Immunotherapy for the treatment of canine transmissible venereal tumor based in dendritic cells pulsed with tumoral exosomes.

Context: Exosomes secreted by tumor cells are a good source of cellular components that stimulate the immune response, such as alarmins (mRNA, tetraspanins (CD9, CD63, CD81), heat-shock proteins, major histocompatibility complex class I molecules) and tumor-associated antigens. These properties permit to pulsed dendritic cells in the immunotherapy for many cancers types. The aim of this study was to demonstrate the use of exosomes derived from canine transmissible venereal tumor (CTVT) as an antigen to pulsed dendritic cells and its administration in dogs with CTVT as treatment against this disease. Material and methods: From primary culture of CTVT cells the exosomes were isolated and characterized by scanning electron microscopy assay, dot blot and protein quantification. The monocytes of each patient were differentiated to dendritic cells (DC) and pulsed with CTVT exosomes (CTVTE). Phagocytosis, tumor size, populations of lymphocytes and IFN-c levels were evaluated. Results: The CTVTE showed a size around 90 nm. CD81, CD63, CD9 and Hsp70 were expressed. Monocytes showed an expression of 85.71% for CD14+, 12.3% for CD80+, 0.1% for CD83+ and 0.8% for DLA-II. In DC 5.1% for CD14+, 86.7% for CD80+, 90.1% for CD83+ and 92.6% for DLA-II and a phagocytosis of 63% was obtained by FITC Dextran test. No side effects were observed in the experimental groups with our therapy. Tumor regression was of 100% at the seventh week, as well as an increase in the level of IFN-γ (142 pg/ml), and CD4+ (28%) and CD8+ (34%) cell percentage. Discusion and conclusion: These results have shown that DC pulsed with tumor exosomes induce regression of the TVT in dogs.

Autologous canine immunotherapy: short-time generated dendritic cells loaded with canine transmissible venereal tumor-whole lysate.

OBJECTIVE: The aim of the present study was to evaluate the therapeutic potential of autologous DCs loaded with whole tumor cell lysate of CTVT generated under a simplified and rapid procedure in vitro production process, in a vulvar submucosal model of CTVT in dogs. MATERIALS AND METHODS: We generated a model of intravulvar CTVT in dogs. A CTVT lysate antigen was prepared according to the method of 1-butanol and after administered with complete Freund's adjuvant via subcutaneous in female healthy dogs and challenge with CTVT cells to corroborate the immunogenicity. Short-time generated dendritic cell pulsed with CTVT whole-lysate was performed, and analyzed by FITC-dextran uptake assay and characterized using anti-canine monoclonal antibodies CD14, CD80, CD83, and DLAII by flow cytometry. Dendritic cell therapy was administered in a frequency of three times every 2 weeks when the CTVT had 4 months of growth and 89 ± 5 cm diameter. The CD3+, CD4+ and CD8+ lymphocytes were determined by flow cytometry, and IFN-γ by ELISA assay. RESULTS AND DISCUSSION: The administration of CTVT whole-lysate resulted in tumor prevention. The short-time generated dendritic cell pulsed with CTVT whole-lysate administration resulted in an efficient reduction and elimination of CTVT, probably due to the increase in lymphocyte populations (CD3+, CD4+, and CD8+), IFN-γ production and tumor infiltrating lymphocytes. CONCLUSION: In conclusion, this study demonstrates the efficacy of immunotherapy based in short-time generated dendritic cell pulsed with CTVT whole-lysate for the treatment of CTVT, and offer veterinary oncologists new alternative therapies to treat this and another malignancy.

Insertion of the LINE-1 element in the C-MYC gene and immunoreactivity of C-MYC, p53, p21 and p27 proteins in different morphological patterns of the canine TVT / Inserção do elemento LINE - 1 no gene C-MYC e imunorreatividade das proteinas C-MYC, p53, p21 e p27 nos diferentes padrões morfológicos do tumor TVT

The canine transmissible venereal tumor (TVT) affects the external genitalia of dogs by the natural transplant of viable tumor cells. Thus, this research aimed to diagnose and characterize TVT morphological patterns, identify the insertion of the LINE-1 element in C-MYC gene, by means of the polymerase chain reaction (PCR), and evaluate the immunohistochemical expression of C-MYC, p53, p21 and p27 proteins. The relationship between C-MYC and p53 proteins and their interference on the expression of p21 and p27 were also studied. For that, 20 samples of naturally occurring TVT were used, subjected to cytopathological, histopathological and immunohistochemical analysis, and to molecular diagnosis of neoplasia. The increased tissue expression and the correlation among C-MYC, p53, p21 and p27 proteins indicate reduction and/or loss of their functionality in the TVT microenvironment, with consequent apoptotic suppression, maintenance of cell growth and progression of neoplasia.(AU)

O tumor venéreo transmissível canino (TVT) afeta a genitália externa de cães pelo transplante natural de células tumorais viáveis. Assim, esta pesquisa teve como objetivo diagnosticar e caracterizar TVT em padrões morfológicos, identificar a inserção do elemento LINE-1 em gene C-MYC, por meio da reação em cadeia da polimerase (PCR), e avaliar a expressão imuno-histoquímica do C-MYC, p53, p21 e p27. A relação entre C-MYC e as proteínas p53 e a sua interferência na expressão de p21 e p27 foram também estudadas. Para isso, foram utilizadas 20 amostras de ocorrência natural de TVT, submetido a exame citopatológico, histopatológica e imuno-histoquímica e ao diagnóstico molecular de neoplasia. A expressão aumentada do tecido e a correlação entre a C-MYC e as proteínas p53, p21 e p27 indicam redução e/ou perda de funcionalidade na TVT em seu microambiente, com consequente supressão apoptótica, manutenção do crescimento celular e progressão da neoplasia.(AU)

Immunology of naturally transmissible tumours.

Naturally transmissible tumours can emerge when a tumour cell gains the ability to pass as an infectious allograft between individuals. The ability of these tumours to colonize a new host and to cross histocompatibility barriers contradicts our understanding of the vertebrate immune response to allografts. Two naturally occurring contagious cancers are currently active in the animal kingdom, canine transmissible venereal tumour (CTVT), which spreads among dogs, and devil facial tumour disease (DFTD), among Tasmanian devils. CTVT are generally not fatal as a tumour-specific host immune response controls or clears the tumours after transmission and a period of growth. In contrast, the growth of DFTD tumours is not controlled by the Tasmanian devil's immune system and the disease causes close to 100% mortality, severely impacting the devil population. To avoid the immune response of the host both DFTD and CTVT use a variety of immune escape strategies that have similarities to many single organism tumours, including MHC loss and the expression of immunosuppressive cytokines. However, both tumours appear to have a complex interaction with the immune system of their respective host, which has evolved over the relatively long life of these tumours. The Tasmanian devil is struggling to survive with the burden of this disease and it is only with an understanding of how DFTD passes between individuals that a vaccine might be developed. Further, an understanding of how these tumours achieve natural transmissibility should provide insights into general mechanisms of immune escape that emerge during tumour evolution.

A tale of two tumours: comparison of the immune escape strategies of contagious cancers.

The adaptive immune system should prevent cancer cells passing from one individual to another, in much the same way that it protects against pathogens. However, in rare cases cancer cells do not die within a single individual, but successfully pass between individuals, escaping the adaptive immune response and becoming a contagious cancer. There are two naturally occurring contagious cancers, Devil Facial Tumour Disease (DFTD), found in Tasmanian devils, and Canine Transmissible Venereal Tumour (CTVT), found in dogs. Despite sharing an ability to pass as allografts, these cancers have a very different impact on their hosts. While DFTD causes 100% mortality among infected devils and has had a devastating impact on the devil population, CTVT co-exists with its host in a manner that does not usually cause death of the dog. Although immune evasion strategies for CTVT have been defined, why DFTD is not rejected as an allograft is not understood. We have made progress in revealing mechanisms of immune evasion for DFTD both in vitro and in vivo, and here we compare how DFTD and CTVT interact with their respective hosts and avoid rejection. Our findings highlight factors that may be important for the evolution of contagious cancers and cancer more generally. Perhaps most importantly, this work has opened up important areas for future research, including the effect of epigenetic factors on immune escape mechanisms and the basis of a vaccine strategy that may protect Tasmanian devils against DFTD.

Gene-expression profiling to identify genes related to spontaneous tumor regression in a canine cancer model.

Microarray transcriptome study in cancer has been commonly used to investigate tumorigenic mechanisms. The unique growth pattern of spontaneous regression (SR) after progressive (P) growth in canine transmissible venereal tumor (CTVT) provides a valuable cancer model to study the genome-wide differences in samples between the two stages of growth. In this study, Affymetrix analysis was performed based on the canine genome to compare the gene expression profiles of CTVT P- and SR-phase tumors. A total of 459 (278 up-regulated and 181 down-regulated) genes were identified as being differentially-expressed during the SR phase by the 2-fold method. Further analysis of these genes revealed that the expression of three genes associated with IL-6 production -TIMD-4, GPNMB and PLTP - was significantly higher in SR-phase tumors than in P-phase tumors; these results were also confirmed by real time RT-PCR in tumor tissues of beagles. In addition, we found that Th17-related genes were over-expressed in the SR phase, suggesting autoimmune responses involvement in tumor regression. Although the interaction between CTVT and host immunity were partially investigated in previous studies, our results enable us to gain new insight into the genes and possible mechanisms involved in tumor regression and reveal potentially useful targets for cancer therapy.

Contagious cancer: lessons from the devil and the dog.

Cancer is generally defined as uncontrollable growth of cells caused by genetic aberrations and/or environmental factors. Yet contagious cancers also occur. The recent emergence of a contagious cancer in Tasmanian devils has reignited interest in transmissible cancers. Two naturally occurring transmissible cancers are known: devil facial tumour disease and canine transmissible venereal tumour. Both cancers evolved once and have then been transmitted from one individual to another as clonal cell lines. The dog cancer is ancient; having evolved more than 6,000 years ago, while the devil disease was first seen in 1996. In this review I will compare and contrast the two diseases focusing on the life histories of the clonal cell lines, their evolutionary trajectories and the mechanisms by which they have achieved immune tolerance. A greater understanding of these contagious cancers will provide unique insights into the role of the immune system in shaping tumour evolution and may uncover novel approaches for treating human cancer.

Chicken HSP70 DNA vaccine inhibits tumor growth in a canine cancer model.

Immunization with xenogeneic DNA is a promising cancer treatment to overcome tolerance to self-antigens. Heat shock protein 70 (HSP70) is over-expressed in various kinds of tumors and is believed to be involved in tumor progression. This study tested a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in an experimental canine transmissible venereal tumor (CTVT) model. Three vaccination strategies were compared: the first (PE) was designed to evaluate the prophylactic efficacy of chHSP70 DNA vaccination by delivering the vaccine before tumor inoculation in a prime boost setting, the second (T) was designed to evaluate the therapeutic efficacy of the same prime boost vaccine by vaccinating the dogs after tumor inoculation; the third (PT) was similar to the first strategy (PE), with the exception that the electroporation booster injection was replaced with a transdermal needle-free injection. Tumor growth was notably inhibited only in the PE dogs, in which the vaccination program triggered tumor regression significantly sooner than in control dogs (NT). The CD4(+) subpopulation of tumor-infiltrating lymphocytes and canine HSP70 (caHSP70)-specific IFN-γ-secreting lymphocytes were significantly increased during tumor regression in the PE dogs as compared to control dogs, demonstrating that specific tolerance to caHSP70 has been overcome. In contrast, no benefit of the therapeutic strategy (T) could be noticed and the (PT) strategy only led to partial control of tumor growth. In summary, antitumor prophylactic activity was demonstrated using the chHSP70 DNA vaccine including a boost via electroporation. Our data stressed the importance of DNA electroporation as a booster to get the full benefit of DNA vaccination but also of cancer immunotherapy initiation as early as possible. Xenogeneic chHSP70 DNA vaccination including an electroporation boost is a potential vaccine to HSP70-expressing tumors, although further research is still required to better understand true clinical potential.

Cancer transmissibility across HLA barriers between immunocompetent individuals: rare but not impossible.

The axiom of human leukocyte incompatibility (HLA) incompatibility has always led scientists to consider cancer transmission between HLA-different individuals impossible. In fact, cancer transmission between individuals represents a frightening possibility in animal populations with limited HLA diversity or for rare cancers exploiting downregulation of HLA expression. We review here evidence from nonhuman models and settings for interhuman transmission.

Immunopathogenic behaviors of canine transmissible venereal tumor in dogs following an immunotherapy using dendritic/tumor cell hybrid.

Canine transmissible venereal tumor (CTVT) is a naturally occurring tumor that can be transmitted between dogs via live tumor cell inoculation. It is also a spontaneous self-regression tumor and its behavior is closely related to host immune responses. Since CTVT had been widely used for tumor models in canine cancers, whether this self-regression may overtake the immunity elicited from an exogenous tumor vaccine remains unclear and certainly worthwhile to be investigated. In this study, we used DCs/tumor hybrids as a tumor vaccine to evaluate the CTVT model. We prepared mature allogeneic dendritic cells from bone marrow and then assessed their phenotype (CD80, CD83, CD86, CD1a, CD11c, CD40 and MHC II), antigen uptake and presenting abilities. Fused dendritic cell/CTVT hybrids were then used as a vaccine, administered three times at two-week intervals via subcutaneous injection near the bilateral auxiliary and inguinal lymph nodes. In comparison with unvaccinated dogs (spontaneous regressed group), within a period of 2.5 months, the vaccinations substantially inhibited tumor progression (p<0.05) and accelerated the rate of regression by a mechanism involving amplification of the host tumor-specific adaptive immune responses and NK cytotoxicity (p<0.001). Pathologic examination revealed early massive lymphocyte infiltration resulting in final tumor necrosis. In addition, there are not any detectable effects on routine physical, body temperature or blood chemistry examinations. In conclusion, our data furnishes a reference value showing that CTVT is a model of potential use for the study of immunity elicited by vaccines against tumors, and also enable early-phase evaluation of the dendritic cell/tumor vaccine in terms of raising host immunity.

Extensive conservation of genomic imbalances in canine transmissible venereal tumors (CTVT) detected by microarray-based CGH analysis.

Canine transmissible venereal tumor (CTVT) is an intriguing cancer that is transmitted naturally as an allograft by transplantation of viable tumor cells from affected to susceptible dogs. At least initially, the tumor is able to evade the host's immune response; thus, CTVT has potential to provide novel insights into tumor immunobiology. The nature of CTVT as a "contagious" cancer, originating from a common ancestral source of infection, has been demonstrated previously by a series of studies comparing geographically distinct tumors at the molecular level. While these studies have revealed that apparently unrelated tumors share a striking degree of karyotypic conservation, technological restraints have limited the ability to investigate the chromosome composition of CTVTs in any detail. We present characterization of a strategically selected panel of CTVT cases using microarray-based comparative genomic hybridization analysis at ~one-megabase resolution. These data show for the first time that the tumor presents with an extensive range of non-random chromosome copy number aberrations that are distributed widely throughout the dog genome. The majority of abnormalities detected were imbalances of small subchromosomal regions, often involving centromeric and telomeric sequences. All cases also showed the sex chromosome complement XO. There was remarkable conservation in the cytogenetic profiles of the tumors analyzed, with only minor variation observed between different cases. These data suggest that the CTVT genome demonstrates a vast degree of both structural and numerical reorganization that is maintained during transmission among the domestic dog population.

Electroporation-mediated IL-12 gene therapy in a transplantable canine cancer model.

Interleukin-12 (IL-12) is effective in treating many types of rodent tumors, but has been unsuccessful in most human clinical trials, suggesting that animal models of more clinical relevance are required for evaluating human cancer immunotherapy. Herein, we report on the effectiveness of gene therapy with plasmid encoding human IL-12 (pIL-12) through in vivo electroporation in the treatment of beagles with a canine tumor, the canine transmissible venereal tumor (CTVT). The optimal electroporation conditions for gene transfer into CTVTs were tested by luciferase activity and determined to be a voltage of 200 V and duration of 50 msec, with the number of shocks set at 10 pulses, and the use of an electrode with 2 needles. Under these conditions, intratumoral administration of as little as 0.1 mg pIL-12 followed by electroporation significantly inhibited the growth of well-established tumors and eventually led to complete tumor regression. Furthermore, local pIL-12 treatment also induced a strong systemic effect that prevented new tumor growth and cured established tumors at distant locations. Intratumoral administration of pIL-12 greatly elevated the IL-12 level in the tumor masses, but produced only a trace amount in the serum. A high level of IFN-gamma mRNA was also detected in the treated tumor masses. pIL-12 gene therapy attracted significantly more lymphocytes infiltrating the tumors, including CD4(+) and CD8(+) T cells, and the surface expression of MHC I and MHC II molecules on CTVT cells was greatly increased after pIL-12 therapy. This treatment also induced apoptosis of the tumor cells as detected by Annexin V. More importantly, delivery of pIL-12 with intratumoral electroporation did not result in any detectable toxicity in the dogs. We conclude that intratumoral electroporation of the pIL-12 gene could cause profound immunologic host responses and efficiently treat CTVT in beagle dogs. The results also indicate that CTVT is an excellent large animal cancer model for testing immunogene therapies mediated by electroporation.

Effects of immunotherapy of IL-6 and IL-15 plasmids on transmissible venereal tumor in beagles.

Canine transmissible venereal tumor (CTVT) is a tumor with low MHC antigen expression and is an ideal tumor model for studying the interactions between host immunity and cancer cells. CTVTs produce high concentrations of TGF-beta to hamper the host immune responses and facilitate their growth progression. However, during the later stages of tumor progression, tumor-infiltrating lymphocytes secrete IL-6. This cytokine antagonizes TGF-beta and restores the IFN-gamma activities in promoting MHC antigen expression, and the NK cytotoxicity that has been repressed by TGF-beta is also activated. In this study, we applied combinatory treatment of IL-6 plasmid and IL-15 plasmid (pIL-6/pIL-15) to CTVT-bearing beagles. IL-6 was used as an anti-TGF-beta cytokine; IL-15 was used to promote NK- and CTVT-specific cytotoxicity. After intratumoral pIL-6/pIL-15 delivery mediated by electroporation, MHC antigen expression on CTVT cells was dramatically increased from in less than 5.9% to up to 34% of the tumor cells. The proportion of CD8(+) T cells infiltrating the tumor was also significantly elevated from 6.96+/-0.23% to 21.63+/-5.40%. In addition, the tumor-specific cytotoxicity was enhanced along with a marked increase in tumor-specific IFN-gamma-producing cells. These immune responses are believed to be the important forces driving the tumor towards regression. The results indicate that pIL-6/pIL-15 combinatory immunotherapy may facilitate a promising and effective means of treating tumors.

Interactions of host IL-6 and IFN-gamma and cancer-derived TGF-beta1 on MHC molecule expression during tumor spontaneous regression.

Many tumors down-regulate major histocompatibility complex (MHC) antigen expression to evade host immune surveillance. However, there are very few in vivo models to study MHC antigen expression during tumor spontaneous regression. In addition, the roles of transforming growth factor betal (TGF-beta1), interferon gamma (IFN-gamma), and interleukin (IL)-6 in modulating MHC antigen expression are ill understood. We previously reported that tumor infiltrating lymphocyte (TIL)-derived IL-6 inhibits TGF-beta1 and restores natural killing (NK) activity. Using an in vivo canine-transmissible venereal tumor (CTVT) tumor model, we presently assessed IL-6 and TGF-beta involvement associated with the MHC antigen expression that is commonly suppressed in cancers. IL-6, IFN-gamma, and TGF-beta1, closely interacted with each other and modulated MHC antigen expression. In the presence of tumor-derived TGF-beta1, host IFN-gamma from TIL was not active and, therefore, there was low expression of MHC antigen during tumor progression. TGF-beta1-neutralizing antibody restored IFN-gamma-activated MHC antigen expression on tumor cells. The addition of exogenous IL-6 that has potent anti-TGF-beta1 activity restored IFN-gamma activity and promoted MHC antigen expression. IFN-gamma and IL-6 in combination acted synergistically to enhance the expression of MHC antigen. Thus, the three cytokines, IL-6, TGF-beta1, and IFN-gamma, closely interacted to modulate the MHC antigen expression. Furthermore, transcription factors, including STAT-1, STAT-3, IRF-1, NF-kappaB, and CREB, were significantly elevated after IL-6 and IFN-gamma treatment. We conclude that the host IL-6 derived from TIL works in combination with host IFN-gamma to enhance MHC molecule expression formerly inhibited by TGF-beta1, driving the tumor toward regression. It is suggested that the treatment of cancer cells that constitutively secrete TGF-beta1 should incorporate anti-TGF-beta activity. The findings in this in vivo tumor regression model have potential applications in cancer immunotherapy.

Transient downregulation of monocyte-derived dendritic-cell differentiation, function, and survival during tumoral progression and regression in an in vivo canine model of transmissible venereal tumor.

Tumors often target dendritic cells (DCs) to evade host immune surveillance. DC injury is reported in many rodent and human tumors but seldom in tumors of other mammals. Canine transmissible venereal tumor (CTVT), a unique and spontaneous cancer transmitted by means of viable tumor cells. CTVT causes manifold damage to monocyte-derived DCs. This cancer provides an in vivo model of cancer to study the role of monocyte-derived DCs during spontaneous regression. Using flow cytometry and real-time reverse-transcription polymerase chain reactions, we compared the expression of surface molecules on monocyte-derived DCs between normal dogs and dogs with CTVT. These markers were CD1a, CD83, costimulatory factors (CD40, CD80, and CD86), and major histocompatability complex classes I and II. In immature DCs (iDCs) and lipopolysaccharide-treated mature DCs (mDCs), the surface markers were mostly downregulated during tumoral progression and regression. The tumor lowered endocytic activity of iDCs, as reflected in dextran uptake, and decreased allogeneic mixed lymphocyte reactions of mDCs. In addition, it decreased the number of monocytes in the peripheral blood by 40%. The tumor substantially impaired the efficiency with which DCs were generated from monocytes and with which mDCs were generated from iDCs. We also found that progression-phase CTVT supernatants that were cultured for 48 h and that contained protein components killed both monocytes and DCs. Additionally, DC numbers were significantly lower in the draining lymph nodes in CTVT dogs than in normal dogs. In conclusion, CTVT caused devastating damage to monocyte-derived DCs; this might be one of its mechanisms for evading host immunity. Reestablishment of monocyte-derived DC activity by the host potentially might contribute to spontaneous tumoral regression. These findings provide insight into the extent of tumoral effects on host immune systems and responses. This information is useful for developing cancer immunotherapies.

Tumor-infiltrating lymphocyte secretion of IL-6 antagonizes tumor-derived TGF-beta 1 and restores the lymphokine-activated killing activity.

IL-6 is a multifunctional cytokine that regulates cell growth, differentiation, and cell survival. Many tumor cells produce TGF-beta1, which allows them to evade CTL-mediated immune responses. IL-6 antagonizes TGF-beta1 inhibition of CD3 cell activation. However, whether IL-6 restores NK activity, which also is suppressed by TGF-beta1, is not known. We used canine transmissible venereal tumor (CTVT), which produces TGF-beta1, as a model to determine whether IL-6 restores lymphokine-activated killer (LAK) activity. During the progression phase, CTVT cells stop expressing MHC molecules. During the regression phase, the number of surface MHC molecules increases dramatically on about one-third of tumor cells. Tumor cells that stop expressing MHC should be targeted by NK cells. In this study, we found that TGF-beta1 secreted by CTVT cells suppressed LAK cytotoxicity. Interestingly, tumor-infiltrating lymphocytes (TIL) isolated from regressing CTVT secrete high concentrations of IL-6 and antagonize the anti-LAK activity of tumor cell TGF-beta1. TIL also produce IL-6 during progression phase, but the concentration is too low to block the anti-LAK activity of TGF-beta1. There is probably a threshold concentration of IL-6 needed to reverse TGF-beta1-inhibited LAK activity. In addition, in the absence of TGF-beta1, IL-6 derived from TIL does not promote the activity of LAK. This new mechanism, in which TIL manufacture high concentrations of IL-6 to block tumor TGF-beta1 anti-LAK activity, has potential applications in cancer immunotherapy and tumor prognosis.

Canine transmissible venereal tumour: cytogenetic origin, immunophenotype, and immunobiology. A review.

Canine transmissible venereal tumour (CTVT) is the only known naturally occurring tumour that can be transplanted as an allograft across major histocompatibility (MHC) barriers within the same species, and even to other members of the canine family, such as foxes, coyotes and wolves. The progression of this tumour is unique in that, it follows a predictable growth pattern. In natural and experimental cases, the growth pattern includes progressive growth phase, static phase and regression phase, and this is followed by transplantation immunity in immunocompetent adults, while metastasis occurs in puppies and immunosuppressed dogs. Because of the uniqueness of CTVT transmission and progression, experimental investigations of various aspects of the biology of CTVT have been used to provide clues to the immunobiology of both animal and human tumours. This review examines the current state of knowledge of the aspects of the cytogenetic origin, immunophenotype, immunobiology and immunotherapy of CTVT.

Canine transmissible venereal tumor cell depletion of B lymphocytes: molecule(s) specifically toxic for B cells.

Canine transmissible venereal tumor (CTVT) is an excellent model for investigating the interaction between host immunity and tumor growth. Although CTVT is an allograft, initially the host immune system is unable to destroy the tumor cells, and the tumor grows progressively for about 4-6 months (P phase). After a short stable phase, the tumor undergoes regression (R phase). In this study, CTVT inoculation significantly reduced the proportion of B lymphocytes among all peripheral blood lymphocytes (PBL), but the proportion of B lymphocytes returned to normal after complete removal of CTVT. Following CTVT inoculation, immunoglobulin concentrations decreased gradually, coincident with B lymphocyte decline. Furthermore, CTVT secreted a soluble, heat- and protease K-sensitive cytotoxic molecule(s) that destroyed peripheral blood B lymphocytes (PBBL) but spared other types of immune cells regardless of whether mitogens, such as IL-2 or Con A, were present. The decrease in the proportion and viability of PBBL was caused by a cytotoxic molecule(s) that induced apoptosis. The molecular weight of the CTVT-derived cytotoxic molecule(s) was 30-100kDa. Human, domestic cat, horse and mouse B cells were also sensitive to the substance.

Effect of tumor infiltrating lymphocytes on the expression of MHC molecules in canine transmissible venereal tumor cells.

Canine transmissible venereal tumor (CTVT) can be allo-transplanted across major histocompatibility complex barriers. The expression of MHC molecules is usually low in the progression (P) stage and then greatly increases during tumor regression (R). We investigated the effects of tumor infiltrating lymphocytes (TIL) on the expression of MHC molecules of CTVT cells. Isolated, viable CTVT cells were inoculated at each of 12 sites (1 x 10(8) CTVT cells per site) on the back of six, mixed-breed dogs. Tumor masses were collected every 2-3 weeks and prepared for histopathologic, immunocytochemistry, flow cytometry and immunoblotting studies. The level of MHC expression on tumor cells from different stages of growth was measured. Initially, expression of MHC I and II molecules in P phase CTVT was low. Twelve weeks post-inoculation (PI), expression increased dramatically and it continued to increase during R phase. Tumor growth slowed after 12 weeks PI and tumors entered R phase around 17 weeks PI. We hypothesize that CTVT evades host immunosurveillance and grows progressively for 12 weeks, when it becomes vulnerable and subject to the host's anti-tumor immune responses. We further demonstrated that R phase, but not P phase, TIL were closely associated with the over-expression of MHC I and II molecules by CTVT cells. The number and proportion of TIL were higher in R phase tumors. Supernatants, from R phase co-cultures (CTVT+TIL) and TIL only, promoted MHC I and II expression on P phase CTVT cells. After culturing alone for 1 month, expression of MHC classes I and II molecules in R phase CTVT cells decreased to the level of P phase CTVT cells. However, the above-mentioned supernatants restored their expression of MHC I and II molecules. In contrast, supernatants from P phase TIL or CTVT cells increased expression slightly or had no effect. Therefore, TIL, not CTVT cells, produce the effective substance (s) to promote the expression of MHC molecules by the tumor cells. Heat treated supernatant was unable to promote the expression of MHC I and II molecules by CTVT cells. In conclusion, TIL isolated from R phase CTVT secreted a heat-sensitive, soluble substance(s) that triggered over-expression of MHC I and II after 12 weeks PI. This caused the tumor to enter R phase and helped stop CTVT growth. Our findings will facilitate the understanding and further investigation of the mechanisms that initiate host immune surveillance against tumors.