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as Myeloid-Derived Suppressor Cellsto the Accumulation of Splenocytes That ActMice Display Aberrant Myelopoiesis Leading

''Sash'' MutantW-shKitdeficient −Mast Cell

Schild, Edgar Schmitt, Marc Becker and Michael StassenThorsten B. Feyerabend, Hans-Reimer Rodewald, Hansjörg Manfred Relle, Ute Distler, Jörg Kuharev, Stefan Tenzer,Döner, Tobias Bopp, Markus Radsak, Markus Hoffmann, Anastasija Michel, Andrea Schüler, Pamela Friedrich, Fatma

ol.1203355http://www.jimmunol.org/content/early/2013/05/01/jimmun

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The Journal of Immunology

Mast Cell–deficient KitW-sh ‘‘Sash’’ Mutant Mice DisplayAberrant Myelopoiesis Leading to the Accumulation ofSplenocytes That Act as Myeloid-Derived Suppressor Cells

Anastasija Michel,* Andrea Schuler,† Pamela Friedrich,* Fatma Doner,*

Tobias Bopp,* Markus Radsak,† Markus Hoffmann,* Manfred Relle,‡ Ute Distler,*

Jorg Kuharev,* Stefan Tenzer,* Thorsten B. Feyerabend,x Hans-Reimer Rodewald,x

Hansjorg Schild,* Edgar Schmitt,* Marc Becker,‡ and Michael Stassen*

Mast cell-deficient KitW-sh ‘‘sash’’ mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect

additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that KitW-sh causes aberrant extramedul-

lary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and

granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of

c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also

highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and

functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able

to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-

bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice,

accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell–independent phenomenon. Thus, the

KitW-sh mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research. The Journal of Immunology,

2013, 190: 000–000.

The receptor tyrosine kinase c-Kit (CD117) and its ligandstem cell factor (SCF) have been intensively studied owingto their multifaceted role in development and hemato-

poiesis (1, 2). These molecules were mapped to the White spottingand Steel loci, respectively, and a variety of mutant alleles havebeen described (3–6).Mice carrying spontaneous loss-of-function mutations at either

Steel or White spotting loci generally show pleiotropic yet similarphenotypes. Pathophysiological manifestations can include mac-

rocytic anemia, sterility, pigmentation defects, and intestinal dis-orders (7).The c-Kit/SCF axis is also crucial for the development of mast

cells, a finding that led to thewidespread use of several strains of c-Kit/SCF mutant mice in mast cell research (8). The compositegenotype KitW/KitW-v causes severe mast cell deficiency (9), yetthese animals are also anemic and infertile. More recently, mastcell–deficient KitW-sh/KitW-sh ‘‘sash’’ mice serve as a commontool, as these animals do not suffer from some disadvantages thatcharacterize the KitW/KitW-v strain (10). Although sash mice alsolack mast cells, melanocytes, and interstitial cells of Cajal, theyare fertile, not anemic, and show normal numbers of gd T cells(11–13).The KitW-sh mutation is an inversion of ∼3.1 Mbp encompass-

ing 27 known genes. However, in contrast to the KitW and KitW-v

mutations,KitW-sh does not alter the coding region ofWhite spottinglocus itself. The 59 breakpoint of this inversion disrupts the Coringene, leading to cardiac hypertrophy, and the 39 breakpoint is lo-cated 72 kbp upstream of the c-Kit transcriptional start site (14,15). Regulatory elements driving the expression of c-Kit in mastcells were mapped within the affected region (16). Thus, it can beassumed that the inversion or additional deletions of cis regulatoryelements prevent mast cell–specific c-Kit expression, which ulti-mately leads to their irreversible demise within a few weeks afterbirth of sash mice (17, 18).The use of Kit mutant mice has been of central importance to

unravel mast cell functions in the immune system and beyond (19,20). However, novel mast cell–deficient strains with unperturbedc-Kit/SCF function challenge some findings obtained in Kit mu-tant mice (21, 22). These inconsistent observations may indicatelimitations of mast cell–dependent mouse strains with mutant Kitalleles (23–25).

*Institute for Immunology, University Medical Center of the Johannes GutenbergUniversity, 55131 Mainz, Germany; †III Medical Department, University MedicalCenter of the Johannes Gutenberg University, 55131 Mainz, Germany; ‡I MedicalDepartment, University Medical Center of the Johannes Gutenberg University,55131 Mainz, Germany; and xDivision for Cellular Immunology, German CancerResearch Center, D-69120 Heidelberg, Germany

Received for publication December 6, 2012. Accepted for publication March 29,2013.

This work was supported by Deutsche Forschungsgemeinschaft Grants STA 984/4-1(to M.S. and M. Radsak), STA 984/1-2 (to M.S.), and Ra 988/4-2 (to M. Radsak andH.S.) the European Research Council (Advanced Grant 233074, to H.-R.R.), and bythe Immunology Center of Excellence Mainz (to M.S.).

Address correspondence and reprint requests to Dr. Michael Stassen, Institute forImmunology, University Medical Center of the Johannes Gutenberg University,Langenbeckstrasse 1, 55131 Mainz, Germany. E-mail address: stassenm@uni-mainz.de

The online version of this article contains supplemental material.

Abbreviations used in this article: CMP, common myeloid progenitor; G-MDSC,granulocytic myeloid-derived suppressor cell; HSC, hematopoietic stem cell; L1C2,line 1 alveolar cell carcinoma; Lin, lineage; L-NMMA, NG-monomethyl-L-arginine;LSK, Lin2Sca-1+c-Kit+; LT-HSC, long-term hematopoietic stem cell; MDSC,myeloid-derived suppressor cell; MEP, megakaryotic/erythroid progenitor; M-MDSC,monocytic myeloid-derived suppressor cell; MPP, multipotent progenitor; MS, massspectrometry; nor-NOHA, Nv-hydroxy-nor-L-arginine; ROS, reactive oxygen spe-cies; SCF, stem cell factor; ST-HSC, short-term hematopoietic stem cell.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203355

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It this study, we demonstrate that the KitW-sh defect causes ex-tramedullary hematopoiesis leading to the accumulation of mye-loid progenitor cells in the spleen of naive sash mice. CD11b+Ly6C+

cells that express intermediate and high levels of Ly6G are alsohighly expanded in sash spleen. Morphologically and functionally,these Ly6G-expressing cells resemble granulocytic myeloid-derivedsuppressor cells (G-MDSC), which typically expand in tumor-bearing hosts. Thus, besides its well-documented roles on mastcell development and survival, the KitW-sh allele affects myelopoi-esis, which may have adverse effects regarding the interpretation ofmast cell–dependent phenomena.

Materials and MethodsMice

C.B6-KitW-sh mice (H-2d) were generated as previously described andbackcrossed at least 12 generations (26). BALB/c wild-type littermateswere obtained from crosses between heterozygous C.B6-KitW-sh/+ mice.Cpa3Cre/+ mice (22) on a BALB/c background and their wild-type litter-mates were obtained from Thorsten Feyerabend (German Cancer ResearchCenter).

All mice were used in accordance with the guidelines of the CentralAnimal Facility of the University of Mainz.

Abs and reagents

CD11b-FITC (M1/70), Ly6G-PE (1A8), CD11b-biotin (M1/70), Ly6C-PerCP-Cy5.5 (HK1.4), CD135-PE (A2F10), CD117-PE-Cy7 (ACK2),CD16/32 (93) , CD34-FITC (RAM34), and CD150-FITC (9D1) were ob-tained from eBioscience. Ly6G-FITC (1A8), CD117-allophycocyanin(ACK2), Sca-1-allophycocyanin (D7), CD8a-biotin (53-6.7), CD5-biotin(53-7.3), CD45R (B220)-biotin (RA3-6B2), CD127-biotin (A7R34), TER-119-biotin, Gr-1-biotin (RB6-8C5), and streptavidin-PerCP were obtainedfrom BioLegend. CD16/32-PE (2.4G2) and CD3ε-biotin (500A2) wereobtained from BD Biosciences. Propidium iodide was from Sigma-Aldrich, and CD4-biotin (H129.19) was by our own production and bio-tinylation.

Flow cytometry analyses and cell sorting

For staining of lineage (Lin) markers, single-cell suspensions from spleenor bone marrow were treated with a mixture of biotinylated Lin marker–specific Abs (to CD3ε, CD4, CD5, CD8a, CD11b, CD45R, CD127, Gr-1,TER-119) and then with streptavidin-PerCP.

For the detection of long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and multipotent progenitors (MPP) (27, 28), spleno-cytes or bone marrow cells were incubated with biotinylated Lin marker–specific Abs (except anti-CD127), then with streptavidin-PerCP, and finallywith fluorescently labeled Abs to CD117, Sca-1, CD150, and CD135.

For the detection of common myeloid progenitors (CMP), megakaryotic/erythroid progenitors (MEP), and granulo-myelomonocytic progenitors(GMP) (29), splenocytes or bone marrow cells were incubated with bio-tinylated Linmarker–specificAbs (except anti-CD11b), thenwith streptavidin-PerCP, and finally with fluorescently labeled Abs to CD117, Sca-1, CD16/32,and CD34.

For analyses of MDSC–like cells, Fc receptors on splenocytes wereblocked with anti-CD16/32 and stained for CD11b, Ly6G, and Ly6C.

For cell sorting, CD4+, CD8+, and B220+ splenocytes were depletedwith DynaBeads (Invitrogen), and then CD11b+ splenocytes were enrichedwith CD11b MACS (Miltenyi Biotec). Fc receptors were blocked usinganti-CD16/32 and cells were finally stained with CD11b, Ly6G, and Ly6C.

Analyses were performed using a FACSCanto or LSR II flow cytometerand FACSDiva software (BD Biosciences). Cell sorting was performedon a FACSAria II with FACSDiva software.

Colony forming and differentiation assays

ColonyGEL 1202 mouse complete medium (Cell Systems) was used ac-cording to the manufacturer’s instructions. In brief, 105 splenocytes wereincubated for 7 d at 37�C in duplicates. Colonies consisting of at least 50cells were counted.

For differentiation assays, ColonyGEL 1202 mouse complete medium orColonyGEL 1201 mouse base medium (Cell Systems) were used. SortedCD11b+Ly6GintLy6C+ cells (105) were cultured in duplicates either incomplete medium or in base medium supplemented with 20 ng/ml M-CSF(PeproTech). On day 7, cells werewashed in PBS, Fc receptors were blocked,

and cells were stained for CD11b, Ly6G, Ly6C, or only with propidium io-dide and phenotype was analyzed via flow cytometry.

Histology

Cytospin preparations were stained with a microscopy Hemacolor set(Merck) according to the manufacturer’s recommendations. Slides wereanalyzed by bright-field microscopy on a Keyence BZ-8000 fluorescencemicroscope.

To assess mast cell numbers, ears were removed, fixed in Roti-Histofix(Roth), and embedded in paraffin. Sections were deparaffinized, rehydrated,and stained with avidin-Alexa Fluor 488 (Invitrogen). Slides were analyzedin GFP channel on a Keyence BZ-8000 fluorescence microscope.

Bone marrow chimeras

BALB/c mice were lethally irradiated with 8.5 Gy from a [137Cs] source(OB58-BA; Buchler Braunschweig). Next day, 5 3 106 bone marrowdonor cells from C.B6-KitW-sh or from BALB/c mice were transferred byi.v. injection. Then, mice were housed under specific pathogen-free con-ditions for a time period of 8 wk before use.

Genotyping

Cells sorted by flow cytometry cells were genotyped according to a pub-lished procedure (15).

Generation of BMDC

Single bone marrow cell suspensions were cultured in IMDM supple-mented with 5% FCS, 1 mM L-glutamine, 1 mM NaPyr, and 50 ng/ml GM-CSF. Medium was changed on days 2 and 4. BMDC were used at day 6 andwere at least 85% CD11c+.

Allogeneic MLR

To explore the suppressive capacity of CD11b+ subpopulations on T cells,sorted cells were given to lymphocytes in a ratio of 1:1, 1:3, or 1:9.

In general, the allogeneic MLR consisted of 5 3 104 lymphocytes fromBALB/c mice as responders and 1.67 3 103 BMDC from C57BL/6 miceas activators. The ratio of BMDC to lymphocytes (1:30) was constant in allexperiments. On day 4, [3H]thymidine (0.5 mCi/well) was added and theincorporation was determined in a liquid scintillation counter (LKBWallac) 20 h later.

The role of reactive oxygen species (ROS), inducible NO synthase, orarginase in the suppression of T cell proliferation was examined by additionof 1000 U/ml catalase (Sigma-Aldrich), 500 mM L-NMMA (NG-mono-methyl-L-arginine; Calbiochem), or 500 mM nor-NOHA (Nv-hydroxy-nor-L-arginine; Calbiochem), respectively. Inhibitors were added to the cocul-tures on day 4; 8 h later [3H]thymidine was added and the incorporation wasdetermined 20 h later.

Line 1 alveolar cell carcinoma–induced tumors

Mice were injected with 105 line 1 alveolar cell carcinoma (L1C2) cells(murine bronchoalveolar carcinoma cell line; H-2d) s.c., two spots in eachflank.

The tumor sizewas determined 2–3wk later as average of four tumors permouse by cross-sectional measurement and multiplication of the two values(square millimeters). Bone marrow chimeras showed moderate progress oftumor development, and therefore their tumor size was determined 4 wkafter the L1C2 injection.

Enrichment of G-MDSC–like cells

Single-cell suspensions of spleen from naive C.B6-KitW-sh mice were pre-pared, and T and B cells were depleted with DynaBeads (CD4, CD8 andB220; Invitrogen). Ly6G+ cells were enriched using the FcR blocking re-agent, anti–Ly6G-biotin, and anti-biotin microbeads (MACS MDSC isola-tion kit; Miltenyi Biotec). The purity of these cells was typically ∼90%Ly6G+ as determined by flow cytometry. These cells (53 106) were injectedi.v. in the tail vein or 2.5 3 106 s.c. between the four tumors on each flank.

Proteolytic digestion for mass spectrometry

Cells were lysed by the addition of lysis buffer (7 M urea, 2 M thiourea, 5mM DTT, 2% CHAPS), followed by sonication. After cell lysis, proteinamounts were determined using the Pierce 660 nm protein assay (ThermoScientific, Rockford, IL). Proteins (20 mg/sample) were digested usinga modified filter-aided sample preparation method (30) using trypsin gold(Promega). Resulting tryptic digest solutions were diluted with aqueous0.1% (v/v) formic acid to a concentration of 200 ng/ml and spiked with 20

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fmol/ml enolase 1 (Saccharomyces cerevisiae) tryptic digest standard(Waters, Manchester, U.K.).

Mass spectrometric analyses

Nanoscale LC separation of tryptic peptides was performed with a nano-Acquity system (Waters) equipped with an HSS-T3 C18 1.8 mm, 75 mm 3250 mm analytical reversed-phase column (Waters) in direct injectionmode as described (31).

Mass spectrometric analysis of tryptic peptides was performed using aSynapt G2-S mass spectrometer (Waters) operated in positive mode elec-trospray ionization with a typical resolution of at least 25,000 full width at halfmaximum using data-independent modes of analysis (32, 33) in combinationwith on-line ion mobility separations (34). The data were postacquisitionlockmass corrected as described (31). In low-energy liquid chromatography/mass spectrometry (MS) mode, data were collected at constant collision en-ergy of 4 eV. In elevated energy MS mode, the collision energy was rampedfrom 25 to 55 eV. One cycle of low and elevated energy data was acquiredevery 1.5 s. All samples were analyzed in four replicates.

Data processing and protein identification

Continuum liquid chromatography/MS data were processed and searchedusing ProteinLynx Global SERVER version 2.5.2 (Waters) using a custom-compiled database containing UniProt reference proteome mouse entriessupplemented with sequence information of enolase-1 (S. cerevisiae), bo-vine trypsin, and human keratins. The initial false-positive rate for proteinidentification was set to 4% based on the search of a 33 randomized data-base (31). The experimental data were typically searched with a 3 ppmprecursor and 10 ppm product ion tolerance with one missed cleavageallowed and fixed carbamidomethyl cysteine and variable methionine oxi-dation set as the modifications. Postidentification analyses, including re-tention time alignment, exact mass retention time clustering, normalization,and label-free quantification (32), were done using an in-house–developedsoftware. The false-positive rate of protein identification was reduced to,0.3% by additional data processing steps including isoform/homology,

replicate, and score filtering. Only proteins with a minimum GlobalSERVER identification score of 250.0 and quantified by two or more uniquelyassigned peptides, with each detected in at least two liquid chromatography/MS runs with a minimum Global SERVER identification score of 5.0 andminimum length of 6 aa, were considered.

Statistical analyses

Statistical differences were determined using the Student t test. A pvalue ,0.05 was considered to be statistically significant. Values for allgroups are expressed as means 6 SD.

ResultsThe KitW-sh mutation causes extramedullary hematopoiesis inthe spleen

Myeloid hyperplasia in the spleen of sash mice due to expansion ofGr-1+ cells was previously reported (15, 35) and was correlatedwith neutrophilia in spleen, bone marrow, and blood in one ofthese studies (15).Following the identification of a traceable polymorphism linked to

the KitW-sh allele, we generated congenic mast cell–deficient mice ona BALB/c background, referred to as C.B6-KitW-sh (26). Our com-parative analyses of C57BL/6-KitW-sh and C.B6-KitW-sh confirmedthe increase of CD11b/Gr-1 double-positive cells in the spleen ofboth strains, which was even more pronounced in C.B6-KitW-sh mice.However, on either genetic background, the numbers of these bonafide neutrophils in bone marrow and blood were unaffected by theKitW-sh mutation (26). This prompted us to investigate the impact ofthe KitW-sh allele on peripheral myelopoiesis in detail.In 10-wk-old sash mice, spleens were enlarged and, on average,

the spleen weight was.3-fold increased compared with wild-type

FIGURE 1. Mast cell–deficient sash mice display

splenomegaly and extramedullary hematopoiesis. (A)

Spleen weights of 10-wk-old BALB/c and C.B6-KitW-sh

mice (n = 10-11). (B) Splenocytes (105 each, in

duplicates) derived from BALB/c (n = 3) and C.B6-

KitW-sh (n = 2) mice were cultured in methylcellulose-

based medium for colony forming cell assays with SCF,

IL-3, IL-6, and erythropoietin. At day 7, colonies

consisting of at least 50 cells were counted. (C) Flow

cytometric analyses (left) of splenocytes and bone

marrow cell suspensions stained with Abs specific for

lineage markers to exclude lineage-committed cells

(CD3ε, CD4, CD5, CD8a, CD11b, CD45R, CD127,Gr-1, TER119). Percentages of Lin2 cells are depicted

(right; n = 4–6).

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littermates (Fig. 1A). Colony-formation assays revealed a strongincrease in CFUs, indicative of extramedullary hematopoiesis (Fig.1B). To dissect this phenomenon, we determined the numbers ofimmature Lin2 cells that do not express cell-surface markerspresent on Lin-committed cells in spleen and bone marrow derivedfrom sash mice and wild-type littermates. As depicted in Fig. 1C,frequencies of Lin2 cells in bone marrow of both strains arecomparable, yet there is a relative expansion of these cells in thespleen of sash mice. As shown in Fig. 2A, Lin2 cells contain bothc-Kit+Sca-12myeloid progenitors and the Lin2Sca-1+c-Kit+ (LSK)

population that encompasses HSC. Compared with C57BL/6 mice(Supplemental Fig. 1A), the expression of Sca-1 is reduced but notabsent on LSK cells from BALB/c mice.HSC can be divided into LT-HSC and ST-HSC (Fig. 2B). MPP

reflect the branch point to both common lymphoid progenitors andCMP, with the latter being able to yield MEP and GMP. GMPfinally differentiate into monocytes and granulocytes (36, 37).Flow cytometric analyses revealed that in the spleen of sash mice,frequencies of LT-HSC, ST-HSC, MPP, CMP, and GMP are in-creased (Fig. 2C). In contrast, numbers of MEP are strongly de-

FIGURE 2. Sash mice develop abberant myelopoiesis characterized by the expansion of MPP, CMP, and GMP in the spleen. (A) Analytical setup. Lin2

splenocytes were stained for c-Kit and Sca-1 to identify myeloid progenitors (Lin2Sca-12c-Kit+) and LSK cells. Myeloid progenitors can be subdivided

into MEP, GMP, and CMP, whereas LSK cells contain LT-HSC, ST-HSC, and MPP. (B) Simplified scheme of hierarchy in myelopoietic progenitor de-

velopment. (C) Lin2 splenocytes were stained according to (A) and percentages of distinct progenitor populations are plotted (n = 4–6). (D) Progenitor

populations shown in (C) plotted according to their geometric mean fluorescence intensity (gMFI) of c-Kit expression.

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creased. This is most likely due to the preferred development ofCMP to GMP. Supplemental Fig. 1B depicts equivalent data fromC57BL/6-KitW-sh mice and their wild-type littermates. Regardingthe expression levels of c-Kit, both populations of HSC and MPPin sash mice are phenotypically inconspicuous (Fig. 2D). How-ever, CMP, GMP, and MEP from the spleen of these animals showreduced expression of c-Kit, indicating deregulation of c-Kit ex-pression during myelopoiesis.

In naive mice, the KitW-sh mutation leads to the intrinsicdevelopment and accumulation of CD11b+Ly6G+Ly6C+

splenocytes

Extramedullary myelopoiesis likely explains the abundance of Gr-1+CD11b+ cells in the spleen of sash mice, as introduced aboveand shown in Fig. 3A.Gr-1 is a myeloid differentiation marker for granulocytes and

belongs to the Ly6 family (38). Staining of CD11b+ splenocytes forLy6C and Ly6G reveals distinct populations (Fig. 3A). Strikingly,populations characterized as Ly6GhighLy6C+ and Ly6GintLy6C+ areenlarged in CD11b+ splenocyte suspensions derived from sash mice(Fig. 3A, 3B). The largest difference can be found for Ly6GintLy6C+

cells, which are virtually absent in wild-type mice (Fig. 3B and gateP6 in the flow cytometry plot of Ly6C versus Ly6G in Fig. 3A).Supplemental Fig. 1C shows equivalent data from C57BL/6-KitW-sh

mice and their wild-type littermates. Within the bone marrow, allthese populations gated in P5, P6, and P7 can also be clearly iden-tified within the CD11b+ population. However, no significant dif-ferences regarding the frequencies of these populations in bonemarrow from wild-type and sash mice are detectable (Fig. 3A).This prompted us to investigate whether the accumulation of

CD11b+Ly6GintLy6C+ cells in the spleen of sash mice (P6 in Fig.3A, 3B) originates from bone marrow emigration. To this end, weperformed quadrupole time-of-flight mass spectrometry followingultraperformance liquid chromatography separation of trypticpeptides derived from CD11b+Ly6GintLy6C+ cells previouslyisolated in high purity by FACS from bone marrow and spleen ofsash mice (Fig. 3C). Interestingly, only 62% of the identified pro-teins are expressed at comparable levels, whereas the remainingproteins are either significantly up- or downregulated. This findingindicates major differences between CD11b+Ly6GintLy6C+ cellsin bone marrow and spleen from sash mice. This technique alsoallowed us to estimate the influence of the sash mutation onprotein expression in CD11b+Ly6GintLy6C+ cells derived fromeither wild-type or sash bone marrow (Fig. 3C). Our results implya drastic influence of the sash mutation, as only ∼70% of theidentified proteins are equally expressed in cells derived fromboth strains.To investigate whether the expansion of the above-described

cell populations P5 and P6 in sash mice is due to an intrinsic defect,we generated chimeras by transferring bonemarrow from sashmiceinto irradiated wild-type recipients. Shown in Fig. 3D is the strongexpansion of Ly6GhighLy6C+ (gate P5) and Ly6GintLy6C+ (gateP6) cells from sash origin in the spleen of wild-type mice. Incontrast, the cells designated as population P7 (Ly6ChighLy6G2)develop in comparable numbers from wild-type and sash donorsand thus were excluded from further analyses. The descent of allthese populations from donor sash bone marrow in wild-typerecipients was verified by genotyping cells isolated by FACS.The wild-type c-Kit allele is absent in the cell populations gated inP5–7 in mice that had been rescued with sash bone marrow fol-lowing irradiation, as only the sash mutation is detectable by PCR(Fig. 3E). Furthermore, in wild-type mice, the presence of radio-resistant mast cells does not influence the expansion of myeloidcells from sash bone marrow (Fig. 3F).

These findings imply that the intrinsic sash defect in bonemarrow cells is both necessary and sufficient to drive aberrantmyelopoiesis in mice with otherwise physiological expression ofc-Kit.

CD11b+Ly6G+Ly6C+ splenocytes from sash mice resembleMDSC

Morphologically, populations gated in P5, P6, and P7 displaydistinct characteristic features (Fig. 4A). Cells in P7 appearmonocyte blast-like, whereas in P5 a polymorphnuclear phenotypeprevails. However, cytospin preparations of splenocytes gated inP6 reveal the abundance of cells with ring-shaped nuclei, whichwere reported to encompass MDSC, although this morphologicalcriterion is not unique to these cells (39, 40).The term MDSC is an operational definition and refers to a het-

erogeneous myeloid population that basically also includes neu-trophils and precursors for monocytes and dendritic cells. Phys-iologically, these cells accumulate under conditions of systemicinsults such as tumors and sepsis and are able to dampen immuneresponses by inhibiting T cell activation (41, 42). Using flowcytometry, we isolated Ly6GhighLy6C+ (gate P5) and Ly6GintLy6C+

(gate P6) cells and demonstrated their ability to potently inhibitallogeneic MLRs in vitro (Fig. 4B). According to the expressionof CD11b, Ly6G, and Ly6C, murine MDSC have been subdividedinto two subpopulations with either typical monocytic (M-MDSC)or granulocytic (G-MDSC) morphology (40, 43). M-MDSC areCD11b+Ly6ChighLy6G2 and produce NO and arginase to inhibitT cell responses, whereas G-MDSC display the phenotype CD11b+

Ly6ClowLy6G+ and release ROS (44, 45). Thus, we next evaluatedthe effects of inhibitors of NO synthase (L-NMMA), arginase (nor-NOHA), and ROS (catalase) on their suppressive activity in allo-geneic MLRs. The presence of either L-NMMA or nor-NOHA onlymarginally affected the suppressive activities of Ly6GhighLy6C+

(gate P5) and Ly6GintLy6C+ (gate P6) cells, whereas catalase sig-nificantly restored proliferation, indicating that production of ROSis of vital importance for suppression to occur (Fig. 4C).Taken together, based on the expression pattern of Ly6C and

Ly6G, the appearance of ring-shaped nuclei, and their ability tosuppress T cell proliferation, the expanded myeloid cell popu-lations in the spleen of naive sash mice resemble G-MDSC.The nature of G-MDSC, their ability to differentiate, and their

relationship to neutrophils are still obscure (45). To investigatethe potential of the G-MDSC–like populations Ly6GhighLy6C+

(gate P5) and Ly6GintLy6C+ (gate P6) to differentiate, we usedmethylcellulose-based media to follow their fate in the presenceof growth factors in vitro. A combination of IL-3, IL-6, SCF, anderythropoietin causes a shift to higher expression of Ly6G; thephenotype almost completely changes from Ly6Gint to Ly6Ghigh

(Fig. 5A). Essentially the same happens with P6 cells isolatedfrom wild-type spleen. However, in the latter case it is obviousthat a significant proportion of cells reduces expression of CD11bbut remains negative for propidium iodide (Fig. 5A). We furtherinvestigated whether the presence of M-CSF enhances the ex-pression of Ly6C, which might indicate a shift from a G-MDSC–to a M-MDSC–like phenotype. However, even in the presenceof M-CSF, only a significant trend toward higher Ly6G expres-sion is observable for P6 cells derived from C.B6-KitW-sh andBALB/c mice (Fig. 5B). Note that under these experimental invitro conditions, G-MDSC–like cells appeared rather long-livedand postmitotic; on day 7, at least 84% of the initially seeded cellsare negative in propidium iodide stainings (Fig. 5A, 5B). Addi-tionally, we also followed the fate of these G-MDSC–like pop-ulations P5 and P6 in allogeneic MLRs on day 4. As depicted inFig. 5C, the vast majority of Ly6GhighLy6C+ cells maintain a sta-

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FIGURE 3. Spleen of naive sash mice display elevated numbers of Ly6G+ cells. (A) CD11b+ splenocytes and bone marrow cells from BALB/c and C.B6-

KitW-sh mice were analyzed by flow cytometry for the expression of Ly6G and Ly6C. Representative histograms of CD11b expression and dot plots of

Ly6G/Ly6C expression in both strains are shown. (B) Total percentages of CD11b+ splenocytes, CD11b+Ly6GhighLy6C+ (P5), CD11b+Ly6GintLy6C+ (P6),

and CD11b+Ly6GlowLy6Chigh (P7) cells are indicated; n = 5. (C) Comparative protein expression pattern of CD11b+Ly6GintLy6C+ (P6) cells isolated by

FACS (purity $ 98%, not shown) from bone marrow (BM) and spleen (SP) of the indicated mouse strains. Tryptic digests where separated by ultra-

performance liquid chromatography and analyzed by quadrupole time-of-flight mass spectrometry. Identified peptides (n = 1062) were used to compare

BALB/c bone marrow–derived CD11b+Ly6GintLy6C+ cells with the corresponding population isolated from C.B6-KitW-sh bone marrow. For comparing

CD11b+Ly6GintLy6C+ cells isolated from C.B6-KitW-sh spleen and bone marrow, 1362 peptides were used. Each measurement was performed in quad-

ruplicates. Representatives of two equivalent biological sample sets are shown. (D) Bone marrow chimeras were generated by irradiation of BALB/c mice

on day 0 and reconstitution with bone marrow cells from congenic C.B6-KitW-sh mice on day 1. Controls received BALB/c bone marrow. Eight weeks later,

CD11b+ splenocytes were analyzed and percentages of CD11b+Ly6GintLy6C+ cells (P6) and CD11b+Ly6GhighLy6C+ cells (P5) are shown (n = 7). (E)

Genotyping of sorted CD11b+ splenocytes derived from chimeric mice was performed using primers specific for c-kit wild-type and sash alleles. (F) Ear

skin sections from bone marrow chimeras were analyzed for the presence of mast cells with fluorescence-conjugated avidin. Some mast cells are indicated

by arrows.

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ble phenotype, whereas Ly6GintLy6C+ cells almost completelyshift toward higher expression of Ly6G, irrespective of whetherthey are derived from C.B6-KitW-sh or BALB/c mice.Collectively, these results indicate that both populations of G-

MDSC–like cells isolated from the spleen of sash mice have alimited developmental capacity and mainly differentiate toward aLy6Ghigh phenotype.

Enhanced growth of transplanted L1C2 tumor cells inC.B6-KitW-sh mice is not due to the absence of mast cells

L1C2 is a cell line derived from a tumor that arose spontaneouslyin a female BALB/c mouse (46). In a first set of experiments, we

compared BALB/c and congenic C.B6-KitW-shmice in their abilityto develop tumors following s.c. inoculation of L1C2. As shownin Fig. 6A, BALB/c mice develop visible and distinct tumorsuntil day 18 following inoculation. However, tumor sizes inC.B6-KitW-sh mice are significantly larger. Subcutaneous tumordevelopment in BALB/c mice is accompanied by slight increases ofspleen weights in the absence of overt metastases. Spleens fromnaive sash mice are variably enlarged but there is no additionalenlargement in tumor-bearing mice (Fig. 6B). Splenomegaly intumor-bearing BALB/c mice is paralleled by the accumulation ofCD11b+ cells (Fig. 6C), which are either Ly6GhighLy6C+ (P5) orLy6GintLy6C+ (P6). This observation is in accordance with thepreviously reported accumulation of MDSC in spleen of tumor-bearing mice. However, depending on the tumor model, MDSCnumbers are highly variable (44). However, there is no furtheraccumulation of cells with MDSC-like phenotype in C.B6-KitW-sh

mice (Fig. 6C).To investigate whether increased tumor burden in sash mice is

due to the absence of mast cells, we generated chimeras followingablation of bone marrow by irradiation. Displayed in Fig. 6D aretumor sizes of irradiated BALB/c mice that either received wild-type or C.B6-KitW-sh bone marrow before inoculation with L1C2.Obviously, the ability to develop larger tumors can be transferredby sash bone marrow, despite the presence of radio-resistant mastcells in wild-type mice (see also Fig. 3F).Additionally, we used mast cell–deficient Cpa3Cre/+ mice on

BALB/c background. In these animals, mast cell deficiency is dueto genotoxic effects of Cre expressed under the control of car-boxypeptidase A3 promoter, whereas the c-Kit/SCF axis is un-impaired (22). According to the expectations, tumor sizes inCpa3Cre/+ mice and their wild-type littermates are comparable(Fig. 6E).Thus, the enhanced growth of L1C2 tumors in sash mice cannot

be ascribed to the absence of mast cells.

Adoptive transfer of G-MDSC–like splenocytes from sash miceto wild-type recipients enhances L1C2 tumor growth

One inherent problem of the operational MDSC definition is theimpossibility to selectively ablate these cells without the risk ofaffecting additional cell populations (47, 48).Furthermore, it is impossible to predict whether G-MDSC–like

cells from sash mutant mice are in every aspect identical to theirwild-type counterparts. Deregulation of c-Kit expression (Fig. 2D)might significantly alter signaling events during the developmentof MDSC. This assumption is also supported by our mass spec-trometry analyses shown in Fig. 3C.Thus, as an equivalent alternative approach to depletion, we

assessed tumor growth in BALB/cmice following adoptive transferof G-MDSC–like cells from sash donors. To this end, we appliedMACS technology to isolate Ly6G+ cells representing the Ly6Gint

and Ly6Ghigh populations, which we collectively and operation-ally termed G-MDSC–like cells (Fig. 7A). As depicted in Fig. 7B,transfer of G-MDSC–like cells in BALB/c mice inoculated withL1C2 cells significantly enhanced tumor growth, irrespective ofwhether the cells were given i.v. or s.c. This experiment un-equivocally supports our notion that aberrant myelopoiesis in sashmice leads to the development of splenocytes with immunosup-pressive function, closely resembling MDSC.

DiscussionNotwithstanding their application in mast cell research, it has tobe considered that c-Kit mutant strains suffer from additionaldefects that may even falsify the results of experiments supposedlyaddressing the role of mast cells.

FIGURE 4. CD11b+Ly6GintLy6C+ and CD11b+Ly6GhighLy6C+ spleno-

cytes from sash mice resemble MDSC. (A) HE-stained cytospin prepara-

tions of the indicated cell populations sorted by flow cytometry. (B)

Allogeneic MLR using 5 3 104 lymph node cells from BALB/c mice as

responders and 1.33 3 103 bone marrow–derived dendritic cells from

C57BL/6 mice as activators (fixed ratio responders/activators, 30:1). Sor-

ted P5 or P6 cells were added in different ratios on day 0 (1:1 [5 3 104],

1:3 [1.67 3 104], and 1:9 [0.55 3 104]). On day 4, [3H]thymidine was

added and the incorporation measured 20 h later. Percentages of inhibition

of proliferation compared with controls without sorted cells are shown. (C)

Experiment was performed as described above with the modification that

on day 4 either catalase, L-NMMA, or nor-NOHA was added. Shown are

representatives of two experiments each performed in triplicates (B, C).

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KitW-sh is a mutation known to block c-Kit expression in some celltypes and to enhance the expression of c-Kit in others. Importantly,in sash mice c-Kit expression is shut off in mast cells, causing mastcell deficiency, whereas the absence of melanocytes might be due toenhanced c-Kit expression at sites of early melanogenesis (10, 11,14, 17, 49). Besides multiple functions in development, the c-Kit/SCF system has essential roles in hematopoiesis during fetal de-velopment and in the adult (1, 50).

In this context, we demonstrate that sash mutant mice developextramedullary myelopoiesis characterized by the accumulation ofHSC, MPP, CMP, and GMP in the spleen. In contrast, frequencies ofMEP are decreased, yet this might be a consequence of higher GMPnumbers, as both cells derive from the same precursor. Interestingly,the expression of c-Kit, measured by flow cytometry, is unimpairedin LT-HSC, ST-HSC, and MPP, but decreased in CMP, GMP, andMEP derived from sash spleen. These findings demonstrate that

FIGURE 5. In vitro, CD11b+Ly6GintLy6C+ cells

develop a Ly6Ghigh phenotype. (A) CD11b+Ly6Gint

Ly6C+ cells (gate P6) were sorted via flow cytometry

from the spleen of wild-type and sash mice and 105

cells were cultured in methylcellulose-based medium in

the presence of growth factors as indicated. On day 7,

cells were analyzed by flow cytometry for the expres-

sion of CD11b. Furthermore, CD11b+ cells were stained

for Ly6G and Ly6C. Viability was determined by pro-

pidium iodide (PI) staining. (B) Experiment was per-

formed as described above but cells were cultured in the

presence of M-CSF. (C) CD11b+Ly6GhighLy6C+ (gate

P5) or CD11b+Ly6GintLy6C+ (gate P6) cells were iso-

lated using flow cytometry from sash or wild-type spleen

and added to an allogeneic MLR in a ratio of 1:1. On

day 4 of coculture, CD11b+ cells were analyzed for the

expression of Ly6C and Ly6G and for viability. Shown

are representatives from two independent experiments

each performed in triplicates.

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the sashmutation broadly affects the expression of c-Kit in precursorcells of the myeloid lineage. Consequently, deregulation of c-Kitexpression causes the accumulation of CD11b+Ly6Gint–highLy6C+

cells in naive mice. These cells phenotypically and functionallyclosely resemble G-MDSC.So far, we can only speculate on the impact of the sash mutation

regarding the generation of these G-MDSC–like cells. The c-Kit/SCF axis was shown to regulate adhesion of HSC and to conferstable interactions between HSC and stroma cells in fetal liver andin bone marrow (51). Thus, reduced expression of c-Kit on mye-loid progenitor cells might promote their exit from the bonemarrow and accumulation in the spleen, where they, at least partly,further develop into G-MDSC–like cells.

Physiologically, MDSC accumulate in lymphoid organs underchronic inflammatory conditions or in tumor-bearing hosts. In thelatter, expansion of MDSC is variable and strongly depends on thetumor model investigated (44). Tumor-derived SCF was reportedto promote the expansion ofMDSC in BALB/c mice. This indicatesthat SCF/c-Kit signals may be critical in the generation of thesecells in mice with uncompromised c-Kit expression (52). Theseresults apparently contradict our own observations of expandedMDSC-like cells in naive sash mice in which c-Kit expression isreduced in MDSC precursors, CMP, and GMP. However, blockadeof SCF production and the effects of the KitW-sh mutation mayhave different impacts on c-Kit signaling intensities and cell fate.Currently, it is a paradigm that MDSC can exert their suppres-

sive activity on T cell function upon exposure to inflammatorymediators. Factors that induce activation of MDSC include IFN-g,IL-3, IL-6, and TGF-b (41). Despite the fact that both tumor-promoting (53) and antitumor activities (54) of mast cells were re-ported, our results allow us to conclude that the growth of L1C2tumor cells is unimpaired by the presence or absence of mast cells.However, the presence of large numbers of G-MDSC–like cells

in naive sash mice might allow their instant activation followingtumor inoculation leading to the suppression of specific T cellresponses, which eventually promotes tumor growth.Mounting evidence suggests that the tumor microenvironment is

capable of expanding and activating MDSC by delivering a host ofimmune mediators. Alternatively, activated T cells are regarded assource for mediators able to activate MDSC. However, activationof MDSC might be critical for tumor progression, as it dampensimmune responses against the tumor (55).Collectively, our data demonstrate that the KitW-sh mutation has

a deep impact on myelopoiesis and is thus not as mast cell–spe-

FIGURE 6. Enhanced growth of transplanted L1C2 tumor cells in C.B6-

KitW-sh mice is not due to the absence of mast cells. (A) BALB/c and C.B6-

KitW-sh mice were inoculated s.c. with L1C2 cells on two spots on each

flank using 105 cells per spot. On day 18, the cross-sectional size (mm2) of

each tumor was determined and expressed as mean size per mouse. Arrows

point to representative tumors; n = 5. (B) On day 18, the spleen weights of

the animals shown in (A) were determined. (C) CD11b+ splenocytes were

analyzed for the expression of Ly6G and Ly6C by flow cytometry. Per-

centages of P5 and P6 populations of L1C2 tumor-bearing or untreated

mice on day 18 are depicted. (D) Chimeras were generated by irradiation

of BALB/c mice on day 0 and reconstitution with bone marrow cells from

congenic C.B6-KitW-sh mice on day 1. Controls received BALB/c bone

marrow. Eight weeks later, mice were inoculated with L1C2 cells and

tumor sizes were determined on day 28; n = 6. (E) BALB/c and Cpa3Cre/+

mice were treated as described in (A); n = 8.

FIGURE 7. Following transfer of G-MDSC–like cells from sash mice

into wild-type recipients, L1C2 tumor growth is enhanced. (A) Splenocytes

from naive C.B6-KitW-sh mice were used for positive selection of Ly6G+

cells as described in the experimental procedures section. Purity of ∼90%was determined by flow cytometry. (B) BALB/c mice were inoculated with

L1C2 cells on day 0. On days 1, 4, 7, and 10 Ly6G+ splenocytes were

isolated using MACS and injected either i.v. into the tail vein (5 3 106) or

s.c. 2.5 3 106 cells into each flank. On day 14, tumor sizes were deter-

mined; n = 5 mice/group.

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cific as it was previously thought. This underpins the need foradditional mast cell–specific and Kit-independent murine modelsin mast cell research.

DisclosuresThe authors have no financial conflicts of interest.

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