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Departments of
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Pathology and
Biology, McMaster University, Hamilton, Ontario, Canada
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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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The first identified member of the B7 costimulatory family of molecules was B7-1 (CD80). Molecular characterization of B7-1 has shown that the molecule is a 44- to 54-kDa member of the Ig superfamily and is the ligand for CD28 and CTLA-4 counter-receptors on T cells (5, 6, 7, 8). B7-1 was first described as an activated B cell marker (9, 10); however, B7-1 expression has since been localized to other APC, including dendritic cells, monocytes, and macrophages. The interaction of B7-1 with CD28 is vital for the amplification and generation of signals necessary for Ag-specific T cell responses and effector functions (11, 12, 13, 14). B7-1 and CD28 interactions have been shown to contribute to Th cell activation and function. In addition, B7-1 has been implicated as a necessary requirement for the generation of CD8+ CTL in the absence of help from CD4+ T cells (15, 16). Inhibiting the interaction of B7-1 with CD28 while allowing Ag-specific interaction can result in T cell anergy, probably by the lack of suitable autocrine growth factor, IL-2, production (as reviewed in 17 . Anergic tumor-infiltrating lymphocytes (TILs)3 have been demonstrated in some tumors. The use of rIL-2 in vitro to overcome anergy has shown that TILs can recognize tumor-specific Ags and be activated by appropriate signals to develop cytotoxicity (18).
Recently, a number of studies have demonstrated potential for the use of the B7 family of costimulators in tumor immunotherapy. The expression of B7 family members in murine tumor models has been shown to activate CD8+ T cells and or CD4+ T cells against the respective tumor cells (6, 16, 19, 20, 21, 22, 23, 24, 25, 26, 27). Moreover, administration of IL-2 has been shown to promote antitumor immunity, presumably by alleviating the anergic block seen in T cells in some tumor models and thereby preventing the onset of anergy (28, 29). We have previously shown (30) that an adenovirus vector expressing human IL-2 administered intratumorally results in approximately 45% regression in a murine adenocarcinoma model. The potential for additive or synergistic effects from the expression of both B7-1 and IL-2 prompted us to investigate the antitumor effects of intratumoral injection of a single adenoviral vector constructed to express both B7-1 and IL-2 from the same cell.
In this study we demonstrate that intratumoral injection of an adenovirus (Ad) vector constructed to express murine B7-1 (Ad5 mB7-1) results in 38% complete regression of tumors, while an Ad vector expressing IL-2 (Ad CAIL-2) (30) demonstrated complete regression in 42% of tumor-bearing mice. In contrast, intratumoral administration of a dicistronic vector expressing both B7-1 and IL-2 (Ad5 mB7-1/hIL-2) resulted in complete regression in 100% of tumor-bearing mice. Cured mice were shown to have generated systemic immunity to a subsequent challenge with fresh tumor cells, and tumor-specific cytotoxic T lymphocyte activity could be detected.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old FVB/n mice were purchased from Taconic Laboratories (Germantown, NY) and housed in a pathogen-free facility until use.
Cell lines
The cell lines used include the following: A549 (American Type Culture Collection, Rockville, MD; CCL-185); WM35, radial phase human melanoma (31); MRC5, human fibroblast cell line (American Type Culture Collection, CCL-171); PyMT, primary polyoma middle T Ag-transformed murine cells obtained from explanted tumors; PTO516, FVB/n kidney-derived cells, 516 MT3 cells derived from PTO516 and stably transformed to express polyoma middle T Ag; 293, human embryonic kidney cells transformed with adenoviral E1 sequences (32); and 293N3S, contact-independent 293 derivative (33). All cell culture reagents were purchased from Life Technologies (Grand Island, NY).
Construction of Ad5 mB7-1
Total RNA was isolated from FVB/n splenocytes using the reagent
Trizol (Life Technologies). RT-PCR was performed using the First Strand
cDNA Synthesis Kit (Life Technologies). Briefly, cDNA was synthesized
using oligo(dT) as the primer. PCR was performed using Vent DNA
polymerase (New England Biolabs, Beverley, MA) and the following
parameters: denaturation at 94°C for 1 min, annealing 55°C for
30 s, and extension at 72°C for 1 min. This was performed using
the following sense and antisense primers designed to anneal to the 5'-
and 3'-ends of the B7 cDNA sequence as deposited in GenBank. The sense
primer 5'-AAGATCTCTCCATTGGCTCTAGATTCCTGGC-3' and the
antisense primer
5'-GAAGATCTGATTGTACCTCATGAGCCACATAATA-3' were designed
to include BglII restriction sites (underlined). The
amplified fragment of 1023 bp was directly ligated into the
EcoRV-digested pDK6 shuttle plasmid (34) to create
pDK6-mB7-1 (Fig. 1
). The shuttle plasmid
pDK6-mB7-1 was amplified and purified by alkaline lysis and cesium
chloride gradient centrifugation. Purified plasmid was then combined
with the rescue plasmid pBHG10 (35) and cotransfected into 293 cell to
produce Ad5 mB7-1.
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The shuttle plasmid containing mB7-1 (pDK6 mB7-1) was sequenced, amplified, and then subjected to the following manipulation to generate a dicistronic construct expressing mB7-1 and hIL-2. The human IL-2 open reading frame was amplified using the following 5'-phosphorylated primers, 5'-TACAGGATGCAACTCCTGTCTTGC-3' (sense) and 5'-CTAATTATCAAGTCAGTGTTGA-3' (antisense). It should be noted that the sense primer is constructed such that the underlined codon (TAC) is the first amino acid immediately after the ATG start codon. The PCR product of 469 bp was then blunt end ligated into the pCITE 2a plasmid (Novagen, Milwaukee, WI), which carries the encephalomyocarditis virus internal ribosomal entry site (IRES). This plasmid was digested with NcoI (recognition site CCATGG) and subsequently blunt ended (blunting was performed with Klenow polymerase large fragment from New England Biolabs) to produce an ATG start codon before ligation with the IL-2 PCR product. Ligation of the IL-2 PCR product with the pCITE 2a plasmid resulted in pCITE—hIL2, which carries the hIL-2 open reading frame driven by the IRES sequence.
The pCITE–hIL-2 plasmid was used as a template for a
second round of PCR. Using the following phosphorylated primer,
5'-TTCCGGTTATTTTCCACCATATTG-3' (IRES sense), and the original hIL-2
antisense primer, a PCR product of 979 bp was amplified corresponding
to the IRES/hIL-2 hybrid molecule. This PCR product was blunt end
ligated into pDK6 mB7-1 digested with SalI (3' to the stop
codon of mB7-1) to produce pDK6 mB7-1/IRES/hIL-2 (Fig. 1
). This plasmid
was then processed in an identical manner to pDK6 mB7-1 (see Ad5 mB7-1)
and cotransfected with pBHG10 to generate Ad5 mB7-1/hIL-2. Both Ad5
mB7-1 and Ad5 mB7-1/hIL-2 were screened using Southern and Northern
techniques to characterize the presence of inserted DNA or the
production of monocistronic (Ad5 mB7-1) or dicistronic mRNAs (Ad5
mB7-1/hIL-2). In the case of Ad5 mB7-1/hIL-2 Western analysis was
performed to detect hIL-2 production. All cloning was confirmed by
sequencing.
Ad CAIL-2 and Ad5 dl70-3
A description of these viruses was provided by Addison (30).
Flow cytometry
Flow cytometric analysis was performed on MRC5, A549, WM35, and PyMT cells to characterize mB7-1 expression from both mB7-1-expressing vectors. Cells were infected for 48 h (MRC5, A549, and WM35) before analysis, and PyMT cells were infected and analyzed over 1 to 3 days. All groups were harvested at specific times, washed in PBS, and incubated with anti-mB7-1 Ab. The PyMT group was also incubated with anti-MHC I or II and anti-B7-2 Abs before flow cytometric analysis. Analysis was performed using a flow cytometer (Becton Dickinson, Mountain View, CA).
hIL-2 assay
MRC5, A549, and WM35 cells were infected at a multiplicity of infection (MOI) of 10 plaque-forming units/cell of either Ad5 CAIL-2 or Ad5 mB7-1/hIL-2. Infected cultures were incubated for 4 days in the case of MRC5 and WM35, and for 5 days in the case of A549 cells. At 24-h intervals, 200-µl aliquots were removed and stored at -70°C for quantification. Secreted hIL-2 was quantitated using the hIL-2 DuoSet Kit (Genzyme Diagnostics, Cambridge, MA).
Tumor cell preparation and vector administration
A transgenic mouse strain (FVB/n) expressing the polyoma middle T (PyMT) Ag under the control of the mouse mammary tumor virus long terminal repeat was the source of the tumor cells used in this study (36). Expression of PyMT Ag results in spontaneous transformation of the mammary epithelium by 8 to 10 wk of age. Tumors were excised from transgenic mice and subjected to enzymatic digestion to generate a single cell suspension (30). The single cell suspension was washed with PBS, and aliquots of 106 tumor cells were injected s.c. into the right hind flank of normal syngeneic FVB/n mice. Palpable tumors (normally 50–75 mm3) arise in these recipients 21 days after initial tumor cell injection, at which time appropriate amounts of virus were injected in a volume of 40 µl. After injection of adenoviral vectors, tumors were monitored weekly using calipers. The volume of the tumor was calculated from the longest diameter and average width, assuming a prolate spheroid. Mice with tumors not responding to vector treatment were killed when the longest diameter exceeded 20 mm. Regressed mice were left for approximately 3 mo and then challenged with 1 x 106 freshly isolated PyMT tumor cells on the left hind flank.
CTL assays
Splenocytes (effectors) were obtained from mice whose tumors had regressed as a result of Ad5 mB7-1, Ad CAIL-2, or Ad5 mB7-1/hIL-2 treatment and cocultured with 516 MT3 cells (stimulators) at a concentration of 1.2 x 105 516 MT3 to 1.2 x 107 splenocytes for 5 days in 12-well dishes. Serial dilutions of the effector cells were incubated in a V-bottom 96-well plate with 5 x 103 516 MT3 or PTO516 target cells. Target cells (106) were labeled with 100 µCi of 51Cr sodium salt for 2 h before coculture with the effector cells. Cells were cocultured for 5 to 6 h, at which time 80 µl of supernatant was removed for counting. The percent specific lysis was calculated as follows: 100 x (experimental cpm - spontaneous cpm)/(maximal cpm - spontaneous cpm).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant adenoviral vectors that express murine B7-1 (Ad5
mB7-1) or both mB7-1 and human IL-2 (Ad5 mB7-1/hIL-2) were constructed
as outlined in Figure 1. Expression cassettes for both mB7-1 or
mB7-1/hIL-2 were inserted into the E1-deleted region of the human
adenovirus type 5 genome. For both constructs, transgenes were flanked
by the murine CMV immediate early promoter (mCMV) and an SV40
polyadenylation signal (SV40). In the expression cassette for Ad5
mB7-1/hIL-2, the encephalomyocarditis virus IRES (37) was placed
between the mB7-1- and the hIL-2-coding sequences, resulting in a
dicistronic DNA fragment. The IRES functions as an internal ribosome
initiation site for translation of hIL-2 in the resulting
dicistronic mRNA.
Expression of the mB7-1 gene and hIL-2 gene (Ad5 mB7-1 and Ad5
mB7-1/hIL-2) was confirmed in human and murine cell lines infected with
each recombinant adenoviral vector. We initially decided to
characterize freshly isolated PyMT cells for the endogenous expression
of crucial immunologic molecules. Single cell suspensions of PyMT tumor
cells were checked by flow cytometry for surface expression of mB7-1,
mB7-2, MHC I and II (Fig. 2,
A–D). No mB7-1 expression could be detected on
uninfected PyMT tumor cells (Fig. 2A). Since no
detectable mB7-1 protein could be demonstrated on PyMT cells, we
proceeded to determine whether the Ad5 mB7-1 and Ad5 mB7-1/hIL-2
vectors could change the PyMT B7-negative phenotype to an
mB7-1-positive phenotype. Figure 2, E and F,
clearly demonstrates that mB7-1 protein was produced and integrated
into the cell membranes of PyMT tumor cells infected with either Ad5
mB7-1 or Ad5 mB7-1/hIL-2. This result confirms that the expression of
mB7-1 on PyMT tumor cells infected with both Ad5 mB7-1 or Ad5
mB7-1/hIL-2 vectors is due to the presence of the mB7-1 transgene (Fig. 2, E and F). WM35, A549, and MRC5 cells
could also be converted from a mB7-1-negative phenotype to a positive
phenotype after infection with the mB7-1-expressing vectors (data not
shown).
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The expression of hIL-2 from Ad5 mB7-1/hIL-2 was
characterized by Western blot analysis. Supernatants from 293 cell
cultures infected with Ad5 mB7-1/hIL-2 and, for comparison, Ad CAIL-2
were size fractionated by SDS-PAGE and Western blotted with an
anti-hIL-2 Ab, demonstrating the presence of two polypeptides of 15
and 17 kDa (data not shown). IL-2 from the dicistronic Ad5 mB7-1/hIL-2
and the monocistronic IL-2 control vector Ad CAIL-2 was quantified by
ELISA (Fig. 3, A–C).
IL-2 levels derived from the Ad5 mB7-1/hIL-2 vector were between 9 and
50 ng/ml compared with hIL-2 levels derived from Ad CAIL-2, which were
between 50 and 625 ng/ml for infections with the same MOI. The
expression observed per 1 x 106 cells with Ad5
mB7-1/hIL-2 was approximately 13-fold lower than that with Ad CAIL-2
(Fig. 3, A–C).
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We have previously shown that intratumoral injection of adenoviral
vectors expressing IL-2 or IL-12 to polyoma middle T tumor-bearing mice
cause regression at a dose of 5 x 108
plaque-forming units (30, 38). We used this same dose for Ad5 mB7-1,
Ad5 mB7-1/hIL-2, and Ad CAIL-2 and compared the effects to those seen
with the E1-deleted control vector, Ad5 dl70-3. Administration of
control virus did not modify the progression of the polyoma middle T
tumor growths in any of the control groups described here or previously
(30, 38). Conversely, treatment with Ad5 mB7-1 resulted in 38% (9 of
24 tumors) total regression of established tumors (Table I), and all the remaining 62% of Ad5
mB7-1-treated tumors showed a pronounced growth retardation, as shown
in Table I. Ad CAIL-2-treated tumors demonstrated different growth or
regression kinetics compared with Ad5 mB7-1. While 42% (10 of 24
tumors) of the Ad CAIL-2-treated mice demonstrated complete regression
(Table I), 37% (9 of 24 tumors) demonstrated a partial growth delay
(19 of 24 tumors, or 79% overall responded), and 21% (5 of 24 tumors)
showed no response to treatment. In contrast, administration of Ad5
mB7-1/hIL-2 caused complete regression of all (100%; n =
38) tumors treated, suggesting at least additive, if not synergistic,
effects between mB7-1 and hIL-2 expressed from the same cell (Table I).
Ad5 dl70-3-treated tumors demonstrated no growth alteration to the
vector; as a result, these mice were usually killed between days 30 and
35 post-tumor cell injection. All mice exhibiting complete regression,
regardless of which vector was used, were tumor free for at least 110
days postinjection.
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Mice that had undergone complete regression were challenged on the
left hind flank with 1 x 106 freshly isolated
polyoma middle T cells on day 110 after the initial vector
administration (see Materials and Methods). None of
the challenged mice developed tumors, and all remained tumor free for
an additional 120 days after challenge (Table II). To ensure the tumor-forming capacity
of these cells, untreated syngeneic mice were included to observe the
kinetics of tumor growth (Table II).
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Two cured mice from each of the following treatments were killed,
and their spleens were removed 120 days after challenge. Splenocytes
prepared from Ad5 mB7-1-treated mice (120 days after challenge)
demonstrated a 13 to 23% specific lysis of 516 MT3 cells at an E:T
cell ratio of 3.3:1 (Fig. 4). Lysis of
PT0516 (no polyoma middle T Ag) was also observed, however at a much
lower percentage. In contrast, splenocytes from Ad CAIL-2-treated mice
(120 days after challenge) demonstrated 57 to 68% specific lysis of
516 MT3 cells and undetectable levels of lysis on PT0516 cells (at E:T
cell ratios of 3.3:1; Fig. 4). Similarly, splenocytes from Ad5
mB7-1/hIL-2-treated mice (120 days after challenge) demonstrated 65 to
73% lysis of 516 MT3 targets and undetectable levels of lysis on
PT0516 cells (at E:T cell ratios of 3.3:1; Fig. 4). When splenocytes
from control mice were used, target cell killing was similar to or less
than that seen for PyMT-negative targets at all effector cell ratios.
This suggests the presence of significant numbers of effector cells
capable of killing PyMT-transformed cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent evidence has demonstrated the ability of certain cytokines to promote tumor rejection, and since the initial IL-2 study by Rosenberg et al. (39), cytokines such as IL-2, IL-4, IL-7, and IL-12 have been studied to assess their potential for mediating tumor rejection in various immunotherapy protocols. Most of these therapies have been limited by the appearance of toxic effects elicited by certain cytokines or the inability of the cytokines under investigation to induce efficient antitumor effector activity (40, 41, 42, 43). TILs and lymphokine-activated killer cells have also been used with only limited success (41, 42, 44). Evidence suggests that genetically modified tumors cells expressing IL-2 or IL-4 either by transfection or retroviral integration can abrogate the ability of tumors to grow (45, 46, 47, 48). We have recently used adenoviral vectors to deliver cytokines such as IL-2, IL-4, and IL-12 intratumorally (30, 38, 49) and have shown that the transient expression of IL-2, IL-4, and IL-12 could augment the recognition of the tumor by the immune system and result in complete regression in a significant fraction of the tumors treated.
In contrast to the use of cytokine mediators, other tumor immunotherapy regimens have focused on the B7 family of costimulatory molecules. B7 family members are required for the transduction of signals that promote T cell activation via B7 ligation with T cell-derived CD28 in the presence of MHC I or II Ag/TCR interactions (50, 51, 52, 53). Transfection of tumor cells with B7-1 has been extensively used and demonstrated to result in only moderate effects on tumor regression (21, 24, 26, 54, 55). More pertinent to this work are the observations of a number of groups that show that B7-1 can be used to augment the activity of IL-12, IL-2, and IL-7 in vivo (56, 57, 58, 59, 60) by positively effecting tumor regression, possibly through interaction with adhesion molecules such as intercellular adhesion molecule-1 (61). Taken together these studies demonstrate that B7-modified tumor cells are capable of delivering, in conjunction with MHC I or II, Ag-specific activation signals to T cells. By far one of the more beneficial effects of B7 enhancement of tumorigenicity is the ability of B7 to directly activate naive CD8+ CTL in the absence of CD4+ help (62).
It is evident from the current literature that the use of cytokines or costimulatory molecules in isolation to modulate the immune response against established tumors is of limited efficacy. The biologic evidence for the activity of IL-2 and B7-1 on tumor rejection prompted us to examine augmentation of the immune response by supplying both B7-1 and IL-2 to the same cell in vivo, reasoning that this might enhance the ability of the immune system to recognize and react against established tumors.
We have demonstrated the efficiency of Ad vectors (Ad5 mB7-1 and Ad5
mB7-1/hIL-2) constructed to express the costimulatory molecule B7-1
(murine) at converting B7-1-negative PyMT tumor cells to a
B7-1-positive phenotype (Fig. 2, A, E, and
F). We have also demonstrated the ability of the
double recombinant vector (Ad5 mB7-1/hIL-2) to produce hIL-2 (Fig. 3);
however, hIL-2 production was at a level 1 log lower than that observed
for the single vector expressing hIL-2 (Ad CAIL-2; Fig. 3) that we had
previously used in this model (30).
Comparison of the effects of intratumoral injection of Ad5 mB7-1 or Ad
CAIL-2 vs Ad5 mB7-1/hIL-2 demonstrated that the combination vector was
much more effective at inducing complete regression in the PyMT model
than either of the vectors expressing mB7-1 or IL-2 alone. Intratumoral
administration of Ad5 mB7-1 demonstrated 37.5% total regression (9 of
24 mice; Table I), with the other 62.5% (15 of 24 mice) showing a
partial response characterized by a drastic reduction in tumor volume
but subsequent relapse into a rapid growth phase (all 24 mice
responded). Similar to our previous data (30), Ad CAIL-2 injection
resulted in 10 of 24 (or 42%) of the animals demonstrating complete
regression and 37% exhibiting partial reduction in tumor volume or a
growth delay (19 of 24 mice responded), with an overall response of
79%. In contrast, Ad5 mB7-1/hIL-2 treated tumors resulted in complete
regression of all tumors treated (100%). Another important observation
demonstrated by Ad5 mB7-1/hIL-2 is the shorter time taken for PyMT
tumors to regress completely. Consistently, all tumors regressed within
14 days of vector administration. On the other hand, tumors undergoing
complete regression by treatment with either Ad5 mB7-1 or Ad CAIL-2
took 19 to 27 days to completely regress.
To determine the ability of all vectors to induce protection from
freshly isolated PyMT tumor cells, we challenged all completely
regressed mice (from Expt. 1, 2, and 3; Table I) with freshly isolated
PyMT tumor cells 110 days after primary tumor injection. Tumor cell
challenge was always administered on the opposite hind flank to the
site of the regressed tumor. All mice that exhibited complete
regression were found to be protected against challenge, demonstrating
that all vectors were capable of generating long lasting systemic
immunity (Table II).
To determine a possible mechanism and the specificity of the effector
function generated during treatment with the vectors, we examined the
CTL activity in the spleen of each completely regressed animal (Fig. 4, A–C). Ad5 mB7-1-treated animals demonstrated 13 to
23% specific lysis on 516 MT3 polyoma middle T-expressing cell line at
an E:T cell ratio of 3.3:1. This is low, but, nevertheless,
significant, since the same spleen cells resulted in only 1% lysis of
the parental line PTO516 that does not express polyoma middle T (Fig. 4A). Conversion of the PyMT tumor from a
B7-1- to a B7-1+ phenotype could enhance the
ability of NK cells to target the tumor cells via a mechanism similar
to that observed by Yeh et al. (63) and Chambers et al. (64).
Therefore, B7-1+ PyMT tumor cells could enhance not only
MHC-restricted effector cells, but also non-MHC restricted NK cells.
The conversion of PyMT cells from a B7-1- to a
B7-1+ population potentially provides an environment for
the activation of at least two effector cell populations (NK and
CD4+/CD8+), resulting in the 100% response
observed for tumors treated with Ad5 mB7-1. Ad5 mB7-1/hIL-2 treatment
results in high PyMT-specific CTL activity (65 to 73% lysis on 516
MT3), as opposed to undetectable lysis on nonspecific targets at an E:T
cell ratio of 3.3:1 (Fig. 4B), similar to that seen
in Ad CAIL-2-treated mice that underwent total regression (Fig. 4C). In contrast to the single vectors, Ad5
mB7-1/hIL-2 encompasses both effects mentioned above for Ad5 mB7-1 and
Ad CAIL-2 into a single system capable of up-regulating IL-2R levels
and providing the autocrine activity of IL-2 directly at the site of
CD28 (T cell): mB7-1 (PyMT tumor cells) ligation. mB7-1 ligation with
CD28 may act to provide the missing signal(s) necessary to reverse
anergy and allow for a greater proliferative response of effector
cells. We know that the hIL-2 levels produced by Ad5 mB7-1/hIL-2 are
13-fold lower than those observed for Ad CAIL-2. Hence, we propose that
hIL-2 and mB7-1 act synergistically to overcome anergy by providing a
microenvironment maximally conducive to effector function and
proliferation.
The findings of this study demonstrate the effectiveness of augmenting the immune response against tumors with adenoviral vectors expressing mB7-1 in combination with hIL-2 (Ad5 mB7-1/hIL-2). Future analyses will determine the effector population activated by the double construct and the effectiveness of this construct in the treatment of other established murine tumors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jack Gauldie, Department of Pathology, 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5.
3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; Ad, adenovirus; hIL, human IL; IRES, internal ribosome entry site; MOI, multiplicity of infection.
Received for publication August 25, 1997. Accepted for publication November 17, 1997.
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elicits CD8+ T cells against the wild-type tumor: correlation with antigen presentation capability. J. Exp. Med. 175:1423.This article has been cited by other articles:
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