Immunity, Vol. 3, p. 19 (18 December 2003) Submitted:
19 November 2003. Accepted: 19 November 2003.
Contributed by: LJ Old
Frequency of NY-ESO-1 and LAGE-1 expression in bladder cancer and evidence of a new NY-ESO-1 T-cell epitope in a patient with bladder cancer
Sacha Gnjatic1, Achim A. Jungbluth1,
Barbara Williamson1, Harry Herr3,
Elisabeth Stockert1*, Guido Dalbagni3,
S. Machele Donat3, Victor E. Reuter4,
Darren Santiago1, Yao-Tseng Chen5,
Dean F. Bajorin2, Gerd Ritter1,
and Lloyd J. Old1
1Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY, USA 2Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 3Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 4Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 5Department of Pathology, Weill Medical College of Cornell University, New York, NY, USA *Deceased September 21, 2002
Cancer-testis (CT) antigens are ideal vaccine targets since their expression is restricted in adult tissues to testicular germ cells and a subset of cancers. The frequency of expression in transitional cell carcinomas (TCCs) of NY-ESO-1, the most immunogenic CT antigen to date, and its closely related gene LAGE-1 was studied. NY-ESO-1 and LAGE-1 antigen expression were found to occur frequently in high-grade TCC tumors. On an MSKCC IRB-approved protocol, 68 patient specimens were collected prospectively at the time of transurethral resection or cystectomy, of which 43 were read pathologically as high-grade tumors (pCIS, pTaG3, pT1, pT2, pT3, and pT4), 8 as low-grade tumors (pTaG1, pTaG2), and 17 as disease-free samples. These 68 samples were analyzed by immunohistochemistry (IHC) and/or RT-PCR. There were also an additional 53 paraffin-embedded specimens studied retrospectively by IHC, of which 39 were high-grade tumors and 14 were low-grade tumors. Cumulatively, our data indicate that NY-ESO-1 and/or LAGE-1 are expressed in 39/82 (48%) high-grade TCC and 3/22 (14%) low-grade TCC samples when analyzed by RT-PCR and/or IHC. Immunological assessment of these patients' sera identified one patient, whose tumor homogeneously expressed NY-ESO-1, which had detectable antibodies against NY-ESO-1 and LAGE-1. Further analysis of this patient, who remains clinically without evidence of disease 24 months after cystectomy for high-grade pT4 disease, revealed T-cell immunity against NY-ESO-1. This patient's T-cell response was determined to be specific for a new NY-ESO-1 epitope, p94-102, in the context of HLA-B35.
In North America, over 50,000 new cases of bladder cancer are reported every year. In the United States, bladder cancer ranks as the fourth most common malignancy among men and as the sixth among women. Approximately 90% of malignant tumors arising in the urinary bladder are of epithelial origin; the majority being transitional cell carcinomas (TCCs) (1). Early stage bladder tumors have been classified into two groups with distinct behavior: "superficial" or invasive (2). Clinically, "superficial" bladder tumors (stages Ta, Tis, and T1) account for 75 to 85% of neoplasms, while the remaining 15 to 25% are invasive of detrusor muscle or beyond (T2, T3, T4) or metastatic at the time of initial presentation. Standard of care in the United States for certain superficial bladder tumors (T1 or Tis) is surgical resection followed by intravesical administration of bacille Calmette-Guerin (BCG). Intravesical BCG as immunotherapy has been employed for the treatment of superficial TCCs for more than 20 years with improved local control and survival (3). For patients who recur with disease after appropriate BCG therapy or patients with invasive disease (into or beyond the muscularis propria of the bladder), the standard of care is radical cystectomy. Unfortunately, over 50% of these patients will recur after initial treatment with radical cystectomy, and the vast majority of these patients will succumb to their disease.
Clinical trials are currently underway to establish the role of adjuvant chemotherapy in the treatment of post-cystectomy patients in an attempt to decrease these high rates of recurrence. However, outside the context of a trial, the treating physician must decide, based on available data, whether or not to treat with adjuvant chemotherapy. At the Memorial Sloan-Kettering Cancer Center (MSKCC), post-cystectomy patients with extravesical or node-positive disease who have a satisfactory performance status, acceptable medical co-morbidities, and who can tolerate cisplatin-based chemotherapy, are offered conventional chemotherapy with four cycles of gemcitabine and cisplatin in the non-protocol setting. However, patients with organ-confined disease or who would be unable to tolerate cisplatin chemotherapy are offered surveillance. At this time, there is no immunotherapy program to treat post-cystectomy patients equivalent to that which exists for the treatment of superficial TCCs.
In an attempt to develop vaccine strategies to immunize TCC patients, we studied tissue samples from TCC patients for the expression of the most immunogenic cancer-testis (CT) antigen to date, NY-ESO-1, and its closely related gene, LAGE-1, by immunohistochemistry (IHC) and/or reverse-transcription polymerase chain reaction (RT-PCR). The NY-ESO-1 antigen was initially identified by serological analysis of recombinant cDNA expression libraries (SEREX) using tumor mRNA and autologous serum from a patient with squamous cell carcinoma of the esophagus. The full-length NY-ESO-1 cDNA was cloned and encodes a protein of 180 amino acids (4). The function of this protein is unknown. The gene encoding NY-ESO-1 has been mapped to chromosome Xq28 (5). A gene, LAGE-1, encoding an antigen closely related to NY-ESO-1 with 94% nucleotide and 87% amino acid homology, has also been mapped to chromosome Xq28 (6).
Since there is increasing evidence that CT antigen expression is correlated with tumors of higher malignant potential, and antibodies and cellular immune responses against NY-ESO-1 have been documented in multiple cancer patients who express the antigen in their tumors (7), we investigated TCC samples for the frequency of expression of NY-ESO-1 and LAGE-1. In a previous study, Kurashige et al. reported that high-grade TCCs frequently expressed NY-ESO-1 mRNA and antibodies against NY-ESO-1 were present in a small subset of these patients (8). Our studies confirm and expand upon these findings.
On an MSKCC IRB-approved protocol, archived pathology samples from previous surgeries or fresh pathology samples from transurethral resections (TURs) or cystectomies from 121 patients who gave informed consented were obtained. Of these 121 patients, 53 archived tumor samples were obtained retrospectively for IHC and 68 fresh patient samples were obtained prospectively for RT-PCR and/or IHC. Of the 53 archived tumor samples, 39 were read pathologically as high-grade tumors (pCIS, pTaG3, pT1, pT2, pT3, and pT4) and 14 as low-grade tumors (pTaG1, pTaG2). Of the 68 fresh tissue samples, 43 were read pathologically as high-grade tumors, 8 as low-grade tumors, and 17 as disease-free samples. In addition, all 121 patient sera were tested for NY-ESO-1 and LAGE-1 specific antibodies by ELISA. NY-ESO-1 and LAGE-1 antibody positive patients were then further HLA-typed and analyzed for NY-ESO-1 specific T-cells.
Our data indicate that NY-ESO-1 and/or LAGE-1 are expressed at a frequency of approximately 30-40% (by RT-PCR and/or IHC) in high-grade TCCs. Of 43 prospectively collected high-grade tumors, 15/43 (35%) were NY-ESO-1 positive by RT-PCR and 17 (40%) were LAGE-1 positive. Ten (23%) samples were positive for both NY-ESO-1 and LAGE-1, whereas 22 (50%) were positive for at least one of the two genes. Thirty-three of the 43 prospectively obtained high-grade tumors were also tested for NY-ESO-1 by IHC and six samples were positive for NY-ESO-1. Thirty-nine retrospectively obtained high-grade tumors were also tested for NY-ESO-1 expression by IHC, and 16 (41%) cases were positive. Overall, 22/72 (31%) high-grade TCC samples were positive for NY-ESO-1 expression by IHC. These data are summarized in Table 1 and Figure 1.
Summary of NY-ESO-1 and LAGE-1 expression in TCCs as determined by IHC and RT-PCR
Of the eight low-grade tumors obtained prospectively, only one tested positive for both NY-ESO-1 and LAGE-1 by RT-PCR. This patient continued to have recurrent low-grade disease with two documented recurrences in a period of ten months. Of the 14 low-grade tumors obtained retrospectively, two samples tested positive for NY-ESO-1 by IHC. One patient was diagnosed as pTaG1 and has been disease-free for 19 months following TUR. The second patient was diagnosed with pTaG2 disease and has had recurrent low-grade disease periodically over a 7-year period with repeated TUR treatments.
There were 3/17 disease-free samples which were found to be NY-ESO-1 and/or LAGE-1 positive by RT-PCR. One patient with LAGE-1 positive results from a cystectomy sample was noted to have had a TUR one month before documenting pT2 disease. A second patient with NY-ESO-1 positive results from a TUR sample continued to have malignant cells reported on urine cytology. A third patient with both NY-ESO-1 and LAGE-1 positive results from a TUR sample also continued to have malignant cells reported on urine cytology. This patient was then found to have invasive disease on repeat TUR 6 months later and pT3 disease on subsequent cystectomy. For the remaining 14 patients with disease-free and NY-ESO-1 or LAGE-1 negative TUR samples, the clinical histories over a one year follow-up period are as follows: 3 patients did not follow-up at MSKCC, 8 patients remained clinically without evidence of disease, 2 patients had malignant cells present on urine cytology, and 1 patient developed recurrent high-grade disease.
We attempted to determine if NY-ESO-1 or LAGE-1 antigen expression in high-grade tumors was a marker for disease recurrence by analyzing clinical information from 69 patients with initial diagnoses of high-grade TCC. Patients were categorized into two groups according to whether they were positive for at least one antigen or negative for both. Patient characteristics are listed in Table 2. Recurrence-free survival curves were estimated by the method of Kaplan-Meier (9) and compared using the log-rank test (10) (Figure 2). From this limited analysis, it appears that there is no difference noted between the patients who tested positive for at least one of the antigens and those who tested negative for both (P = 0.70). The data is summarized in Table 3.
Summary of recurrence-free survival (RFS) for 69 patients with high-grade TCC
All 121 patients had their sera tested for antibodies against NY-ESO-1 and LAGE-1. Sixty-six patients were pathologically without evidence of disease at the time of blood collection, eight patients had metastatic disease at the time of blood collection, and fifty-two patients had their blood collected at the time of TUR or cystectomy (as treatment for active disease). Of those patients who were treated surgically, 43 had high-grade disease and 8 had low-grade disease. Only one patient, who was clinically without evidence of disease at the time of blood collection due to radical cystectomy for pT4 disease 8 months before, was found to have antibodies against NY-ESO-1 and LAGE-1. We collected and tested this patient's serum at different time points, including 8 months post-cystectomy, 10 months post-cystectomy and 22 months post-cystectomy. At each time point, ELISA tests demonstrated high antibody titers against NY-ESO-1 (Figure 3) and LAGE-1 (data not shown). This patient's tumor was noted to be strongly positive for NY-ESO-1 with greater than 75% of the tumor cells staining for NY-ESO-1 by IHC (Figure 4). Upon further analysis, this patient was found to have T-cells reactive against a novel NY-ESO-1 epitope in the context of HLA-B35. This patient, who has an approximately 50% chance of relapse, continues to remain clinically disease-free, in the absence of chemotherapy treatment, 24 months after cystectomy.
Antibodies to NY-ESO-1 in patient UC-098 at various time points. Antibodies to NY-ESO-1 at 8 , 10 and 22 months after cystectomy are shown, as compared to a known positive control and negative control.
Expression of NY-ESO-1 in patient UC-098 with invasive transitional cell carcinoma. Homogeneous expression of NY-ESO-1 in urinary bladder was detected with monoclonal antibody ES121.
We established from earlier monitoring studies of cancer patients that there was a correlation between antibody and CD8+ T cell responses to NY-ESO-1 (11). To determine if seropositive patient UC-098 followed the same pattern, we presensitized his CD8+ T cells with NY-ESO-1 peptide 157-165, a known HLA-A2-restricted epitope. Surprisingly, although this patient was determined to be HLA-A2 positive, no specific response could be detected to this peptide. To determine whether patient UC-098 responded to other epitopes, we stimulated CD8+ T cells with a 30-mer peptide from NY-ESO-1, polypeptide 80-109. This polypeptide was previously shown to be processed by antigen presenting cells into multiple epitopes with various HLA restriction (12). A specific T cell response was observed in ELISPOT assays, indicating that an epitope within region 80-109 of NY-ESO-1 was processed and recognized (Figure 5A). To further map this epitope, 18-mer peptides with sequences spanning the central NY-ESO-1 region were used as targets of the CD8+ T cells. Two overlapping peptides, 85-102 and 91-108, were recognized, indicating that the epitope was included within region 91-102 (Figure 5A). Finally, shorter peptides were tested as potential targets, and peptides 93-101, 93-102 and 94-102 were the minimal sequences detectable by ELISPOT (Figure 5B).
Mapping of the epitope within region 80-109 of NY-ESO-1. ELISPOT assay with CD8+ T cells from patient UC-098 presensitized with NY-ESO-1 peptide 80-109 and tested against histocompatible B-EBV targets pulsed with peptides at 10 ÁM (A and B) or at various concentrations (C).
In titration assays, peptide 94-102 appeared to be the better epitope, since it was still recognized by CD8+ T cells at concentrations as low as 100 pM (Figure 5C). This epitope was recently described as restricted by HLA-B51 (13). However, since UC-098 did not express HLA-B51, we sought to define the restriction element used for the observed CD8+ T cell response by testing partially histocompatible targets for their capacity to present peptide 94-102 (Figure 6). Only HLA-B35 positive targets were recognized when pulsed with peptide 94-102, demonstrating a new restriction element for this epitope. Interestingly, one HLA-B51 positive target was also recognized when pulsed with 94-102, albeit to a lower extent, showing potential cross-reactivity between HLA-B35 and HLA-B51 for peptide presentation (Figure 6). Finally, we wanted to confirm that peptide 94-102 was naturally processed from full-length NY-ESO-1 in antigen-presenting cells. We found that CD8+ T cells specific for peptide 94-102 could recognize target cells infected with vaccinia virus recombinant for NY-ESO-1 (Figure 7A). We also tested HLA-B35 positive tumor cell lines SK-MEL-139, which expresses NY-ESO-1, and SK-MEL-106, which does not. Even though both tumor cell lines could present peptide 94-102 when pulsed exogenously, SK-MEL-139 was not spontaneously recognized by T cells in the absence of peptide (Figure 7B). More NY-ESO-1+ HLA-B35+ targets need to be tested to determine if the lack of recognition is due to a specific processing defect by SK-MEL-139.
Identification of the restriction element responsible for the observed T cell response. ELISPOT assay with CD8+ T cells from patient UC-098 presensitized with NY-ESO-1 peptide 80-109 and tested against partially histocompatible B-EBV targets pulsed or not with NY-ESO-1 peptide 94-102. The complete class I haplotype from UC-098 is indicated on top, matching target alleles are shown in bold and italic print.
NY-ESO-1 peptide 94-102 is naturally processed from the full-length protein in antigen-presenting cells. ELISPOT assay with CD8+ T cells from patient UC-098 presensitized with NY-ESO-1 peptide 80-109 and tested against histocompatible B-EBV targets pulsed with NY-ESO-1 peptides, or infected with vaccinia virus recombinant for NY-ESO-1 or wild-type (A); or tested against melanoma cell lines pulsed or not with NY-ESO-1 peptide 94-102 (B).
From our analyses, NY-ESO-1 and LAGE-1 are expressed more frequently by high-grade TCCs. We found expression of NY-ESO-1 and LAGE-1 to be approximately 30-40% in high-grade TCCs. This information correlates with previous studies documenting that these antigens are associated with more aggressive disease. These results also correlate with the previous report from Kurashige et al. (8) demonstrating expression of NY-ESO-1 mRNA in 44% of grade 3 TCCs. Our studies further recognized NY-ESO-1 positive and LAGE-1 positive patient samples which were pathologically read as low-grade disease or disease-free. These patients were then noted to have evidence of recurrent disease. It should also be pointed out that the urine cytologies also continued to demonstrate malignant cells in these patients who developed recurrent disease. Statistical analyses of our limited data set did not demonstrate a difference in recurrence-free survival for patients who were NY-ESO-1 or LAGE-1 antigen positive versus patients who were antigen negative. We plan to further explore the issue of whether NY-ESO-1 and/or LAGE-1 can be used, in addition to pathological diagnoses, as a predictive marker of disease in future prospective clinical trials.
In contrast to the study by Kurashige et al., we did not detect antibodies to NY-ESO-1 or LAGE-1 in any of our patients with high-grade TCCs, with the exception of UC-098. This difference in the frequency of seropositivity may be due to the bulk of disease present at the time of sera collection. Since MSKCC is primarily a referral center, the majority of our patients were treated with TURs prior to their initial visit in our clinic. Therefore, most of our patients had minimal disease at the time of serum collection. This residual disease may not be sufficient to maintain antibody titers high enough to be detected by ELISA. However, this does not explain the obvious antibody and T-cell immunity against NY-ESO-1 noted in our disease-free patient UC-098. In the paper by Kurashige et al., there is also an antibody positive patient (TCC44) who is noted to be disease-free.
This is now the second TCC patient to be reported to have long-term immunity to NY-ESO-1 associated with disease-free survival. This is in contrast to other tumor types where the presence of NY-ESO-1 antibody appears to be antigen driven and disappears with tumor removal or regression of disease secondary to therapy. It is clear that these discrepancies need to be investigated further.
Another point raised by our data relates to the fact that patient UC-098 had a CD8+ T cell response directed to NY-ESO-1 peptide 94-102 and restricted by HLA-B35, rather than against the major HLA-A2 peptide 157-165. It is not known whether the HLA-B35-restricted response is dominant over HLA-A2, or whether it is the only T cell activity detectable at this sampling time point. Interestingly, NY-ESO-1 peptide 94-102 was also shown to be presented by HLA-B51 (13). HLA-B51 and -B35 have very similar peptide binding motifs, and their cross-reaction was recently observed for an HIV-derived peptide (14). Similar super-type presentations were also observed for MAGE-A1 and MAGE-A3 peptides recognized by CD8+ clones in either HLA-A1 or HLA-B35 restriction (15, 16). In addition, the presence of tumor infiltrating lymphocytes to NY-ESO-1 restricted by HLA-B35 has been found in a melanoma patient (17).
The immunogenicity and expression pattern of NY-ESO-1 makes it an ideal candidate for vaccine therapy in TCC patients. Early TCC (pT1 or Tis) is already treated with immunotherapy consisting of BCG, which leads to a cure rate of approximately 90%. Previous randomized trials in superficial TCC (pT1 or pTis) have shown that tumor resection followed by intravesical BCG results in survival rates superior to surgery alone and surgery plus chemotherapy. In contrast, more advanced TCC (pT2 or greater) is predominantly treated with radical cystectomy alone with cure rates of approximately 50%. Perioperative chemotherapy may be beneficial but the degree of benefit is controversial because this elderly population is poorly tolerant of chemotherapy. New strategies to treat high-risk TCC are clearly warranted.
Based on our data and prior reports of peptide-vaccine generated immunity against NY-ESO-1, we developed a clinical trial whereby post-cystectomy patients with high-grade TCC, expressing NY-ESO-1 and/or LAGE-1, will be eligible for vaccination with the NY-ESO-1 protein. The full-length NY-ESO-1 protein allows us to enroll patients with any HLA type and will further enable us to define other NY-ESO-1 epitopes. Patients will also receive BCG and GM-CSF as immunological adjuvants. NY-ESO-1 specific antibodies and T-cells will be measured to document the immunity generated against NY-ESO-1.
BCG, bacille Calmette-Guerin; IHC, immunohistochemistry; MSKCC, Memorial Sloan-Kettering Cancer Center; TCC, transitional cell carcinoma; TUR, transurethral resection
The authors wish to thank Dexter David, Department of Medicine at MSKCC, for assistance with data management and Jennifer Bacik, Department of Epidemiology and Biostatistics at MSKCC, for assistance with statistical analyses. The authors also wish to express their gratitude to the late Dr. Elisabeth Stockert for her guidance and support.
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Patients and tissue samples
There were 126 patients, 28 females and 98 males. The patients ranged in age from 34 to 90, with a median age of 71. The pathology samples were collected from 26 cystectomies and 100 TURs. Tissue samples from sixty-nine patients with high-grade TCC were obtained either retrospectively or prospectively as part of an MSKCC IRB-approved tissue acquisition protocol. All patients gave informed consent.
Patients were categorized into two groups according to whether they were positive for at least one antigen or negative for both. Recurrence-free survival curves were estimated by the method of Kaplan-Meier and compared using the log-rank test.
5 µm cuts of standard formalin-fixed paraffin-embedded tissue specimens were applied to slides for immunohistochemistry (Fisherbrand Superfrost/Plus Slides) and heated at 60°C for 2 hours to ensure adherence. The slides were then deparaffinized in xylene and rehydrated in a series of graded alcohols. Immunohistochemical detection was carried out with monoclonal antibody ES121 to NY-ESO-1. Before IHC staining, heat-based antigen retrieval was performed by heating the slides in DAKO high pH solution (DAKO, Carpinteria, CA). ES121 was applied overnight at 4°C and then detected with the Envision Plus system (DAKO). Diaminobenzidine tetrahydrochloride (Biogenex, San Ramon, CA) was used as the chromogen. Counterstaining was done with a hematoxylin solution. Any type of immunoreactivity of the tumor cells was interpreted as positive.
mRNA was isolated using the TriReagent LS kit (Molecular Research Center, Cincinnati, OH). The mRNA preps were quantitated by measuring the OD at 260 nm. Reverse transcription was performed using 4 µg of mRNA and PCR was carried out using 200 ng of cDNA. Previously described gene-specific oligonucleotide primers for NY-ESO-1 (4) and LAGE-1 [5'-CTGCGCAGGATGGAAGGTGCCCC-3' (sense); 5'-GGCTTAGCGCCTCTGCCCTG-3' (antisense)] were used to perform RT-PCR consisting of 35 amplification cycles in a thermal cycler at an annealing temperature of 60°C. Samples were then analyzed by gel electrophoresis.
Recombinant NY-ESO-1 and LAGE-1 protein solution (100 µl/well) at a concentration of 1 µg/ml in coating buffer (15 mM Na2CO3, 30 mM NaHCO3, pH 9.6, with 0.02% NaN3) was added to TC microwell plates 60 X 10 (Nunc, Roskilde, Denmark) overnight at 4°C. The plates were then washed with PBS and incubated for 2 h at room temperature with 10 µl/well serum dilutions in 2% BSA . After washing, 10 µl/well diluted secondary antibody in 2% BSA (goat anti-human IgG-AP; Southern Biotechnology, Birmingham, AL) was added to the plates, which were then incubated for 1 h at room temperature. The plates were then washed, incubated with 10 µl/well substrate solution (Attophose substrate; JBL Scientific, San Louis Obispo, CA) for 25 min at room temperature, and immediately read (CytoFluor 2350; Millipore, Bedford, MA). Sera were then tested over a range of serial fourfold dilutions from 1:100 to 1:100,000. A positive reaction is defined as an OD value of a 1:400 diluted serum that exceeds the mean OD value of sera from normal donors (n=70) by three standard deviations (18).
Peptides and viral vectors
Synthetic NY-ESO-1 30-mer polypeptide 80-109 (ARGPESRLLEFYLAMPFATPMEAELARRSL), nonamer and decamer peptides included in 80-109, and peptide 157-165 (SLLMWITQC) were obtained from Bio-Synthesis (Lewisville, Texas), with a purity of >90% as determined by mass spectrometry. Wild-type vaccinia virus (v.v.WT) and vaccinia virus recombinant for full-length NY-ESO-1 (v.v.ESO) have been described previously (19).
In vitro sensitization of CD8+ T cells
CD8+ T lymphocytes from patient UC-98 were separated from peripheral blood lymphocytes (PBLs) by antibody-coated magnetic beads (Dynabeads; Dynal, Oslo, Norway) and seeded into round-bottomed 96-well plates (Corning, NY) at a concentration of 5x105 cells per well in RPMI medium 1640 supplemented with 10% human AB serum (NABI, Boca Raton, FL), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 1% nonessential amino acids. As antigen presenting cells (APCs), PBLs depleted of CD8+ T cells were pulsed with 10 µM peptide overnight at 37°C in 250 µl serum-free medium (X-VIVO-15, Bio-Whittaker). Pulsed APCs were then washed, irradiated and added to the plates containing CD8+ T cells at a concentration of 1x106 APCs per well. After 8 hours, IL-2 (10 U/ml, Roche Molecular Biochemicals, IN) and IL-7 (20 ng/ml, R&D systems, MN) were added to the culture wells, and this step was repeated every three to four days, until the cells were harvested for testing.
EBV-transformed B lymphocytes and melanoma cell lines SK-MEL-106 and SK-MEL-139 cultured in RPMI medium 1640 supplemented with 10% FCS (Summit Biotechnology), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 1% nonessential amino acids were used as target cells. For every cell type, HLA class I allele expression was determined by high-resolution DNA typing. For ELISPOT assays, target cells were pulsed overnight with 10 ÁM peptide or infected with 30 pfu/cell v.v.WT or v.v.ESO in 250 µl X-VIVO-15 (Bio-Whittaker).
For ELISPOT assays, flat-bottomed, 96-well nitrocellulose plates (MultiScreen®-HA; Millipore, Bedford, MA) were coated with IFN-gamma mAb (2 µg/ml, 1-D1K; Mabtech, Stockholm, Sweden) and incubated overnight at 4°C. After washing with RPMI, the plates were blocked with 10% human AB type serum for 2 h at 37°C. Presensitized CD8+ T cells (5x104 and 1x104) and 5x104 targets cells (peptide-pulsed or v.v.ESO infected EBV-B, or tumor cells) were added to each well and incubated for 20 h in RPMI medium 1640 without serum. The plates were then washed thoroughly with water containing 0.05% Tween 20 to remove cells, and IFN-gamma mAb (0.2 µg/ml, 7-B6-1-biotin; Mabtech) was added to each well. After incubation for 2 h at 37°C, the plates were washed and developed with streptavidin-alkaline phosphatase (1 µg/ml; Mabtech) for 1 h at room temperature. After washing, substrate (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; Sigma) was added and the plates incubated for 5 min. After final washes, plate membranes displayed dark-violet spots that were counted under the microscope.
Ludwig Institute for Cancer Research
New York Branch of Human Cancer Immunology at Memorial Sloan-Kettering Cancer Center
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