Peter I. Schrier

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma.

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented byHLA-B7 and is encoded by a new gene that we have namedRAGE1. No expression ofRAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.

Detection and Functional Analysis of CD8+ T Cells Specific for PRAME: a Target for T-Cell Therapy

Purpose: Preferentially expressed antigen on melanomas (PRAME) is an interesting antigen for T-cell therapy because it is frequently expressed in melanomas (95%) and other tumor types. Moreover, due to its role in oncogenic transformation, PRAME-negative tumor cells are not expected to easily arise and escape from T-cell immunity. The purpose of this study is to investigate the usefulness of PRAME as target for anticancer T-cell therapies. Experimental Design: HLA-A*0201-subtyped healthy individuals and advanced melanoma patients were screened for CD8+ T cells directed against previously identified HLA-A*0201-binding PRAME peptides by IFN-γ enzyme-linked immunosorbent spot assays and tetramer staining. PRAME-specific T-cell clones were isolated and tested for recognition of melanoma and acute lymphoid leukemia (ALL) cell lines. PRAME mRNA expression was determined by quantitative real-time reverse transcription-PCR. Results: In 30% to 40% of healthy individuals and patients, PRA100-108-specific CD8+ T cells were detected both after in vitro stimulation and directly ex vivo after isolation by magnetic microbeads. Although CD45RA− memory PRA100-108-specific T cells were found in some individuals, the majority of PRA100-108-tetramer+ T cells expressed CD45RA, suggesting a naive phenotype. PRA100-108-tetramer+ T-cell clones were shown to recognize and lyse HLA-A*0201+ and PRAME+ melanoma but not ALL cell lines. Quantitative real-time reverse transcription-PCR showed significantly lower PRAME mRNA levels in ALL than in melanoma cell lines, suggesting that PRAME expression in ALL is below the recognition threshold of our PRA100-108-tetramer+ T cells. Conclusion: These data support the usefulness of PRAME and in particular the PRA100-108 epitope as target for T-cell therapy of PRAME-overexpressing cancers.

Renal-cell carcinoma-specific lysis by cytotoxic T-lymphocyte clones isolated from peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Melanoma and renal‐cell carcinoma (RCC) are generally considered to be relatively immunogenic tumor types in humans. In the case of melanoma, many major histocompatibility complex (MHC) class I‐restricted tumor‐specific cytotoxic T lymphocytes (CTL) have been isolated from either tumor‐infiltrating lymphocytes (TIL) or autologous peripheral blood lymphocytes (PBL). In contrast, such CTL have only incidentally been described in the case of RCC. It has often been reported that TIL lines isolated from RCC display non‐MHC‐restricted and non‐specific activity. Here, we report the isolation and characterization of tumor‐specific CTL from PBL of one RCC patient and from TIL of another RCC patient. CTL clones 263/17 and 263/45, isolated from the PBL of patient LE‐9211, were restricted by HLA‐B7. CTL clone 5E, isolated from the TIL of patient LE‐8915, was restricted by HLA‐B37. The autologous RCC cell lines were efficiently lysed by the CTL clones, whereas normal epithelial cells of the proximal tubuli matched for the restriction element and K562 were not. From a panel of allogeneic RCC cell lines, CTL 5E recognized MZ‐1940‐RCC. Reactivity to allogeneic RCC sharing HLA‐B7 was also observed with CTL 263/17 and 263/45, both of which could lyse the HLA‐B7‐positive cell line MZ‐1851‐RCC. Our data provide evidence that common tumor antigens are recognized by CTL on RCC.

Transcriptional suppression of HLA-B expression by c-Myc is mediated through the core promoter elements

In melanoma, HLA class I expression is suppressed by overexpression of the c-myc oncogene. This suppression has severe consequences for the recognition of these tumor cells by the immune system of the organism. We show here that transcription of the HLA-B locus, which is mainly affected by c-Myc, is downmodulated at the level of initiation of transcription. The transcriptional activity of various HLA-B reporter constructs was tested in a melanoma cell line with low endogenous c-myc expression and in transfectants with high stable and transient c-myc expression. We demonstrated that the responsive region can be mapped to the core promoter region of HLA class I, ruling out any effects of c-myc overexpression on the enhancer A or enhancer B regions. The region subject to downregulation is confined to a 43 base pair fragment encompassing the CCAAT and TATA elements. By coupling this region to a heterologous viral enhancer, we showed that the downmodulation by c-Myc is independent of the presence and nature of an enhancer. These results suggest a mechanism in which c-Myc downregulates the expression of HLA class I genes by interfering with the basal level of transcription.

C-Myc Represses Transiently Transfected HLA Class I Promoter Sequences not Locus-Specifically

Overexpression of the c-myc oncogene is frequently accompanied by downregulation of Major Histocompatibility Complex (MHC, HLA in humans) class I antigens. In human melanoma c-myc overexpression downmodulates HLA-B expression, whereas HLA-A is hardly affected. Repression of HLA-B is mediated through the core promoter, containing a CAAT-box and a non-conventional TATA-box. We show evidence that in transient transfection assays the HLA-A2 and HLA-B7 promoters are repressed by c-myc to the same extent. Therefore, other sequences of the HLA-A and HLA-B genes, possibly intron/exon sequences, should contribute to the locus B-specificity of the downregulation. Furthermore, c-myc does not seem to alter binding of protein complexes to the CAAT- or TATA-box of HLA-B7 or HLA-A2 in gel retardation assays. Comparison of promoters repressed by c-myc reveals a weak consensus sequence of the initiator (Inr) element: TCA(+1)YYYNY. The presence of a TCA sequence in the initiator region of the MHC class I promoter makes downregulation by c-myc through the Inr likely. We speculate that the Inr contributes to MHC class I promoter activity by stimulating recruitment of TFIID to the weak, non-conventional TATA-box, thereby making it susceptible to repression by c-myc through the Inr.

Uv-induced N- ras mutations are T-cell targets in human melanoma

Human cutaneous melanoma is heterogeneous with respect to the genetic aberrations involved and the genes altered are potential targets for the immune system. The incidence of cutaneous melanoma is known to be linked to UV peak exposure, and the N-ras oncogene is clearly one of the genes involved in the UV carcinogenesis in melanoma. It is mutated in a significant proportion of melanomas and therefore may serve as a target for T cells. Here, we report that an human leukocyte antigen-A2 binding peptide CLLDILDTAGL, encompassing the frequently found 61 -Leu mutation in N -ras, induces cytotoxic T lymphocytes from healthy donor blood that lyse 61 -Leu N-ras transfected melanoma cells. Furthermore, we have found an association between the presence of N-ras mutations and clinical response to immunotherapy with interleukin-2 plus interferon in a group of stage IV melanoma patients. Although the overall survival of these patients was not affected by the N-ras status of their melanomas, these studies suggest that mutated N-ras may provide a target for cytotoxic T lymphocytes in melanoma patients.

N‐ras oncogene expression changes the growth characteristics of human melanoma in two independent SCID‐hu mouse models

Fifteen percent of all human melanomas carry mutations in ras genes, the majority of which are located in codon 61 of the N‐ras gene. However, the biological significance of these mutations is as yet unknown. In this study, we investigated the influence of N‐ras oncogene products mutated in codon 61 on the growth characteristics of human melanoma in vivo by establishing 2 SCID‐hu mouse xenotransplantation models. Tumors grown in SCID mice injected with human melanoma carrying activated N‐ras genes were significantly larger (p < 0.004) than tumors grown in animals injected with the appropriate control transfectants. Additionally, tumors with N‐ras point mutations clearly showed a more pleomorphic phenotype than the control groups. Our results, obtained in 2 independent SCID‐hu xenotransplantation models, suggest that mutated N‐ras oncogene expression may be an important factor influencing growth characteristics of human melanoma without altering metastatic potential. These novel in vivo model systems provide a tool for further study of the biology of mutated ras in melanoma and should also prove useful for testing new and improved treatment strategies for human melanoma carrying mutated ras genes.