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Ludwig Institute for Cancer Research, Brussels Branch, and Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of the several P815 Ags that CTLs can recognize, P815AB and P815E were found to be immunodominant 3, 6 . P815AB Ag has been studied extensively 4, 5, 7, 8, 9 , and has been the focus of a number of studies in recent years as a model for designing and optimizing antitumor CTL responses 10, 11, 12, 13 . P815E however, had yet to be defined. We set out to identify this Ag by screening cells transfected with a cDNA library derived from tumor P815 with P815E-specific CTLs. We report here that unlike P815AB, which is produced by tumor-specific gene activation, P815E results from a point mutation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Syngeneic DBA/2 mice were raised in specific pathogen-free conditions. All mice used in these experiments were between 10 and 12 wk old.
Peptides
All peptides were synthesized in house on solid phase using fluorenylmethoxycarbonyl for transient NH2-terminal protection 14 . Peptides were characterized by mass spectrometry and stored at 20 mg/ml in DMSO at -20°C.
Cell lines
P1 is a clonal line isolated from a permanent cell line derived from the transplantable P815-X2 subline 15 . P198, P89, and P35 are immunogenic tumor cell lines (tum-) produced by treatment of P1 with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine 15 . The cell lines P1.istA-B- (P1.204), P1.istA-B-iscC-D-, L1210, L1210.P1A, and L1210.P1A.B7-1 have been described previously 3, 16 . V4D6 is a DBA/2 mastocytoma cell line produced by infection of an IL-3-dependent mast cell line with a retrovirus containing the v-Ha-ras gene 17 . The L1210.P1E.B7-1 tumor cell line was obtained by transfecting L1210 with the B7-1 cDNA cloned into plasmid pEFBOS 18 , into which the puromycin resistance gene had been introduced. Transfected cells were selected using 1.25 µg/ml of puromycin, cloned by limiting dilution, and transfected with the gene P815E that was cloned into pcDNA3.1 (Invitrogen, San Diego, CA). Transfected cells were selected with 2.5 mg/ml G418 and cloned by limiting dilution. The L1210.P1E cell line was produced by transfecting L1210 with the gene P815E cloned in pcDNA3.1, and positive clones were selected for as described above. All transfections were done by electroporation. All cell lines except V4D6 were cultured in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FCS in a humidified, 8% CO2 atmosphere at 37°C. V4D6 was cultured in Iscove’s medium supplemented with 10% FCS.
CTL clones
To obtain CTL clones 89/62 and 351.1.E5, spleen cells (3 x 106) from mice immunized 4–15 wk previously with living P815 tum- variant P89 or P35 cells were cultured in a mixed lymphocyte-tumor cell culture (MLTC)3 with 3 x 105 stimulator cells (P1.istA-B-iscC-D- Ag-loss variant). The MLTC was established in 15 ml of DMEM supplemented with L-arginine HCl (116 mg/l), L-asparagine (36 mg/l), L-glutamine (216 mg/l), glucose (4.5 g/l final concentration), 10 mM HEPES, 5 x 10-5 M 2-ME, and 10% FCS. Cultures were maintained at 37°C in a humidified atmosphere containing 8% CO2. After 7 days, MLTC cells were cloned by limiting dilution in 96-well round-bottom plates as follows: Limiting numbers of cells were plated with 7 x 105 irradiated (2000 rads) DBA/2 spleen cells and 3 x 103 irradiated (5000 rads) stimulator cells in a final volume of 150 µl of MLTC medium supplemented with supernatant from secondary mixed leukocyte culture (50% v/v). The plates were incubated for 7 days at 37°C in a humidified atmosphere containing 8% CO2; subsequently aliquots from each microculture were tested for cytolytic activity against the appropriate target cells.
CTL clones were maintained in culture by in vitro stimulation twice per week in 2 ml of MLTC medium supplemented with HAT (10-4 M hypoxanthine, 3.8 x 10-7 M aminopterin, and 1.6 x 10-5 M 2-deoxythymidine) as well as 50% secondary MLC supernatant containing 105 P1.azar hypoxanthine aminopterin and 2-deoxythymidine-sensitive cells and 5 x 106 irradiated (3000 rads) splenocytes.
Chromium release assay
Chromium release assays were performed as described previously 19 . Briefly, CTLs were incubated with 2000 51Cr-labeled cells at various E:T ratios in 96-well conical microplates in a final volume of 200 µl. The supernatants were collected after a 4-h incubation at 37°C for the measurement of the chromium released from lysed cells.
Lytic units (LU)
One LU is defined as the number of effector cells that lyse 50% of 104 target cells in 4 h. This was estimated by the means of the regression (1-e-kx) from the specific chromium release obtained at different E:T ratios chosen in the linear range of the lysis curve. Results are expressed as LU/106 cells.
Peptide-pulsed assay
For peptide-pulsed assays, chromium-labeled V4D6 cells were incubated for 30 min at 37°C with various concentrations of peptide in X-vivo 10 medium (Whittaker Bioproducts, Walkersville, MD) before the addition of CTLs. For peptide inhibition assays, chromium-labeled V4D6 cells were incubated for 30 min at 37°C in X-vivo 10 medium with various concentrations of competitor peptides followed by incubation with P815E peptide GYCGLRGTGV at a concentration known to induce maximal lysis by anti-P815E CTLs. The concentration of P815E peptide inducing maximum lysis was determined on the same day of the competition assay. The cells were incubated with peptide for 30 min at 37°C, washed, and resuspended in 100 µl of fresh X-vivo medium; next, CTLs were added to achieve an E:T ratio of 5:1. Chromium release was measured after 2 h.
Inhibition by class I-specific Abs
A total of 15,000 P198 tum- cells were incubated together with a 1/10 dilution of hybridoma supernatant containing anti-class I Abs (SF1-1.1.1 for Kd (ATCC HB159) and 28.14.8 S for Ld (ATCC HB27)) for 4 h at 37°C before the addition of 200 CTLs (clone 89/62 or clone 351.1.E5) per well. Cultures were incubated overnight, and supernatant was collected for an assessment of TNF production using the TNF-sensitive WEHI-164 clone 13 cells 20 in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay 21 .
cDNA library
Total cellular RNA was prepared from P1 cells by the guanidine-isothiocyanate procedure 22 . Poly(A)+ mRNA was purified by chromatography on an oligo(dT) cellulose column (Pharmacia, Uppsala, Sweden) as described previously 22 . cDNA was synthesized with the Superscript Choice System (Life Technologies) using oligo(dT)18 primers containing a NotI restriction site followed by ligation to BstXI adapters (Stratagene, La Jolla, CA), phosphorylation, and digestion with NotI. Size-fractionated cDNA were ligated into the BstXI and NotI sites of pcDNA3.1 downstream from the CMV promoter. Recombinant plasmids were electroporated into Escherichia coli Top10F’ and selected by ampicillin (100 µg/ml). Plasmid cDNA from pools of 100 transformed colonies were extracted using the QIAprep 8 plasmid kit (Qiagen, Hilden, Germany).
Screening of transfected COS cells
A total of 15,000–20,000 COS cells were seeded in
96-well flat-bottom microwells in a total volume of 100 µl of DMEM
supplemented with 10% FCS on the day before transfection. The gene
encoding the murine H-2Kd 23 was cloned into plasmid
pcDNA1.1/Amp, and COS cells were transiently transfected by the
DEAE-dextran method 24 with 70 ng of DNA from the H-2Kd
construct alone or together with 
100 ng of DNA from the cDNA pools.
Recognition of transfected COS cells was determined by TNF secretion of
CTL 89/62. Briefly, at 48 h after the transfection, 1000 CTLs were
added to each well of a 96-well flat-bottom microtiter plate containing
the transfected COS cells. The plates were incubated overnight at
37°C in an humidified, 8% CO2 atmosphere, after which
time supernatants were collected and assessed for the presence of TNF
as described above.
DNA sequencing and homology search
DNA sequencing was performed by the dideoxy-chain termination method either manually (Thermo Sequenase cycle sequencing kit, Amersham, Cleveland, OH) or on an Applied Biosystems Prism 310 Genetic Analyzer (Perkin Elmer Applied Biosystems, Norwalk, CT) using specific oligonucleotides as primers. The computer search for sequence homology was performed with BLAST (blast@ncbi.nlm.nih.gov) (National Library of Medicine, National Institutes of Health, Bethesda, MD).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P815-specific CTLs were obtained previously by in vitro
stimulation of splenocytes from mice immunized with P815. Some CTL
clones showed lytic activity against an Ag-loss variant that had lost
the expression of the Ags P815A, P815B, P815C, and P815D (Fig. 1
A and 3 . The additional
Ag was named P815E.
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B).
The anti-H-2Kd Ab SF1-1.1.1 was found to inhibit
P815-induced TNF production by both CTL clones 89/62 and 351.1.E5,
indicating that P815E was recognized on the MHC class I molecule
H-2Kd. Identification of a cDNA encoding P815E Ag
A directional cDNA library using poly(A)+ mRNA derived
from the P815 subline P1 was prepared as described in Materials
and Methods. We divided the library into 2300 pools of 100
recombinant clones and prepared DNA for transfection into COS cells
together with the cDNA coding for H-2Kd as described above.
After 24 h of incubation with the CTL clone 89/62, the level of
TNF in the culture supernatant was assessed for each pool. One pool was
shown to induce TNF production by the CTLs. This pool was subcloned,
and DNA from single colonies were transfected as described above and
tested for TNF induction. We found only one cDNA (B9.2) out of 1600
single colonies tested that induced the production of TNF from CTL
89/62 when transfected together with the class I restriction element
H-2Kd (Fig. 2). The very low
frequency of clones that were positive for the cDNA of interest
appeared to result from the relatively slower growth rate of bacteria
containing this cDNA.
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The cDNA encoding P815E was 1326 bp long and was found to be 79%
homologous with the bovine gene coding for methionine sulfoxide
reductase (MsrA), an enzyme involved in the reduction of protein
methionine sulfoxide residues and methyl sulfoxide compounds (GenBank
accession number U37150) 25 . The full murine MsrA sequence has not
yet been published; however, a search within the expressed sequence tag
bank produced almost the entire murine MsrA sequence from normal mouse
tissue. Comparison of the compiled murine MsrA gene and the gene
encoding P815E showed complete identity except for a single discrepancy
at nucleotide position 704. This point mutation was confirmed by direct
sequence analysis of the PCR product from normal mouse liver cDNA,
showing that a guanine nucleotide in the normal gene was replaced by an
adenine in the gene encoding P815E. The mutation resulted in an amino
acid change at position 221 of the protein sequence, where a glycine
was replaced by an arginine (Fig. 3). It
appears thus far that the mutation is unique to mastocytoma P815, as
sequence analyses of the PCR products from six other mastocytomas and
three mouse leukemias were all negative for the P815E mutation.
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Identification of the P815E antigenic peptide
To determine whether the mutation was responsible for the
recognition by CTL 89/62, we synthesized peptides that contained the
modified residue and corresponded to the binding motif of the class I
restriction element H-2Kd. This motif is characterized by a
tyrosine at position 2 and by a leucine, isoleucine, or valine at
positions 9 or 10 26 . A decamer fulfilling the above criteria was
identified at position 216–225 (Fig. 3) of the P815 murine MsrA
protein sequence (GYCGLRGTGV). This peptide and a peptide corresponding
to the normal protein sequence (GYCGLGGTGV) were synthesized and tested
for recognition by CTL 89/62 (Fig. 4A). Only the peptide
containing the changed residue was recognized by the CTL. However, the
normal peptide was able to bind to H-2Kd, as increasing
concentrations of both this peptide and a peptide known to bind to
H-2Kd inhibited lysis of P815E-pulsed target cells (Fig. 4B). Therefore, we conclude that a nucleotide mutation in
the murine MsrA gene of P815 gives rise to a new antigenic epitope
recognized by CTLs generated in vivo.
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We have observed previously that a proportion of mice immunized
i.p with living cells of leukemia line L1210 transfected with both the
gene P1A and the cDNA coding for the costimulatory molecule
B7-1 (L1210.P1A.B7-1 cells) were protected against challenge with live
P815 tumor cells 27 . Following the identification of P815E, we were
interested in testing both the relevance of P815E as a tumor-rejection
Ag and the effectiveness of immunizing mice against two Ags expressed
on the same tumor. Therefore, we transfected L1210 with the cDNAs
coding for P815E and B7-1. Having established that P815E-specific CTLs
could be generated in mice by immunization i.p. with 106
live L1210.P1E.B7-1 cells (Fig. 5), we
challenged immunized mice 3 wk later with 4 x 105
live P815 cells. Surprisingly, we found that the survival rate of mice
immunized against P815E was similar to the nonimmunized controls (Fig. 6). Thus, contrary to P815AB, immunization against P815E did not appear
to protect mice against P815 challenge, even though immunization with
the Ags P815AB and P815E appears to produce CTL responses of similar
magnitudes (Fig. 5). Moreover, we observed that immunization against
both the P815AB and P815E Ags did not produce better protection than
that induced by immunization against P815AB alone (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The P815E gene codes for MsrA, an enzyme that is capable of reducing methionine sulfoxide residues in proteins and is therefore thought to be important in controlling the oxidative stress induced by aerobic metabolism 36, 37 . Enzymatic and gene expression studies of normal mouse and rat tissues has shown ubiquitous expression of this gene, although immunohistochemistry with MsrA-specific Abs has shown the specific localization of this protein to the retinal pigmented epithelium, alveolar macrophages, renal medulla, neurons, and central nervous system 36, 38 . It is thought that these regions and cell lineages are subjected to higher metabolic rates, leading to oxidative environments and thereby requiring higher levels of MsrA 38 . There is no evidence to suggest that the MsrA mutation we have found in P815 has an oncogenic function. This is in line with the unique occurrence of this mutation in P815.
Recently, immunization against P815AB was shown to protect mice against
challenge with live P815 cells, but resistance to tumor growth was
evident only in 50% of the immunized animals 27 . This result
prompted us to ask whether immunizing mice against additional P815 Ags
could improve this protection, and the identification and
characterization of P815E Ag presented us with an opportunity to
address this question. However, we found immunization against P815E
alone to be completely ineffective in protecting mice against live
tumor challenge, despite the fact that Ag-specific CTL activity was of
a similar magnitude to that seen in mice immunized with P815AB.
Immunizing mice against both the P815E and P815AB Ags did not improve
resistance to tumor growth compared with P815AB alone.
The lack of protection observed after immunization with P815E Ag has relevance to a recent proposal that unique Ags resulting from mutations are likely to be more useful for cancer immunotherapy than tumor-specific shared Ags 39, 40 . This proposal is based on two main arguments. The first is that shared Ags, being encoded by normal genes expressed in normal male germline cells, may generate some level of tolerance that prevents the most effective CTLs from coming into play. We agree that this possibility cannot be excluded a priori and believe that it is necessary to test it experimentally. The second argument is based on the past observation that immunizing animals with methylcholanthrene-induced tumors confers protection against the immunizing tumor but not against other methylcholanthrene-induced tumors 41 . However, it is likely that, like the methylcholanthrene-induced tumor P815, these tumors carry several Ags, some of which may be unique and some of which may be shared. As a result, every tumor probably carries a unique set of Ags, and this alone could explain why protective immunization was effective on the same tumor and not on others.
An experiment directly addressing the protective ability of a shared Ag was performed by Ramarathinam et al., who reported that immunization with P815 failed to protect mice against the tumors J558 and MethA, both of which share the P815AB Ag 42 . However, they report that there is a certain degree of protection against J558 and, here again, the better protection against P815 may be due to several Ags not shared by MethA and J558. We have observed that immunization with tumor L1210 expressing P815AB Ag can induce prolonged resistance to challenge with tumor P815. This protection can be attributed solely to the P815AB Ag, because P1A-transgenic mice, which are tolerant to P815AB, are not protected by immunization. Protection has also been observed after immunization with DNA encoding the P815AB Ag 13 . Further, contrary to the notion that unique Ags may be more useful for immunotherapy, we found that immunization with the unique Ag P815E did not induce resistance to live P815 tumor challenge, even though it can induce a specific CTL response.
The reason why no protection was observed after immunization with P815E
despite a good induction of CTLs is not clear. One explanation might be
that the Ag is present on P815 cells at a low level or in an unstable
form, which could prevent effective lysis in vivo. In support of this
possibility, we have observed that anti-P815E CTLs lyse P815
targets in vitro with less efficiency than P815AB-specific CTLs (Fig. 1; C. Uyttenhove, unpublished observations). The fact that earlier
studies detected significant levels of anti-P815E CTLs in
P815-bearing animals implies that a low level of Ag expression on P815
cells would still be sufficient to stimulate the CTLs to proliferate
3, 6 .
We do not wish to suggest that unique Ags will be ineffective for tumor immunotherapy, but our results indicate that each Ag needs to be assessed individually, not only for its ability to induce lymphocyte responses, but also for its ability to induce tumor rejection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Benoît J. Van den Eynde, Ludwig Institute for Cancer Research, 74 avenue Hippocrate, UCL 7459, 1200 Brussels, Belgium. E-mail address:
3 Abbreviations used in this paper: MLTC, mixed lymphocyte-tumor cell culture; LU, lytic unit(s); MsrA, methionine sulfoxide reductase.
Received for publication September 21, 1998. Accepted for publication December 7, 1998.
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References |
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-fluorenylmethysoxycarbamylamino-acid on polymide supports: synthesis of substance P and of acyl carrier protein 65–74 decapeptide. J. Chem. Soc. Perkin Trans. I 1:538.
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) or
the normal peptide (
). Nonamers lacking the first or the last
residue were also tested but were not recognized as well as the decamer
by CTL 89/62 (data not shown). CTLs were tested at an E:T ratio of 5:1.
B, Both the normal peptide (GYCGLGGTGV) (
) was included as a negative control (44). CTL 89/62 was tested at
an E:T ratio of 5:1.
