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*
Department of Oncology and Surgical Sciences, Oncology Section, Padova, Italy;
Experimental Immunology Branch, National Cancer Institute-National Institutes of Health, Bethesda, MD 20892; and
Laboratory of Experimental Immunology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) were ruled out as candidate effectors for the
suppression mechanism. The immortalized myeloid lines represent a
novel, useful tool to shed light on the molecules involved in the
differentiation of myeloid-related suppressors as well as in the
inhibitory pathway they use to control T lymphocyte
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The myeloid suppressor cells have been defined in the past as natural
suppressors, and constitute a cell population with an uncertain
phenotype (7). Even though several data suggested that
they belonged to the monocyte-macrophage lineage, discrepancies in the
marker distribution among the suppressor cells have been reported
(6, 8, 9, 10). We found that MSC isolated from the spleens of
immunocompromised mice were positive for CD31 Ag, a marker of
granulocyte/monocyte mouse precursors. These
CD11b+/Gr-1+/CD31+
splenocytes retained their inhibitory properties when cultured in vitro
in standard medium, but suppressive functions could be modulated by
cytokine exposure (3). Exposure to IL-4 increased
MSC-inhibitory activity, while culture in the presence of IL-4 and
GM-CSF induced their differentiation into a nonadherent population of
dendritic cells (DC). Exposure to type I Th cytokines, such as IL-12 or
the combination TNF- and IFN-
, also abrogated the suppressive
activity by inducing differentiation into macrophage-like cells able to
sustain T lymphocyte activation. A common
CD31+/CD11b+/Gr-1+
myeloid progenitor can thus be isolated from the lymphoid organs of
immune compromised mice that gives rise to accessory cells capable of
either activating or inhibiting the function of
CD8+ T lymphocytes, depending on the cytokines
present during the progenitor maturation/differentiation process
(3).
To investigate the molecular mechanisms governing the MSC differentiation and suppressive properties, we immortalized CD11b+/Gr-1+ cells selected by panning from the splenocytes of immunosuppressed mice. Immortalization was conducted by repeated cycles of infection with an ecotropic retrovirus encoding the oncogenes v-myc and v-raf. The immortalized cell lines shared many of the monocyte/macrophage markers (CD11b, F4/80, CD14, CD11c), but differed from previously characterized macrophage lines for their ability to suppress lymphocyte proliferation and cytotoxic response induced by alloantigens. Following an initial screening of the various immortalized cell lines, we finally selected two stable MSC lines: MSC-1 and MSC-2. The first line retained its inhibitory properties, while the second strongly up-regulated its suppressive functions after exposure to the cytokine IL-4. MSC-1 and IL-4-pretreated MSC-2, but not untreated MSC-2, were also able to suppress the function of Ag-specific CTL clones by triggering their apoptotic cascade.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CT-26, a BALB/c (H-2d) carcinogen-induced, undifferentiated colon carcinoma, and MBL-2 (H-2b), a Moloney virus-induced lymphoma, were previously described (2, 3). The mouse mammary adenocarcinoma, TS/A (H-2d), was kindly provided by Dr. G. Forni (University of Turin, Turin, Italy). NIH J2 Leuk is a monocyte/macrophage cell line immortalized from bone marrow of C3H/HeJ mice (11, 12). L1210 (H-2d) lymphocytic leukemia, J774A.1 macrophage, and NIH-3T3 fibroblast cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). E88 CTL clone and the adenocarcinoma C26 (H-2d) were kindly provided by Dr. M. Rodolfo (Istituto Nazionale Tumori, Milan, Italy). Cells were maintained in medium 1 consisting of DMEM (Life Technologies, Paisley, U.K.) supplemented with 2 mM L-glutamine, 10 mM HEPES, 20 µM 2-ME, 150 U/ml streptomycin, 200 U/ml penicillin, and 10% heat-inactivated FBS (Life Technologies).
Generation and culture of immortalized MSC lines
A panning technique was used to enrich Gr-1+ cells from the spleens of immunosuppressed BALB/c mice. Briefly, 60-mm petri dishes (Falcon 1016, Cockeysville, MD) were coated for 3 h with 10 µg/ml of the anti-Ly-6G mAb, RB6-8C5 (ATCC; purified from ascites), washed, and blocked with 10 mg/ml BSA in PBS. Splenocytes (3 ml, 2.5 x 107 cells) were dispensed into petri dishes and after 1 h at 37°C, the dishes were washed and medium 1 was added to the attached cells. Cells were incubated for an additional 6 h and detached by gentle pipetting with PBS plus 2 mM EDTA. Gr-1+ splenocytes were infected with a retrovirus encoding the viral oncogenes v-myc and v-raf using 50% supernatant from NIH J2 Leuk cells (11, 12). After 24 h, cells were collected and exposed to a second round of infection under the same conditions. Cells were then resuspended in medium 2 consisting of RPMI 1640 (Euroclone, Paignton-Devon, U.K.), 10% heat-inactivated FBS (BioWhittaker, Walkersville, MD), 2 mM L-glutamine, 1 mM sodium pyruvate, 20 µM 2-ME, 150 U/ml streptomycin, and 200 U/ml penicillin. For the first 3 wk, 10 ng/ml mouse GM-CSF (PeproTech, Rocky Hill, NJ) was added to the culture together with 25% conditioned medium from NIH-3T3, then replaced by 25% conditioned medium from the same immortalized cells. After 1 mo, cells were grown in medium 2 without GM-CSF and conditioned medium. The MSC-1 line originated from Gr-1+ splenocytes from mice bearing a 1-mo-old TS/A tumor, while the MSC-2 line originated from Gr-1+ splenocytes from mice immunized 6 days earlier with a recombinant vaccinia virus encoding mouse IL-2 (1). Cell lines were subcloned by limiting dilution using GM-CSF and conditioned medium as supplements to medium 2 during the first stages. The cell lines have been frozen, thawed, and grown in medium for more than 1 year, with no sign of stable changes in their phenotype and function, although fluctuation in some marker expression levels has been occasionally detected. The uncloned lines, and one clone each from MSC-1 and 2 were used throughout these studies. Data obtained with the subclones were identical with those obtained with the parental lines.
Peptides and cytokines
SPSYVYHQF, representing the aa 423–431 of the gp70 envelope protein of the endogenous AKR623 retrovirus presented in association with H-2 Ld (13), was synthesized and purified by Neosystem (Strasburg, France). Mouse rGM-CSF, and IL-4 (PeproTech) were resuspended in PBS containing 1% mouse serum (Sigma, St. Louis, MO). Human rIL-2 (Euro Cetus-Chiron, Milan, Italy) was resuspended in RPMI with 20% FBS.
Evaluation of CTL response
MLC were set up in 24-well plates by coincubating in a total
volume of 1 ml/well 2.5 x 106 splenocytes
from 8- to 12-wk-old female BALB/c mice (H-2d),
an equal number of -irradiated C57BL/6 splenocytes
(H-2b), and (unless otherwise stated) 3% (of the
total number of cells) -irradiated MSC or J774A.1 control cells. In
cell separation experiments, splenocytes were cultured at the same cell
concentration as above in 24-well plates (Falcon) containing a culture
chamber insert with 0.45-µm pores (Millipore, Bedford, MA).
Alternatively, 105 E88 CTL were stimulated with
0.75 x 105 C26 and 3 x
106 BALB/c splenocytes, both irradiated, in a
final volume of 1 ml/well of medium 2 containing 10 U/ml rIL-2 and
0.15 x 106 -irradiated MSC or J774A.1
cells. After 5 days, the cultures were tested for the ability to lyse
peptide-pulsed or allogeneic targets in a 5-h
51Cr release assay using 2 x
104 target cells previously labeled with 100
µCi Na51CrO4 for 60 min.
The percentages of specific lysis were calculated from triplicate
samples using the formula: (experimental cpm - spontaneous
cpm)/(maximal cpm - spontaneous cpm) x 100. LU per
106 effector cells were calculated by dividing
106 by the number of cells giving 30% specific
lysis. LU/106 cells were then used to calculate
the LU/culture from the number of viable cells recovered in the
cultures.
Proliferation assays
BALB/c splenocytes (2 x 105
cells/well) were cultured in 96-well round-bottom plates (Falcon) and
stimulated with an equal number of -irradiated C57BL/6 splenocytes.
Various numbers of -irradiated MSC or J774A.1 cells were added in a
total volume of 200 µl/well of medium 2. After 3 or 5 days of
incubation, cultures were pulsed with 1 µCi/well
[3H]TdR (NEN, Life Science Products, Boston,
MA), and [3H]TdR incorporation was measured by
scintillation counting. Data are expressed as cpm (mean ± SD) of
triplicate cultures.
Abs, cytometric analysis, and apoptosis detection
FITC- or PE-labeled mAb recognizing mouse CD11b (Mac-1), CD14,
Mac-2, Mac-3, H-2Kd, H-2Dd,
CD80 (B7-1), CD30, CD40,
I-Ad/I-Ed, CD3, CD4, CD8,
and isotype-matched controls were purchased from PharMingen (San Diego,
CA); FITC- or PE-labeled mAbs recognizing mouse Ly-6G (Gr-1), or CD86
(B7-2), were from Immunokontact (Bioggio, Switzerland); rat mAbs
recognizing mouse F4/80, MOMA-2, 88a, CD31, ER-MP58, DEC-205 were from
BMA Biomedicals (Augst, Switzerland). PE donkey anti-rat
F(ab')2 (Jackson ImmunoResearch, West Grove, PA)
or TC streptavidin (DakoPatts, Glostrup, Denmark) were used for
secondary labeling. Biotinylated rat anti-mouse CD11c mAb (N418;
ATCC) was a gift of U. Grohmann (University of Perugia, Perugia,
Italy). The mAb 2.4G2 (anti-mouse CD16/CD32; PharMingen) was used
to block binding to FcR. Cells were treated with 2.4G2, stained with
appropriate amounts of mAb (1–10 µl/106
cells), and analyzed using a Coulter XL flow cytometer (Coulter
Electronics, Hialeah, FL) equipped with a 488-nm Argon Ion Laser
(Coherent, Innova). At least 10,000 cells were collected for each
histogram, and dead cells were gated out based on their scatter
properties.
FITC-conjugated annexin-V (Boehringer Mannheim, Indianapolis, IN) was used to detect apoptosis. Briefly, 106 cells were stained with a PE-conjugated anti-CD8 mAb (PharMingen) and with FITC-conjugated annexin-V in 100 µl of labeling solution and incubated for 10–15 min, according to manufacturer’s protocol. Then 0.4 ml of incubation buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) was added, and stained cells (15,000 or more) were analyzed by flow cytometry.
RNase protection assay
The Multiprobe RNase Protection Assay (PharMingen) was performed according to the manufacturer’s directions with few modifications.
Hybridization. [33P]UTP (70–80 µCi/reaction) was used to synthesize the probe, and 1.5 x 106 cpm was added to each hybridization reaction. After synthesis and addition of yeast tRNA and EDTA (final volume 50 µl), 25-µl aliquots of the reaction were placed on two TE Micro Select D G25 spin columns (5'-3'; Boulder, CO) and the probe purified by centrifugation for 30 s at 12,000 rpm in a microcentrifuge.
RNase inactivation. A cocktail was prepared containing 200 µl Ambion RNase inactivation reagent (Ambion, Austin, TX), 50 µl ethanol, 5 µg yeast tRNA, and 1 µl Ambion GycoBlue coprecipitate per RNA sample. A total of 250 µl of the inactivation cocktail was pipetted into 1.5-ml microcentrifuge tubes, and the individual RNase-treated samples were added to each tube. The samples were mixed well, placed at -70°C for 30 min, and centrifuged for 15 min in a room temperature microcentrifuge at 14,000 rpm. The supernatants were poured off, a sterile cotton swap was used to remove excess liquid from the tube, and the pellet was resuspended in 3 µl of PharMingen sample buffer before gel electrophoresis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain large, homogeneous populations of MSC,
Gr-1+ cells were isolated from the spleens of
immunosuppressed mice and immortalized by retroviral transduction. We
infected this population containing the myeloid suppressors with an
ecotropic retrovirus expressing myc and raf
oncogenes that was used in previous studies to immortalize successfully
mouse macrophages and DC (11, 12). After screening for the
ability to suppress proliferative and cytotoxic responses, two cell
lines, MSC-1 and MSC-2, were selected for further study. The phenotypes
of these lines indicate that they, like the J774A.1 nonsuppressive
control line, belong to the monocyte/macrophage lineage (Table I
). Moreover, although the MSC lines were
derived from cells expressing Gr-1, a marker for immature myeloid
progenitors and mature polymorphonuclear cells, they lost this marker
during the immortalization process, as did
Gr-1+/CD11b+ splenocytes
when put into culture (1, 2). None of the cell lines
examined expressed the CD3, CD4, or CD8 T cell markers, and class II
MHC expression was low in all three lines, but could be strongly
up-regulated by IFN- (data not shown). The MSC-1 and 2 lines showed
significant differences in several markers; most notably MSC-1 showed
decreased CD14 and MAC-3 expression, and increased DEC-205 when
compared with MSC-2 and J774 cells. These differences did not reflect
the distinct origins of the two lines because clones with the same
phenotypes as MSC-1 and MSC-2 were isolated from both sources of
myeloid cells (not shown). We suppose that these differences rather
reflect the arrest at particular stages of myeloid differentiation
stochastically achieved through the immortalization process.
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The immunosuppressive properties of the MSC lines were evaluated
by adding them to MLCs. As seen in Fig. 1, alloreactive CTL recognizing
H-2b target cells were generated in a standard
MLC, and in MLC in which J774A.1 cells were present. By contrast,
addition of only 3% MSC-1 resulted in a complete abrogation of
cytolytic activity of the MLC. Although MSC-2 were not constitutively
suppressive in this assay, they acquired the ability to abrogate the
cytolytic activity of alloreactive CTL following 2 days pretreatment
with mouse IL-4 (Fig. 1), and they retained their suppressive function
for at least 7 days after removal of IL-4 (data not shown).
Pretreatment of J774A.1 cells with IL-4 failed to induce the
suppressive phenotype (not shown), implying that the MSC represent a
specialized macrophage subset. Indeed, MSC-2, but not J774A.1 cells
expressed the -chain of the IL-4R (IL-4R), while the common
-chain was present in both lines; MSC-2 also constitutively
expressed the IL-7R (Fig. 2A). The MSC-2 line underwent
phenotypic changes following exposure to IL-4, the most important of
which are summarized in Fig. 2B. IL-4 up-regulated some
costimulatory molecules such as CD80, and CD40; this together with the
basal expression of CD86 (not shown) questions the loss of
costimulation as the main mechanism for T lymphocyte suppression.
Interestingly, an increase in CD4 expression was also observed in
response to IL-4, but there was only a modest augmentation in
I-Ad/I-Ed, although the
same treatment causes MHC II up-regulation in mature mouse monocytes
(14).
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To evaluate the involvement of soluble factors in MSC-dependent
suppression, we set up transwell experiments in which MSC lines were
cultured either together with, or in close proximity to, an allogeneic
MLC. The effectors were then tested in a cytotoxicity assay against
syngeneic (CT-26) or allogeneic (MBL-2) targets. As shown in the box in
Fig. 1
, no inhibition of the MLC was observed when the IL-4-treated
MSC-2 line was separated by the transwell membrane. Cell-cell contact
was thus required for the suppressive activity of MSC-2 line.
Suppression of alloreactive CTL depends on antiproliferative and proapoptotic activity of MSC lines
Suppression of CTL activity could occur at either the generation
or effector phases. To determine whether MSC blocked proliferation,
BALB/c splenocytes were stimulated with allogeneic cells in the
presence of J774A.1 or MSC-2. When tested on day 3, MSC-2, but not
J774A.1 cells inhibited the proliferation of alloreactive T
lymphocytes, and this effect was not dependent upon previous exposure
to IL-4 (Fig. 3A). On day 5,
IL-4-treated MSC-2 almost completely abrogated the MLC
proliferation (Fig. 3B), whereas J774A.1 and untreated MSC-2
partially blocked proliferation. However, this inhibition of T
lymphocyte growth was not sufficient to impair the generation of a
cytolytic response against allogeneic targets, as shown in Fig. 1.
MSC-1 also completely suppressed alloreactive T cell proliferation
(Fig. 4). Like the MSC-2-induced
suppression, this effect required cell-cell contact because the
suppression was not observed when the MSC-1 were separated from the MLC
by transwells. Increasing the number of MSC-1 to a 1:1 ratio with the
effector cells in the MLC did not result in suppression across the
transwell membrane, thus making less likely the involvement of soluble
suppressive factors either released at low doses or unstable in the
culture medium.
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).
Apoptosis was detected as the percentage of CD8+
lymphocytes staining with FITC annexin-V, a protein with high affinity
for phosphatidylserine, which is translocated from the inner to the
outer leaflet of the plasma membrane in the initial phases of apoptosis
(16). Compared with J774A.1, apoptosis was clearly
increased when CTL were activated in the presence of MSC-1 and
IL-4-pretreated MSC-2 lines. This cell death correlated with the
drastic reduction in the number of LU recovered in the cultures of CTL
E88. Interestingly, a high proportion of apoptotic
(annexin-V+) CTL had decreased expression of
CD8.
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It has been shown that both TNF- and Fas-L are required to
induce apoptosis of CD8+ T cells, whereas Fas-L
alone induces apoptosis of most activated CD4+ T
cells (17). These death signals engage specific receptors
that assemble caspase-triggering protein complexes that, in turn,
regulate cell death (18, 19). By contrast, TNF-related
apoptosis-inducing ligand (TRAIL) inhibits T lymphocyte activation by
blocking cell cycle progression (20) However, neither Fas
ligand, lymphotoxin-ß (a member of TNF family), or TRAIL was
expressed by the MSC-2 line (Fig. 6).
Although some basal transcription of TNF- occurred, this was
down-modulated by IL-4. In contrast, Fas, TNFR, and CD30 (Fig. 2B), another member of the TNFR family, are present in
IL-4-treated MSC-2. IL-4 therefore appears to make MSC-2 more
susceptible to death signals. However, the death domain-containing
proteins (TNFR-associated death domain protein, Fas-associated death
domain protein, and receptor interacting protein) that convey the death
signal from the membrane-associated receptors are apparently
down-modulated by IL-4 (Fig. 6A). TGF-ß, a cytokine with
several inhibitory functions (21), did not seem to be
responsible for T lymphocyte suppression because it was expressed
equally in suppressive and nonsuppressive lines (Fig. 6); moreover, a
mAb neutralizing the biological activities of TGF-ß1, 2, or 3 did not
reverse the suppression of T lymphocyte activation mediated by MSC
lines (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We show that MSC lines prevent the generation of allo-specific CTL by
blocking T cell proliferation, and induce apoptosis in Ag-stimulated
CTL clones. The mechanism by which MSC lines suppress T cell
proliferation will be discussed in the accompanying
paper,3 but the transwell experiments
demonstrate clearly that contact between MSC and splenocytes is
required for proliferative arrest and apoptosis. Some of the most
common apoptosis-inducing molecules expressed on cell membranes,
including TRAIL, Fas ligand, TNF-, and lymphotoxin-ß can be ruled
out based on their lack of expression in MSC (current results and
(1)). In experiments not shown, we also eliminated the
involvement of several soluble molecules that might have been released
by MSC lines following contact with Ag-activated T lymphocytes and that
are known to be inhibitory in some systems. Thus, the failure of
neutralizing Abs or inhibitors to reverse suppression ruled out IL-10,
TGF-ß, PGs, and hydrogen peroxide as mediators of suppression.
Production of indoleamine 2, 3-dioxygenase by activated macrophages and
DC has been shown also to inhibit T cell proliferation through
tryptophan consumption, and it was proposed as a mechanism for the
regulation of immune responses (26, 27). However,
inhibition of T lymphocyte functions by MSC lines could not be
prevented using the indoleamine 2, 3-dioxygenase inhibitor
1-methyl-DL-tryptophan or by adding a tryptophan excess to
the MLC (not shown). In the accompanying paper, we show that MSC use
two mechanisms to block T cell proliferation.4 In
allo-specific responses, MSC block early events, including IL-2
production, by a contact-dependent mechanism. Mitogen-driven responses,
however, are suppressed by a mechanism requiring NO and IFN-. This
latter mechanism does not interfere with IL-2 production or
up-regulation of early activation markers on T cells, but does impair
the IL-2 signaling pathway. The induction of NO production in MSC lines
requires contact with activated splenocytes, and the difference between
suppressive and nonsuppressive macrophages appears to be that only the
MSC produce NO under these conditions.4
The differences found among the MSC lines probably reflect a
heterogeneity in the
Gr-1+/CD11b+ progenitors.
Phenotypic and functional analyses indicate that at least two different
myeloid stages can be distinguished for the suppressor cells. MSC-1 is
the prototype of immature monocytic suppressor cells that does not
require preactivation with cytokines to exert an inhibitory activity on
T lymphocytes, while MSC-2 is a more mature cell that requires IL-4
pretreatment. It was recently shown that IL-4 causes alternative
activation of macrophages (28). Alternatively activated
macrophages can be distinguished, phenotypically, by the expression of
a distinctive set of molecules including antiinflammatory cytokines
such as IL-1R antagonist and receptors used in innate immunity.
Functionally, they can induce tolerance and down-regulate inflammatory
responses (28). In one report, IL-4-stimulated human
macrophages inhibited mitogen-mediated proliferation of
CD4+ lymphocytes by an as yet unknown mechanism
(29). Our experiments suggest a relationship between our
myeloid suppressors and the alternatively activated macrophages,
particularly in the case of MSC-2, which not only gains the suppressive
phenotype in response to IL-4 (Figs. 1, 3, and 5), but also
up-regulates the expression of IL-1R antagonist (not shown). An
increase in the proinflammatory cytokine, IL-6, following IL-4
treatment (Fig. 6), however, implies a more complex pattern of IL-4
activity on the MSC-2 line.
IL-4 exerts an important role in the control of immune responses in
vivo. When transgenically expressed in the pancreatic ß-cells of
nonobese diabetic mice or in the CNS of SJL/J mice, IL-4 prevented
autoimmune diabetes and experimental allergic encephalomyelitis
development, respectively (30, 31). APC alternatively
activated by IL-4 might thus play a role in controlling the response of
T lymphocytes to Ags presented by peripheral tissues. It has been shown
that peripheral tolerance to MHC class I-restricted self Ags occurred
by two simultaneous processes: apoptotic elimination of self-reactive
CD8+ T lymphocytes, and clonal anergy associated
with down-regulation of the ß TCR and CD8 (32, 33).
Interestingly, in our studies, both events, i.e., apoptosis and
down-regulation of the CD8 molecule, were observed in vitro when cloned
CTL were activated by the Ag in the presence of MSC lines (Fig. 5).
However, the MSC lines express very few MHC class II molecules, and
therefore most certainly lack the capacity to present self or allo Ags
to CD4+ T cells. Moreover, they inhibit
proliferative responses of both CD4+ and
CD8+ T cells indiscriminately, regardless of
whether Ag or mitogen is used in stimulation (data not shown). It is
therefore unlikely that the normal counterparts of the MSC lines
interact with T cells in an Ag-specific manner, but instead, they most
probably serve as homeostatic regulators that temper responses of all T
cells during periods of intense, sustained antigenic challenge.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Vincenzo Bronte, Department of Oncology and Surgical Sciences, Oncology Section, Via Gattamelata 64, 35128 Padova, Italy.
3 A. Mazzoni, V. Bronte, E. Apolloni, P. Serafini, P. Zanovello, A. Visintin, J. H. Spitzer, J. A. Titus, and D. M. Segal. Myeloid suppressor lines inhibit T cell responses by NO dependent and independent mechanisms. Submitted for publication.
4 Abbreviations used in this paper: MSC, myeloid suppressor cell; DC, dendritic cell; TRAIL, TNF-related apoptosis-inducing ligand.
Received for publication July 7, 2000. Accepted for publication September 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Eur. J. Immunol. 24:1441.[Medline]
. J. Immunol. 143:491.[Abstract]
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P. C. Rodriguez, D. G. Quiceno, J. Zabaleta, B. Ortiz, A. H. Zea, M. B. Piazuelo, A. Delgado, P. Correa, J. Brayer, E. M. Sotomayor, et al. Arginase I Production in the Tumor Microenvironment by Mature Myeloid Cells Inhibits T-Cell Receptor Expression and Antigen-Specific T-Cell Responses Cancer Res., August 15, 2004; 64(16): 5839 - 5849. [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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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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