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Journal of Clinical Oncology, Vol 24, No 36 (December 20), 2006: pp. 5725-5734 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.05.3314 Immunosurveillance and Survivin-Specific T-Cell Immunity in Children With High-Risk Neuroblastoma
From the Abramson Family Cancer Research Institute; Hematology-Oncology Division, Department of Medicine; Department of Pathology and Laboratory Medicine; Division of Oncology, Department of Pediatrics; and Department of Pathology, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA; Department of Pathology, Children's Hospital; and the Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA Address reprint requests to Robert H. Vonderheide, MD, DPhil, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, 551 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104; e-mail: rhv{at}mail.med.upenn.edu
PURPOSE: Tumor immunosurveillance influences oncogenesis and tumor growth, but it remains controversial whether clinical failure of immunosurveillance is a result of lymphocyte dysfunction or tumor escape. In this study, our goal was to characterize the physiology of tumor immunosurveillance in children with high-risk neuroblastoma (HR-NBL). PATIENTS AND METHODS: Immunohistopathologic studies were carried out on 26 tumor samples from a cohort of HR-NBL patients diagnosed at Children's Hospital of Philadelphia for the 2-year period from May 2003 to May 2005. Blood from nine HLA-A2+ patients in this cohort was analyzed for T cells specific for the antiapoptotic protein survivin. RESULTS: Survivin protein was expressed by 26 of 26 tumors. In HLA-A2+ patients, circulating cytotoxic T lymphocytes (CTLs) specific for survivin were detected by peptide/major histocompatibility complex tetramer analysis in the blood of eight of nine children with HR-NBL at the time of diagnosis. Rather than being selectively rendered anergic in vivo, circulating survivin-specific CTLs were highly functional as shown by cytotoxicity and interferon gamma enzyme-linked immunospot assays in six of nine patients. Survivin-specific CD107a mobilization by T cells was found in five of five patients. By immunohistochemistry, tumor-infiltrating T cells were few or absent in 26 of 26 tumors. CONCLUSION: Children with HR-NBL harbor robust cellular immune responses to the universal tumor antigen survivin at the time of diagnosis, but intratumoral T cells are strikingly rare, suggesting a failure of cellular immunosurveillance. Efforts to develop novel therapies that increase T-cell trafficking into tumor nests are warranted.
Dynamic interactions between tumor cells and lymphocytes in vivo can influence oncogenesis, tumor growth, and clinical outcome in patients.1-3 In particular, the presence of lymphocytes within tumors independently predicts prognosis.4-7 Extensive mouse and human studies indicate that cytotoxic T lymphocytes (CTLs) are a prime mediator of tumor immunosurveillance.8,9 It remains controversial, however, whether clinical failure of immunosurveillance is a result of lymphocyte dysfunction or tumor escape. On one hand, tumor escape mechanisms may permit evasion from even the most vigorous immune responses, but on the other hand, immune function of patients may be globally and markedly depressed, leaving a tumor immunologically unaffected that might otherwise be rejected.10-12 In children, the biology of tumor immunosurveillance is particularly poorly understood. Recent data demonstrate that pediatric cancer patients preserve a functional pool of antigen-specific memory T cells and have an exceedingly low rate of life-threatening infection, despite large tumor burdens and chemotherapy.13,14 We hypothesized that, to the extent children with cancer exhibit immune resiliency, there may be therapeutically important features of tumor immunosurveillance in this population. As a model, we studied patients with neuroblastoma (NBL).15 Certain clinical, pathologic, and genetic parameters define patients with high-risk NBL (HR-NBL), for whom prognosis is poor despite aggressive treatment.16,17 Lymphocytes have been noted in biopsy samples of NBL,18-21 but the function and specificity of these T cells are unknown. Indeed, recognition of NBL by T cells has been considered unlikely by some, given that major histocompatibility complex (MHC) expression by NBL cells is minimal.22-24 We reconsidered this notion in light of findings that link NBL biology to expression of survivin,25,26 an antiapoptotic protein that has been studied as a widely expressed tumor-associated antigen recognized by CTL.27-30 Survivin (BIRC5, 17q25) is a member of the inhibitor of apoptosis gene family that also regulates mitosis.31 It is expressed in virtually every human cancer, whereas in normal tissue, its expression is low or absent.32,33 Survivin-specific CTLs have been detected at low frequency in melanoma, leukemia, and breast cancer.27-30 Vaccination of tumor-bearing mice against survivin results in tumor rejection.34-36 We recently reported initial evidence for survivin-specific CTLs in patients with HR-NBL.37 In the current study, we evaluated T-cell immunosurveillance in HR-NBL by characterizing survivin as a universally expressed tumor antigen in a prospective cohort of patients.
Blood Samples, Tumor Specimens, and Cell Lines Patients were enrolled after informed consent on institutional review board–approved Children's Hospital of Philadelphia protocols for the treatment of HR-NBL (patients with stage III or IV disease with unfavorable biology features). Blood and tumor samples were obtained at diagnosis. Additional tumor samples were obtained at the time of resection surgery after induction chemotherapy and used to prepare RNA. For one patient, the tumor cell line (NSJ3) was established, which exhibited morphology consistent with NBL, has been maintained in culture for more than 25 passages, and forms tumors in immunocompromised mice. Blood samples from normal donors were obtained after institutional review board–approved informed consent. Peripheral-blood mononuclear cells (PBMCs) were isolated by Ficoll centrifugation. Allogeneic NBL cell lines were obtained from Dr Garrett Brodeur (Children's Hospital of Philadelphia) and T2 cells were from the American Type Culture Collection.
Immunohistochemistry
Plasmids and Vectors
RNA Preparation
Establishment of T-Cell Cultures Using RNA-Electroporated CD40-Activated B Cells
Tetramer, Enzyme-Linked Immunospot Assay, and Chromium Release Analysis
CD107a Mobilization Assay
RT-PCR and Western Blot for Survivin
Survivin Expression in HR-NBL Immunohistochemistry was used to evaluate survivin protein expression from diagnostic tumor specimens from 26 patients with HR-NBL (stage III or IV with unfavorable biology features). Mean age at diagnosis was 2.9 years (range, 9 months to 14 years). Cytoplasmic and nuclear staining for survivin protein was identified in 26 of 26 samples within tumor nests (Fig 1). Cytoplasmic staining was observed in the vast majority of tumor cells, whereas stromal elements within tumors appeared negative for survivin staining. Nuclear staining, although more intense, was restricted to a subset of tumor cells within individual nests.
Survivin-Specific CTL Responses in HR-NBL To determine whether patients with HR-NBL have a cellular immune response to survivin, we prospectively evaluated all 18 HR-NBL patients diagnosed at Children's Hospital of Philadelphia for the 2-year period from May 2003 to May 2005 and treated on a high-dose chemotherapy and tandem stem-cell transplantation protocol.16 Peripheral-blood CD8+ T lymphocytes were obtained at diagnosis and analyzed for survivin-specific functional responses in the nine patients expressing the most common MHC class I allele, HLA-A2 (Table 1). Patient T cells were stimulated in vitro with autologous CD40-B loaded with full-length survivin mRNA (v GFP mRNA as a negative control) and assayed for tumor cytotoxicity. We found that, in six (67%) of nine patients, T cells stimulated with CD40-B loaded with survivin mRNA efficiently lysed autologous NBL tumor cells or HLA-matched allogeneic NBL cells but did not lyse HLA-mismatched or MHC class I–negative allogeneic NBL cells (Fig 2 A and Table 1). Each target examined expressed survivin, as determined by RT-PCR and Western blot (data not shown). T cells from the same patients that were stimulated with CD40-B loaded with GFP mRNA failed to lyse tumor targets (Fig 2C and Table 1). Normal donor HLA-A2+ T cells stimulated with CD40-B loaded with full-length survivin mRNA (n = 6) also failed to lyse NBL tumors, even if HLA matched (Table 1).
Survivin specificity of patient T cells was further examined by measuring CD107a mobilization in vitro in response to incubation with tumor. CD107a is mobilized from vesicles to the cell surface during degranulation and T-cell–mediated cytolysis.40,41 For each of five patients tested, T cells stimulated with CD40-B loaded with survivin mRNA efficiently mobilized CD107a during incubation with HLA-matched allogeneic NBL cells but not during incubation with MHC class I–negative allogeneic tumor cells or media alone (Table 1). For one patient (patient A), T cells stimulated with CD40-B loaded with survivin mRNA efficiently mobilized CD107a during incubation with autologous NBL tumor cells but not during incubation with MHC class I–negative allogeneic tumor cells or media alone (Fig 3A). We then combined CD107a measurements with peptide/MHC tetramer analysis and found, for two HLA-A2+ patients tested, that the vast majority of CD107a+ T cells simultaneously labeled with tetramers specific for the Sur1M2 epitope (shown for patient A in Fig 3A). In contrast, less than 1% of CD107a-negative T cells labeled with Sur1M2 tetramer (Fig 3A).
We then used CD40-activated B cells electroporated with total RNA from NBL tumor cells, rather than survivin mRNA, to stimulate patient T cells to determine whether survivin expression in NBL is able to stimulate survivin-specific CTLs when cross presented on antigen-presenting cells (APCs). Each NBL tumor or tumor cell line from which total tumor RNA was generated expressed survivin by Western blot and RT-PCR (data not shown). We found that, in six (67%) of nine patients, T cells stimulated with CD40-B loaded with autologous total NBL RNA (n = 1) or allogeneic total NBL RNA (n = 5) lysed autologous NBL tumor cells or HLA-matched allogeneic NBL cells but did not lyse HLA-mismatched or MHC class I–negative allogeneic NBL cells (Fig 2B and data not shown). Lysis was dependent on interaction with MHC class I because neutralizing antibody to MHC class I blocked the effect by more than 50% (Fig 2D). Patient T cells stimulated with CD40-B loaded with autologous tumor mRNA efficiently mobilized CD107a after incubation with autologous tumor but not with MHC class I–negative allogeneic tumor cells or media alone (Fig 3B). The majority of CD107a+ T cells in these experiments labeled with Sur1M2 tetramer, whereas less than 1% of CD107a– T cells labeled with Sur1M2 tetramer (Fig 3B). Similarly, in another patient, T cells stimulated with allogeneic total NBL RNA mobilized CD107a after incubation with HLA-matched allogeneic tumor cells but not MHC class I–negative tumor cells or media alone (data not shown).
For patient T-cell cultures stimulated with CD40-B electroporated with either survivin mRNA or total tumor RNA, interferon gamma (IFN-
Survivin-Specific CTLs Are Found at Diagnosis in HR-NBL Patients Given the universal overexpression of survivin in HR-NBL tumors and an immunodominance of the Sur1M2 survivin epitope in HLA-A2+ patients with the disease, we then used tetramers and ELISPOT analysis to determine whether Sur1M2-specific CD8+ T cells were detectable in unstimulated PBMCs from our cohort of HR-NBL patients, using samples obtained at diagnosis. In eight (89%) of nine HLA-A2+ HR-NBL patients in our cohort, Sur1M2-specific CD8+ T cells were readily identified in patient PBMCs (Fig 5 and Table 2); however, Sur1M2 tetramer staining was minimal in HLA-A2+ normal donors (n = 8), HLA-A2– normal donors (n = 5), and HLA-A2– NBL patients (n = 9; Fig 5 and Table 2). There were significantly more Sur1M2-specific CD8+ T cells in NBL HLA-A2+ patients than in HLA-A2– NBL patients or HLA-A2+ or HLA-A2– normal donors (P < .05 for each comparison; Fig 5B).
In addition, in six (67%) of nine HLA-A2+ HR-NBL patients in our cohort, T cells capable of secreting IFN-  in response to Sur1M2 peptide were readily detected by ELISPOT analysis among fresh PBMCs, whereas minimal reactivity was seen with PBMCs alone or in response to negative control peptide (Table 2). In contrast, IFN- secretion to Sur1M2 peptide was minimal in unmanipulated peripheral blood from HLA-A2+ normal donors (n = 8), HLA-A2– normal donors (n = 2), and HLA-A2– NBL patients (n = 9; Table 2 and data not shown).
Lymphocytic Infiltration of HR-NBL Tumors at Diagnosis
Appropriately processed material from diagnostic biopsies was not available for tetramer or ELISPOT analysis, given the multiple scientific and clinical priorities of the national HR-NBL biology protocol. However, for two HLA-A2+ patients with HR-NBL in our cohort, single cell suspensions were prepared from tumor samples obtained at the time of definitive surgical resection. In each case, CD45+ CD3+ T cells were identified by flow cytometry as a rare subpopulation of cells (< 5% of all viable cells), but Sur1M2-specific CD8+ T cells were not detected (data not shown).
In this study, we evaluated the physiology of tumor immunosurveillance in children with NBL. We found that universal overexpression of the antiapoptosis protein survivin in HR-NBL tumors is associated in most patients with survivin-specific CD8+ T cells that are readily identified in peripheral blood at diagnosis but not in normal individuals. Moreover, in HLA-A2+ patients with HR-NBL, the vast majority of this response was devoted to the Sur1M2 epitope of survivin. By several experimental measurements, survivin-specific CD8+ T cells were fully functional in vitro. However, it seems unlikely from our histopathologic findings that survivin-specific CTLs mediate productive immunosurveillance in vivo, at least not in patients presenting with clinically evident disease. In every sample, T cells only rarely infiltrated tumor nests, despite a lymphoid reaction in the surrounding stroma. Thus, there seems to be a discrepancy between functional, tumor-specific T cells in the periphery and minimal lymphocyte activation in the tumor nest. In contrast to tumor-specific T cells in melanoma42 or in certain animal models,11 circulating survivin-specific T cells in HR-NBL have not been selectively rendered anergic in vivo. Multiple mechanisms may dictate the lack of T-cell infiltration into HR-NBL lesions. One possibility is weak or absent MHC expression by NBL tumor cells that results in suboptimal presentation of tumor antigen to specific T cells and impaired intratumoral T-cell activation. Using immunohistochemistry for MHC class I expression, we found that NBL tumor cells in all samples we studied were negative for MHC class I (unpublished results), which is consistent with findings from previous studies.22-24 In contrast, the majority of cell lines established from HR-NBL are MHC class I positive (unpublished results), potentially suggesting their origin from an MHC class I–positive clone. However, the selective pressures influencing the outgrowth of NBL tumor cells in culture are unknown.43 It remains possible that downregulation of MHC class I expression on NBL in vivo represents, in part, the consequence of immunoediting of originally MHC class I–positive NBL tumor cells in the face of tumor-specific CTLs.2 Other tumor-derived factors may also play a role in the inhibition of T-cell immunosurveillance in HR-NBL, including, for example, inhibitory cytokines or chemotactic factors. In addition, death ligand expression by NBL may lead to premature death of infiltrating T cells. Each of these mechanisms would lead to undetectable T cells by immunohistochemistry of clinically apparent tumors.44 The presence of survivin-specific T cells in patients with HR-NBL suggests that priming to survivin in vivo occurs via a process of antigen cross presentation.45,46 During this process, dendritic cells and other professional APCs take up apoptotic or necrotic tumor material, degrade tumor-derived proteins into short peptides via the proteasome, and ultimately present peptide-MHC complexes on the surface of the cells.47 NBL cells themselves are unlikely to act as APCs because, in vivo, these tumor cells do not seem to express MHC class I, a necessary contributor for the critical signal 1 for CTL activation. NBL cells also do not express costimulatory molecules such as CD80 or CD86, which are required for signal 2 for T-cell activation (unpublished results). At the surface of a dendritic cell, however, engagement of a specific T-cell receptor by peptide-MHC complexes activates CTL.48 In adults with cancer, tumor antigen cross presentation may be limited because of dysfunction or deficiency of patient APCs.49-52 In children with HR-NBL, our evidence for survivin-specific T-cell immune responses implies preserved function of APCs. It remains to be studied whether survivin-specific T-cell immunity is a feature of low-risk NBL. These findings suggest new opportunities for immunotherapy in NBL. Although vaccines for children with cancer remain under investigation,53,54 vaccine efforts in general are aimed at priming CTL responses, which in HR-NBL may already be ongoing and robust. Our data suggest that the development of strategies to modulate negative immune checkpoints or upregulate tumor MHC expression would be important in HR-NBL. A clinical trial testing adoptive transfer of activated lymphocytes after stem-cell transplantation is underway at our institution.55
The authors indicated no potential conflicts of interest.
We thank Carl June, MD, Garrett Brodeur, MD, James Riley, PhD, and Soldano Ferrone, MD, PhD, for their helpful discussions.
Supported by National Cancer Institute Grants No. R21-CA110516 (S.A.G.) and R01 CA113783 (R.G.C.), and grants from Alex's Lemonade Stand (J.M.M.), the Damon Runyon Cancer Research Fund (R.H.V.), the Alliance for Cancer Gene Therapy (R.H.V.), and the Pennsylvania Department of Health (R.H.V.). The Pennsylvania Department of Health specifically disclaims responsibility for any analysis, interpretations, or conclusions. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
