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*
Surgery Branch and
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and
Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, MD 20814
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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 deactivation of immune responses is conducted, in part, by suppressive and regulatory circuits that result in apoptosis of lymphocytes (1). Mechanisms for the induction of apoptosis in T lymphocytes include "propriocidal cell death" and "clonal exhaustion." The propriocidal form of death has been shown to occur upon TCR engagement in lymphocytes previously exposed to IL-2. This mechanism can control the extent of T cell activation by eliminating a portion of newly dividing, Ag-reactive lymphocytes (2, 3) and is mediated through the engagement of receptors for Fas and TNF (4, 5, 6, 7).
Cytotoxic effector cells can be rapidly and specifically deleted in mice exposed to high doses of a particular strain of rapidly disseminating lymphocytic choriomeningitis virus (LCMV)4. A phenomenon termed "clonal exhaustion" is due to the apoptotic death of T cells after restimulation through their TCR, resulting in a chronic virus carrier state in which there is a complete absence of memory T cells (8, 9, 10). Thus, a functional state of tolerance may be the result of Ag persistence at a time when restimulation of lymphocytes programs them to die.
T cells may receive instructions to die from a population of cells called "natural suppressors" (NS). This poorly defined population of nonlymphoid cells may be derived from the monocyte-macrophage lineage. In experimental animal models, NS cells have been reported to suppress the generation of CTL independently of Ag and MHC restriction and may play a role in the inhibition of B cells and Th cells, as measured by allo- and Ag-specific proliferative responses (11, 12, 13, 14, 15). NS cells have been reported to be responsible for the suppression of immune responses associated with cancer as well as infectious diseases like Trypanosoma and Salmonella (11, 16, 17).
In this communication, we explore the immune deactivation that follows infection or immunization. We found that immunization strategies that have been optimized for their ability to elicit cytotoxicity elicit the weakest secondary immune responses upon early restimulation. We explore the immune sequelae after immunization with a vaccinia virus (VV)-encoding IL-2 and the model Ag, ß-galactosidase (ß-gal), an immunogen previously found to be highly effective in the generation of a potent primary immune response (18).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CT26.WT, the ß-gal-expressing CT26.CL25, and EL4 thymoma and its ß-gal-expressing (LacZ transfected) subclone, E22, have been described (18, 19). The first two cell lines are H-2d, while EL4 and E22 are H-2b. BSC-1 cells (CCL26, American Type Culture Collection, Manassas, VA) and HeLa S3 (CCL2.2, American Type Culture Collection) were used to prepare all the VV stocks. Cell lines were maintained in culture media consisting of RPMI 1640, 10% heat-inactivated FBS (Biofluids, Rockville, MD), 0.03% L-glutamine, 100 mg/ml streptomycin, 100 mg/ml penicillin, and 50 mg/ml gentamicin sulfate (National Institutes of Health Media Center, Bethesda, MD). CT26.CL25 and E22 were maintained in culture media containing 400 µg/ml G418 (Life Technologies, Grand Island, NY). BSC-1 and HeLa S3 were maintained in DMEM.
Recombinant vaccinia viruses
All rVV used in this study were generated by insertion of the foreign genes into the VV thymidine kinase gene by homologous recombination (20). Virus preparations were propagated from plaque-purified crude virus stocks, as described previously (21). Briefly, 175-cm2 flasks of HeLa S3 or BSC-1 cells were infected with 5 plaque-forming units (PFU)/cell and incubated at 37°C for about 72 h. Infected cells were harvested and centrifuged at 1000 x g for 10 min. Cells were resuspended in 10 mM Tris (pH 9.0) and lysed by 30 strokes of a dounce homogenizer. Nuclei and cell debris were partially removed by centrifugation for 5 min at 1000 x g, viral particles were collected after purification by centrifugation over a sucrose cushion, and stocks were aliquoted and stored at -80°C.
Viral concentrations were determined by plaque titration on BSC-1
cells. rVV used in a single experiment were titered concurrently to
maximize accuracy. Preparation of rVV expressing the influenza
A/PR/8/34 nucleoprotein (NP), NP-rVV, was previously described (22).
Murine IL-2 cDNA was amplified by PCR from pBMGNeomIL2 and ligated into
the SmaI-BamHI site of vaccinia expression
vector, pMJ601, which contains the lacZ gene under the
control of the natural p7.5 early promoter (18). IFN-
was inserted into the VV genome using a similar procedure, as reported
(23). In the VJS6 construct, the Escherichia coli lacZ gene
was under the control of the natural p7.5 early/late
promoter element from plasmid pSC65 (24); this construct was named
ß-gal-rVV for simplicity. Wild-type (WT) VV strain WR was kindly
provided by J. Yewdell and J. Bennink (National Institute of Allergy
and Infectious Diseases, Bethesda, MD).
Peptides
The following synthetic peptides were synthesized by Peptide Technologies (Washington, D.C.) to a purity of greater than 99% as determined by HPLC and amino acid analysis: TPHPARIGL (amino acids 876–884 of ß-gal, H-2Ld-restricted (25)), DAPIYTNV (amino acids 96–103 of ß-gal, H-2Kb-restricted, (26)).
Antibodies
FITC- or phycoerythrin-labeled mAb recognizing mouse CD8, CD4,
CD11b (Mac-1), Lyt-6G (Gr-1), and the isotype-matched controls were
purchased from PharMingen (San Diego, CA). Concentrations used for
cytofluorometry ranged between 1 and 12 µg/106 cells
depending on the Ab. The mAb 24G.2 (CD16/CD23, PharMingen), which
reacts with a common epitope of the extracellular domain of the mouse
FcRII/FcRIII, was used to block the nonspecific binding of mAb
during staining. For the in vitro and in vivo depletion studies, mAb
were extensively dialyzed against PBS to remove the sodium azide.
Evaluation of CTL responses
Eight- to 12-wk-old female BALB/c, (Animal Production Colonies,
Frederick Cancer Research Facility, National Institutes of Health,
Frederick, MD), C57BL/6J, or MRL-lpr/lpr mice
(The Jackson Laboratory, Bar Harbor, ME) were immunized with various
doses (5 x 106 to 2 x 107
PFU/mouse) of different rVV. The spleens were collected on day 6 after
immunization, separated into a single-cell suspension, and tested for
their ability to lyse ß-gal-positive targets in a 6-h
51Cr release assay as previously described (18). Briefly,
2 x 106 target cells were incubated with 200 µCi
Na51CrO4 (51Cr) for 90 min
(together with 1 µg/ml of peptide or 100 µl of crude VV-WT
preparation, where designated). After labeling, the targets were washed
and diluted to 105 viable cells/ml. Targets were then
plated at 0.1 ml/well in 96-well plates (104 cells/well)
and effectors were added at the indicated ratio. Plates were incubated
for 6 h before harvesting. The amount of 51Cr released
was determined by -counting and the percentage of specific lysis was
calculated from triplicate samples using the formula: [(experimental
cpm - spontaneous cpm)/(maximal cpm - spontaneous cpm)] x
100. In some experiments, splenocytes, homogenized to a single cell
suspension, were cultured at 5 x 106 cells/ml in
75-cm2 flasks (Costar, Cambridge, MA) with 30 ml of RPMI
1640 containing 10% FCS (Biofluids), 0.1 mM nonessential amino acids,
1 mM sodium pyruvate (Biofluids), and 5 x 10-5 M
2-ME (Life Technologies, Rockville, MD). For the in vitro stimulation,
the peptide (1 µg/ml) or irradiated tumor cells at a
responder-to-stimulator ratio of 40:1 were added to the cultures. After
6 days, effectors were harvested and tested in a 6-h 51Cr
release assay, as indicated above. In cell separation experiments,
splenocytes were cultured at the same cell concentration in 24-well
plates (Costar) containing a culture chamber insert with 0.4-µM pores
(Millipore, Bedford, MA).
In vivo studies
In protection studies, BALB/c mice (5/group) were immunized with 5 x 106 PFU of rVV, boosted on day 6 with various Ags, and inoculated i.v. 21 days after the boosting with 5 x 105 tumor cells. Negative controls were always included and consisted of mice inoculated with only the vehicle used to resuspend the Ag. Mice were sacrificed on day 12 following tumor inoculation and lung metastases were enumerated in a coded, blind fashion.
Detection of apoptosis in lymphocyte subpopulations
A modification of the method described by Sherwood and Schimke (27) was employed. Briefly, 106 cells were stained with a FITC-conjugated anti-CD8 or anti-CD4 mAb (PharMingen) for 30 min at 4°C in FACS buffer (Ca2+- and Mg2+-free HBSS containing 0.5% BSA and 0.02% sodium azide). Cells were washed three times in cold FACS buffer and fixed with the addition of 70% ice-cold ethanol. After 1 h incubation at 4°C, cells were washed twice with PBS and resuspended in propidium iodide (PI) staining solution (PBS containing 100 µg/ml RNase A and 50 µg/ml PI, both from Sigma, St. Louis, MO). Flow cytometry was performed on a Becton Dickinson (San Jose, CA) FACScan using a 488 Argon Laser. Data analysis was performed on either the Cellfit (Becton Dickinson) or Modfit (Verity Software House, Topsham, ME) software packages. In some experiments, PI staining was compared with staining with the TUNEL method used by the in situ cell death detection kit (Boehringer Mannheim, Indianapolis, IN), obtaining comparable results (not shown).
Isolation of splenic populations
A panning technique employing flasks coated with mouse anti-rat Abs (T-25 AIS MicroCell, AIS, Santa Clara, CA) was used to deplete specific populations from spleens. Spleens were depleted of red cells with ACK lysis buffer (Biofluids) and resuspended in HBSS containing 1 mM EDTA and 10% mouse serum (HBSS-EDTA-MS). The remaining cells were placed on ice, incubated for 30 min with the primary Ab at a concentration of 10 µg/107 cells, and washed three times with cold HBSS-EDTA. Next, the cells were resuspended in ice-cold HBSS-EDTA-MS at a concentration of 4 x 107/ml, transferred to flasks coated with the secondary Ab (anti-rat), and incubated for 1 h at 4°C. Finally, the nonadherent cells were dislodged and collected as a negative fraction. Fluorescence labeling confirmed >95% depletion. The cells that remained attached to the flasks were provided with RPMI 1640 complete medium and incubated for further studies. Adherent cells were recovered by gentle scraping in Versene solution (1:5000, Biofluids). In some experiments, 2 h adherence to plastic or ingestion of carbonyl iron (Myloclear, Cedarlane Laboratories, Accurate Chemical and Scientific Corp., Westbury, NY) followed by magnetic sorting were used to deplete spleens of mature monocytes/macrophages. CD8+ lymphocytes were separated through affinity columns (R & D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The percentage of CD8+ cells after enrichment was usually 80–90%.
Statistical analysis
The Wilcoxon-Mann-Whitney U test was used to examine the null hypothesis of identity of ranks between two sets of data. All the p values were reported as two-sided.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an effort to increase the immunogenicity of recombinant
anti-cancer vaccines, we previously inserted a variety of cytokine
genes into the genome of a ß-gal-expressing rVV (18). Insertion of
the gene-encoding IL-2 into a construct-encoding ß-gal significantly
augmented primary cytolytic T lymphocyte responses specific for VV.
Conversely, insertion of the gene-encoding IFN- had the opposite
effect of decreased CTL activity (Fig. 1
A). However, when an aliquot
of the same splenic preparation was evaluated for the generation of
ß-gal-specific cytotoxicity after a 6-day incubation in vitro with
the ß-gal-immunodominant peptide (TPHPARIGL), a nearly opposite
result was obtained in which IL-2-rVV-treated mice showed greatly
diminished CTL responses, while IFN--rVV-treated mice exhibited
potent cytotoxicity (Fig. 1C). Thus, while splenocyte
cultures derived from mice that had been primed in vivo with rVV
containing the genes for IL-2 and ß-gal showed no cytotoxicity
against ß-gal-positive tumor cells upon restimulation with the
ß-gal peptide, cultures from mice infected with virus-encoding
IFN- with ß-gal displayed excellent killing capabilities. Mice
immunized with ß-gal-rVV, which did not encode any cytokine, showed
only a weak cytolytic response.
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B), restimulation of
these same splenocytes with peptide for 6 days in vitro resulted in the
complete disappearance of cytotoxic activity (Fig. 1D). On
the other hand, no primary ß-gal-specific cytolysis was observed in
mice primed with the IFN--containing virus, but splenocytes from
these mice were specifically cytolytic after identical stimulation with
peptide (Fig. 1, B vs D). The phenomenon of
apparent suppression of cytolytic activity was observed only upon
"early" restimulation: when splenocytes were harvested 14 days
after immunization with the panel of rVV-containing cytokine genes and
stimulated with peptide in vitro, all groups mounted efficient CTL
responses (data not shown). A trivial explanation for the apparent lack of CD8+ T cell function was "fratricide," i.e., the killing of one lymphocyte by another after the addition of soluble peptide. However, the cytolytic capacity of IL-2rVV-primed splenocytes was also eliminated by restimulation with tumor cells that had been transfected with the ß-gal gene (CT26.CL25) rather than pulsed with peptide (not shown).
Lack of a cytolytic response is not associated with irreversible damage of CD8+ T cells
Induction of an unresponsive state by hyperstimulation of the
immune response has been explained in other models by several
mechanisms such as anergy, propriocidal apoptosis, and clonal
exhaustion, which all focus on properties inherent to T lymphocytes. We
thus set out to separate and characterize the CD8+ T
lymphocytes using negative separation methods that eliminated other
cells from the cultures, without ligation of differentiation Ags on the
CD8+ cells. However, when purified populations of
CD8+ lymphocytes derived from day 6 spleens of
IL-2rVV-inoculated mice were cultured together with naïve
splenocytes depleted of CD8+ cells and pulsed with ß-gal
peptide, excellent CTL responses were observed (Fig. 1E).
Indeed, comparable responses were generated irrespective of the rVV
inoculated 6 days before the cultures were established with the
expected exception of the ß-gal-negative virus, NP-rVV. Thus, the
suppression observed was not a characteristic of the T cells, but was
dependent on some other element(s) in the splenic population.
Suppression of CD8+ T lymphocyte activity occurs in vivo and is long-lived
To evaluate the effects of "early" boosting on the suppression
of immune responses in vivo, we employed a tumor challenge model. We
have previously established the importance of CD8+ T cells
in the effective immune response to CT26.CL25 (19, 28). Mice were
immunized with either carrier (HBSS) or with rVV-encoding ß-gal in
combination with IL-2 or IFN-, boosted 6 days later with ß-gal
protein, then challenged 3 weeks after the boost with a syngeneic
murine carcinoma-expressing ß-gal. The protection from tumor
challenge was nearly complete in mice receiving an initial inoculum of
IFN--rVV (Fig. 2A).
However, while the IL-2-rVV was also protective when boosted with an
irrelevant protein, OVA, tumor protection was abrogated in mice that
were initially primed with the IL-2-rVV and boosted with ß-gal
protein. Similar results were obtained in IL-2-rVV-immunized mice when
a recombinant fowlpox virus (FPV)-expressing ß-gal was used as a
booster (Fig. 2B). FPV-ß-gal and, to a lesser extent, the
ß-gal protein were immunogenic because they induced protection from
tumor challenge when used alone (p = 0.001 and
0.005, respectively, Fig. 2, A and B). Thus, the
suppression could be observed in vivo, was Ag-specific, and was
relatively long-lived.
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We hypothesized that the unresponsive state observed above could
be due to anergy or apoptosis. To explore the death rate under various
conditions of restimulation, we employed a double-staining protocol
designed to evaluate the percentage of hypodiploid (apoptotic) cells
among lymphocytes that were positive for CD8 (Fig. 3). In splenocyte cultures from mice
primed with V69 rVV, the mean percentage of apoptosis upon in vitro
stimulation was 22.4%. This baseline value of apoptosis was not Ag
specific, because V69 encoded the influenza NP gene in lieu of
LacZ (NP-rVV) and did not prime CTL responses (Fig. 1). The
number of apoptotic cells after stimulation with antigenic peptide rose
to 46.0% in cultures derived from mice that had been immunized with
VJS6, which encodes ß-gal without the heterologous addition of
cytokines. Levels of apoptosis comparable to negative controls were
detected in cultures derived from IFN--rVV immunized mice. Apoptosis
of CD8+ T cells was significantly increased in mice
immunized with the IL-2-rVV. Similar patterns of apoptotic death was
observed in CD8+ cultures that were restimulated with tumor
cells expressing the ß-gal Ag; however, CD4+ cells
present in the same experiment did not follow the same pattern of
apoptosis (not shown). Thus, apoptotic death appeared to be limited to
the CD8+ compartment and was not a generalized death of all
lymphocytes present in the culture.
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We next investigated the molecular mechanism causing apoptosis of
CTL in acutely infected mice. The production of soluble factors
secreted by the suppressive splenocytes was ruled out by mixing
splenocytes from mice infected with different rVV in diffusion
chambers. The experiments clearly showed that cell-cell contact was
required for the immunosuppressive effect, because suppression of CTL
generation only occurred when the suppressor-containing population was
not separated by a membrane from the population responsive to peptide
stimulation (Fig. 4).
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, top), with IL-2-rVV or
IFN--rVV. As before, primary and secondary responses against the
tumor cell line, EL-4, VV-WT infected EL-4, or the ß-gal-expressing
line, E22, were tested in 51Cr release assays (Fig. 5, bottom). An identical pattern of suppression was observed in
the Fas-deficient mice as in normal C57BL/6J mice. This generation of
the same IL-2-rVV-induced suppression in MRL-lpr/lpr mice
indicated that the cell membrane-associated mechanism did not involve
Fas-FasL interaction.
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The lack of response in the whole spleens taken from
IL-2-rVV-infected mice could be explained with either the absence of
competent APC or with the presence of regulatory suppressor elements
distinct from CD8+ lymphocytes. The first possibility was
addressed by mixing experiments in which we found that splenocytes from
IL-2-rVV-infected mice could suppress the response of IFN--rVV mice
even when mixed at a 1:2 ratio before culture with ß-gal peptide (not
shown). Additionally, Fig. 4 shows a similar mixing experiment of
splenocytes from IL-2-rVV-infected mice and IFN--rVV-infected mice,
which demonstrates the same pattern of suppression. These findings
disfavored the hypothesis of insufficient APC function and supported
the presence of suppressive elements.
To identify candidate cells with suppressive activity, we
cytofluorometrically evaluated surface markers on the spleen cells of
naive mice or mice immunized with IL-2-rVV or IFN--rVV. As
can be seen in the upper panels of Fig. 6, both Mac-1 and Gr-1 were present on
the surfaces of a population of cells that was increased in the spleens
of mice that had been immunized with IL-2-rVV. Gr-1 is a marker that is
normally expressed by granulocytes, monocytes, and immature myeloid
precursors in the bone marrow but is expressed at a very low level in
the spleens of normal mice (29). This population of cells expressing
both Ags was significantly increased (p <
0.01) in the spleens of IL-2-rVV-immunized mice but not in mice
immunized with IFN--rVV (means ± SD of three experiments were:
3.03 ± 0.87, 2.92 ± 0.22, and 7.93 ± 0.68 for
naïve, IFN--rVV- and IL-2-rVV-inoculated mice,
respectively). Moreover, in vivo depletion with anti-Gr-1 mAb but
not with the control mAb reduced the double-positive cells in the
spleens of mice inoculated with IL-2-rVV to levels comparable to those
detected in naïve or in IFN--rVV-immunized mice. The
depletion was even more effective in vitro (Fig. 6, bottom).
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To ascertain whether these cells were suppressing the generation
of CTL in our cultures, different mAbs were used to deplete, in vitro
or in vivo, specific populations before restimulation of the spleens of
mice infected 6 days earlier with rVV immunogens. As shown in Fig. 7A, depletion of cells
positive for Gr-1 or Mac-1 completely restored the capacity of cells to
mount cytolytic responses. Cytotoxicity recovered in Ab-depleted
cultures was similar to that obtained with a population of
CD8+ lymphocytes enriched from the same spleens or with the
other positive controls included in the assay (IFN--rVV-immunized
mice or mice immunized 14 days earlier with IL-2-rVV). An
isotype-matched Ab (rat IgG2b) did not produce the same effect, ruling
out an in vitro artifact related to the experimental protocol.
Moreover, a second Ab directed against a mouse macrophage Ag, Mac-3,
only restored 20% of the CTL activity seen in the cultures of
CD8+ enriched populations (not shown). The data shown in
Fig. 7A further supports the hypothesis that the suppression
of CD8+ cell responses is not a characteristic of the T
cells, but rather is a quality of some other splenocyte component. In
vivo depletion of Gr-1+ cells by repeated i.p. inoculations
of the Ab during the first days of infection with IL-2-rVV resulted in
a complete recovery of the deficient CTL response, while no effect was
observed with the control IgG2b (Fig. 7B). Thus,
Mac-1+/Gr-1+ cells appear to mediate the
suppression of CD8+ T cells in vitro and in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C) and was almost complete at higher
doses of 2 x 107 PFU/mouse (data not shown). The
apoptotic death of CD8+ T cells and coincident loss of
cytolytic capacity was not invariably associated with IL-2-rVV
infection as it was absent when the dose was lowered to 5 x
104 PFU/mouse (not shown). Moreover, a
Mac-1+/Gr-1+ cell-dependent suppression of the
generation of alloreactive CTL was observed in the spleens of mice
acutely infected with VV-WT (data not shown). We have shown that Ag has
been cleared by day 6 after infection (N.P.R. and V.B.,
unpublished observation). The artificial reintroduction of the same Ag
at a time when Ag has been cleared by the ongoing immune response may
mimic the long-term persistence of Ag seen when a particular strain of
LCMV is used (8). In our experiments, the reintroduction of Ag was
accomplished either by in vivo reinfection with a recombinant virus or
purified protein or by in vitro restimulation with an immunodominant
peptide. "Exhaustion" of the immune response may not be due to a
failure of the T cell compartment but instead may be a consequence of
the feedback circuit that regulates the intensity of immune stimulation
in the face of persistent Ag. It is well documented that, at the apex of their increase, lymphocytes are unable to respond to mitogenic activation, as seen in the paradigmatic example of LCMV infection (30). In fact, further stimulation is associated with the induction of apoptosis (9). Based on our data and on the work of others, the following scenario seems likely: during the first encounter with an infectious agent, professional APC present the Ag to naïve lymphocytes. Activated lymphocytes migrate from lymphoid organs to the peripheral tissues via the circulation and start eliminating the antigenic challenge. Activation of silent, autoreactive lymphocytes recognizing cross-reactive epitopes can occur (31, 32, 33). Local tissue destruction, and the systemic toxicities of cytokines may be harmful and could potentially trigger a circuit leading to autoimmune destruction of tissues. Proportional to the intensity of the immune response, a population of granulocyte-macrophage precursors is mobilized from the bone marrow that is capable of differentiating into a suppressive population. Through a contact-dependent mechanism, the macrophage-like regulatory cells induce apoptosis of previously activated lymphocytes, down-modulating the immune response.
The kinetics of the induction of Mac-1+/Gr-1+ cells following inoculation with rVV were coincident with the functional suppression of the generation of cytolytic responses upon antigenic restimulation. The earliest detection of an increase of Mac-1+/Gr-1+ cells was on day 4, and baseline levels of splenic Mac-1+/Gr-1+ cells were measured by day 14, although some minor variability in these kinetics was observed (data not shown). The delay observed between the peak of CTL activity and the induction of the Mac-1+/Gr-1+ suppressor cells could allow for the destruction of viable pathogen and elimination of the antigenic stimulation for the future "memory" pathogen-specific T cells.
CD8+ lymphocytes activated in the presence of
Mac-1+/Gr-1+ cells are induced to die and show
clear signs of apoptosis, such as the appearance of hypodiploid nuclei
(Fig. 3) or the incorporation of labeled-nucleotides by the enzyme
terminal deoxynucleotidyltransferase (TdT), employed in the
TUNEL method (not shown). The percentage of CD8+
lymphocytes undergoing apoptosis after in vitro exposure to ß-gal
peptide was unexpectedly high (nearly 60% after the background is
subtracted in the ß-gal-expressing IL-2-rVV group). The most likely
explanation for this high apoptotic rate could be the extremely high
numbers of precursors that have been revealed by recent studies
employing ELISPOT assays and class I tetramers (34, 35). In addition,
there may also be nonspecific apoptotic death of "bystander" cells
interacting with the Mac-1+/Gr-1+ cells.
The molecular mechanism causing apoptosis of CD8+ T cells
is not known at present. In experiments with diffusion chambers, we
found that cell-cell contact was required for loss of cytolytic
capability and apoptosis to occur (Fig. 4). Fas Ag (CD95) seemed a
likely candidate for the observed cell contact-dependent
immunosuppression. The FasR is a cell surface protein that is a member
of the TNFR family, and it is expressed on many cells in the immune
system and other tissues. Its main function is to trigger apoptosis
when complexed with FasL (CD95L) (36, 37). The activation of mature
human T cells sensitizes them to Fas-mediated apoptosis (38, 39). This
is important in the regulation of the immune response and the
maintenance of self-tolerance as illustrated by patients with a
deficiency in FasR known as autoimmune lymphoproliferative syndrome
(ALPS). These patients exhibit massive T cell expansions resulting in
lymphadenopathy and splenomegaly as well as varied autoimmune responses
(40, 41). However, the cell membrane-associated mechanism responsible
for apoptosis of CD8+ T cells in our system did not involve
Fas-FasL as indicated by the observation of the same IL-2-rVV-induced
suppression in MRL-lpr/lpr mice, the murine counterpart to
ALPS (Fig. 5). This finding is consistent with the observations of
Lohman et al. (42) that in vivo there were significant numbers of
apoptotic cells in the spleens of lpr/lpr mice after LCMV
infection. This indicates that Fas is not required for the immune
down-regulation of the CD8+ T lymphocyte response after
acute LCMV infection. Further, TNF/TNFR, and CTLA-4/B7-1/B7-2
interactions were not responsible for the effect as a wide range of
concentrations of blocking Abs against TNF-
or CTLA-4 were
completely ineffective or detrimental to the generation of a CTL
response (V.B. and N.P.R., unpublished observations).
The Mac-1+/Gr-1+ cells we describe here are currently being further characterized and are comprised mostly of monocytes and a population of myeloid precursors. These latter cells closely resemble the cells previously designated "natural suppressor" (NS) cells. Comparison of the functional qualities of these cells with those of NS cells is interesting. Phenotypically, NS cells lack the usual markers of mature B cells, T cells, or macrophages and do not kill classical NK targets. They have been found in several environments involving intense hematopoiesis such as neonatal/newborn spleens, adult bone marrow, adult spleens after total lymphoid irradiation, during growth of some tumors, after treatment with cyclophosphamide, or during graft-vs-host-disease (43, 44, 45, 46). We have recently characterized an identical population of suppressor elements in mice bearing large tumor burdens (V.B. and N.P.R., manuscript in preparation).
We and others have recently demonstrated that IL-2 can profoundly enhance the activity of CD8+ T cells after immunization (18). These findings have been extended to the clinic in cancer immunotherapy trials where the addition of adjuvant IL-2 to synthetic peptide immunogens results in enhanced tumor destruction. However, at the same time we observed an apparent disappearance of Ag-specific T cells from the circulation (47). Based on the data presented, one might speculate that the disappearance of these anti-tumor T cells may be due to a suppressive population of Mac-1+/Gr-1+ cells.
In conclusion, we have characterized the function of a population of cells involved in a novel feedback pathway that directs the death of T lymphocytes. Immunizations that are separated by intervals that are too short to allow for the activities of regulatory cells to subside may be detrimental for the immune responses that we seek to elicit. The induction or isolation of these cells could represent a powerful tool useful in the control of autoimmunity and transplant rejection.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Oncology and Surgical Sciences, Oncology Section, Via Gattamelata 64, 35128, Padova, Italy. E-mail address:
3 Address correspondence and reprint requests to Dr. Nicholas P. Restifo, Building 10, Room 2B42, National Cancer Institute, 10 Center Drive, MSC 1502, Bethesda, MD 20892-1502; E-mail address:
4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; VV, vaccinia virus; FPV, fowlpox virus; NS, natural suppressor; NP, nucleoprotein; ß-gal, ß-galactosidase; ALPS, autoimmune lymphoproliferative syndrome; FasL, Fas ligand; PFU, plaque-forming unit; WT, wild-type.
Received for publication April 9, 1998. Accepted for publication July 14, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T lymphocytes for apoptosis. Nature 353:858.[Medline]
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T. J. Stewart, D. J. Liewehr, S. M. Steinberg, K. M. Greeneltch, and S. I. Abrams Modulating the Expression of IFN Regulatory Factor 8 Alters the Protumorigenic Behavior of CD11b+Gr-1+ Myeloid Cells J. Immunol., July 1, 2009; 183(1): 117 - 128. [Abstract] [Full Text] [PDF]
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J. S. Pufnock and J. L. Rothstein Oncoprotein Signaling Mediates Tumor-Specific Inflammation and Enhances Tumor Progression J. Immunol., May 1, 2009; 182(9): 5498 - 5506. [Abstract] [Full Text] [PDF]
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S. Ostrand-Rosenberg and P. Sinha Myeloid-Derived Suppressor Cells: Linking Inflammation and Cancer J. Immunol., April 15, 2009; 182(8): 4499 - 4506. [Abstract] [Full Text] [PDF]
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A. W. Mailloux and M. R. I. Young NK-Dependent Increases in CCL22 Secretion Selectively Recruits Regulatory T Cells to the Tumor Microenvironment J. Immunol., March 1, 2009; 182(5): 2753 - 2765. [Abstract] [Full Text] [PDF]
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H. Li, Y. Han, Q. Guo, M. Zhang, and X. Cao Cancer-Expanded Myeloid-Derived Suppressor Cells Induce Anergy of NK Cells through Membrane-Bound TGF-{beta}1 J. Immunol., January 1, 2009; 182(1): 240 - 249. [Abstract] [Full Text] [PDF]
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 B. Zhang, Y. Zhang, N. A. Bowerman, A. Schietinger, Y.-X. Fu, D. M. Kranz, D. A. Rowley, and H. Schreiber Equilibrium between Host and Cancer Caused by Effector T Cells Killing Tumor Stroma Cancer Res., March 1, 2008; 68(5): 1563 - 1571. [Abstract] [Full Text] [PDF]
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 P.-Y. Pan, G. X. Wang, B. Yin, J. Ozao, T. Ku, C. M. Divino, and S.-H. Chen Reversion of immune tolerance in advanced malignancy: modulation of myeloid-derived suppressor cell development by blockade of stem-cell factor function Blood, January 1, 2008; 111(1): 219 - 228. [Abstract] [Full Text] [PDF]
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C. E. Clark, S. R. Hingorani, R. Mick, C. Combs, D. A. Tuveson, and R. H. Vonderheide Dynamics of the Immune Reaction to Pancreatic Cancer from Inception to Invasion Cancer Res., October 1, 2007; 67(19): 9518 - 9527. [Abstract] [Full Text] [PDF]
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 J. E. Talmadge Pathways Mediating the Expansion and Immunosuppressive Activity of Myeloid-Derived Suppressor Cells and Their Relevance to Cancer Therapy Clin. Cancer Res., September 15, 2007; 13(18): 5243 - 5248. [Abstract] [Full Text] [PDF]
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C. M. Paulos, A. Kaiser, C. Wrzesinski, C. S. Hinrichs, L. Cassard, A. Boni, P. Muranski, L. Sanchez-Perez, D. C. Palmer, Z. Yu, et al. Toll-like Receptors in Tumor Immunotherapy Clin. Cancer Res., September 15, 2007; 13(18): 5280 - 5289. [Abstract] [Full Text] [PDF]
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I. Fricke, N. Mirza, J. Dupont, C. Lockhart, A. Jackson, J.-H. Lee, J. A. Sosman, and D. I. Gabrilovich Vascular Endothelial Growth Factor-Trap Overcomes Defects in Dendritic Cell Differentiation but Does Not Improve Antigen-Specific Immune Responses Clin. Cancer Res., August 15, 2007; 13(16): 4840 - 4848. [Abstract] [Full Text] [PDF]
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 J. W. Rasmussen, J. Cello, H. Gil, C. A. Forestal, M. B. Furie, D. G. Thanassi, and J. L. Benach Mac-1+ Cells Are the Predominant Subset in the Early Hepatic Lesions of Mice Infected with Francisella tularensis Infect. Immun., December 1, 2006; 74(12): 6590 - 6598. [Abstract] [Full Text] [PDF]
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 P. Serafini, K. Meckel, M. Kelso, K. Noonan, J. Califano, W. Koch, L. Dolcetti, V. Bronte, and I. Borrello Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function J. Exp. Med., November 27, 2006; 203(12): 2691 - 2702. [Abstract] [Full Text] [PDF]
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A. V. Ezernitchi, I. Vaknin, L. Cohen-Daniel, O. Levy, E. Manaster, A. Halabi, E. Pikarsky, L. Shapira, and M. Baniyash TCR {zeta} Down-Regulation under Chronic Inflammation Is Mediated by Myeloid Suppressor Cells Differentially Distributed between Various Lymphatic Organs J. Immunol., October 1, 2006; 177(7): 4763 - 4772. [Abstract] [Full Text] [PDF]
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K. C. McKenna and J. A. Kapp Accumulation of Immunosuppressive CD11b+ Myeloid Cells Correlates with the Failure to Prevent Tumor Growth in the Anterior Chamber of the Eye J. Immunol., August 1, 2006; 177(3): 1599 - 1608. [Abstract] [Full Text] [PDF]
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R. Yang, Z. Cai, Y. Zhang, W. H. Yutzy IV, K. F. Roby, and R. B.S. Roden CD80 in Immune Suppression by Mouse Ovarian Carcinoma-Associated Gr-1+CD11b+ Myeloid Cells. Cancer Res., July 1, 2006; 66(13): 6807 - 6815. [Abstract] [Full Text] [PDF]
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X. Song, Y. Krelin, T. Dvorkin, O. Bjorkdahl, S. Segal, C. A. Dinarello, E. Voronov, and R. N. Apte CD11b+/Gr-1+ Immature Myeloid Cells Mediate Suppression of T Cells in Mice Bearing Tumors of IL-1{beta}-Secreting Cells J. Immunol., December 15, 2005; 175(12): 8200 - 8208. [Abstract] [Full Text] [PDF]
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S. Kusmartsev, S. Nagaraj, and D. I. Gabrilovich Tumor-Associated CD8+ T Cell Tolerance Induced by Bone Marrow-Derived Immature Myeloid Cells J. Immunol., October 1, 2005; 175(7): 4583 - 4592. [Abstract] [Full Text] [PDF]
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L. Brys, A. Beschin, G. Raes, G. H. Ghassabeh, W. Noel, J. Brandt, F. Brombacher, and P. D. Baetselier Reactive Oxygen Species and 12/15-Lipoxygenase Contribute to the Antiproliferative Capacity of Alternatively Activated Myeloid Cells Elicited during Helminth Infection J. Immunol., May 15, 2005; 174(10): 6095 - 6104. [Abstract] [Full Text] [PDF]
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S. Kusmartsev and D. I. Gabrilovich STAT1 Signaling Regulates Tumor-Associated Macrophage-Mediated T Cell Deletion J. Immunol., April 15, 2005; 174(8): 4880 - 4891. [Abstract] [Full Text] [PDF]
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S. P. Zehntner, C. Brickman, L. Bourbonniere, L. Remington, M. Caruso, and T. Owens Neutrophils That Infiltrate the Central Nervous System Regulate T Cell Responses J. Immunol., April 15, 2005; 174(8): 5124 - 5131. [Abstract] [Full Text] [PDF]
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 C. De Santo, P. Serafini, I. Marigo, L. Dolcetti, M. Bolla, P. Del Soldato, C. Melani, C. Guiducci, M. P. Colombo, M. Iezzi, et al. Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination PNAS, March 15, 2005; 102(11): 4185 - 4190. [Abstract] [Full Text] [PDF]
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K. P. A. MacDonald, V. Rowe, A. D. Clouston, J. K. Welply, R. D. Kuns, J. L. M. Ferrara, R. Thomas, and G. R. Hill Cytokine Expanded Myeloid Precursors Function as Regulatory Antigen-Presenting Cells and Promote Tolerance through IL-10-Producing Regulatory T Cells J. Immunol., February 15, 2005; 174(4): 1841 - 1850. [Abstract] [Full Text] [PDF]
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T. Ghansah, K. H. T. Paraiso, S. Highfill, C. Desponts, S. May, J. K. McIntosh, J.-W. Wang, J. Ninos, J. Brayer, F. Cheng, et al. Expansion of Myeloid Suppressor Cells in SHIP-Deficient Mice Represses Allogeneic T Cell Responses J. Immunol., December 15, 2004; 173(12): 7324 - 7330. [Abstract] [Full Text] [PDF]
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S. Kusmartsev, Y. Nefedova, D. Yoder, and D. I. Gabrilovich Antigen-Specific Inhibition of CD8+ T Cell Response by Immature Myeloid Cells in Cancer Is Mediated by Reactive Oxygen Species J. Immunol., January 15, 2004; 172(2): 989 - 999. [Abstract] [Full Text] [PDF]
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E. Chiffoleau, T. Kobayashi, M. C. Walsh, C. G. King, P. T. Walsh, W. W. Hancock, Y. Choi, and L. A. Turka TNF Receptor-Associated Factor 6 Deficiency during Hemopoiesis Induces Th2-Polarized Inflammatory Disease J. Immunol., December 1, 2003; 171(11): 5751 - 5759. [Abstract] [Full Text] [PDF]
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C. Melani, C. Chiodoni, G. Forni, and M. P. Colombo Myeloid cell expansion elicited by the progression of spontaneous mammary carcinomas in c-erbB-2 transgenic BALB/c mice suppresses immune reactivity Blood, September 15, 2003; 102(6): 2138 - 2145. [Abstract] [Full Text] [PDF]
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S. Kusmartsev, F. Cheng, B. Yu, Y. Nefedova, E. Sotomayor, R. Lush, and D. Gabrilovich All-trans-Retinoic Acid Eliminates Immature Myeloid Cells from Tumor-bearing Mice and Improves the Effect of Vaccination Cancer Res., August 1, 2003; 63(15): 4441 - 4449. [Abstract] [Full Text] [PDF]
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 S. Kusmartsev and D. I. Gabrilovich Inhibition of myeloid cell differentiation in cancer: the role of reactive oxygen species J. Leukoc. Biol., August 1, 2003; 74(2): 186 - 196. [Abstract] [Full Text] [PDF]
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M. Dupuis, M. de Jesus Ibarra-Sanchez, M. L. Tremblay, and P. Duplay Gr-1+ Myeloid Cells Lacking T Cell Protein Tyrosine Phosphatase Inhibit Lymphocyte Proliferation by an IFN-{gamma}- and Nitric Oxide-Dependent Mechanism J. Immunol., July 15, 2003; 171(2): 726 - 732. [Abstract] [Full Text] [PDF]
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A. D. Billiau, S. Fevery, O. Rutgeerts, W. Landuyt, and M. Waer Transient expansion of Mac1+Ly6-G+Ly6-C+ early myeloid cells with suppressor activity in spleens of murine radiation marrow chimeras: possible implications for the graft-versus-host and graft-versus-leukemia reactivity of donor lymphocyte infusions Blood, July 15, 2003; 102(2): 740 - 748. [Abstract] [Full Text] [PDF]
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Y. Liu, J. A. Van Ginderachter, L. Brys, P. De Baetselier, G. Raes, and A. B. Geldhof Nitric Oxide-Independent CTL Suppression during Tumor Progression: Association with Arginase-Producing (M2) Myeloid Cells J. Immunol., May 15, 2003; 170(10): 5064 - 5074. [Abstract] [Full Text] [PDF]
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V. Bronte, P. Serafini, C. De Santo, I. Marigo, V. Tosello, A. Mazzoni, D. M. Segal, C. Staib, M. Lowel, G. Sutter, et al. IL-4-Induced Arginase 1 Suppresses Alloreactive T Cells in Tumor-Bearing Mice J. Immunol., January 1, 2003; 170(1): 270 - 278. [Abstract] [Full Text] [PDF]
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A. B. Geldhof, J. A. Van Ginderachter, Y. Liu, W. Noel, G. Raes, and P. De Baetselier Antagonistic effect of NK cells on alternatively activated monocytes: a contribution of NK cells to CTL generation Blood, December 1, 2002; 100(12): 4049 - 4058. [Abstract] [Full Text] [PDF]
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 O. Goni, P. Alcaide, and M. Fresno Immunosuppression during acute Trypanosoma cruzi infection: involvement of Ly6G (Gr1+)CD11b+ immature myeloid suppressor cells Int. Immunol., October 1, 2002; 14(10): 1125 - 1134. [Abstract] [Full Text] [PDF]
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A. Mencacci, C. Montagnoli, A. Bacci, E. Cenci, L. Pitzurra, A. Spreca, M. Kopf, A. H. Sharpe, and L. Romani CD80+Gr-1+ Myeloid Cells Inhibit Development of Antifungal Th1 Immunity in Mice with Candidiasis J. Immunol., September 15, 2002; 169(6): 3180 - 3190. [Abstract] [Full Text] [PDF]
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A. Mazzoni, V. Bronte, A. Visintin, J. H. Spitzer, E. Apolloni, P. Serafini, P. Zanovello, and D. M. Segal Myeloid Suppressor Lines Inhibit T Cell Responses by an NO-Dependent Mechanism J. Immunol., January 15, 2002; 168(2): 689 - 695. [Abstract] [Full Text] [PDF]
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L. I. Terrazas, K. L. Walsh, D. Piskorska, E. McGuire, and D. A. Harn Jr. The Schistosome Oligosaccharide Lacto-N-neotetraose Expands Gr1+ Cells That Secrete Anti-inflammatory Cytokines and Inhibit Proliferation of Naive CD4+ Cells: A Potential Mechanism for Immune Polarization in Helminth Infections J. Immunol., November 1, 2001; 167(9): 5294 - 5303. [Abstract] [Full Text] [PDF]
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O. Atochina, T. Daly-Engel, D. Piskorska, E. McGuire, and D. A. Harn A Schistosome-Expressed Immunomodulatory Glycoconjugate Expands Peritoneal Gr1+ Macrophages That Suppress Naive CD4+ T Cell Proliferation Via an IFN-{gamma} and Nitric Oxide-Dependent Mechanism J. Immunol., October 15, 2001; 167(8): 4293 - 4302. [Abstract] [Full Text] [PDF]
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D. I. Gabrilovich, M. P. Velders, E. M. Sotomayor, and W. M. Kast Mechanism of Immune Dysfunction in Cancer Mediated by Immature Gr-1+ Myeloid Cells J. Immunol., May 1, 2001; 166(9): 5398 - 5406. [Abstract] [Full Text] [PDF]
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E. Apolloni, V. Bronte, A. Mazzoni, P. Serafini, A. Cabrelle, D. M. Segal, H. A. Young, and P. Zanovello Immortalized Myeloid Suppressor Cells Trigger Apoptosis in Antigen-Activated T Lymphocytes J. Immunol., December 15, 2000; 165(12): 6723 - 6730. [Abstract] [Full Text] [PDF]
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V. Bronte, E. Apolloni, A. Cabrelle, R. Ronca, P. Serafini, P. Zamboni, N. P. Restifo, and P. Zanovello Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor capable of activating or suppressing CD8+ T cells Blood, December 1, 2000; 96(12): 3838 - 3846. [Abstract] [Full Text] [PDF]
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S. Salvadori, G. Martinelli, and K. Zier Resection of Solid Tumors Reverses T Cell Defects and Restores Protective Immunity J. Immunol., February 15, 2000; 164(4): 2214 - 2220. [Abstract] [Full Text] [PDF]
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I. Angulo, F. G. de las Heras, J. F. Garcia-Bustos, D. Gargallo, M. A. Munoz-Fernandez, and M. Fresno Nitric oxide-producing CD11b+Ly-6G(Gr-1)+CD31(ER-MP12)+ cells in the spleen of cyclophosphamide-treated mice: implications for T-cell responses in immunosuppressed mice Blood, January 1, 2000; 95(1): 212 - 220. [Abstract] [Full Text] [PDF]
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V. Bronte, D. B. Chappell, E. Apolloni, A. Cabrelle, M. Wang, P. Hwu, and N. P. Restifo Unopposed Production of Granulocyte-Macrophage Colony-Stimulating Factor by Tumors Inhibits CD8+ T Cell Responses by Dysregulating Antigen-Presenting Cell Maturation J. Immunol., May 15, 1999; 162(10): 5728 - 5737. [Abstract] [Full Text] [PDF]
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