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Immunology |
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 min
(5)
to several hours (2)
. Furthermore,
the complex formation prevents p53 from transcriptionally activating
genes that induce cell cycle arrest (6
, 7)
or apoptosis
(8
, 9)
. In contrast, complex formation of p53 with the
human papillomavirus type 16 E6 (HPV16 E6) oncoprotein leads to an
increased ubiquitin-dependent and proteasome-mediated degradation of
p53 (10)
. Over the past years, several laboratories
have investigated the feasibility of p53-directed immunotherapy of
cancer (11, 12, 13, 14, 15, 16)
. A study by Gnjatic et al.
(17)
showed that overexpression of p53 in breast
adenocarcinomas and melanoma was required for recognition by
peptide-induced wtp53-specific CTLs. It was postulated that the
overexpression of p53 in tumor cells, in contrast to the low and
ubiquitous expression in normal tissue (18)
, creates a
therapeutic window allowing wtp53-specific CTLs to eradicate a tumor
without damage to normal tissue. This notion was supported by our study
in which wtp53-specific CTLs, raised in p53-/- mice, eradicated
p53-overexpressing tumors in p53+/+ mice without detectable damage to
normal tissue (16)
. Therefore, experimental data reported
thus far indicated that the efficacy of such immunotherapy would
require the tumor to express greatly elevated levels of p53 as
determined by IHC. This latter parameter is routinely used as a
diagnostic marker in cancer treatment (19)
. In the present study, we investigated whether T-cell-mediated immunotherapy of cancer exploiting p53 as a target antigen would indeed require the tumor to express greatly elevated levels of p53, or whether more modest expression levels would suffice to sensitize such tumors for immune attack by CTLs. Analysis of a panel of murine tumors, expressing different p53 steady-state levels, for sensitivity to p53-specific CTLs revealed that tumors expressing low levels of p53 could also be efficiently lysed by these CTLs. Accordingly, overexpression of p53 by the tumor is not a prerequisite for therapeutic efficacy of adoptively transferred p53-specific CTLs in tumor-bearing mice. Comparison of Ad5- and HPV16-transformed cells revealed that, in inverse correlation with p53 steady-state levels, HPV16-transformed cells are efficiently lysed by p53-specific CTLs, whereas their Ad5-transformed counterparts are not killed. Complex formation with the Ad5E1B protein sequesters and stabilizes p53 in Ad5-transformed cells, whereas in HPV16-transformed cells, p53 is rapidly degraded by the proteasome. Our data suggest that the rate of p53 processing rather than the p53 steady-state levels determine whether a given tumor is a good target for CTL-mediated immunotherapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-IFN (Pepro Tech; Northampton, England) for
48 h before use. Isolation and culture of the CTL clones used in
this study have been described elsewhere [wtp53-specific CTL clone
1H11 (16)
, HPV16 E7-specific CTL clone 9.5
(27)
, and Ad5E1B-specific CTL clone 0.1 C2
(28)
].
Adoptive Transfer of wtp53-specific CTLs.
wtp53-specific CTL clone 1H11 (2.0 x 107) was i.v. injected in either tumor-bearing or
nonchallenged p53+/+ C57Bl/6 nude mice and C57Bl/6 immunocompetent
mice. CTL administration was accompanied by 6 x 105 IU of rIL-2 emulsified in 50% IFA,
administered s.c. (on the day of adoptive transfer and 1 week later).
Control mice received either IL-2 alone or nothing.
Eu3+ Release Cytotoxicity Assay, Cytokine ELISA.
Experimental procedures to measure cell-mediated cytotoxicity in a
Eu3+ release assay have been described elsewhere
(29)
. For cytokine ELISA, supernatant of specifically
restimulated T-cell cultures was harvested after 3 days. Production of -IFN by the T cells was measured by a sandwich ELISA
performed in maxisorp plates (Nunc, Roskilde, Denmark) using
antimouse--IFN-specific Abs [clones R4–6A2 (capture) and
biotinylated XMG1.2 (detection); PharMingen, San Diego, CA],
streptavidin-conjugated poly-horseradish peroxidase (Central Laboratory
for Blood Transfusion, Amsterdam, the Netherlands), and
2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (Sigma, St.
Louis, MO) as a substrate. Absorbance at 415 nm was measured using
kineticalc 2.12 software in an EL312e Biokinetics ELISA plate reader
(Biotek Instruments, Winooski, VT).
IHC.
Tumor cells were spun down on a slide, air-dried, permeabilized, fixed
with acetone, and washed in PBS. The cells were incubated at
room temperature with 15 µg of Ab pAb 122 (30)
reactive against p53. The cells were then labeled with a biotinylated
second Ab directed against one of the p53 Abs. They were subsequently
incubated with a freshly prepared biotinylated horseradish
peroxidase/streptavidine complex (DAKO, Glostrup, Denmark) and
developed with 3,3'-diaminobenzidine, which forms a very
stable brown end product. This was followed by a Mayer
Heamatoxylin nuclear staining.
Immunoprecipitations.
Cell labeling and immunoprecipitations were performed as described
previously (4)
with minor modifications. In brief, for
each immunoprecipitation, 2 x 106
cells were labeled for 1 h with 500 µCi
EXPRE35S35S protein
labeling mix (New England Nuclear) in 2 ml of methionine-free medium
containing 5% of dialyzed FCS (Life Technologies, Inc.), after which
cell lysates were prepared (t = 0 h), or
cells were washed once with standard culture medium and chased for
indicated periods (t = 1 h,
t = 4 h). Before lysis, cells were washed once with
PBS, after which they were lysed in 1 ml of immunoprecipitation buffer
(30 mM HEPES/NaOH (pH 7.8), 140
mM NaCl, 1% Triton X-100,
phenylmethylsulfonyl fluoride, and trypsin inhibitor).
Immunoprecipitations were performed with protein G Sepharose (Pierce)
precoated with the Abs of choice. pAb 240 and pAb 246 were less
efficient in immunoprecipitating p53 from lysates than was pAb
122. This difference is most likely related to the weaker affinity of
protein G for murine IgG1 subclass Ab (pAb 240, pAb 246) than
for murine IgG2b subclass Ab (pAb 122; see Pierce
manual4
). Separation of immunoprecipitates on SDS polyacrylamide gels was
performed as described previously (4)
. The gels were
analyzed using a Molecular Dynamics PhosphoImager 445 SI with
Imagequant 4.2 software.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As reported by us previously (16)
, tumor
lines that were generated through transfection of a mutant murine
p53 gene (val135), are efficiently lysed by
p53-specific CTLs (Fig. 1, B and C)
. This
correlates with the fact that these cells express very high levels of
p53. The mutagen-induced MC38 colon carcinoma was also lysed by CTLs,
albeit at lower efficiency (Fig. 1E)
. This cell line also
expresses significant levels of p53, which are encoded by endogenous
copies of the p53 gene rather than by transfected
p53 genes. The p53-specific CTLs were found to lyse FBL-3
erythroleukemia cells with a comparable efficiency, whereas EL-4
thymoma cells were lysed even more efficiently. This
observation is striking because these latter two cells showed only
barely detectable steady-state levels of p53 (Fig. 1, D and F)
. These data indicate that high p53 expression levels as
detected by IHC are not a prerequisite for efficient killing by
p53-specific CTLs.
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and very low in HPV16-transformed cells
(Fig. 2, C and D)
. Intriguingly, the sensitivity
to lysis by p53-specific CTLs did not correlate with p53 expression
levels, in that the HPV16-transformed cells were lysed more efficiently
(Fig. 2, C and D)
than their Ad5-transformed
counterparts (Fig. 2, A and B)
. The poor
sensitivity of the Ad5-transformed cells was not attributable to
overall resistance to CTL lysis, inasmuch as these cells were
efficiently lysed by Ad5E1B-specific CTLs (Fig. 2, A and B)
as well as by p53-specific CTLs when exogenously loaded
with the relevant peptide epitope (not shown). To confirm that T-cell
receptor triggering, rather than overall sensitivity of targets to
lysis, dominated the outcome of our experiments, we tested the
secretion of -IFN (31)
by the p53-specific CTLs on
incubation with HPV16- or Ad5-transformed cells. Also in this assay,
the p53-specific CTLs are better triggered by the HPV16-transformed
cells despite the fact that steady-state p53 levels in these cells are
very low (Fig. 2E)
. Because in Ad5-transformed cells the p53
protein is reported to be very stable, whereas in HPV16-transformed
cells p53 was shown to be rapidly degraded, our data suggest that
degradation rate rather than steady-state levels determines whether
these cells constitute good targets for p53-specific CTLs.
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; t = 0 h) revealed that all of the cells
expressed p53, but that the synthesis rates of p53 differed greatly
between these cells. Fig. 3
shows different exposures of the same gel
(see Fig. 3
legend for details). The p53 + H-ras-transformed cells, which were generated through
transfection of a mutant p53 gene, showed, by far, the
highest p53 synthesis rates. MC38, FBL-3, and EL-4 synthesized
intermediate amounts of p53, whereas synthesis rates were very low in
Ad5- and HPV16-transformed cells.
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, Lanes
1 and 2). This is a result of the fact that these cells
express the temperature-sensitive mutant p53-val135, for which, at
37°C, wild-type and mutant conformations were shown to coexist
(34)
. Also in MC38 lysates, both wild-type and mutant
p53 were detected (Fig. 3
, Lanes 7 and 8).
Sequence analysis of the mutant p53 allele from these cells
revealed that it encodes a gene product carrying a glycine-to-valine
substitution at amino acid position 242 (data not shown). Only pAb 246
was capable of precipitating p53 from lysates of FBL-3, EL-4, and Ad5-
and HPV16-transformed cells (Fig. 3
, Lanes 14,
20, 26, and 32), which indicates that
these cell lines exclusively express wtp53.
The results obtained with pAb 240 and pAb 246 correspond well with the
immunoprecipitations from pulse-chase labeled cells (Fig. 3
,
t = 1 h, t = 4 h).
Quantification of the bands at different time points showed that the
half-life of p53 in FBL-3 and EL-4 was approximately 30 min. This is in
accordance with previously reported characteristics of wtp53
(5)
as well as with the fact that only pAb 246, but not
pAb 240, precipitates p53 from these cells. HPV16-transformed cells
show a greatly decreased p53 half-life in that all of the p53 is
degraded within 1 h (Fig. 3
, Lane 29), consistent with
the reported enhancement of p53 turnover by HPV16E6 (2)
.
In contrast, p53 turnover in Ad5-transformed cells is negligible (Fig. 3
, Lanes 22–24), which is attributable to the fact that it
is sequestered into stable complexes by the Ad5E1B
Mr 55,000 protein
(4)
. Of note, the presence of coprecipitated Ad5E1B bears
witness to the presence of such complexes in these cells. Finally, p53
is very stable in p53 + Ras MEC (Fig. 3
, Lanes 4–6) and in MC38 (Lanes 10–12), in
accordance with the observation that these cells express mutant
p53. Estimated overall p53 half-life in these cells is 4 h.
It is highly conceivable that the decrease in p53 in these cells is
attributable to turnover of the p53 fraction that exhibits the
wild-type conformation, as previously reported for other cell lines in
which these two forms of p53 coexists (34)
.
Taken together, the turnover rates of p53 in the different cells are in
accordance with the wild-type and/or mutant forms of p53 detected in
these cells. Furthermore, the immunoprecipitation data can account for
the steady-state levels of p53 as detected by IHC (Figs. 1
and 2)
. The
cell lines with low p53 turnover rates accumulate high levels of
intracellular p53, whereas cell lines with high p53 turnover show very
little p53 in IHC (Table 1)
. From this summary of the data, it can also be seen that recognition
of the cells by p53-specific CTLs does not show strong correlation with
p53 steady-state levels, as exemplified by the observation that
Ad5-transformed cells are poorly recognized. Neither does CTL
recognition require elevated p53 synthesis rates, because HPV16 MEC,
FBL-3, and EL-4 are efficiently recognized despite modest p53 synthesis
levels. Instead, Table 1
reveals that these CTLs especially recognize
target cells that display relatively high p53 turnover rates (HPV16
MEC, FBL-3, and EL-4), or cells that combine increased p53 half-life
with increased p53 synthesis levels. This is particularly true for
p53 + H-ras MEC and, to a lesser
extent, for MC38. In conclusion, elevated synthesis and/or steady-state
levels of p53 are not a prerequisite for recognition of tumor cells by
p53-specific CTLs, whereas high p53 turnover is an important factor
determining sensitivity of tumor cells to p53-specific CTLs.
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and 2)
suggest that the in vivo efficacy of these
CTLs may not be limited to tumor cells that express greatly elevated
levels of p53. We, therefore, challenged mice with tumor cells
expressing moderately increased (MC38) or barely detectable
steady-state levels (FBL3) of p53 (Fig. 1, D and E)
, and analyzed whether treatment of mice with adoptively
transferred anti-wtp53 CTLs and rIL-2 could prevent the outgrowth of
these tumors. In accordance with our observation that the CTLs can lyse
both of these tumor cells in vitro (Fig. 1, D and E)
, administration of these CTLs resulted in tumor-free
survival of the majority of the mice challenged with either of the
tumor cells (Figs. 4
and 5)
. These results imply that CTL-mediated immunotherapy that targets
p53-derived epitopes can be applied to tumors displaying a wide range
of p53 expression levels.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to HPV16-transformed cells, several other tumors (MC38, FBL-3, EL-4) expressing moderate-to-low levels of p53 were efficiently recognized by the wtp53-specific CTLs. Of these, FBL-3 and EL-4 also show high p53 turnover rates, which supports our notion that this parameter is of a high predictive value with respect to the recognition by p53-specific CTLs. The situation with MC38 is more complicated in that this tumor, like the p53-transfected tumors 4J and 5D, features a combination of increased p53 synthesis with increased p53 stability. Apparently, this setting also feeds sufficient p53 into the proteasome degradation pathway. Taken together, our data argue that overexpression of p53 can elevate, but does not necessarily result in an elevated presentation of, p53-derived peptides in surface MHC class I molecules. On the other hand, modest and even low steady-state p53 levels can suffice to sensitize tumors for immune attack by wtp53-specific CTLs, provided that the p53 turnover rates are high. Ultimately, techniques establishing the amount of p53-derived peptides presented by MHC class I molecules, like peptide/MHC-specific Abs (37) , will determine whether tumors constitute good targets for p53-directed CTL-mediated immunotherapy. However, these techniques are currently not available. We conclude that determination of p53 steady-state expression levels in tumors, as analyzed with IHC, is a valuable diagnostic tool, but that caution should be taken to use p53 expression as an inclusion criterion for clinical immune intervention protocols that target p53 because patients who might benefit from such an intervention might otherwise be excluded.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work has been supported by the
Dutch Cancer Society (Grant 96-1352) and by the Dutch Organization for
Scientific Research (Grant 920-03-094). G. M. J. B. is recipient of
a Dutch Cancer Society Fellowship. 
2 To whom requests for reprints should be
addressed, at Department of Immunohematology and Blood
Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA
Leiden, the Netherlands. Phone: 31-71-5263845; Fax: 31-71-5216751;
E-mail: R.Offringa{at}Immunohematology.MedFac.LeidenUniv.nl
3 The abbreviations used are: wtp53,
wild-type p53; MEC, mouse embryo cell; B6MEC, MEC of C57BL/6 origin;
IL, interleukin; rIL, recombinant IL; IFA, incomplete Freund’s
adjuvant; IHC, immunohistochemistry; Ab, antibody.
4 Pierce Technical Manual for Immunopure
Immobilized Protein G9, Rockford, IL.
Received 3/27/00. Accepted 8/ 4/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) the peptide are shown. For graphs
B–F, peptide-pulsed (• and lightfaced
lines) and -nonpulsed (
and boldfaced lines, the specific lysis of the tumor
cell line of interest by wtp53-specific CTL clone 1H11. Two
p53-transfected tumor cell lines were tested:
B, 4J (n = 8);
C, 5D (n = 6); three
in vivo induced tumors were tested:
D, FBL-3 (n = 4);
E, MC38 (n = 3); and
F, EL-4 (n = 7).
n, number of independent tests performed (each test was
performed in triplicate); abscissa, E:T ratio;
ordinate, percentage of specific lysis.
and
lightfaced lines) was a control for the lysability of
the Ad5E1-transformed tumor cells. C–D, CTL clone 9.5
recognizing the HPV16E7-derived epitope RAHYNIVTF (
