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*
Ludwig Institute for Cancer Research, Brussels Branch and Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium; and
Department of Urology, Cliniques Universitaires Saint Luc, Université Catholique de Louvain, Brussels, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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So far, four groups of human tumor Ags have been defined. The first group consists of tumor-specific Ags, such as those encoded by genes MAGE-1 (1, 2, 3), MAGE-3 (4, 5, 6), BAGE (7), GAGE (8), and RAGE (9). These genes are expressed in a variety of tumors but not in most normal tissues, in which they are expressed only in cells belonging to immune-privileged organs, such as the germ cells in the testis and some placenta cells, which appear to be devoid of MHC molecules. The second group is composed of differentiation Ags, such as tyrosinase (10, 11), Melan-A/MART-1 (12, 13), gp100 (14, 15), tyrosinase-related protein 1 (16), and tyrosinase-related protein 2 (17). These Ags are expressed in normal melanocytes and most melanomas. The third group comprises Ags encoded by mutated genes, such as MUM-1 (18), cyclin-dependent kinase 4 (19), ß-catenin (20), and HLA-A2 (21). These genes are expressed ubiquitously, and the Ag results from a tumor-specific mutation in the coding region. Finally, the fourth group consists of Ags such as HER2-neu that are present in normal tissues but are expressed in tumors at substantially higher levels (22).
In addition to melanoma and RCC, bladder tumors appear to be sensitive to immunologic control, as suggested by the remarkable results of the intravesical instillation of Bacillus of Calmette-Guérin (BCG) in superficial bladder carcinomas (23). The exact mechanism of the antitumor effect of BCG is unclear, but BCG locally induces a dramatic inflammatory reaction that may generate optimal conditions for the activation of tumor-specific T cells. Although lytic activities have been detected in populations of lymphocytes infiltrating bladder cancers, CTLs specifically directed against such tumors have not been characterized (24, 25).
We derived a cell line from a surgical specimen of transitional cell carcinoma of the bladder, and we used it to stimulate autologous blood lymphocytes. We observed lytic activity that was specifically directed against the autologous tumor cell line, but only after stimulation with autologous tumor cells transfected with the B7-1 cDNA. We cloned these T lymphocytes by limiting dilution and obtained a panel of tumor-specific CTL clones that recognized three different Ags. We report here the molecular characterization of one of these Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The bladder carcinoma cell line LB831-BLC was derived from the primary invasive bladder tumor (pT3, g3) of a 65-yr-old Caucasian patient, LB831 (HLA-A2403, -A3, -B4403, -B4901, -Cw0401, and -Cw07). The caryotype of the cell line performed at passage 7 showed that the number of chromosomes varied from 56 to 144, confirming that the LB831 cell line was a tumor line. LB905-BLC is a bladder carcinoma cell line derived from an HLA-Cw07 patient. BB64-RCC is a RCC cell line and MZ2-MEL is a melanoma cell line; both were derived from HLA-B4403 patients. The tumor cells were cultured in Iscove’s medium (Life Technologies, Gaithersburg, MD) containing 10% human serum or 10% FCS (Life Technologies) in an 8% CO2 incubator. The lymphoblastoid cell line LB831-EBV was derived from the PBLs of patient LB831 with 1 µg/ml of cyclosporin A (Sandoz, Basel, Switzerland) and 20% (v/v) of supernatant of EBV-transformed B95-8 cells using standard techniques. This cell line was grown in RPMI 1640 medium (Life Technologies) containing 10% FCS in a 5% CO2 incubator. LG2-EBV cells were grown in Iscove’s medium containing 10% FCS in an 8% CO2 incubator. PBL-PHA cells were prepared by stimulating PBLs with 0.1% (v/v) PHA (Difco, Detroit, MI) and 100 U/ml of IL-2 (Eurocetus, Amsterdam, The Netherlands) and cultured in an 8% CO2 incubator in Iscove’s medium containing 10% human serum. The class I-negative human B cell line, C1R (26), transfected with an HLA-B4403 cDNA (27, 28) was provided by K. Fleischhauer (Istituto Scientifico H.S. Rafaele, Milan, Italy) and grown in RPMI 1640 medium supplemented with 10% FCS in a 5% CO2 incubator. All media were supplemented with L-arginine (116 µg/ml), L-asparagine (36 µg/ml), L-glutamine (216 µg/ml), streptomycin (0.1 mg/ml), and penicillin (200 U/ml).
Antitumor CTL clones
The blood mononuclear cells of patient LB831 were isolated by
Lymphoprep (Nycomed, Oslo, Norway) density-gradient centrifugation and
stored at -80°C. An autologous mixed lymphocyte tumor cell culture
(MLTC) was performed as described previously (29) or using irradiated
B7-1-transfected LB831-BLC cells as stimulators and
CD8+ T lymphocytes as responders. CD8+ T
lymphocytes were sorted with magnetic beads that had been covalently
coupled to anti-CD8 Abs (magnetic-activated cell sorter, Miltenyi
Biotec, Bergisch-Gladbach, Germany). Irradiated non-CD8+
cells were added to the mixed culture during the first stimulation.
Stimulator cells were treated with IFN-
for 48 h before the
stimulation. On day 21, lymphocytes from the culture were cloned by
limiting dilution in Iscove’s medium supplemented with IL-2 (50 U/ml).
The long-term culture of CTL clones was conducted as described
previously (29).
Cytotoxicity assay
The lytic activity of CTLs was tested in a chromium release assay as described previously (30). Briefly, 1000 chromium-labeled cells in 100 µl were incubated in 96-well microplates with an equal volume of CTLs at different E:T ratios. Chromium release was measured after 4 h of incubation. For the peptide assay, labeled LG2-EBV cells were incubated for 30 min at 37°C with various concentrations of peptides. CTLs were subsequently added, and chromium release was measured as described above. For the competition assay, the C1R-B4403 cells were labeled in the presence of the human-specific class I HLA mAb W6/32 (anti-HLA class I (31)) at a 1/40 dilution of the hybridoma cell culture media. Cells were incubated for 30 min at room temperature with various concentrations of competitor peptides in X-vivo 10 medium (Whittaker Bioproducts, Walkersville, MD) followed by incubation under the same conditions with the tyrosinase peptide SEIWRDIDF. Cells were then washed and processed for the chromium assay with CTL 22/31 (32) as described above. Chromium release was measured after 2 h.
CTL stimulation assay
A total of 3000 CTLs were added to microwells containing stimulator cells in 100 µl of Iscove’s medium supplemented with 10% human serum and 50 U/ml of IL-2. The supernatant was collected after 18 h, and its TNF content was determined by testing the cytotoxic effect on WEHI-164 clone 13 cells (33) in a 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyl tetrazolium bromide colorimetric assay (34). Inhibition with W6/32, B1–23–2 (anti-HLA-B and -C), B9.4.1 (anti-CD8), 13B8.2 (anti-CD4, donated by D. Olive, Institut National de la Santé et de la Recherche Médicale U119, Marseille, France), GAPA-3 (anti-HLA-A3), and C7709A2.6 (anti-HLA-A24) mAbs was performed by adding a 1/30 dilution of ascites to the test.
Construction of the cDNA library
Total RNA (tRNA) was extracted from LB831-BLC cells using the guanidine isothiocyanate procedure (35). Poly(-A)+ RNA that had been enriched by an oligo(dT) cellulose column was converted to cDNA with the Superscript Choice System (Life Technologies) using an oligo(dT) primer containing an NotI site at its 5' end. The cDNA was ligated to HindIII adaptors (Stratagene, La Jolla, CA), phosphorylated, digested with NotI, and inserted into the HindIII and NotI sites of the expression vector pCEP4 (Invitrogen, San Diego, CA). Recombinant plasmids were electroporated into Escherichia coli Top10F' and selected with ampicillin (100 µg/ml). The library was divided into pools of 100 cDNA clones. Each pool of bacteria was amplified, and plasmid DNA was extracted using the QIAprep 8 plasmid kit (Qiagen, Hilden, Germany).
Cloning of the HLA cDNA from LB831-BLC
tRNA was extracted as described above from the LB831 tumor sample. Reverse transcription was performed on 2 µg of tRNA in a 20 µl reaction volume containing 4 µl of 5x reverse transcriptase buffer (Life Technologies), 1 µl of 10 mM deoxynucleoside triphosphate, 2 µl of a 20 µM solution of oligo(dT)15 primer, 20 U of RNasin (Promega, Madison, WI), 2 µl of 0.1 M DTT, and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies). The reaction was incubated at 42°C for 60 min. One-tenth of the cDNA product was subsequently supplemented with 10 µl of 10x polymerase buffer, 4 µl of 2.5 mM deoxynucleoside triphosphate, 2.5 µl of 20 µM primer solutions, 2.5 U of Pfu DNA polymerase (Stratagene), and water to a final volume of 100 µl. PCR amplification was performed using the forward primer 5'-ACTGGGCGGATCCGGACTCAGAATCTCCCCAGACGCCGAG and the reverse primer 5'-ACTGCCCGAATTCTCTCAGTCCCTCACAAGGCAGCTGTC that hybridize to the 5' and 3' untranslated regions of all HLA class I genes, respectively, and that contain a BamHI site (forward primer) or an EcoRI site (backward primer) (36). An initial denaturation step that was performed at 94°C for 4 min was followed by 35 cycles of amplification (1 min at 94°C, 5 s at 62°C, and 3 min at 75°C) and by a final elongation step at 75°C for 10 min. The PCR products were digested with EcoRI and BamHI, inserted into pcDNA3, and fully sequenced to verify their identity.
PCR assay for sequencing of the mutation
tRNA was extracted from LB831-EBV cells as described previously. Reverse transcription was performed on RNA from LB831-BLC and LB831-EBV cells as described above. PCR amplification was performed with DNA polymerase Dynazyme (Finnzymes Oy, Helsinki, Finland), the sense primer MGU1 (5'-GCTCAGATGATGTGGTTGAT located at position 591–610 of KIAA0205), and the antisense primer MGU10 (5'-ACTGTTGGTTTCCTGTATCC located at position 1044–1063 of KIAA0205). The PCR conditions were 5 min at 94°C followed by 35 cycles consisting of 1 min at 94°C, 1 min at 57°C, and 1 min at 72°C and by a final elongation step at 72°C for 15 min. The PCR products were subsequently purified on a Sepharose CL6B column and sequenced with the primer MGU3 (5'-TTGAATGCACTTGTAGCACA located at position 891–910 of KIAA0205). The PCR products were cloned into the pCR3.1 vector using the Bidirectional Eukaryotic TA Cloning Kit (Invitrogen), and individual clones were sequenced with the primer MGU3. The RNA integrity of LB831-EBV cells was checked by reverse transcription and amplification of the ß-actin mRNA.
PCR assay for expression of the KIAA0205 gene in normal tissues
Both tRNA extraction and RT-PCR amplification were performed on normal human tissues as described above. An additional step of DNA digestion was performed after the RNA extraction to eliminate any contaminating genomic DNA. The PCR reaction was performed with the sense primer MGU2 (5'-AATTACAGGAGCAGAGATCG located at position 735–754 of KIAA0205) and the antisense primer MGU10. The PCR products were then analyzed on a 1.5% agarose gel.
PCR assay for expression of the mutation in tumors
tRNA extraction and RT-PCR amplification were performed as described above. The PCR reaction was performed for 35 cycles with the sense primer MGU1 and the antisense primer MGU11 (5'-CTCCTACGGTGACCTTGACA located at position 1356–1375 of KIAA0205). The annealing temperature was 59°C. The reaction was then divided into two aliquots, one of which was digested with SspI. The PCR products were subsequently analyzed on a 1.5% agarose gel. The nonmutated PCR product is 784 base pairs (bp). The presence of the mutation at position 1023 creates an SspI site around that position (the mutated and normal sequences located at position 1023–1028 are AATATT and GATATT, respectively), and two fragments of 432 bp and 352 bp are then generated after digestion with SspI. RNA integrity was checked by reverse transcription and amplification of the ß-actin mRNA.
Transfection of 293 cells expressing EBV nuclear Ag (EBNA)-1 (293-EBNA)
Transient transfection was performed with the lipofectamine reagent (Life Technologies). Briefly, 5 x 104 293-EBNA cells in a flat-bottom 96-well dish were transfected with 100 ng of plasmid DNA from a pool of the cDNA library or with 100 ng of plasmid containing P-035-1 cDNA, 60 ng of plasmid pcDNA3 containing the HLA-B4403, and 2.5 µl of lipofectamine. Transfected cells were tested in a CTL stimulation assay after 24 h.
DNA sequence analysis
DNA sequencing was performed by the dideoxy chain termination method (Thermo Sequenase cycle sequencing kit, Amersham, Cleveland, OH) using specific oligonucleotides as primers. The computer search for sequence homology was performed with the database located at blast@ncbi.nlm.nih.gov (National Library of Medicine, National Institutes of Health, Bethesda, MD).
Stable transfection of tumor cell lines
The bladder carcinoma cell line LB831-BLC was transfected by the calcium phosphate precipitation method as described previously (37). A total of 1 x 106 cells were transfected with 20 µg of plasmid pEF-BOSpuro-PL3 containing the cDNA B7-1. Briefly, B7-1 was amplified using the sense primer 5'-GGGTCCAAATTGTTGGCTTTCACT and the antisense primer 5'-GAAGAATGCCTCATGATCCCCA in a PCR reaction with cDNA from cell line LB23-EBV as a template. The PCR conditions were similar to those described previously, except that the annealing temperature was 55°C, and the extension was performed for 3 min. The B7-1 insert was then cloned into plasmid pEF-BOSpuro-PL3, which was derived from pEF-BOS (38) by the insertion of a puromycin resistance gene and a polylinker. Puromycin-resistant LB831-BLC cells were selected in 0.8 µg/ml puromycin (Sigma, St. Louis, MO) and then cloned by limiting dilution. RCC BB64-RCC was transfected by the calcium phosphate precipitation method with plasmid pEF-BOSpuro-PL3 containing the P-035-1 insert and selected in puromycin as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To derive CTLs directed against bladder tumor cells, we first adapted to culture tumor cells from a surgical sample of invasive bladder carcinoma. We then stimulated autologous blood lymphocytes with irradiated cells from this tumor line, called LB831-BLC, in the presence of IL-2. After three weekly stimulations, the T cell culture exerted a weak lytic activity against K562 NK target cells but no specific activity against autologous tumor cells (data not shown).
To increase the stimulatory capacity of LB831-BLC cells, we transfected them with the cDNA of the B7-1 costimulatory molecule. We cloned the transfected population by limiting dilution and selected a single cell clone, LB831-BLC-B7-1, based on its high surface expression of B7-1. We stimulated autologous CD8+ blood mononuclear cells with irradiated LB831-BLC-B7-1 cells in the presence of IL-2 and autologous irradiated feeder cells. After three weekly restimulations with freshly irradiated LB831-BLC-B7-1 cells and IL-2, the culture showed weak yet reproducible lytic activity on the autologous tumor cells. This lytic activity was specific, since the same level of lysis was reached in the presence of an excess of unlabeled K562 NK target cells (data not shown). This finding indicated that cytotoxic T cells specifically directed against LB831-BLC human tumor cells were present in the MLTC.
To analyze more precisely the cytotoxic response and further
characterize the Ags recognized by the T cells, we cloned the bulk MLTC
by limiting dilution. We obtained a panel of CD8+ T cell
clones, including clones 360b/40, 360b/41, and 360b/52, which lysed
autologous LB831-BLC tumor cells but not K562 or autologous
EBV-transformed B cells (Fig. 1
). A
heterologous bladder tumor line, LB905-BLC, was also lysed by CTL
360b/52 but was not lysed by the two other clones.
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). All clones specifically produced TNF
when stimulated with LB831-BLC tumor cells. This production of TNF was
completely blocked in the presence of the anti-HLA class I mAb
W6/32. Anti-CD8 but not anti-CD4 Abs also inhibited the release of
TNF. The CTL clone 360b/40 was blocked by an Ab directed against HLA-B
or -C molecules (Fig. 2). This observation indicates that this CTL
recognizes an Ag, LB831-A, which is presented by HLA molecule B4403,
HLA-B4901, HLA-Cw04, or HLA-Cw07. CTL clone 360b/41 was not inhibited
by the anti-B or -C Ab, indicating that it is restricted by one of
the two HLA-A molecules of patient LB831. A mAb against HLA-A3, but not
a mAb against HLA-A24, blocked the production of TNF by CTL 360b/41,
indicating that this CTL recognizes a second Ag, LB831-B, which is
presented by HLA-A3. TNF secretion by the third CTL clone, 360b/52, was
blocked by the anti-B or -C Ab. In agreement with the lysis data,
we found that this clone produced TNF when stimulated with heterologous
LB905-BLC tumor cells. This production was also inhibited by the Ab
against HLA-B or -C molecules. Because CTL 360b/52, as opposed to CTL
360b/40, recognizes LB905-BLC, it defines an Ag different from LB831-A,
which we called LB831-C. Since HLA-Cw07 is the only B or C molecule
that is common to both LB831-BLC and LB905-BLC, this molecule is
probably the restricting molecule for CTL 360b/52. Unfortunately, this
CTL clone could not be maintained in a long-term culture, thereby
preventing further analysis of Ag LB831-C.
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To identify the Ag recognized by CTL 360b/40, we prepared a
directional cDNA library with poly(A)+ RNA from the
LB831-BLC tumor cell line. We cloned the library into plasmid pCEP4
downstream from the CMV promoter. The plasmid also contains the EBV
origin of replication and the sequence-encoding EBNA-1, which
transactivates the EBV replication origin allowing high copy episomal
replication of the plasmid when transfected into mammalian cells. We
divided the cDNA library into 582 pools of 100 recombinant clones and
prepared DNA from each pool. As described above, CTL 360b/40 appeared
to be restricted by HLA-B4403, -B4901, -Cw04, or -Cw07. Therefore, we
used PCR amplification to clone the cDNAs coding for each of these
molecules. We transiently transfected human 293-EBNA with DNA from each
cDNA pool and from the HLA-B4403 cDNA. We added CTLs after 24 h
and measured the production of TNF in the supernatant. One cDNA pool
was able to induce the production of TNF by CTL 360b/40. We then
subcloned the bacteria corresponding to this pool and similarly
transfected DNA from individual colonies. One cDNA, P-035-1, was able
to induce TNF secretion by CTLs when transfected with the HLA-B4403
cDNA (Fig. 3A). P-035-1
cDNA did not stimulate CTL 360b/40 when transfected without B4403 cDNA,
suggesting that Ag LB831-A consists of a peptide encoded by P-035-1 and
presented by HLA-B4403. Overexpression of the transiently transfected
cDNA was not required for CTL recognition, as an HLA-B4403+
RCC line stably transfected with P-035-1 cDNA was lysed by CTL 360b/40
(Fig. 3B).
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P-035-1 cDNA is 1378 bp long, and its sequence is almost identical
to a cDNA sequence reported in GenBank under the name KIAA0205 (GenBank
accession number D86960). This cDNA has been isolated from an acute
myeloblastoid leukemia line called KG-1. Nothing is known about the
expression profile of this gene in normal tissues. It is 6253 bp long
and contains an open reading frame of 1110 nucleotides from position
228 to 1337 (Fig. 4). Our cDNA is
identical to the region of KIAA0205 located between positions 426 and
1766, except in position 1023, in which G is replaced by A. In
addition, the last 37 nucleotides of our cDNA are not found in
KIAA0205. The region of KIAA0205 that is downstream from position 1766
might correspond to unspliced intron sequences, even though it does not
start with a donor splice site. Alternatively, the region might
correspond to an unrelated sequence accidentally linked to the first
sequence during the construction of the cDNA library.
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).
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The nucleotide substitution found in P-035-1 at position 1023 of
the KIAA0205 sequence changes one amino acid at position 266 of the
putative protein of 370 residues. This sequence difference could result
either from allelic polymorphism, since the two sequences come from
different individuals, or from a tumor-specific mutation in P-035-1. To
test the latter possibility, we cloned the equivalent cDNA from
autologous normal LB831-EBV cells. We amplified a 472-bp fragment
encompassing position 1023 by PCR using either tumoral cDNA from
LB831-BLC or normal cDNA from LB831-EBV as a template and then
sequenced the PCR products (Fig. 6).
Position 1023 of the normal sequence read G as in KIAA0205,
demonstrating that the nucleotide substitution resulted from a
tumor-specific mutation. Position 1023 of the tumoral sequence read
both A and G, as in P-035-1 and KIAA0205, respectively, indicating that
only one of the two alleles of the gene was mutated in the tumor
cells.
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, we observed two
bands of the expected sizes after SspI digestion of the PCR
products derived from both the original tumor and the cultured cell
line, indicating that the mutation had occurred in vivo. This finding
was further confirmed when we sequenced the PCR product: 5 of 20 clones
obtained after ligation of the PCR product from the original tumor in a
plasmid vector harbored the mutated gene. We then looked for the
mutation in a series of other tumor samples by PCR amplification
followed by SspI digestion. We did not find the mutation in
any of the 60 bladder carcinoma samples, 26 melanoma samples, 12 RCC
samples, and 55 other tumor samples tested. Therefore, this mutation
appears to be unique to LB831-BLC.
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To determine whether the mutation is responsible for the
recognition of LB831-BLC cells by CTL 360b/40, we tested synthetic
peptides encoded by the mutated region for CTL recognition. The
mutation changes residue 266 of the KIAA0205 protein from aspartic acid
to asparagine. The consensus motif for binding to HLA-B4403 is a
glutamic acid at position 2 and a hydrophobic residue at position 9 or
10 (39). Since residue 263 of KIAA0205 is a glutamic acid and residue
270 is a tryptophan, peptide 262–270 most likely binds to HLA-B4403.
We then synthesized the normal (AEPIDIQTW) and the mutated (AEPINIQTW)
versions of peptide 262–270. We pulsed HLA-B4403+
EBV-B cells with these peptides and tested them for recognition by CTL
360b/40. As shown in Figure 8A, the mutated peptide was
efficiently recognized by the CTLs, whereas the normal peptide was not.
As expected from the presence of the binding motif, the normal peptide
can bind to HLA-B4403, since increasing amounts of this peptide reduce
the binding of a control peptide to HLA-B4403 (Fig. 8B). We conclude that the mutation found in position
1023 of the KIAA0205 gene in LB831-BLC tumor cells creates a
new epitope in peptide 262–270 and is therefore responsible for the
recognition of the tumor cells by CTL 360b/40.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although a significant proportion of bladder tumors express the genes MAGE, BAGE, and GAGE (42), which were initially characterized on melanoma, nothing is known about the Ags that are actually recognized by autologous CTLs on bladder tumors. We show here that our CTL clones recognize three different tumor Ags. This finding is in line with the observations concerning melanoma and RCC, where the autologous CTL response was found to be directed against a number of distinct Ags (43, 44, 45, 46, 47). This diversity of tumor Ags may prove essential for the success of cancer immunotherapy. Simultaneous immunization against several Ags may increase the efficacy of cancer vaccines and reduce the emergence of escaping variants arising through a loss of Ag expression.
The function of the KIAA0205 protein is not known. Its ubiquitous expression might reflect a housekeeping function. It contains no signal peptide nor any known targeting signal to specific organelles, and might therefore be located in the cytosol.
Several mutations that generate antigenic peptides recognized by CTLs on human tumors appear to play a role in oncogenesis. This is the case for cyclin dependent kinase 4, whose mutation prevents binding to its inhibitor p16 and therefore constitutively drives the cell into the cycle (19). This also appears to be the case for the mutated ß-catenin, which is stabilized and thereby aberrantly activates gene transcription by constitutively forming activating complexes with transcription factor LEF-1 (20, 48). We also recently reported a mutation generating an antigenic peptide in caspase-8, a protease involved in the transduction of the apoptotic signal through the FAS and the TNF receptor (41). The ability of the mutated caspase-8 to transduce the apoptotic signal appears to be reduced relative to its normal counterpart. Because the function of the KIAA0205 protein is unknown, it is very difficult to determine whether the mutation reported here also has an oncogenic effect. Preliminary experiments of transfection into NIH-3T3 cells failed to detect a transforming property for the mutated KIAA0205.
In the last five years, a number of human tumor Ags that are recognized by class I-restricted CTLs have been characterized, mainly on melanoma and RCC (49). This work has allowed the development of new clinical trials targeted specifically against defined tumor Ags. Such trials are currently being evaluated in melanoma (50). The results presented here represent the first molecular characterization of a tumor Ag recognized by CTLs on a bladder tumor. They indicate that the genetic approach aimed at cloning the gene encoding the antigenic target of previously established CTLs, which was initially developed in melanoma, is also efficient in bladder cancer and will hopefully lead to the characterization of clinically useful Ags in this tumor type.
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Acknowledgments |
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Footnotes |
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2 Present address: SmithKline Beecham Biologicals, Rue de l’Institut 89, 1330 Rixensart, Belgium.
3 Address correspondence and reprint requests to Dr. Benoît J. Van den Eynde, Ludwig Institute for Cancer Research, Brussels Branch and Cellular Genetics Unit, Université Catholique de Louvain, Avenue Hippocrate 74, UCL 7459, B-1200 Brussels, Belgium.
4 Abbreviations used in this paper: RCC, renal cell carcinoma; BCG, Bacillus of Calmette-Guérin; MLTC, mixed lymphocyte tumor cell culture; tRNA, total RNA; bp, base pair; EBNA, EBV nuclear Ag.
Received for publication January 7, 1998. Accepted for publication February 6, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A.G.S. Buggins, N. Lea, J. Gaken, D. Darling, F. Farzaneh, G.J. Mufti, and W.J.R. Hirst Effect of Costimulation and the Microenvironment on Antigen Presentation by Leukemic Cells Blood, November 15, 1999; 94(10): 3479 - 3490. [Abstract] [Full Text] [PDF]
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J. Bilsborough, A. Van Pel, C. Uyttenhove, T. Boon, and B. J. Van den Eynde Identification of a Second Major Tumor-Specific Antigen Recognized by CTLs on Mouse Mastocytoma P815 J. Immunol., March 15, 1999; 162(6): 3534 - 3540. [Abstract] [Full Text] [PDF]
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), the NK
cell-sensitive line K562 (
), and the heterologous bladder tumor line
LB905-BLC (
) were used as target cells. Chromium release was
measured after 4 h.
