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*
Laboratorio di Immunologia dei Tumori and Cancer Immunotherapy and Gene Therapy Program,
Unità di Epidemiologia, and
Università Vita e Salute, Istituto Scientifico H San Raffaele, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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),
pulsed with apoptotic cells primes tumor-specific CTLs and confers
protection against a tumor challenge. Our findings demonstrate that
tumor cells undergoing apoptosis are, though scarcely, immunogenic in
vivo, outline the different roles of M and DCs in the physiologic
clearance of unwanted cells, and have implications in designing
immunomodulating vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)3 in the
activation of a specific immune response. Monocytes/M, upon
engulfment of apoptotic cells, release immunoregulatory factors like
IL-10 and TGF-ß (4, 5), and, after phagocytosis of monocytes
committed to apoptosis by viral infection, do not elicit a specific CTL
response (6). On the other hand, dendritic cells (DCs) pulsed with
apoptotic monocytes activated in vitro virus-specific CTLs (6).
Therefore, depending on the scavengers involved in the clearance of
apoptotic cells, the result in terms of specific immune response may be
opposite. Furthermore, massive cellular destruction enhances
cross-presentation of peripheral self Ags in vivo (7). We reasoned that
the balance between turning off and on of the immune system against the
dying cells might be influenced by the relative amounts of cells
undergoing apoptosis at a given time, and by the APCs involved in their
clearance. To investigate the immunogenic potential of apoptotic cells in vivo, we used the highly tumorigenic (minimal tumorigenic dose: 103 cells) Rauscher virus-induced H-2b T cell lymphoma RMA (8, 9). RMA cells easily undergo apoptosis either by UV irradiation or by mitomycin C treatment, followed by serum deprivation (3).
In this study, we show that apoptotic tumor cells can be immunogenic in vivo, but are less immunogenic than nonreplicating (NR) live tumor cells. The immunogenicity of apoptotic cells appears to be tightly dependent on the APCs involved in processing and presentation of the antigenic material contained in the apoptotic cells, and on the balance among immunomodulatory cytokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 and IL-10-/- (10) female mice, 6- to 8-wk old (Charles River, Calco, Italy), were housed in a pathogen-free animal facility and treated in accordance with the European Community guidelines and the Institute Ethical Committee. Apoptosis was induced either by treatment with mitomycin C (50 µg/ml for 30 min at 37°C) followed by serum deprivation or by UV irradiation, and verified as previously described (3). Cells were injected 24 and 48 h after mitomycin C treatment or UV irradiation. As previously reported (3), at that time, >95% of the cells were in advanced apoptosis, as detected by staining with annexin V and analysis of the DNA content (data not shown). Necrosis was induced by exposing the cells to 3–5 cycles of rapid freezing/thawing in H2O (4 x 107/ml) until cell membrane integrity was lost. NR cells were obtained by treatment with mitomycin C (11) and injected immediately thereafter.
Phagocytosis assays
Phagocytosis of apoptotic cells was assessed by flow cytometry
(3). Briefly, mice were injected i.p. with 107
apoptotic (Apo) or NR-RMA cells, which were previously labeled with the
PKH2-GL aliphatic green fluorochrome, according to the manufacturer’s
instructions (Sigma, St. Louis, MO), and sacrificed at different time
points. Peritoneal cells were collected and analyzed by flow cytometry
after staining with anti-MAC-1 PE-conjugated mAb (PharMingen, San
Diego, CA) or immediately after addition of the vital dye ethidium
bromide (EB). EB allows to discriminate between phagocytes, which
actually engulfed apoptotic cells, and phagocytes, which bind to but do
not internalize them. Living cell membranes exclude the dye, which,
therefore, does not stain internalized apoptotic cells. M that
phagocytosed apoptotic cells are identified as green fluorescent cells,
whereas those that bound to, but do not internalize apoptotic cells,
are identified as double positive cells. In parallel, peritoneal cells
from mice injected with unlabeled Apo- or NR-RMA cells were stained
with anti-MAC-1 PE-conjugated mAb, anti-CD80, anti-CD86,
anti-MHC class I, anti-IAb, and
anti-CD40 FITC-conjugated mAbs (PharMingen) and analyzed by flow
cytometry.
Immunization models
M and DCs were derived by culturing bone marrow precursors
for 7–10 days in culture medium containing 20 ng/ml murine recombinant
M-CSF or GM-CSF (3, 12). M and DCs were pulsed overnight with a 1:5
ratio of Apo-RMA cells. As assessed by flow cytometry, >95% of the
cells cultured in the presence of either one of the two growth factors
expressed MAC-1, Kb, and Db
membrane molecules. The cells collected from the plates cultured in the
presence of GM-CSF also expressed the classical DC markers N418,
IAb, CD40, CD80, and CD86 (data not shown). After
apoptotic cell pulsing, DCs showed a phenotypic pattern of mature cells
(13). The phenotypic pattern of M was not modified by the overnight
incubation with apoptotic cells (data not shown). For immunization,
peritoneal cells were collected 20 h after the injection of
107 Apo-RMA cells or PBS, as described above.
Mice were injected i.p. or s.c. with 0.5 ml PBS or the indicated number
of NR-, Apo-, necrotic (Necro) RMA cells, Apo-EL4G cells, 2 x
105 M or DCs, or peritoneal cells from the
corresponding number of mice. Fourteen days later, mice were either
sacrificed and their spleen cells collected, or challenged s.c. in the
opposite flank with 104 RMA cells and checked for
tumor appearance every other day. Tumor appearance and size were
evaluated by measuring two perpendicular diameters by a caliper every
other day. Animals were scored positive when the mean of the two
perpendicular diameters of the tumor was >2 mm. Mice with no palpable
tumor 8 wk after tumor challenge were scored negative and rechallenged
in the opposite flank with 104 RMA cells. Cell
depletion experiments with mAbs and carragenan were conducted as
previously described (11, 14). When indicated, mice were injected i.p
with 1 µg of GM-CSF 30 min before injection of Apo-RMA cells.
In vitro CTLs induction
T-enriched cells were prepared from the spleens of vaccinated mice as described (15), resuspended in RPMI 1640 containing 10% heat inactivated FCS, 50 µM 2-ME, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (culture medium) and coincubated with 3 x 106 mitomycin C-treated RMA cells at an effector-stimulator ratio of 10:1. After 4 days of culture, blasts were isolated on a lympholyte-M gradient (Cedarlane, Hornby, Ontario, Canada), cultured for an additional day in medium supplemented with 20 IU/ml human rIL-2 and tested for cytolytic activity in a 4-h 51Cr release assay (11).
Ab and cytokine titration
Sera were collected by cardiac puncture 20 h (for cytokine titration), or, for IgG titration, 2 wk (for the first injection) or 1 wk (for the second injection), after immunization and frozen. For anti-RMA IgG titration, targets (103 51Cr-labeled RMA or Jurkat cells) were added to V-bottom wells of microtiter plates, incubated for 1 h at 37°C with serial dilution of normal or immune sera, and washed. Rabbit complement (1:40 dilution; Cedarlane) was added to each well, and plates were incubated for 2 h at 37°C. A total of 100 µl/well were collected and counted in a gamma counter. Cytokine titration was performed by ELISA kits following the manufacturers instructions (Endogen, Woburn, MA).
Statistical analysis
Statistical analyses were performed using the Log-Rank, Wilcoxon, and Fisher-Exact tests. Comparison of survival curves was considered statistically significant for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RMA is a well-characterized tumor model, whose immunogenicity is
strongly biased by the expression of virus-related T cell epitopes
(16). A single injection of 
106 NR-RMA cells
protects H-2b mice from a further challenge with
RMA cells (17). We recently reported that Apo-RMA cells were swiftly
phagocytosed by H-2b peritoneal M, and that a
subpopulation of engulfing M was able to present Apo-RMA-derived
epitopes to RMA-specific CTLs (3). To verify whether Apo-RMA cells were
able to activate in vivo a tumor-specific immune response, we injected
C57BL/6 mice with increasing numbers of Apo-RMA cells and measured the
ability of their T-enriched spleen cells (15), after in vitro
restimulation, to kill 51Cr-labeled RMA cells.
Indeed, mice injected with Apo-RMA cells developed a tumor-specific CTL
response (Fig. 1
A, first
panel). This response was not due to an in vitro priming of
anti-RMA CTLs, since spleen cells from PBS-injected animals never
killed RMA targets (Fig. 1A, fourth panel).
Twenty-fold more Apo-RMA than NR-RMA cells were necessary to induce
similar responses (Fig. 1A, first and
second panels). RMA cells killed by necrosis
(Necro-RMA) never elicited tumor-specific CTLs (Fig. 1A,
third panel), therefore excluding that immunogenicity was
due to soluble Ags released by the dying cells.
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B).
Jurkat cells, a human T cell leukemia line, were never recognized (data
not shown).
To investigate the in vivo efficacy of the immune response recruited by
Apo-RMA cells, immunized mice were challenged s.c. with
104 RMA cells. A significant protection and delay
in tumor appearance was evident at Apo-RMA cells doses
107 (Table I).
Doubling the dose of apoptotic cells increased the percentage of
protected animals, even though it never reached the gold standard
obtained by immunization with 0.5 x 106
NR-RMA cells (100% protection; Table I). The immune response elicited
by apoptotic cells is endowed with the characteristics of a mature
immune response including specificity, since apoptotic EL-4G cells, a
syngeneic chemically induced T cell lymphoma, did not protect against
RMA, and memory, since all immunized animals rejected a second
challenge of RMA (Table I). Necro-RMA cell administration did not
elicit any protective response (Table I).
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).
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Results in Figs. 1 and 2 and Table I demonstrate that cells
undergoing apoptosis may elicit an immune response that stems from the
interaction among all components of the adaptive immune response (i.e.,
CTLs, Th, and B cells), and requires the direct involvement of
professional APCs. M are the best-characterized scavenger phagocytes
(1). Their ability to cross-prime T lymphocytes may be quenched by the
fact that apoptotic cell clearance by M associates with secretion of
immunoregulatory factors, such as IL-10 and TGF-ß (4, 5). IL-10, in
particular, negatively affects the maturation and Ag
processing/presentation potential of DCs (20, 21) and skews DC
precursors toward a M-like scavenger pathway (22). Conversely,
proinflammatory cytokines, like IL-1ß, trigger DC maturation (20, 21). DCs, which also recognize and internalize apoptotic cells,
elicited in vitro virus-specific CTLs (6). Therefore, we measured the
concentration of IL-10 and IL-1ß in the sera of mice immunized with
the subimmunogenic (i.e., 5 x 106) or the
immunogenic (i.e., 107) dose of Apo-RMA cells,
and compared them with the data obtained in mice immunized with NR-RMA
cells. Indeed, we found that efficacy or failure of our vaccination
procedure correlated with IL-10 and IL-1ß concentration in the sera
of vaccinated animals (Fig. 3A). Protection elicited by
107 apoptotic cells associated with high IL-10
(p < 0.03 when compared with PBS-injected
animals) and IL-1ß (p < 0.008) secretion.
However, when IL-10 (p < 0.001) exceeded by
far IL-1ß levels, as in the sera of mice injected with 5 x
106 apoptotic cells, vaccination did not elicit
any response. NR-RMA cells (5 x 105) did
not induce IL-10 secretion above the background level (i.e., mice
injected with PBS), while constantly triggering the release of IL-1ß
(p < 0.01). Besides the actual concentration
of each cytokine, in animals injected with both NR or high numbers of
Apo-RMA cells, the ratio between IL-10 and IL-1ß was comparatively
low. Fig. 3B reports the mean among the ratios between IL-10
and IL-1ß concentration in the sera of each single vaccinated animal.
On the contrary, in mice injected with low numbers of apoptotic cells
(i.e., 5 x 106), IL-10 exceeded by far the
IL-1ß concentration (Fig. 3B). No substantial difference
in serum levels of IFN-
or TNF-
was detected among animals
injected with Apo-RMA cells (5 or 10 x
106/animal) or PBS (8, 8, and 7 animals,
respectively) (data not shown).
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and Table I),
efficiently elicited RMA-specific CTLs (Fig. 4A) and protected 40% of the
mice challenged with RMA cells (Fig. 4B). When the dose of
immunogen was reduced to 0.5 x 106 cells,
NR-RMA elicited a strong anti-RMA CTL response, whereas Apo-RMA
cells still failed to elicit CTLs in IL-10-/-
mice (Fig. 4A), suggesting that multiple factors contribute
to modulate the immunogenicity of apoptotic cells.
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To verify whether peritoneal M were indeed involved in the
clearance of apoptotic cells, green-fluorescent Apo-RMA cells were
injected i.p. As assessed by flow cytometry of peritoneal cells, 4 and
20 h after injection, 74.8 ± 2.5 and 86.1 ± 1.6%,
respectively, of MAC-1+ peritoneal cells were
actively phagocytosing apoptotic cells, and few labeled apoptotic cells
were not internalized (<5%). In Fig. 5,
representative experiments of in vivo phagocytosis 20 h after i.p.
injection of Apo- (panels A and B) or
NR-RMA cells (panels C and D) are
reported. At FACS analysis, apoptotic cells are depicted as
PKH2-GL+/EB+; NR-RMA cells,
which do not internalize EB, are PKH2-GL+ only.
Massive phagocytosis of Apo-RMA cells is demonstrated by the almost
complete absence of double positive cells (Fig. 5A). In
fact, internalized apoptotic cells are not stained by EB, but make
phagocytes PKH2-GL+. Fig. 5B shows
that most of the MAC-1+ cells (which represent
the 51 ± 6% of the entire peritoneal population) are actively
phagocytosing apoptotic cells. Most phagocytosing
MAC-1+ cells expressed very low amounts of
membrane MHC class II, CD80, and CD86 molecules (data not shown), a
phenotype characteristic of M. Altogether, these data are strongly
suggestive for a massive and unique involvement of peritoneal M in
the phagocytosis of apoptotic cells. Fig. 5, C and
D, shows that NR-RMA cells are still present in the
peritoneum of injected animals 20 h after inoculum. Because NR-RMA
cells are still alive, they do not internalize EB and, therefore, do
not allow identification of phagocytosis by scavenger cells. However,
the fact that most of the MAC-1+ cells are not
PKH2-GL+, strongly suggests that peritoneal M
are not phagocytosing NR-RMA cells. Less than 10% double positive
cells in Fig. 5D may represent either NR-RMA cells attached
to the surface of MAC-1+ cells, or NR-RMA cells
undergoing secondary apoptosis (Fig. 5C), eventually phagocytosed
by M.
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), therefore giving an indirect evidence
of the immunomodulatory role of M in the induction of a specific
immune response against tumor cells undergoing programmed death.
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in the induction of an
immune response against Apo-RMA cells, peritoneal cells were collected
from 10 C57BL/6 mice injected 20 h before with
107 Apo-RMA cells or PBS, pooled and injected
immediately after i.p. into syngeneic animals. No protection against a
challenge of RMA cells was obtained in both groups of animals (data not
shown).
Finally, we derived M and DCs from bone marrow precursors (3, 12)
and verified their ability, after pulsing with Apo-RMA cells, to elicit
a tumor-specific immune response in vivo. Injection of Apo-RMA-pulsed
DCs resulted in CTL priming (Fig. 7A) and protection against a
challenge with RMA cells (Fig. 7B). As previously reported
(3), M efficiently internalized identical numbers of Apo-RMA cells
(data not shown), but neither elicited a specific immune response (Fig. 7A), nor allowed protection against a challenge with RMA
cells (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We demonstrate here that apoptotic cells may be immunogenic in vivo, although with a much lower potential than live cells. Our data suggest that this complex event is regulated by several factors, including the number of cells undergoing programmed death, the APCs involved in engulfment, processing, and presentation of Ags contained in the apoptotic cells, and the cytokines released in the microenvironment.
In wild-type H-2b mice, at least
107 Apo-RMA cells were required to elicit an
RMA-specific immune response. The dose of Ag is 20-fold higher than the
one needed for activation of a specific immune response by NR-RMA
cells. The decreased immunogenicity might be correlated with decreased
cell viability, while apoptotic cells may just represent a middle
immunogenic population between viable cells and dead cells. This
possibility is supported by the analysis of the in vivo phagocytosis of
Apo- and NR-RMA cells. Although Apo-RMA cells are readily phagocytosed
by scavenger cells and 20 h after injection <5% apoptotic cells
are still floating in the peritoneal fluid, most of the NR-RMA cells
are still alive by that time and eventually available for more
efficient APC processing/presentation. It has been reported that the
death of tumor cells by apoptotic pathway inversely correlates with
their immunogenicity (25). Apoptosis requires the activation of a
well-characterized family of cysteine aspartate proteases (26). Loss of
immunogenicity of cells undergoing apoptosis may depend on the cleavage
of relevant epitopes. The immunogenicity of RMA cells mostly depends on
a single immunodominant viral CTL epitope (27), which is not altered in
NR-RMA cells (16). The fact that CTLs specific for the immunodominant
CTL epitope of RMA (16) kill a subset of M-engulfing Apo-RMA cells
(3) suggests that the immunodominant epitope is still present also in
the antigenic repertoire of Apo-RMA cells. Experiments are in progress
to verify whether apoptotic cells have a qualitative and/or
quantitative alteration of their antigenic repertoire.
M are considered the most efficient scavenger cells (1). Our in
vitro and in vivo data (Ref. 3, and this study) suggest that, in our
system, M are the scavenger cells mostly involved in phagocytosis of
apoptotic tumor cells. Upon injection of 107
Apo-RMA cells, >85% of the peritoneal M were actively
phagocytosing dying cells, with <5% (i.e., < 0.5 x
106) apoptotic cells, which were not
internalized. However, neither unpurified peritoneal cells from animals
injected with Apo-RMA cells nor Apo-RMA-pulsed M were able to elicit
a specific immune response in vivo. Several considerations may explain
the failure of Apo-RMA-pulsed M to elicit a specific immune
response. Processing of the dying cells by M may lead to the
production and presentation of an altered epitope repertoire (see
above). The engulfment of Apo-RMA cells in M does not trigger
activation and up-regulation of MHC and costimulatory molecules.
Finally, M, upon engulfment of apoptotic cells may release
immunomodulatory factors, such as IL-10 and TGF-ß (4, 5). Indeed, we
found that IL-10 was consistently released in the serum of animals
immunized with apoptotic tumor cells. Moreover, we found that apoptotic
tumor cells were more immunogenic in IL-10-/-
mice. M are not the unique producers of immunomodulatory cytokines,
and we cannot exclude that apoptotic cells elicit different T cell
differentiation and response (Th1/Th2), or the activation of T
regulatory cells (28). The fact that IL-10-/-
mice injected with 5 x 105 Apo-RMA cells,
the equivalent immunogenic dose of NR-RMA cells, are not protected
against a challenge of RMA cells suggests that other factors contribute
to determine the scarce immunogenicity of apoptotic cells.
DCs, the most potent APCs (20, 21), efficiently internalize apoptotic
tumor cells and undergo maturative changes (13). Indeed, and to our
knowledge this is the first report, DCs pulsed with apoptotic cells
efficiently primed in vivo a tumor-specific CTL response and allowed
rejection of a challenge with RMA cells. We have no direct evidence of
the in vivo capture of apoptotic cells by DC, and we are currently
investigating this issue. In preliminary experiments, we found that two
of five animals injected s.c. with Apo-RMA cells were protected by a
challenge in the opposite flank with RMA cells. Although the protection
is relatively higher than the one obtained by i.p. injection of the
same number of Apo-RMA cells (40% and 29%, respectively), the results
are much lower than what expected by bypassing the peritoneal M. We
cannot exclude, however, that other mechanisms in distinct tissues
operate similarly to the one described by us.
Our results suggest that a prevalence of professional phagocytes (i.e.,
M) may favor Ag sequestration and paralysis of the immune response
via release of soluble factors. IL-10 is a major factor contributing to
limiting apoptotic cell immunogenicity in vivo. In conditions of normal
tissue turnover, this is likely to be the most common occurrence,
favoring the noninflammatory, nonimmunogenic clearance of cells
undergoing apoptosis. When the apoptotic cell meal overcomes scavenger
phagocytes, immature DCs are possibly recruited. The balance between
IL-10 and IL-1ß may favor their further maturation and migration to
secondary lymphatic tissues, where activation of a specific immune
response takes place.
However, in particular pathophysiologic conditions, apoptotic cells act as a source of Ag. The ability of DCs to prime in vivo specific T lymphocytes after internalization of apoptotic cells reconciles the physiologic removal of unwanted cells undergoing apoptosis without inflammation (1), with the involvement of apoptotic cells in other phenomena, such as cross-priming (6, 29, 30) and autoimmunity (31, 32). These results have implications in defining vaccination strategies. Manipulation of the mechanisms regulating apoptosis (33) as well as the balance between pro- and antiinflammatory cytokines (20, 21) may have direct/indirect effects on the APCs involved in processing and presentation of Ags from apoptotic cells and in switching the immune system on or off.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matteo Bellone, Laboratorio di Immunologia dei Tumori, Istituto Scientifico H San Raffaele, Via Olgettina 60, I-20132, Milan, Italy. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; M, macrophage; Apo, apoptotic; NR, nonreplicating; Necro, necrotic; EB, ethidium bromide.
Received for publication December 22, 1998. Accepted for publication April 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), 5 x 106 (
), 107 (
), 2
x 107 (
) Apo-RMA (first panel), NR-RMA
(second panel), Necro-RMA (third panel)
cells or PBS (
; fourth panel) were cultured for 5
days in the presence of RMA cells and tested in 4-h
51Cr-release assays. Curves represent the cytotoxicity
against RMA. Lysis of YAC-1 (H-2k/d) and EL-4G
(H-2b) targets was always <5% (data not shown). Values
are expressed as percentage (average of triplicates) of specific
51Cr release at the indicated E:T ratios. B,
Sera were collected 2 wk after the first injection (left
panel, 1x) or 1 wk after the second injection (right
panel, 2x) of Apo-RMA (•), NR-RMA (
