By
From the * Department of Immunohematology and Blood Bank, University Hospital Leiden, 2300 RC Leiden, the Netherlands; Department of Obstetrics and Gynaecology, Free University Hospital,
1007 MB Amsterdam, The Netherlands; § Department of Pathology, University Hospital Leiden,
2300 RC Leiden, The Netherlands; and
Cancer Immunology Program, Cardinal Bernadin Cancer
Center, Loyola University of Chicago, Maywood, Illinois 60153
The tumor suppressor protein p53 is overexpressed in close to 50% of all human malignancies.
The p53 protein is therefore an attractive target for immunotherapy. Cytotoxic T lymphocytes (CTLs) recognizing a murine wild-type p53 peptide, presented by the major histocompatibility
complex class I molecule H-2Kb, were generated by immunizing p53 gene deficient (p53 /
)
C57BL/6 mice with syngeneic p53-overexpressing tumor cells. Adoptive transfer of these
CTLs into tumor-bearing p53 +/+ nude mice caused complete and permanent tumor eradication. Importantly, this occurred in the absence of any demonstrable damage to normal tissue.
When transferred into p53 +/+ immunocompetent C57BL/6 mice, the CTLs persisted for
weeks in the absence of immunopathology and were capable of preventing tumor outgrowth.
Wild-type p53-specific CTLs can apparently discriminate between p53-overexpressing tumor cells and normal tissue, indicating that widely expressed autologous molecules such as p53 can
serve as a target for CTL-mediated immunotherapy of tumors.
The efficacy of virus-specific CTLs to eradicate virus-induced tumors has been well documented (1).
However, since the majority of tumors are not virus-
induced, characterization of tumor-associated antigens encoded by cellular genes is important for the development of
new immunotherapeutic strategies. Target antigens on nonvirally-induced tumors recognized by CTLs were recently
identified, notably in patients with melanoma (6). The
fact that these antigens are lineage- or tumor-specific, limits the
use of these targets in immunotherapy to a small group of cancers. On the other hand, the expression of some of these antigens on normal melanocytes demonstrates that self antigens
can serve as targets for CTL-mediated destruction of tumors.
Mutations in the gene encoding the tumor suppressor
protein p53 are found in ~50% of all human malignancies
(19). Recently, a direct link has been established between
mutational hot spots in the p53 gene leading to its overexpression, and carcinogenic metabolites derived from agents
in cigarette smoke (20). In normal cells, p53 induces a cell
cycle arrest, allowing DNA to be checked for irregularities,
thereby guarding the integrity of the genome (21). Mutation of p53 abolishes its function as a suppressor of the cell
cycle, promoting the escape of transformed cells from the
normal restriction of controlled growth. Since these mutations, causing overexpression of p53, are present in a wide
variety of cancers (22), a large group of patients would benefit from p53 directed immunotherapy. One could
consider mutant p53 sequences as target antigens for tumor-specific CTLs. However, p53 mutations occur at many
different sites in the p53 molecule, necessitating identification of the site of mutation in each patient before therapy.
Furthermore, not all mutations are contained in MHC-binding CTL epitopes. If, in contrast, wild-type (wt)1 p53
sequences are used, the entire sequence of the p53 protein is available for properly processed immunogenic T cell epitopes. We hypothesized that the altered expression of p53, seen in
many cancers, leads to modified processing and presentation of wt p53-derived peptides by MHC class I molecules.
Recently, wt p53 peptide-specific CTL were generated from
human and murine responding lymphocytes, some of which
recognized p53-overexpressing tumors in vitro (26).
Here, we report the in vivo eradication of established
p53-overexpressing tumors in C57BL/6 p53 (+/+) nude
mice by a well-defined wt p53-specific CTL clone in the
absence of any demonstrable immunopathology. These
CTLs, generated in p53-deficient mice and recognizing the murine wt p53-derived epitope AIYKKSQHM (amino acids [aa] 158-166) presented by the MHC class I molecule
H-2Kb, were also capable of preventing the outgrowth of a
more aggressive p53-overexpressing tumor in immunocompetent p53 (+/+) C57BL/6 mice.
Mice.
C57BL/6 (B6, H-2b) mice were obtained either from
the Netherlands Cancer Institute (Amsterdam, The Netherlands)
or from IFFA Credo (Larbresle, France), C57BL/6 nu/nu (B6
nude, H-2b) mice were obtained from Bomholtgard (Ry, Denmark), and the p53 knockout (p53 Cell Lines.
Mouse embryo cells (MECs) of C57BL/6 origin
(B6MECs) were transformed by transfection with the following
oncogenes: Xhoc3, Adenovirus type 5 E1 (2); C3, HPV16 E + L
and EJras (36); 4J, mut.p53 + H-ras; 5A and 5D, mut.p53 + N-ras;
and 6J3, mut.p53 + fos (van Hall, T., M.P.M. Vierboom, C.J.M.
Melief, and R. Offringa, manuscript in preparation). The tumor cell
lines 4J, 5A, 5D, and 6J3 express high levels of a murine p53 containing a mutation at position 135 (37). Cell line EL-4 originates
from an H-2b thymoma (38). The cell line "Koko" was generated
from a solid tumor that arose spontaneously in a p53 knockout
mouse. The following CTL clones, all of C57BL/6 origin, were
used: anti-MCF1233 CTL clone BTM (provided by F. Ossendorp,
University Hospital Leiden, Leiden, The Netherlands; reference
39), anti-Ad5E1A CTL clone 5 (2), and anti-Ad5 E1B CTL
clone 100B6 (5). Mouse cell lines were cultured in Iscove's modified
Dulbecco medium (Gibco Biocult, Glasgow, UK) supplemented
with 8% FCS, penicillin (100 IU/ml), and Generation of Tumor Cell Line-specific CTL Cultures.
Tumor cell
line-specific CTL cultures were generated as described previously
(2, 39). In brief, spleen cells from B6 and p53 Peptides.
Peptides were generated by solid phase strategies on
an Abimed AMS 422 multiple peptide synthesizer (Abimed, Langenfeld, Germany) by repeated cycles in which addition of Fmoc
protected aa to a resin of polystyrene was alternated with an
Fmoc-deprotection procedure using Fmoc chemistry. The purity
of the peptides was determined by reverse phase HPLC and was
found to be routinely >90% pure. Peptides were dissolved in
DMSO (final DMSO concentration 0.25%) and diluted in 0.9%
NaCl to a peptide concentration of 2 mg/ml and stored at
MHC Class I Peptide Binding Assay.
The MHC class I peptide binding assay using the processing defective cell line RMA-S
and the peptide competition assay were performed as described
(41). In brief, RMA-S cells were cultured for 36 h at 26°C in culture medium in the presence of 2% human pool serum to increase
the cell surface expression of "empty" class I molecules (42). In
96-well plates, RMA-S cells (2.5 × 105/well) were cultured in
serum-free medium (40 µl/well) and peptide (10 µl/well) for 4 h
at 37°C. Subsequently, the cells were washed and stained for analysis on a FACscan® flow cytometer (Becton Dickinson, Mountain View, CA) using, in the first step, a monoclonal antibody
against H-2Kb (B8.24.3; reference 43) and, in the second step,
FITC-labeled goat anti-mouse F(ab Peptide Competition Cytotoxicity Assay.
For the competition assay, competitor peptides were aliquoted in triplicate in 96-well
U-bottom plates at 10 times the final concentration, 10 µl/well.
To each well, 10 µl of 50 pM of the reference peptide MCF1233
574-581 (KSPWFTTL; reference 39) was added. Triplicate wells
were included that contained either no peptide or reference peptide only. EL-4 target cells (103 cells/well) were labeled with Europium (Eu3+) and added to the peptides (40 µl/well). After 60 min of incubation, 5 × 103 CTL clone BTM cells (39) were
added to the wells for another 4 h of incubation before supernatants were collected. By interpolation of the data obtained with
each competitor peptide at different concentrations, we calculated the peptide concentration that inhibits 50% of the maximal
CTL lysis in the presence of the reference peptide alone (IC50).
Transporter Associated with Processing Translocation Assay.
This
assay was performed as described (44, 45). In brief, RMA cells
were washed in incubation buffer and permeabilized during 10 min at 37°C with 2.5 IU/ml streptolysin O. Subsequently, radioiodinated reporter peptide (TVNKTERAY) was added to the
permeabilized cells together with titrated amounts of competitor
peptide and ATP during 5 min at 37°C. Glycosylated reporter
peptide was recovered with Con A Sepharose and quantified by Eu3+ Release Cytotoxicity Assay.
Experimental procedures to
measure cell-mediated cytotoxicity were performed in an Eu3+
release assay as described elsewhere (46). In brief, varying numbers of effector cells (harvested on Ficoll if necessary) were added
to 103 Eu3+ labeled target cells in 100 µl of culture medium in
96-well U-bottomed plates. After 4 h incubation time, 20 µl culture supernatant was collected and mixed with 200 µl Enhancer
Solution (Wallac, Turku, Finland). Measurement of the samples
took place in a fluorometer (1234 Delfia; Wallac). The mean percentage specific lysis of triplicate wells was calculated as follows:
percent specific lysis = ([cpm experimental release Transfection of COS-7 Cells.
Transient transfection of COS-7
cells was performed as described elsewhere (5, 12). In short, 100 ng of plasmid pcDNA/Amp-p53 containing mut.p53 (37) together with 100 ng of plasmid pcDNAI/Amp-Db or pcDNAI/
Amp-Kb were transfected by the DEAE-dextran-chloroquine
method into 104 COS-7 cells (47). Plasmid pcDNA/Amp-p53
harbors the p53 and plasmids pcDNAI/Amp-Db and pcDNAI/
Amp-Kb harbor the H-2Db and H-2Kb genes, respectively. The
COS-7 cells were incubated in 100 µl IMDM containing 10%
FCS for 48-72 h at 37°C, after which 1,500 CTLs in 25 µl
IMDM containing 50 CU rIL-2 were added. Upon specific stimulation of the CTL, TNF- Cold Target Inhibition Cytotoxicity Assay.
Koko cells were loaded
with peptide at a concentration of 50 µg/ml for 2 h. Cells were
washed five times to remove unbound peptide. Effector cells
were preincubated with 5 × 104 unlabeled "blocking" cells (50 times excess) for 60 min at 37°C, before addition of labeled cells.
After a subsequent 4-h incubation period with labeled cells, the
supernatant was collected.
Adoptive Transfer of Anti-p53-specific CTLs.
wt p53-specific CTL
clone 1H11 (2.0 × 107) was intravenously injected in either tumor-bearing or nonchallenged p53-competent C57Bl/6 nude
mice and C57Bl/6 immunocompetent mice in combination with
105 CU rIL-2 administered subcutaneously in IFA (on the day of
adoptive transfer and one week later). Control mice received
nothing or an irrelevant clone together with rIL-2. The recovery
of the intravenously injected anti-p53 CTL clone 1H11 from the
spleen was tested at the indicated time point in an Eu3+ release assay.
Winn Type Assay.
The tumor cell 5D was injected in the
peritoneum at indicated doses. On the same day 2.0 × 107 wt
p53-specific CTL clone 1H11 or a control clone 9.5, recognizing the HPV16 E7-derived epitope RAHYNIVTF (36), was injected intraperitoneally together with 105 CU rIL-2 administered
subcutaneously in IFA (on the day of adoptive transfer and one
week later).
wt p53-specific CTLs
were generated by immunizing C57BL/6 p53-deficient
(p53
To identify the p53 epitope recognized by these CTLs, the murine
wt p53 aa sequence (48) was screened for the presence of peptides matching the H-2Kb peptide binding motif (49).
A total of 17 peptides were selected and tested for their capacity to bind and stabilize H-2Kb using the RMA-S cell
binding assay (50). Seven peptides were identified that bound
to H-2Kb (Table 1, UC50 column). The ability of these
peptides to compete with a known H-2Kb-binding epitope
MCF1233 574-581, thereby inhibiting the recognition of
this peptide by CTL clone BTM, demonstrates direct binding to the murine MHC class I molecule H-2Kb (41). Table 1, CC50 column). Translocation by transporters associated with processing, as shown by their capacity to compete for transport of a reference peptide in a transporter associated with processing translocation assay (44), indicates that
these peptides, when properly processed in the cytoplasm,
can enter the endoplasmic reticulum lumen where they
bind to the MHC class I molecules (Table 1, IC50 column).
These peptides were subsequently tested for their ability to
sensitize Eu3+-labeled Koko cells for lysis by the p53-specific CTL clone 1H11. Only the peptide AIYKKSQHM, derived from the wt sequence of p53 (aa 158-166), was recognized by the CTLs (Fig. 2 A). Titrated amounts of
length variants of this peptide were tested to establish the
optimal length. The peptide AIYKKSQHM (aa 158-166),
recognized at a concentration range of 0.1-1 pM (Fig. 2 B),
was the optimal length peptide and one of the best binders
(Table 1). Koko cells loaded with the peptide AIYKKSQHM were able to specifically block the recognition
of the p53-overexpressing tumor cell lines 4J (Fig. 2 C) and
5D (data not shown) in a cold target inhibition cytotoxicity
assay, demonstrating that this peptide is the naturally processed epitope presented by 4J and 5D.
Table 1.
MHC Class I Kb-binding, Peptide Competition, and
TAP-dependent Transport of Seven wt p53 Peptides
To establish whether this
epitope is commonly expressed, mutant p53-transformed
cells and other tumor cells, with no known p53 overexpression, were tested for recognition. The lysis of HPV16 transformed tumor cell line C3 (36; Fig. 3 A) and the thymoma EL-4 (data not shown) by CTL clone 1H11 was comparable to the lysis of other p53-overexpressing lines 4J, 5A,
5D, and 6J3 (Fig. 3 A). Strikingly, nontransformed B6MEC
and Con A-stimulated T cell blasts were efficiently recognized by CTL clone 1H11 (Fig. 3 A), demonstrating the
potential cross-reacting ability of these p53-specific CTLs to
nonmalignant cells. On the other hand, freshly isolated thymocytes (Fig. 3 B) and freshly isolated spleen cells (data not
shown) are not lysed by these CTLs.
Since these wt
p53-specific CTLs cross-reacted on nontransformed cells,
we then assessed whether mice carrying a functional p53
gene would survive adoptive transfer of these potentially autoreactive CTLs and whether these CTLs could eradicate established 4J tumors in these mice without overt immunopathology. Adoptively transferred wt p53-specific CTLs were
retrieved from the spleen of B6 p53 +/+ nude mice up to
3 mo after intravenous administration (Fig. 4, A and B), but
not from splenocytes of untreated B6 nude mice (Fig. 4 C).
No signs of autoimmune-induced damage were observed in mice that had recieved wt p53-specific CTLs. We subsequently tested the possibility of eradicating established 4J
tumors by adoptive transfer of wt p53-specific CTLs. Tumors grew progressively in untreated mice (Fig. 5 A) and in
mice injected with an irrelevant CTL clone recognizing an
Ad5 E1B-derived epitope (5; Fig. 5 B). Adoptive transfer
by intravenous infusion of the wt p53-specific clone 1H11
resulted in complete and permanent (>5 mo) tumor eradication in mice with small- (average size = 41 mm3; Fig. 5
C) and medium- (average size = 140 mm3; Fig. 5 D) sized
tumors. Intratumoral injection of similar numbers of wt
p53-specific CTLs in combination with rIL-2 also led to the complete eradication of medium-sized tumors (data not
shown). Even large established 4J tumors (average size = 427 mm3) were eradicated in three out of six mice (Fig. 5
E) after intravenous infusion of the wt p53-specific clone
1H11. Mice that had rejected p53-induced tumors after
treatment with wt p53-specific CTLs retained long-term
tumor-specific CTL immunity, since wt p53-specific CTLs could be retrieved from spleens of these animals one month
after CTL treatment (Fig. 6, A and B).
To investigate whether the
wt p53-specific CTLs would also persist in immunocompetent p53 +/+ C57BL/6 mice or would be deleted in the
presence of a T cell compartment absent in C57BL/6 nude mice, p53-specific CTLs were transferred into p53 +/+
C57BL/6 immunocompetent mice and the spleens of these
mice were assayed for p53-specific activity after 14 d. wt p53-specific CTLs, recognizing the peptide and 4J tumor cells,
could be retrieved from spleens of C57BL/6 p53 +/+ immunocompetent mice at day 14 (Fig. 7 A) and up to at least
7 wk after adoptive transfer of these CTLs (data not
shown). We also evaluated the ability of these wt p53-specific CTLs to prevent the outgrowth of the more aggressive
tumor, 5D, transformed by mutant p53 and N-ras, in a
Winn assay (Table 2). The 5D tumor, in contrast to the 4J
tumor, is tumorigenic in immunocompetent syngeneic B6
mice. In the group of mice challenged intraperitoneally with 5D and simultaneously treated with control CTL
clone 9.5, recognizing an HPV16 E7-derived epitope, 12 out of 12 animals developed a progressively growing tumor
and died within 3 wk. In the group of mice challenged intraperitoneally with 5D and treated with the wt p53-specific CTL clone 1H11, only 1 out of 12 developed a progressively growing tumor, thus demonstrating in vivo
activity against the tumor without demonstrable autoimmune pathology.
/
; H-2b) mice were obtained from GenPharm (Mountain View, CA; 35) and held under
specific pathogen-free conditions. Since offspring could only be
obtained by crossing a p53 heterozygous female (p53 +/
) with
a p53 male (p53
/
), the mice had to be analyzed for their p53
status by PCR analysis. For the PCR analysis two primer sets
were used. Primer set A consists of a forward primer binding to
the neomycin resistance gene (5
GCA TCG CCT TCT ATC
GCC TTC TTG AC 3
, neo fwd), with which the wt p53 gene
was destroyed, and a reverse primer that binds to a sequence in
exon 5 (5
ATC ACC ATC GGA GCA GCG CTC ATG 3
,
p53 exon 5 rev). Primer set A gives a band of 120 bp only when
the neomycin gene is present. Primer set B consists of a forward
primer binding to a sequence in intron 4 of the wt p53 gene (5
CAG TCC TCT CTT TGC TGG CTC GCT CT 3
, p53 intron 4 fwd), which is deleted by the insertion of the neomycin
resistance gene in the wt p53 gene. This sequence is only present
in an intact wt p53 gene. The same reverse primer used in primer
set A was used for primer set B. Primer set B gives a band of 180 bp only when the wt p53 gene is present.
-mercaptoethanol (2 × 10
5 M) at 37°C in humidified air containing 5% CO2.
/
mice were
taken 3 wk after the second immunization with 107 irradiated 4J
cells treated with IFN-
(20 U/ml for 48 h) and brought into
culture after enriching for T cells via passage over a nylon wool
column. 5 × 104 responder cells were cocultured with 5 × 103
irradiated and IFN-
-treated 4J cells in a total volume of 100 µl/
well in 96-well U-bottomed plates. Bulk cultures were restimulated in vitro with irradiated and IFN-
-treated 4J cells once a
week, for 6 wk, which resulted in the CTL line 8. CTL clones,
of which one was clone 1H11, were obtained by limiting dilution
of the 4J-specific bulk culture at wk 3 as described (2). Long-term cultures were grown in medium with 10% FCS and 1.5% culture
supernatant from PMA/Con A-stimulated rat spleen cells (40) and
25 CU (150 IU) rIL-2/ml (Eurocetus, Amsterdam, The Netherlands). All CTL clones and lines obtained had the marker profile
Thy-1+, CD4
, and CD8+. All of the in vitro and in vivo experiments were done with CTL clone 1H11 unless indicated otherwise.
80°C.
)2 fragments.
counting. The IC50 was calculated by determining the concentration of competitor peptide that decreased the maximal amount
of recovered glycosylated reporter peptide by half.
cpm spontaneous release]/[cpm maximum release
cpm spontaneous release]) × 100. The spontaneous release of the Eu3+-labeled target
cells was <30% in all experiments. The figures shown are representative for experiments done in duplicate.
is released in the supernatant, as
measured in a bioassay with the TNF-
-sensitive cells WEHI-164 clone 13 (5). Percent WEHI cell death was calculated by the following formula: percent specific lysis = (1
[OD 550-650 in sample wells/OD 550-650 in wells containing untransfected
COS-7 cells and CTLs]) × 100.
Generation of wt p53-specific CTL.
/
) mice with p53-overexpressing 4J tumor cells. The tumor cell 4J expresses high levels of mutant p53 at
the RNA level as tested on a Northern blot, and at the
protein level p53 as shown by cytospin staining (data not
shown). Spleen cells of mice immunized and boosted with
irradiated IFN-
-treated 4J cells were restimulated in vitro
with 4J, and the antigen specificity of the resulting bulk
CTL cultures was analyzed. The CTL specifically lysed 4J,
but not the tumor cell line Koko derived from a p53
/
mouse (Fig. 1 A). Specific recognition of p53 was tested by incubating these CTLs with COS-7 cells transiently transfected with cDNAs encoding one of the two MHC class I
molecules, H-2Kb or H-2Db with or without mutant p53.
Specific recognition was assayed by TNF-
production.
Fig. 1 B shows that the CTL bulk cultures recognize
COS-7 cells transfected with mutant p53 in an MHC class I H-2Kb-restricted manner. Several CTL clones, displaying
the same specificity, were isolated from this bulk culture by
limiting dilution. Subsequent experiments were performed
with CTL clone 1H11, which is a representative of the
clones recognizing p53.
Fig. 1.
p53 specificity and Kb restriction of bulk culture of tumor-specific CTLs. (A) Recognition of 4J cells (filled squares) and not of Koko cells
(open circles) by a 2-wk-old CTL bulk culture, named line 8, in a Eu3+ release cytotoxicity assay (46; Genzyme, Cambridge, MA). (B) CTL bulk culture line 8, specifically recognizing 4J, recognizes COS-7 cells transfected
with plasmids containing the genes of H-2Kb and mutant p53 (37).
[View Larger Version of this Image (13K GIF file)]
Kb binding peptides
UC50
CC50
IC50
µg/ml
µg/ml
µg/ml
aa 119-127: VMCYSPPL
18
12
2.5
aa 122-130: TYSPLNKL
20
14
6
aa 123-131: YSPPLNKLF
4
0.5
>100
aa 127-134: LNKLFCQL
4
2
7
aa 158-166: AIYKKSQHM
2.7
0.3
4.5
aa 222-229: AGSEYTTI
100
18
70
aa 227-234: TTIHYKYM
7
1.1
6
SV9: FAPGNYPAL
0.1
0.2
ND
Seven wt p53-derived were characterized in an MHC class I binding
assay (column 1; reference 42), peptide competition cytotoxicity assay
(column 2; reference 41), and TAP-dependent translocation assay (column 3; reference 44). (UC50) Peptide concentration resulting in 50% of
the maximal upregulation of H-2Kb in the presence of the known
H-2Kb-binding peptide Sendai virus NP 324-332 (61). (CC50) Peptide concentration that inhibits 50% of the maximal lysis by CTL clone BTM recognizing the reference peptide KSPWFTTL derived from the MCF1233 virus (39). (IC50) Concentration of competitor peptide that
decreased the maximal amount of recovered glycosylated reporter peptide by 50% (44, 45). Indicated in bold are so-called anchor residues in
the H-2Kb-binding motif described by Falk et al. (49).
Fig. 2.
Peptide specificity (A-C)
and sensitivity (B) of p53-specific CTL
clone 1H11 on peptide-pulsed target
cells (A and B) and tumor cells (C). (A)
A representative clone, 1H11, derived
from line 8 by limiting dilution, was
tested for lytic activity of Eu3+-labeled
p53 /
Koko cells pulsed with one of
seven H-2Kb binding wt p53 peptides
(Table 1). Targets were unloaded Koko cells (open circles) or Koko cells loaded
with peptides (given in aa sequence):
VMCTYSPPL (open triangles), TYSPPLNKL (filled triangles), YSPPLNKLF (open squares), LNKLFCQL (filled squares), AIYKKSQHM
(filled circles), AGSEYTTI (open diamonds), TTIHYKYM (filled diamonds).
As a control, Koko cells pulsed with Kb-binding MCF1233-derived peptide KSPWFTTL (39) was taken along (asterisks). (B) Koko cells were incubated with titrated amounts of length variants of the wt mouse p53
epitope AIYKKSQHM (aa 158-166). CTL clone 1H11 was added after
a preincubation of 30 min at an E/T ratio of 10:1. The following length
variants were tested: AIYKKSQHM (closed circles; aa 158-166), AIYKKSQHMT (open triangles; aa 158-167), AIYKKSQH (open squares; aa
158-165); IYKKSQHM (open circles; aa 159-166); AMAIYKKS (closed
triangles; aa 156-163). (C) Cold target blocking of the p53-overexpressing tumor cell 4J was tested by preincubation of the CTL clone 1H11
with the following unlabeled cells: no cells (open squares), koko cells (open
circles), Koko cells pulsed with the wt p53 epitope AIYKKSQHM (closed
circles; aa 158-166) and 4J cells (closed triangles). After 60 min, labeled 4J
cells were added.
[View Larger Version of this Image (24K GIF file)]
Fig. 3.
Lytic ability of wt p53-specific CTL clone 1H11 on a panel
of normal and tumor cells. (A) A panel of Eu3+-labeled C57BL/6-derived
cell lines were tested for lysis by p53-specific CTL clone 1H11 in a cytotoxicity assay. The following targets were tested for recognition: MEC
(filled circles); C3: HPV16 + EJras (open triangles); 5A (open diamonds) and
5D (filled diamonds): p53 + N-ras; 6J3: p53 + fos (open squares); 4J: p53 + H-ras (filled squares); and Con A-stimulated spleen cells (filled triangles). The p53 /
Koko cell line (open circles) was taken along as the negative
control. (B) Lack of recognition of freshly isolated thymocytes (open triangles) by the p53-specific CTL clone 1H11 in a 51Cr-cytotoxicicty assay
(29). Thymocytes are recognized by the p53-specific CTL clone 1H11
when pulsed with the wt p53 peptide AIYKKSQHM (filled triangles). p53
/
Koko cells, with (filled circles) and without (open circles) this peptide,
were taken along as a positive control for recognition of the peptide.
[View Larger Version of this Image (15K GIF file)]
Fig. 4.
Long-term survival of adoptively transferred wt p53-specific
CTLs in B6 nude mice. Recovery of wt p53-specific CTLs (clone 1H11)
3 mo after adoptive transfer of these CTLs into C57BL/6 nude mice as
tested in a Eu3+ release assay (46). Spleen cells of mice that had received wt p53-specific CTLs in the presence (A) or absence of 6 × 105 IU IL-2
(B), and naive (C) mice were restimulated in vitro with 4J and tested for
their peptide specificity on Eu3+-labeled p53 /
Koko cells (open circles), p53
/
Koko cells pulsed with the wt p53 peptide AIYKKSQHM
(filled circles), and recognition 4J (filled squares) cells after 7 d of culture.
[View Larger Version of this Image (13K GIF file)]
Fig. 5.
Tumor eradication
by adoptive transfer of wt p53-specific CTLs. C57BL/6 nude
mice were subcutaneously challenged with 107 4J tumor cells.
After 3 wk, CTL treatment of
established tumors was started.
Treatment consisted of intravenous adoptive transfer of 2 × 107
CTL clone 1H11 in 300 µl PBS
on day 0, and a subcutaneous injection of 6 × 105 IU rIL-2 (Cetus Corp., Emeryville, CA) in 50% incomplete Freund's adjuvant (IFA) on days 0 and 7. Tumor growth is given as a percentage relative to the size of the
tumor when the treatment was started. Mice were given no treatment (A; n = 5), treatment with an irrelevant clone recognizing an Ad5E1B epitope (B;
n = 4; reference 5), or treatment with the wt p53-specific CTL clone 1H11 (C-E). Mice treated with the wt p53-specific clone 1H11 consisted of three groups: mice bearing small tumors (C; 100% = 41 mm3; n = 6), medium-sized tumors (D; 100% = 140 mm3; n = 4), and large tumors (E; 100% = 427 mm3; n = 6) at the start of treatment.
[View Larger Version of this Image (12K GIF file)]
Fig. 6.
Recovery of wt p53-specific CTLs from nude mice after
successful tumor eradication. wt p53-specific CTL clone 1H11 was retrieved from the spleens of C57BL/6 nude mice 1 mo after tumor eradication as tested in a Eu3+ release assay. CTLs were administered intravenously (A) or intratumorally (B). A nontreated naive mouse (C) was taken
along as a negative control. Spleen cells were restimulated in vitro with 4J
cells and tested for their peptide specificity on p53 /
Koko cells (open
circles), Koko cells pulsed with the p53 peptide AIYKKSQHM (filled circles), and recognition of 4J cells (filled squares) after 7 d of culture.
[View Larger Version of this Image (13K GIF file)]
Fig. 7.
Recovery of wt p53-specific CTL activity after adoptive
transfer of CTLs into immunocompetent mice. wt p53-specific CTL
clone 1H11 was recovered from the spleens of C57BL/6 p53 +/+ immunocompetent mice 14 d after the CTLs were administered intavenously (A), as tested in a Eu3+ release assay. A nontreated C57BL/6
p53 +/+ immunocompetent mouse (B) was taken along as a negative
control. Spleen cells were restimulated in vitro with 4J and tested for their
peptide specificity on p53 /
Koko cells (open circles), Koko cells pulsed
with the p53 peptide AIYKKSQHM (filled circles), and recognition of 4J
cells (filled squares) after 7 d of culture.
[View Larger Version of this Image (16K GIF file)]
3 mo after adoptive transfer of the p53-specific CTL clone 1H11 into nontumor-bearing nude animals and one month after successful eradication of small- and medium-sized tumors, tissues
(liver, kidney, spleen, small intestine, large intestine, lung,
stomach, heart, brain, skin, lymph nodes, and bone marrow) were collected and examined microscopically (hematoxylin and eosin stained). Examination of coded samples
by two independent investigators showed no evidence of
immunopathology in normal tissues of these nude mice
(Fig. 8). An increased infiltrate of mononuclear cells was
observed in normal tissue of cured mice. This is probably caused by the administration of rIL-2, since mice treated
with an irrelevant clone also show this infiltrate (data not
shown). Similarly, tissues from C57BL/6 p53 +/+ immunocompetent mice did not show evidence of immunopathology at times when, after adoptive transfer, the wt p53-specific CTLs could be recovered (Fig. 7 A). Staining of
spleen and skin for CD4 and CD8 showed no difference between treated animals and nontreated control animals
(data not shown).
The search for widely expressed tumor antigens as targets for MHC class I-restricted CTLs is of great importance for the development of T cell-mediated immunotherapy of cancer. The tumor suppressor protein p53 is potentially such an antigen, with altered expression in up to 50% of all human tumors (19). In the present study we demonstrate that CTLs recognizing a murine wt p53-derived epitope were able to eradicate a p53-overexpressing tumor in p53 +/+ B6 nude mice in the absence of demonstrable immunopathology.
wt p53-specific CTLs were generated by immunizing
C57BL/6 p53 /
mice with syngeneic p53-overexpressing 4J tumor cells. As expected, a wt p53-derived sequence
was found to serve as an excellent epitope, the mutant p53
area being devoid of MHC class I-binding motif carrying
sequences (49). The wt p53-specific CTLs recognized the
H-2Kb-binding epitope AIYKKSQHM (aa 158-166; Fig.
2 A) with high affinity (Fig. 2 B), comparable to previously
published CTL clones against viral epitopes that were able
to eradicate established tumors (2, 4, 5).
C57BL/6 nude and immunocompetent mice carrying
the wt p53 gene survived the adoptive transfer of wt p53-specific CTLs. These potentially autoreactive CTLs persist
normally in p53 +/+ mice (Fig. 4, A and B, and 7 A)
without damage to normal tissues (Fig. 8 C). This persistence of large amounts of self-reactive T cells was elegantly
shown in a study of Ohashi et al. (51). They demonstrated that CTLs expressing a transgenic TCR recognizing a lymphocytic choriomeningitis virus glycoprotein-derived epitope
remained functionally unresponsive towards islet cells expressing the transgene lymphocytic choriomeningitis virus
glycoprotein. Similar observations were made by Goverman et al. (52), who showed that animals with large amounts
of functionally, autoreactive T cells expressing the transgenic TCR specific for the naturally expressed myelin basic
protein can be present without causing experimental allergic encephalomyelitis. However, in both models the transgenic TCR-carrying T cells can be activated to cause autoimmunity (51, 52). In contrast, the wt p53-specific CTLs
were active in tumor-bearing C57BL/6 nude mice and
eradicated established tumors (Fig. 5, C-E) without autoimmunity. In this respect, our study corroborates observations in Friend leukemia virus envelope (FLVenv) transgenic mice
in which adoptively transferred FLVenv-autoreactive T
cells can eradicate FLVenv expressing tumor cells without
damage to normal tissue expressing this artificial autoantigen (53).
A simple explanation for the observed tumor selectivity can be the increased expression of the p53 protein resulting from the p53 mutation. Alternatively, the lack of "danger" signals delivered by normal tissues (54) might protect against the destruction by the potentially autoreactive wt p53-specific CTL. In fact, in normal tissues, homeostatic mechanisms apparently control tissue damage by potentially autoreactive T cells. Only powerful inflammatory stimuli can provoke autoaggression mediated by these otherwise dormant T cells, as illustrated by the examples of experimental allergic encephalomyelitis (55, 56) and adjuvant arthritis (57). The explanation why, in this particular instance, no autoimmune tissue damage occurs despite the infusion of a large number of activated cloned CTLs may lie in insufficient antigen display by the MHC class I molecules in combination with lack of proper costimulation (54) and downregulatory chemokine and cytokine conditions (55).
Recent reports show the induction of wt p53 peptide-specific responses with cross-reactivity on endogenously p53-expressing targets (28, 29, 31). However, the in vivo relevance of these responses remains to be demonstrated. In reports in which wt p53 was used as an immunogen (30, 58), therapeutic effects were found consisting of eradication of established tumors or protection against a subsequent tumor challenge. However, the mechanism of antitumor activity in these studies remains to be clarified. In our model, a well-defined high affinity CD8+ CTL clone recognizing a wt p53 epitope eradicated established tumors in B6 nude mice and prevented the outgrowth of a more aggressive tumor injected simultaneously in the peritoneal cavity in B6 immunocompetent mice.
To generate these CTLs, responding lymphocytes from p53 knockout mice were used for the obvious reason that these mice are not tolerant of p53. Since p53 is a crucial protein serving as a checkpoint in the cell cycle of stimulated T cells (59) which are open to attack by wt p53-specific CTLs (Fig. 3 A), one of the major challenges is to try to break tolerance of wt p53 and to generate high affinity wt p53-specifc CTLs in normal p53 +/+ individuals.
Activation of CTLs to autoantigens seems feasible in cancer patients as evidenced by the recent analyses of responses against melanoma-associated antigens and against p53 (11, 16, 31). From the blood of healthy donors, CTLs reactive against the autoantigen tyrosinase and against wt p53 can be aroused from their unresponsive state by appropriate in vitro stimulation (29, 31, 60). Our data support the idea to use widely expressed tumor-associated antigens such as p53 for CTL-mediated immunotherapy.
Address correspondence to Cornelis J.M. Melief, Department of Immunohematology and Blood Bank, University Hospital Leiden, Bldg, 1, E3-Q, PO box 9600, 2300 RC Leiden, The Netherlands. Phone: 31-71-5263800; FAX: 31-71-5216751.
Received for publication 19 February 1997 and in revised form 6 June 1997.
H.W. Nijman was supported by The Netherlands Organization for Scientific Research grant 900-716-075.We would like to thank T. Ottenhof and B. Roep for critically reading the manuscript. G. Schijf and K. Goris were very helpful in technical assistance and maintaining the mice.
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