In this follow-up to our pilot trial, we observed regression of metastatic RCC in 38% of patients who underwent a nonmyeloablative allogeneic HSCT for metastatic RCC. Although clinical evidence suggests that T cells mediate graft-versus-RCC effects, difficulty in generating tumor lines from patients undergoing transplantation has hampered in vitro studies aimed at characterizing the antigens targeted by donor T cells. In this study, we provide insight into the mechanisms accounting for RCC regression after nonmyeloablative HSCT. We found evidence suggesting that both broadly expressed mHas and antigens restricted to the tumor are targets for donor T cells isolated from patients with evidence for a graft-versus-RCC effect. Using PBMCs collected from both responding patients, we succeeded in expanding the CTLs, which were cytotoxic to patient autologous RCC cells in vitro. In one of these responders (LYO), the CTLs had a cytotoxicity pattern consistent with recognition of an antigen that was not restricted to the tumor, lysing both patient autologous LYO-LCL cells and LYO-RCC cells but not donor LYOD-LCL cells. Following limiting dilution cloning, we identified a CD8+
T cell clone with a similar cytotoxicity pattern that lysed both LYO-RCC cells and LYO-LCL cells but not donor LYOD-LCL cells. These findings are consistent with donor T cell recognition of mHa expressed broadly on both tumor cells and hematopoietic cells of the recipient. Tykodi et al. (4
) previously demonstrated that mHa-specific T cell clones cytotoxic to RCC cells in vitro could be isolated from both responding and nonresponding patients with metastatic kidney cancer following an allogeneic HSCT. Such mHas are known to be expressed on a variety of hematological malignancies and are thought to be dominant targets of transplanted donor T cells mediating GVL effects. Our data confirm that mHas absent in the donor but present in the patient are expressed on patient RCC cells. The isolation of an mHa-specific T cell clone from a responding patient that killed patient RCC cells in vitro suggests that donor T cells were primed in vivo to this antigen after HSCT and provide evidence that mHas may be a target for a graft-versus-RCC effect. The detection of mHa-specific CTLs from a responding patient that kill patient RCC cells in vitro is also consistent with the clinical observation that GVT effects occur more frequently in patients who develop GVHD.
In this analysis, we also detected RCC-reactive T cells by ELISPOT in the first several months after HSCT in all 4 patients. However, in the 2 nonresponders, these T cells were detected only transiently. In contrast, in the 2 patients who had tumor regression, RCC-reactive T cells persisted in the blood over a prolonged interval. These findings suggest factors that eliminate or sustain RCC-reactive T cells that expand after HSCT may have a critical impact on the ability to generate graft-versus-RCC tumor effects.
In patient SAUJ, who had a GVT effect against metastatic RCC associated with survival of more than 4 years after HSCT, CD8+ CTL lines and T cell clones were expanded from blood collected after transplantation that had tumor-specific cytotoxicity in vitro. Using cDNA expression cloning, we identified 2 transcripts (CT-RCC-8 and CT-RCC-9) encoding an antigen recognized by HLA-A11–restricted RCC-reactive T cells expanded from a responding patient after HSCT. We subsequently identified a 10-amino-acid peptide antigen (CT-RCC-1) that was encoded from the shared common sequence region of CT-RCC-8 and CT-RCC-9 that was recognized by a T cell clone derived from these RCC-reactive CTLs. Remarkably, CT-RCC-1–specific CTLs recognized approximately 50% of RCC cell lines that expressed HLA-A11 but not patient fibroblasts or patient LCL cells, suggesting this antigen is commonly expressed in RCC tumors at levels that induce CTL killing but not in normal tissues. Semiquantitative RT-PCR for expression of CT-RCC-8 and -9 and real-time PCR for their shared common sequence region showed that these transcripts were expressed at variable levels in fresh kidney cancer samples and in greater than 50% of cultured RCC cell lines but not in pooled cDNA from normal tissues including the kidneys and testis nor in a number of other non-RCC tumor lines.
The nucleotide sequences of CT-RCC-8 and -9 matched with genomic sequences of an HERV-E located on chromosome 6q that was not previously known to be expressed in human cells. HERVs exist widely within the human genome as proviruses, with most being transcriptionally inactive (6
). Recently, some HERVs were identified to have transcriptionally active components with biological activity expressed widely in human tissues; Seifarth et al. (8
) showed that an HERV-E clone, 4-1, had active transcription in many normal tissues. In contrast, the transcriptional products derived from the HERV-E that we identified were selectively expressed in RCC and were not detected in normal tissues.
HERV-K transcripts have been found to be expressed in tissue malignancies including melanoma, teratocarcinoma, and prostate cancer as well as hematological malignancies (9
). Although more than 50 HERV-Es are estimated to exist in the human genome, this report is, to our knowledge, only the second to identify an HERV-E transcription product expressed in tumor cells (13
) and the first to identify an HERV-E expressed in RCC. Furthermore, unlike HERV-K expression, which can be detected not only in tumors but also in normal tissues such as the testis, skin cells (12
), and blood cells (10
), the sequences derived from this HERV-E appear to be selectively expressed in RCC cells, with virtually undetectable levels of expression in normal tissues, making them potentially ideal targets for tumor immunotherapy.
Recently, CTLs recognizing HERV-K–derived peptides that kill tumor cells in vitro have been identified in a few cancer patients (12
). Similarly, we isolated and expanded a T cell clone from the blood of a responding transplant patient that recognized an HERV-E–derived antigen and killed patient RCC cells and HLA-A11+
RCC cells in vitro. The HLA-A11–restricted 10-mer peptide named CT-RCC-1 was identified to be the target antigen of these CTLs that had RCC-specific cytotoxicity. Tetramer analysis showed that CT-RCC-1–specific T cells were absent at baseline but were detected after HSCT following regression of metastatic disease, suggesting that this antigen had immunogenicity in vivo. To the best of our knowledge, this is the first report to identify a T cell population recognizing an HERV-derived antigen with expression restricted to tumor cells.
Because the phenotypic frequency of HLA-A11 expression is low (15
), immunotherapy approaches targeting the CT-RCC-1 antigen through tumor peptide vaccination or the adoptive infusion of CT-RCC-1–specific CTLs would be limited to a minority of patients with metastatic kidney cancer. The full protein products translated from CT-RCC-8 and -9 and other genes derived from this HERV-E have not yet been defined. It is possible, therefore, that other immunogenic peptides derived from this HERV could be expressed on more common HLA class I molecules, a finding that potentially would broaden the application of immunotherapy approaches targeting antigens derived from this HERV to a greater percentage of patients with metastatic RCC.
The factors regulating expression of the CT-RCC antigens in RCC are currently unknown. However, the observation that CT-RCC-8 and CT-RCC-9 were not detected in a variety of hematological malignancies and other solid tumors suggests that genetic mutations specific to RCC (i.e., the von Hippel–Lindau [VHL] gene) may in part regulate expression of transcripts derived from this HERV.
Clear-cell RCC is the dominant histological subtype of kidney cancer, accounting for approximately 75% of the cases of spontaneously occurring RCC. Loss of function of the VHL tumor suppressor gene from a mutation or as a result of promoter hypermethylation occurs in nearly 80% of these tumors, which ultimately leads to increased protein expression of HIF-1α and -2α, resulting in overexpression of more than 150 client genes that play a fundamental role in tumor progression, spread, and response to hypoxia (prevalent in tumor tissues) (16
). Recent data suggest that clear-cell carcinoma may represent the subtype of kidney cancer that is immunoresponsive, with some studies reporting virtually no responses to IL-2–based immunotherapy occurring in patients with non-clear-cell RCC (17
). Although it represented the dominant histological subtype in our transplant trial, it is nonetheless interesting that GVT effects following HSCT were only observed in patients with clear-cell RCC (48% cumulative response rate), with no responses seen in the 14 patients with non-clear-cell tumors. One hypothesis to explain these findings would be that loss of VHL function occurring in clear-cell tumors may result in aberrant expression of CT-RCC HERV-derived transcription products, shown here to be a target for transplanted donor T cells, which theoretically could also be a target for autologous T cells. A histological review of the RCC cell lines and fresh RCC tissues used in experiments presented in this article showed all to be clear-cell carcinomas, with more than half expressing HERV-E transcripts. Furthermore, limited preliminary data from an ongoing study of fresh tumors suggest that this HERV-E may have transcriptional activity limited to the clear-cell variant of kidney cancer (unpublished observations), which is intriguing given the track record for this tumor being the immunoresponsive subtype of RCC.
The unavailability of fresh RCC cells in the majority of patients who received transplants in this study precluded an RT-PCR–based analysis to correlate tumor HERV-E expression with a GVT effect. The development of antibodies that recognize the CT-RCC-1 antigen and/or other peptides and proteins derived from this HERV would permit such an analysis, where immunohistochemistry on paraffin-preserved tumor samples could be used to discern HERV expression. Furthermore, because HLA-A11, the MHC class I restricting allele for the CT-RCC-1 antigen, is present in only a minority of the population, including a small subset of transplanted patients in this transplant series, an analysis correlating response to the expansion of CD8+ CT-RCC-1–specific T cells could not be performed. The identification of other immunogenic peptides derived from CT-RCC-8 and -9 and other gene sequences of this HERV-E that are expressed on more common HLA class I molecules would allow for a study correlating disease response to the generation of CD8+ T cells recognizing HERV-E antigens. Further, it would permit an analysis to discern whether HERV-E antigens represent a target antigen of tumor-infiltrating lymphocytes in fresh kidney cancer tumors.
In conclusion, this study provides insight into the immunological mechanisms accounting for regression of RCC following allogeneic HSCT and provides the first evidence to our knowledge that GVT effects against this malignancy may be associated with tumor-specific immune responses to antigens expressed on RCC cells. The expansion of CT-RCC antigen–specific T cells in a patient who had a GVT effect associated with prolonged regression of metastatic RCC suggests that gene products derived from HERV-E are immunogenic in vivo and may be a novel target for RCC immunotherapy.