Immunosurveillance and Survivin-Specific T-Cell Immunity ... - CiteSeerX

VOLUME 24 䡠 NUMBER 36 䡠 DECEMBER 20 2006

JOURNAL OF CLINICAL ONCOLOGY

O R I G I N A L

R E P O R T

Immunosurveillance and Survivin-Specific T-Cell Immunity in Children With High-Risk Neuroblastoma Christina M. Coughlin, Mark D. Fleming, Richard G. Carroll, Bruce R. Pawel, Michael D. Hogarty, Xiaochuan Shan, Barbara A. Vance, Jarish N. Cohen, Sonya Jairaj, Elaina M. Lord, Michael H. Wexler, Gwenn-ae¨l H. Danet-Desnoyers, Jack L. Pinkus, Geraldine S. Pinkus, John M. Maris, Stephan A. Grupp, and Robert H. Vonderheide From the Abramson Family Cancer Research Institute; HematologyOncology 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. Submitted December 14, 2005; accepted October 5, 2006. 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. 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: [email protected]. © 2006 by American Society of Clinical Oncology 0732-183X/06/2436-5725/$20.00 DOI: 10.1200/JCO.2005.05.3314

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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. J Clin Oncol 24:5725-5734. © 2006 by American Society of Clinical Oncology

INTRODUCTION

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 lifethreatening 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 5725

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Coughlin et al

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 survivinspecific 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. PATIENTS AND METHODS 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 Tissues were processed, sectioned, and stained in laboratories accredited by the Joint Commission on Accreditation of Healthcare Organizations. Immunoperoxidase studies were performed using paraffin sections incubated with antibodies to survivin (1:100; R&D Systems, Minneapolis, MN), CD3 (1:400; DakoCytomation, Carpinteria, CA), CD4 (1:200, 4B12; Novocastra, Newcastle upon Tyne, United Kingdom), CD8 (1:100; C8/144B), CD20 (1: 500; L26), and perforin (1:100; DakoCytomation). For each antibody, standard quality control procedures were undertaken to optimize antigen retrieval, primary antibody dilution, secondary antibody detection, and other factors for both signal and noise. Specificity of the survivin antibody was demonstrated by specific recognition of human recombinant survivin in direct enzyme-linked immunosorbent assays and Western blots. For immunochemistry of lymphoid antigens, positive control staining was performed using sections from a standard lymph node in each case. As few as one positively labeled T cell can be detected per section under these and other highly optimized conditions.38 Plasmids and Vectors The in vitro transcription template vector, pVA67D, was generated using the pGEM-TE polymerase chain reaction (PCR) cloning plasmid (Promega, Madison, WI) by subcloning an oligonucleotide containing 67 adenine residues and 3⬘ internal NheI and HindIII sites immediately after the stretch of adenines into the SalI and NsiI sites of a circularized pGEM-TE. To eliminate upstream translation initiation from 5⬘-ATG sites, the plasmid containing the poly A tail was digested with ApaI and EcoRI, filled in with DNA polymerase Klenow fragment, and ligated to form pVA67D. cDNAs for survivin and eGFP genes were amplified by reverse transcriptase (RT) -PCR and cloned into the

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Fig 1. Survivin expression in high-risk neuroblastoma (HR-NBL). Paraffin sections of HR-NBL tumors at diagnosis were stained with antisurvivin antibody. Representative data from two patients among 26 analyzed are shown. (A) Patient A had a stroma-rich tumor, and (B) patient B had a stroma-poor tumor. Boxes indicate areas of survivin-positive tumor nests. Stromal areas (ⴱ) are survivin negative.



Survivin-Specific T-Cell Immunity in HR-NBL

EcoRI and/or SacI sites of pVA67D to generate antigen-specific in vitro transcription templates.

t test. Chromium release assays were performed as described.37 Standard deviation was less than 5% for each experimental point.

RNA Preparation mRNA was prepared from template plasmids by in vitro transcription using the T7 RNA polymerase promoter in a reaction containing an m7guanine cap (mMessage mMachine kit; Ambion, Austin, TX), treated with DNaseI to remove plasmid sequences, and purified by acidic phenol:chloroform extraction and RNeasy column separation (Qiagen, Valencia, CA). Total cellular RNA was prepared from cell lines using RNeasy columns (Qiagen). RNA samples from tumor resection specimens were prepared using either beta-mercaptoethanol or guanidine lysis and column purification.

CD107a Mobilization Assay Effector CTL lines in 100 ␮L of media (107 cells/mL) were mixed with 100 ␮L of tumor cells at an effector-to-target ratio of 2:1, with 2 ␮L of anti-CD107a antibody (Research Diagnostics, Concord, MA) and 20 nmol/L monensin (Sigma, St Louis, MO). After 5 hours at 37°C, cells were stained with monoclonal antibody and tetramers.

Establishment of T-Cell Cultures Using RNA-Electroporated CD40-Activated B Cells CD40-activated B cell lines (CD40-B) were established, and RNA electroporation was performed as described.37 Survivin and eGFP mRNA was used at 2 to 5 ␮g/sample, and tumor RNA was used at 10 ␮g/sample initially and then at 2 ␮g/sample for restimulations. Tetramer, Enzyme-Linked Immunospot Assay, and Chromium Release Analysis Peptide/MHC tetramer for the HLA-A2–restricted Sur1M2 epitope of survivin (LMLGEFLKL)28 and negative control HLA-A2 tetramer were purchased from Immunomics (San Diego, CA), and analysis was performed as previously described.37 Enzyme-linked immunospot (ELISPOT) assays were performed as described37 using peptide-loaded T2 cells as stimulators at a responder-to-stimulator ratio of 2:1. Results are shown as the mean of triplicates ⫾ 1 standard deviation, and statistics were performed using the unpaired

RT-PCR and Western Blot for Survivin RT-PCR was performed as described.26 For Western blotting, lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and survivin expression was analyzed using a survivin-specific polyclonal antibody (Novus Biologicals, Littleton, CO).

RESULTS

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

Table 1. Immune Function of HLA-A2⫹ Patient and Normal Donor T Cells Stimulated With CD40-B Cells Loaded With Survivin Versus GFP mRNA CD107 Mobilization (%)§

Cytotoxicity (%)‡ T Cells Stimulated With Survivin mRNA–Loaded CD40-B Cells Donor Patient A B C D E F G H I Normal donor 1 2 3 4 5 6

T Cells Stimulated With GFP mRNA–Loaded CD40-B Cells

T Cells Stimulated With Survivin mRNA– Loaded CD40-B Cells

Age

Stageⴱ

N-myc†

SK-NRA

SK-NAS

SH-SY5Y

SK-NRA

SK-NAS

SH-SY5Y

SK-NRA

SH-SY5Y

20 months 3 years 21 months 14 years 9 months 5 years 3 years 2 years 2 years

4 4 4 4 4s 4 4 4 4

Amp Amp Amp Non-Amp Amp ND Non-amp Amp Amp

42.3 28.2 21.3 10.7 21.3 37.5 25.5 17.4 18.3

9.0 13.2 4.4 6.9 4.4 2.7 0.4 11.8 9.7

8.9 2.6 5.5 14.6 5.5 3.3 5.5 2.7 0.5

10.3 3.2 8.5 4.0 8.5 12.1 1.2 3.2 1.4

5.9 9.9 0.2 2.6 4.2 2.7 0.0 10.2 2.1

5.1 4.0 4.2 7.7 0.2 3.0 0.0 4.0 1.9

3.4 2.4 2.2 ND ND ND 1.3 ND 1.9

0.6 0.5 0.8 ND ND ND 0.5 ND 0.6

6.1 3.0 6.1 8.0 0.2 11.1

6.0 3.9 6.0 10.5 0.5 16.5

3.9 3.6 3.9 4.5 0.9 10.6

5.5 7.0 5.5 7.3 0.9 4.1

4.1 11.8 4.1 8.3 6.7 7.8

4.0 10.4 4.0 2.1 1.3 6.2

Abbreviations: CD40-B, CD40-activated B cells; Amp, amplified; ND, not determined; NBL, neuroblastoma; MHC, major histocompatibility complex. ⴱ Staging was performed according to the International Neuroblastoma Staging System.15 Stage 4s NBL is localized primary disease in infants, with metastases limited to skin, liver, and/or bone marrow (⬍ 10% of cells). †Amplification status of the N-myc gene is crucial in risk stratification of patients with NBL. ‡Patient and normal donor T cells were stimulated with CD40-B cells loaded with survivin mRNA or GFP mRNA and tested for cytotoxicity in chromium release assays against three NBL cell lines. SK-NRA cells are HLA-A2⫹; SK-NAS cells are HLA mismatched but MHC class I positive; and SH-SY5Y are MHC class I negative. Percent specific lysis at effector-to-target ratios of 30:1 is shown. Criterion for positivity was specific lysis of SK-NRA ⬎ 20%. Standard deviation was ⬍ 5%. §Patient T cells were stimulated with CD40-B cells loaded with survivin mRNA and tested for CD107a mobilization when incubated with two NBL lines. The percentage of CD107a⫹ cells among CD3⫹ T cells is shown. Criterion for positivity was ⬎ 1% CD107a⫹ CD3⫹ T cells.

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A

30:1

3:1

B

20

3:1

10:1

20

3:1

C

10:1

15

3:1

10:1

30:1

E:T Ratio

15

3:1

10:1

10:1

30:1

E:T Ratio

15

30:1

E:T Ratio

3:1

10:1

30:1

E:T Ratio

Specific Lysis (%)

60

Targets (all survivin-positive) NSJ3 autologous tumor cells SK-NRA (HLA-A2+) SK-NAS (HLA-A2-neg, MHC class I-pos) 20

SH-SY5Y (MHC class I-neg) NSJ3 + anti-MHC class I mAb 3:1

10:1

30:1

E:T Ratio Fig 2. Survivin as a tumor-associated antigen in high-risk neuroblastoma (HR-NBL). HLA-A2⫹ peripheral-blood mononuclear cells from HR-NBL patients were stimulated with CD40-activated B cells (CD40-B) loaded with (A) survivin, (B and D) NBL tumor (autologous tumor RNA for patient A and allogeneic tumor RNA for patients B and C), or (C) GFP mRNA. Chromium release cytotoxicity assays are shown. (left column) Patient A; (middle column) patient B; (right column) patient C. E:T ratio, effector-totarget ratio.

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 HRNBL 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 5728

SH-SY5Y 0.1%

CD107+

2.7%

0.1%

CD107-

Fig 3. CD107a mobilization assays are shown, in which cells were gated on CD3⫹ CD4⫺ CD14⫺ cells, and the percentage of CD3⫹ CD107a⫹ cells is indicated. Cells were then gated on CD3⫹ CD107a⫹ versus CD3⫹ CD107a⫺ cells, and the percentage of Sur1M2 tetramer-positive cells is indicated. (A) CD40-activated B cells (CD40-B) loaded with survivin mRNA. (B) CD40-B loaded with autologous neuroblastoma mRNA. E:T ratio, effector-to-target ratio.

30

D

40

0.1%

NSJ3 (autologous)

CD3 E:T 2:1

45

30

CD107-

59%

20

3:1

Specific Lysis (%)

30

40

30:1

45

Specific Lysis (%)

45

B media

E:T Ratio

E:T Ratio

0.1%

30:1

60

40

30:1

10:1

E:T Ratio

Specific Lysis (%)

40

CD107a 3:1

60

Specific Lysis (%)

Specific Lysis (%)

30:1

2.2%

CD3 E:T 2:1

15

E:T Ratio

60

Specific Lysis (%)

10:1

0.1%

CD107+

survivin tetramer

10:1

E:T Ratio

0.1%

87%

NSJ3 (autologous)

SH-SY5Y

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 2A 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

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B 400 P = .03

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300 200

*

100 0 T Cells only

Tax

Sur1M2

Spots Per 50,000 Cells

3:1

15

30

CD107a

15

30

Spots Per 50,000 Cells

30

media

45

Specific Lysis (%)

45

Specific Lysis (%)

Specific Lysis (%)

45

survivin tetramer

A

* 400 P = .02 300 200

*

100 0 T Cells only

Tax

Sur1M2

Fig 4. Interferon gamma enzyme-linked immunospot assays are shown, in which T2 cells pulsed with HTLV-1 tax peptide (LLFGYPVYV)39 or Sur1M2 peptide were used as stimulators. (A) T cells stimulated with CD40-activated B cells (CD40-B) loaded with survivin mRNA. (B) T cells stimulated with CD40-B loaded with autologous neuroblastoma mRNA. Bars represent 1 standard deviation. JOURNAL OF CLINICAL ONCOLOGY



A

Negative Tetramer

Sur1M2 Tetramer

HLA-A2+ patient A

HLA-A2+ patient B

HLA-A2+ patient C

HLA-A2- patient

0.40%

0.42%

0.64%

0.03%

0.03%

0.05%

0.02%

0.02%

Fig 5. Survivin-specific cytotoxic T lymphocytes identified in unmanipulated peripheral T cells. (A) Labeling of CD8⫹ T cells from four patients using survivin Sur1M2 tetramer versus negative control. (B) Summary results for Sur1M2 tetramer analysis from HLA-A2– and HLA-A2⫹ normal donors and high-risk neuroblastoma (HR-NBL) patients. Bars represent mean percentage; positivity is defined as more than 0.1%. Statistical comparisons based on unpaired t test.

CD8

B

P = .001 P = .001 P = .001

CD8+ Sur1M2 Tetramer+ (%)

1.0

0.1

0.01 A2-

A2+

Normal Donors

A2-

A2+

HR-NBL Patients

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 antigenpresenting 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 5729

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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-␥) ELISPOT analysis demonstrated strong reactivity to Sur1M2 peptide (shown for patient A in Fig 4; similar data for two other patients tested not shown). As negative controls, IFN-␥ secretion by T cells alone or T-cell cultures challenged with a negative control peptide was minimal. HLA-A2⫹ normal donor T cells stimulated with CD40-B loaded with survivin mRNA (n ⫽ 6) failed to secrete IFN-␥ alone or in response to Sur1M2 or negative control peptide (data not shown). 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 HLAA2⫹ 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 We then evaluated lymphocytes associated with NBL tumor in vivo by performing immunohistochemistry for lymphoid antigen expression on diagnostic tumor biopsies. We studied 26 patients presenting with HR-NBL, including patients whose peripheral-blood lymphocytes were examined in vitro for survivin-specific T-cell reactivity. None of the patients had been diagnosed with opsoclonusmyoclonus-ataxia syndrome, which is associated with unique lymphoid features.21 CD8⫹ and CD4⫹ T-cell infiltration into NBL tumor nests was minimal or undetectable in each patient (Fig 6). CD20⫹ B cells and CD56⫹ natural killer cells within the tumor were also rare or undetectable (data not shown). Nevertheless, in most patients (23 of 26 patients; 88%), lymphocytes were evident in the peritumoral stroma, particularly in the fibrovascular bundles at the

Table 2. Survivin Specificity and Function of Freshly Isolated T Cells From HLA-A2⫹ Patients and Normal Donors IFN-g Secretion by ELISPOT† Tetramer Analysis (%) Donor Patient A B C D E F G H I Normal donor 1 2 3 4 5 6 7 8

ⴱ

Sur1M2 (No.)

Tax (No.)

Sur1M2

Negative

Mean

SD

Mean

SD

P

0.40 0.42 0.64 0.07 0.46 0.67 0.43 0.42 0.48

0.03 0.05 0.02 0.02 0.04 0.03 0.02 0.05 0.02

57 153 121 25 40 185 138 78 98

8 34 72 35 7 41 41 14 21

9 32 26 18 28 35 74 39 78

5 10 12 18 16 12 23 33 15

.04 .03 .04 .90 .04 .04 .02 .09 .10

0.05 0.04 0.04 0.06 0.05 0.10 0.06 0.09

0.02 0.04 0.02 0.04 0.04 0.04 0.04 0.04

16 21 18 15 4 14 37 29

6 6 4 4 4 6 11 16

18 20 20 15 5 4 42 14

4 5 2 5 3 2 18 6

.52 .73 .39 .95 .46 .01 .59 .07

Abbreviations: IFN-g, interferon gamma; ELISPOT, enzyme-linked immunospot assay; SD, standard deviation; PBMC, peripheral blood mononuclear cell. ⴱ Results are shown as the percentage of tetramer-positive cells among CD8⫹ T cells in fresh PBMCs. Criteria for positivity were Sur1M2 tetramer ⬎ 0.10% and negative tetramer ⱕ 0.05%. †For IFN-g ELISPOT, peptide-loaded T2 cells were used as stimulators. Unstimulated, fresh PBMCs were used as responders. Results are shown as mean number of spots per 2 million input PBMCs ⫾ 1 SD. Criteria for positivity were ⬎ 30 Sur1M2 spots and P ⬍ .05 compared with tax peptide negative control.

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A

B

CD8

CD4

C

D

CD8

CD4

E

F

CD8

CD4

Fig 6. Rare tumor-infiltrating lymphocyte populations in high-risk neuroblastoma tumors at diagnosis. Immunohistochemical results are shown for anti-CD8 (A, C, E), anti-CD4 (B, D, F), and antiperforin (G, corresponding to boxed area in E). (A and B) Patient A; (C and D) patient B; and (E, F, and G) patient J. Red arrows indicate peritumoral T cells; black arrows indicate intratumoral T cells. Magnification is ⫻20, except ⫻100 for inset. Similar results were obtained for 26 patients.

G

Perforin

edges of tumor nests (Figs 6E and 6F). These lymphocytes were largely CD3⫹ T cells, including both CD4⫹ and CD8⫹ cells of which the majority of the latter expressed perforin (Fig 6G). In a few samples, organized lymphoid tissue and B-cell follicles were observed at the edges of tumor nests, but even in these cases, intratumoral lymphocytes were minimal.

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, 5731

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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). DISCUSSION

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 REFERENCES 1. Burnet F: Cancer: A biological approach. BMJ 1:841-847, 1957 2. Dunn GP, Old LJ, Schreiber RD: The immunobiology of cancer immunosurveillance and immunoediting. Immunity 21:137-148, 2004 3. Boon T, van Baren N: Immunosurveillance against cancer and immunotherapy: Synergy or antagonism? N Engl J Med 348:252-254, 2003 4. Clemente CG, Mihm MC Jr, Bufalino R, et al: Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer 77:1303-1310, 1996 5. Naito Y, Saito K, Shiiba K, et al: CD8⫹ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. Cancer Res 58: 3491-3494, 1998 6. Schumacher K, Haensch W, Roefzaad C, et al: Prognostic significance of activated CD8(⫹) T cell infiltrations within esophageal carcinomas. Cancer Res 61:3932-3936, 2001 5732

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 HRNBL, 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

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32. Ambrosini G, Adida C, Altieri DC: A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 3:917-921, 1997 33. Velculescu VE, Madden SL, Zhang L, et al: Analysis of human transcriptomes. Nat Genet 23: 387-388, 1999 34. Zeis M, Siegel S, Wagner A, et al: Generation of cytotoxic responses in mice and human individuals against hematological malignancies using survivin-RNA-transfected dendritic cells. J Immunol 170:5391-5397, 2003 35. Siegel S, Wagner A, Schmitz N, et al: Induction of antitumour immunity using survivin peptidepulsed dendritic cells in a murine lymphoma model. Br J Haematol 122:911-914, 2003 36. Xiang R, Mizutani N, Luo Y, et al: A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res 65:553-561, 2005 37. Coughlin CM, Vance BA, Grupp SA, et al: RNA-transfected CD40-activated B cells induce functional T-cell responses against viral and tumor antigen targets: Implications for pediatric immunotherapy. Blood 103:2046-2054, 2004 38. Cattoretti G: Standardization and reproducibility in diagnostic immunohistochemistry. Hum Pathol 25:1107-1109, 1994 39. Kannagi M, Shida H, Igarashi H, et al: Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells. J Virol 66:2928-2933, 1992 40. Betts MR, Brenchley JM, Price DA, et al: Sensitive and viable identification of antigen-specific CD8⫹ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281:65-78, 2003 41. Rubio V, Stuge TB, Singh N, et al: Ex vivo identification, isolation and analysis of tumorcytolytic T cells. Nat Med 9:1377-1382, 2003 42. Lee PP, Yee C, Savage PA, et al: Characterization of circulating T cells specific for tumorassociated antigens in melanoma patients. Nat Med 5:677-685, 1999 43. Israel M, Thiele C: Tumor cell lines of the peripheral nervous system. Atlas Human Tumor Cell Lines 3:43-78, 1994

44. Drake CG, Jaffee E, Pardoll DM: Mechanisms of immune evasion by tumors. Adv Immunol 90:5181, 2006 45. Heath WR, Carbone FR: Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol 19:47-64, 2001 46. Rock KL: The ins and outs of crosspresentation. Nat Immunol 4:941-943, 2003 47. Thomas AM, Santarsiero LM, Lutz ER, et al: Mesothelin-specific CD8(⫹) T cell responses provide evidence of in vivo cross-priming by antigenpresenting cells in vaccinated pancreatic cancer patients. J Exp Med 200:297-306, 2004 48. Clark CE, Vonderheide RH: Getting to the surface: A link between tumor antigen discovery and natural presentation of peptide-MHC complexes. Clin Cancer Res 11:5333-5336, 2005 49. Almand B, Resser JR, Lindman B, et al: Clinical significance of defective dendritic cell differentiation in cancer. Clin Cancer Res 6:1755-1766, 2000 50. Mohty M, Jarrossay D, Lafage-Pochitaloff M, et al: Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment. Blood 98:3750-3756, 2001 51. Hoffmann TK, Muller-Berghaus J, Ferris RL, et al: Alterations in the frequency of dendritic cell subsets in the peripheral circulation of patients with squamous cell carcinomas of the head and neck. Clin Cancer Res 8:1787-1793, 2002 52. Orsini E, Guarini A, Chiaretti S, et al: The circulating dendritic cell compartment in patients with chronic lymphocytic leukemia is severely defective and unable to stimulate an effective T-cell response. Cancer Res 63:4497-4506, 2003 53. Rousseau RF, Hirschmann-Jax C, Takahashi S, et al: Cancer vaccines. Hematol Oncol Clin North Am 15:741-773, 2001 54. Mackall CL, Helman LJ: Targeting pediatric malignancies for T cell-mediated immune responses. Curr Oncol Rep 2:539-546, 2000 55. Laport GG, Levine BL, Stadtmauer EA, et al: Adoptive transfer of costimulated T cells induces lymphocytosis in patients with relapsed/refractory non-Hodgkin’s lymphoma following CD34-selected hematopoietic cell transplantation. Blood 102:20042013, 2003

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Acknowledgment We thank Carl June, MD, Garrett Brodeur, MD, James Riley, PhD, and Soldano Ferrone, MD, PhD, for their helpful discussions.

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Authors’ Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest.

Author Contributions Conception and design: Christina M. Coughlin, Mark D. Fleming, Richard G. Carroll, Stephan A. Grupp, Robert H. Vonderheide Financial support: Richard G. Carroll, John M. Maris, Stephan A. Grupp, Robert H. Vonderheide Provision of study materials or patients: Christina M. Coughlin, Mark D. Fleming, Richard G. Carroll, Bruce R. Pawel, Michael D. Hogarty, Xiaochuan Shan, John M. Maris, Stephan A. Grupp, Robert H. Vonderheide Collection and assembly of data: Christina M. Coughlin, Mark D. Fleming, Richard G. Carroll, Bruce R. Pawel, Michael D. Hogarty, Xiaochuan Shan, Barbara A. Vance, Jarish N. Cohen, Sonya Jairaj, Elaina M. Lord, Michael H. Wexler, Gwenn-ae¨l H. Danet-Desnoyers, Jack L. Pinkus, Geraldine S. Pinkus, Stephan A. Grupp, Robert H. Vonderheide Data analysis and interpretation: Christina M. Coughlin, Mark D. Fleming, Richard G. Carroll, Bruce R. Pawel, Michael D. Hogarty, Xiaochuan Shan, Barbara A. Vance, Jarish N. Cohen, Sonya Jairaj, Elaina M. Lord, Michael H. Wexler, Gwenn-ae¨l H. Danet-Desnoyers, Jack L. Pinkus, Geraldine S. Pinkus, John M. Maris, Stephan A. Grupp, Robert H. Vonderheide Manuscript writing: Christina M. Coughlin, Mark D. Fleming, Richard G. Carroll, Jack L. Pinkus, Geraldine S. Pinkus, Stephan A. Grupp, Robert H. Vonderheide Final approval of manuscript: Christina M. Coughlin, Mark D. Fleming, Richard G. Carroll, Bruce R. Pawel, Michael D. Hogarty, Xiaochuan Shan, Barbara A. Vance, Jarish N. Cohen, Sonya Jairaj, Elaina M. Lord, Michael H. Wexler, Gwenn-ae¨l H. Danet-Desnoyers, Jack L. Pinkus, Geraldine S. Pinkus, John M. Maris, Stephan A. Grupp, Robert H. Vonderheide

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