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Laboratoire d’Immunologie Cellulaire, Institut Gustave-Roussy, Villejuif, France
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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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Also, some Ags recognized by tumor-reactive T cells may be generated by alternatively processed transcripts including intronic sequences, as in the case of MUM1 (11), N-acetylglucosaminyltransferase-V (15), or gp100 (16). The T cell-mediated surveillance of the integrity of the cell may extend beyond the genome and its intronic regions and focus also on peptides coded by an alternative open reading frame (ORF) located within the primary ORF, as in the case of gp75/tyrosinase-related protein-1 (TRP-1) (17) and NY-E50-1 (18). There are only a few examples of the usage of alternative ORFs in eukaryotes reported in the literature, and the biologic significance of the corresponding gene products is unknown. Nonetheless, it may be speculated that these products may serve as antigenic targets of the Ag-processing machinery to increase the efficiency of immune surveillance. There is an increasingly apparent relationship between abnormal translational control of gene expression (as for c-myc or fibroblast growth factor-2) (19, 20, 21) and cancer; thus, the identity of immunogenic peptides in tumors may very well extend beyond the ones derived from the primary ORF. Understanding the mechanisms by which alternative ORFs are translated in tumor cells may have important implications in tumor immunology.
This study demonstrates that a peptide derived from an alternative ORF of the intestinal carboxyl esterase (iCE) gene is recognized by an HLA-B7-restricted RCC-reactive T cell clone. The reactive tumor-infiltrating lymphocytes (TILs) are amplified in situ at the tumor site. Unexpectedly, this alternative ORF is initiated from a cryptic non-AUG (ACG) codon.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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K562 cells and the EBV-transformed B cell line from patient 1 were cultured in medium consisting of RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 1% L-glutamine (200 mM,) 1% sodium pyruvate (200 mM), 1% HEPES, 5% FCS, and penicillin (50 international units (IU)/ml) (Life Technologies). WEHI-164 clone 13 (W13) was kindly provided by Dr. Benoît J. Van den Eynde Bruxelles, Belgium; COS-7 cells were cultured in RPMI 1640 (Seromed, Biochrom KG, Berlin, Germany) supplemented with 1% L-glutamine (200 mM), 1% sodium pyruvate (200 mM), 1% HEPES, 5% FCS, and penicillin (50 IU/ml).
Patients and establishment of RCC cell lines
RCC cell lines were established as described previously (22). Primary tumors were obtained from untreated patients who had undergone radical nephrectomy. The RCC-1 cell line was established from patient 1 (HLA A1, A32, B7, B12–44, Cw5, Cw7), a 56-year-old male with a clear and granular renal cell carcinoma without metastasis. After surgery, fragments were processed by enzymatic digestion, and tumor cell suspensions were plated in complete RCC medium (22). The tumor cell lines RCC-2 (HLA A1, A3, B7, B8, Cw7, Cw7), RCC-3 (HLA A1, A29, B22, B15–62/63, Cw1, Cw7–17), RCC-4 (HLA A3, A19–29, B7, B12–44, Cw7, Cw16), RCC-5 (HLA A1, A3, B7, B22–56, Cw1, Cw7), RCC-6 (HLA A9–24, A32, B12–44, B18, Cw5, Cw5), RCC-7 (HLA A1, A28–68, B8, B40–60, Cw3, Cw7), and RCC-8 (HLA A2, A10–25, B18, B13, Cw8, Cw6), which were derived from the primary tumor of patients 2, 3, 4, 5, 6, 7, and 8, respectively, were maintained in complete RCC medium.
Generation of CTLs from TILs of patient 1
Autologous TILs were generated from a thawed suspension of dissociated tumor cells. An autologous mixed lymphocyte-tumor cell culture (MLTC) was performed as follows: on day 1, dissociated tumor cells were seeded at 2 x 106 TILs in 6-well flat-bottom plates (Falcon, Becton Dickinson, NJ) in RPMI 1640 (Life Technologies) containing 1% L-glutamine (200 mM), 1% sodium pyruvate (200 mM), 8% human AB serum (Institut Jacques Boy, S.A, Reims, France), penicillin (50 IU/ml) supplemented with 5% T cell growth factor (TCGF), and 50 IU/ml of human IL-2 (rIL-2) (Roussel Uclaf, Romainville, France), hereafter referred to as MLTC complete medium. MLTC complete medium was removed every 3 days as needed and replaced with new MLTC complete medium. On days 7, 15, and 21, 2 x 106 TILs were restimulated with 2 x 105 irradiated (100 Gy) autologous tumor cells seeded in 6-well flat-bottom plates with MLTC complete medium. On day 15, TILs were tested for cytotoxic activity against the autologous RCC-1 and K562 cell lines, characterized for surface phenotype by direct immunofluorescence, and cloned by limiting dilution. TILs were seeded to 0.6–600 cells/well in 96 V-shaped microwell plates (Nunc, Roskilde, Denmark) preseeded with irradiated autologous tumor cells (1 x 104/well) as stimulators and irradiated allogeneic PBLs (8 x 104/well) and irradiated EBV-transformed B cells (2 x 104/well) as feeder cells in a total volume of 200 µl of MLTC complete medium. Every 3 days, 60 µl of supernatant were removed from each well and replaced by 60 µl of fresh medium. Clones were screened for cytotoxicity in a standard 4-h chromium release assay. Every 7–10 days, CTL clones were restimulated with the allogeneic feeder cell line and the autologous tumor cell line as described above.
Cytotoxicity assay
The cytolytic activity of the CTLs was assessed in a standard 51Cr release assay as described previously (22). Target cells (RCC cell lines, K562) were labeled for 1 h with 50–100 µCi of 51Cr (DuPont New England Nuclear, Boston, MA) at 37°C. A total of 2 x 103 cells were seeded into 96-microwell plates in 100 µl of RPMI 1640 supplemented with 5% FCS. Effector cells were added to the wells at different E:T ratios ranging from 40:1 to 0.1:1. For an inhibition of lysis by mAbs, target cells were preincubated for 2 h in the presence of a saturating mAb concentration before the addition of effector cells. The 96-microwell plates were incubated at 37°C for 4 h, and supernatants were collected and counted for the release of 51Cr. For blocking cytotoxicity or TNF production, the following mAbs were used: W6/32, a pan-MHC class I mAb, and B1.23.2 (ME1), an HLA-B/C-specific mAb.
Transfection of COS-7 cells and screening of transfectants
Transfection experiments were performed with COS-7 cells using
the DEAE-dextran-chloroquine method (5, 7, 23). At 3 days before
transfection, COS-7 cells were seeded in 96-microwell, flat-bottom
plates at 5 x 103 cells/well in 150 µl of
RPMI 1640 containing 20% FCS. Transfection experiments were performed
in duplicate in two different microwell plates. For transfection,
medium was discarded and replaced by 30 µl of transfection mixture
containing 35 µg of DEAE-dextran (Sigma) and 0.1 mM chloroquine
(Sigma), with 100 ng of plasmid DNA representing a pool of 
200
recombinant clones from the cDNA library and 100 ng of the autologous
HLA-B*0702 plasmid. COS-7 cells were incubated for 4 h
at 37°C, medium was removed, and cells were incubated for 2 min in
1x PBS buffer containing 10% of DMSO solution. Cells were washed once
in 1x PBS buffer and incubated with RPMI 1640/10% FCS for 2 days.
After 2 days, transfected COS-7 cells were tested for their ability to
stimulate the production of TNF by the 3B8 clone, as assessed with the
WEHI assay.
Transfected COS-7 cells were tested for their ability to stimulate the production of TNF (24). A total of 2 x 103 CTLs (clone 3B8) were added to 96-microwell, flat-bottom plates containing transiently transfected COS-7 cells in 100 µl of RPMI 1640/10% FCS. Each supernatant was collected after 18 h, and its TNF content was determined by testing its cytotoxic effect on WEHI-164 clone 13 cells (25) in an MTT colorimetric assay.
Complementarity determining region-3 (CDR3) size analysis
CDR3 size analyses of TCRBV gene segments expressed by the CTL clone 3B8 or found in blood or tumor fragments were performed as described previously (22). The procedure used for CDR3 size analysis includes independent RT-PCR amplifications of TCRBV-BC fragments (26) followed by a runoff of the PCR products using nested fluorescent TCRBC or TCRBJ primers (27) and size determination of fluorescent runoff products by electrophoresis on an automated DNA sequencer ABI 373 (Perkin-Elmer Applied Biosystems, Foster City, CA) using Immunoscope software (28). Because the 5' and 3' primer positions are fixed, variations in the size of the runoff products are only due to differences in the length of CDR3 regions. Each peak is characterized by its position (CDR3 size) and an intensity of fluorescence (arbitrary fluorescence unit or fluorescence unit). The graphs representing CDR3 size patterns were standardized at 100% for the highest peaks. In blood from healthy donors, most profiles reflecting CDR3 size diversity in a given Vß subfamily displayed five to eight peaks at 3-nucleotide (nt) intervals with a nearly Gaussian distribution (21). Dominant peaks were defined as high-intensity signals with a dramatic decrease in other CDR3 signals.
Construction of the cDNA library
Poly(A)+ RNA was extracted from the RCC-1 cell line using a maxi Message Marker kit (R&D Systems, Abingdon, U.K.) according to the manufacturer’s instructions. First-strand cDNA was synthesized with the Superscript Choice System (Life Technologies, Gaithersburg, MD) using an oligo(dT) primer containing a NotI site at its 5' end followed by second-strand cDNA synthesis. Blunt-end cDNAs were ligated to semiBstXI adapters (Invitrogen, San Diego, CA), digested with NotI, and subsequently fractioned by chromatography on Sephacryl S-500 HR columns. cDNA size fractions were subcloned into the BstXI and NotI sites of the pcDNAI expression vector. Recombinant plasmids were electroporated into Escherichia coli MC1061/P3, and bacteria were selected on Luria-Bertani-agar plates with ampicillin (50 µg/ml) and tetracycline (10 µg/ml). In screening experiments, the RCC-1 cDNA library was divided into 400 pools of 200 cDNA clones. Each pool of bacteria was amplified, and plasmid DNA was extracted using the alkaline lysis method (29).
Isolation of full-length iCE cDNA and of truncated or point-mutated iCE cDNA
Total RNA was extracted from an RCC cell line according to the guanidine-isothiocyanate/cesium chloride centrifugation procedure (30). Reverse transcription was performed on 5 µg of total RNA in a reaction volume of 20 µl using a cDNA Cycle Kit (Invitrogen) according to the manufacturer’s instructions. A total of 1 µl of the cDNA reaction was used in a PCR using Taq DNA polymerase (Perkin-Elmer). For the amplification of human iCE cDNA (31), the following primers were used: primer P1, 5'-CCCAAGCTTGGTGAATAGCAGCGTGTCCGC-3' (nt -28 to -48, sense) and primer P2, 5'-TGCTCTAGAAGGGAGCTACAGCTCTGTGTG-3' (nt 1666–1687, antisense). PCR conditions were 10 min at 95°C followed by 30 cycles of amplification (94°C for 1 min, 60°C for 2 min, and 72°C for 3 min with a final extension for 10 min at 72°C). The PCR product obtained was digested by HindIII and XbaI and subsequently subcloned into the HindIII and XbaI sites of the pcDNAI expression vector for sequencing. The numbers in parentheses represent the nucleotide numbers complementary to the iCE cDNA published sequence (GenBank accession no. Y09616). iCE site-directed mutants were prepared by encoding the desired point mutation in overlapping oligonucleotide primers and generating the mutants by PCR (32). The sequencing of PCR products was performed with a DNA sequencing kit (ABI Prism, Perkin-Elmer Applied Biosystems).
Northern blot analysis
Total RNA was extracted from various primary tumors using the
guanidinium isothiocyanate/cesium chloride centrifugation technique
(30). RNA poly(A)+ was prepared as described
above from RCC cell lines and untransformed renal cell lines. A total
of 5 µg of poly(A)+ RNA or 10 µg of total RNA
were subjected to electrophoresis in a 1.2% agarose formaldehyde gel
and transferred to Hybond-N+ (Amersham, Little
Chalfont, U.K.) nylon membranes. The RNA blot was hybridized both with
a 2C2 cDNA fragment corresponding to nt 1033–2009 of the published
human iCE cDNA sequence (31) and with GAPDH cDNA as probes. All probes
were labeled with [
-32P]dCTP (3000 Ci
mmol-1) using the Prime-IT II Random Primer
labeling kit (Stratagene, La Jolla, CA). Hybridization was performed at
48°C for 16 h.
Membranes were washed twice with 2x SSC at 52°C, washed once for 15 min with 0.2 SSC/0.1% SDS, and subsequently autoradiographed or analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first stimulated TILs from patient 1 with irradiated autologous
tumor cells in the presence of low-dose IL-2 and TCGF (22). After 15
days of MLTC, a specific cytolytic activity against the autologous
tumor cells was detected (31% of lysis at a 40:1 E:T ratio); TILs were
cloned by limiting dilution in the presence of autologous tumor cells,
EBV-transformed B cells, and allogeneic PBLs, with addition of IL-2 and
TCGF. We isolated a TCRß+
CD8+ clone, termed 3B8, which lysed the
autologous RCC cell line but not the NK-sensitive K562 target cells.
The cytotoxicity of clone 3B8 against the autologous RCC-1 cell line
was blocked with W6/32 mAb (Fig. 1
A). In both cytotoxicity
(Fig. 1B) and TNF production (data not shown) assays, all
allogeneic HLA-B7+ RCC cell lines (RCC-2, RCC-4,
and RCC-5 in Fig. 1B) and none of the
HLA-B7- RCC cell lines (RCC-3, RCC-6, RCC-7, and
RCC-8) were recognized by CTL clone 3B8. The autologous EBV-transformed
B cell line or PHA blasts were not recognized by 3B8 (data not shown).
Therefore, it was concluded that the Ag recognized by 3B8 is presented
by the HLA-B7 molecule and appears to be commonly expressed in RCC cell
lines. The six HLA class I molecules from RCC-1 were isolated by RT-PCR
(33), cloned in pcDNAI, and sequenced. The nucleotide sequence of
autologous HLA-B7 cDNA led us to identify the allele involved as being
HLA-B*0702. Transfection of this HLA allele in two
HLA-B7- allogeneic RCC cell lines was sufficient
to induce recognition (TNF secretion) by CTL clone 3B8, confirming that
this clone led us to identify a shared Ag that is expressed by all RCC
(data not shown).
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For the 3B8 clone, a signal was obtained with only one of the 24
Vß subfamily primers (TCRVB5) and
only one of the 13 TCRBJ primers (TCRBJ1S2)
tested. CDR3 size distribution analysis showed that the
TCRBV5J1S2 clonotype of 3B8 was dominant in the tumor (as
shown with TCRBV5-BC primers in Fig. 2A and with
TCRBV5-BJ1S2 primers for a more refined analysis in Fig. 2B), whereas such a clonotype was not found in PBMCs (a
nearly Gaussian CDR3 length distribution with TCRBV-BC
primers, see Fig. 2A). This result strongly suggests that
the 3B8 clone was expanded specifically at the tumor site as shown
previously by cDNA sequencing in several cases (14, 34, 35, 36).
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A cDNA library was constructed in the pcDNAI expression vector
from RNA extracted from the RCC-1 cell line. The cDNA library was
divided into 400 pools of 200 recombinant plasmids; each pool was
cotransfected in duplicate in COS-7 cells with the expression vector
pcDNAI containing the cDNA encoding for autologous
HLA-B*0702. COS-7 cells were then tested for their ability
to stimulate the production of TNF by 3B8. After 48 h,
cotransfected COS-7 cells were incubated for 24 h with 3B8; the
concentration of TNF in the culture supernatants was measured by its
cytotoxic effect on WEHI cells. The amounts of TNF found in the
supernatants ranged from 8 to 11 pg/ml except for two pairs of higher
duplicates (14 and 15 pg/ml). For each pool of bacteria corresponding
to these candidate wells, plasmid DNA was extracted and subcloned. A
second screening was performed by transfecting COS-7 cells with 50
pools of 50 recombinant plasmids extracted from positive duplicates.
Finally, a third screening in COS-7 cells led us to the isolation of
two identical cDNA clones (cDNA clones 2C2 and 3G7) that transferred
the expression of the Ag in HLA-B7+ COS-7 cells.
The results obtained with these cDNA clones are shown in Fig. 3A.
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Identification of the antigenic peptide
To delimit the minimal nucleotidic region coding for the antigenic
peptide, various truncated cDNAs corresponding to the iCE coding region
were obtained from cDNA clone 2C2 (Fig. 4). These cDNA fragments subcloned into
the pcDNAI expression vector were transfected into COS-7 cells together
with pcDNAI containing the autologous HLA-B*0702 cDNA. A
minimal nucleotidic coding region was located between nt 763-1033. To
reduce the nucleotidic sequence coding for the Ag, several truncated
cDNAs were obtained by PCR amplification; these truncated cDNAs were
cotransfected with the HLA-B*0702 allele in COS-7 cells.
COS-7 cells transfected with a fragment ranging from nt 763 to 855 were
recognized by CTL 3B8 but not with a fragment ranging from nt 763 to
834 (Fig. 4), indicating that the peptide coding region was located
between nt 763 and 855. After examination of the corresponding amino
acid sequence, all possible nonamers and decamers were synthesized and
tested for their ability to render autologous EBV-transformed B cells
sensitive to lysis by 3B8. None of them were found to be positive at
10-4 or 10-5 M.
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HLA-A2 binding peptide Ags are known to up-regulate the expression
of HLA-A2 molecules on T2 cells (37). Similarly, we used
HLA-B*0702-transfected T2 cells (38) to analyze the binding
ability and stability of the iCE peptide (Fig. 6). The binding of the iCE peptide was
stable over time at 50 mM for 
4 h, in contrast to the control
HLA-A2-restricted hsp 70 peptide (14).
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To determine the tissue distribution of iCE mRNAs, a human
RNA Master blot (Clontech, Palo Alto, CA) consisting of a nylon
membrane to which poly(A)+ RNAs from 50 human
tissues have been immobilized in separate dots was hybridized with a
32P-labeled cDNA clone, 2C2, as a probe. iCE mRNA
was detected in the liver, kidney, small intestine, colon, and heart
and was weakly expressed in the pituitary gland, adrenal gland,
prostate, and stomach (data not shown). No signal was found in fetal
tissues, bone marrow, peripheral leukocytes, lung, and brain. To
identify the mRNA species, a Northern blot was prepared with RNA
poly(A)+ from various RCC cell lines and
untransformed renal cell lines (Fig. 7A) as well as total RNA
extracted from various primary tumors (i.e., renal tumors, melanoma,
bladder tumors, neuroblastoma, and colon tumors) (Fig. 7B).
The RNA blot was hybridized with a cDNA probe corresponding to nt
1033–2009 of the 2C2 sequence. As shown in Fig. 7A, two
mRNA species (4.5 kb and 3.5 kb) described previously by Schwer et al.
(31) were detected in RCC carcinoma cell lines as well as in
untransformed renal cells. In renal primary tumors a single mRNA
transcript (3.5 kb) was detectable, whereas no iCE transcript was
detected in primary tumors of different histotypes (Fig. 7B). Although an additional-2.2 kb transcript has been
reported (31) in the small intestine and liver, no such transcript was
detected in the various cell lines or primary tumors tested. Thus, the
iCE protein is encoded in RCC tumors by a predominantly expressed
single mRNA species (3.5 kb).
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A stop codon was first introduced at position 807 of the
full-length iCE cDNA (Fig. 8A)
just before the nonamer encoding sequence to confirm that the peptide
recognized in the cytotoxicity assays is indeed encoded by the
corresponding sequence in COS-7 transfection assays. This point
mutation (mutant A) abolishes CTL 3B8 recognition following
cotransfection with HLA-B*0702 into COS cells (Fig. 8B). We subsequently mutated the natural AUG translation
initiation site at position 3; this point mutant (mutant B) was still
recognized (Fig. 8B), indicating that neither the natural
iCE amino acid sequence nor a chimeric sequence resulting from
programmed translational frameshifting (that is, for iCE slippage of
the ribosome one codon forward) and recoding of the downstream sequence
(39, 40) is coding for the recognized peptide.
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B, none of these mutations
abolishes recognition by CTL 3B8. These results strongly suggest that a
cryptic non-AUG codon is used in iCE cDNA as an alternative translation
initiation site.
To delimit the minimal nucleotidic region coding for this cryptic
non-ATG codon, we introduced stop codons that would interrupt the +1
ORF at different positions upstream from the antigenic peptide (between
positions 428 and 809), with point mutations at positions 466 (mutant
E), 519, 666, and 786 of the full-length iCE cDNA (Fig. 8A).
All four of these mutants abolished CTL 3B8 recognition following
cotransfection (see the result of mutant E for position 466 in Fig. 8B). A minimal nucleotidic region was then located between
nt 428 and 466. We subsequently searched for possible non-ATG codons
(CTG, ACG) in this short sequence and found only one, an ACG codon at
position 440. Mutation of this codon to ACT (mutant F) abolished CTL
3B8 recognition (Fig. 8B). Thus, the first non-AUG codon in
the +1 ORF was used to initiate the translation process.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results reveal that a novel mechanism may be involved in generating T cell epitopes. An alternative ORF induced by a cryptic non-AUG codon leading to a +1 translational reading frame was shown here to encode a tumor Ag recognized by TILs. In two other examples, gp75/TRP-1 (17) and NY-E50-1 (18), peptides recognized by TILs are coded by an alternative ORF located within the primary ORF. A mechanism by which the alternative ORF is translated has been suggested for gp75/TRP-1 (17), where recognition was affected by the presence of an internal AUG preceding the epitope. In addition to this ribosomal scanning mechanism, ribosomal frameshifting (39, 40) has been suggested for the production of T cell epitopes (41); however, in iCE, this possibility was excluded by the mutation of the natural ATG translation initiation site, which did not affect peptide recognition. In fact, the presence of the first cryptic internal translation initiation site (an ACG codon at position 440) in the +1 alternative ORF of iCE was enough to direct the expression of sufficient amounts of iCE peptide for T cell activation in vitro as well as in vivo (i.e., leading to in situ T cell clonal expansion). The leaky scanning model, in which ribosomes occasionally bypass the first AUG with a poor Kozak consensus sequence and initiate translation at a downstream translation initiation site may apply to iCE because of the presence of a pyrimidine at position +4 in place of a purine.
To our knowledge, this is the first example of an epitope coded by a non-ATG-defined alternative ORF and recognized by tissue-reactive T cells in human disease. It is not known whether low levels of expression of this alternative ORF in vivo may result in a failure to induce T cell tolerance to these products, leading to recognition in normal adult tissues. In the present study, untransformed HLA-B7+ renal cell lines established in vitro were in fact recognized in cytotoxicity assays by the TIL-derived 3B8 clone (data not shown). It has been shown that non-ATG-defined alternative initiations of translation of the fibroblast growth factor-2 molecule are induced in stressed or transformed cells compared with ATG-defined ones (20). Similarly, the expression of non-ATG-initiated forms of iCE may be up-regulated in tumors, leading to the in situ clonal expansion of the corresponding TILs, as observed in the present study. This alternative iCE ORF would in the latter case represent an interesting tumor Ag for use in the immunotherapy of patients with hepatocarcinoma or colon or renal adenocarcinoma (tumors that may express iCE mRNA). More generally, these findings also raise the possibility that alternative ORFs induced by non-AUG codons in the three translational reading frames may encode T cell epitopes in some human diseases, such as cancer or autoimmune disorders. Short ORFs could be found in large numbers, and this may greatly increase the repertoire of nonmutated T cell epitopes recognized in adult tissues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frédéric Triebel, Laboratoire d’Immunologie Cellulaire, Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France. E-mail address:
3 Abbreviations used in this paper: RCC, renal cell carcinoma; ORF, open reading frame; TIL, tumor-infiltrating lymphocyte; CDR3, complementarity determining region-3; iCE, intestinal carboxyl esterase; MLTC, mixed lymphocyte-tumor cell culture; TCGF, T cell growth factor; nt, nucleotide; IU, international units; TRP-1, tyrosinase-related protein-1.
Received for publication December 14, 1998. Accepted for publication April 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-w29/Vß1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur. J. Immunol. 22:1261.[Medline]
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A. Moreau-Aubry, S. Le Guiner, N. Labarriere, M.-C. Gesnel, F. Jotereau, and R. Breathnach A Processed Pseudogene Codes for a New Antigen Recognized by a Cd8+ T Cell Clone on Melanoma J. Exp. Med., May 1, 2000; 191(9): 1617 - 1624. [Abstract] [Full Text] [PDF]
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N. Guerra, M. Guillard, E. Angevin, H. Echchakir, B. Escudier, A. Moretta, S. Chouaib, and A. Caignard Killer inhibitory receptor (CD158b) modulates the lytic activity of tumor-specific T lymphocytes infiltrating renal cell carcinomas Blood, May 1, 2000; 95(9): 2883 - 2889. [Abstract] [Full Text] [PDF]
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