Catia Traversari

Structure, Chromosomal Localization, and Expression of 12 Genes of the Mage Family

We reported previously that human geneMAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with aMAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12MAGE genes are located in the q terminal region of chromosome X. LikeMAGE-1, the 11 additionalMAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that ofMAGE-1. The coding sequences of theMAGE genes predict the same main structural features for allMAGE proteins. In contrast, the promoters and first exons of the12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of eachMAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of theMAGE family includingMAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes

Human melanoma line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocytes (CTL). As a first step towards the cloning of the gene coding for one of these antigens, we tried to obtain transfectants expressing the antigen. The DNA recipient cell was a variant of MZ2-MEL which had been selected with a CTL clone for the loss of antigen E. It was cotransfected with genomic DNA of the original melanoma line and with selective plasmid pSVtkneoβ. Geneticin-resistant transfectants were obtained at a frequency of 2 × 10−4. These transfectants were then screened for their ability to stimulate the production of tumor necrosis factor by the anti-E CTL clone. One transfectant expressing antigen E was identified among 70 000 drug-resistant transfectants. Its sensitivity to lysis by the anti-E CTL was equal to that of the original melanoma cell line. When this transfectant was submitted to immunoselection with the anti-E CTL clone, the resulting antigen-loss variants were found to have lost several of the transfected pSVtkneoβ sequences. This indicated that the gene coding for the antigen had been integrated in the vicinity of pSVtkneoβ sequences, as expected for cotransfected DNA.

Rexpression of HLA class I antigens and restoration of antigen-specific CTL response in melanoma cells following 5-aza-2'-deoxycytidine treatment.

Cell surface expression of HLA class I/peptide complexes on tumor cells is a key step in the generation of T‐cell‐based immune responses. Several genetic defects underlying the lack of HLA class I expression have been characterized. Here we describe another molecular mechanism that accounts for the complete absence of HLA class I molecule expression in a tumor line (MSR3‐mel) derived from a melanoma patient. Hypermethylation of the MSR3‐mel DNA, specifically of HLA‐A and ‐B genes, was identified, which resulted in loss of HLA class I heavy chain transcription. Treatment of MSR3‐mel cells with the demethylating agent 5′‐aza‐2′‐deoxycytidine (DAC) allowed HLA‐A and ‐B transcription, restoring cell surface expression of HLA class I antigens and tumor cell recognition by MAGE‐specific cytotoxic T lymphocytes. The MSR3‐mel line was obtained from a metastatic lesion of a nonresponding patient undergoing MAGE‐3.A1 T‐cell‐based peptide immunotherapy. It is tempting to speculate that the hypermethylation‐induced lack of HLA class I expression is the cause of the impaired response to vaccination. This study provides the first evidence that DNA hypermethylation is used by human neoplastic cells to switch off HLA class I genes, thus providing a new route of escape from immune recognition.

High homogeneity of mage, bage, gage, tyrosinase and Melan-A/Mart-1 gene expression in clusters of multiple simultaneous metastases of human melanoma: Implications for protocol design of therapeutic antigen-specific vaccination strategies

Human melanoma cells express several antigens which are recognized by autologous and specific CTL clones in association with HLA‐class‐I molecules. Many of these antigens represent suitable targets for tumor immunotherapy, since their expression in human melanoma cells is common and highly specific. In order to achieve real clinical success with therapeutic vaccination strategies, one important requirement is the expression of the target antigen by all the tumor lesions of a patient. We have studied this issue by assessing, through an RT‐PCR approach, the expression of MAGE‐1, MAGE‐2, MAGE‐3, BAGE, GAGE‐1/2, Tyrosinase and Melan‐A/MART‐1 genes in 17 clusters of simultaneous in‐transit or regional lymph‐node metastases collected from 15 stage‐III and 1 stage‐IV (AJCC/UICC pTNM system) melanoma patients. In 14 out of 17 clusters of simultaneous metastatic lesions (82%), the homogeneity in the pattern of gene expression within the cluster was complete. Heterogeneity within the same cluster was observed in only 3 out of 17 clusters (18%) and represented only minor features. Our data reveal that, in AJCC‐stage‐III melanoma patients, different but simultaneous metastatic lesions express the same pattern of antigen‐coding genes. These observations have 2 main clinical implications: (i) the antigenic characterization of one single and easily accessible lesion allows identification of optimal targets for an active antigen‐specific immunotherapy treatment; (ii) almost all the metastatic lesions are expected to be hit by the immune response eventually induced against the tumor antigen. Moreover, these data suggest that active specific immunotherapy directed against MAGE‐1, MAGE‐3, BAGE, GAGE‐1/2, Melan‐A/MART‐1 and Tyrosinase antigens could be exploited as an adjuvant treatment to surgery in high‐risk AJCC‐stage‐III‐melanoma patients. Int. J. Cancer 77:200–204, 1998.© 1998 Wiley‐Liss, Inc.

Characterization of antigenic peptides presented by HLA-B44 molecules on tumor cells expressing the gene MAGE-3

The amino acid sequence of the protein encoded by the gene MAGE‐3was screened for peptides containing the binding motif for HLA‐B44. Nine peptides were synthesized, and their binding affinity for HLA‐B*4402 and ‐B*4403 was analyzed in an HLA class I α‐chain refolding assay. Four peptides with binding affinity for HLA‐B*4403 were chosen for in vitro cytotoxic T‐lymphocyte induction assays using as antigen‐presenting cells peptide‐pulsed, autologous activated B lymphoblasts from a healthy, B*4403+ donor. Peptide‐specific effectors could be raised only against one peptide, M3‐167. Cytotoxic T lymphocytes specific for this peptide were also able to recognize melanoma cell lines expressing HLA‐B44 and the gene MAGE‐3, strongly suggesting that M3‐167is a naturally processed MAGE‐3‐encoded epitope presented by HLA‐B44.M3‐167 is a 1 amino acid N‐terminal extension of M3‐168, a naturally processed epitope MAGE‐3‐encoded epitope presented by HLA‐A1 that has been previously described. TAP binding studies of these 2 peptides revealed that the TAP affinity of M3‐167 is about 9‐fold higher than that of M3‐168. M3‐167 or a longer precursor could be transported into the endoplasmatic reticulum, where it could be trimmed for presentation by HLA‐A1 or ‐B44 molecules. Taken together, our data suggest that M3‐167 could be an immunodominant peptide encoded by the gene MAGE‐3.

Multiple mechanisms are responsible for the alteration in the expression of HLA class I antigens in melanoma

Dear Sir, Fonsatti et al. in this issue have offered some criticisms of our article on the role of methylation in the turn-off of MHC class I antigens in the MSR3 melanoma cell line. In our report,1 we described the role of methylation only as one more mechanism among those reported to date that lead to HLA total loss (Phenotype no. I). We did not intend to claim that the behavior shown by cell line MSR-3 was the rule, and indeed it may be the exception, as Fonsatti et al. note. In fact, other mechanisms ( 2m inactivation, TAP/LMP downregulation, altered DNA-binding factors and oncogene overexpression) are observed more frequently and also contribute to a similar phenotype.2–6 In particular, alterations caused by those mechanisms involving structural alterations to 2m lead to irreversible losses that will not respond to treatment with IFNor demethylation. We proposed a role for methylation in MSR3-mel only after careful evaluation of the cells to rule out 2m mutation, genomic loss, nuclear transcription factors downregulation and loss of inducibility by cytokines. The conclusions Fonsatti et al. reach after their study of cell lines with total loss (Table I, p. XX) cannot be sustained unless structural mechanisms are ruled out because some of these mechanisms are likely to have occurred in at least some of their lines. Moreover, the special behavior of MSR3-mel after demethylation may result from the fact that HLA genes in this cell line are hypermethylated, as shown in Southern blot analyses. Do the authors have information regarding the methylation state of the HLA genes in the lines they studied? This information is fundamental to observe the effects of 5-AZACdR. In our study, we reconciled our findings with those of earlier studies on the role of methylation in regulating HLA class I gene expression by recalling that a minimum level of methylation may be needed to maintain HLA class I antigen expression.7 This is why our findings for level of expression were clear in mRNA analyses but less evident in assays to demonstrate surface expression. To better document HLA class I expression, we used INFtreatment in studies designed to observe the recognition of MAGE-specific cytotoxic T lymphocytes. We feel that the arguments presented by Fonsatti et al. would have been more solid, and more directly comparable to our findings, if they had reported mRNA levels rather than FACs analyses. The authors also claim that HLA-A2 expression is not recovered in lines in which this antigen has been lost. From this they deduce that hypermethylation does not represent a mechanism used to turn off HLA-A2 expression. Have the authors taken into account the possibility of a structural alteration in the HLA-A2 gene, as a result, for example, of loss of heterozygosity associated with chromosome 6p21? Their findings point in this direction, unless the pattern of HLA-A2 methylation is assumed to differ somehow from the methylation of other HLA antigens that were induced in the demethylation assays. This would account for the lack of response to 5-AZA-CdR. In this connection, our experience with HLA allelic losses suggests that most such instances are due to loss of heterozygosity (HLA haplotypic loss), mutational events or aberrant splicing.8–13 Fonsatti et al. should determine the mechanism of HLA-A2 loss in the cell lines they tested before they undertake experiments to recover the expression of this molecule. In some experiments, we used 3 M and up to 10 M 5-AZA. Cell line MSR3-mel was resistant to prolonged culture in the presence of 5-AZA-CdR for periods longer than those reported in our article. Cultures were initiated and maintained at both participating laboratories, and no toxic effects were reported during the study. We note, however, that this behavior may have reflected hypermethylation not only of the HLA genes but also of many other genes. This would probably lead to the particular behavior of the cell line during demethylation, in which both cisand trans-acting factors may modify not only HLA expression but also many other characteristics. In conclusion, we have emphasized that multiple mechanisms are responsible for HLA class I defects observed in tumor cells.14,15 and that the characteristics of the tumor tissue, and in particular the degree of conservation of different components of the antigen presentation machinery, need to be determined in detail before immunotherapeutic decision-making processes can be placed on a firmer footing. Finally, we suggest that the treatment of demethylating agents should be attempted only after the intrinsic mechanisms involved in downregulation of MHC antigens have been identified and verified. Yours sincerely, Francisco RUIZ-CABELLO, Catia TRAVERSARI and Federico GARRIDO

Monoclonal antibodies against NIH 3T3 cells transformed by human thyroid carcinoma DNA

First cycle transformants of NIH 3T3 cells transfected with metastatic human thyroid carcinoma DNA were used as immunogen to obtain monoclonal antibodies (MAbs) against normal and transformation-related antigens. The transformed cell line (M33) was shown to contain Alu sequences. Two MAbs were selected on the basis of their differential reactivity toward untreated NIH 3T3 cells or the transformed M33 cell line. By immunofluorescence, immunoelectronmicroscopy and biochemical analysis, the first MAb (MTr1) was demonstrated to recognize an epitope on cytoskeletal filaments of proliferating murine fibroblasts. Similar MTr1-labelled filaments were also found to accumulate into cytoplast-like structures spontaneously produced by M33 cells. The characterization by immunofluorescence of MTr2, the second MAb, indicates that it recognizes a specific human antigen associated with normal thyroid epithelial cells and differentiated thyroid tumors.