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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 Offringa
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 165:869-877; ;
doi: 10.4049/jimmunol.165.2.869
http://www.jimmunol.org/content/165/2/869
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
urine tumor models are essential and powerful tools in
tumor immunological research. During the last decade, the molecular identity of a number of tumorassociated CTL epitopes has been elucidated (for review, see Refs.
1–3). 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 carcinogeninduced 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
M
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 (15–17). 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.
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands
Materials and Methods
Received for publication February 28, 2000. Accepted for publication April 25, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Rienk Offringa, P.O. Box 9600,
2300 RC Leiden, The Netherlands. E-mail address: [email protected].
leidenuniv.nl
Copyright © 2000 by The American Association of Immunologists
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
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.
0022-1767/00/$02.00
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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 tumorassociated 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 ␣- and ␤-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. The Journal of Immunology, 2000, 165:
869 – 877.
870
RMA, EL-4, AND MBL-2 LYMPHOMAS SHARE A UNIQUE CTL EPITOPE
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 PMAstimulated 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 ␣␤-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]) ⫻ 100. Measurement of secreted TNF-␣ by stimulated CTL was performed with a bioassay, using
WEHI 164 clone 13 cells, as described previously (25). The percentage of
TNF-␣ released in triplicate wells was calculated as follows: % TNF-␣
release ⫽ [(A550 – 650 experimental wells ⫺ A550 – 650 wells containing medium only)/(A550 – 650 wells containing 500 pg/ml TNF-␣ ⫺ A550 – 650 wells
containing medium only)] ⫻ 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 ␮g/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␣ and V␤ gene usage were previously published (28). The downstream primer used for the TCR C␣ region was
5⬘-TGG CGT TGGTCT CTT TGA AG-3⬘, resulting in a product of ⬃400
bp, and the primer for the C␤ 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 B824-3-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 ␤2-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 ␮m internal diameter ⫻ 90 ␮m 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 ⫻ 75 ␮m, 5-␮m 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 ⫻
105 mbar), and the collision voltage was ⬃30 V (for similar procedures, see
Ref. 29, 30).
Results
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. 1, A and B). However, some
RMA-specific cultures failed to recognize this epitope and the
env189 –196 epitope (Fig. 1, 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. 1, 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. 1, C and D) also failed to recognize
Moloney MuLV-infected cells (Fig. 1E), whereas RMA tumor
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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 ␮M 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.
The Journal of Immunology
871
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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 ␮g/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– 83specific CTL (F), or env189 –196-specific CTL (G) was measured in a WEHI bioassay.
872
RMA, EL-4, AND MBL-2 LYMPHOMAS SHARE A UNIQUE CTL EPITOPE
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.
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.
Anti-tumor CTL selectively recognize T lymphoma cell lines
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 ⫻ 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 antitumor CTL (data not shown). Subsequently, Db-binding peptides
from 120 ⫻ 109 RMA cells were separated by reverse phase
HPLC. Anti-tumor CTL selectively recognized four fractions (Fig.
3), 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. 4), 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
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. 2B).
MuLV-induced T cell lymphomas (RMA and MBL-2), MuLVinduced pro-B cell lymphomas (33A3 and 33H2), and an MuLVinduced erythroleukemia (FBL-3) were all lysed, whereas chemically induced EL-4 cells that do not express FMR MuLV Ags were
not lysed (Fig. 2B). In contrast, anti-tumor CTL clones only recognized the T cell lymphomas RMA, MBL-2 and EL-4 (Fig. 2A),
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. 2A). 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
Purification and sequencing of the novel CTL epitope
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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.
The Journal of Immunology
873
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. 4) led to the following four coeluting
peptides: NKGENAQAI, NKGENAQAL, KNGENAQAI, and
KNGENAQAL. These peptides comply very well with the pub-
lished 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.
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.
5A). However, when the peptides were titrated, the recognition of
the peptides carrying a C-terminal leucine (L) decreased markedly
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.
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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 ⫻ 109
RMA cells were fractionated by reverse phase HPLC and were loaded on chromium-labeled HeLa.Db cells. f, 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 ⫻ 106 RMA cells. The right y-axis shows the percentage of acetonitrile in each HPLC fraction,
as indicated by the solid line.
874
RMA, EL-4, AND MBL-2 LYMPHOMAS SHARE A UNIQUE CTL EPITOPE
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. 5A). The variation at the Nterminus (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. 5B 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. 5B). The peptides ending with
isoleucine showed stronger binding, suggesting that this accounts
for the fact that they are better targets for CTL recognition.
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
FIGURE 6. Adoptive transfer of CTL prevents RMA tumor outgrowth. GagL75– 83-specific CTL (cl1) and antitumor 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. Tumorfree survival rates did not change after the last day shown.
Comparable results were observed in tumor-bearing mice
receiving CTL i.p.
the efficacy of adoptively transferred CTL against RMA in tumorbearing 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. 6). No difference in tumor
protection was detected between mice receiving the CTL i.v. (Fig.
6) or i.p. (not shown). All mice receiving IL-2 only or saline developed progressively growing tumors.
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 I), the NKGENAQAI and NKGENAQAL peptides as well as the KNGENAQAI and KNGENAQAL peptides
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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 chromiumlabeled 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.
The Journal of Immunology
875
Table I. Peptide vaccination induces protective responses against RMA
tumorsa
Long-Term Protected Mice
Injected Peptides
PBS
molHb
gagL75–83 ⫹ molH
Env189–196 ⫹ molH
NKGENAQAI/Ld ⫹ molH
KNGENAQAI/L ⫹ molH
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.
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 V␤ region (V␤12), including fully identical CDR3
regions (43) (TCR ␤-chain sequence accession no. AF020206).
This prompted us to analyze the different tumor cells in our laboratory for their TCR ␤- and ␣-chains. By TCR-specific PCR analysis and subsequent DNA sequencing we confirmed the identical
V␤ usage in these cell lines. Furthermore, we found that all recognized tumor cell lines also shared the usage of one TCR V␣
gene (V␣10), as shown in Fig. 7 for RMA, MBL-2, and EL-4. By
sequencing the V␣-J␣-C␣ region we established that these tumors
shared identical junctional regions. This TCR ␣ 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. 7).
The common origin of these cell lines is most likely the result of
a cross-contamination during in vitro culture or in vivo passage
c
2/8 (25%)
0/8 (0%)
8/8 (100%)
NDe
3/7 (42%)
3/7 (42%)
Second experiment
0/8 (0%)
0/8 (0%)
8/8 (100%)
0/8 (0%)
3/8 (37%)
1/8 (12%)
a
B6 mice were injected s.c. with 100 ␮g peptide together with 50 ␮g T helper
peptide (molH) in IFA 2 wk before RMA tumor challenge (250 cells i.p.).
b
molH; T helper epitope encoded by Friend, Moloney, and Rauscher MuLV env
gene (EPLTSLTPRCNTAWNRLKL: Ref. 14).
c
Number of mice surviving/total number of mice.
d
A total of 50 ␮g of I-containing peptide was mixed with 50 ␮g of L-containing
peptide.
e
ND, not determined.
many years ago, as described for other widely used cell lines (34 –
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 ␣ or ␤ sequences or by
MuLV sequences, suggesting that it is encoded by an as yet unknown cellular gene.
Discussion
The exclusive expression of a unique CTL epitope by RMA,
MBL-2, and EL-4 in combination with their identical TCR ␣- and
␤-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. 2A) 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 ␣ and ␤ 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.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 7. RMA, MBL-2, and EL-4 express the same TCR V␣ and V␤
genes. RT-PCR using a panel of V␣- and V␤-specific primers revealed that
RMA, MBL-2, and EL-4 cells expressed V␣10 and V␤12 gene segments.
Shown are PCR products (400 bp for V␣ and 200 bp for V␤ 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␣3 and V␤8 gene segments,
and 1H11 is a control CTL clone expressing the V␣10 gene (42). Con A
cDNA was derived from 4-day Con A (5 ␮g/ml)-stimulated spleen cells,
containing all TCR variable genes. Not shown are tumor cells lines that
also generated PCR products with V␣10 and V␤12 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).
First experiment
876
RMA, EL-4, AND MBL-2 LYMPHOMAS SHARE A UNIQUE CTL EPITOPE
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 antitumor 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 ␣ and ␤ 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.
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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,
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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 mideighties. Of note, the Friend MuLV-induced erythroleukemia
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