Decline in MHC class I expression with increasing thickness of cutaneous melanomas in standard-strain transgenic mouse models.

Major histocompatibility complex (MHC) class I proteins are required for the formation of complexes with antigenic peptides that enable cytotoxic T lymphocytes (CTLs) to recognize and lyse target cells. The frequent loss of MHC class I expression reported in human melanomas and melanoma cell lines may therefore be an obstacle to CTL-based immunotherapy. We have investigated the expression of MHC class I proteins in the cutaneous melanomas of Tyr-SV40E (C57BL/6 strain) transgenic mice in order to evaluate their potential as experimental models for immunotherapy. The SV40 large T (LT) oncoprotein, which is expressed exclusively in the melanocytic lineages of these mice, was used as a marker for flow cytometric analysis of the parenchymal (potential target) cells of 35 freshly dissociated samples from 28 primary tumours. All the tumours were ulcerated and exceeded the Breslow thickness indicative of a poor clinical prognosis in human melanoma. Using antibodies against H-2D(b) and H-2K(b) class I proteins, the LT antigen-positive cells were found to have high levels of both these MHC class I molecules in the thinnest tumours (2 mm), whereas the levels tended to decline with increasing tumour thickness. Among the tumours > 4 mm thick, five had no detectable MHC class I expression. Unexpectedly, the apparent loss of H-2D(b) and H-2K(b) proteins was observed not only in LT-positive cells but also in LT-negative cell populations. Expression of both H-2D(b) and H-2K(b) was restored in tumours derived from a class I-low melanoma cell line by treatment of the hosts with interferon-gamma. These results implicate a regulatory defect as a principal cause of the loss of MHC class I antigens, as noted by others in some human tumours, and they demonstrate that this loss is remediable, even in advanced stages of melanomas.

The underwhite (uw) locus acts autonomously and reduces the production of melanin.

The mouse has provided several significant models for hypopigmentation disorders, including the major forms of albinism. Mutations at the mouse underwhite locus confer one of the most severe hypopigmentation phenotypes, similar to mutations at the pink-eyed dilution locus that is a model for type 2 oculocutaneous albinism. A melanocyte cell line established from underwhite mutant mice failed to pigment under conditions that support pigment production in wild-type melanocytes and melanoblasts from underwhite skin graft transplants failed to produce melanin in normal skin, demonstrating that the action of the gene encoded by the underwhite locus is intrinsic to melanocytes. Mice with mutations at the underwhite locus and either the pink-eyed dilution locus or the melanocortin receptor 1 locus exhibited more severe hypopigmentation than either mutation alone, suggesting that the actions of these genes are independent. These results demonstrate that the underwhite locus is a major determinant of mammalian pigmentation.

Comparative decreases in tyrosinase, TRP-1, TRP-2, and Pmel 17/silver antigenic proteins from melanotic to amelanotic stages of syngeneic mouse cutaneous melanomas and metastases.

Malignant cutaneous melanomas and metastases were taken directly from in situ lesions of genetically identical (C57BL/6 strain) Tyr-SV40E transgenic mice, and samples were analyzed by Western immunoblotting with antisera specific for the COOH terminus of each of four melanocytic proteins. These were tyrosinase, TRP-1, TRP-2, and Pmel 17/silver. Of the 13 melanomas examined, there were 5 melanotic primary tumors, 5 amelanotic primary tumors, and 3 amelanotic metastases. The melanotic tumors expressed all of the markers to some extent. In contrast, the amelanotic tumors lacked detectable levels of one, two, or three of the proteins, except for an apparently amelanotic tumor sample in which all were expressed, but in which some melanotic cells were likely to have been present. Thus, despite some variability, there is clearly a downward trend in the presence of these proteins as the tumors become amelanotic, a pigmentary change associated with ongoing malignant progression. In the amelanotic tumors, tyrosinase was most often deficient, whereas TRP-2 was most often persistently expressed. These results, obtained from melanomas of syngeneic origin, indicate that tumors in the relatively early stages of malignancy might be more responsive than later-stage tumors to immunotherapy involving an ensemble of antigenic peptides of the tested gene products. Moreover, TRP-2 peptides may be especially useful for therapeutic intervention at the later stages.

Differences in latency and inducibility of mouse skin melanomas depending on the age and anatomic site of the skin.

To determine whether the occurrence of skin melanoma is influenced by the age or the anatomic source of the skin in melanoma-susceptible transgenic mouse models, skin was grafted from donors of different ages or from different anatomic sites to a standard (lateral trunk) site in adult recipients of the same transgenic strain. In 27 grafts of neonatal body skin, melanomas arose with a significantly shorter latency than in 37 grafts of older body skin. The difference may reflect not only the larger number of extrafollicular melanocytes in a given area of neonatal skin but also their unusually high mitotic activity shortly after birth and the influence of other growing skin cells nearby. Each of these body-skin grafts usually developed a single tumor situated near the graft edge. Because maximal wound healing occurs at the edge of such full-thickness skin grafts, melanocytes near the edge would receive the highest exposure to growth factors and cytokines associated with wound healing. In contrast to these results, grafts of snout skin yielded many melanomas, each originating from melanocytes within a vibrissa follicle rather than at the graft edge. The relatively strong local tumorigenic stimulus may be attributable to intrafollicular growth factors normally involved in whisker growth. The above-described experiments support the conclusion that agents in the immediate skin environment of the melanocyte, in addition to the state of the melanocyte itself, contribute to melanoma formation.

Up-regulation of specific tyrosinase mRNAs in mouse melanomas with the c2j gene substituted for the wild-type tyrosinase allele: utilization in design of syngeneic immunotherapy models.

The expression of cell-specialization genes is likely to be changing in tumor cells as their differentiation declines. Functional changes in these genes might yield unusual peptide epitopes with anti-tumor potential and could occur without modification in the DNA sequence of the gene. Melanomas undergo a characteristic decline in melanization that may reflect altered contributions of key melanocytic genes such as tyrosinase. Quantitative reverse transcriptase-PCR of the wild-type (C) tyrosinase gene in transgenic (C57BL/6 strain) mouse melanomas has revealed a shift toward alternative splicing of the pre-mRNA that generated increased levels of the Delta1b and Delta1d mRNA splice variants. The spontaneous c2j albino mutation of tyrosinase (in the C57BL/6 strain) changes the pre-mRNA splicing pattern. In c2j/c2j melanomas, alternative splicing was again increased. However, while some mRNAs (notably Delta1b) present in C/C were obligatorily absent, others (Delta3 and Delta1d) were elevated. In c2j/c2j melanomas, the percentage of total tyrosinase transcripts attributable to Delta3 reached approximately 2-fold the incidence in c2j/c2j or C/C skin melanocytes. The percentage attributable to Delta1d rose to approximately 2-fold the incidence in c2j/c2j skin, and to 10-fold that in C/C skin. These differences provide a basis for unique mouse models in which the melanoma arises in skin grafted from a C/C or c2j/c2j transgenic donor to a transgenic host of the same or opposite tyrosinase genotype. Immunotherapy designs then could be based on augmenting those antigenic peptides that are novel or overrepresented in a tumor relative to the syngeneic host.

Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: a projected basis for immunotherapy.

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase-PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Delta1b and Delta1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Delta1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Delta1d reached 4.0% as compared with 0. 8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Delta1b and Delta1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.

Accelerated growth of melanomas after specific immune destruction of tumor stroma in a mouse model.

Destruction of the entire stroma in a tumor could provide a stringent test of the prospects for tumor eradication in a single treatment. This possibility was investigated by experimental immune destruction of the stroma in a mouse melanoma model. Melanomas were first produced by grafting skin from transgenic C57BL/6 females of high-melanoma susceptibility to low-susceptibility transgenic males so that the malignant cells would be genetically female and the stromal cells genetically male. Subcutaneous transplant lines were then established from the melanotic and the amelanotic zones of such a melanoma and were carried in transgenic male hosts to ensure the male composition of the stroma. Thus, the male-specific H-Y histocompatibility antigen, which is ubiquitously expressed on male cells, could provide the target for an immune attack against the stroma. The transplant lines were next passaged once in transgenic females preimmunized against the H-Y antigen by having received and rejected a graft of C57BL/6 nontransgenic male skin. The antistromal immune response of these hosts did not prevent recovery of the tumors, which required a substantially prolonged latency. However, after retransplantation to nonimmunized males and females, the latency was markedly shortened from the original level. Thus, the treatment had indirectly selected for more rapidly growing tumor cells and hastened malignant progression.

Expression of plasminogen activators and plasminogen activator inhibitors in cutaneous melanomas of transgenic melanoma-susceptible mice.

The Tyr-SV40E transgenic mouse model of malignant skin melanoma has been used here to generate melanomas in genetically identical (C57BL/6) mice for analysis of the plasminogen activator (PA) system during tumor development and progression. Twenty-two melanocytic lesions were examined by in situ zymography for PA activity and by immunohistochemistry for concomitant visualization of PA proteins; these lesions encompassed 3 nevi and 19 primary melanomas ranging from melanotic through mixed tumors to amelanotic tumors. Although urokinase-type plasminogen activator (u-Pa) activity was not detected at premalignant stages, it began to appear early in tumorigenesis and became more prominent in later stages of a majority of the tumors. The activity was largely attributable to the endothelium of sprouting capillaries and to a lesser degree to granulocytes, fibroblastic cells, and occasional melanoma cells within tumors. Tissue-type plasminogen activator (t-PA) was undetectable or low in all cases. Of the inhibitors (PAI), PAI-1 was seen in endothelial and fibroblastic cells and in the extracellular matrix, whereas PAI-2 occurred in only one case and was melanoma cell associated. Eleven additional melanomas were analyzed by reverse transcription-PCR for PA expression in RNA extracts from relatively large tumor samples. These were obtained from eight primary melanomas and three metastases, again spanning melanotic, mixed, and amelanotic cases. From four of the mixed primary tumors with distinct melanotic and amelanotic zones, the respective components were propagated separately in transgenic hosts as s.c. transplants to obtain data for clearly identifiable melanotic versus amelanotic parts. u-PA and PAI-1 mRNAs were expressed in all. t-PA expression varied greatly and was notably high in several amelanotic tumors or tumor components, possibly as a result of large blood vessels, as such vessels were seen to be t-PA positive in normal tissue. The u-PA activity in sprouting capillaries may indicate a role in neoangiogenesis. Therefore, according to these mouse models, u-PA may indirectly be a potential therapeutic target against melanoma progression.

Changes in expression of putative antigens encoded by pigment genes in mouse melanomas at different stages of malignant progression.

Cutaneous melanomas of Tyr-SV40E transgenic mice (mice whose transgene consists of the tyrosinase promoter fused to the coding regions of simian virus 40 early genes) strikingly resemble human melanomas in their development and progression. Unlike human melanomas, the mouse tumors all arise in genetically identical individuals, thereby better enabling expression of specific genes to be characterized in relation to advancing malignancy. The products of pigment genes are of particular interest because peptides derived from these proteins have been reported to function as autoantigens with immunotherapeutic potential in some melanoma patients. However, the diminished pigmentation characteristic of many advanced melanomas raises the possibility that some of the relevant products may no longer be expressed in the most malignant cells. We have therefore investigated the contributions of several pigment genes in melanotic vs. relatively amelanotic components of primary and metastatic mouse melanomas. The analyses reveal marked differences within and among tumors in levels of mRNAs and proteins encoded by the wild-type alleles at the albino, brown, slaty, and silver loci. Tyrosinase (the protein encoded by the albino locus) was most often either absent or undetectable as melanization declined. The protein encoded by the slaty locus (tyrosinase-related protein 2) was the only one of those tested that was clearly present in all the tumor samples. These results suggest that sole reliance on targeting tyrosinase-based antigens might selectively favor survival of more malignant cells, whereas targeting the ensemble of the antigens tested might contribute toward a more inclusive and effective antimelanoma strategy.

Ultraviolet radiation-induced malignant skin melanoma in melanoma-susceptible transgenic mice.

A new mouse model of UV radiation-induced melanoma is described. Unlike previous models, melanoma induction requires only short-term irradiation, does not require the application of chemical carcinogens, and does not cause any other tumors. The model takes advantage of the fact that Tyr-SV40E (C57BL/6 strain) transgenic mice are all melanoma-susceptible, and that different inbred lines are susceptible to different extents. Four-day-old mice of a moderately susceptible line (line 9 transgenic homozygotes) were exposed for 20 min/day to 328 mJ/cm2 to UVB (280-320 nm wavelength, comprising 70% of the total irradiance) for up to 4 consecutive days. Melanocytic lesions resembling macules, nevi, or early melanomas gradually appeared in the irradiated mice that were not seen in unirradiated transgenic controls of similar age. To afford ongoing observation beyond the short life span of line 9 homozygous mice, skin samples containing a total of 26 selected lesions were grafted at 20 weeks after UV radiation to longer-lived unirradiated hosts of transgenic line 12, in which melanoma susceptibility is low. Ten lesions in the grafts became melanomas; all melanomas had ulcerated and two had metastasized. At the stages examined, all the tumors were deeply melanotic. The remaining 16 lesions were still indolent when the experiment was terminated at 57 weeks post-UV radiation. The present protocol lends itself to variations in the choice of transgenic line, the age of the treated mice, and the intensity and duration of ultraviolet light; appropriate combinations of these variables would be expected to yield melanomas in relatively long-lived transgenic mice without skin grafting. The new model provides an opportunity to determine the melanoma action spectrum, to characterize at the molecular level the melanomas induced by ultraviolet light in comparison with those of other origins, and to investigate in vivo the photoprotective role of melanin.

Lens development in a dominant X-linked congenital cataract of the mouse.

Xcat is a recently identified mouse mutation causing X-linked dominant congenital cataract. The mutation is of particular interest as a possible animal model for the human X-linked cataract syndrome. Using light microscopy, we examined the histological changes of mutant lenses at selected intervals between embryonic (E) day 14 and postnatal (P) day 21. At E14, primary fiber formation completely fills the former lens vesicle in both normal and mutant mice, but in affected animals the primary fibers are irregularly arranged and show small foci of cellular disintegration. Progressive degeneration of primary fibers occurs from E15 to E18 and, during late gestation, secondary lens fibers also begin to degenerate. The lens epithelium and newly differentiated fibers, however, show no evident abnormality. Postnatally, most of the lens substance becomes amorphous; the cataractous process terminates in rupture of the posterior lens capsule by P21. Analysis of crystallin and cytoskeletal proteins of postnatal cataractous lenses revealed no significant abnormalities when compared to normal lenses. The observed sequence of histological changes indicates that the Xcat mutation affects the differentiation of lens fiber cells at some point after their initial elongation.

Comparison of the abilities of MHC-compatible bone marrow cells and lymph node cells to induce tolerance of skin allografts in rats.

A comparison has been made of the abilities of bone marrow cells and lymph node cells to induce tolerance of skin when inoculated into neonatal rats known to differ only with regard to non-MHC incompatibilities, including putative skin-specific (Skn) antigens. Each recipient received 50 x 10(6) cells, and tolerance was confirmed by the permanent acceptance of donor-strain neonatal heart tissue transplanted to the pinna of the ear. In 5 of the 8 MHC-compatible strain combinations tested, BMC were significantly more effective than LNC in inducing tolerance of skin, whereas in one situation LNC proved more efficient. Although the results are in accord with the occurrence of Skn antigens in rats, it appears that at least some of the antigens involved must also be expressed by BMC or LNC, but not equally by both of these tissues. The results also provide evidence that in rats, as in mice, the MHC can play a major role in determining the response to, and/or the immunogenicity of, Skn antigens.

Transgenic mouse model of malignant skin melanoma.

Tyr-SV40E transgenic mice are specifically susceptible to melanoma due to expression of the oncogene in pigment cells. Mice of the more susceptible lines die young of early-onset eye melanomas, when skin melanomas are still infrequent and benign. To surmount this obstacle, skin from donors of two high-susceptibility lines was grafted to Tyr-SV40E hosts of a low-susceptibility line of the same inbred strain, thereby enabling the skin to outlive the donors and continue to grow in immunocompetent but tolerant hosts. Unexpectedly, donor pigment cells in all the grafts soon selectively proliferated close to areas of greatest wound healing, forming a dense black tracery, especially at the outer rim of the grafts. These lesions slowly grew radially within the grafts, producing irregular greyish patches. Local vertical thickenings then appeared and developed into small melanomas, which soon ulcerated through the epidermis. The tumors rapidly enlarged and became deeply invasive. Discrete black nevi also arose, with many becoming larger and distinctly blue, but those not near areas of pronounced wound healing did not progress to malignancy. In this first series, malignant melanoma resulted in all the grafts from the more susceptible of two donor lines and in some grafts from the other line. Distant metastases occurred in some cases from each line. Most tumors were hypomelanotic and heterogeneous, with lobes or areas differing in melanization. The results strongly suggest that growth factors and cytokines--known to be produced in wound repair--are triggering the growth and malignant conversion of these genetically susceptible melanocytes and that in the graft situation we are merely witnessing a caricature--a usefully exaggerated manifestation of the true events underlying the genesis of melanomas. The striking resemblance to the human malignancy, the genetic uniformity and different susceptibilities of the transgenic lines, and the experimental possibilities in the grafted mice all make them an excellent model of the disease.

Histopathogenesis of malignant skin melanoma induced in genetically susceptible transgenic mice.

Animal models of human malignant skin melanoma were created in melanoma-susceptible inbred-strain transgenic mice by grafting skin from donors of high-susceptibility lines to hosts of a low-susceptibility line, thereby overcoming the problem of early death of the more susceptible animals from eye melanomas. As already described [Mintz, B. & Silvers, W. K. (1993) Proc. Natl. Acad. Sci. USA 90, 8817-8821], melanocytes within the grafts selectively proliferated in close proximity to areas of greatest wound healing, presumably in response to mitogenic factors from cells contributing to wound repair. An orderly sequence of externally visible events culminated in malignant melanoma. We examine here the histogenetic concomitants of these changes and find that they define a stepwise sequence strikingly comparable to that leading to human cutaneous melanoma. Moreover, the histological details suggest some of the underlying mechanisms. While the early lesions are first seen in the superficial dermis in the mouse, and in the basal layer of the epidermis in the human, both progress by radial growth followed by vertical growth. Melanocytic hyperplasia resulted in nests of densely melanized fusiform cells which were losing their dendrites. Some discrete lesions in the deep dermis appeared as blue nevi. As radial proliferation advanced, cellular atypia increased and the previously independent melanocytes cohered closely and formed a small solid tumor; the cells were usually then hypomelanotic or amelanotic. Ulceration of tumor through the epidermis occurred early. The tumor mass grew rapidly in the deep dermis and invaded and destroyed subcutaneous tissue and muscle. Primary tumors in the skin were often heterogeneous, with lobules or regions differing in pigmentation or atypia. However, the cells in circulating emboli, or in metastases in lymph nodes and lungs, appeared relatively homogeneous. These genetically uniform transgenic mouse models provide experimental access to the multistage genesis of melanoma.

Priming and cross-priming for H-Y in female mice.

Although female mice (H-2b and H-2g) that are responders to H-Y are able to cross-prime for this antigen, their ability to do so varies from strain to strain and can be influenced by whether they are exposed to H-Y via a subcutaneous hind footpad inoculation of male cells or via a male skin graft. On the other hand, females (H-2k) that are low responders to H-Y only can be sensitized to male skin isografts following a hind footpad inoculation of syngeneic male cells.

Major-histocompatibility-complex restricted and nonrestricted autoimmune effector mechanisms in BB rats.

To investigate whether the immunologic mechanisms of autoimmune pancreatic beta-cell destruction are MHC restricted, we examined the relative vulnerability of islet allografts from a panel of MHC-compatible and -incompatible donors to autoimmune damage after transplantation to spontaneously diabetic BB recipients. To circumvent a potentially confounding allograft response to the foreign islet graft, we utilized two strategies: (1) pretransplant in vitro culture of islets to delete intraislet APCs; and (2) induction of islet donor-specific immunologic tolerance in diabetes-prone BB rats. Experiments employing organ culture to prevent rejection demonstrated that MHC-incompatible grafts were significantly less vulnerable to autoimmunity than MHC-compatible grafts. In contrast, when we used the model of immunologic tolerance to exclude rejection, both MHC-compatible and -incompatible islet grafts were equally susceptible to autoimmune damage. The reason for this discrepancy has not been defined fully but may be related to our observation that tolerant BB animals exhibit increased peripheral blood NK-cell activity. NK cells are known to be cytotoxic to islets in vitro and could play a role in a non-MHC-restricted diabetogenic response in vivo. We conclude that both MHC-restricted and nonrestricted mechanisms are capable of contributing to anti-beta-cell autoimmunity in BB rats.

Major histocompatibility complex restriction and cross-priming of H-Y antigen in rats.

Although female rats can be sensitized to H-Y-incompatible male skin isografts following exposure to MHC-incompatible male lymphoid cells, these cells are not as effective as MHC-compatible male cells. Evidence is presented that the effectiveness of the MHC-incompatible cells is a consequence of crosspriming and that such crosspriming only occurs if these cells are rejected.