|
|
|
|
|
|
|
||
Brief Definitive Report |
breathna{at}nantes.inserm.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Key Words: peptide • epitope • processing • tumor immunity • CTL
Most of the antigens characterized to date are HLA class I restricted and presented by melanoma cells. Many of them are derived from normal or mutated gene products obtained by classical pathways of gene expression 1. However, an increasing number of reports describe antigens resulting from the use of nonclassical pathways of gene expression, when unexpected events take place during transcription, splicing, or translation 6. Thus, the NA17-A antigen 7 is translated from a transcript obtained by activation of a cryptic promoter within intron sequences of the N-acetylglucosaminyltransferase-V gene. Reverse strand transcription of a housekeeping gene gives rise to an antigen recognized by CTLs on a human kidney tumor 8. Intron retention during splicing is responsible for generation of some antigens from the gp100 9 and tyrosinase-related protein (TRP)-2 10 genes. Use of alternative open reading frames (ORFs) for translation can also create antigens, as described for a breast and melanoma-shared antigen 11, a gp75TRP-1 antigen 12, a renal cell carcinoma antigen derived from the intestinal carboxyl esterase gene 13, and an antigen derived from the LAGE-1 gene 14.
We describe here a new tumor antigen generating an HLA class I–restricted CTL response against melanoma. Surprisingly, the antigen is coded for by a processed pseudogene. Expression of the "living" counterpart of the pseudogene does not lead to a corresponding CTL response. This difference between the two genes provides insights into how a gene coding for a tumor antigen was created.
HLA Transfection in Tumor Cell Lines.
Library Screening.
Transfection of COS-7 Cells.
T Cell Stimulation Assay.
Production of Modified cDNAs.
Reverse Transcriptase–PCR Assays.
Peptide Stimulation Assays.
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
CTLs can recognize antigens presented by human tumor cells. In the hope that manipulating this CTL response to combat cancer will prove possible, a number of groups have started to identify the antigens in question (for review see reference 1). The results of several clinical trials of both adoptive immunotherapy 2 and vaccination 345 based on this work suggest that the effort is worthwhile.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Cell Lines.
Melanoma cell lines (M) were established from metastatic tumor fragments from different patients. IPC 277/5 and IGR 1/54 melanoma cell lines were gifts from C. Aubert (INSERM U119, Marseille, France). The FM2.29 cell line was a gift from J. Zeuthen (Danish Cancer Society Research Center, Copenhagen, Denmark). DAUV melanoma cell line and mouse fibrosarcoma WEHI 164 clone 13 used for TNF assays was obtained from T. Boon (Ludwig Institute for Cancer Research, Brussels, Belgium). T cell clone M88.7 was derived from tumor infiltrating lymphocytes of patient M88 (A*0201, A*0301, B*4002, B*1302, Cw*02022, Cw*0602) as described previously 15.
Cells were seeded in 96-well plates and incubated until 80% confluent before addition of expression vector (100 ng) and Lipofectamine (0.5 µl) in serum-free medium. After 12–15 h, the transfection mixture was replaced with complete medium. Transfected tumor cell lines were used for stimulation of T cell clone M88.7 36–48 h after transfection.
Poly A+ RNA was obtained from M88 cells using the FastTrack 2.0 mRNA extraction kit (Invitrogen Corp.). mRNA was converted to cDNA using a cDNA synthesis kit (Stratagene Inc.). The cDNA obtained was inserted into pCI-neo (Promega). Recombinant plasmids were electroporated into Escherichia coli XL2-Blue MRF' (Stratagene Inc.). For screening, 1,500 pools of 100 ampicillin-resistant bacteria per pool were constituted. Plasmid DNA was extracted by the alkaline lysis method from each pool 16. For genomic gene cloning, a human cosmid library (provided by A. Haunauer, IGBMC, Strasbourg, France) was hybridized with the 2.7-kb NA88-A cloned cDNA. Selected regions of the positive cosmid identified were sequenced using the Applied Biosystems 373 DNA sequencer with the DNA sequencing Kit Dye terminator (Perkin Elmer).
DEAE–dextran-chloroquine transfection was as described 17. In brief, 1.5 x 104 COS-7 cells were transfected with 100 ng of the HLA B*1302 expression vector and 100 ng of a pool of the cDNA library. Transfectants were tested after 48 h for their ability to stimulate TNF production by T cells. One positive pool was identified, and 500 individual plasmids obtained from it tested for the ability to trigger T cell clone TNF release as described for pools.
T cells (2.5 x 103 per well) were added to COS-7 cells or tumor cells 24–48 h after transfection. Culture supernatants were harvested 6 h later and tested for TNF content by measuring culture supernatant cytotoxicity to WEHI 164 clone 13 in a colorimetric assay 18.
Truncated NA88-A cDNAs were generated using standard techniques 16 and cloned into pcDNA3 (Invitrogen Corp.) or into pCI-neo. pNA88-A Trp, pNA88-A Ser, and pHPX42B Stop were generated using the QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene Inc.).
Total RNA was extracted by the guanidinium–cesium chloride procedure 16 and used for cDNA synthesis 19. Some samples of tumor and normal tissue cDNA were provided by F. Brasseur (Ludwig Cancer Institute, Brussels, Belgium). To test for genomic DNA contamination, cDNA samples were used for PCR with primers designed to amplify the MAGE-1 genomic gene. None of the samples used here gave a positive result in this test. For amplification of NA88-A cDNA, PCR assays were performed with primers 5'-ccttgtggttcgagtccc-3' and 5'-GGACACGGCCACAGGACC-3' for 35 cycles (1 min at 94°C, 1 min at 62°C, and 1 min 30 s at 72°C). Under these conditions, primers will not amplify HPX42B cDNA. PCR products were size fractionated and blotted to Hybond-N+ (Amersham Pharmacia Biotech) before hybridization with the cloned NA88-A cDNA. Samples were normalized for RNA and cDNA integrity and quantity by PCR amplification of human β-actin cDNA 19.
T cell clone M88.7 was stimulated with different concentrations of peptide (Leiden University Medical Center) in the presence or absence of 10 µg/ml brefeldin-A (Sigma-Aldrich). For stimulation without brefeldin-A, culture supernatants were harvested 6 h later and tested for TNF content. For stimulation with brefeldin-A, after 6 h cells were fixed for 10 min with PBS containing 4% paraformaldehyde, washed twice, and stored at 4°C. Fixed, stimulated cells were stained for cytokine. In brief, cells were stained with the PE-conjugated anti–IL-2 mAb (MQ1-17H12; BD PharMingen) at a concentration of 5 µg/ml for 30 min at room temperature. Reagent dilutions and washes were made with PBS containing 0.1% BSA (Sigma-Aldrich) and 0.1% saponin (Sigma-Aldrich). After staining and washes, cells were resuspended in PBS and analyzed on a FACScanTM (Becton Dickinson).
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
The M88.7 T Cell Clone Recognizes an Antigen Presented by HLA B*1302.
The CD8+ T cell clone M88.7, isolated from a tumor-invaded lymph node of melanoma patient M88, has no lytic activity but is able to secrete TNF and IL-2 on stimulation by autologous M88 melanoma cells (data not shown). To identify the restricting molecule, a panel of 12 melanoma cell lines not recognized by M88.7 T cells was transiently transfected with expression vectors for the different HLA molecules of the M88 patient. After transfection with the HLA B*1302 vector (pHLA B*1302), 6 of the 12 lines stimulated TNF release by M88.7 T cells (Fig. 1 A).
|
The NA88-A cDNA was sequenced (EMBL/GenBank/DDBJ accession no. AF164963). Expression of the NA88-A gene was then studied by reverse transcriptase (RT)-PCR using primers 400 and 970 (Fig. 2 A) chosen from the sequence. All six melanoma cell lines known to produce antigen (i.e., those that stimulated TNF production by M88.7 T cells after transfection with pHLA B*1302; see Fig. 1 A) yielded an RT-PCR product of the size expected for the NA88-A product. These RT-PCR products hybridized to NA88-A cDNA (data not shown). The remaining six cell lines known to not produce antigen (i.e., those unable to stimulate TNF production by M88.7 T cells, even after transfection with pHLA B*1302; Fig. 1 A) yielded no such RT-PCR product (data not shown).
|
NA88-A RNA Is Similar to the Homeoprotein-encoding HPX42B mRNA.
There was no long ORF on the 2.7-kb NA88-A cDNA, although it did contain an AATAAA polyadenylation signal and terminal poly A tail. While the sequence was new, its first 1,048 nucleotides proved to be 
90% identical to two noncontiguous parts (separated by an Alu family member) of the 3' untranslated region (UTR) of the previously reported HPX42B cDNA (Fig. 2 A; similar sequences are shown in black). HPX42B cDNA was originally obtained in a search for new homeobox genes, using degenerate PCR on cDNA derived from a purified population of CD34+ human hemopoietic cells 20. The similarity between the NA88-A and HPX42B sequences suggested that the cloned NA88-A cDNA could be part of the 3' UTR of a homeoprotein-encoding gene related to HPX42B. To test this hypothesis, we decided to isolate the genomic NA88-A gene by screening a human cosmid library.
A Processed Pseudogene Encodes NA88-A RNA.
We isolated one NA88-A cosmid and sequenced part of it to search for sequences coding for a HPX42B-like protein. The NA88-A sequence determined is shown in Fig. 2 B, aligned with the corresponding HPX42B gene sequence. (A region of chromosome 10 including the HPX42B has been sequenced. Comparison of this sequence with the HPX42B cDNA shows that the gene contains two introns. Their positions, but not their sequences, are marked in Fig. 2 B.) The NA88-A and HPX42B sequences are very similar. However, relative to the HPX42B gene, the NA88-A gene contains a number of mutations. One point mutation converts codon 22 of the HPX42B gene (tgg) into a premature stop codon (tga; boxed in Fig. 2 B). Relative to the HPX42B gene, the NA88-A gene also contains a number of deletions and insertions. While several of these remove or add codons without changing the reading frame, two of the deletions (of 1 and 80 bp) and one insertion (of 2 bp) do change it. As a result of all these mutations, the NA88-A gene no longer codes for a homeoprotein.
Further comparison of the genes shows that the NA88-A gene lacks equivalents of the HPX42B gene's introns (Fig. 2B and Fig. C). These observations strongly suggest that the NA88-A gene is a processed pseudogene, resulting from chromosomal integration of part of a retrotranscript of HPX42B mRNA. For splicing of the first HPX42B intron, the HPX42B mRNA employed to generate the NA88-A gene appears to have used an alternative 3' splice site (3'SS in Fig. 2B and Fig. C). We cannot tell how the second intron was removed, as the NA88-A gene contains a large deletion covering the region corresponding to the junction between HPX42B exons 2 and 3. One hallmark of processed pseudogenes is the presence of an A-rich sequence corresponding to the poly A tail of the original mRNA. However, as the NA88-A gene lacks sequences corresponding to the end of the HPX42B mRNA's 3' UTR (Fig. 2 A), this A-rich sequence is not expected to be present in the NA88-A pseudogene. In conclusion, our data show that the NA88-A antigen is coded for (a) by a processed pseudogene and (b) by sequences derived from the 3' UTR of the HPX42B mRNA.
Identification of the Peptide Recognized by M88.7 T Cells.
To identify the region of the 2,729-bp NA88-A cDNA coding for the antigenic peptide, we tested a number of subfragments for their ability to direct antigen production. TNF was produced by M88.7 T cells exposed to COS-7 cells cotransfected with pHLA B*1302 and vectors containing the NA88-A cDNA region 1–722, but not by M88.7 T cells exposed to COS-7 cells cotransfected with pHLA B*1302 and vectors containing the NA88-A cDNA region 1–700 (Fig. 3 A). These results positioned the end of the sequence coding for the antigenic peptide between nucleotides 700 and 722. Peptides covering all three reading frames encoded by the cDNA region 684–730 were synthesized (Fig. 3 A) and tested for their capacity to induce TNF production by M88.7 T cells. Only one of them led to significant TNF production (peptide 1, RMTQGQHFLQKV; Fig. 3 B).
|
HPX42B mRNA Expression Does Not Lead to T Cell Stimulation.
An alignment of the HPX42B and NA88-A cDNA sequences covering the region of the NA88-A sequence coding for the antigenic peptide is shown in Fig. 4 A. Part of the 3' UTR of the HPX42B cDNA can potentially encode the peptide MTQGQHFSQKV, which differs from the NA88-A antigenic peptide by a single substitution, serine (underlined) for leucine. To test if HPX42B mRNA expression leads to stimulation of M88.7 T cells, COS-7 cells were cotransfected with pHLAB*1302 and pHPX42B (Fig. 4 B), an expression vector containing the entire HPX42B cDNA. Transfected cells were unable to stimulate TNF production by M88.7 T cells (Fig. 4 C). However, COS-7 cells cotransfected with pHLAB*1302 and a point-mutated NA88-A expression vector coding for the serine-containing HPX42B peptide (pNA88-A Ser; Fig. 4 B) did stimulate TNF production by M88.7 T cells (Fig. 4 C), albeit less well than cells transfected with wild-type pNA88-A. Thus, although both NA88-A RNA and HPX42B mRNA code for a peptide capable of stimulating M88.7 T cells, only NA88-A RNA expression leads to peptide presentation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In contrast, the NA88-A's "parent" gene, the HPX42B gene, is transcribed in a variety of normal tissues (our unpublished observations). The 3' UTR of HPX42B mRNA has codons for a peptide that can be recognized by M88.7 T cells. However, unlike for NA88-A RNA, expression of HPX42B mRNA does not lead to T cell stimulation. One reason is that antigenic peptides must be excised from a precursor of 125 amino acids (which begins MTQGQHFSQKVWKCPEWE...; antigenic peptide in bold). Thus, expression of a mutated HPX42B mRNA, in which the Trp codon immediately following the peptide's terminal Val codon, is replaced by a stop codon, will lead to T cell stimulation. The absence of M88.7 T cell stimulation after wild-type HPX42B mRNA expression can be explained if downstream flanking residues stop appropriate peptides from being produced in quantities sufficient for presentation. Proteolytic cleavage COOH-terminal to the peptide's Val residue may be inefficient. Other cleavages may be favored that lead to peptides containing MTQGQHFSQKV but that cannot be presented productively to M88.7 T cells, due to a problem with peptide stability, TAP (transporter associated with antigen processing) transport, or MHC loading. Alternatively, the downstream residues could provoke cleavage within the MTQGQHFSQKV sequence itself. In favor of these suggestions, a number of reports exist 2122232425262728 describing the influence of flanking residues on epitope production and/or presentation.
None of the above problems is likely to adversely affect production of NA88-A antigenic peptide. The peptide MTQGQHFLQKV is the direct translation product of a short ORF (a stop codon follows the Val codon), and antigenic peptides can be derived from it after at most minimal NH2 terminal trimming. In addition to avoiding potential problems with precursor processing (see above), the lack of any significant processing requirement should limit the level of transcription and translation required to produce sufficient levels of peptide for T cell stimulation. This could be important, as the NA88-A RNA contains no primary ORF, but rather many short ORFs, and there is no obvious reason why the ORF coding for the antigenic peptide should be chosen for translation rather than any of the others. Translation of the antigenic peptide's ORF is thus likely to be inefficient.
In conclusion, the NA88-A gene codes for a melanoma-specific antigen today, in large part because, during its evolution, a point mutation transformed a Trp codon into a stop codon. This significantly augmented production of antigenic peptide.
|
Acknowledgments |
|---|
This work was supported in part by the Ligue Nationale contre le Cancer (axe "Immunologie des Tumeurs"), the Ligue des Deux Sèvres, the Ligue de Loire Atlantique, and the Association pour la Recherche contre le Cancer.
Submitted: 22 February 2000
Accepted: 13 March 2000
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Van den Eynde B.J. & Van der Bruggen P.. T cell defined tumor antigens, Curr. Opin. Immunol., 9, 1997, 684–693.[Medline]
Rosenberg S.A., Yannelli J.R., Yang J.C., Topalian S.L., Schwartzentruber D.J., Weber J.S., Parkinson D.R., Seipp C.A., Einhorn J.H. & White D.E.. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2, J. Natl. Cancer Inst., 865, 1994, 1159–1166.
Marchand M., Weynants P., Rankin E., Arienti F., Belli F., Parmiani G., Cascinelli N., Bourlond A., Vanwijck R. & Humblet Y.. Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3, Int. J. Cancer, 63, 1995, 883–885.[Medline]
Rosenberg S.A., Yang J.C., Schwartzentruber D.J., Hwu P., Marincola F.M., Topalian S.L., Restifo N.P., Dudley M.E., Schwarz S.L. & Spiess P.J.. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma, Nat. Med., 4, 1998, 321–327.[Medline]
Nestle F.O., Alijagic S., Gilliet M., Sun Y., Grabbe S., Dummer R., Burg G. & Schadendorf D.. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells, Nat. Med., 4, 1998, 328–332.[Medline]
Mayrand S.M. & Green W.R.. Non-traditionally derived CTL epitopesexceptions that prove the rules?, Immunol. Today., 19, 1998, 551–556.[Medline]
Guilloux Y., Lucas S., Brichard V.G., Van P.A., Viret C., De Plaen E., Brasseur F., Lethe B., Jotereau F. & Boon T.. A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyltransferase V gene, J. Exp. Med., 183, 1996, 1173–1183.
Van Den Eynde B.J., Gaugler B., Probst-Kepper M., Michaux L., Devuyst O., Lorge F., Weynants P. & Boon T.. A new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription, J. Exp. Med., 190, 1999, 1793–1800.
Robbins P.F., El Gamil M., Li Y.F., Fitzgerald E.B., Kawakami Y. & Rosenberg S.A.. The intronic region of an incompletely spliced gp100 gene transcript encodes an epitope recognized by melanoma-reactive tumor-infiltrating lymphocytes, J. Immunol., 159, 1997, 303–308.[Abstract]
Lupetti R., Pisarra P., Verrecchia A., Farina C., Nicolini G., Anichini A., Bordignon C., Sensi M., Parmiani G. & Traversari C.. Translation of a retained intron in tyrosinase-related protein (TRP)2 mRNA generates a new cytotoxic T lymphocyte (CTL)-defined and shared human melanoma antigen not expressed in normal cells of the melanocytic lineage, J. Exp. Med., 188, 1998, 1005–1016.
Wang R.F., Johnston S.L., Zeng G., Topalian S.L., Schwartzentruber D.J. & Rosenberg S.A.. A breast and melanoma-shared tumor antigenT cell responses to antigenic peptides translated from different open reading frames, J. Immunol., 161, 1998, 3598–3606.[Medline]
Wang R.F., Parkhurst M.R., Kawakami Y., Robbins P.F. & Rosenberg S.A.. Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen, J. Exp. Med., 183, 1996, 1131–1140.
Ronsin C., Chung-Scott V., Poullion I., Aknouche N., Gaudin C. & Triebel F.. A non-AUG-defined alternative open reading frame of the intestinal carboxyl esterase mRNA generates an epitope recognized by renal cell carcinoma-reactive tumor-infiltrating lymphocytes in situ, J. Immunol., 163, 1999, 483–490.
Aarnoudse C.A., van den Doel P.B., Heemskerk B. & Schrier P.I.. Interleukin-2-induced, melanoma-specific T cells recognize CAMEL, an unexpected translation product of LAGE-1, Int. J. Cancer., 82, 1999, 442–448.[Medline]
Pandolfino M.C., Viret C., Gervois N., Guilloux Y., Davodeau F., Diez E. & Jotereau F.. Specificity, T cell receptor diversity and activation requirements of CD4+ and CD8+ clones derived from human melanoma-infiltrating lymphocytes, Eur. J. Immunol, 22, 1992, 1795–1802.[Medline]
Maniatis T., Fritsh E.F. & Sambrook J., Molecular Cloning, 2nd ed, 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYpp. 1,659.
Brichard V., Van Pel A., Wolfel T., Wolfel C., De Plaen E., Lethe B., Coulie P. & Boon T.. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas, J. Exp. Med., 178, 1993, 489–495.
Hansen M.B., Nielsen S.E. & Berg K.. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill, J. Immunol. Methods., 119, 1989, 203–210.[Medline]
Labarriere N., Diez E., Pandolfino M.C., Viret C., Guilloux Y., Le G.S., Fonteneau J.F., Dreno B. & Jotereau F.. Optimal T cell activation by melanoma cells depends on a minimal level of antigen transcription, J. Immunol., 158, 1997, 1238–1245.[Abstract]
Moretti P., Simmons P., Thomas P., Haylock D., Rathjen P., Vadas M. & D'Andrea R.. Identification of homeobox genes expressed in human haemopoietic progenitor cells, Gene., 144, 1994, 213–219.[Medline]
Theobald M., Ruppert T., Kuckelkorn U., Hernandez J., Haussler A., Ferreira E.A., Liewer U., Biggs J., Levine A.J. & Huber C.. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope, J. Exp. Med., 188, 1998, 1017–1028.
Bergmann C.C., Tong L., Cua R., Sensintaffar J. & Stohlman S.. Differential effects of flanking residues on presentation of epitopes from chimeric peptides, J. Virol., 68, 1994, 5306–5310.
Del Val M., Schlicht H.J., Ruppert T., Reddehase M.J. & Koszinowski U.H.. Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein, Cell., 66, 1991, 1145–1153.[Medline]
Eisenlohr L.C., Yewdell J.W. & Bennink J.R.. Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes, J. Exp. Med., 175, 1992, 481–487.
Niedermann G., Butz S., Ihlenfeldt H.G., Grimm R., Lucchiari M., Hoschutzky H., Jung G., Maier B. & Eichmann K.. Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules, Immunity., 2, 1995, 289–299.[Medline]
Ossendorp F., Eggers M., Neisig A., Ruppert T., Groettrup M., Sijts A., Mengede E., Kloetzel P.M., Neefjes J. & Koszinowski U.. A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation, Immunity., 5, 1996, 115–124.[Medline]
Shastri N., Serwold T. & Gonzalez F.. Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C-terminal flanking residues, J. Immunol., 155, 1995, 4339–4346.[Abstract]
Yellen-Shaw A.J., Wherry E.J., Dubois G.C. & Eisenlohr L.C.. Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein, J. Immunol., 158, 1997, 3227–3234.[Abstract]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| TABLE OF CONTENTS |
|
