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* Program in Molecular Immunology, Institute of Molecular Medicine and Genetics, and Departments of
Medicine and
Pediatrics, Medical College of Georgia, Augusta, GA 30912
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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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Recently, we have identified a natural immunosuppressive mechanism that prevents maternal T cell-mediated rejection of murine allogeneic fetuses (5, 6). These studies revealed that indoleamine 2,3-dioxygenase (IDO)3 activity normally contributes to maintenance of maternal T cell tolerance to fetal alloantigens because pregnant mice exposed to the IDO-specific inhibitor 1-methyl-tryptophan selectively rejected allogeneic fetuses, whereas syngeneic fetuses developed to term. The IDO mechanism is used by cultured human macrophages and dendritic cells to suppress in vitro T cell proliferation (7, 8). Several recent reports extend the potential biologic significance of the IDO mechanism to murine immunoregulatory CD8+ dendritic cells because their ability to suppress delayed-type hypersensitivity responses to tumor-associated peptides was blocked by exposure to 1-methyl-tryptophan (9, 10, 11). These data suggest that physiologic cells expressing IDO inhibit the generation of T cell responses in vivo. To explain these phenomena we hypothesized that proximity to cells expressing IDO inhibits T cell activation, possibly due to localized depletion of the essential amino acid tryptophan (7, 12). However, pharmacological approaches using an inhibitor of IDO activity to moderate T cell responses in vivo do not permit unequivocal mechanistic interpretations of the observed functional outcomes.
To complement pharmacological studies and to further address relationships between IDO activity and inhibition of T cell responses, we used two molecular genetic strategies to enhance IDO activity in transfected cell lines and in new strains of transgenic mice. In the current study, we directly evaluated the hypothesis that enhanced IDO activity in cells or tissues inhibits T cell responses. We report that the potency of allogeneic T cell responses elicited by IDO-transfected cells in vitro and in vivo was reduced significantly and that allogeneic T cell responses elicited after T cell adoptive transfer were less potent in recipient transgenic mice that overexpressed IDO in tissue microenvironments.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Full-length murine IDO cDNA was isolated from IFN-
-stimulated
RAW cells using standard RT-PCR and DNA cloning procedures. IDO cDNA
was cloned into pGEM T-Easy (Promega, Madison, WI) and completely
sequenced as described previously (13). A full-length
(1.2-kb) IDO cDNA fragment was digested with NotI enzyme and
cloned into NotI-cut pcDNA-3 cDNA expression vector
containing CMV promoter elements.
Cell lines and transfection
A total of 2 x 107 MC57G (ATCC No. CRL2295; gift from Dr. D. Moskofidis, Immag, MCG) or MB49 (gift from Dr. J. Leonard, Genetics Institute, Cambridge, MA) tumor cells were electroporated in the presence of 20 µg of linearized (NruI, BglI) pcDNA3-IDO or pcDNA-3 vector DNA using a setting of 320 mV/975 µF on an electroporation machine (Bio-Rad, Hercules, CA). Cells were cultured in IMDM medium supplemented with 10% FCS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) (IMDM complete) for 2 days to recover. Cells were harvested and plated in 96-well plates and cloned by limiting dilution in IMDM complete medium supplemented with 500 µM tryptophan and 1200 µg/ml G418 (Life Technologies, Rockville, MD). After 3 wk of selection, single clones were selected and analyzed.
Mice
All mice used for these studies were bred under specific pathogen-free conditions at the Medical College of Georgia. CBK transgenic mice express an H-2Kb transgene on the inbred CBA strain genetic background (14). BM3 (14) and A1 (15) transgenic mice are TCR-transgenic mice containing large cohorts of H-2Kb-specific CD8+ T cells or male (H-Y) Ag-specific CD4+ T cells on the inbred CBA strain genetic background. IDO-transgenic mice were generated by the staff of the Medical College of Georgia Transgenic Unit. Briefly, rDNA was microinjected into fertilized oocytes from inbred CBA/Ca strain parents using standard procedures. The DNA construct (MI) was prepared by ligating a murine full-length IDO cDNA into the cloning site of the cDNA expression cassette pDOI, which uses promoter elements from a murine MHC class II gene (16). Two transgenic (MI) founder mice were identified by Southern blot/in situ hybridization using a transgene specific probe, and mice were mated with CBA/Ca strain partners to establish two separate transgenic lineages, 31 and 33. Recipient mice for adoptive transfer experiments were generated by intercrossing MI transgenic (heterozygous) mice (line 33) with CBK transgenic (homozygous) mice and selecting double (MI x CBK) and single (CBK) transgenic littermates by genotypic analyses.
Western blotting
The pcDNA-3-IDO-transfected MC57G clones and vector-transfected clones were cultured to confluency. Total protein was extracted using RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 ng/ml PMSF, 66 ng/ml aprotinin). Samples were briefly sonicated (sonicator from Fisher Scientific, Pittsburgh, PA), avoiding heating and bubbling. Total protein was assayed using protein assay reagents (Pierce, Rockford, IL). Samples containing 50 µg of total protein lysate were separated on 12% SDS-PAGE. IDO protein was detected using a polyclonal Ab preparation from rabbits immunized with one of two synthetic murine IDO C-terminal peptides and anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA). Actin protein was detected using a mouse anti-actin mAb (Chemicon International, Temecula, CA) and anti-mouse IgG-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) using standard blotting and detection techniques. In vivo IDO expression was assessed by removing spleens from MI transgenic and control CBA mice and placing them in 2 ml of ice-cold RIPA buffer. Samples were homogenized using a Powergen-125 homogenizer (Fisher Scientific). Homogenized samples were processed and blotted as described above.
HPLC analysis
A total of 105 transfected MC57G or MB49
cells were seeded into 96-well plates in 200 µl of IMDM complete
medium containing G418 (1 mg/ml). After 3 days, 75 µl of culture
medium was removed and extracted with 1.4 ml of HPLC-grade methanol.
Precipitated proteins were removed by centrifugation and supernatants
were dried. Samples were reconstituted with 100 µl of HPLC-grade
deionized water, and 20 µl of sample was injected into a C18 column
(Luna C18(2), 250 x 4.6 mm, 5 µm; Phenomenex, Torrance,
CA). Samples were eluted with a gradient of water:acetonitrile (0–80%
acetonitrile) over 20 min. Standard concentration curves were prepared
using mixtures of kynurenine and tryptophan. IDO activities in spleens
of MI transgenic and CBA mice were assessed by culturing
106 splenocytes in 200 µl of IMDM complete
medium in 96-well plates for 3 days in the presence of IFN- (200
U/ml). Seventy-five microliters of culture medium was analyzed as
described above.
Mixed lymphocyte cultures
Semiconfluent IDO-construct or vector-only transfected MC57G cells were harvested, washed twice to remove G418, and seeded (5, 2.5, and 1.2 x 104 cells/well) into 96-well plates in 100 µl of IMDM complete medium with 2-ME (50 µM). Splenocytes from TCR transgenic BM3 mice (14) were stained with 1 µM CFSE (Molecular Probes, Eugene, OR) in PBS for 35 min at 37°C and washed twice with medium. A total of 105 BM3 splenocytes in 100 µl/well were used as responders.
Immunizations
CBA/Ca mice received a total of two, four, or six i.p. injections at biweekly intervals of MC57G-Vo, MC57G-24, MC57G-26 (single cell suspensions of 107 cells in 200 ul), or PBS (200 ul). Four days after the last injection, spleens were harvested. Lymphocytes were used as responders (5 x 105 cells/well) in an MLR with irradiated C57BL/6 splenocytes (3000 rad, 1.25 x 105 cells/well). Cultures were performed in triplicate wells using 96-well flat-bottom plates in 200 µl of IMDM complete medium with 2-ME (50 µM). Proliferation was assessed by adding [3H]thymidine (1 µCi/well) during the last 8–12 h of a 4-day culture. H-2b-specific cytolytic activity mediated by elicited T cells was assessed in chromium release assays using MC57 targets after coculture for 3 days with B6 splenocytes according to standard procedures (17).
Flow cytometric analyses
After coculture, cells were harvested, washed with PBS, and
stained with PE-conjugated anti-mouse CD8
Ab (BD PharMingen, San
Diego, CA) for 1 h. Cells were washed with PBS and analyzed using
a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). CD69
expression by T cells was assessed by staining cocultures with
PE-conjugated anti-mouse CD69 and APC-conjugated anti-mouse
CD8 (BD PharMingen) and subjecting them to flow cytometry.
Histograms and dot plots were produced from gated live lymphocyte
populations using light scatter parameters. Semiconfluent MC57G IDO-
and vector-transfected cells were harvested, stained with
FITC-conjugated mAb to mouse H-2Kb (Caltag
Laboratories, Burlingame, CA), and analyzed by flow cytometry as
described above.
Apoptosis of T cells was assessed by flow cytometry 72 h after coculture. Cells were harvested and stained with CD8-PE, propidium iodide, and FITC-conjugated annexin V (BD PharMingen). Annexin V histograms were generated for gated CD8+ T cell populations. TCR expression by BM3 CD8+ T cells 72 h after coculture was assessed by staining cells with anti-TCR clonotypic Ab (Ti98 biotin), CyChrome-streptavidin (BD PharMingen).
T cell adoptive transfer
A total of 107 splenocytes from BM3 mice
in 200 µl of PBS were injected into the tail veins of recipient mice.
Mice were sacrificed 96 h after adoptive transfer and single cell
suspensions were prepared from spleen. RBCs were removed by osmotic
lysis and cells were counted using a Coulter (Seattle, WA) counter.
Donor T cells were detected by staining cell preparations with
CD4-FITC, CD8-PE, and anti-clonotypic Ab (Ti98 conjugated with
biotin) as described previously (14). Stained cells were
analyzed by flow cytometry (FACSCalibur; BD Biosciences) using
CellQuest analysis software (BD Biosciences). A total of 2 x
107 splenocytes from A1 female TCR-transgenic
mice (15) were transferred into recipient male and female
mice, which were sacrificed 90 h later. A1 donor T cells were
detected using anti-CD4 and anti-V
8.1/2 mAbs (BD PharMingen)
as described previously (15). Donor T cell numbers in
spleen, mesenteric, and axillary lymph nodes were measured 20 h
after adoptive transfer to assess whether enhanced IDO expression
affected trafficking of donor splenocytes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MC57G fibrosarcoma cell lines (H-2b
haplotype) were selected for this study because they elicit potent
H-2Kb-specific T cell responses in vitro and do
not express IDO constitutively (data not shown). After electroporation
to introduce rDNA containing CMV promoter elements linked to murine IDO
cDNA sequences, we isolated a series of IDO-transfected MC57G clones
and screened them for IDO gene transcription, protein expression, and
enzyme activity (Fig. 1
). Cell lysates
prepared from IDO-transfected MC57G clones contained a single band
stained by rabbit polyclonal anti-murine IDO peptide-specific Abs,
which was the same size as IDO protein detected in murine epididymis
(Fig. 1A and data not shown). IDO-transfected cell lines
catabolized tryptophan and produced kynurenine, a metabolite produced
by oxidative catabolism of tryptophan (Fig. 1B).
IDO-transfected and vector-transfected clones expressed comparable
levels of surface H-2Kb Ag (Fig. 1C).
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IDO-transfected MC57G cells were cocultured with splenocytes from
BM3 TCR-transgenic mice that were prestained with the tracking dye
CFSE. BM3 mice contain large cohorts of
H-2Kb-specific CD8+ T cells
(14). After culture for 24–72 h, cocultures were stained
with anti-CD8 mAbs and analyzed by flow cytometry to assess the
total number of CD8+ T cells and the number of
times they had divided (Fig. 2). The
number of CD8+ T cells did not change after
72 h when cocultured with IDO-transfected MC57G cells (Fig. 2A). In contrast, CD8+ T cell numbers
increased 2.5-fold over the same period when cultured with
vector-transfected MC57G cells after an initial lag phase. Flow
cytometric analyses of CFSE staining profiles revealed that few
CD8+ T cells still exhibited CFSE staining
intensities comparable with undivided (naive) BM3 T cells (M1 marker;
Fig. 2B), indicating that most CD8+ T
cells had divided one to four times when cocultured with
vector-transfected MC57G cells for 72 h. In contrast, most
CD8+ T cells cocultured with IDO-transfected
MC57G clones for the same time exhibited CFSE staining intensities
comparable with undivided BM3 T cells. Based on these outcomes, we
estimated that 
80% of naive T cells did not divide at all in the
presence of IDO-transfected cells and that the rest (20%) divided
once only. Similar outcomes were observed when BM3 T cells were
cocultured with cloned IDO-transfected MB49 bladder carcinoma cells,
another H-2b-haplotype cell line (data not
shown). Thus, T cell proliferation was limited in the presence of
IDO-transfected tumor cells.
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We evaluated whether incubation with IDO-transfected tumor cells
induced expression of T cell activation markers. BM3 splenocytes were
cocultured with IDO-transfected and vector-transfected tumor cells,
stained with anti-CD69, anti-CD8, and anti-TCR (Ti98) mAbs,
and analyzed by flow cytometry (Fig. 3).
After 48 h, before proliferation began in control cultures, almost
all CD8+ T cells coexpressed CD69, irrespective
of whether they were cultured with IDO-transfected or
vector-transfected MC57G cells (Fig. 3, top panels).
Similarly, the ability of tumor cells to induce CD71 expression was not
affected by IDO expression (data not shown). We also evaluated TCR
expression levels on T cells using an anticlonotypic mAb (Ti98),
because TCR down-regulation occurs when naive (resting)
CD8+ T cells from BM3 transgenic mice are
activated (14). The number of cells expressing high levels
of TCR was reduced substantially and comparably after 72 h of
coculture with IDO-transfected and vector-transfected cells (Fig. 3, middle panels). These outcomes indicated that IDO expression
by tumor cells expressing Ag had no effect on their ability to activate
naive BM3 T cells by the criteria of inducing expression of activation
markers or TCR down-regulation.
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15–20%) of CD8+ T
cells stained with annexin V in cocultures with IDO-transfected or
vector-transfected MC57G tumor cells, whereas very few T cells
incubated alone stained with annexin V (Fig. 3
, lower
panels). Thus, coculture with IDO-transfected MC57G tumor cells
did not increase the proportion of T cells undergoing apoptosis.
However, coculture with MC57G tumor cells enhanced annexin V staining
of T cells before cell division occurred, probably due to tumor cell
growth and nutrient consumption. After 72 h, the proportions of
CD8+ T cells stained with annexin V were
substantially higher in cocultures with MC57G tumor cells and showed
wide variation in multiple experiments (40–70%, data not shown).
However, increased annexin V staining of T cells was also observed at
later times when BM3 splenocytes were incubated alone, showing that
spontaneous T cell death rates were also higher. Similar results were
obtained when T cells were stained with propidium iodide to assess T
cell viability in cocultures (data not shown). These outcomes revealed
that expression of IDO by Ag-presenting tumor cells did not cause T
cells to die faster via apoptosis, especially before T cells started
dividing in the first 48 h of coculture. After prolonged
coculture, it was difficult to detect specific effects of
IDO-transfectants on T cell viability due to enhanced annexin V
staining in all cultures. IDO-transfected tumor cells inhibit alloreactive T cell responses in vivo
CBA (H-2k haplotype) mice were injected
twice weekly (for 1–3 wk) with allogeneic IDO-transfected (clone 24 or
26) or vector-transfected MC57G (H-2b) tumor
cells to assess whether IDO-transfected cells moderated in vivo T cell
responses to H-2b alloantigens.
H-2b-specific T cell responses were assessed by
coculturing splenocytes from immunized mice (responders) with
irradiated splenocytes from C57BL/6 (H-2b
stimulators) mice (Fig. 4). Elicited
H-2b-specific proliferative responses were
significantly less potent when responders originated from mice injected
with IDO-transfected clone 26 tumor cells, compared with responses from
mice injected with PBS (p 
0.02). Similarly,
IDO-transfectant clone 24 elicited less potent
H-2b-specific responses after two or four
injections (p 0.02). After six injections of
clone 24 cells, proliferative responses were not significantly reduced
relative to control mice injected with PBS, although the trend for
reduced responses in IDO-transfectant-treated mice was still apparent
in this group. Assays for T cell cytolytic function executed in
parallel after 3 days of mixed lymphocyte culture (with irradiated B6
splenocytes) revealed similar trends in reduced killing of MC57 target
cells when elicited T cells originated from mice exposed to
IDO-transfected MC57 cells (data not shown). However, exposure to
IDO-transfectants did not completely abrogate
H-2b-specific cytolytic functions, indicating
that immunization with IDO-transfected MC57G tumor cells did not
eliminate all H-2b-specific cytolytic precursors
in mice. These outcomes suggest that the pool of
H-2b-specfic T cells was reduced when CBA mice
were exposed to IDO-transfected MC57G cells before mixed lymphocyte
culture. This effect was not an innate quality of MC57G cells per se
because H-2b-specific proliferative responses
were comparable when responder splenocytes originated from naive
(PBS-treated) CBA mice or from mice exposed to vector-transfected MC57G
tumor cells. This suggested that IDO expression by MC57G cells was a
critical factor and that MC57G cells, in common with other tumor cell
lines, were not very immunogenic. These outcomes indicated that
IDO-expressing H-2b tumor cells reduced the
potency of T cell responses to H-2b alloantigens
substantially below the level elicited when responders originated from
naive CBA mice.
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To further evaluate whether increased IDO activity inhibited in
vivo T cell responses, we adapted an experimental system in which
H-2Kb-specific CD8+ T cells
from donor BM3 TCR transgenic mice mounted potent responses to
recipient H-2Kb alloantigen after adoptive
transfer (5, 14). For these experiments, we used
IDO-transgenic (MI) mice expressing increased IDO activity due to
expression of murine IDO cDNA linked to promoter elements derived from
a murine MHC class II gene (see Materials and Methods).
Phenotypic characterization of splenocytes isolated from MI transgenic
mice lines 31 and 33 revealed increased amounts of IDO protein (Fig. 1D) and IDO enzyme activity (Fig. 1E). Recipient
mice expressing H-2Kb alloantigen on the MI
transgenic background were generated by intercrossing MI line 33, which
expressed the highest amount of IDO protein in spleen (Fig. 1D), with H-2Kb (CBK) transgenic mice
and identifying double- (MI x CBK) and single-transgenic (CBK)
offspring by genotype analysis. BM3 splenocytes were injected into
these recipients and numbers of donor T cells
(Ti98+CD8+) present in
recipient spleen were assessed by flow cytometry.
Ninety-six hours after adoptive transfer, numbers of donor BM3
CD8+ T cells detected in spleens of
double-transgenic (MI x CBK) recipients were substantially less
(66%) than in spleens of CBK recipients (Fig. 5A). Comparable numbers of BM3
T cells (stained with the tracking dye CFSE) were detected in these
lymphoid tissues from recipient CBA and MI transgenic mice, indicating
that enhanced IDO expression did not alter the anatomical distribution
of injected T cells (data not shown). As expected, adoptive transfer of
BM3 splenocytes into CBA recipients elicited no T cell responses.
Similar outcomes were obtained when male (H-Y) Ag-specific
CD4+ T cells from A1 TCR transgenic donors
(15) were transferred into male and female MI transgenic
mice (Fig. 5B). Approximately 33% fewer H-Y-specific donor
CD4+ T cells were elicited in MI transgenic male
mice expressing enhanced IDO activity. These outcomes indicated that
enhanced IDO activity in MI transgenic mice inhibited
alloantigen-specific T cell responses after T cell adoptive transfer
in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The majority of T cells cultured with IDO-transfected MC57G tumor cells did not divide, even though they exhibited features of activated T cells, because expression of CD69, CD71 was induced and TCR levels were down-regulated. These outcomes suggest that the ability of MC57G tumor cells to deliver signals through the TCR/CD3 complex was not affected by IDO expression. This further implies that regulation of T cell proliferation in the presence of IDO-transfected tumor cells occurred after the majority of T cells entered the cell cycle and before completion of the first cell cycle. These outcomes with IDO-transfected cells recapitulate previous data showing that human macrophages expressing IDO blocked T cell cycle progression (7). However, previous studies with human myeloid cells expressing IDO relied exclusively on pharmacological inhibition of IDO activity. The studies reported here extend previous observations by showing that genetic manipulations leading to IDO expression in APCs also lead to inhibition of elicited T cells responses.
We could not assess whether T cells entered S-phase when cocultured with IDO-transfected MC57G tumor cells because background incorporation of thymidine by tumor cells was not reliably blocked by irradiation or use of mitomycin C in our experimental system (data not shown). However, data from experiments in which T cells were activated in the absence of the essential amino acids tryptophan or isoleucine/leucine showed that the ability of human and murine T cells to enter S-phase is exquisitely and selectively dependent on the presence of tryptophan midway through the G0-S phase transition (data not shown). The precise mechanisms whereby T cell cycle progression depends on the availability of tryptophan remain obscure. Nevertheless, the data we report here are the first direct test of the hypothesis that genetic manipulations to enhance IDO expression in APCs lead to inhibition of T cell responses.
The eventual outcomes of physiologic immune responses depend critically on the functional status of dendritic cell subsets that either promote or suppress T cell responses (18, 19). Though much is known about mechanisms used by dendritic cells to generate effector T cells, less is known about critical processes they use to suppress or deviate T cell responses after Ag-specific activation. Many recent reports document a role for dendritic cells in immunoregulatory, rather than immunostimulatory, phenomena (3, 4, 20). However, details of the cellular, molecular, and biochemical mechanisms underlying these immunoregulatory phenomena remain obscure. Puccetti and colleagues (10, 11) have shown that murine splenic CD8+ dendritic cells are potent suppressors of delayed-type hypersensitivity responses to tumor-associated peptides presented by immunogenic CD8- dendritic cells. The immunosuppressive properties of these CD8+ dendritic cells were blocked by 1-methyl-tryptophan, the same competitive inhibitor of IDO enzyme activity that blocked maternal T cell tolerance of fetal allografts during murine pregnancy (5, 6). More recently, this group has shown that murine macrophages and dendritic cells express IDO and that CD8+ dendritic cells, but not macrophages, express IDO constitutively (21). Moreover, CD8+ dendritic cells increased the rate of CD4+ T cell apoptosis, providing a potential mechanistic explanation for the immunosuppressive effects mediated by this dendritic cell subset in vivo (11, 21). In contrast, we observed no increase in the rate of T cell apoptosis when BM3 T cells were cocultured with IDO-transfected cells, although tumor cell growth in cocultures enhanced T cell apoptosis and precluded rigorous investigation at later times. Similarly, human T cells cultured with immunosuppressive macrophages or dendritic cells did not exhibit enhanced apoptosis (7, 8). These discrepancies might arise due to differences in the experimental systems used to assess the impact of IDO activity on T cell activation. For example, CD8+ T cells from BM3 TCR transgenic mice and human T cells may be inherently more resistant than CD4+ T cell clones to apoptosis. In addition, MC57G tumor cells, unlike CD8+ dendritic cells, may not deliver apoptotic signals to T cells. Nevertheless, the ability to inhibit T cell clonal expansion after activation, with or without subsequent apoptosis, provides a mechanistic explanation for the observed link between physiological expression of IDO and suppression of T cell responses in vivo. Clonal expansion of Ag-specific T cells is an obligatory step in generating effective physiologic immune responses due to the very low frequency of Ag-specific T cells present in naive T cell repertoires. Hence, the ability of APCs expressing IDO to block or inhibit T cell clonal expansion during the afferent phase would have a major impact on the potency of immune responses in vivo. In summary, data presented in this report suggest that genetic manipulations to force IDO expression may result in enhanced ability of cells and tissues to suppress T cell responses in vivo after transplantation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew L. Mellor, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, CA 2006, Augusta, GA 30912-2000. E-mail address: amellor{at}mail.mcg.edu
3 Abbreviations used in this paper: IDO, indoleamine 2,3-dioxygenase.
4 D. H. Munn, M. D. Sharma, J. R. Lee, S. C. Antonia, R. Burgess, C. Slingluff, and A. L. Mellor. Immunoregulatory dendritic cells expressing indoleamine, 2,3 dioxygenase associated with human tumors. Submitted for publication.
Received for publication November 7, 2001. Accepted for publication February 1, 2002.
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inhibits presentation of a tumor/self peptide by CD8- dendritic cells via potentiation of the CD8+ subset. J. Immunol. 165:1357.+ dendritic cells expressing indoleamine 2,3-dioxygenase. J. Immunol. 167:708.+ dendritic cells. Int. Immunol. 14:65.This article has been cited by other articles:
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A. Boasso, J.-P. Herbeuval, A. W. Hardy, S. A. Anderson, M. J. Dolan, D. Fuchs, and G. M. Shearer HIV inhibits CD4+ T-cell proliferation by inducing indoleamine 2,3-dioxygenase in plasmacytoid dendritic cells Blood, April 15, 2007; 109(8): 3351 - 3359. [Abstract] [Full Text] [PDF]
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A. Curti, S. Pandolfi, B. Valzasina, M. Aluigi, A. Isidori, E. Ferri, V. Salvestrini, G. Bonanno, S. Rutella, I. Durelli, et al. Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25- into CD25+ T regulatory cells Blood, April 1, 2007; 109(7): 2871 - 2877. [Abstract] [Full Text] [PDF]
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L. Zhu, F. Ji, Y. Wang, Y. Zhang, Q. Liu, J. Z. Zhang, K. Matsushima, Q. Cao, and Y. Zhang Synovial Autoreactive T Cells in Rheumatoid Arthritis Resist IDO-Mediated Inhibition J. Immunol., December 1, 2006; 177(11): 8226 - 8233. [Abstract] [Full Text] [PDF]
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X. Zheng, J. Koropatnick, M. Li, X. Zhang, F. Ling, X. Ren, X. Hao, H. Sun, C. Vladau, J. A. Franek, et al. Reinstalling Antitumor Immunity by Inhibiting Tumor-Derived Immunosuppressive Molecule IDO through RNA Interference J. Immunol., October 15, 2006; 177(8): 5639 - 5646. [Abstract] [Full Text] [PDF]
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S. Agaugue, L. Perrin-Cocon, F. Coutant, P. Andre, and V. Lotteau 1-Methyl-Tryptophan Can Interfere with TLR Signaling in Dendritic Cells Independently of IDO Activity J. Immunol., August 15, 2006; 177(4): 2061 - 2071. [Abstract] [Full Text] [PDF]
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G. D. Basu, T. L. Tinder, J. M. Bradley, T. Tu, C. L. Hattrup, B. A. Pockaj, and P. Mukherjee Cyclooxygenase-2 Inhibitor Enhances the Efficacy of a Breast Cancer Vaccine: Role of IDO J. Immunol., August 15, 2006; 177(4): 2391 - 2402. [Abstract] [Full Text] [PDF]
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M. S. von Bergwelt-Baildon, A. Popov, T. Saric, J. Chemnitz, S. Classen, M. S. Stoffel, F. Fiore, U. Roth, M. Beyer, S. Debey, et al. CD25 and indoleamine 2,3-dioxygenase are up-regulated by prostaglandin E2 and expressed by tumor-associated dendritic cells in vivo: additional mechanisms of T-cell inhibition Blood, July 1, 2006; 108(1): 228 - 237. [Abstract] [Full Text] [PDF]
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H. Fujigaki, K. Saito, F. Lin, S. Fujigaki, K. Takahashi, B. M. Martin, C. Y. Chen, J. Masuda, J. Kowalak, O. Takikawa, et al. Nitration and Inactivation of IDO by Peroxynitrite J. Immunol., January 1, 2006; 176(1): 372 - 379. [Abstract] [Full Text] [PDF]
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R. Potula, L. Poluektova, B. Knipe, J. Chrastil, D. Heilman, H. Dou, O. Takikawa, D. H. Munn, H. E. Gendelman, and Y. Persidsky Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis Blood, October 1, 2005; 106(7): 2382 - 2390. [Abstract] [Full Text] [PDF]
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 J. Tsukada, A. Ozaki, T. Hanada, T. Chinen, R. Abe, A. Yoshimura, and M. Kubo The role of suppressor of cytokine signaling 1 as a negative regulator for aberrant expansion of CD8{alpha}+ dendritic cell subset Int. Immunol., September 1, 2005; 17(9): 1167 - 1178. [Abstract] [Full Text] [PDF]
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C. M. Cham and T. F. Gajewski Glucose Availability Regulates IFN-{gamma} Production and p70S6 Kinase Activation in CD8+ Effector T Cells J. Immunol., April 15, 2005; 174(8): 4670 - 4677. [Abstract] [Full Text] [PDF]
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S. Glennie, I. Soeiro, P. J. Dyson, E. W.-F. Lam, and F. Dazzi Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells Blood, April 1, 2005; 105(7): 2821 - 2827. [Abstract] [Full Text] [PDF]
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A. Boasso, J.-P. Herbeuval, A. W. Hardy, C. Winkler, and G. M. Shearer Regulation of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA-synthetase by CTLA-4-Fc in human CD4+ T cells Blood, February 15, 2005; 105(4): 1574 - 1581. [Abstract] [Full Text] [PDF]
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A. L. Mellor, P. Chandler, B. Baban, A. M. Hansen, B. Marshall, J. Pihkala, H. Waldmann, S. Cobbold, E. Adams, and D. H. Munn Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase Int. Immunol., October 1, 2004; 16(10): 1391 - 1401. [Abstract] [Full Text] [PDF]
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 C. Hucke, C. R. MacKenzie, K. D. Z. Adjogble, O. Takikawa, and W. Daubener Nitric Oxide-Mediated Regulation of Gamma Interferon-Induced Bacteriostasis: Inhibition and Degradation of Human Indoleamine 2,3-Dioxygenase Infect. Immun., May 1, 2004; 72(5): 2723 - 2730. [Abstract] [Full Text] [PDF]
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D. H. Munn, M. D. Sharma, and A. L. Mellor Ligation of B7-1/B7-2 by Human CD4+ T Cells Triggers Indoleamine 2,3-Dioxygenase Activity in Dendritic Cells J. Immunol., April 1, 2004; 172(7): 4100 - 4110. [Abstract] [Full Text] [PDF]
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 K. A. Swanson, Y. Zheng, K. M. Heidler, T. Mizobuchi, and D. S. Wilkes CDllc+ Cells Modulate Pulmonary Immune Responses by Production of Indoleamine 2,3-Dioxygenase Am. J. Respir. Cell Mol. Biol., March 1, 2004; 30(3): 311 - 318. [Abstract] [Full Text] [PDF]
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N. A. Begum, K. Ishii, M. Kurita-Taniguchi, M. Tanabe, M. Kobayashi, Y. Moriwaki, M. Matsumoto, Y. Fukumori, I. Azuma, K. Toyoshima, et al. Mycobacterium bovis BCG Cell Wall-Specific Differentially Expressed Genes Identified by Differential Display and cDNA Subtraction in Human Macrophages Infect. Immun., February 1, 2004; 72(2): 937 - 948. [Abstract] [Full Text] [PDF]
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A. L. Mellor, B. Baban, P. Chandler, B. Marshall, K. Jhaver, A. Hansen, P. A. Koni, M. Iwashima, and D. H. Munn Cutting Edge: Induced Indoleamine 2,3 Dioxygenase Expression in Dendritic Cell Subsets Suppresses T Cell Clonal Expansion J. Immunol., August 15, 2003; 171(4): 1652 - 1655. [Abstract] [Full Text] [PDF]
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 U. Grohmann, F. Fallarino, R. Bianchi, C. Orabona, C. Vacca, M. C. Fioretti, and P. Puccetti A Defect in Tryptophan Catabolism Impairs Tolerance in Nonobese Diabetic Mice J. Exp. Med., July 7, 2003; 198(1): 153 - 160. [Abstract] [Full Text] [PDF]
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A. L. Mellor and D. H. Munn Tryptophan Catabolism and Regulation of Adaptive Immunity J. Immunol., June 15, 2003; 170(12): 5809 - 5813. [Full Text] [PDF]
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A. M. Mackler, E. M. Barber, O. Takikawa, and J. W. Pollard Indoleamine 2,3-Dioxygenase Is Regulated by IFN-{gamma} in the Mouse Placenta During Listeria monocytogenes Infection J. Immunol., January 15, 2003; 170(2): 823 - 830. [Abstract] [Full Text] [PDF]
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 D. H. Munn, M. D. Sharma, J. R. Lee, K. G. Jhaver, T. S. Johnson, D. B. Keskin, B. Marshall, P. Chandler, S. J. Antonia, R. Burgess, et al. Potential Regulatory Function of Human Dendritic Cells Expressing Indoleamine 2,3-Dioxygenase Science, September 13, 2002; 297(5588): 1867 - 1870. [Abstract] [Full Text] [PDF]
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and 
) or vector
transfectants (
) over time. B, CFSE staining profiles
of viable CD8+ cells after coculturing BM3 splenocytes
alone (upper left panel) or with MC57G
vector-transfected cells (lower left panel) or
IDO-transfected clones (right panels) for 72 h.
Bars indicate percentage of undivided (M1, CFSEhigh) and
divided (M2, CFSElow) CD8+ T cells. Data
are representative of >10 experiments. Similar results were obtained
when cocultures were analyzed at slightly earlier times (66 h).
