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*
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and
Department of Oncology and Surgical Sciences, Oncology Section, Azienda Ospedaliera, Padova, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
a contact-dependent stimulus. Experiments with inducible NO synthase
knockout mice demonstrated that the inhibition of T cell proliferation
by CD11b+Gr-1+ cells in the spleens of
immunosuppressed mice is also dependent upon NO, indicating that the
MSC lines accurately represent their normal counterparts. The
distinctive capacity of MSC to generate suppressive signals when
encountering activated T cells defines a specialized subset of myeloid
cells that most likely serve a regulatory function during times of
heightened immune activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
in macrophages and
in some macrophage lines. Because homogeneous populations of MSC are difficult to isolate in large numbers from mice, we have taken the approach of immortalizing MSC to generate cloned cell lines. In a previous report (27) we isolated Gr-1+CD11b+ cells from the spleens of mice that were immunosuppressed by tumor or virus and infected them with a retrovirus encoding the v-myc and v-raf viral oncogenes. Cloned lines obtained from these cells lost expression of Gr-1 in culture but did express several monocyte/macrophage markers such as CD11b, F4/80, CD86, and CD11c. All cloned lines retained the capacity to inhibit T cell proliferation and the generation of alloreactive CTL, in contrast to several phenotypically similar macrophage lines that lacked this function. In this paper we have examined the mechanism by which these cells block T cell activation. In vitro experiments as well as studies using iNOS knockout mice clearly show that NO plays a critical role in the suppression of immune responses by MSC lines. NO operates at the level of the IL-2R signaling pathway, inhibiting Janus kinase (JAK)1 and JAK3, STAT5, extracellular signal-regulated kinase (Erk), and Akt activation by IL-2. The secretion of NO by MSC lines is tightly controlled, requiring surface and soluble signals from activated T cells. As such, MSC serve as sensors for T cell activation and differ from nonsuppressive macrophage lines, which fail to release NO when encountering activated T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation, culture, and phenotypes of MSC lines have been described previously (27). Briefly, adherent Gr-1+CD11b+ cells were isolated from the spleens of BALB/c mice that were found to be immunosuppressed during the growth of the mammary adenocarcinoma TS/A tumor or following immunization with an IL-2-producing recombinant vaccinia virus (10). These cells were immortalized with a retrovirus encoding the v-myc and v-raf oncogenes, and cell lines designated MSC-1 and MSC-2 were established from the tumor-bearing and vaccinia-treated mice, respectively. The two lines were cloned by limiting dilution, and one representative clone from each line was used throughout this study. The control BALB/c alveolar macrophage line, MH-S, was obtained from American Type Culture Collection (Manassas, VA).
Proliferation assays
Single-cell suspensions were prepared from spleens of 6- to
12-wk-old female mice, and RBC were removed with ACK Lysing Buffer
(Biofluids, Rockville, MD). BALB/c splenocytes (4 x
105 cells/well) were stimulated with 5 µg/ml
Con A (Sigma-Aldrich, St. Louis, MO) in the presence of irradiated (60
Gy) MSC lines or MH-S cells (syngeneic to the responder cells). Assays
were performed in 96-well plates, 200 µl/well in RPMI 1640 containing
10% FCS, glutamine, antibiotics, and 55 µM 2-ME. Unless stated
otherwise, the numbers of MSC or MH-S cells were 3% of the total
number of splenocytes. After 1 or 2 days of stimulation with Con A,
cells were pulsed for 16 h with 5 µCi/ml
[3H]thymidine, harvested with a Tomtec
harvester (Tomtec, Orange, CT), and counted using a beta scintillation
counter. Data are expressed as counts per minute (mean ± SE) of
triplicate cultures. In some experiments splenocytes from DO11.10
TCR-transgenic mice (BALB/c background), specific for chicken OVA
peptide residues 323–339 in the context of I-Ad
(28), were used as responders. Splenocytes (4 x
105/well) were stimulated in the presence or the
absence of MSC lines with 1 µM antigenic peptide for 2 days and then
pulsed with [3H]thymidine as described above.
Some experiments were performed in 24-well Transwell plates
(Costar, Cambridge, MN) in a final volume of 800 µl.
Splenocytes (1.8 x 106/600 µl) were in
the lower compartment of the well, and MSC lines or MH-S cells
(0.54 x 105/200 µl) were in the upper
compartment. Cells were cultured with Con A for 2 days, then pulsed
with [3H]thymidine as described above. Where
indicated, 200 Cetus units/ml human rIL-2 (Hoffmann-LaRoche, Nutley,
NJ) or 0.2 mM
NG-monomethyl-L- and
D-arginine (L- and
D-NMMA; Alexis, San Diego, CA) were added to
cultures. Neutralizing anti-mouse IFN- mAb (clone 37895.11; R&D
Systems, Minneapolis, MN) was used at 30 µg/ml.
Flow cytometry
Cells were surface stained using the following mAbs:
PE-anti-CD25 (IL-2R -chain), PE-anti-CD132 (cytokine common
-chain), FITC-anti-CD122 (IL-2R 
-chain), FITC-anti-CD69,
PE-anti-Gr-1, and FITC-anti-CD11b. All mAbs were purchased from
BD PharMingen (San Diego, CA), except for the anti-CD25 (Caltag
Laboratories, Burlingame, CA). Staining was performed in the presence
of saturating levels of the anti-FcR mAb, 2.4G2 (BD PharMingen).
Ten thousand cells were acquired for each sample with a FACScan flow
cytometer (BD Biosciences, San Jose, CA). Dead cells were gated out
based on their scatter properties. For cell cycle analysis, cells were
fixed in a 0.5% formaldehyde solution in HBSS for 15 min at 4°C,
washed once in HBSS, and incubated overnight at 4°C in 0.1% sodium
citrate and 0.1% Triton X-100 containing 25 µg/ml propidium iodide
and 0.1 mg/ml RNase A. Twenty thousand cells were acquired for each
sample. Doublets and other aggregates were gated out using the BD
Biosciences Doublet Discrimination Module.
IL-2, IFN-, and NO measurements
Levels of IL-2 or IFN- in culture supernatants were assayed
using ELISA kits from BioSource International (Camarillo, CA) and R&D
Systems, respectively. The detection limit was 13 pg/ml for IL-2 and 2
pg/ml for IFN-. Treatment of MSC lines or MH-S with IFN- was
performed in 24-well plates. After irradiation, cells (0.54 x
105 cells/800 µl) were stimulated with titrated
amounts of recombinant mouse IFN- (PeproTech, Rocky Hill,
NJ), and after a 48-h incubation at 37°C NO levels were measured in
supernatants. NO production was measured as the nitrite concentration
using the Griess assay. Supernatants (100 µl) were added to 100 µl
of a 1/1 mixture of 1% sulfanilamide dihydrochloride and 0.1%
naphthylethylenediamine dihydrochloride (Sigma-Aldrich) in 2.5%
H3PO4. Plates were
incubated at room temperature for 10 min, and absorbance at 550 nm was
measured using a microplate reader. Nitrite contents were calculated
with reference to a sodium nitrite standard curve. The detection
threshold was 1 µM.
Immunoprecipitation and immunoblotting
In some studies, nylon wool-enriched T cells (
75%
CD3+) were cultured for 24 h with Con A and
MSC, for an additional 4 h with MSC in the absence of serum and
Con A, followed by 15 min with or without human rIL-2 (200 U/ml). In
other experiments T cells (>95% CD3+) were
negatively selected from lymph nodes using anti-mouse Ig-coated
magnetic beads (Polysciences, Warrington, PA). After 2-day stimulation
with Con A, cells were washed in CO2-acidified
RPMI 1640 to remove bound cytokines, rested for 24 h in RPMI 1640
plus 1% FCS, treated for 2 h with 350 µM
S-nitroso-N-acetylpenicillamine (SNAP; Molecular
Probes, Eugene, OR) or with an equivalent volume of solvent (DMSO), and
incubated for 5 min with or without IL-2. In both cases lymphocytes
were lysed for 10 min on ice with 0.4% Nonidet P-40 in 20 mM Tris-HCl
(pH 8), 10% glycerol, 50 mM NaF, 2 mM EDTA, 137 mM NaCl, 1 mM PMSF, 10
µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM
Na3VO4 and clarified by
centrifugation. Aliquots containing 200 µg of protein (Bio-Rad
Protein Assay; Bio-Rad, Richmond, CA) were precleared and
immunoprecipitated using Ab bound to protein A- or protein G-Sepharose.
Immunoprecipitates were resolved by 6% SDS-PAGE, transferred to a
nitrocellulose membrane, and Western blotted first for the
phosphorylated protein, then after stripping in 0.1 M glycine, 0.1%
SDS, and 1% Tween 20, pH 2.2, for 1 h at room temperature for
total protein. For Erk1/2, Akt, and STAT5, 30 µg of lysate were
Western blotted directly. Proteins were detected by ECL (Amersham
Pharmacia Biotech, Piscataway, NJ).
The following Abs were used for immunoprecipitations: rabbit anti-STAT5b (R&D Systems), anti-JAK3 mAb (Upstate Biotechnology, Lake Placid, NY), and anti-JAK1 mAb (Transduction Laboratories, Lexington, KY). The following Abs were used for blotting: HRP-PY-20 anti-phosphotyrosine (for phospho-STAT5b; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-STAT5b (R&D Systems), 4G10 anti-phosphotyrosine (for phospho-JAK3; Upstate Biotechnology), rabbit anti-phospho-JAK1 (Calbiochem, San Diego Ca), rabbit anti-phospho-mitogen-activated protein kinase (MAPK) (Erk1/2; New England Biolabs, Beverly, MA), rabbit anti-Erk1 (Transduction Laboratories), rabbit anti-phospho-Akt (Thr308) rabbit anti-Akt (New England Biolabs), rabbit anti-JAK3 (Upstate Biotechnology), anti-JAK1 mAb (Transduction Laboratories), and rabbit anti-phospho-STAT5 (Zymed Laboratories, San Francisco, CA). HRP anti-rabbit or anti-mouse Ig were used as developing reagents.
Experiments with iNOS knockout mice
Four- to 6-wk-old B6;129P-Nos2tm1Lau (iNOS-/-) female mice and B6129PF2/J wild-type (wt) controls were purchased from The Jackson Laboratory (Bar Harbor, ME). Recombinant mouse GM-CSF (PeproTech) was administered twice a day over a 3-day period (5 µg/mouse i.p. twice daily). Splenocytes were then collected, and their phenotype and proliferative responses to Con A were determined as described above. Data were analyzed using Student’s t test. Values of p < 0.05 were considered insignificant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the inhibitory capacities of MSC lines, BALB/c
splenocytes were treated with Con A in the presence of titrated numbers
of MSC. As shown in Fig. 1
A,
cloned MSC potently inhibited proliferation, with as few as 3% of
cells causing complete suppression. By contrast, cells from the
phenotypically similar macrophage line MH-S (and J774, not shown) were
noninhibitory, indicating that not all myeloid lines are suppressive.
In addition to their capacities to block mitogen-induced proliferation,
the MSC lines were also effective inhibitors of peptide-specific
responses (Fig. 1B) as well as allogeneic responses, as
shown previously (27). FACS analysis of propidium
iodide-stained lymphocytes indicated that the MSC blocked proliferation
of mitogen-stimulated T cells by preventing them from entering the cell
cycle (data not shown). This block was observed after 24 and 48 h
of stimulation and at 24 h was reversible, because splenocytes
that had been stimulated with Con A for 24 h in the presence of
suppressor lines retained the capacity to proliferate following removal
of MSC (Fig. 1C). Thus, during the first day of stimulation,
the MSC lines induced an arrest in the cell cycle but did not kill the
T cells.
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We next asked whether MSC-mediated suppression occurs at early or
later stages of T cell activation. As indicators of early,
TCR-dependent events, we measured IL-2
secretion and CD25 and CD69 surface expression. The data of Table I and
Fig. 2 show that the MSC lines do not
block IL-2 secretion or CD25 and CD69 up-regulation, suggesting that
MSC lines do not block the early stages of T cell activation.
Therefore, we hypothesized that the MSC lines might operate at a later
stage in activation, e.g., at the level of IL-2R signaling. To test
whether limiting levels of IL-2 could account for the suppression,
excess IL-2 was added on the first day of culture to Con A-activated
splenocytes and suppressor cells; however, splenocytes still failed to
proliferate (data not shown). Lymphocytes in both suppressed and
control cultures expressed comparable levels of the three IL-2R
subunits, CD25, CD122, and CD132 (Fig. 2), suggesting that the site of
suppression was downstream of the IL-2/IL-2R interaction. Three
signaling pathways are known to be activated by IL-2: JAK/STAT,
Ras/MAPK, and phosphoinositol 3-kinase/Akt (29). To
evaluate whether there was an impairment in IL-2 signaling, T cells
were stimulated with Con A in the presence or the absence of MSC-1
cells or control MH-S cells for 24 h, washed, and rested in
serum-free medium for 4 h. Cells were then incubated for 15 min
with rIL-2 and lysed, and representative proteins from each pathway
were tested for activation. As shown in Fig. 3A, all three pathways were,
in fact, impaired in MSC-suppressed T cells, as indicated by the lack
of phosphorylation of STAT5b, Erk1/2, and Akt in response to IL-2.
Stripping and reblotting with anti-STAT5b, anti-Erk1/2, and
anti-Akt Abs indicated that equal amounts of protein were loaded in
each lane. These data provide direct evidence that the MSC lines
disrupt all known pathways activated by the IL-2R.
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Because NO is known to be involved in immunosuppression in several
systems, we asked whether it also plays a role in MSC-mediated
suppression. As shown in Fig. 4, a
selective inhibitor of NOS, L-NMMA, reversed the
MSC-mediated block of proliferation, while its inactive enantiomer,
D-NMMA, had no effect. Reversal occurred in cultures
activated with either mitogen (Fig. 4A) or OVA peptide (Fig. 4B), and relatively high levels of
NO2-, an end product of NO
decomposition, were detected in both types of culture (Table II and data not shown) when MSC lines,
but not MH-S cells, were present. Moreover, addition of NO (in the form
of the NO donor, SNAP) to the cultures at concentrations similar to
those produced by the MSC resulted in blocked proliferation (data not
shown) and impaired phosphorylation of proximal components of the IL-2
signaling pathway (Fig. 3
B). NO had the most dramatic effect
on STAT5, but it clearly inhibited JAK3 and, to a lesser extent, JAK1
phosphorylation. Thus, NO is an essential component of MSC-mediated
suppression, and NO by itself is able to mediate suppression.
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and direct contact between
MSC and lymphocytes
Because IFN- up-regulates iNOS in several types of myeloid
cells, we asked whether it was involved in the immunosuppression
mediated by our MSC lines. As shown in Fig. 4A, a blocking
anti-IFN- Ab reversed suppression, indicating that IFN- is
required for MSC-dependent suppression. Next, IFN- was added
directly to the MSC to determine whether it would induce NO secretion.
As shown in Fig. 5 (upper
panel), IFN- did induce a dose-dependent release of NO
from the MSC, but even in the presence of 1000 ng/ml IFN-, MSC
released only half the amount of NO as when cultured with
mitogen-activated splenocytes (Fig. 5, lower panel), which,
by comparison, produced 13 ng/ml IFN-. On the other hand, the MH-S
line secreted lower levels of NO than the MSC at all concentrations of
IFN- tested and did not show an increase in the mitogen-activated
cocultures (Fig. 5). Finally, we observed that the same neutralizing
anti-IFN- mAb that reversed the MSC-dependent inhibition of
proliferation by 50% (Fig. 4A) also inhibited NO
production by 30% (data not shown).
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is required for NO production but that
in the mixed cultures there are additional factors that boost the
release of NO from the MSC lines. To determine whether these factors
involve cell-cell contact, Con A-stimulated splenocytes and MSC were
cultured in different compartments of Transwell plates, separated by a
membrane that prevented direct intercellular contact while allowing
diffusion of soluble mediators. Table II shows that despite the
presence of similar levels of IFN- in both the same well and
Transwell configurations, NO production and suppression of mitogenic
responses occur only if MSC are physically in contact with lymphocytes.
Thus, in addition to IFN-, other signals mediated by direct contact
between MSC and lymphocytes are required to achieve the levels of NO
required for suppression. CD11b+Gr-1+ cells from iNOS knockout mice are not immunosuppressive
In the above studies we used immortalized cell lines to show that
immune suppression by
CD11b+Gr-1+ MSC occurs by
an NO-dependent mechanism. To determine whether this mechanism, in
fact, applies to immunosuppressed mice, we treated
iNOS-/- and wt mice with GM-CSF to induce the
migration of MSC to the lymphoid organs (1). After 3-day
treatment with GM-CSF, the percentages and numbers of
CD11b+Gr-1+ cells were
enhanced in the spleens of both iNOS-/- and wt
mice relative to those in PBS-treated controls (Table III). Despite the presence of comparable
numbers of cells with the MSC phenotype in both
iNOS-/- and wt mice, immunosuppression was
observed only in wt mice (Fig. 6). Thus,
Con A-induced proliferation of splenocytes from wt, GM-CSF-treated mice
was significantly lower than proliferation of wt splenocytes from mice
treated with PBS. Moreover, addition of the iNOS inhibitor,
L-NMMA, to splenocytes from wt, GM-CSF-treated mice
significantly increased proliferation. By contrast, splenocytes from
GM-CSF-treated, iNOS-/- mice proliferated
normally in response to Con A, and L-NMMA had no effect on
proliferation. These results confirm that NO is an essential component
of immunosuppression mediated by normal MSC found in the spleens of
GM-CSF-treated mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is noteworthy that the cloned MSC are extremely potent inhibitors of
T cell proliferation, but that inhibition, at least for the first
24 h, is reversible. Therefore, the immediate function of the MSC
is to prevent activated T cells from entering the cell cycle without
killing the cells. Presumably, the block in proliferation would
eventually lead to apoptotic cell death, and, in fact, we have shown
previously (27) that after a 5-day coculture, MSC lines
induce apoptosis in a CTL clone as measured by annexin V binding. The
MSC lines do not express many of the known death-signaling molecules,
including Fas ligand (Fas-L), TNF-related apoptosis-inducing
ligand, TNF-, and lymphotoxin- (27), and we
have performed experiments in lpr mice that allowed us to
exclude the involvement of Fas-Fas-L pathways in suppression induced by
MSC in vivo (10). Moreover, an anti-TNF Ab failed to
restore the myeloid-induced immune unresponsiveness. Therefore,
apoptosis of T cells after prolonged suppression most likely results
from the proliferative block rather than from a death signal mediated
by Fas-L or TNF-.
The mechanism by which the MSC lines block mitogen and peptide-driven proliferation is well defined by our studies. The MSC lines do not impair the early events triggered by TCR cross-linking; in fact, suppressed lymphocytes release IL-2 and up-modulate activation markers such as CD25 and CD69. Instead, they prevent the IL-2R from transducing signals. Thus, although suppressed T cells express normal levels of the three subunits of the IL-2R, they fail to proliferate or activate STAT5, Erk1/2, and Akt, three components of the IL-2 signaling pathway (29), in response to IL-2. We have demonstrated that NO released by MSC is both necessary and sufficient to inhibit Con A and peptide-stimulated T cell proliferation. Evidence has been accumulating that NO modulates the biological functions of a number of intracellular signaling proteins (30) either directly by S-nitrosylation of critical cysteine residues (31) or indirectly by activation of guanylyl cyclase (32). Several kinases, including members of the JAK family, MAPKs, phosphoinositol 3-kinase, and Akt, have been reported to be sensitive to NO regulation (30), and it is likely that the MSC block T cell proliferation by acting upon multiple members of the IL-2 signaling pathway. One protein that is known to be affected by NO is JAK3, the kinase upstream of STAT5 in the IL-2R signaling pathway. In one study direct exposure of JAK3 to NO resulted in its oxidation and loss of enzymatic activity (33), and in another NO altered JAK3 activity through guanylate cyclase (34). Our results are consistent with either of these possibilities and further indicate that JAK1 is sensitive to regulation by NO. Because JAK1 and -3 are the first kinases in the IL-2 signaling pathway, it is also possible that all the effects of NO on components of the IL-2 pathway that we observed are due to the impaired activities of the JAKs.
Synthesis of NO in macrophages is catalyzed by iNOS, whose expression
is up-regulated by a number of cytokines, including IFN-, TNF-,
and IL-2 (35). Our data indicate that IFN- together
with a cell-mediated signal from activated splenocytes are necessary
for generating both the full suppressive activity of MSC and high
levels of NO secretion. These signals are produced by activated T
cells, and in the absence of an activation signal, T cells do not
stimulate NO production in MSC (A. Mazzoni, unpublished observation).
The cell surface interactions that increase NO secretion by MSC (but
not MH-S cells) have not been defined. We have observed that an
agonistic anti-CD40 mAb can synergize with IFN- to induce high
levels of NO secretion by the MSC, as described in other systems
(24, 36). However, ligation of CD40 by CD154 is not
required for suppression, because 1) a blocking anti-CD154 mAb was
unable to reverse suppression and 2) the MSC retained the capacity to
suppress Con A-induced proliferation of splenocytes from CD154 knockout
mice (A. Mazzoni, unpublished observations). Thus, we cannot say what
role, if any, CD40 plays in the generation of NO by the MSC lines.
An important question is whether the immortalized lines reflect normal myeloid suppressor lines. These lines were derived from cells that were clearly shown to be responsible for immunosuppression in vivo, and they retain their suppressive functions in culture. Moreover, they are phenotypically similar to splenic MSCs, although all MSC lines show small phenotypic differences from each other and from the primary MSC, possibly reflecting clonal variation within the bulk MSC population. However, the most convincing evidence that the MSC lines accurately reflect their normal counterparts comes from the iNOS knockout experiments. Our results predict that in cases where MSC mediate immunosuppression, such suppression would be lacking in iNOS-deficient mice. We found that cells with the MSC phenotype could be recruited to the spleens of iNOS-/- mice by repeated injections of GM-CSF, indicating that iNOS is not required for maturation or migration of these cells. However, as predicted, immunosuppression was not observed in the iNOS knockout mice, indicating that NO is an essential mediator of immunosuppression in vivo. Interestingly, in two autoimmune settings, experimental autoimmune encephalomyelitis (37, 38) and uveitis (39), the severity of the disease is increased in iNOS-deficient mice, suggesting once again the role of NO as negative feedback in the control of immune responses.
Angulo et al. (24) have recently described a population of natural suppressor cells isolated from bone marrow that is very similar to our MSC lines in phenotype and function. Therefore, it is likely that the MSC are bone marrow derived, and that they leave the bone marrow and migrate to the lymphoid organs upon receiving an appropriate signal, one of which is GM-CSF. Interestingly, the migration signal need not originate from the lymphoid tissues and may derive from immune stress in peripheral tissues. Once the MSC arrive at the lymphoid tissues they can serve as sensors of T cell activation, producing NO in response activation signals, which, in turn, impairs the IL-2 signaling pathway, blocking T cell proliferation. The MSC appear to be a functionally distinct subset, in that other myeloid lines, such as MH-S and J774, corresponding to normal alveolar and splenic macrophages, respectively, fail to produce NO in response to activated T cells and therefore fail to block proliferation. It is also possible that MSC differ from other myeloid lineages in their capacity to enter lymphoid organs in response to intense immune stimulation and that their distinct suppressive capacity reflects their immature phenotype. In either case the unique capacity of MSC to populate lymphoid organs and block T cell proliferation in response to activation signals suggests that they play an important role in controlling immune responses during times of heightened immune activity.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David M. Segal, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 4B36, Bethesda, MD 20892-1360. E-mail address: dave_segal{at}nih.gov
3 Abbreviations used in this paper: MSC, myeloid suppressor cell; Fas-L, Fas ligand; iNOS, inducible NO synthase; JAK, Janus kinase; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; NMMA, N-monomethyl arginine; SNAP, S-nitroso-N-acetylpenicillamine; wt, wild type.
Received for publication February 8, 2001. Accepted for publication November 13, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), after which
proliferation was measured by [3H]thymidine
incorporation. MH-S cells were used as a negative control (
). An
asterisk indicates proliferation in the absence of MSC or MH-S cells.
Similar results were obtained in 10 independent experiments.
B, Inhibition of peptide-specific responses. DO11.10
TCR-transgenic splenocytes were stimulated with 1 µM
OVA323–339 peptide in the presence or the absence of 3%
MSC or MH-S cells, as indicated. Incorporation of
[3H]thymidine was measured after 2 days. Data are
representative of seven different experiments. C,
Reversibility of suppression by MSC lines. BALB/c splenocytes were
stimulated with Con A for 3 days in the presence of no additional cells
(Con A), MSC-1 cells (Con A + MSC-1), or MH-S cells (Con A + MH-S).
Cells were cultured together for the entire stimulation period (filled
bars) or cultured together for 24 h (hatched bars), after which
splenocytes (nonadherent cells) were transferred (without washing) to a
fresh plate and cultured for 48 additional h. Cells were pulsed with
[3H]thymidine between days 2 and 3. Similar results were
obtained using MSC-2 cells. Data are representative of three different
experiments.
