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*
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21287
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
CD25+ cells improves the effectiveness of Lm-E7 treatment,
suggesting that TGF- and CD25+ cells are in part
responsible for this suppressive response. CD4+ T cells
from mice immunized with Lm-E7 are capable of suppressing the ability
of Lm-LLO-E7 to induce the regression of TC-1 when transferred to
tumor-bearing mice. These studies demonstrate the complexity of
L. monocytogenes-mediated tumor immunotherapy targeting
the human tumor Ag, HPV-16 E7. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Listeria monocytogenes infection is a classic model for the induction of a protective cellular immune response (7). As an intracellular pathogen, L. monocytogenes has direct access to the cytoplasm of APC. This ability to access the cytoplasm is largely due to the hemolytic activity of listeriolysin O (LLO) (8). LLO, a 529-aa protein with hemolytic activity, is secreted by Listeria and perforates the phagosomal membrane, allowing the bacterium to escape the vacuole and enter the cytoplasm. The hemolytic domain of LLO resides in the C-terminus of the protein. Proteins secreted by L. monocytogenes during this intracellular phase of its life cycle are effectively targeted by the cellular immune system (9). We have taken advantage of L. monocytogenes to target proteins to the cellular immune system by engineering the bacterium to secrete influenza nucleoprotein, NP, a model tumor Ag. Treatment of mice bearing tumors expressing NP with the NP-secreting Listeria recombinant (Lm-LLO-NP) resulted in the regression of Ag-bearing tumors (10, 11, 12). Although the influenza NP is a useful model Ag, it is not expressed by human tumors. Hence, we have turned our attention to relevant tumor-specific Ags, the HPV-16 proteins E6 and E7 that are constitutively expressed in HPV-16-associated tumors (6). E6 and E7 expression is sufficient to immortalize murine or human cells (13, 14). For example, the poorly immunogenic tumor, TC-1, is derived from murine lung cells immortalized with HPV-16 E6 and E7 (13, 15, 16, 17). TC-1 provides a model in which a human tumor-associated protein (E7), the expression of which is driven by the endogenous E7 promoter, serves as a target for immunotherapy.
Here we describe two recombinant L. monocytogenes (rLm) strains, Lm-LLO-E7 and Lm-E7, that express and secrete E7. These recombinants differ enormously in their effectiveness as E7-specific tumor immunotherapeutics. Lm-LLO-E7 induces an immune response capable of causing the regression of established TC-1 tumors, whereas the other, Lm-E7, induces a response that suppresses anti-TC-1 immunity. In this study we have investigated the immune responses induced by the rLm strains and have established a system useful for comparing effective and ineffective induction of tumor immunity by recombinant L. monocytogenes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA).
Cell lines
The C57BL/6 syngeneic TC-1 tumor was immortalized with HPV-16 E6 and E7 and transformed with the c-Ha-ras oncogene (13). TC-1 expresses low levels of E6 and E7 and is highly tumorigenic. TC-1 was grown in RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µM nonessential amino acids, 1 mM sodium pyruvate, 50 µM 2-ME, 400 µg/ml G418, and 10% National Collection Type Culture-109 medium at 37° with 10% CO2.
L. monocytogenes strains and propagation
The Listeria strains used in the E7 tumor Ag studies
are Lm-LLO-E7 (hly-E7 fusion gene in an episomal expression
system), Lm-E7 (single-copy E7 gene cassette integrated into
Listeria genome), Lm-LLO-NP (hly-NP fusion
gene in an episomal expression system), and Lm-Gag (single-copy HIV-1
Gag gene cassette integrated into the chromosome). Lm-LLO-NP, also
known as DP-L2028 (18), and Lm-Gag, also known as ZY-18
(19), have been previously described. E7 was amplified by
PCR using the primers 5'-GGCTCGAGCATGGAGATACACC-3'
(XhoI site is underlined) and
5'-GGGGACTAGTTTATGGTTTCTGAGAACA-3' (SpeI site is
underlined) and ligated into pCR2.1 (Invitrogen, San Diego, CA). E7 was
excised from pCR2.1 by double digest with XhoI and
SpeI and ligated into pGG-55. The expression system, pGG-55,
is modeled on pDP-2028 (18). The hly-E7 fusion
gene and prfA are cloned into pAM401, a multicopy shuttle
plasmid, generating pGG-55. The hly promoter drives the
expression of the first 441 aa of the hly gene product, LLO,
which is joined by the XhoI site to the E7 gene. The result
is a hly-E7 fusion gene that is transcribed and secreted as
LLO-E7. By deleting the hemolytic C-terminus of LLO we have removed the
hemolytic activity in the fusion protein. The pluripotential
transcription factor, prfA, is also included on pGG-55. By
transforming a prfA negative strain of Listeria,
XFL-7 (a kind gift from Dr. Hao Shen, University of Pennsylvania), with
pGG-55 we select for the retention of the plasmid in vivo (Fig. 2
). The
hly promoter and gene fragment were generated using
primers
5'-GGGGGCTAGCCCTCCTTTGATTAGTATATTC-3'
(NheI site is underlined) and
5'-CTCCCTCGAGATCATAATTTACTTCATC-3' (XhoI
site is underlined). The prfA gene was PCR
amplified using primers
5'-GACTACAAGGACGATGACCGACAAGTGATAACCCGGGATCTAAATAAATCCGTTT-3'
(XbaI site is underlined) and
5'-CCCGTCGACCAGCTCTTCTTGGTGAAG-3' (SalI
site is underlined). Lm-E7 was generated by introducing an expression
cassette containing the hly promoter and signal sequence
driving the expression and secretion of E7 into the orfZ
domain of the L. monocytogenes genome. E7 was amplified by
PCR using the primers 5'-GCGGATCCCATGGAGATACACCTAC-3'
(BamHI site is underlined) and
5'-GCTCTAGATTATGGTTTCTGAG-3' (XbaI site is
underlined). E7 was then ligated into the pZY-21 shuttle vector. The
resulting plasmid, pZY-21-E7, is an expression system that includes the
previously described expression cassette inserted in the middle of a
1.6-kb sequence that corresponds to the orfX, Y, Z domain of
the L. monocytogenes genome. L. monocytogenes
strain 10403S was transformed with pZY-21-E7. The homology domain
allows for insertion of the E7 gene cassette into the orfZ
domain by homologous recombination. Clones were screened for
integration of the E7 gene cassette into the orfZ domain.
Bacteria were grown in brain heart infusion medium with (Lm-LLO-E7 and
Lm-LLO-NP) or without (Lm-E7 and ZY-18) chloramphenicol (20 µg/ml).
Bacteria were frozen in aliquots at -80°C.
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Primers were synthesized by Operon Technologies (Alameda, CA) and were resuspended in Tris-EDTA and stored at -20°C.
Synthetic peptides
Synthetic peptides were HPLC purified. Peptides were resuspended in DMSO or PBS (2 mg/ml) as solubility allowed.
Western blotting
Listeria strains were grown in Luria-Bertoni medium at 37°C and were harvested at the same OD measured at 600 nm. The supernatants were TCA precipitated and resuspended in 1x sample buffer supplemented with 0.1 N NaOH. Identical amounts of each cell pellet or each TCA-precipitated supernatant were loaded on 4–20% Tris-glycine SDS-PAGE gels (NOVEX, San Diego, CA). The gels were transferred to polyvinylidene difluoride and probed with an anti-E7 mAb (Zymed Laboratories, South San Francisco, CA). The secondary Ab was HRP-conjugated anti-mouse (Amersham Pharmacia Biotech, Little Chalfont, U.K.). Blots were developed with Amersham ECL detection reagents and exposed to Hyperfilm (Amersham Pharmacia Biotech).
Measurement of tumor growth
Tumors were measured every other day with calipers spanning the shortest and longest surface diameters. The mean of these two measurements was plotted as the mean tumor diameter in millimeters against various time points. Mice were sacrificed when the tumor diameter reached 20 mm. Tumor measurements for each time point are shown only for surviving mice.
Effects of Listeria recombinants on established tumor growth
Six- to 8-wk-old C57BL/6 mice (Charles River) received 2 x 105 TC-1 cells s.c. on the left flank. One week following tumor inoculation the tumors had reached a palpable size, 4–5 mm in diameter. Groups of eight mice were then treated with 0.1 LD50 i.p. Lm-LLO-E7 (107 CFU), Lm- E7 (106 CFU), Lm-LLO-NP (107 CFU), or Lm-Gag (5 x 105 CFU) on days 7 and 14 unless otherwise stated, or the mice were left untreated.
51Cr release assay
C57BL/6 mice, 6–8 wk old, were immunized i.p. with 0.1LD50 Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag or were left untreated. Ten days postimmunization spleens were harvested. Splenocytes were established in culture with irradiated TC-1 cells (100:1, splenocytes:TC-1) as feeder cells. Following 5 days of in vitro stimulation, splenocytes were used in a standard 51Cr release assay. Briefly, splenocytes were cultured with the following targets: EL-4, EL-4/E7, or EL-4 pulsed with E7 H-2b peptide (RAHYNIVTF) (20). E:T cell ratios were 80:1, 40:1, 20:1, 10:1, 5:1, and 2.5:1. All dilutions were performed in triplicate. Following a 4-h incubation at 37°C, cells were pelleted, and 50 µl supernatant was removed from each well. The samples were assayed with a Wallac 1450 scintillation counter (Gaithersburg, MD). The percent specific lysis was determined as [(experimental counts per minute - spontaneous counts per minute)/(total counts per minute - spontaneous counts per minute)] x 100.
TC-1-specific proliferation
C57BL/6 mice were immunized with 0.1 LD50 and boosted by i.p. injection 20 days later with 1 LD50 Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag. Six days after the boost the spleens were harvested from immunized and naive mice. Splenocytes were established in culture at 5 x 105/well in flat-bottom 96-well plates with 2.5 x 104, 1.25 x 104, 6 x 103, or 3 x 103 irradiated TC-1 cells/well as a source of E7 Ag. Splenocytes were also established without TC-1 cells or with 10 µg/ml Con A. The cells were pulsed 45 h later with 0.5 µCi [3H]thymidine/well. Plates were harvested 18 h later using a Tomtec harvester 96 (Orange, CT), and proliferation was assessed with a Wallac 1450 scintillation counter. The change in counts per minute was determined as experimental counts per minute - no Ag counts per minute.
Flow cytometric analysis
C57BL/6 mice were immunized i.v. with 0.1 LD50 Lm-LLO-E7 or Lm-E7 and boosted 30 days later. Three-color flow cytometry for CD8 (53-6.7, PE conjugated), CD62 ligand (CD62L; MEL-14, APC conjugated), and E7 H-2Db tetramer was performed using a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson, Mountain View, CA). Splenocytes harvested 5 days after the boost were stained at room temperature with H-2Db tetramers loaded with the E7 peptide (RAHYNIVTF) or a control (HIV-Gag) peptide. Tetramers were initially provided by Dr. Larry R. Pease (Mayo Clinic, Rochester, MN) and subsequently by the National Institute of Allergy and Infectious Diseases Tetramer Core Facility and the National Institutes of Health AIDS Research and Reference Reagent Program. The tetramers were used at a 1/200 dilution. Cells were analyzed as described above comparing tetramer+, CD8+, CD62Llow cells generated by Lm-E7 or Lm-LLO-E7 immunization.
Depletions of specific immune components
CD8+ cells, CD4+
cells and IFN-
were depleted in TC-1-bearing mice by injecting the
mice with 0.5 mg 2.43 (11), GK1.5 (11), or
xmg1.2 (21) mAb, respectively, on days 6, 7, 8, 10, 12,
and 14 post-tumor challenge. CD4+ and
CD8+ cell populations were reduced by 99% as
measured by flow cytometric analysis. Also, CD25+
cells and TGF- were depleted from TC-1-bearing mice. The
CD25+ cells were depleted by i.p. injection of
0.5 mg/mouse anti-CD25 mAb (PC61, a gift of Andrew J. Caton
(22)) on days 4 and 6 after tumor challenge. TGF- was
depleted by i.p. injection of the anti-TGF- mAb (2G7, a gift
from H. I. Levitsky), into TC-1-bearing mice on days 6, 7, 8, 10, 12,
14, 16, 18, and 20 following tumor challenge. Mice were treated with
107 Lm-LLO-E7 or Lm-E7 on day 7 following tumor
challenge. Tumor growth was measured as described above.
Adoptive transfer
Donor C57BL/6 mice were immunized and boosted 7 days later with 0.1 LD50 Lm-E7 or Lm-Gag. The donor splenocytes were harvested and passed over nylon wool columns to enrich for T cells. CD8+ T cells were depleted in vitro by incubating with 0.1 µg 2.43 anti-CD8 mAb for 30 min at room temperature. The labeled cells were then treated with rabbit complement. The donor splenocytes were >60% CD4+ T cells, as determined by flow cytometric analysis. TC-1 tumor-bearing recipient mice were immunized with 0.1 LD50 7 days post-tumor challenge. CD4+-enriched donor splenocytes (107) were transferred 9 days after tumor challenge to each recipient mouse by i.v. injection. Tumor growth was measured as described previously.
Statistics
For comparisons of tumor diameters, the mean and SD of tumor
size for each treatment group were determined, and statistical
significance was determined by Student’s t test
(23). In all experiments, p 
0.05 was
considered significant. The p values are reported in the
figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have designed and constructed two rLm strains that express and
secrete the HPV-16 E7 gene product. The constructs differ in their
expression system as well as in the form of the secreted E7 tumor Ag.
Lm-E7 has a single copy of the E7 gene integrated into the genome,
which expresses the E7 protein preceded only by the LLO signal sequence
to ensure secretion of E7 (Fig. 1A). Lm-LLO-E7 uses a
multicopy episomal expression system to secrete a fusion protein
consisting of a nonfunctional LLO joined at the C-terminus to E7 (Fig. 1B). The rLm construct, Lm-E7, is modeled after the Lm-Gag
recombinant that has previously been demonstrated to induce effective
anti-viral immunity (19, 24, 25). Lm-LLO-E7 is modeled
after the Lm-LLO-NP rLm strain that has shown remarkable effectiveness
as an immunotherapeutic targeting the artificial tumor Ag, NP
(10, 11, 12). Lm-LLO-E7 expresses and secretes a 67-kDa LLO-E7
fusion protein, and Lm-E7 secretes E7 that migrates at approximately 14
kDa, as verified by anti-E7 Western blot (Fig. 2
). The virulence of Lm-LLO-E7 and Lm-E7
is significantly decreased compared with that of the wild-type strain
10403S, but is similar to that of the respective control strains,
Lm-LLO-NP and Lm-Gag. We hypothesized that the expression system may
influence the effectiveness of the rLm as a tumor therapeutic.
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Lm-E7 and Lm-LLO-E7 were compared for their abilities to impact on
TC-1 growth. Subcutaneous tumors were established on the left flank of
C57BL/6 mice. Seven days later tumors had reached a palpable size of
4–5 mm in diameter. The mice were treated on days 7 and 14 with 0.1
LD50 Lm-E7, Lm-LLO-E7, or, as controls, Lm-Gag
and Lm-LLO-NP. While Lm-E7 had no effect on tumor growth compared with
the Lm-Gag control, Lm-LLO-E7 induced complete regression of 75% of
established TC-1 tumors (Fig. 3). The
slowing of TC-1 growth in Lm-E7-treated mice compared with naive
controls is clearly due to innate immune mechanisms, since the isogenic
control, Lm-Gag, slows tumor growth to the same extent.
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To determine whether Lm-LLO-E7 was more effective than Lm-E7 at
inducing an E7-specific CTL response, we compared CTL levels induced by
the two recombinants with a 51Cr release assay
using syngeneic EL-4 target cells. The results, shown in Fig. 4, demonstrate that both strains induce
similar levels of E7-specific lytic activity. EL-4/E7 and EL-4 pulsed
with the peptide, RAHYNIVTF, were effectively lysed by splenocytes
from Lm-E7- or Lm-LLO-E7-immunized mice, while splenocytes from control
immunized mice produced little or no lysis. Similarly, EL-4 without the
E7 peptide failed to be lysed during the duration of the
51Cr release assay.
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To further analyze the abilities of the two recombinants to induce
E7-specific CD8+ T cells, mice were immunized and
boosted with Lm-E7 or Lm-LLOE7, and their splenocytes were stained with
H-2Db tetramers loaded with the E7 peptide. When
activated CD8+ T cells were analyzed for tetramer
staining, Lm-E7 and Lm-LLO-E7 induced similar levels of
tetramer-positive T cells (Fig. 5).
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are necessary for TC-1
regression
In the NP model system both CD8+ T cells and
IFN- were necessary for the regression of established NP-bearing
tumors (11, 21). When CD8+ T cells
were depleted by the in vivo administration of Ab 2.43 following TC-1
challenge, Lm-LLO-E7 had little or no impact on tumor growth (Fig. 6A). Likewise, when IFN-
was depleted using the Ab xmg1.2 following TC-1 challenge, Lm-LLO-E7
had little or no impact on tumor growth (Fig. 6B). Depletion
of CD8+ T cells or IFN- had no effect on the
incapacity of Lm-E7 to influence the growth of TC-1 in C57BL/6 mice
(data not shown).
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Depletion of CD4+ cells significantly
decreases the effectiveness of Lm-LLO-E7 treatment on TC-1-bearing
mice. None of eight mice depleted of CD4+ cells
exhibited complete tumor regression, while five of eight nondepleted,
Lm-LLO-E7-treated mice had complete tumor regression (Fig. 7A). Surprisingly, depletion
of CD4+ cells in Lm-E7 mice improved the
anti-TC-1 response. Following depletion, three of eight
Lm-E7-treated mice had complete regression of established TC-1 tumors
(Fig. 7B). Also, mice with growing tumors in the
Lm-E7-treated, CD4+-depleted group showed slower
tumor growth compared with the nondepleted, Lm-E7 mice. The difference
in tumor size in the CD4+-depleted compared with
the undepleted group was statistically significant
(p < 0.001) on day 27 (Fig. 7B). The effects
demonstrated by the depletion experiments are not simply due to an
inability of the depleted animals to clear the Lm-LLO-E7 infection, as
the rLm are largely cleared by innate immunity. SCID mice infected with
107 Lm-LLO-E7 rapidly reduced numbers of
Lm-LLO-E7 in the spleen to below detectable levels within 2 days
following infection (G. R. Gunn, et al., unpublished
observations).
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The dramatic difference in the effects of
CD4+ T cells induced by Lm-E7 vs Lm-LLO-E7 on
tumor growth prompted us to explore the induction of T cells by the rLm
strains. We assessed the TC-1-specific proliferative response of
splenocytes from rLm-immunized mice. Proliferation of primed
splenocytes in response to stimulation with exogenous Ag is a measure
of Ag-specific immunocompetance. Ag-specific proliferation is largely
mediated by the release of IL-2 from T cells responding to the Ag
presented by APCs (26). The results indicate that
splenocytes from Lm-LLO-E7-immunized mice proliferate when exposed to
irradiated TC-1 cells as a source of E7 (Fig. 8). Conversely, splenocytes from Lm-E7
and rLm control immunized mice exhibited little or no proliferative
response to TC-1 cells. Lm-LLO-E7-induced proliferation was evident at
splenocyte to TC-1 ratios of 20:1, 40:1, 80:1, and 160:1. These data
suggest that Lm-E7 does not induce a Th cell response to the E7 Ag
expressed by TC-1.
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A population of CD4+ T cells expressing the
marker CD25 (IL-2R
) has been implicated in the maintenance of
immunological self-tolerance (27, 28, 29). Furthermore,
CD4+CD25+ T cells have been
shown to contribute to tumor growth by suppressing anti-tumor
immune responses (30). We hypothesized that this
suppressive population is associated with the lack of T cell help in
mice treated with Lm-E7. To address this question we depleted mice of
CD25+ cells with an anti-CD25 mAb. One
difficulty associated with this depletion is that the IL-2R is
up-regulated on activated T cells. Therefore, it was necessary to
deplete the CD25+ cells from naive, tumor-bearing
mice before priming this subset with Lm-E7. TC-1-bearing mice were
treated with anti-CD25 or the control Ab,
anti--galactosidase. While the depletion of
CD25+ cells had no effect on the growth of the
TC-1 tumors in naive mice, anti-CD25-treated mice receiving Lm-E7
exhibited significantly slower tumor growth than the
anti--galactosidase-treated controls (which were also treated
with Lm-E7; Fig. 9). These data suggest
that the CD4+CD25+
population may be playing a role in the poor anti-tumor immune
response generated by Lm-E7 treatment. However, in contrast to the
depletion of CD4+ T cells, the depletion of
CD25+ cells in Lm-E7-treated mice did not result
in the mice becoming tumor free. This may be due to the efficiency
and/or timing of the depletions or to the fact that there are other
CD4+ CD25- T cells that
have suppressive effects.
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in vivo greatly improves the efficacy of Lm-E7
treatment
TGF- has been implicated in the escape of tumors in
immunocompetent hosts (31, 32). Furthermore, TGF- was
reported to be secreted by CD4+,
CD25+, suppressive T cells and was necessary for
the immune suppressive function of these cells (33). We
depleted TGF- to ascertain its role in the tumor escape exhibited in
TC-1-bearing mice treated with Lm-E7. TC-1-bearing mice were treated
with an anti-TGF- mAb (2G7) before and after administration of
Lm-E7 or Lm-LLO-E7. While treatment with 2G7 had no apparent effect on
mice that received Lm-LLO-E7 (six of eight mice were tumor free without
2G7 and seven of eight were tumor free with 2G7), anti-TGF-
treatment had a profound effect on mice receiving Lm-E7 (Fig. 10). On day 21, the day after the last
administration of 2G7, five of eight Lm-E7 mice were tumor free, while
none of eight of the Lm-E7 group that did not receive 2G7 were tumor
free. However, soon after Ab administration was stopped, three of the
tumors that had regressed in response to Lm-E7/2G7 treatment grew out
(Fig. 10). These data suggest that TGF- plays a major role in the
suppression of the Lm-E7-mediated, anti-TC-1 response.
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We hypothesized that the suppressive anti-TC-1 response
induced by Lm-E7 treatment could be recapitulated in Lm-LLO-E7-treated
mice by cell transfer. To address this hypothesis we immunized and
boosted donor C57BL/6 mice with Lm-E7 or the control, Lm-Gag.
Splenocytes were harvested from the donor mice and enriched for
CD4+ T cells. These
CD4+-enriched splenocytes were transferred
(107 cells/recipient mouse) to TC-1-bearing mice
that had been treated with Lm-LLO-E7. While transfer of
CD4+ splenocytes from Lm-Gag-immunized donor mice
had no effect on Lm-LLO-E7 immunotherapy, CD4+
cells from Lm-E7-immunized donors largely abrogated the anti-TC-1
immune response, with two of eight mice tumor free compared with six of
eight tumor free in the other Lm-LLO-E7-treated groups (Fig. 11). These results demonstrate that
Lm-E7 not only fails to induce a potent Th cell response, but instead
induces a CD4+-suppressive population capable of
disrupting the anti-TC-1 immune response induced by Lm-LLO-E7.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
that Lm-LLO-E7 does not secrete more E7 than Lm-E7 when cultured in
vitro. However, since the expression of many of the
PrfA-regulated L. monocytogenes virulence factors
is up-regulated in vivo, it is possible that Lm-LLO-E7 expresses more
of the LLO-E7 fusion protein in vivo than does Lm-E7. It is also
conceivable that multiple copies of prfA could increase the
virulence of Lm-LLO-E7, thereby providing a higher Ag load and longer
Ag exposure to responding T cells. We have evidence that this is not
the case, since Lm-LLO-E7 has an approximately 10-fold higher
LD50 than Lm-E7. Therefore, neither expression
level of E7 nor the virulence increase resulting from multiple copies
of prfA appears to explain the difference in anti-tumor
immune responses provoked by the two recombinant bacteria. Lm-LLO-E7 and Lm-E7 express and secrete E7 in different forms. Lm-LLO-E7 expresses a large fusion protein made of a nonhemolytic LLO joined at its C-terminus to E7. In contrast, Lm-E7 expresses E7 preceded only by the LLO signal sequence and the first several amino acids of LLO. The listerial Ag, LLO, is very efficiently processed and presented to the immune system via MHC class I (34). LLO has been shown to be the dominant target of CD8+ T cells in mice immunized with wild-type L. monocytogenes (35). Work with another secreted listerial protein, p60, shows that manipulation of the N-terminus amino acid can drastically effect the processing and presentation of this secreted Ag, an example of the N-end rule (36). However, we designed Lm-E7 to ensure the efficient processing of the LLO signal sequence by including the first several amino acids of LLO after the signal sequence and preceding E7. Therefore, since both constructs encode the same N-terminus for the recombinant E7 protein, the N-end rule does not explain the vast difference in anti-tumor immunity induced by the two strains. Since the hemolytic domain of LLO was deleted from the LLO-E7 fusion protein, it is also not the case that the LLO portion of the LLO-E7 fusion protein alters the location or distribution of the protein by virtue of the hemolytic activity of LLO. It is, however, possible that a domain present in the LLO-E7 fusion protein that is absent in the E7 protein may influence the processing and presentation of LLO-E7 in such a way that it induces a superior anti-tumor response.
The experiments we have described in this paper demonstrate that
CD8+ T cell responses to the immunodominant
peptide RAHYNIVTF are induced by both Lm-E7 and Lm-LLO-E7 to
similar levels. However, it is possible that the form of the Ag may be
influencing the emergence of subdominant epitopes in the animals
immunized by the fusion protein. We do not believe that this is the
case because the level of CTL activity in the spleen of mice immunized
by Lm-LLO-E7, as measured by 51Cr release assays
(see
Figs. 3–5), is identical for RAHYNIVTF-pulsed targets and for
tumors expressing E7 as an endogenous Ag. However, such assays are only
crude indicators of the precursor frequency of epitope-specific CTL.
Therefore, we have not ruled out that Lm-LLO-E7 allows the emergence of
cryptic epitopes within the E7 sequence.
Lm-LLO-E7 and Lm-E7 induce greatly different TC-1-specific
proliferative responses. The proliferation assay shown in Fig. 5
demonstrates a very considerable difference in this functional quality
of the Lm-LLO-E7- and Lm-E7-induced responses. The proliferative
response of Lm-LLO-E7-immunized splenocytes in response to irradiated
TC-1 cells demonstrates that the E7 response induced by Lm-LLO-E7 can
be recalled by endogenous levels of E7. Proliferation in response to an
exogenous source of Ag is a conventional measure of Ag-specific T cell
help. Since Lm-E7 fails to elicit a TC-1-specific proliferative
response, this suggests poor induction of an E7-specific,
CD4+ T cell response by Lm-E7.
It is also possible that the inclusion of the LLO fusion and/or the lesser virulence of Lm-LLO-E7 effectively promotes a potent CD4+, E7-specific T cell response. The majority of phagocytosed L. monocytogenes are killed and degraded in the phagosomal-lysosomal compartment (37). Peptides generated by phagocytosis and degradation in the phagosomal-lysosomal compartment can be efficiently presented by MHC class II molecules to CD4+ T cells. The hly gene (that encodes for the LLO hemolysin) is up-regulated in the phagosome (38). Since they use the same promoter, the LLO-E7 fusion protein and E7 are also probably up-regulated, while Lm-LLO-E7 and Lm-E7 are in the phagosome. Nevertheless, it also may be that by including LLO in a fusion to E7 we have altered the processing of E7 in the phagosomal compartment compared with the E7 secreted by Lm-E7. The fact that Lm-LLO-E7 is less virulent than Lm-E7 and can be given in higher quantities may also influence the CD4+ T cell response, since the initial Ag load is approximately 10-fold larger than that for Lm-E7. Therefore, with the combination of a higher early Ag load and more efficient processing and presentation, Lm-LLO-E7 may induce a potent CD4+ T cell response, whereas Lm-E7 does not.
It should be noted that the lack of E7-specific Th cell responses in
mice immunized with Lm-E7 did not impact on the ability of this vaccine
to induce conventional E7-specific CTL responses (Fig. 4). This is not
surprising, since Th1 responses are undoubtedly provided in abundance
by responses to listerial Ags expressed by the vector. However,
E7-specific CD4+ effector cells clearly play a
very important role in competent anti-tumor immunity, as was
demonstrated by the depletion experiments shown in Fig. 8. Thus, the
efficacy of rLm vaccines engineered to express E7 appears to correlate
with the type of CD4+ T cell immunity induced by
these vectors.
Finally, the role of CD4+ T cells in the
regression of TC-1 seems to be complex. Depletion of
CD4+ cells in TC-1-bearing mice treated with
Lm-LLO-E7 predictably diminishes the ability of these mice to reject
their tumors. We have shown similar results in the influenza NP model,
where depletion of CD4+ cells weakened, but did
not completely abrogate, the anti-tumor response (11).
It is likely that these CD4+ T cells supply some
of the IFN- that is necessary for tumor regression
(39). However, depletion of CD4+ T
cells in mice treated with Lm-E7 improved the anti-TC-1 response
(Fig. 8). This suggests that the CD4+ T cell
response induced by Lm-E7 treatment is detrimental to the overall
anti-tumor response. The adoptive transfer data (Fig. 11) suggest
that Lm-E7 elicits CD4+ T cells that actively
respond with a suppressive phenotype and that the deletion of the
CD4+ compartment removes this suppression. The
lack of an in vitro proliferative response from splenocytes isolated
from Lm-E7-treated mice supports the suppression hypothesis, since
CD4+, CD25+-suppressive T
cells, as their designation suggests, fail to elicit proliferative
responses to Ag-specific stimulation (29). We and others
have also shown that depletion of CD25+ cells
improves tumor-specific immune responses (30) (
Figs. 3–11). The CD4+ T cell suppression is probably
mediated at least in part by the immune suppressive action of TGF-
(Fig. 10). Studies are currently underway to determine the exact
mechanism of this suppression.
Our results demonstrate that Lm-LLO-E7 is capable of inducing a potent anti-tumor response that targets the HPV E7 protein. As this protein is present in HPV-16-infected cells and in most cervical tumors, Lm-LLO-E7 may prove to be an effective immunotherapeutic in humans. We have also shown that CTL induction alone is a poor indicator for effectiveness of an anti-tumor vaccine. Ag-specific inhibition of tumor growth may be a better indicator of potential clinical effectiveness. It has been noted that there are no reliable surrogate immune markers for anti-tumor efficacy (40). A positive correlation between an in vitro measurable immune parameter and in vivo tumor regression would provide a powerful catalyst to rationally designed immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yvonne Paterson, Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076. E-mail address: yvonne{at}mail.med.upenn.edu
3 Abbreviations used in this paper: HPV, human papilloma virus; CD62L, CD62 ligand; rLm, recombinant L. monocytogenes; LLO, listeriolysin O; Lm-E7, rLm strain that secretes HPV-16 E7; Lm-Gag, rLm strain that secretes HIV-1 Gag; Lm-LLO-E7, rLm strain that secretes a fusion protein consisting of HPV-16 E7 plus part of the hemolysin of L. monocytogenes; Lm-LLO-NP, rLm strain that secretes a fusion protein consisting of the nucleoprotein of influenza strain A/PR/8/34 plus part of the hemolysin of L. monocytogenes; NP, nucleoprotein.
Received for publication November 29, 2000. Accepted for publication September 24, 2001.
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) monoclonal antibody. Cancer Res. 59:3128.1 cDNA escapes immune surveillance. Proc. Natl. Acad. Sci. USA 87:1486. expression inhibits cell proliferation in vitro, yet increases tumorigenicity and tumor growth of Meth A sarcoma cells. Cancer Res. 53:4391.-dependent inhibition of tumor angiogenesis by tumor-infiltrating CD4+ T cells requires tumor responsiveness to IFN-gamma. J. Immunol. 166:2276.This article has been cited by other articles:
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H. Starks, K. W. Bruhn, H. Shen, R. A. Barry, T. W. Dubensky, D. Brockstedt, D. J. Hinrichs, D. E. Higgins, J. F. Miller, M. Giedlin, et al. Listeria monocytogenes as a Vaccine Vector: Virulence Attenuation or Existing Antivector Immunity Does Not Diminish Therapeutic Efficacy J. Immunol., July 1, 2004; 173(1): 420 - 427. [Abstract] [Full Text] [PDF]
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C. Peters, X. Peng, D. Douven, Z.-K. Pan, and Y. Paterson The Induction of HIV Gag-Specific CD8+ T Cells in the Spleen and Gut-Associated Lymphoid Tissue by Parenteral or Mucosal Immunization with Recombinant Listeria monocytogenes HIV Gag J. Immunol., May 15, 2003; 170(10): 5176 - 5187. [Abstract] [Full Text] [PDF]
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cpm is defined as (experimental cpm) - (no-TC-1 control).
Depicted is one of two identical experiments.
