By
From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
Elevated levels of the p53 protein occur in ~50% of human malignancies, which makes it an
excellent target for a broad-spectrum T cell immunotherapy of cancer. A major barrier to the
design of p53-specific immunotherapeutics and vaccines, however, is the possibility that T cells
may be tolerant of antigens derived from wild-type p53 due to its low level of expression in
normal thymus and lymphohemopoetic cells. The combination of p53 deficient (p53/
) and
p53+/+ HLA-A2.1/Kb transgenic mice was used as a model to explore the possibility that A2.1restricted cytotoxic T lymphocytes (CTL) are functionally tolerant of self peptides derived
from the wild-type p53 tumor suppressor protein. A2.1-restricted CTL specific for a naturally processed p53 self-epitope spanning residues 187-197 were completely aborted in p53+/+ as
opposed to p53
/
transgenic mice. In contrast, CTL specific for a second self-epitope spanning residues 261-269 of the murine p53 sequence were detected in both p53
/
and p53+/+
A2.1/Kb transgenic mice. However, the avidity of the CTL effectors obtained from p53+/+
mice was 10-fold lower than that obtained from p53
/
mice, again suggesting elimination of
CTL with high avidity for the A2.1-peptide complex. The circumvention of functional tolerance of high avidity CTL may therefore be a necessary prerequisite for optimizing immunotherapy against A2.1-restricted wild-type p53 epitopes in humans.
Peptides presented by class I MHC molecules and derived from normal self-proteins that are expressed at elevated levels by cells from a wide variety of human (Hu)1 malignancies provide, in theory, potential target antigens for a
broad-spectrum, CTL-based immunotherapy of cancer. The
Hu p53 tumor suppressor protein is an excellent source for
such general, tumor-associated and class I MHC-bound
CTL epitopes as its expression is markedly upregulated in
many different types of tumor cells. Such overexpression
correlates with the presence of mutation within p53 that inactivates its normal activity in tumor supression (1, 2). Due
to the diversity of mutations in p53 that can arise in tumors, if this protein is to serve as a general tumor antigen, it
would be necessary to target peptides representative of the wild-type (WT) sequence. However, since it is also expressed at low level in some types of normal tissues, such as
thymus, spleen, and lymphohemopoetic cells (3), WT p53
derived self-MHC-self-peptide complexes may also represent thymic and/or peripheral tolerogens, thereby preventing immune responses (6). This is particularly true for
class I MHC-peptide complexes expressed by bone marrow derived cells in the thymus, as such expression would
cause negative selection of immature thymic T cells with
high avidity for self-MHC-self-peptide complexes (6).
In the case of p53, this intrathymic deletion of potentially
self-p53-reactive T cells could result in a peripheral T cell
repertoire purged of CTL precursors with sufficient avidity to recognize natural WT p53 epitopes presented by class I
MHC molecules on tumor cells. Several studies in mice
and man, however, have provided conflicting results of
whether class I MHC-restricted CTL are functionally tolerant of WT p53 (15).
We have recently devised an experimental strategy (23)
that exploits species differences between Hu and murine (Mu)
WT p53 protein sequences (24) to circumvent Hu WT
p53-specific T cell tolerance and obtain HLA-restricted CTL
specific for epitopes from Hu p53. HLA-A2.1 To explore the possibility that class I MHC-restricted
CTL are tolerant of WT p53, we took advantage of p53 Mice.
The p53 Peptides.
The selection of synthetic peptides representing sequences within the Hu WT p53 protein as well as their binding
affinities for A2.1 have been reported (23). Synthetic WT p53
peptides that were homologous and nonhomologous to Mu WT
p53 sequences and had either high (Hu p53.65-73, 129-137, 187195, 264-272, and Mu p53.261-269) or intermediate (Hu p53.149157, 187-197, 210-218, 255-264, and 322-330) A2.1-binding affinity were used in these studies (Table 1) (23). Peptides were
synthesized by the core facility of TSRI using a synthesizer
(model 430A; Applied Biosystems Foster City, CA). Purity was
ascertained by mass spectrometry and reverse-phase HPLC analysis on a Vydac C18 column (Hesperia, CA).
Table 1.
Immunogenicity of A2.1-binding WT p53 Peptides in p53 (A2.1
)
transgenic (Tg) mice (25) were used to generate A2.1restricted CTL with high avidity for endogenously processed, nonhomologous Hu WT p53 peptides. The Hu
WT p53-specific CTL obtained were able to lyse a broad
variety of p53-overexpressing Hu tumor cells yet did not
recognize nontransformed Hu targets (23). Hu p53 peptides that were homologous to Mu WT p53 sequences did
not induce CTL responses capable of lysing tumor cells, regardless of whether these peptides had high, intermediate,
or low binding affinity to A2.1 (23). Considering that not
all of the nonhomologous Hu WT p53 peptides that bound
A2.1 were capable of inducing a CTL response (23), this
left unresolved the issue of whether self tolerance, or some
other mechanism, was responsible for nonresponsiveness to
homologous p53 peptides.
/
mice (26) backcrossed onto HLA-Tg mice expressing a
chimeric class I molecule, A2.1/Kb, which contains the
-1
and
-2 domains of the HLA-A2.1 molecule, and the
-3
domain of Mu H-2Kb (23, 25, 27, 28). The expression of
the chimeric A2.1/Kb molecule allows A2.1-restricted Mu
T cells to interact sufficiently with the
-3 domain of the
transgene product via their Mu CD8 coreceptor during
cognate recognition of A2.1-peptide complexes in thymus
and periphery (25, 27). Furthermore, the Ag processing and presentation machinery in A2.1/Kb-Tg mice and Hu
cells are sufficiently similar to present the same CTL epitopes (23, 25, 28, 34, 35). Also, the TCR repertoire in man
and A2.1/Kb-Tg mice has been demonstrated to be sufficiently diverse to respond to the same A2.1-peptide complexes (23, 36). The p53
/
A2.1/Kb mice do not express Mu WT p53 protein (26) and therefore would not be
tolerant of self-p53 epitopes presented by A2.1. Although p53 is required for thymocyte apoptosis induced by ionizing radiation (26, 39) or adenosine deaminase deficiency
(40), apoptotic cell death of thymocytes and mature T cells
as a result of exposure to compounds that mimic TCR engagement during negative selection has been shown to be
independent of p53 expression (26, 41). Except for a putative effect due to the lack of expression of self-MHC-p53
peptide complexes, repertoire selection of T cells in p53
/
and p53+/+ A2.1/Kb mice is anticipated to be identical. In
this report, we compared the capability of p53
/
and
p53+/+ A2.1/Kb-Tg mice to mount A2.1-restricted CTL
responses after immunization with A2.1-binding WT p53
peptides that were either homologous or nonhomologous
in sequence between Hu and Mu WT p53.
/
mice (26) were obtained from Tyler Jacks
(Massachusetts Institute of Technology) and mated with line 6 A2.1/Kb-Tg (23, 25, 28). Progeny were interbred, and offspring screened for mice that were p53
/
and expressed A2.1/Kb. Line
22 A2.1-Tg mice were H-2b/b and heterozygous for the transgene, which contains the
-1, -2, and -3 domain of A2.1 (23,
25). C57BL/6 mice were purchased from the breeding colony of
The Scripps Research Institute (TSRI). Mice were propagated
and maintained under specific pathogen free conditions in our vivarium at TSRI. All experimental procedures were performed
according to the National Institutes of Health Guide for the Care
and Use of Laboratory Animals.
/
A2.1/Kb-Tg and p53+/+A2.1/Kb-Tg Mice
E/T
Percent specific lysis
WT p53 peptide
p53
/
A2.1/Kb-Tg
p53+/+ A2.1/Kb-Tg
Position
Sequence
T2A2Kb
T2A2Kb + Pe*
T2A2Kb
T2A2Kb + Pe
Hu 65-73
RMPEAAPPV
30
5
44
4
31
10
3
16
2
13
Hu 129-137
ALNKMFCQL
30
3
2
4
5
3
6
2
3
0
Hu 149-157
STPPPGTRV
30
4
57
2
52
3
1
11
1
8
Hu 187-195
GLAPPQHLI
30
3
3
5
1
3
1
0
1
1
Hu 187-197
GLAPPQHLIRV
30
1
84
1
3
3
1
32
1
0
Hu 210-218
NTFRHSVVV
30
4
4
4
4
3
0
2
1
1
Hu 255-264
ITLEDSSGNL
30
2
1
0
1
3
0
0
2
0
Hu 264-272
LLGRNSFEV
30
3
42
3
57
3
1
10
1
14
Mu 261-269
LLGRDSFEV
30
1
82
1
45
3
1
37
0
16
Hu 322-330
PLDGEYFTL
60
11
12
ND
ND
6
7
1
ND
ND
Responder spleen cells from peptide-primed p53 /
and p53+/+ A2.1/Kb-Tg mice were restimulated in vitro with the indicated priming peptide.
After 6 d, effector cells were assayed for cytotoxicity against T2A2Kb targets and the same cells pulsed with the priming peptide at 10
6 M. Data are
representative for at least two independent experiments for each peptide and a total of at least four mice per strain and peptide. Amino acid residues
that are homologous to the MuWT p53 sequence are in bold type. Nonhomologous residues are underlined.
*
Pe, peptide.
Cell Lines. Previously described cell lines and transfectants used in these studies included T2-A2.1/Kb (T2A2Kb), Jurkat-A2.1 (JA2), the H-2b/b thymoma line EL4, EL4-A2.1/Kb (EA2Kb), the naturally A2.1-expressing, p53-deficient osteosarcoma line Saos-2, and this same line transfected with a Hu mutant p53 gene, Saos2/143 (23, 25, 27, 42, 43). To obtain EA2Kb.1p53 transfectants, EL4 cells were cotransfected with 10 µg of plasmid containing a genomic clone of A2.1/Kb (27) and 2 µg of pC53-4.2N3 containing a Hu mutant p53 cDNA linked to the neomycin resistance gene (44), as described (27). The corresponding parental and p53-transfected lines expressed similar levels of A2.1 (Saos-2, Saos2/143) or A2.1/Kb (EA2Kb, EA2Kb.1p53) as detected by flow cytometry (27). The 10(3) Balb/c fibroblast cell line, which lacks endogenous Mu p53 and a transfectant of the line that expresses a Mu p53 gene containing a mutation at residue 215, were kindly provided by Drs. Dirk Dittmer and Arnold Levine (Princeton University) (42, 45).
Peptide Priming of p53/
and p53+/+ A2.1/Kb-Tg Mice and
Propagation of CTL.
Mice were injected s.c. at the base of the
tail with 100 µg of the indicated WT p53 peptide and 120 µg of
the I-Ab-binding synthetic T helper peptide representing residues
128-140 of the hepatitis B virus core protein (36) emulsified in
100 µl of IFA (23). After 10 d, spleen cells of primed mice were cultured with irradiated A2.1/Kb-Tg LPS-activated spleen cell
stimulators that had been pulsed with the priming WT p53 peptide at 5 µg/ml and Hu
2-microglobulin at 10 µg/ml in complete
RPMI media (RPMI1640 containing 10% vol/vol fetal calf serum, 25 mM Hepes, 2 mM glutamine, 5 × 10
5 M
-mercaptoethenol and 50 µg/ml gentamycin) (23, 25, 28). After 6 d, the resultant effector cells were assayed in a 4-h 51Cr-release assay (27)
at various E/T ratios for lytic activity against T2A2Kb and EL4 targets that had been pulsed with either the priming WT p53 peptide, an unrelated A2.1-binding peptide, or no peptide. Immunization of mice with a given peptide as well as the subsequent effector cell cultures and 51Cr-release assays were performed simultaneously for p53
/
and p53+/+ A2.1/Kb-Tg mice. A polyclonal, A2.1-restricted CTL line (p53
/
A2Kb 187) derived from
peptide-primed p53
/
A2.1/Kb mice and specific for Hu WT
p53.187-197 was established by weekly restimulation of effector
CTL with irradiated JA2 cells that had been pulsed with 5 µg of the
indicated p53 peptide, irradiated C57BL/6 spleen filler cells, and 2%
(vol/vol) rat Con A supernatant (23, 27). The polyclonal, A2.1restricted CTL line (A2 149) specific for Hu WT p53.149-157
has been established from peptide-primed A2.1-Tg mice as previously reported (23). CTL clones specific for Hu WT p53.187-197
(p53
/
A2Kb 187 clone 4) and 149-157 (A2 149 clone 5) were derived by limiting dilution (46) of effector cell populations originally obtained from peptide primed p53
/
A2.1/Kb and A2.1Tg mice, respectively. Stimulators used for CTL cloning were
peptide-pulsed JA2 cells. Lysis of EA2Kb and EA2Kb.1p53 as well
as cytokine- and noncytokine-pretreated Saos-2 and Saos-2/143
targets by Hu WT p53-specific CTL lines and clones was determined in a 5-h 51Cr-release assay (27). Cytokine-pretreated targets had been exposed for 20 h to both rIFN-
(R&D Systems,
Minneapolis, MN) at 20 ng/ml and rTNF-
(R&D Systems) at 3 ng/ml (23). Anti-A2.1 inhibition was performed by exposure of
51Cr-labeled target cells to the anti-A2.1 mAb PA2.1 at saturating, nontoxic concentrations (23, 47).
Origin and Use of Vaccinia Recombinants. The vaccinia recombinant, vv-A2.1, which expresses the HLA-A2.1 molecule (48) was kindly provided by Dr. M. Nishimura (Surgery Branch, National Cancer Institute). The vPE16 (49) vaccinia recombinant, which expresses the gp160 of the human immunodeficiency virus type 1, was kindly provided by Drs. Jack Bennink and Jonathan Yewdell (National Institute of Allergy and Infectious Diseases). Target cells were infected as monolayers with 20 PFU per cell for 1 1/2 h in RPMI containing 0.1% BSA. Virus was removed, and cells incubated 4 h in complete RPMI. Monolayers were disrupted by trypsinization, and cells labeled with 51Cr as previously described (27).
Spleen cells from mice primed
with homologous and nonhomologous A2.1-binding WT
p53 peptides were restimulated with peptide in vitro and
tested for an A2.1-restricted, peptide-specific CTL response.
As reported, p53+/+ A2.1/Kb-Tg mice could mount an A2.1restricted CTL response specific for several nonhomologous
Hu WT p53 peptides, including 65-73, 149-157, and 264272, the latter two of which have been shown to represent naturally processed CTL epitopes (Table 1) (23). Also as reported, these mice failed to develop A2.1-restricted CTL
specific for the remaining homologous and nonhomologous
Hu WT p53 peptides tested, including 129-137, 187-195, 187-197, 210-218, and 255-264 (23). As anticipated, p53/
A2.1/Kb mice responded to the same three nonhomologous
Hu p53 peptides recognized by p53+/+ A2.1/Kb mice, demonstrating a comparable level of lytic activity. A lack of A2.1restricted CTL responses by p53
/
A2.1/Kb-Tg mice was
also observed with the remaining nonhomologous peptides, as well as with three out of four of the homologous
WT p53 peptides (187-195, 255-264, and 322-330) which
are identical in sequence in Hu and Mu p53 (Table 1). In
contrast, however, a strong A2.1-restricted, peptide-specific CTL reponse was induced in p53
/
as opposed to
p53+/+ A2.1/Kb-Tg mice after priming with the homologous WT p53.187-197 peptide (Table 1 and Fig. 1 A). Also,
the amount of lysis obtained with responder spleen cells
from p53
/
A2.1/Kb-Tg mice immunized with the Mu
WT p53.261-269 peptide was about twofold higher as
compared with CTL derived from p53+/+ A2.1/Kb-Tg
mice (Table 1). Mu WT p53.261-269 is identical with Hu
p53.264-272 at all but one amino acid residue, and has an
equivalent high affinity for A2.1 (23). Significantly, the
concentration of Mu WT p53.261-269 peptide required to
obtain half-maximum lysis of T2A2Kb targets by CTL derived from p53
/
versus p53+/+ A2.1/Kb-Tg mice was
~10-fold lower (5.2 × 10
8 M versus 4.6 × 10
7 M) (Fig.
1 C). This was in contrast to the comparable avidity demonstrated by p53+/+ and
/
A2.1-restricted CTL specific for
the nonhomologous Hu WT p53 epitopes 149-157 and
264-272 (Fig. 1, B and D). As may be anticipated from lack
of tolerance against the Hu WT 264-272 peptide, there is
no cross-reactive recognition of the Mu 261-269 peptide
by the Hu WT 264-272-specific CTL, and vice-versa.
The peptide specificity and the A2.1 restriction of the
CTL reponses shown in Table 1 and Fig. 1 were evidenced
by the failure of these CTL both to lyse T2A2Kb cells
pulsed with unrelated A2.1-binding peptides, and to respond to A2.1/Kb-negative targets pulsed with the priming peptide (data not shown). It is of interest that A2.1-restricted
CTL derived from p53/
A2.1/Kb-Tg mice and specific
for the homologous 11-mer peptide p53.187-197 did not
recognize the 9-mer peptide p53.187-195, even though
the 9-mer had an 1.4-fold higher binding affinity for A2.1
(23) (data not shown).
A prerequisite for tolerance induction by any self
peptide is its ability to be endogenously processed and
transported into the endoplasmic reticulum for association
with class I MHC molecules (50). To determine whether the
homologous WT p53.187-197 peptide is actually presented
as naturally processed T cell epitope by A2.1 on the surface
of Mu and Hu cells, both a peptide-specific, polyclonal
CTL line (p53/
A2Kb 187) and a CTL clone (p53
/
A2Kb 187 clone 4) were established from p53
/
A2.1/KbTg mice and tested for p53-specific recognition of Mu and
Hu cell lines transfected with Hu p53, EA2Kb.1p53 and
Saos-2/143, respectively (Fig. 2, A and C). As a positive control for p53-specific, A2.1-restricted lysis, the polyclonal CTL line A2 149 and the A2 149 CTL clone 5, both
of which were derived from A2.1-Tg mice and specific for
the natural CTL epitope Hu WT p53.149-157 (23), were
included in these studies (Fig. 2, B and D). Comparison of
the levels of lysis of the p53 transfectants relative to the parental target lines indicated that both WT p53.187-197 and
149-157 were endogenously processed and presented by
Mu and Hu cells. Recognition was A2.1-restricted, as lysis
was inhibited by an A2.1-specific antibody (data not shown).
To obtain substantial Ag-specific lysis of A2.1-expressing
Saos-2/143 cells by p53
/
A2Kb 187 CTL clone 4 as opposed to A2 149 CTL clone 5, targets had to be pretreated
by IFN-
and TNF-
(Fig. 2, C and D), a method that is
known to facilitate TCR-mediated Ag recognition and target cell lysis by increasing the numbers both of MHC-peptide complexes and of adhesion molecules expressed on the
cell surface (51, 52). The failure of the particular p53
/
A2Kb 187 CTL clone 4 to lyse untreated Saos-2/143 cells
was consistent with the previous finding that CTL from
A2.1/Kb-Tg mice are at a disadvantage in recognition of
cells expressing A2.1 as compared with A2.1/Kb, due to
the inability of Mu CD8 to interact with the Hu
-3 domain of the A2.1 molecule (23, 25, 27, 28, 31). However, due to their in vivo selection and stimulation in the absence of a sufficient participation by Mu CD8, CTL from A2.1Tg mice, such as A2 149 clone 5, express TCRs with unusually high affinity for the relevant A2.1-peptide complex
and thus require less peptide Ag for their CD8-independent target cell recognition (23, 25, 53). A2 149 CTL clone
5 as opposed to p53
/
A2Kb 187 CTL clone 4 was therefore able to lyse untreated Saos-2/143 targets.
The Mu p53.261-269 Self-peptide is Presented by A2.1 on Murine Cells.
Previous studies had demonstrated the Hu
analogue of the Mu p53.261-269 peptide was endogenously processed and presented in association with A2.1
(23). However, as the Mu and Hu peptides differed at one
residue (Table 1) it was possible this difference affected the
presentation of the Mu peptide, resulting in incomplete tolerance induction. This could explain its recognition in
both p53-deficient and -sufficient animals. To determine if
the Mu WT p53.261-269 peptide was presented by A2.1
on Mu cells, a transfectant of the mouse 10(3) cell line
which expresses high levels of Mu p53, was infected with a
recombinant strain of vaccinia virus that expresses the A2.1
molecule, vv-A2.1. As a control for the effect of viral infection, cells were infected by a recombinant strain encoding the gp160 of HIV-1 (vPE16). As presented in Fig. 3,
the 10(3) Mu p53 transfectant expressing the A2.1 molecule was specifically lysed by a CTL population specific for
the Mu 261-269 peptide epitope. Neither the p53-deficient parental line, 10(3), nor the 10(3) Mu p53 transfectant infected with vPE16 rather than vv-A2.1 were lysed
by these same effectors. Therefore, the Mu WT p53.261269 peptide is presented by cells that express both Mu p53
and A2.1.
The combination of p53/
and p53+/+ A2.1/Kb-Tg
mice was used as a model to determine if normal levels of
expression of WT p53 self-peptides presented by A2.1 result in functional tolerance. The studies demonstrate that
the A2.1-restricted CTL response to the homologous WT
p53.187-197 self-peptide was eliminated by self tolerance
in p53+/+ A2.1/Kb-Tg mice. Also, comparison of the levels
of lysis by effector cells specific for the Mu WT p53.261269 self-peptide indicates that CTL with a significantly lower
avidity were recruited in p53+/+ relative to the p53
/
strain of A2.1/Kb-Tg mice. These findings are consistent
with a scenario in which functional tolerance of class I
MHC-restricted T cells to WT p53 results in either elimination of all p53-specific CTL (as with p53.187-197) or
elimination of CTL with high avidity for the relevant class I
MHC-peptide complexes (as with p53.261-269), while
those with low avidity persist. Recent studies demonstrate
that low avidity T cells can escape the negative selection
process and emerge into the periphery (54).
However, the data further suggest that gaps in the T cell repertoire that are independent of p53 expression also occur, as not all of the homologous and nonhomologous WT p53 peptides with high or intermediate A2.1-binding affinity were capable of inducing a CTL response in either strain of Tg mice. Such gaps may reflect lack of expression or positive selection of TCRs of the relevant specificity, or deletion attributable to the presence of crossreactive epitopes contributed by other proteins (10, 11, 58).
It is of interest that the WT p53.187-197 specific CTL
obtained from p53/
A2.1/Kb mice do not lyse EA2Kb
targets unless they are transfected with p53 and contain
high levels of the protein. Similarly, the EA2Kb targets
were not lysed by the Mu 261-269-specific CTL (data not shown). As is the case in normal lymphohemopoetic cells,
EL4 cells express only low levels of WT p53 (Hernandez,
J., and L.A. Sherman, unpublished observation). Therefore,
these results support our previous findings that normal levels of expression of p53 are insufficient for recognition by
CTL raised against p53 peptides (23). Nevertheless, these
same low numbers of class I MHC-peptide complexes are
sufficient to achieve tolerance of high avidity CTL, as observed in these studies. Previous reports have demonstrated that the amount of Ag required for negative selection is less than that required for recognition by effector T cells (61, 62).
Several Hu WT p53 peptides, such as 149-157, 187-197, and 264-272, that have been shown in this and a previous study (23) to be immunogenic in Tg mice and to represent naturally processed CTL epitopes, have also been demonstrated to stimulate Hu PBL primarily in vitro and to induce an A2.1-restricted, peptide-specific CTL response (19). Whereas CTL from Tg mice were able to lyse Hu p53 transfectants and/or Hu tumor cells (23), this has not been reported for CTL from Hu PBL (19), thereby raising the possibility that the latter represent low avidity T cells that have survived p53-specific tolerance induction. This hypothesis is supported by our observation that A2.1restricted CTL with apparently higher avidity for the Mu WT p53.261-269 self-peptide were aborted in p53+/+ A2.1/Kb-Tg mice, whereas low avidity CTL could be induced. It should be emphasized that Mu WT p53.261-269 differs by only one conservative amino acid exchange from the naturally processed Hu WT p53.264-272 T cell epitope and is bound by A2.1 with an equivalent high affinity (23).
In contrast to our results with Mu WT p53.261-269 and to studies with Hu PBL (19), we could not detect A2.1restricted CTL with even low avidity for the WT p53.187197 T cell epitope in p53+/+ A2.1/Kb-Tg animals. The molecular and biological reasons for these discrepancies are as yet unclear. As demonstrated by the data in Figs. 2 and 3, both peptides are endogenously presented in association with A2.1 on the surface of Mu cells. However, we cannot exclude the possibility that they are endogenously presented at different densities. It is possible the Mu WT p53.261269 epitope is processed and/or presented less efficiently than the p53.187-197 epitope. This could result in less efficient tolerance induction, but would not necessarily explain the difference in responsiveness to p53.187-197 by mouse and human CTL. Alternatively, the lower A2.1 binding affinity of p53.187-197 relative to Mu p53.261-269 (23) in conjunction with the low level of expression of the A2.1/Kb transgene relative to the level of expression of conventional MHC molecules (28), could be directly or indirectly involved. For example, the primary in vitro selection of Hu PBL derived, peptide-specific CTL involved repeated cycles of restimulation with Hu stimulators expressing high levels of A2.1 and pulsed with high concentrations of synthetic peptide in the presence of exogenous T cell growth promoting cytokines (19). Such conditions may promote expansion of low avidity CTL specific for peptides with intermediate A2.1-binding affinity, such as p53.187-197 (19, 63). However, the in vivo priming of A2.1/Kb-Tg mice expressing low levels of A2.1/Kb, followed by one cycle of in vitro restimulation of responder spleen cells with peptide-pulsed A2.1/Kb stimulators (in the absence of exogenous cytokines) may not be sufficient to induce T cells with low avidity for peptides that represent intermediate (WT p53.187-197) as opposed to high (Mu WT p53.261-269) A2.1 binders. It may be anticipated that low avidity T cells would be the ones most dependent for their stimulation on large numbers of MHC-peptide complexes, which in turn, would be fewer for peptides with lower affinity for A2.1. Future experiments will determine if altered experimental conditions can lead to induction of murine CTL specific for p53.187-197. Alternatively, A2.1-positive humans and these A2.1/Kb-Tg mice that have been demonstrated to be sufficiently similar in selecting A2.1-restricted T cells specific for peptides with high binding affinity to A2.1 could differ considerably from each other in their selection of low avidity T cells specific for self-peptides with intermediate A2.1-binding affinity (38).
In several recent papers, the immune response of BALB/c
mice to a Mu p53 peptide was contrasted with that same
peptide containing a point mutation (Mu p53.234-240 and
236 M to I) (15). Although immunization with peptide
in adjuvant resulted in responsiveness only to the mutated
sequence (15, 16), a response to the WT peptide was obtained when these same mice were injected with dendritic cells pulsed with peptide (17). These results indicated that it
was more difficult to obtain a response to the WT versus
the mutant p53 sequence, consistent with an effect on the
repertoire due to self tolerance. By using p53/
mice, our
results have directly demonstrated the influence of self tolerance on T cell recognition of p53.
In summary, this study shows functional tolerance of high avidity CTL specific for naturally processed WT p53 peptides presented by A2.1. The data also demonstrate the flexibility of WT p53-specific tolerance, as the extent by which low avidity CTL survived the induction of self-tolerance varied between the particular class I MHC-p53 peptide combinations. These residual low avidity CTL, however, could provide an opportunity for immunotherapy of tumors that express high levels of p53 (17, 18). Given the variability of the effect observed on the repertoire by selftolerance to different peptides, as well as variability of responsiveness due to different modes of immunization, it is likely that the success of the immunotherapy directed towards self-proteins will require careful examination of responses to each MHC-peptide complex. Furthermore, given the fact that low level expression of p53, such as that observed in normal cells and tumors expressing nonmutant p53, such as EL4, is insufficient for cell lysis, it is further likely that the level of presentation of p53 peptides by individual tumors will also determine the success of such immunotherapy.
Address correspondence to Linda A. Sherman, The Scripps Research Institute, Department of Immunology, IMM-15, 10666 North Torrey Pines Road, La Jolla, CA 92037. M. Theobald's present address is Department of Hematology, Johannes Gutenberg-University, Mainz, D-55101, Germany.
Received for publication 25 September 1996 and in revised form 19 December 1996.
M. Theobald is a fellow of the Stipendienprogramm Infektionsbiologie provided by the German Cancer Research Center (DKFZ) and funded by the German Ministry for Education and Research (BMBF). J. Hernandez is the recipient of a fellowship funded by the Spanish Ministry for Education and Science (EX 94 05269499). J. Lustgarten is the recipient of an American Cancer Society fellowship. These studies were supported by National Institutes of Health Grants CA 57855 and CA 25803 to L.A. Sherman.We thank Tyler Jacks for providing p53/
mice, and Arnold J. Levine (Princeton University) for providing
Saos-2/143 transfectants and the 10(3) cell lines. We thank Patty Krier and Carol Wood for excellent secretarial assistance.
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