Cell Transplantation, Vol. 20, pp. 941–954, 2011 0963-6897/11 $90.00 + .00Printed in the USA. All rights reserved. DOI: 10.3727/096368910X540621Copyright 2011 Cognizant Comm. Corp. E-ISSN 1555-3892

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In Vivo Induction of Myeloid Suppressor Cells and CD4+Foxp3+ TRegulatory Cells Prolongs Skin Allograft Survival in Mice

D. Adeegbe,* P. Serafini,*† V. Bronte,‡§ A. Zoso,* C. Ricordi,* and L. Inverardi*¶

*Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, FL, USA†Department of Microbiology and Immunology, Sylvester Comprehensive Cancer Center, Miller School of Medicine,

University of Miami, Miami, FL, USA‡Verona University Hospital and Department of Pathology, Immunology Section, University of Verona, Verona, Italy

§Istituto Oncologico Veneto, Padova, Italy¶Division of Endocrinology Diabetes and Metabolism, Miller School of Medicine, University of Miami, Miami, FL, USA

Natural CD4+Foxp3+ T regulatory (Treg) cells can promote transplantation acceptance across major histo-compatibility complex (MHC) barriers, while myeloid-derived suppressor cells (MDSCs) inhibit effector T-cell responses in tumor-bearing mice. One outstanding issue is whether combining the potent suppressivefunction of MDSCs with that of Treg cells might synergistically favor graft tolerance. In the present study,we evaluated the therapeutic potential of MDSCs and natural Treg cells in promoting allograft tolerance inmice by utilizing immunomodulatory agents to expand these cells in vivo. Upon administration of recombi-nant human granulocyte-colony stimulating factor (G-CSF; Neupogen), or interleukin-2 complex (IL-2C),Gr-1+CD11b+ MDSCs or CD4+Foxp3+ Treg cells were respectively induced at a high frequency in the periph-eral lymphoid compartments of treated mice. Interestingly, induced MDSCs exhibited a more potent sup-pressive function in vitro when compared to MDSCs from naive mice. Furthermore, in vivo coadministrationof Neupogen and IL-2C induced MDSCs at percentages that were higher than those seen when either agentwas administered alone, suggesting an additive effect of the two drugs. Although treatment with either IL-2C or Neupogen led to a significant delay of MHC class II disparate allogeneic donor skin rejection, thecombinatorial treatment was superior to either alone. Importantly, histological assessment of surviving graftsrevealed intact morphology and minimal infiltrates at 60 days posttransplant. Collectively, our findingsdemonstrate that concurrent induction of MDSCs and Tregs is efficacious in downmodulating alloreactiveT-cell responses in a synergistic manner and highlight the therapeutic potential of these naturally occurringsuppressive leukocytes to promote transplantation tolerance.

Key words: Transplantation; Tolerance; Suppression; Myeloid-derived suppressor cells (MDSCs)

INTRODUCTION within the tumor microenvironment and either persis-tence or progression of tumor growth (29,33). In humanstudies, equivalent populations of cells have also beenTumorigenesis is a complex phenomenon that has

been studied utilizing animal models with the attempt reported in cancers such as head and neck squamous cellcarcinoma (3).to understand its pathogenesis. Once established, tumors

utilize several strategies to evade recognition by the im- Depending on the model of investigation, MDSCshave been described as expressing various cellular mark-mune system (8,42). One such strategy is the recruitment

of myeloid-derived suppressor cells (MDSCs) through ers (15). In mice, they are typically contained in theCD11b+ leukocyte subset and express varying levels ofgranulocyte-macrophage colony stimulating factor (GM-

CSF) and granulocyte-colony stimulating factor (G- Gr-1 (Ly6G/C), a cell surface molecule with heteroge-neous distribution that is used to delineate their differen-CSF) that is produced by tumor cells (15,42). The preva-

lence of these suppressor cells in and around the tumor tiation stages (7,50). They also express F4/80 (23), andinterleukin-4 receptor α (IL-4Rα) (12,16). Unlike con-microenvironment consequently contributes to tumor es-

cape, as several animal models have demonstrated a cor- ventional CD11b+ macrophages, these cells express lowlevels of major histocompatibility complex (MHC) classrelation between increased frequencies of these cells

Received March 30, 2010; final acceptance October 4, 2010. Online prepub date: November 5, 2010.Address correspondence to L. Inverardi, Diabetes Research Institute, Miller School of Medicine, 1450 NW 10th avenue, Miami, FL 33136, USA.Tel: 305-243-5347; Fax: 305-243-4404; E-mail: LInverar@med.miami.edu

941

942 ADEEGBE ET AL.

II, CD40, and B7.1/2 costimulatory molecules, a pheno- Antibodies and Fluorescent Activated Cell Sorter(FACS) Analysistype that is distinct from that of immature and mature

antigen-presenting cells (4,41,51). The potent suppress- FITC-conjugated monoclonal antibodies (mAbs) toive activity of MDSCs is revealed by in vitro studies in CD4 (Gk1.5), Gr-1 (RB6-8C5), CD40 (HM40-3), CD80which they effectively inhibit the proliferation of either (GL1), IA/E (NIMR-4), F4/80 (BM8), phycoerythrinantigen- or mitogen-activated T lymphocytes (16,43). (PE)-conjugated mAbs to Gr-1 (Ly6C/6G), CD11b (M1/

As the understanding of the mechanisms by which 70), CD124, peridinin chlorophyll protein complexMDSCs suppress effector immune responses is gradu- (PerCP)-anti Gr-1 (RB6-8C5), allophycocyanin (APC)-ally unfolding, certain key proteins have been identified conjugated CD11b (M1/70), and CD25 (PC61) were allas crucial to their suppressive function. For example, ex- purchased from BD Pharmingen (San Diego, CA). PE-pression of IL-4Rα is paramount to arm MDSCs with anti-Foxp3 (FJK16s) was obtained from eBiosciencesthe ability to function as suppressors, with the most po- (San Diego, CA) and was used in intracellular FACStent cells residing within the Ly-6C-high subset (11,16). analysis following the manufacturer’s instructions.In addition, these cells express high levels of nicotin- FACS analysis was performed using FACs Calibur andamide adenine dinucleotide phosphate (NADPH), phago- CellQuest software. For cultured cells, 7AAD dye wascytic oxidase (heme oxygenase-1), nitric oxide synthase utilized to exclude dead cells from the analysis. Typi-(NOS2), and arginase-1, enzymes that are involved in cally 30,000–50,000 events were collected per sample.processes that lead to the production of nitric oxide

Cell Purification and In Vitro Studies(NO), peroxynitrite, reactive oxygen species (ROS), orto the depletion of the semiessential amino acid L-argi- To isolate unfractionated T cells, spleen cell suspen-nine (9,24,25,46). The resulting metabolites affect T-cell sions were maintained in complete RPMI-1640 mediumintracellular signaling by lowering the CD3-ζ chain, supplemented with 5% fetal calf serum (FCS). Cellsblocking IL-2R activated pathways, changing the affin- were washed twice with Hank’s balanced salt solutionity of the T-cell receptor (TCR)/MHC peptide complex, (HBSS) and incubated with Thy1.2 magnetic-activatedor inducing apoptosis (6,13,34,36,38,40). cell separation (MACS) microbeads, respectively, in

Although there are extensive studies on the role of HBSS containing 5% FCS and 2 mM EDTA for 15 minMDSCs in downmodulating immunity in tumor models, at 4°C. After incubation, cells were positively selectedlittle is known about their role in nontumor settings. on MS MACS separation columns (Miltenyi, Biotec,While it is well established that natural CD4+Foxp3+ Auburn, CA). MDSCs from normal or Neupogen-treatedTreg cells are efficacious at promoting tolerance across mice were purified from spleen cell suspensions afterMHC barriers in bone marrow and solid tissue trans- incubating with PE-conjugated Gr-1 and anti-PE mi-plantation settings, there are scant data about the role crobeads using a similar protocol as above. For carboxy-of MDSCs in modulating time and/or severity of graft fluorescein succinimidyl ester (CFSE) dilution assays,rejection. In recent reports, MDSCs were implicated in purified T cells were labeled with CFSE at 2.5 µM con-animal models of kidney allograft tolerance (12) and centration and incubated at 37°C for 10 min. Cells (1 ×were also found to delay rejection of antigen-mismatched 106) were cultured with 1 × 106 antigen-presenting cellsskin grafts (10,53). Given their potent inhibitory effect (APCs) (T-cell depleted, mitomycin-treated splenocytes)in tumor-bearing mice, one outstanding issue is whether in the presence or absence of 50 µl anti-CD3 supernatantcombining the potent suppressive function of both natu- (2C11) in 24-well plates for 48 h. MDSCs were thenral CD4+Foxp3+ Treg cells and MDSCs might favor added at 1:1 ratio to responding T cells. In other experi-transplantation tolerance. Thus, the present study was ments, purified T cells were isolated from naive BALB/designed to evaluate the therapeutic potential of MDSCs c mice, or mice treated with Neupogen and/or IL-2 com-and natural Treg cells in promoting allograft tolerance plex and then incubated with C57BL/6 APCs (1 × 106)by utilizing immunomodulatory agents to expand these in the presence or absence of MDSCs (as indicated) ob-cells in vivo. tained from Neupogen-treated mice. [3H]Thymidine was

added to cultures in the last 18 h of a 96-h culture.MATERIALS AND METHODS

In Vivo Mouse StudiesMice

C57BL/6, BALB/c, C3H/HeJ, and B6(C)-H-2- Mice were injected daily with recombinant human G-CSF (Neupogen, Amgen Inc., Thousand Oaks, CA at 5AB1bm12/KhEgj were obtained from Jackson Labora-

tory (Bar Harbor, ME). All experiments were approved µg/mouse) subcutaneously (SC) once a day for 5–14days unless otherwise specified. G-CSF was diluted inby the Institutional Animal and Use Committee

(IACUC) at the University of Miami and performed fol- 5% dextrose and 1% mouse serum albumin (vehicle).As control, mice received vehicle or PBS. IL-2 complexlowing the IACUC guidelines.

MYELOID SUPPRESSOR CELLS PROMOTE TOLERANCE 943

(IL-2C) containing 0.5 µg recombinant mouse IL-2 regimen was then utilized to determine whether chronictreatment affects the duration of MDSCs induction and(eBioscience, San Diego, CA) and 5 µg rat anti-mouse

IL-2 (JES6-12A1, R&D Systems, MN) in 100 µl PBS persistence. Upon analysis, elevated levels of MDSCscould be observed in the spleen of treated mice for morewas injected into the peritoneal cavity of each mouse

for 5 consecutive days and twice a week thereafter (48) than 1 week after cessation of the 14-day administrationregimen (Fig. 1E). These data indicate that Gr-1+CD11b+throughout the duration of the study for skin graft recipi-

ents. Skin grafting was performed as previously de- cells are effectively induced in mice using recombinanthuman G-CSF and highlight how chronic administrationscribed (1). Grafts were monitored every other day and

scored as rejected when >80% or more of the original can enhance MDSCs persistence.graft tissue had become necrotic as assessed by visual

The Phenotype of Neupogen-Induced MDSCs Isexamination.Largely Indistinguishable From That in Naive Mice

Histological Assessment In this study, it remains a possibility that MDCSs in-Donor skin tissues were harvested from recipient duced with Neupogen exhibit a distinct phenotype that

mice at indicated time points posttransplant and fixed in is different from that seen in naive mice. To investigateparaformaldehyde. Tissue sections (5 µm thick) were this issue, MDSCs isolated from the spleen of Neupo-then stained with hematoxylin-eosin after paraffin em- gen-treated mice were analyzed. We show that they ex-bedding. press little to no MHC class II molecules, B7.1 (CD80)

costimulatory molecule, and moderate level of F4/80,Statistical Analysis similar to MDSCs from tumor-free normal mice (Fig.

Data are expressed as mean ± SEM unless otherwise 2A). However, a marginal increase was noted for CD40indicated. A two-tailed Student’s t-test was used to de- expression on induced MDSCs relative to their naivetermine the significance between two groups as speci- counterparts. In addition, IL-4Rα, which has been corre-fied in the results. Values of p < 0.05 were considered lated with suppressive function (16,42), was higher onsignificant. Bonferroni correction test was performed for MDSCs from Neupogen-treated mice than that onall compared groups at indicated confidence levels to MDSC from naive mice (Fig. 2A, B, Table 1), suggest-further evaluate the validity of the significance in differ- ing that Neupogen not only expanded MDSCs but alsoences as measured by the Student’s t-test. induced their activation. We also found that expression

of the CXCR2 and CCR4 chemokine receptors wasRESULTS

much higher on MDSC from treated mice, consistentIn Vivo Administration of Human Recombinant G-CSF with the idea of systemic recruitment and trafficking that(Neupogen) Induces MDSCs in Mice result in higher frequencies at multiple peripheral sites

(data not shown). These data suggest that Neupogen ad-To begin to investigate whether these suppressor cellsare efficacious in promoting tolerance to alloantigens, ministration not only increases MDSCs number but

could also favor their maturation towards a more sup-we treated mice with recombinant human G-CSF (Neu-pogen), the administration of which has been reported pressive phenotype.to increase the fraction of cells that express Gr-1, one of

Inhibition of T-Cell Response by Neupogen-Induced MDSCsthe phenotypic markers of MDSCs (37). A short course(5 days) of treatment with different doses of Neupogen We then sought to determine whether MDSCs from

Neupogen-treated mice are functionally capable of sup-led to induction of Gr-1+CD11b+ cells in the spleen ofBALB/c recipient mice, which was remarkable at the 5 pressing polyclonal and alloreactive T-cell responses.

Unfractionated T cells stimulated with anti-CD3 prolif-and 10 µg dosage 1 day posttreatment (Fig. 1A–C). Thiseffect was transient, as only modest increase above con- erate extensively as revealed by CFSE dilution of both

the CD4+ and CD8+ subsets within 3 days of culturetrol levels in the MDSC frequency was noted at 3 and 7days after treatment (Fig. 1A, C). From these data, we (Fig. 3A). This proliferative response is inhibited upon

addition of MDSCs isolated from Neupogen-treateddetermined that a dosage of 5 µg/recipient was optimalat inducing MDSCs and was chosen for subsequent mice with many responder cells (�38%) having under-

gone only one or two rounds of cell division (Fig. 3A).treatments. To further evaluate the efficacy of in vivoNeupogen administration, we analyzed, concurrently, The addition of spleen cells, in contrast, failed to yield

similar outcomes, demonstrating that the MDSCs sup-multiple tissues including peripheral blood, spleen, bonemarrow (Fig. 1D), and lymph nodes (data not shown). pression is unlikely due to nonspecific depletion of

growth factors in the cultures. To further evaluateSimilar to the spleen, significant increases in the fractionof Gr-1+CD11b+ MDSCs were observed. As this induc- MDSC suppressive function, we set up a mixed leuko-

cytes reaction (MLR) in the presence or absence oftion appears to be short lived, a 2-week daily injection

944 ADEEGBE ET AL.

Figure 1. Kinetics of myeloid-derived suppressor cell (MDSC) induction in Neupogen-treated mice. BALB/c mice were injectedSC with recombinant human granulocyte-colony stimulating factor (G-CSF; Neupogen) at 1, 5, and 10 µg (A) or 5 µg/mouse(B–E) for 5 (A–D) or 14 (E) consecutive days. Control mice received vehicle or PBS. At different time points as indicated in thegraphs, mice were sacrificed and flow cytometry analysis was then performed to evaluate the frequency of MDSCs, identified bycoexpression of CD11b and Gr-1. (A) Frequency of MDSCs in the spleen of mice treated with the indicated doses of Neupogen 1,3, and 7 days after the last injection. (B) Representative fluorescence-activated cell sorting (FACS) plot 1 day posttreatment in thespleens of mice that received 5 µg of Neupogen (lower dot plot) or of control mice that received PBS (upper dot plot). (C) Absolutenumbers of Gr-1+CD11b+ cells in the spleen of control versus Neupogen-treated mice at 1, 3, and 7 days posttreatment. (D)Summary of MDSC frequency in the peripheral blood (PB), bone marrow (BM), and the spleen (SPLN) of treated mice 1, 3, and7 days after the last treatment. (E) Summary of MDSC frequency 1–2 weeks after the last injection following chronic (14 consecu-tive days) administration of Neupogen. Data represent the mean ± SEM for 2–5 mice/time point.

MYELOID SUPPRESSOR CELLS PROMOTE TOLERANCE 945

Figure 2. Phenotype of MDSCs in mice treated with Neupogen. BALB/c mice were treated with 5 µg Neupogen (recombinanthuman G-CSF) for 5 days. Multicolor FACS analysis was performed to establish the phenotype of induced MDSCs after gating onGr-1+CD11b+ cells in the spleens of treated mice harvested 3 days after treatment. (A). Representative histogram plots of majorhistocompatibility complex (MHC) class II, CD40, CD80, F480, and IL-4Rα expression on gated Gr-1+CD11b+ cells in the spleenof naive (dashed lines) or Neupogen-treated mice (solid thick lines) analyzed 3 days after treatment. Isotype staining is depicted insolid thin line. (B) Mean fluorescent intensity (MFI) of IL-4Rα expression on gated MDSCs in the spleen (SPLN) and peripheralblood (PB) of naive versus Neupogen-treated BALB/c mice. Data are representative from three independent experiments.

MDSCs. Analysis of T-cell cultures stimulated with al- the suppressive capacity of Neupogen-induced MDSCsboth in a polyclonal antigen stimulation setting and inlogeneic APCs revealed a significant reduction (p =

0.001) in proliferation of responder cells in the presence an alloreactive response, which is highly relevant intransplantation settings.of MDSCs, while addition of control syngeneic spleen

cells resulted in higher proliferative responses (Fig. 3B).In Vivo Induction of MDSCs and Treg Cells PromotesInterestingly, on a per cell basis, MDSCs isolated fromSurvival of Skin Allograftstreated mice inhibited the MLR response more potently

than those derived from naive unmanipulated mice (Fig. Currently, there is paucity of data addressing MDSC-suppressive functions in transplantation settings. Be-3C). This finding supports our hypothesis that Neupogen

promotes MDSCs differentiation toward a more sup- cause both Treg and MDSCs have the potential to pre-vent or attenuate alloreactive T-cell responses (1,10,12,pressive phenotype. Collectively, these data demonstrate

Table 1. Summary of Mean Fluorescent Intensity (MFI) Values for the Expressionof Indicated Cell Surface Molecules on Naive Versus Induced MDSCs as Describedin Figure 2A

MHC Class II CD40 CD80 F4/80 IL-4Rα

Naive 32.5 ± 2.5 86.0 ± 4.0 26.5 ± 1.5 50.5 ± 1.5 6.8 ± 0.05Induced 29.0 ± 0.5 134.6 ± 17.3 33.0 ± 0.5 42.3 ± 6.4 24.3 ± 0.9

Values are MFI ± SEM.

946 ADEEGBE ET AL.

Figure 3. Suppression of T-cell proliferative response by Neupogen-induced MDSCs. (A) BALB/c carboxyfluorescein succinimidylester (CFSE)-labeled T cells (1 × 106) were cultured with BALB/c antigen-presenting cells (APCs; 1 × 106) and stimulated with 0.5µg α-CD3 in 24-well plates for 3 days. MDSCs isolated from Neupogen-treated mice or spleen cells from normal mice were addedto the cultures at a 1:1 ratio to the responding T cells. Proliferation of labeled T cells was then assessed by evaluating CFSEdilution as measured by FACS analysis after gating on viable 7AAD−, CD4+, or CD8+ T cells. Representative FACS plots of CFSEintensity on day 0 and day 3 of cell culture from three independent experiments are shown. (B) MDSCs from treated mice suppressalloreactive T-cell response in vitro. BALB/c T cells (1 × 105) were cocultured with C57BL/6 APCs (1 × 105) in 96-well roundbottom plates for 4 days in the presence or absence of MDSCs (1 × 105) purified from the spleen of Neupogen-treated BALB/cmice or control spleen cells from normal mice. Thymidine was added to the triplicate wells in the last 18 h of culture. p-Valuesfor statistically significant difference between the indicated pairs of experimental groups as determined by Student’s t-test areshown (p < 0.05) and these differences remained statistically significant after performing Bonferroni correction test for comparisonsto the no MDSC group at a 0.025 significance level. (C) Percent inhibition by induced versus naive MDSCs. Purified MDSCsisolated from naive or Neupogen-treated mice were added to cultures as described in (B) at indicated numbers. Percent inhibitionwas calculated from 3H proliferation counts in the presence of MDSC as a percentage of total counts in the absence of MDSCs.Data from two independent experiments are shown.

18,20,26,31) (Fig. 3B, C), we hypothesized that increas- periments with mice treated with Neupogen and/or IL-2C utilizing donor skin that is disparate in only theing their numbers in vivo by administration of Neupo-

gen (for MDSCs) or IL-2C (for Treg cells) (48), alone MHC class II constituents (i.e., bm12 to C57BL/6) aswell as unrelated third party that is both MHC class Ior in combination might downregulate antidonor immu-

nity and tip the balance in favor of tolerance. To test and II disparate (i.e., C3H to C57BL/6). In C57BL/6recipients, treatment with Neupogen (Fig. 4B) resultedthis hypothesis, we performed skin transplantation ex-

MYELOID SUPPRESSOR CELLS PROMOTE TOLERANCE 947

in a significant delay of MHC class II disparate (bm12) strated that MDSCs can induce Treg expansion (19,27,44), treatment with Neupogen alone did not significantlybut not C3H allogenic donor skin rejection [p = 0.0045,

mean survival time (MST) of 40 vs. 16 days] when com- increase Treg fractions in the peripheral blood, spleen,and lymph nodes (Fig. 5A). Administration of IL-2Cpared to the control PBS-treated group (Fig. 4A). Inter-

estingly, the administration of the IL-2 complex also de- alone or in combination with Neupogen, however, ledto expansion of CD4+Foxp3+ Treg cells in these periph-layed significantly (p = 0.002) the loss of bm12 donor

skin (MST of 50 vs. 20 days for controls) (Fig. 4C), eral sites when compared to PBS controls (Fig. 5A).While Neupogen alone resulted in an elevation ofan effect that was even more pronounced when it was

combined with Neupogen (MST of 74) (p = 0.0032) MDSC fraction in treated mice, the percentage of MDSCswas not affected in mice treated with IL-2 complex, sug-(Fig. 4D). Although the rejection of fully allogeneic

C3H donor skin was comparable in all groups, these gesting that the latter acts specifically to promote theexpansion of Treg cells and not MDSCs (Fig. 5B). Inter-data highlight an effect of the combinatorial treatment

in promoting prolonged survival across limited MHC estingly, an additive effect was noted for MDSC frac-tions in mice treated with a combination of both IL-2barriers when the two naturally occurring suppressor cell

subsets are induced to elevated levels in the periphery. complex and Neupogen in all three compartments (Fig.5B). One possible effect of the increased frequency of

Delayed Allograft Rejection Correlates With both of these naturally occurring suppressor cells in cir-Attenuated T-Cell Response in Mice Treated culation is that T-cell response is dampened in treatedWith the Combination of Neupogen and IL-2 Complex mice. To evaluate T-cell response to alloantigens in

vitro, T cells purified from C57BL/6 mice that wereAt the time of graft rejection, all transplant recipientswere further analyzed to determine frequencies of MDSCs treated with Neupogen and/or IL-2C were stimulated in

vitro with allogeneic APCs in the presence or absenceand Treg cells. Although previous findings have demon-

Figure 4. Kinetics of skin graft rejection in an MHC class II disparate skin graft model. Eight-to12-week-old C57BL/6 mice received three contiguous skin grafts from C57BL/6, B6bm12, andC3H donor mice, respectively. Mice were then treated with Neupogen (5 µg) and/or IL-2 complexdaily for the first 5 days beginning at day 0 and thereafter three times a week for Neupogen ortwice a week for IL-2 complex throughout the duration of experiment. The control group receivedPBS. All recipient mice were monitored every other day until graft rejection, defined as >80% lossof tissue. Kinetics of graft rejection for MHC class II disparate bm12 donor skin and full MHCmismatch C3H donor skin in control C57BL/6 or treated recipients is shown. Treatments and themean survival time (MST) of allogeneic skin grafts are listed within each panel of the figure.

948 ADEEGBE ET AL.

MYELOID SUPPRESSOR CELLS PROMOTE TOLERANCE 949

of α-CD3. Upon analysis, the proliferative response of ing (H&E) on donor bm12 skin tissues that were excisedfrom C57BL/6 transplant recipients treated with PBST cells derived from all groups as indicated after poly-

clonal stimulation was comparable (Fig. 5C). In MLR (controls), Neupogen, and/or IL-2C. Analysis at day 12posttransplant revealed that in the control PBS groupresponses, however, proliferation by T cells obtained

from mice treated with both Neupogen and IL-2 com- there was massive cellular infiltration and tissue dam-age, which precedes graft loss between 15 and 20 daysplex was significantly reduced in contrast to that of T

cells obtained from mice treated with PBS, Neupogen, for rejecting control mice. In contrast, intact epidermis,hair follicles, and minimal infiltrates were observed inor IL-2 complex alone (Fig. 5D). These data suggests

that alloantigen-reactive T-cell subsets in mice treated all experimental mice (Fig. 6A). Remarkably, at 60 daysposttransplant, 2/4 of the mice treated with IL-2 com-with both agents remained in a somewhat suppressed

state ex vivo and thus only partially responded to alloan- plex and 4/5 of the mice treated with Neupogen/IL-2complex still retained 50–70% of donor graft (data nottigen stimulation.

One of several mechanisms that is implicated in shown). Histological evaluation of bm12 graft retrievedfrom these mice at day 60 posttransplant revealed mod-MDSC-mediated suppression of antitumor immunity is

the downregulation of the ζ chain of the CD3 complex erate infiltration, but with a mostly intact epidermallayer and preserved hair follicles, suggesting minimalthat results in diminished activation of tumor-specific T

cells (15). Due to the prevalence of high fractions of pathological damage to the overall tissue (Fig. 6B).Taken together, these data further support the overallMDSCs in mice treated with Neupogen, we investigated

whether this mechanism is operative in our system. observation that loss of bm12 donor skin is delayed intreated mice, possibly due to inhibition of effector cellAfter a short course of Neupogen and/or IL-2 complex

treatment, analysis of the splenic T cells revealed a infiltration into the graft or impairment of effector func-tion at the graft site.downregulation of the ζ chain in Neupogen or IL-2 com-

plex-treated mice that was more striking with the combi-DISCUSSIONnation of the two agents (Fig, 5E, F). This result is con-

sistent with the diminished alloreactive responses by T Current treatments for restraining the immune re-sponses against allografts often utilize strong immuno-cells from treated mice and supports the notion that im-

paired T-cell activation resulting from suboptimal CD3 suppressive biological agents that can sometimes lead todamaging side effects and generate a global depressionsignaling likely contributes to lowered T-cell prolifera-

tion. of the immune system. As various regulatory and/orsuppressor cells that target multiple immune perturba-

Histological Assessment of Skin Grafts Reveals tions are being uncovered, there is increasing interest inMarkedly Reduced Cellular Infiltration the utilization of such naturally occurring suppressivein Treated Recipients cells as therapeutic alternatives to promoting transplan-

tation tolerance. Herein, we explored the potential ofIn addition to visual evaluation and scoring of donorskin grafts, we performed hematoxylin and eosin stain- two suppressive leukocyte subsets, CD4+Foxp3+ T cells

FACING PAGE

Figure 5. Induction of MDSCs and CD4+Foxp3+ Treg cells upon coadministration of Neupogen and IL-2 complex. C57BL/6 skintransplant recipients as described in Figure 4 were sacrificed at time of graft rejection and the peripheral blood, spleen, and lymphnodes (LN) were utilized for multicolor FACS analysis to determine the fraction of CD4+Foxp3+ or Gr-1+CD11b+ cells. Summaryof (A) CD4+Foxp3+ Treg or (B) Gr-1+CD11b+ MDSC percentages in the peripheral blood (PB), spleen (SPLN), and lymph nodes(LN) in the groups that received the indicated treatments. (C, D) C57BL/6 mice were treated for 5 consecutive days with Neupogenand/or IL-2 complex, followed by a maintenance dose of Neupogen (three times) and/or IL-2 complex (twice) for 1 week. Threedays after last treatment, mice were sacrificed and T cells (1 × 105) purified from the spleens of these mice were cultured withBALB/c (left) or C3H (right) APCs in the presence (C) or absence (D) of α-CD3 for 3 or 4 days, respectively, in 96-well roundbottom plates. Thymidine was added to the triplicate wells in the last 18 h of culture. Summary of T-cell proliferation based onthymidine incorporation from one representative of two independent experiments is shown. p-Values for statistically significantdifference between the indicated pairs of experimental groups as determined by Student’s t-test are shown (p < 0.05) and thedifference between the IL-2C and IL-2C + Neu in mice cultured with C3H APCs remained statistically significant (and other valueswere nearly significant) after performing Bonferroni correction test for comparisons to the IL-2C + Neu group at a 0.0167 signifi-cance level. (E, F) Evaluation of the CD3-ζ chain in treated mice. Mice were treated with Neupogen or IL-2 complex (IL-2C)alone or in combination for 5 days. Multicolor FACS analysis was performed to establish the expression of the CD3-ζ chain on Tcells in the spleens of treated mice harvested 2 days after treatment. (E) Representative histogram plots and (F) summary of meanfluorescent intensity (MFI) of ζ chain expression on gated CD4+ (left panel) and CD8+ (right panel) cells in the spleen of indicatedgroups of treated mice. Data in (F) are a composite of 3–4 mice per group.

950 ADEEGBE ET AL.

Figure 6. Histological evaluation of skin grafts. Donor C57BL/6 bm12 skin were harvested at (A) 12 days from C57BL/6 micethat were treated as indicated or at (B) 60 days for the IL-2 complex or in combination with Neupogen-treated group only. Tissuesections (5 µm thick) were stained with hematoxylin and eosin (H&E) after paraffin embedding. Scale bars: 125 µm. e; epidermis,d; dermis.

and myeloid-derived suppressor cells, to facilitate toler- may be instrumental in creating a “tolerogenic” environ-ment. Thus, treatments targeted at induction or traffick-ance in a murine model of MHC disparate skin trans-

plantation. A key finding in this study is that donor skin ing of MDSCs to lymph nodes need to be explored. Ofnote is that our choice of human G-CSF for these experi-graft rejection is significantly delayed when the fraction

of these two regulatory cells are increased in circulation ments is in part based on our findings that both mouseand human G-CSF were superior in inducing MDSCs inas potentiated by IL-2 complex and recombinant human

G-CSF. vivo compared to GM-CSF (data not shown). We chosehuman G-CSF for these experiments due to its readyIn many experimental tumor models, a high fraction

of MDSCs are observed in the tumor microenvironment availability and its potential use in additional investiga-tions that may utilize human cells in a murine model.and this correlates with an abundance of immunomodu-

latory cytokines such as G-CSF, GM-CSF, and IL-10, Although the dose of the human G-CSF utilized in thisstudy is higher than currently administered in patientswhich are secreted by tumor cells demonstrating the role

of these biological agents in the recruitment of MDSCs after chemotherapy, it is important to reiterate that ourinvestigations were performed in mice. So, the observa-(15,29). Our finding that administration of recombinant

human G-CSF (Neupogen) led to several fold induction tion that mouse G-CSF induced levels of MDSCs thatwere higher than human G-CSF in one of our limitedof MDSCs in multiple tissues supports such observa-

tions and is consistent with reports of others where GM- experiments (not discussed) is not surprising. Thus, wehave reason to believe that current clinical dosage ofCSF and/or G-CSF have been utilized to mobilize cells

of the myeloid lineage in rodents (28,37,43). Although Neupogen or even lower may be efficacious for promot-ing the induction of MDSCs in humans due to speciesignificant large numbers of MDSCs relative to unmani-

pulated animals were measured in the peripheral blood, specificity. Moreover, because interindividual differ-ences may exist in the metabolism and catabolism ofspleen, and bone marrow, the fold increase is less dra-

matic and somewhat unremarkable in the lymph nodes. this cytokine (32), further studies in humans are neces-sary to establish the minimal dose of Neupogen capableAs a critical site for T-cell priming, lymph node MDSCs

MYELOID SUPPRESSOR CELLS PROMOTE TOLERANCE 951

of mobilizing and differentiating MDSCs for therapeutic sponse that, for instance, correlates with the ex vivoanalysis of CD3 ζ chain expression on T cells isolatedpurposes.

An outstanding issue is whether the induced MDSCs from treated mice, it is somewhat contradictory to the invivo observations in which C3H donor skin graft wasare different in phenotype and function from the natu-

rally occurring cells. Given the heterogenicity of MDSCs, not delayed in mice treated with both Neupogen and IL-2 complex. Our in vitro study represents an isolatedwe cannot rule out the possibility that other minor im-

mature myeloid progenitor cellular subsets reside within event involving alloreactive T-cell response, particu-larly, through the direct allorecognition pathway. Inthe induced population. Nevertheless, it is quite clear

from our assessments that the induced MDSCs are vivo, however, multiple effector mechanisms have beenimplicated in acute and/or chronic graft rejection. Be-largely similar in phenotype to the typical Gr-1+CD11b+

cells in naive mice. The higher expression of IL-4Rα sides the indirect pathway, which is also crucial forchronic graft rejection, there is growing evidence thatexpression on the former, however, may indicate a more

potent suppressive function, consistent with the observa- some humoral (high-affinity alloantibodies) and even in-nate (NK cells) immune response contribute to graft re-tion that MDSCs from Neupogen-treated mice inhibited

T-cell proliferation better than those derived from naive jection (22,30). Thus, the apparent disparity between de-pressed alloreactive T-cell response in vitro and themice when tested in an MLR (Fig. 3C). Our evaluation

of the induced MDSCs in the present study also revealed absence of a delay in fully allogeneic donor graft maylikely be explained by such additional effector mecha-they express little to no MHC class II molecules and

other canonical costimulatory molecules, and, as such, nisms that regulatory cells need to overcome to promotetolerance.are unlikely to function and contribute to professional

and classical antigen presentation and T-cell activation. It is unclear at present whether this conditioning regi-men results in a generalized immunosuppression similarAlthough we previously showed (44), using a transgenic

system, that MDSCs can cross-present antigen specifi- to drugs that are currently utilized to dampen the im-mune response in transplantation settings or if it is acally to Tregs, it is extremely difficult to confirm or

deny this hypothesis in this experimental setting because graft specific anergy. Our findings demonstrate that thesetreatment regimen are unlikely to induce a “wholesale”skin alloantigens are unknown and CD4 T cells with this

particular specificity are not easily detectable. anergy as proliferation to polyclonal stimulator was onlyminimally diminished in vitro within the bulk T-cellOur data indicate that both CD4+ and CD8+ T cells

were effectively suppressed in the presence of poly- population. Although the mechanisms of MDSC sup-pression in this study are yet to be elucidated, it remainsclonal anti-CD3 stimulation, consistent with the report

by De Wilde et al. (10). The capacity of these MDSCs a possibility that the induced MDSCs may modify T-cellphenotype, using mechanisms dependent on L-arginineto prevent alloantigen-mediated T-cell response was

demonstrated in an MLR and further extended to in vivo metabolism and/or disrupting the CD3 complex (14,15,35,39,45,47,49), which ultimately results in lowered ef-studies where MHC class II disparate bm12 donor skin

graft rejection was delayed significantly in the presence fector function. The finding that the ζ chain expressionby T cells within the spleen of mice treated with eitherof induced MDSCs. In naive mice, the kinetics of allo-

geneic skin graft rejection is not altered even in the pres- Neupogen or more remarkably in combination with IL-2 complex is downregulated is consistent with thisence of a small fraction of circulating MDSCs, suggest-

ing that utilization of these cells to favor transplantation notion. It remains to be determined the possible involve-ment of Treg cells in this process as IL-2 complex treat-tolerance will likely require protocols geared at expand-

ing their numbers in vivo. Our findings thus provide ment, similar to Neupogen treatment alone, led to lowerexpression of the ζ chain. Alternatively, MDSCs maysome proof of principle to achieving such goals, at least

in a somewhat stringent animal model of MHC-mismatched induce the apoptosis of some of the alloreactive T cellsthat are in proximity within a microenvironment (15)transplantation. Interestingly, combinatorial treatment

that also increases the frequency of CD4+Foxp3+ Treg and this may explain the significantly depressed prolifer-ative response by T cells from treated mice. Anothercells in parallel with MDSCs strikingly led to more ro-

bust delay of MHC class II disparate donor skin, sug- possibility is that when present in higher than normalfrequencies within a niche, they may competitively pre-gesting an additive suppressive effect on host alloreac-

tive T cells. Consistent with this idea, T cells isolated vent APCs from gaining sufficient access to T cells, thusincreasing the threshold required for optimal T-cell acti-from mice treated with both IL-2 complex and Neupo-

gen proliferate less in the presence of allogeneic APCs vation and differentiation. Although we favor the ideathat our approach utilizing pharmacologic agents to in-when compared to those derived from mice receiving

single treatments (Fig. 5D). Although this in vitro find- duce MDSCs and Tregs in vivo may be less harsh onthe immune system than commonly used immunosup-ing suggests some attenuation of alloreactive T-cell re-

952 ADEEGBE ET AL.

plantation tolerance. J. Immunol. 176(12):7149–7153;pressive drugs, additional experiments are currently un-2006.der way to evaluate these possibilities.

2. Ahmadzadeh, M.; Rosenberg, S. A. IL-2 administrationAccumulation of suppressive T cells has been pre- increases CD4+ CD25(hi) Foxp3+ regulatory T cells in

viously noted in long-term tolerated grafts (17). Histo- cancer patients. Blood 107(6):2409–2414; 2006.3. Almand, B.; Clark, J. I.; Nikitina, E.; van Beynen, J.;logical assessment revealed minimal infiltration of in-

English, N. R.; Knight, S. C.; Carbone, D. P.; Gabrilovich,flammatory cells within donor skin from IL-2 complex/D. I. Increased production of immature myeloid cells inNeupogen-treated recipients when retrieved during thecancer patients: A mechanism of immunosuppression in

acute phase of graft rejection, but over time (day 60) cancer. J. Immunol. 166(1):678–689; 2001.some inflammation became evident. One possible expla- 4. Apolloni, E.; Bronte, V.; Mazzoni, A.; Serafini, P.;

Cabrelle, A.; Segal, D. M.; Young, H. A.; Zanovello, P.nation for our histological findings is that T-cell differ-Immortalized myeloid suppressor cells trigger apoptosisentiation is initially effectively prevented in drainingin antigen-activated T lymphocytes. J. Immunol. 165(12):lymph nodes, but with time, chronic graft rejection coin-6723–6730; 2000.

cides with activated T cells escaping complete suppres- 5. Asiedu, C.; Andrades, P.; Ray, P. D.; George, J. F.;sion and migrating to grafts. The near absence of severe Thomas, J. M. IL-10 and IL-4 in skin allograft survival

induced by T-cell depletion plus deoxyspergualin. Celldamage and necrosis of “tolerated” grafts even in theTransplant. 17(6):713–720; 2008.presence of moderate level of leukocytic infiltrates may

6. Bingisser, R. M.; Tilbrook, P. A.; Holt, P. G.; Kees, U. R.be an indication that the induced suppressive cells alsoMacrophage-derived nitric oxide regulates T cell activa-

migrate to the graft to actively curb host–antidonor im- tion via reversible disruption of the Jak3/STAT5 signalingmune response. Additional experiments are planned to pathway. J. Immunol. 160(12):5729–5734; 1998.

7. Bronte, V.; Apolloni, E.; Cabrelle, A.; Ronca, R.; Serafini,investigate these issues.P.; Zamboni, P.; Restifo, N. P.; Zanovello, P. Identifica-To date, only a few reports have explored the possi-tion of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitorble involvement of MDSCs in transplantation tolerancecapable of activating or suppressing CD8(+) T cells.

(10,12,31,53). Our investigation differs from that of oth- Blood 96(12):3838–3846; 2000.ers in that recipient mice did not undergo extensive con- 8. Bronte, V.; Chappell, D. B.; Apolloni, E.; Cabrelle, A.;

Wang, M.; Hwu, P.; Restifo, N. P. Unopposed productionditioning or were not subjected to cellular ablation pro-of granulocyte-macrophage colony-stimulating factor bytocols (5) that deplete potential effector cells. We reliedtumors inhibits CD8+ T cell responses by dysregulatingon the potential for naturally occurring suppressor cellsantigen-presenting cell maturation. J. Immunol. 162(10):

to inhibit “allo” immunity by utilizing Neupogen, a drug 5728–5737; 1999.that is widely used in the clinic to promote myeloid cell 9. Bronte, V.; Serafini, P.; Mazzoni, A.; Segal, D. M.; Zano-

vello, P. L-arginine metabolism in myeloid cells controlssurvival, expansion, and differentiation, as well as IL-2T-lymphocyte functions. Trends Immunol. 24(6):302–306;complex, which effectively boosts the expansion of Treg2003.cells, similar to IL-2 therapy (2,52) or superagonist

10. De Wilde, V.; Van Rompaey, N.; Hill, M.; Lebrun, J. F.;CD28 antibody treatment (21). While these observations Lemaitre, P.; Lhomme, F.; Kubjak, C.; Vokaer, B.; Olden-indicate that both MDCSs and Treg cells can “coopera- hove, G.; Charbonnier, L. M.; Cuturi, M. C.; Goldman,

M.; Le Moine, A. Endotoxin-induced myeloid-derivedtively” promote transplantation tolerance, it is of interestsuppressor cells inhibit alloimmune responses via hemeto investigate ways to expand these cells in an antigen-oxygenase-1. Am. J. Transplant. 9(9):2034–2047; 2009.specific context as this provides the benefit of tolerance

11. Dolcetti, L.; Peranzoni, E.; Ugel, S.; Marigo, I.; Fernandezto MHC antigens while allowing immune response to Gomez, A.; Mesa, C.; Geilich, M.; Winkels, G.; Traggiai,foreign pathogens. E.; Casati, A.; Grassi, F.; Bronte, V. Hierarchy of immu-

nosuppressive strength among myeloid-derived suppressorIn summary, our findings highlight the potency ofcell subsets is determined by GM-CSF. Eur. J. Immunol.MDSCs in combination with Treg cells to inhibit allo-40(1):22–35; 2010.graft rejection and may provide therapeutic strategies to

12. Dugast, A. S.; Haudebourg, T.; Coulon, F.; Heslan, M.;induction and maintenance of transplantation tolerance. Haspot, F.; Poirier, N.; Vuillefroy de Silly, R.; Usal, C.;In addition, our model provides a platform to investigate Smit, H.; Martinet, B.; Thebault, P.; Renaudin, K.;

Vanhove, B. Myeloid-derived suppressor cells accumulatetolerance to vascularized solid tissues in broader trans-in kidney allograft tolerance and specifically suppress ef-plantation experiments as well as to evaluate mecha-fector T cell expansion. J. Immunol. 180(12):7898–7906;nisms by which both cells synergistically promote toler-2008.

ance. 13. Duhe, R. J.; Evans, G. A.; Erwin, R. A.; Kirken, R. A.;Cox, G. W.; Farrar, W. L. Nitric oxide and thiol redoxACKNOWLEDGMENT: This work was supported by the Dia-regulation of Janus kinase activity. Proc. Natl. Acad. Sci.betes Research Institute Foundation.USA 95(1):126–131; 1998.

14. Dupuis, M.; De Jesus Ibarra-Sanchez, M.; Tremblay,REFERENCESM. L.; Duplay, P. Gr-1+ myeloid cells lacking T cell pro-tein tyrosine phosphatase inhibit lymphocyte proliferation1. Adeegbe, D.; Bayer, A. L.; Levy, R. B.; Malek, T. R.

Cutting edge: Allogeneic CD4+CD25+Foxp3+ T regula- by an IFN-gamma- and nitric oxide-dependent mecha-nism. J. Immunol. 171(2):726–732; 2003.tory cells suppress autoimmunity while establishing trans-

MYELOID SUPPRESSOR CELLS PROMOTE TOLERANCE 953

15. Gabrilovich, D. I.; Nagaraj, S. Myeloid-derived suppres- 29. Marigo, I.; Dolcetti, L.; Serafini, P.; Zanovello, P.; Bronte,V. Tumor-induced tolerance and immune suppression bysor cells as regulators of the immune system. Nat. Rev.

Immunol. 9(3):162–174; 2009. myeloid derived suppressor cells. Immunol. Rev. 222:162–179; 2008.16. Gallina, G.; Dolcetti, L.; Serafini, P.; De Santo, C.;

Marigo, I.; Colombo, M. P.; Basso, G.; Brombacher, F.; 30. Martin, F. Transplantation: It may take two to tango andto treat. Immunol. Cell Biol. 86(2):107–108; 2008.Borrello, I.; Zanovello, P.; Bicciato, S.; Bronte, V. Tu-

mors induce a subset of inflammatory monocytes with im- 31. Morecki, S.; Gelfand, Y.; Yacovlev, E.; Eizik, O.; Shabat,Y.; Slavin, S. CpG-induced myeloid CD11b+Gr-1+ cellsmunosuppressive activity on CD8+ T cells. J. Clin. Invest.

116(10):2777–2790; 2006. efficiently suppress T cell-mediated immunoreactivity andgraft-versus-host disease in a murine model of allogeneic17. Graca, L.; Cobbold, S. P.; Waldmann, H. Identification

of regulatory T cells in tolerated allografts. J. Exp. Med. cell therapy. Biol. Blood Marrow Transplant. 14(9):973–984; 2008.195(12):1641–1646; 2002.

18. Hanash, A. M.; Levy, R. B. Donor CD4+CD25+ T cells 32. Morstyn, G.; Campbell, L.; Souza, L. M.; Alton, N. K.;Keech, J.; Green, M.; Sheridan, W.; Metcalf, D.; Fox, R.promote engraftment and tolerance following MHC-mis-

matched hematopoietic cell transplantation. Blood 105(4): Effect of granulocyte colony stimulating factor on neutro-penia induced by cytotoxic chemotherapy. Lancet1828–1836; 2005.

19. Huang, B.; Pan, P. Y.; Li, Q.; Sato, A. I.; Levy, D. E.; 1(8587):667–672; 1988.33. Murdoch, C.; Muthana, M.; Coffelt, S. B.; Lewis, C. E.Bromberg, J.; Divino, C. M.; Chen, S. H. Gr-1+CD115+

immature myeloid suppressor cells mediate the develop- The role of myeloid cells in the promotion of tumour angi-ogenesis. Nat. Rev. Cancer 8(8):618–631; 2008.ment of tumor-induced T regulatory cells and T-cell an-

ergy in tumor-bearing host. Cancer Res. 66(2):1123– 34. Nagaraj, S.; Gupta, K.; Pisarev, V.; Kinarsky, L.; Sher-man, S.; Kang, L.; Herber, D. L.; Schneck, J.; Gabrilov-1131; 2006.

20. Joffre, O.; Santolaria, T.; Calise, D.; Al Saati, T.; Hudri- ich, D. I. Altered recognition of antigen is a mechanismof CD8+ T cell tolerance in cancer. Nat. Med. 13(7):828–sier, D.; Romagnoli, P.; van Meerwijk, J. P. Prevention of

acute and chronic allograft rejection with CD4+CD25+ 835; 2007.35. Nagaraj, S.; Schrum, A. G.; Cho, H. I.; Celis, E.; Gabri-Foxp3+ regulatory T lymphocytes. Nat. Med. 14(1):88–

92; 2008. lovich, D. I. Mechanism of T cell tolerance induced bymyeloid-derived suppressor cells. J. Immunol. 184(6):21. Kitazawa, Y.; Fujino, M.; Li, X. K.; Xie, L.; Ichimaru,

N.; Okumi, M.; Nonomura, N.; Tsujimura, A.; Isaka, Y.; 3106–3116; 201036. Otsuji, M.; Kimura, Y.; Aoe, T.; Okamoto, Y.; Saito, T.Kimura, H.; Hunig, T.; Takahara, S. Superagonist CD28

antibody preferentially expanded Foxp3-expressing nTreg Oxidative stress by tumor-derived macrophages sup-presses the expression of CD3 zeta chain of T-cell recep-cells and prevented graft-versus-host diseases. Cell Trans-

plant. 18(5):627–637; 2009. tor complex and antigen-specific T-cell responses. ProcNatl. Acad. Sci. USA 93(23):13119–13124; 1996.22. Kroemer, A.; Xiao, X.; Degauque, N.; Edtinger, K.; Wei,

H.; Demirci, G.; Li, X. C. The innate NK cells, allograft 37. Pan, L.; Delmonte, Jr., J.; Jalonen, C. K.; Ferrara, J. L.Pretreatment of donor mice with granulocyte colony-stim-rejection, and a key role for IL-15. J. Immunol. 180(12):

7818–7826; 2008. ulating factor polarizes donor T lymphocytes toward type-2 cytokine production and reduces severity of experimen-23. Kusmartsev, S.; Gabrilovich, D. I. STAT1 signaling regu-

lates tumor-associated macrophage-mediated T cell dele- tal graft-versus-host disease. Blood 86(12):4422–4429;1995.tion. J. Immunol. 174(8):4880–4891; 2005.

24. Kusmartsev, S.; Nagaraj, S.; Gabrilovich, D. I. Tumor- 38. Rivoltini, L.; Carrabba, M.; Huber, V.; Castelli, C.;Novellino, L.; Dalerba, P.; Mortarini, R.; Arancia, G.;associated CD8+ T cell tolerance induced by bone mar-

row-derived immature myeloid cells. J. Immunol. 175(7): Anichini, A.; Fais, S.; Parmiani, G. Immunity to cancer:Attack and escape in T lymphocyte-tumor cell interaction.4583–4592; 2005.

25. Kusmartsev, S.; Nefedova, Y.; Yoder, D.; Gabrilovich, Immunol. Rev. 188:97–113; 2002.39. Rodriguez, P. C.; Ochoa, A. C. Arginine regulation byD. I. Antigen-specific inhibition of CD8+ T cell response

by immature myeloid cells in cancer is mediated by reac- myeloid derived suppressor cells and tolerance in cancer:Mechanisms and therapeutic perspectives. Immunol. Rev.tive oxygen species. J. Immunol. 172(2):989–999; 2004.

26. Lee, 4th, M. K.; Moore, D. J.; Jarrett, B. P.; Lian, 222:180–191; 2008.40. Rodriguez, P. C.; Zea, A. H.; Culotta, K. S.; Zabaleta, J.;M. M.; Deng, S.; Huang, X.; Markmann, J. W.; Chiaccio,

M.; Barker, C. F.; Caton, A. J.; Markmann, J. F. Promo- Ochoa, J. B.; Ochoa, A. C. Regulation of T cell receptorCD3zeta chain expression by L-arginine. J. Biol. Chem.tion of allograft survival by CD4+CD25+ regulatory T

cells: Evidence for in vivo inhibition of effector cell pro- 277(24):21123–21129; 2002.41. Schmielau, J.; Finn, O. J. Activated granulocytes andliferation. J. Immunol. 172(11):6539–6544; 2004.

27. MacDonald, K. P.; Rowe, V.; Clouston, A. D.; Welply, granulocyte-derived hydrogen peroxide are the underlyingmechanism of suppression of t-cell function in advancedJ. K.; Kuns, R. D.; Ferrara, J. L.; Thomas, R.; Hill, G. R.

Cytokine expanded myeloid precursors function as regula- cancer patients. Cancer Res. 61(12):4756–4760; 2001.42. Serafini, P.; Borrello, I.; Bronte, V. Myeloid suppressortory antigen-presenting cells and promote tolerance

through IL-10-producing regulatory T cells. J. Immunol. cells in cancer: Recruitment, phenotype, properties, andmechanisms of immune suppression. Semin. Cancer Biol.174(4):1841–1850; 2005.

28. MacDonald, K. P.; Rowe, V.; Filippich, C.; Thomas, R.; 16(1):53–65; 2006.43. Serafini, P.; Carbley, R.; Noonan, K. A.; Tan, G.; Bronte,Clouston, A. D.; Welply, J. K.; Hart, D. N.; Ferrara, J. L.;

Hill, G. R. Donor pretreatment with progenipoietin-1 is V.; Borrello, I. High-dose granulocyte-macrophage col-ony-stimulating factor-producing vaccines impair the im-superior to granulocyte colony-stimulating factor in pre-

venting graft-versus-host disease after allogeneic stem cell mune response through the recruitment of myeloid sup-pressor cells. Cancer Res. 64(17):6337–6343; 2004.transplantation. Blood 101(5):2033–2042; 2003.

954 ADEEGBE ET AL.

44. Serafini, P.; Mgebroff, S.; Noonan, K.; Borrello, I. My- K. F.; Roden, R. B. CD80 in immune suppression bymouse ovarian carcinoma-associated Gr-1+CD11b+ my-eloid-derived suppressor cells promote cross-tolerance in

B-cell lymphoma by expanding regulatory T cells. Cancer eloid cells. Cancer Res. 66(13):6807–6815; 2006.50. Youn, J. I.; Nagaraj, S.; Collazo, M.; Gabrilovich, D. I.Res. 68(13):5439–5449; 2008.

45. Sinha, P.; Clements, V. K.; Bunt, S. K.; Albelda, S. M.; Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J. Immunol. 181(8):5791–5802; 2008.Ostrand-Rosenberg, S. Cross-talk between myeloid-de-

rived suppressor cells and macrophages subverts tumor 51. Zea, A. H.; Rodriguez, P. C.; Atkins, M. B.; Hernandez,C.; Signoretti, S.; Zabaleta, J.; McDermott, D.; Quiceno,immunity toward a type 2 response. J. Immunol. 179(2):

977–983; 2007. D.; Youmans, A.; O’Neill, A.; Mier, J.; Ochoa, A. C. Ar-ginase-producing myeloid suppressor cells in renal cell46. Szuster-Ciesielska, A.; Hryciuk-Umer, E.; Stepulak, A.;

Kupisz, K.; Kandefer-Szerszen, M. Reactive oxygen spe- carcinoma patients: A mechanism of tumor evasion. Can-cer Res. 65(8):3044–3048; 2005.cies production by blood neutrophils of patients with la-

ryngeal carcinoma and antioxidative enzyme activity in 52. Zhang, H.; Chua, K. S.; Guimond, M.; Kapoor, V.;Brown, M. V.; Fleisher, T. A.; Long, L. M.; Bernstein,their blood. Acta Oncol. 43(3):252–258; 2004.

47. Talmadge, J. E. Pathways mediating the expansion and D.; Hill, B. J.; Douek, D. C.; Berzofsky, J. A.; Carter,C. S.; Read, E. J.; Helman, L. J.; Mackall, C. L. Lympho-immunosuppressive activity of myeloid-derived suppres-

sor cells and their relevance to cancer therapy. Clin. Can- penia and interleukin-2 therapy alter homeostasis ofCD4+CD25+ regulatory T cells. Nat. Med. 11(11):1238–cer Res. 13(18 Pt. 1):5243–5248; 2007.

48. Tang, Q.; Adams, J. Y.; Penaranda, C.; Melli, K.; Piaggio, 1243; 2005.53. Zhang, W.; Liang, S.; Wu, J.; Horuzsko, A. Human inhibi-E.; Sgouroudis, E.; Piccirillo, C. A.; Salomon, B. L.; Blue-

stone, J. A. Central role of defective interleukin-2 produc- tory receptor immunoglobulin-like transcript 2 amplifiesCD11b+Gr1+ myeloid-derived suppressor cells that pro-tion in the triggering of islet autoimmune destruction. Im-

munity 28(5):687–697; 2008. mote long-term survival of allografts. Transplantation86(8):1125–1134; 2008.49. Yang, R.; Cai, Z.; Zhang, Y.; Yutzy, 4th, W. H.; Roby,