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The Journal of Immunology, 2000, 165: 869-877.
Copyright © 2000 by The American Association of Immunologists

Identification of a Novel Tumor-Specific CTL Epitope Presented by RMA, EL-4, and MBL-2 Lymphomas Reveals Their Common Origin

Thorbald van Hall, Jeroen van Bergen, Peter A. van Veelen, Margriet Kraakman, Lukas C. Heukamp, Frits Koning, Cornelis J. M. Melief, Ferry Ossendorp and Rienk Offringa1

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
C57BL/6 mice generate a vigorous H-2Db-restricted CTL response against murine leukemia virus (MuLV)-induced tumors. For many years it has been suggested that this response is directed to an MuLV-encoded peptide as well as to a nonviral tumor-associated peptide. Recently, a peptide from the leader sequence of gag was demonstrated to be the MuLV-derived epitope. Here we describe the molecular identification of the tumor-associated epitope. Furthermore, we show that the CTL response against this epitope can restrict the outgrowth of MuLV-induced tumors in vivo. The epitope is selectively presented by the MuLV-induced T cell tumors RBL-5, RMA, and MBL-2 as well as by the chemically induced T cell lymphoma EL-4. Intriguingly, these tumors share expression of the newly identified epitope because they represent variants of the same clonal tumor cell line, as evident from sequencing of the TCR {alpha}- and ß-chains, which proved to be identical. Our research shows that all sources of RBL-5, RMA, RMA-S, MBL-2, and EL-4 tumors are derived from a single tumor line, most likely EL-4.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Murine tumor models are essential and powerful tools in tumor immunological research. During the last decade, the molecular identity of a number of tumor-associated CTL epitopes has been elucidated (for review, see Refs. 1, 2, 3). This knowledge has been widely exploited to test the effectiveness as well as the drawbacks of Ag-based immunotherapy against cancer.

CTL epitopes presented by murine tumors that are induced by viruses or viral oncogenes are generally virus encoded (4). Next to these immunogenic epitopes four additional classes of tumor-specific T cell epitopes have been described. First, several carcinogen-induced murine tumors were shown to express unique CTL epitopes that arose from point mutations in cellular genes. The critical event was not a change in the expression of these genes, but substitution of an amino acid in a peptide that resulted in increased MHC binding and/or T cell recognition. Examples of such CTL epitopes have been found at the surface of the murine mastocytoma line P815 and its mutagenized variants (5). Second, experiments with the P815 system revealed that ectopic expression of cellular genes could lead to presentation of epitopes derived from these genes (P815A/B Ag) (6). By now, many other members of this so-called testis cancer family of tumor Ags have been identified in human tumors (7). A third class of tumor-specific CTL epitopes commonly found in murine tumors is encoded by (re)activated endogenous retroviral sequences that are integrated in the cellular genome. For instance, the widely used EL-4 lymphoma expresses a CTL epitope derived from an endogenous mouse mammary tumor virus (MMTV)2 envelope protein (8), whereas the C26 colon carcinoma was found to express an epitope encoded by an endogenous murine leukemia virus (MuLV) (9). Induced expression of these sequences appears to be the result of DNA demethylation (10, 11). A fourth category of tumor-associated epitopes, which has originally been defined in human melanomas, concerns tissue lineage-specific Ags such as the melanocyte-specific Ags (12). A murine counterpart has been found in the B16 melanoma (13).

MuLV are naturally occurring retroviruses that induce hematological tumors in mice. The crucial role of CTL as well as Th cells in the rejection of MuLV-induced tumors has been firmly established (14). Widely used tumor cell lines transformed by antigenically related MuLV are the Friend MuLV-induced erythroleukemia FBL-3, the Moloney MuLV-induced T cell lymphoma MBL-2, and the Rauscher MuLV-induced T cell lymphoma RBL-5 and its derivatives RMA and RMA-S. For these tumors, two CTL epitopes and one Th epitope were molecularly identified and were all found to be of viral origin (15, 16, 17). Interestingly, the CTL response against the MBL-2 tumor was shown to include an additional specificity directed to a nonviral Ag (18). Although the existence of this nonviral tumor epitope was postulated in the eighties, its identity remained an enigma despite the fact that several laboratories have since attempted to identify this epitope. In the present paper we describe the molecular identification of this epitope that is selectively expressed by certain T cell lymphomas. In addition, we provide evidence that these tumor cell lines originate from the same clonal cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and mice

All cell lines used in this study were derived from C57BL/6 (B6, H-2b) mice. RMA and RMA-S cell lines are T cell lymphomas derived from the Rauscher MuLV-induced RBL-5 cell line (19). MBL-2 and FBL-3 are isolated from a Moloney and a Friend MuLV inoculated C57BL/6 mouse, respectively (20). EL-4 is a dimethylbenzanthracene-induced thymoma cell line (21). Transformed mouse embryo cells are primary embryonic cells transfected with plasmids encoding activated oncogenes (22). B16 melanoma cells and MCF1233 MuLV-induced B cell lymphomas 771 and 786 have been described previously (13, 23). The Moloney MuLV-induced pro-B cell lymphomas 33H2 and 33A3 were provided by Dr. M. Schilham (Leiden University Medical Center, Leiden, The Netherlands). FRE.Db and FRE.Kb are stable transfectants of the Fisher rat embryo cell line. HeLa.Db and T2.Db cells are stable H-2Db transfectants of the human cervical carcinoma cell line HeLa and T cell hybrid T2, respectively. Freshly isolated sarcomas were obtained by i.m. injections of the Moloney murine sarcoma and leukemia virus complex as previously described (24). All cell lines were cultured in IMEM (BioWhittaker Europe, Verviers, Belgium) supplemented with 8% heat-inactivated FCS (Life Technologies, Breda, The Netherlands), 2 mM L-glutamine (ICN Biomedicals, Costa Mesa, CA), 100 IU/ml penicillin (Yamanouchi Pharma, Leiderdorp, The Netherlands), and 30 µM 2-ME (Merck, Darmstadt, Germany) at 37°C in humidified air with 5% CO2.

C57BL/6 mice were bred and obtained from the TNO-PG breeding facility (Leiden, The Netherlands). C57BL/6 nu/nu mice were obtained from IFFA Credo (Zeist, The Netherlands). All mice were kept under specific pathogen-free conditions in the animal facility of Leiden University Medical Center.

Generation and analysis of CTL clones

The env-specific CTL clone 10B6 was generated by immunization of a B6.CH-2bm13 mouse with Moloney MuLV; it recognizes the SSWDFITV epitope presented by H-2Kb (env; aa 189–196) as described previously (15). CTL clones specific for the tumor epitope and the CCLCLTVFL epitope (gag leader; aa 75–83), presented by H-2Db, were derived from spleen cells of C57BL/6 (B6) mice immunized with irradiated RMA tumor cells. Bulk cultures were restimulated weekly with irradiated RMA cells and irradiated naive spleen cells as feeders in complete culture medium supplemented with 2% (v/v) conditioned medium from Con A- and PMA-stimulated rat splenocytes. After 3–5 wk the conditioned medium was gradually replaced by 10 Cetus units of recombinant human IL-2 (Eurocetus, Amsterdam, The Netherlands). CTL clones were obtained by standard limiting dilution and were weekly restimulated with RMA cells, feeders, and IL-2. All obtained CTL clones expressed {alpha}ß-TCR and CD8 as tested by flow cytometry.

The cytolytic activity of CTL clones was measured by means of a chromium (51Cr) release assay, as described previously (14). The mean percent specific lysis of triplicate wells was calculated as follows: % specific lysis = ([cpm experimental release - cpm spontaneous release]/[cpm maximum release - cpm spontaneous release]) x 100. Measurement of secreted TNF-{alpha} by stimulated CTL was performed with a bioassay, using WEHI 164 clone 13 cells, as described previously (25). The percentage of TNF-{alpha} released in triplicate wells was calculated as follows: % TNF-{alpha} release = [(A550–650 experimental wells - A550–650 wells containing medium only)/(A550–650 wells containing 500 pg/ml TNF-{alpha} - A550–650 wells containing medium only)] x 100.

Moloney virus infection

In vitro virus infections were performed with Abelson Moloney virus collected from supernatant of NIH-3T3 Abelson virus nonproducer cells (ANN-1), which were productively infected with cloned Moloney MuLV (26). Batches of virus-containing supernatants were collected after 24 h. Virus batches were stored at -80°C. FRE transfectants were infected with Abelson Moloney MuLV by culturing the cells for 3–4 days in complete culture medium containing 50% (v/v) virus supernatant from ANN-1 cells with 10 µg/ml polybrene (Sigma-Aldrich, Zwyndrecht, The Netherlands).

MHC class I-peptide binding assay

The binding capacity of peptides to H-2Db was determined using the RMA-S binding assay as previously described (27). Briefly, RMA-S cells were cultured for 36 h at 26°C and were added to serial dilutions of peptide. After 4 h of incubation at 37°C, cells were washed and stained with the mAb 28.14.8S specific for H-2Db and FITC-labeled goat-anti-mouse Ig. Fluorescence was determined using a FACScan cytometer (Becton Dickinson, Mountain View, CA). The fluorescence index was calculated as follows: FI = (mean fluorescence with peptide/mean fluorescence without peptide).

RT-PCR of TCR genes

Total RNA from 107 tumor cells was isolated using TRIzol according to the manufacturer’s recommendation (Life Technologies). cDNA was generated by oligo(T)-primed RNA using AMV reverse transcriptase (Promega, Madison, WI). The reaction was heat inactivated, diluted in water, and stored at 20°C until usage. PCR incubation temperatures were 95, 58, and 72°C subsequently (1 min for all steps, 30 cycles). Primer sequences for determination of TCR V{alpha} and Vß gene usage were previously published (28). The downstream primer used for the TCR C{alpha} region was 5'-TGG CGT TGGTCT CTT TGA AG-3', resulting in a product of ~400 bp, and the primer for the Cß region was 5'-CTT GGG TGG AGT CAC ATT TCT C-3', resulting in a product of ~200 bp. PCR products were directly cloned using a TOPO TA cloning kit (Invitrogen, San Diego, CA) and were sequenced using standard procedures.

Purification of eluted peptides

Peptides were eluted out of purified H-2Db or H-2Kb molecules as previously described (29). Briefly, MHC class I molecules were purified by affinity chromatography with 28-14-8S-coupled (Db-specific Ab) or B8-24-3-coupled (Kb-specific Ab) cyanogen bromide-activated Sepharose 4B beads (Pharmacia LKB, Uppsala, Sweden). Peptides were eluted by acid treatment and were separated from the heavy chains and ß2-microglobulin by filtration over a 10-kDa cutoff Centricon filter (Amicon, Lexington, MA). Peptides were fractionated using reverse phase micro C2C18 HPLC (Smart System, Pharmacia). Buffer A was 0.1% trifluoroacetic acid in water; buffer B was 0.1% trifluoroacetic acid in acetonitrile.

Mass spectrometry

Electrospray ionization mass spectrometry was performed on a hybrid quadrupole time-of-flight mass spectrometer (Q-TOF, Micromass, Manchester, U.K.) equipped with an on-line nanoelectrospray interface (capillary tip, 20 µm internal diameter x 90 µm outer diameter) with an approximate flow rate of 250 nl/min. This flow was obtained by splitting of the 0.4 ml/min flow of a conventional high pressure gradient system using an Acurate flow splitter AC-400-VAR (LC-Packings, Amsterdam, The Netherlands). Injections were made with a dedicated micro/nano HPLC autosampler (FAMOS, LC-Packings). The analytical HPLC column was packed with PEPMAP (15 cm x 75 µm, 5-µm particle size; LC-Packings). The gradient went from 10% B to 90% B in 30 min (A: 95/5/1, v/v/v, water/methanol/acetic acid; B: 10/90/1, v/v/v, water/methanol/acetic acid). Mass spectra were recorded from a mass of 50–2000 Da every second with a resolution of 5000 FWHM. The resolution allows direct determination of the monoisotopic mass, also from multiple charged ions. In the MS/MS mode, ions were selected with a window of 2 Da with the first quadrupole, and fragments were collected with high efficiency with the orthogonal time-of-flight mass spectrometer. The collision gas applied was argon (4 x 105 mbar), and the collision voltage was ~30 V (for similar procedures, see Ref. 29, 30).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of CTL specific for a tumor-associated epitope on RMA

The CTL response against MuLV-induced lymphomas of the Friend, Moloney, and Rauscher (FMR) types in C57BL/6 (H-2b) is predominantly H-2Db restricted (15, 24). A peptide derived from the gag leader sequence was identified as the major target epitope (gagL75–83)(16). In addition, a subdominant H-2Kb-restricted response was described toward a viral env-derived peptide, env189–196 (15). By analysis of several independent T cell cultures from RMA-immunized mice we isolated CTL lines with the previously described gagL75–83 specificity (Fig. 1Go, A and B). However, some RMA-specific cultures failed to recognize this epitope and the env189–196 epitope (Fig. 1Go, C and D). To determine whether these CTL recognized other virus-encoded peptides we tested Moloney MuLV-infected FRE.Db and FRE.Kb cells. GagL75–83- and env189–196-reactive CTL specifically recognized MuLV-infected FRE.Db and FRE.Kb cells, respectively (Fig. 1Go, F and G), indicating proper MuLV infection as well as MHC class I processing and presentation. In contrast, CTL that did not recognize one of the defined viral epitopes (Fig. 1Go, C and D) also failed to recognize Moloney MuLV-infected cells (Fig. 1GoE), whereas RMA tumor cells were efficiently recognized. In the remainder of this manuscript we will refer to these CTL as anti-tumor CTL. Also, transiently expressed cDNAs encoding viral gag, pol, or env genes in HeLa.Db and HeLa.Kb cells consistently failed to sensitize these cells for recognition by these CTL, while the transfected cells were stimulatory for anti-env189–196 and anti-gagL75–83 CTL (data not shown). Taken together, our data suggested that the anti-tumor CTL are directed against a nonviral epitope expressed by RMA cells. Several groups have reported CTL with comparable, unknown specificity (16, 31, 32), and the involved epitope has long been searched for. We therefore set out to identify the cognate peptide of these CTL.



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  		FIGURE 1. RMA presents two distinct Db-restricted CTL epitopes. Mice were immunized twice with irradiated RMA cells, and bulk cultures were generated by restimulation of splenocytes in vitro in the presence of RMA. The specificity of the T cell cultures was analyzed after two restimulations. A–D, Cytolytic activity against chromium-labeled RMA, HeLa.Db, and HeLa.Db cells loaded with 5 µg/ml gagL75–83 peptide. Of the eight RMA-specific T cell cultures, five reacted to the gagL75–83 peptide, whereas three did not. Two of each type are shown: ln24 (A), ln42 (B), ln17 (C), and ln26 (D). E–G, Comparison of three types of RMA-specific CTL, as tested in a TNF secretion assay using the indicated target cells. Fischer rat embryo cells (FRE) stably transfected with H-2Db or Kb were infected with Moloney MuLV where indicated (MoMuLV). TNF secretion by anti-tumor CTL (ln 26; E), gagL75–83-specific CTL (F), or env189–196-specific CTL (G) was measured in a WEHI bioassay.

 
Anti-tumor CTL selectively recognize T lymphoma cell lines

Several CTL clones were derived from bulk cultures displaying the anti-tumor reactivity. A large panel of syngeneic murine tumor cells of different tissue origin was examined for recognition by these clones in cytotoxicity assays and cytokine release assays. CTL clones specific for the viral gagL75–83 peptide lysed all cell lines containing the FMR MuLV, because this peptide sequence is conserved in this MuLV type. This recognition was independent of the lymphoid lineage of the transformed cell lines (Fig. 2GoB). MuLV-induced T cell lymphomas (RMA and MBL-2), MuLV-induced pro-B cell lymphomas (33A3 and 33H2), and an MuLV-induced erythroleukemia (FBL-3) were all lysed, whereas chemically induced EL-4 cells that do not express FMR MuLV Ags were not lysed (Fig. 2GoB). In contrast, anti-tumor CTL clones only recognized the T cell lymphomas RMA, MBL-2 and EL-4 (Fig. 2GoA), as well as the RBL-5 cell line, the parental cell of RMA (data not shown). The B cell lymphomas and erythroleukemia cells were not lysed (Fig. 2GoA). This recognition pattern suggested that anti-tumor CTL recognize a nonviral Ag that is selectively expressed by T cell lymphomas. In accordance with this idea, mouse embryo cells transformed by a variety of oncogenes, B16 melanoma cells, and MCF MuLV-induced B cell lymphomas 771 and 786 were not recognized by the anti-tumor CTL (not shown). In addition, freshly isolated sarcomas that were induced by i.m. inoculation of the Moloney MuLV/Moloney sarcoma virus complex did not express the tumor epitope, whereas these sarcomas did express the Moloney virus encoded epitopes (data not shown). Finally, we tested syngeneic Con A-activated T cell blasts as a source of nontransformed T cells. These cells were not recognized by our anti-tumor CTL (data not shown). In conclusion, our anti-tumor CTL recognize an epitope that is selectively expressed on the widely used T cell lymphomas RMA/RBL-5, MBL-2, and EL-4 and that is most likely of nonviral origin.



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  		FIGURE 2. Anti-tumor CTL selectively recognize T cell lymphomas. Cytolytic activity of anti-tumor CTL (A) and gagL75–83-specific CTL (B) against a panel of lymphoid tumors was tested in a chromium release assay. Long term CTL clones were used (cl26 and cl1, respectively) that had been propagated in vitro for at least 4 mo. RMA, MBL-2, and EL-4 are tumors of the T cell lineage. 33H2 and 33A3 are Moloney MuLV-positive pro-B cell lymphomas, and FBL-3 is a Friend MuLV-induced erythroleukemia. The precise origins of the tumor cell lines are described in Materials and Methods. One representative experiment of three is shown.

 
Purification and sequencing of the novel CTL epitope

We set out to identify the epitope of the anti-tumor CTL through biochemical purification and sequencing by mass spectrometry. Peptides were eluted from immunopurified Db and Kb molecules from 20 x 109 MBL-2 cells and fractionated by reverse phase HPLC. This showed that a Db-binding peptide, present in a fraction eluting from the column at a low acetonitrile concentration (~15%), was capable of sensitizing target cells for lysis by anti-tumor CTL (data not shown). Subsequently, Db-binding peptides from 120 x 109 RMA cells were separated by reverse phase HPLC. Anti-tumor CTL selectively recognized four fractions (Fig. 3Go), which were pooled and subsequently separated by reverse phase HPLC using a methanol gradient. A single fraction sensitized target cells for lysis by the CTL (data not shown). In a third HPLC run, the active fraction was further separated on a nano-LC column that was connected to a mass spectrometer. Twelve fractions were collected in a 96-well plate, and only one fraction was recognized by anti-tumor CTL. Nevertheless, five detectable peptide masses were present in this fraction, and collision spectra (MS/MS) of all these were recorded (data not shown). Interpretation of one of the MS/MS spectra (Fig. 4Go), with an ion mass of 472.8 (2+), yielded the partial six-amino acid C-terminal sequence (D/E)NA(K/Q)A(I/L). However, ambiguities were left for the sequence of the amino acids at the N-terminus. Peptide mixes were synthesized containing the sequence XXXENAKA(I/L) and XXXENAQA(I/L), where several different amino acids were placed on the first three positions (X). Testing these mixes with anti-tumor CTL revealed that only a glutamine (Q) at position 7 resulted in strong recognition. Further testing of peptides with various amino acids on position 4 containing random amino acids on the first three positions showed by far the strongest CTL response to the peptides with a glutamic acid (E) on this position. In contrast, the order and character of the first three amino acids did not have any impact on the CTL recognition of the peptide. Comparison between the MS/MS spectra of several active synthetic candidates and the MS/MS spectrum of the eluted peptide determined in the active fraction (Fig. 4Go) led to the following four coeluting peptides: NKGENAQAI, NKGENAQAL, KNGENAQAI, and KNGENAQAL. These peptides comply very well with the published binding motif for H-2Db (33). Importantly, we found no matches for either of the candidate sequences in the different available protein and DNA databases. This indicates that the epitope recognized by our anti-tumor CTL is most likely derived from an as yet unknown gene.



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  		FIGURE 3. Purification and recognition of RMA eluted peptides by anti-tumor CTL. Peptides that were eluted from H-2Db molecules of 120 x 109 RMA cells were fractionated by reverse phase HPLC and were loaded on chromium-labeled HeLa.Db cells. {blacksquare}, Specific lysis by anti-tumor CTL (ln17) as indicated on the left y-axis. In this experiment duplicate wells were incubated with an E:T cell ratio of 7.5:1. The specific lysis of RMA cells was 55%. Each test well contained peptide material corresponding to 4 x 106 RMA cells. The right y-axis shows the percentage of acetonitrile in each HPLC fraction, as indicated by the solid line.

 


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  		FIGURE 4. MS/MS fragmentation spectrum of the candidate peptide. Peptide fragments detected after collision (30 V) of precursor ion mass of 943.5, present as m/z 472.8 (2+). Peaks with m/z 187.6 and 156.3 were considered not to belong to m/z 472.8, because these masses were found in MS/MS spectra of other peptide candidates as well. Individual N-terminal b and C-terminal y ions are depicted with their corresponding masses in the figure. The top line indicates the deduced amino acid sequence. The leucine (L) at the C-terminus could represent an isoleucine (I) as well.

 
CTL recognition and binding affinity of synthetic peptide candidates

Because biochemical analysis did not provide further indications in favor of one of the peptide candidates, all four were tested in CTL recognition and MHC class I binding assays. At high peptide concentrations (high nanomolar range) no differences in lysis were observed among the four peptides by anti-tumor CTL clones (Fig. 5GoA). However, when the peptides were titrated, the recognition of the peptides carrying a C-terminal leucine (L) decreased markedly faster than that of the peptides with an isoleucine (I) at this position. The concentration needed for half-maximal lysis of the L peptides was at least 10-fold higher than the concentration needed for the I-containing peptides (Fig. 5GoA). The variation at the N-terminus (NK/KN)GENAQAI or (NK/KN)GENAQAL of the peptide did not influence recognition by the CTL. Peptide binding studies with the four peptides were performed with a widely used assay that measures the stabilization of MHC class I molecules at the surface of TAP-deficient RMA-S cells by exogenous loading of synthetic peptides (27). Fig. 5GoB shows that all four peptides can be considered as intermediate to strong Db-binding peptides compared with several known Db-binding CTL epitopes that have been tested in our laboratory (27). However, a reproducible 5-fold difference was observed between the peptides with isoleucine or leucine as C-terminal residue (Fig. 5GoB). The peptides ending with isoleucine showed stronger binding, suggesting that this accounts for the fact that they are better targets for CTL recognition.



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  		FIGURE 5. CTL recognition and MHC class I binding of four epitope candidates. A, Synthetic peptides were serially diluted and loaded on chromium-labeled T2.Db cells. Specific chromium release due to lysis by anti-tumor CTL (cl17) is indicated. An E:T cell ratio of 20:1 was used. The lysis of T2.Db cells in the absence of peptide was 5%. B, Binding of synthetic peptides to H-2Db molecules on the surface of RMA-S cells at several concentrations. Indicated is the fluorescence index of H-2Db expression levels in the presence vs the absence of peptide. Both experiments were repeated three times with similar outcome.

 
In vivo responses toward the newly identified epitope

The efficacy and specificity of the CTL response against the newly identified epitope were further analyzed in vivo. First, we tested the efficacy of adoptively transferred CTL against RMA in tumor-bearing mice. Nude mice were injected i.p. with RMA tumor cells, leading to progressive tumor burden within 3 wk. Long-lasting tumor protection was observed for all mice that were treated with the gagL75–83-specific CTL and for approximately half the mice that received the anti-tumor CTL (Fig. 6Go). No difference in tumor protection was detected between mice receiving the CTL i.v. (Fig. 6Go) or i.p. (not shown). All mice receiving IL-2 only or saline developed progressively growing tumors.



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  		FIGURE 6. Adoptive transfer of CTL prevents RMA tumor outgrowth. GagL75–83-specific CTL (cl1) and anti-tumor CTL (cl26) were adoptively transferred into syngeneic nude mice. CTL were injected (i.v.) on the same day as 103 RMA tumor cells (i.p.) and 105 Cetus units rIL-2 (s.c.) emulsified in IFA. After 1 wk mice received an additional depot of IL-2. Each group contained five mice. Mice were sacrificed after progressive development of ascites. Tumor-free survival rates did not change after the last day shown. Comparable results were observed in tumor-bearing mice receiving CTL i.p.

 
Subsequently, the effect of peptide vaccination was examined in immunocompetent C57BL/6 mice in a prophylactic immunization model. Immunization with the gagL75–83 peptide emulsified in IFA was previously shown to protect mice against a subsequent challenge with RMA tumor cells (14). Moreover, the protective effect was greatly enhanced when a specific Th epitope from MuLV was included in the peptide vaccine (14). Here, we compared peptide vaccines containing the gagL75–83 peptide, the env187–196 peptide, or the newly identified CTL epitope together with the Th peptide (referred to as molH). In two independent experiments (Table IGo), the NKGENAQAI and NKGENAQAL peptides as well as the KNGENAQAI and KNGENAQAL peptides were combined in one vaccine. Injection of these peptides in combination with the helper peptide resulted in more efficient tumor protection than vaccination with the helper peptide alone. In contrast, addition of the subdominant env187–196 CTL epitope did not improve the tumor protective capacity. Taken together, these data show that anti-tumor CTL exhibit in vivo tumoricidal activity in both peptide-vaccinated mice and an adoptive immunotherapy setting. Moreover, CTL that are induced by the different synthetic peptides are capable of recognizing the naturally presented peptide on the surface of RMA. The slightly stronger protective effect of vaccination with the NKGENAQA(I/L) peptides suggests, but does not prove, that the asparagine residue represents the N-terminus of the natural CTL epitope.


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  		Table I. Peptide vaccination induces protective responses against RMA tumorsa

 
RMA, MBL-2, and EL-4 all express identical TCR chains

We found the tumor-derived CTL epitope to be expressed by three different T cell lymphomas (RMA, MBL-2, and EL-4). These tumors are widely considered to be independently derived, distinct cell lines. Therefore, we considered this novel epitope to be a transformation-associated Ag that is exclusively expressed in the T cell lineage. By coincidence, we were informed by C. G. Brooks (Newcastle, U.K.) about the fact that these tumor cells expressed the same TCR Vß region (Vß12), including fully identical CDR3 regions (43) (TCR ß-chain sequence accession no. AF020206). This prompted us to analyze the different tumor cells in our laboratory for their TCR ß- and {alpha}-chains. By TCR-specific PCR analysis and subsequent DNA sequencing we confirmed the identical Vß usage in these cell lines. Furthermore, we found that all recognized tumor cell lines also shared the usage of one TCR V{alpha} gene (V{alpha}10), as shown in Fig. 7Go for RMA, MBL-2, and EL-4. By sequencing the V{alpha}-J{alpha}-C{alpha} region we established that these tumors shared identical junctional regions. This TCR {alpha} sequence is filed in the GenBank database (accession no. AF218247). We excluded the possibility of cross-contamination of cell lines within our laboratory, because we confirmed these findings with cell samples obtained from several other laboratories (not shown, see Fig. 7Go). The common origin of these cell lines is most likely the result of a cross-contamination during in vitro culture or in vivo passage many years ago, as described for other widely used cell lines (34, 35, 36). Importantly, these findings also imply that the epitope described here represents a unique epitope rather than a T-cell-lineage-specific tumor Ag. The sequence of this novel CTL epitope is not comprised within the clonotypic TCR {alpha} or ß sequences or by MuLV sequences, suggesting that it is encoded by an as yet unknown cellular gene.



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  		FIGURE 7. RMA, MBL-2, and EL-4 express the same TCR V{alpha} and Vß genes. RT-PCR using a panel of V{alpha}- and Vß-specific primers revealed that RMA, MBL-2, and EL-4 cells expressed V{alpha}10 and Vß12 gene segments. Shown are PCR products (400 bp for V{alpha} and 200 bp for Vß genes) from cDNA of the indicated T cell cultures together with a 100-bp marker. Clone 3 (cl 3) is a control CTL clone expressing V{alpha}3 and Vß8 gene segments, and 1H11 is a control CTL clone expressing the V{alpha}10 gene (42 ). Con A cDNA was derived from 4-day Con A (5 µg/ml)-stimulated spleen cells, containing all TCR variable genes. Not shown are tumor cells lines that also generated PCR products with V{alpha}10 and Vß12 primers: RBL-5, RMA-S, and the transfectants RMA.MUC1 (Imperial Cancer Research Fund, Guy’s Hospital, London, U.K.) and RMA-S.B7 (Karolinska Institute, Stockholm, Sweden).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The exclusive expression of a unique CTL epitope by RMA, MBL-2, and EL-4 in combination with their identical TCR {alpha}- and ß-chains have unequivocally revealed that these widely used cell lines have a common origin. Cross-contamination during in vitro culture or in vivo passage of these cell lines most likely lies at the basis of this phenomenon. Early analyses of the immune responses to these tumors have determined both virus-directed and tumor cell-directed CTL (18). Recently, an important virus-derived CTL epitope was identified (16). In the present paper we describe identification of the tumor-associated CTL epitope as determined by mass spectrometry. The nonamer peptide NKGENAQAI most likely represents the natural epitope sequence, although we cannot exclude that the order of the first two residues is reversed and/or that the C-terminal residue is leucine instead of isoleucine.

The newly identified CTL epitope was selectively expressed on the transformed T cells RBL-5/RMA, MBL-2, and EL-4. B cell lymphomas and murine tumors of other origin were not recognized by these CTL. The fact that this epitope was presented by T cell lymphomas regardless of the transforming agent (MuLV induced as well as chemically induced; see Fig. 2GoA) suggested that this epitope originated from a T cell differentiation Ag. However, in close collaboration with the laboratory of C. G. Brooks (Newcastle, U.K.) we discovered that all cell lines recognized by our CTL express identically rearranged TCR {alpha} and ß genes. This indicates that these cell lines, which have widely been considered as distinct and independently isolated cell lines, represent cross-contaminants of the same cell line. Importantly, this cross-contamination was demonstrated in two independent laboratories, which never exchanged cell lines. Furthermore, the same TCR profiles were found in cell samples obtained from yet other laboratories. The cell lines that are involved in this widespread cross-contamination are MBL-2, EL-4, and RBL-5 together with its derivatives RMA and RMA-S. Importantly, several of these cell lines display very distinct phenotypes, such as the TAP deficiency in RMA-S cells that leads to low expression of MHC class I on the cell surface and the absence of FMR type MuLV proteins from EL-4.

In itself, the fact that such widely used cell lines have been cross-contaminated is not surprising, since this has been described for other commonly used human tumor cell lines such as the HeLa cervical carcinoma cell line (34, 37). Nevertheless, this report together with that of Brooks and co-workers (see Footnote 3) presents the first case of such widespread cross-contamination of murine cell lines, which is even more striking because these cell lines are among the most widely employed murine tumor lines for immunological research. Although it is very difficult to make a reliable reconstruction of the events that resulted in the cross-contamination, there are two basic possibilities. First, all cell lines may be derived from EL-4, a chemically induced cell line generated in 1945 (21). Secondary infection with Rauscher or Moloney MuLV of EL-4 could have resulted in RBL-5 and MBL-2. Transduction of such retroviruses can readily occur in laboratories where such retroviruses and cell lines are propagated in parallel. Virus infection could also have occurred by in vivo passage of these cells, which was a commonly used procedure to propagate tumors several years ago. Hence, we have observed that Rauscher and Moloney MuLV, the reported transforming agents of RMA and MBL-2, respectively, have strong sequence homology in immunologically relevant regions. It has never been carefully checked whether the integrated MuLV gene copies in these tumor cell lines display clear differences. Alternatively, all cell lines may be derived from the same Rauscher/Moloney MuLV-induced tumor, with EL-4 being a virus-loss variant. This latter possibility we consider unlikely (see below). Importantly, screening of cell lines from several laboratories confirmed that this cross-contamination is widespread, implicating that it took place many years ago. The fact that the RBL-5 cell line, which gave rise to RMA and RMA-S in the eighties, is also involved in the cross-contamination establishes the idea that the cross-contamination must have taken place before the mid-eighties. Of note, the Friend MuLV-induced erythroleukemia FBL-3 is a distinct cell line and has no relationship to the EL-4/RBL-5/MBL-2 tumor lines, because it obviously does not express TCR genes.

The finding that all tumor cell lines that present the novel tumor-specific CTL epitope are all derivatives of one common tumor cell line implies that this epitope is uniquely expressed by one tumor rather than a lineage-specific Ag. Similar CTL epitopes were shown to be derived from point-mutated cellular genes and have especially been found in murine tumors that were induced by potent carcinogens (5, 38, 39). In this respect it is of interest to note that EL-4 cells are obtained from a dimethylbenzanthracene-treated mouse, a compound known to efficiently induce DNA mutations (40, 41). The previously identified MMTV-derived CTL epitope expressed on EL-4 was also shown to differ at one critical amino acid position compared with known MMTV sequences. Interestingly, this epitope was found to be shared by EL-4 and RMA (8). These considerations are in favor of the hypothesis that RBL-5 and MBL-2 are derivatives of EL-4, rather than the reverse (see above). Homology searches in the available databases for our peptide sequence, allowing minor amino acid variation, did not yield a gene of interest. We have excluded the joining region of the TCR {alpha}- or ß-chain as expressed on the tumor cells and known (endogenous) viral sequences, e.g., MuLV and MMTV as the possible genes that might encode this peptide. Furthermore, our attempts to isolate the gene encoding this CTL epitope through expression screening of cDNA libraries failed. Recently, we successfully employed this approach to clone a novel CTL epitope expressed on certain murine tumor cells (25). Extensive screening of >60,000 cDNA clones from a size-selected cDNA library did not result in identification of a positive clone. In summary, this CTL epitope is most likely generated by a point mutation in an unknown cellular gene.

Our in vivo experiments have shown that the CTL against the newly identified epitope play a clear role in the protective anti-tumor response. Nevertheless, the CTL response against the viral gagL75–83 epitope appears to be more efficient in tumor eradication in prophylactic peptide vaccinations as well as in therapeutic adoptive transfer setting. One possible explanation for this is the fact that RMA shows high expression of its MuLV genes and therefore presents virus-derived MHC class I peptides to a higher extent than peptides derived from cellular genes. Alternatively, the amounts of CTL precursors specific for the involved peptides may differ. Peptide vaccination with the helper peptide only can result in complete protection against this MHC class II-negative tumor (14); however, in these experiments this protective capacity was only sufficient for a significant delay of tumor growth. These results indicate that the CTL response to the novel epitope contributes markedly to the total CTL response against these tumors.

In conclusion, the Db-binding peptide NKGENAQAI or a very similar sequence is the RMA-specific CTL epitope that has long been searched for. The identification of this peptide together with the expression of identical TCR {alpha} and ß rearrangements revealed that RBL-5/RMA, MBL-2, and EL-4 have a common origin.


   Acknowledgments
 
We thank Dr. Marco Schilham for supplying the cell lines; Drs. Colin Brooks, Rolf Kiessling, Lars Franksson, and Max Petersson for sharing unpublished data; Drs. Bert Hiemstra, Willemien van Benckhuijsen, and Jan Wouter Drijfhout for peptide synthesis; and Dr. Rene Toes for critically reading this manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Rienk Offringa, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Back

2 Abbreviations used in this paper: MMTV, mouse mammary tumor virus; MuLV, murine leukemia virus; B6, C57BL/6; FMR, Friend-, Moloney-, and Rauscher-type MuLV; FRE, Fischer rat embryo; MS, mass spectrometry. Back

Received for publication February 28, 2000. Accepted for publication April 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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