DNA has identified four MHC class I sequences from Saha-UA , Saha-UB and Saha-UC and one sequence from Saha-UD present in the DFT2 genome ( Pye et al . , 2016b ) . We find that DFT2 cells express all five genomic MHC class I sequences , SahaI*35 ( Saha-UA ) , SahaI*90 ( Saha-UB ) SahaI*74/88 ( unassigned ) , SahaI*27 ( Saha-UC ) and SahaI*32 ( Saha-UD ) loci ( Table 1 and Figure 5 ) . We also identified a sixth expressed sequence ( SahaI*27–1 ) that differs from SahaI*27 by one non-synonymous substitution . Alignment of MHC class I sequences from DFT2 and DFT1 cells ( when treated with IFNγ ) show that across exon 2 of the heavy chain DFT1 and DFT2 share expression of four MHC class I alleles , SahaI*27 , SahaI*90 , SahaI*35 and SahaI*27–1 , with only SahaI*46 unique to DFT1 and SahaI*74/88 unique to DFT2 ( Table 1 ) . Using gene specific primers we were able to confirm that both DFT2 and DFT1 cells also express Saha-UK ( non-polymorphic ) ( Table 1 and Figure 2 ) . To assess the relative expression level of each of the classical MHC class I alleles in DFT2 cells , PCR amplicons were cloned and >60 clones were sequenced , resulting in 61 classical MHC class I sequences from DFT2 . Of these sequences the most abundant is SahaI*27 , representing 22 ( 36% ) of the clones sequenced ( Table 1 ) . SahaI*27–1 and SahaI*35 were represented by 13 clones each ( 21% ) , while SahaI*74/88 and SahaI*90 were the least abundant , with 9 ( 15% ) and 4 ( 6% ) clones respectively . These results indicate that DFT2 cells do not express MHC class I alleles equally and that the dominant allele is SahaI*27 . Given the dominant expression of SahaI*27 by DFT2 cells we determined the prevalence of SahaI*27 and other MHC class I alleles in three host devils . Exon 2 of MHC class I transcripts were amplified from the mRNA of spleen samples from three devils infected with DFT2 ( TD_RV , TD_818 and TD_SN ) ( Table 1 ) . Alignment of the sequences demonstrates that the host devils share SahaI*32 ( Saha-UD ) , Saha-UK and SahaI*27 with DFT2 . In addition , TD_818 and TD_SN share SahaI*27–1 and either SahaI*35 or SahaI*90 with DFT2 ( Table 1 and Figure 5 ) . The MHC class I alleles that are expressed by DFT2 cells but not by the hosts are of particular interest as they are likely to be immunogenic upon cell transmission . SahaI*74/88 is unique to the DFT2 cell lines and differs from SahaI*27 and SahaI*27–1 found in the host animals by a single non-synonymous substitution at position 59 or position 76 , which are not predicted to interact with the TCR ( Figure 5B ) . The class I alleles with lower expression on DFT2 , SahaI*90 and SahaI*35 , share between 91 and 97% amino acid identity with the host alleles . To identify any further polymorphic sites that may have been missed when selecting clones for sequencing and to validate the sequences found in the hosts we used two approaches . First , we amplified and directly sequenced the MHC class I products from TD_RV , TD_818 and TD_SN to compare the chromatograms produced from sequencing pooled transcripts . This analysis did not reveal any additional polymorphic sites ( Figure 5—figure supplement 1 ) . Second , we used the sequences identified in Table 1 to conduct targeted searches of the genomes of TD_RV , TD_818 and TD_SN ( Stammnitz et al . , 2018 ) to confirm the presence of these alleles in genomic DNA .
Here we show that DFT2 cells express classical and non-classical MHC class I molecules both in vitro and in vivo . This is in stark contrast to DFT1 , a likely older and more widespread contagious cancer in the Tasmanian devil , which has lost expression of MHC class I ( Siddle et al . , 2013 ) . However , DFT2 cells express the non-polymorphic , non-classical MHC class I molecule ( Saha-UK ) likely reducing the immunogenicity of these tumour cells . Further , the classical MHC class I allele with the highest expression in DFT2 cells is shared with host devils . Finally , we show that MHC class I expression varies among DFT2 tumours in vivo , suggesting that MHC class I negative subclones could emerge as DFT2 transmits more widely . Our results demonstrate that DFT2 cells express MHC class I molecules , in contrast to DFT1 and the contagious cancer circulating among dogs , CTVT . DFT1 cells lack surface expression of MHC class I due to loss of β2m , TAP1 and TAP2 transcripts ( Siddle et al . , 2013 ) . However , loss of MHC class I in DFT1 is reversible upon treatment with the inflammatory cytokine IFNγ ( Siddle et al . , 2013 ) and a small number of individuals have been found to have a successful immune response against the tumour ( Pye et al . , 2016a ) . Similarly , CTVT is thought to down-regulate MHC expression during transmission , but expression can be upregulated on CTVT cells during a tumour specific immune response from the host ( Yang et al . , 1987 ) . In contrast , we show that DFT2 cells express cell surface β2m molecules in addition to classical and non-classical class I heavy chains , indicating that functional MHC class I are present on the surface of DFT2 cells . The expression of a non-classical MHC class I molecule , Saha-UK , on DFT2 cells could reduce the immunogenicity of the tumour cells . Saha-UK is expressed in some DFT2 tumours in vivo and the Saha-UK heavy chain is expressed at similar levels to the three classical MHC class I genes ( amplified as a group ) in vitro . While antibodies specific for Saha-UD are not available , RT-PCR indicates it is also expressed , but at a very low level compared to Saha-UK . Non-classical MHC class I molecules can be inhibitory ligands for NK receptors and some human cancers down-regulate classical MHC class I and overexpress non-classical MHC class I to avoid a cytotoxic immune response ( Kochan et al . , 2013 ) . While the function of Saha-UK and Saha-UD are not known ( Siddle et al . , 2009 ) , neither are highly polymorphic ( Saha-UK is monomorphic [Siddle et al . , 2010] ) and unlikely to elicit an allogeneic T cell response in host devils . However , further investigation is needed to determine the function of these molecules and the functional classification of Tasmanian devil MHC class I molecules as ‘classical’ or ‘non-classical’ . DFT2 cells also express classical MHC class I alleles belonging to the Saha-UA , -UB and -UC loci . Of these classical MHC class I alleles , SahaI*27 is the most abundant in DFT2 cells and is also found in the three host devils , reducing the immunogenicity of DFT2 cells in these animals . The primers designed in this study should amplify all known DFT1 and DFT2 MHC class I alleles with equal efficiency , suggesting that this allele represents an MHC class I locus with dominant expression . As well as being found in the host devils , this allele is highly prevalent in at least one devil population ( Lane et al . , 2012 ) and the high expression levels in DFT2 may be facilitating tolerance of the tumour in this population of devils . The similarity of the MHC class I alleles between DFT2 and host devils may reflect a shared geographical range and genotype pool to the cancer founder's ( Stammnitz et al . , 2018 ) . Thus , the frequency of this allele among the wider population may impact the ability of DFT2 to spread further . The heterogenic expression of MHC class I molecules in DFT2 biopsies suggests that MHC class I expression is not fixed and may be gradually lost in DFT2 . While some MHC class I alleles are shared between DFT2 and its hosts , the tumour does have a number of unique alleles . SahaI*74/88 differs to SahaI*27 by only one amino acid that is not predicted to interact with the TCR , but SahaI*90 and SahaI*35 have a number of unique sites when compared to host MHC class I alleles . While these alleles have lower expression on DFT2 than SahaI*27 , their presence may still initiate an immune response in some host animals , providing a selective pressure for MHC class I loss . It is possible that , like single organism tumours , DFT2 is undergoing immunoediting ( Dunn et al . , 2002 ) , which can operate to down-regulate specific MHC class I alleles that are immunogenic ( McGranahan et al . , 2017 ) or select for mutations that remove or modulate total MHC class I expression . However , while four of the six primary DFT2 tumours examined have CD3 positive cells in the tumour mass , which may indicate an immune response , the infiltration is notably present in DFT2_812 , which is negative for classical MHC class I and could be expected to be CD3 negative . Thus , further detailed analysis is needed to define the host immune response to DFT2 and its role in shaping tumour evolution . While DFT1 cells currently circulating in the population are MHC class I negative , it is possible that DFT1 down regulated MHC class I expression after the tumour became transmissible and that initial transmission events were among individuals that shared prevalent MHC class I alleles . This is supported by previous studies that have found reduced polymorphism in devil MHC and microsatellite loci ( Jones et al . , 2007; Siddle et al . , 2007 ) . In addition , a hemizygous deletion of β2m has recently been identified in a DFT1 cell line , perhaps indicating past selective pressure for MHC class I loss in this older transmissible tumour ( Stammnitz et al . , 2018 ) . Similarly , CTVT is thought to have emerged in an old world dog and the genetic structure of a dog pack would have favoured the emergence of CTVT ( Murgia et al . , 2006 ) . However , the presence or absence of MHC antigens is likely not singly responsible for determining the ability of the host to respond to the tumour . DFT2 may also be manipulating its microenvironment by release of immune suppressive cytokines , such as Transforming Growth Factorβ , which can drive metastasis ( Tauriello et al . , 2018 ) and in conjunction with PD1/PD-L1 interactions can inhibit and exclude T cells from a tumour ( Mariathasan et al . , 2018 ) . In addition , recruitment of T regulatory cells ( Curiel et al . , 2004; James et al . , 2010 ) or immunosuppressive myeloid cells ( Almand et al . , 2001 ) can also negatively impact the T cell responses to tumours . DFT1 is now at least 21 years old and CTVT is predicted to be over 10 , 000 years old ( Murchison et al . , 2014 ) , whereas DFT2 was first discovered in 2014 and is most likely a recently emerged tumour ( Pye et al . , 2016b ) . As such , DFT2 provides an opportunity to study the early evolution of a contagious cancer . The emergence of a contagious cancer that can transmit while maintaining MHC class I expression indicates that loss of MHC is not necessary for transmission , but as the tumour encounters the immune system of genetically disparate hosts subclones that have down-regulated MHC may be selected . Our results predict that loss of MHC class I is already occurring , perhaps due to structural mutations or epigenetic changes . Loss of MHC antigens could allow rapid dissemination of DFT2 through the population , impacting an already vulnerable species .
Six wild Tasmanian devils were either trapped or found dead from road trauma or other causes . Tissue biopsies and fine needle aspirates were either collected post mortem or from live devils that were subsequently released . All animal procedures were performed under a Standard Operating Procedure approved by the General Manager , Natural and Cultural Heritage Division , Tasmanian Government Department of Primary Industries , Parks , Water and the Environment or under University of Tasmania Animal Ethics Committee Permit A0014976 . Sample information is shown in Supplementary file 1 . Tumour and spleen samples were collected as described in Supplementary file 1 and were formalin fixed ( 10% neutral buffered formalin ) and then paraffin embedded . The DFT1 cell line , DFT1_4906 , and fibroblast cell line , Fibroblasts_Salem , have been described elsewhere ( Murchison et al . , 2012 ) . Three cell lines derived from DFT2 primary tumours ( DFT2_RV , DFT2_SN and DFT2_TD549 ) were established from fine needle aspirates collected in culture medium and cultured at 35°C and 5% CO2 . Cell lines DFT1_4906 , DFT2_RV and DFT2_SN were grown in RPMI 1640 with L-glutamine ( Gibco; ThermoFisher Scientific ) with 10% heat inactivated foetal bovine serum ( FBS ) ( Gibco; ThermoFisher Scientific ) and penicillin/streptomycin ( 100 units/ml penicillin and 0 . 1 mg/ml streptomycin ) ( Gibco; ThermoFisher Scientific ) . Fibroblasts_Salem was cultured in DMEM with high glucose and L-glutamine ( Gibco; ThermoFisher Scientific ) with 10% FBS ( Gibco; ThermoFisher Scientific ) and penicillin/streptomycin ( 100 units/ml penicillin and 0 . 1 mg/ml streptomycin ) ( Gibco; ThermoFisher Scientific ) . Cells were passaged at 80–90% confluency at a ratio of 1:3 using trypsin ( 0 . 05% ) to detach cells . DFT1 cells were treated with recombinant devil IFNγ derived from a transfected cell line ( CHO_SahaIFNy ) as previously described ( Siddle et al . , 2013 ) . Cells were incubated on ice with anti-devil β2m antibody supernatant for 30 min , followed by secondary antibody ( 2 μg/ml goat anti-Mouse IgG ( H + L ) Alexa Fluor 488 Conjugate; ThermoFisher Scientific ) for 30 min . The specificity of the antibody was confirmed by incubating 50 μg/ml recombinant devil β2m protein ( Siddle et al . , 2013 ) with anti-devil β2m antibody on ice for 30 min prior to staining . Cells were analysed on BD FACSCalibur and data analysed using FlowJo software . MHC class I transcripts were aligned and translated into protein sequences using CLC workbench . Two regions were identified , in the α1 domain and the α3 domain , where the amino acid sequenced of the Saha-UA , -UB and –UC sequences are highly similar to each other , with only one amino acid change , but contain low similarity to Saha-UK and Saha-UD . Using these sequences the following peptides were synthesised for immunisations , WMEKVQDVDPGYWE against Saha-UA , -UB and -UC and RITHRTHPDGKVTL against Saha-UK . Mice were immunised subcutaneously with a mixture of GERBU adjuvant and 50 μg of either WMEKVQDVDPGYWE-C or RITHRTHPDGKVTL-C coupled to diphtheria toxoid via the N-terminal cysteine . Three days later spleen lymphocytes were fused with the SP2 cell line using PEG as fuseogen . Hybridomas were selected based on reactivity in ELISA against both N- and C-terminal coupled peptide and subsequently screened against Tasmanian devil fibroblast cells and verified against recombinant expressed MHC class I by western blot as described below . Cell pellets were lysed on ice for 30 min at 4 × 107 cells/ml lysis buffer ( 150 mM NaCl , 100 mM TrisCl , 1 mM MgCL2 and 1% digitonin ) and the lysates clarified . 10 μl of cell lysate was added to 15 μl loading buffer ( 500 μl 2X Lamelli sample buffer , 50 μl β-mercaptoethanol and 450 μl dd-H2O ) and heated to 95°C 10 min . Samples were run on 12% ProtoGel ( National Diagnostics Protogel 30% ) using Laemmli buffers and Fisherbrand Vertical Gel Tank . Proteins were transferred to nitrocellulose blotting membrane ( Amersham Protran GE Healthcare Life Sciences ) in transfer buffer ( 25 mM Tris , 190 mM glycine and 20% ( w/v ) methanol ) using Mini Trans-Blot Cell ( Bio-Rad ) . The membrane was blocked for 45 min with 150 mM NaCl , 0 . 1% Tween 20 , 4% milk powder , 50 mM TrisCL , pH 8 and incubated with primary antibodies at 4°C overnight , washed with 150 mM NaCl , 0 . 1% Tween 20 , 4% milk powder , 50 mM TrisCL , pH 8 , and incubated with secondary antibody ( IRDye 680RD Goat anti-Mouse IgG ( H + L ) ) for 30 min at room temperature before washing as above . Membranes were visualised using the Li-cor Odyssey scanner . The specificity of the antibodies was determined using full length recombinant devil MHC class I heavy chain proteins for Saha-UC ( SahaI*27 ) and Saha-UK . SahaI*27 and Saha-UK were amplified from devil fibroblast cDNA using primer Saha349 and Saha350 ( Saha-UC ) and Saha335 and Saha351 ( Saha-UK ) ( Supplementary file 2 ) . The subsequent amplicons were cloned into the pET22b+ vector ( Novagen ) and transformed into Rosetta pLysS cells ( Novagen ) according to the manufacturer’s instructions and the transcripts were sequenced in both directions . Bacterial colonies containing pET22B+-SahaUC and pET22B+-SahaUK were cultured to OD600 0 . 6 and protein expression was induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside ( IPTG ) . Bacterial cells were pelleted and resuspended in solubilisation buffer ( 8 M Urea , 50 mM Mes pH 6 . 5 , 0 . 1 mM EDTA and 1 mM DTT ) and the lysates clarified . The total protein in lysates was measured using Bradford Reagent following the manufacturers instructions . 20 μg protein was loaded onto a 12% gel and electrophoresis and blotting were performed as described above . Membranes were incubated with the undiluted supernatant from primary antibodies α-UK_15-29-1 and α-UA/UB/UC_15-25-18 at 4°C overnight . DFT1 and DFT2 primary tumours were fixed in 10% ( mass/vol ) PBS-buffered formalin solution for 2 to 4 d . Tissues were processed and embedded in paraffin blocks and cut onto coated slides at 4–5 μm thickness . Sections were deparaffinized in xylene and rehydrated through graded alcohol . Antigen retrieval was performed by water bath ( 95°C ) in citrate buffer solution ( 10 mM citric acid , 0 . 05% Tween20 pH 6 ) for 40 min followed by cooling for 15 min . Endogenous peroxidase was blocked by incubation of slides with 0 . 3% H2O2 ( Sigma Aldrich ) and non-specific protein binding was blocked with 10% ( mass/vol ) goat serum . Sections were incubated with primary antibody ( list of antibodies in Supplementary file 3 ) at 4°C overnight . Peroxidase-coupled secondary antibody ( Envision kit; Dako ) was used to detect primary antibody binding following the manufacturers instructions . Sections were counterstained with haematoxylin ( vector hematoxylin nuclear counterstain Gill’s Formula ) for 4 min , differentiated in 2% ( mass/vol ) acetic acid and blued in 0 . 2% ( mass/vol ) ammoniated water . Sections were dehydrated through graded alcohol to xylene and cover-slipped . Images were captured using the Nikon Eclipse 400 microscope , Retiga 2000R camera and Q-capture pro seven computer software . The Nucleospin RNA II kit ( Macherey-Nagel ) was used to extract RNA from cell lines according to the manufacturer’s instructions . 1 μg of RNA was reverse transcribed to cDNA using Thermofischer Scientific RevertAid Premium Reverse Transcriptase ( 200 U ) with 1X RT buffer , 0 . 5 mM dNTPs , 20 pm oligodT primer ( Promega ) and nuclease free H2O to a total volume of 20 μl . Primer set one was designed to amplify the classical MHC class I ( Saha-UA , -UB and –UC ) by aligning all available MHC class I sequences from the Tasmanian devil and embedding primers in conserved regions . Primer set one amplifies the three loci as a group , due to the high level of sequence similarity between the classical class I genes , gene specific PCRs for these loci were not possible . Primer set two was designed to amplify the non-classical MHC class I ( Saha-UK ) . All primers are listed in Supplementary file 2 . RT-qPCR was carried out for RPL13A , β2m , classical MHC class I ( Saha-UA , -UB and -UC ) and non-classical MHC class I ( Saha-UK ) on the StepOnePlus Real-Time PCR system ( ThermoFisher Scientific ) using PrecisionPLUS qPCR MasterMix ( Primerdesign ) with primers at 5 mM each following the cycling protocol; 95°C 2 min , followed by 40 cycles of 95°C for 15 s and 60°C for 1 min . RPL13A was used as the reference gene and data analysis was performed using the relative standard curve method for comparing gene expression ( DFT1_4906 = 1 ) . All cDNA samples were tested in triplicate and controls with no cDNA template were included . DFT2 at four dilutions ( 250 , 50 , 10 , 2 and 0 . 4 ng ) was used to create a standard curve of amplification for RPL13A with an R2 of 0 . 999 and 97 . 14% efficiency . Recombinant DNA at five dilutions ( 0 . 2 , 0 . 04 , 0 . 008 , 0 . 00016 and 0 . 000032 ) was used to create a standard curve of amplification of MHC class I Saha-UK and MHC class I Saha-UA , UB and UC with R2 values of 0 . 991 and 0 . 997% and 105 . 2 and 111 . 4% efficiency respectively . IFNγ treated DFT1 ( 4906 ) cells at five dilutions ( 250 , 50 , 10 , 2 and 0 . 4 ng ) was used to create a standard curve of amplification for β2m with an R2 of 0 . 994 and 91 . 4% efficiency . The Nucleospin RNA II kit ( Macherey-Nagel ) was used to extract RNA from DFT2 cell lines , DFT1 cells after treatment with IFNγ , and host samples ( TD_RV , TD_SN and TD_818 ) . The NucleoSpin totalRNA FFPE kit ( Macherey-Nagel ) was used to extract RNA from formalin fixed tumour tissues according to the manufacturer’s instructions . 1 μg of RNA was reverse transcribed to cDNA as described previously . Three primer sets were used to amplify all classical and non-classical MHC class I alleles . Primer set one was used to amplify Saha-UA , -UB and –UC products as a group , primer sets 2 and 3 were used to amplify Saha-UK and Saha-UD genes specifically ( for PCR conditions see Supplementary file 4 ) . All products were purified using NucleoSpin Gel and PCR Clean-up ( Macherey-Nagel ) , cloned into a pJET plasmid ( CloneJET; ThermoFisher Scientific ) . Plasmid DNA from individual clones was isolated and sequenced using T7 primer . PCR , cloning and sequencing was performed in triplicate for the DFT2 cells and duplicate for the spleen samples . For primer set 1 between 15 and 65 clones were sequenced for each PCR , for primer sets 2 and 3 between 4 and 6 clones were sequenced . To assess the relative abundance of each of the classical MHC class I alleles within DFT2 cells 65 clones were isolated and sequenced using primer set 1 . To validate the MHC class I alleles , Saha-UA , -UB and -UC genes were amplified using primer set one as described above and resulting PCR products ( containing all MHC class I alleles for these genes ) were sequenced directly using these primers . The resulting chromatograms were used to identify polymorphic sites in the transcripts and to confirm the validity of alleles . In addition , MHC class I alleles for RV , 818 and SN were searched against genomes generated for these animals ( Stammnitz et al . , 2018 ) to validate their presence in the genome . All sequences were quality checked and analysed in CLC Genomic Workbench . Residues in the MHC class I sequences predicted to interact with the peptide or the TCR were identified by aligning to HLA-A2 ( Bjorkman et al . , 1987 ) .
Devil Facial Tumour 2 ( DFT2 ) is a recently discovered contagious cancer circulating in the Tasmanian devil ( Sarcophilus harrisii ) , a species which already harbours a more widespread contagious cancer , Devil Facial Tumour 1 ( DFT1 ) . Here we show that in contrast to DFT1 , DFT2 cells express major histocompatibility complex ( MHC ) class I molecules , demonstrating that loss of MHC is not necessary for the emergence of a contagious cancer . However , the most highly expressed MHC class I alleles in DFT2 cells are common among host devils or non-polymorphic , reducing immunogenicity in a population sharing these alleles . In parallel , MHC class I loss is emerging in vivo , thus DFT2 may be mimicking the evolutionary trajectory of DFT1 . Based on these results we propose that contagious cancers may exploit partial histocompatibility between the tumour and host , but that loss of allogeneic antigens could facilitate widespread transmission of DFT2 .
While cancer cells typically cannot spread between individuals , there are a few examples of contagious tumours . Remarkably , two examples have emerged in a single species , the Tasmanian devil , a marsupial carnivore . These tumours are known as Devil Facial Tumour 1 ( or DFT1 ) and Devil Facial Tumour 2 ( or DFT2 ) ; both cause tumours round the faces of infected devils . Since it emerged in the 1990s , DFT1 has killed 60–90% of the devil population . Preserving the few remaining healthy devils has been a major challenge for conservationists . This challenge became more urgent in 2014 , when the second contagious cancer , DFT2 , was discovered circulating in the population . Contagious cancers are rare because the immune system usually eliminates cells coming from outside the body . Each healthy cell carries molecules known as the major histocompatibility complex ( MHC ) that act as a barcode showing where each cell comes from . The immune system uses these molecules to help it tell the difference between the body’s own cells and those from elsewhere . DFT1 can avoid the immune system and spread to new hosts because it has lost MHC . In 2014 , scientists identified a new Tasmanian devil cancer named DFT2 , but it was unclear how this second cancer evades the immune system as it spreads from host to host . Caldwell et al . have now examined the MHC on cells from DFT2 cancers , including cells grown in the laboratory and cells taken from cancer biopsies . Biochemical tests showed that the DFT2 cells do carry MHCs , but that the MHC barcodes of DFT2 are similar to those of the devils infected with the disease . This finding may explain how the cancer can spread undetected in these animals , because the immune system does not recognize it as coming from outside the body . Further analyses also reveal that the cancer cells are slowly evolving to lose their MHCs . This means DFT2 could , with time , become as contagious as DFT1 . These two contagious cancers threaten the future of the Tasmanian devil . As top predators , devils are key to the ecosystem in Tasmania and their preservation is vital . While DFT2 provides a unique opportunity to study an emerging cancer as it develops , this research will also help to protect the devils and may lead to effective vaccines . These results could also reveal how other cancers avoid the immune system and may help to detect them during treatment . In addition , there are many similarities between contagious cancer cells and organ transplants . Understanding the role of MHC in DFT2 could lead to better ways to prevent rejection following transplants .