T Cells Favoring Tumor Growth CCR5-Dependent Recruitment of Regulatory Myeloid-Derived Suppressor Cells Mediate Tumor-Infiltrati

Tumor-Infiltrating Monocytic Myeloid-Derived Suppressor Cells Mediate CCR5-Dependent Recruitment of Regulatory T Cells Favoring Tumor Growth This information is current as of September 26, 2021. Eva Schlecker, Ana Stojanovic, Christian Eisen, Christian Quack, Christine S. Falk, Viktor Umansky and Adelheid Cerwenka J Immunol 2012; 189:5602-5611; Prepublished online 14 November 2012; Downloaded from doi: 10.4049/jimmunol.1201018 http://www.jimmunol.org/content/189/12/5602 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2012/11/14/jimmunol.120101 Material 8.DC1 References This article cites 49 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/189/12/5602.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Tumor-Inﬁltrating Monocytic Myeloid-Derived Suppressor Cells Mediate CCR5-Dependent Recruitment of Regulatory T Cells Favoring Tumor Growth

Eva Schlecker,* Ana Stojanovic,* Christian Eisen,† Christian Quack,‡ Christine S. Falk,‡ Viktor Umansky,x,{ and Adelheid Cerwenka*

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of myeloid cells in cancer patients and tumor- bearing mice that potently inhibits T cell responses. During tumor progression, MDSCs accumulate in several organs, including the tumor tissue. So far, tumor-infiltrating MDSC subpopulations remain poorly explored. In this study, we performed global gene expression profiling of mouse tumor-infiltrating granulocytic and monocytic (MO-MDSC) subsets compared with MDSCs from

peripheral blood. RMA-S lymphoma–infiltrating MO-MDSCs not only produced high levels of NO and arginase-1, but also Downloaded from greatly increased levels of chemokines comprising the CCR5 ligands CCL3, CCL4, and CCL5. MO-MDSCs isolated from B16 melanoma and from skin tumor–bearing ret transgenic mice also expressed high levels of CCL3, CCL4, and CCL5. Expression of CCR5 was preferentially detected on regulatory T cells (Tregs). Accordingly, tumor-infiltrating MO-MDSCs directly attracted high numbers of Tregs via CCR5 in vitro. Intratumoral injection of CCL4 or CCL5 increased tumor-infiltrating Tregs, and deficiency of CCR5 led to their profound decrease. Moreover, in CCR5-deficient mice, RMA-S and B16 tumor growth was delayed emphasizing the importance of CCR5 in the control of antitumor immune responses. Overall, our data demonstrate that chemo- http://www.jimmunol.org/ kines secreted by tumor-infiltrating MO-MDSCs recruit high numbers of Tregs revealing a novel suppressive role of MDSCs with potential clinical implications for the development of cancer immunotherapies. The Journal of Immunology, 2012, 189: 5602–5611.

ultiple mechanisms of immune suppression accompa- 7). Several mechanisms for their direct suppressive activity on nying tumor development have been reported (1). T cells were reported, such as the production of reactive oxygen M Alterations in myelopoiesis that occur during tumor species (ROS) and nitrogen species and arginase-1 (8) or by de- growth lead to the accumulation of myeloid-derived suppressor priving cysteine from the environment (9). So far, most studies cells (MDSCs). MDSCs represent a heterogeneous population of investigated the accumulation, subset distribution, and regulatory myeloid cells at different stages of differentiation (2, 3). In mice, role of MDSCs in bone marrow, blood, and spleen. Comparatively by guest on September 26, 2021 MDSCs are characterized by the coexpression of the cell-surface little is known about their function within the tumor tissue of solid markers Gr-1 and CD11b. Within this population, two MDSC malignancies including lymphoma and melanoma. subsets with distinct morphological features have been identified Regulatory T cells (Tregs) represent another suppressive pop- comprising monocytic MDSCs (MO-MDSCs) that express high ulation that potently inhibits T cell and NK cell function in cancer levels of Ly6C and F4/80 and granulocytic MDSCs (PMN- patients and tumor-bearing mice (10–12). CD4+CD25+ Tregs ex- MDSCs) expressing Ly6G and no F4/80 (4, 5). MDSCs potently pressing the transcription factor Foxp3 inhibit autoimmune re- inhibit T cell proliferation and cytokine production in vitro (3, 6, sponses and promote tumor progression (13, 14). Accordingly, the depletion of Tregs results in improved antitumor immune responses and delays tumor growth in many tumor models (15). High numbers *Innate Immunity, Research Program Tumor Immunology, German Cancer Research ofTregsintumorssuchaslymphoma(16)areoftenassociatedwith Center, Heidelberg D-69120, Germany; †Division of Stem Cells and Cancer, German Cancer Research Center, Heidelberg D-69120, Germany; ‡Immune Monitoring Unit, poor prognosis for cancer patients. Several mechanisms of Treg National Center for Tumor Diseases, German Cancer Research Center, Heidelberg D- recruitment to tumors were reported. In breast and prostate cancer 69120, Germany; xSkin Cancer Unit, German Cancer Research Center, Heidelberg D- { patients, CCL22 present in the tumor microenvironment mediated 69120, Germany; and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Treg trafficking in a CCR4-dependent manner (17, 18). Recently, it Mannheim, Heidelberg 68167, Germany was shown that tumor-derived CCL28 mediates CCR10-dependent Received for publication April 5, 2012. Accepted for publication October 3, 2012. Treg recruitment in ovarian cancer (19). In a mouse model of This work was supported by the Deutsche Krebshilfe (109174 to A.C.). pancreatic adenocarcinoma that produces CCL5, increased Treg The sequences presented in this article have been submitted to Gene Expression numbers were associated with CCR5 expression by Tregs (20). Omnibus under accession number GSE41620 (http://www.ncbi.nlm.nih.gov/geo/ However, whether additional cells, like tumor-infiltrating immune query/acc.cgi?acc=GSE41620). cells, mediate CCR5-dependent Treg recruitment remains elusive. Address correspondence and reprint requests to Dr. Adelheid Cerwenka, German Recent evidence demonstrates that MDSCs and Tregs interact Cancer Research Center, DKFZ/D080, Im Neuenheimer Feld 280, D-69120 Heidel- berg, Germany. E-mail address: [email protected] with each other to build a suppressive network during antitumor The online version of this article contains supplemental material. immune responses. In this context, it was shown that CD40 (21), Abbreviations used in this article: iNOS, inducible NO synthase; KO, knockout; CD80 (22), and arginase-1 expression (23) by MDSCs are in- MDSC, myeloid-derived suppressor cell; MO-MDSC, monocytic myeloid-derived volved in the induction of Treg development and T cell tolerance suppressor cell; PMN-MDSC, granulocytic myeloid-derived suppressor cell; ROS, in spleen, bone marrow, and ascites. It has been recently reported reactive oxygen species; Treg, regulatory T cell; wt, wild-type. that CCL5 gene expression in blood-derived MDSCs was down- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 regulated by treatments with tolerating regimen affecting the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201018 The Journal of Immunology 5603 intragraft localization of Tregs (24). Whether tumor-infiltrating Chemotaxis assay MDSCs interact with Tregs within the tumor tissue of solid Tregs from spleens of naive C57BL/6-CD45.1+ mice or CCR52/2 mice tumors and influence each other’s accumulation and recruitment were enriched by magnetic beads using CD4+CD25+ isolation kit (Miltenyi remains to be investigated. Biotec; purity $95%) and stimulated with 5 mg/ml anti-CD3/CD28 mAbs In our study, we performed global gene arrays of tumor-infiltrating overnight in serum-free primary cell medium (RPMI with 1% L-glutamine, MDSC subsets to gain insight in their function within the tumor 1% penicillin, 1% streptomycin, 1% nonessential amino acids, 1% sodium pyruvate, and 50 mM 2-ME). This preactivation increased the percentages tissue. Our data demonstrate that tumor-infiltrating MO-MDSCs of CCR5+ Tregs from 7 to 40% (data not shown). Tumor-infiltrating produce high levels of the CCR5 ligands, CCL3, CCL4, and CCL5 MDSC subsets from C57BL/6 mice expressing CD45.2 were sorted by and directly attract Tregs in a CCR5-dependent manner. Accordingly, FACS and placed in 600 ml primary cell medium in the lower wells of an in CCR5-deficient mice, decreased Treg levels were detected in the HTS Transwell-24 (Corning) with a 5-mm pore size. After 18 h, preactivated Tregs (2 3 105 in 200 ml) were added to the upper chamber and tumor tissue that was associated with delayed tumor growth. Overall, cultured for 5 h. Migrated Tregs were harvested from the lower chamber our results reveal the recruitment of Tregs as a novel suppressive and stained with anti-CD45.1 mAbs. After staining, Accudrop Counting mechanism mediated by tumor-infiltrating MO-MDSCs. Beads (BD Biosciences) were added, and the numbers of cells were determined by CD45.1 expression using the FACSCalibur flow cytometer (BD Biosciences). Purified Tregs from CCR52/2 mice were labeled with Materials and Methods CFSE, and numbers of migrated cells were distinguished by CFSE labeling Mice and tumor models after addition of Accudrop Counting Beads (BD Biosciences). C57BL/6 (wild-type [wt]) mice were purchased from Charles River Lab- Morphological analysis oratories (Sulzfeld, Germany, and Erembodegem, Belgium). CCR52/2 mice (at least 10 backcross generations) and congenic C57BL/6-CD45.1+ Cytospins of FACS-sorted MDSC subsets were prepared using poly-L-ly- Downloaded from mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and sin–coated microscopic glass slides (Sigma-Aldrich). Cells were fixed in bred in our animal facility. Mice (C57BL/6 background) that overexpress 4% paraformaldehyde in PBS for 10 min. The nuclei were stained with the human ret transgene in melanocytes under the control of the mouse Mayer’s Hematoxylin (DakoCytomation), and counterstaining of the cy- metallothionein I promoter enhancer were kindly provided by Dr. I. tosol was performed with 10% eosin Y (Applichem). Images were ac- Nakashima (Chubu University, Aichi, Japan) (25). Mice were housed in quired using the Olympus BX51 microscope with 3200 original specific pathogen–free conditions and used in experiments at 6–8 wk of age. magnification (Olympus).

All experiments were performed according to local animal experimental http://www.jimmunol.org/ Characterization of MDSC subsets ethics committee guidelines (Regierungspra¨sidium, Karlsruhe, Germany). The C57BL/6 syngenic MHC class I–deficient lymphoma cell line RMA-S Arginase activity. A total of 6 3 105 FACS-sorted MDSCs were washed in and the melanoma cell line B16BL6 (American Type Culture Collection, PBS. Pellets were resuspended in 100 ml lysis buffer (0.4% Triton X-100 ATCC) were cultured in RPMI 1640 and DMEM, respectively, with 10% with 10 mM Tris-HCl and protease inhibitors) for 30 min at 4˚C. Arginase FCS, 1% L-glutamine, 1% penicillin, and 1% streptomycin (Invitrogen). activity in the supernatants was measured using the QuantiChrom arginase RMA-S (5 3 105 or 106 as indicated) or B16 (5 3 105) cells were inoc- assay kit (BioAssay Systems). ulated into the right flank of mice. Tumors were measured every second or NO production. A total of 105 FACS-sorted MDSCs were cultured in third day with a caliper, and the volume (length 3 width 3 height) was 200 ml primary cell medium with or without 1 mg/ml LPS (5). Super- calculated. natants were collected after 24 h, and nitrite concentrations were analyzed according to manufacturer’s instructions (Cayman Chemical). After 10 Cell preparation and flow cytometry min, the absorbance at 540 nm was measured. Nitrite concentrations were by guest on September 26, 2021 Peripheral blood was collected, and erythrocytes were lysed. Tumors were determined by comparing the absorbance values for the test samples to removed, cut into pieces, and digested in 0.5 mg/ml hyaluronidase type V a standard curve generated by serial dilution of 0.25 mM sodium nitrite. and 0.5 mg/ml DNase I (both Sigma-Aldrich) for 30 min at 37˚C. Living ROS production. Single-cell suspensions from blood and tumor were in- cells were enriched using Lympholite M gradients (Cedarlane Laborato- cubated at 37˚C in PBS containing 2.5 mM oxidative-sensitive dye DCFDA ries). The following Abs were used for flow cytometry: anti–Gr-1 (RB6- (Invitrogen) with or without 30 ng/ml PMA for 30 min. Subsequently, cells 8C5), anti-CD11b (M1/70), anti-F4/80 (BM8), anti-Ly5.1 (A20), anti-CD3 were stained with anti–Gr-1 and anti-F4/80 mAbs and analyzed by flow (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-Foxp3 (FJK-16s), cytometry. anti-CCR5 (HM-CCR5), and Armenian hamster IgG (HTK888). Staining was performed in PBS with 1% FCS, 10% Fc block (supernatant from anti- Bioplex protein array mouse CD16/32 hybridoma, clone 2.4G2), and 0.02% sodium azide for A total of 5 3 104 FACS-sorted MDSCs were cultured in 100 ml primary 30 min at 4˚C. For CCR5 staining, cells were preincubated with anti-CCR5 cell medium for 24 h. Supernatants were collected and further diluted 1:1 mAb for 1 h at 37˚C. For intracellular staining of Foxp3, cells were fixed with Bioplex Sample Diluent (Bio-Rad). Chemokine release was simul- and stained using Foxp3 staining buffer set (eBioscience). Labeled cells taneously determined with the Bioplex System (Bio-Rad) according to the were analyzed on an FACSCalibur (BD Biosciences) by using the Cell- manufacturer’s instructions. Standard curves and concentrations were Quest program (BD Biosciences). Before cell sorting of tumor-infiltrating calculated with Bioplex Manager 4.1.1 (Bio-Rad) on the basis of the five- MDSC subpopulations by FACS, cells were pre-enriched via anti–Gr-1- parameter logistic plot regression formula. APC and anti-APC beads (Miltenyi Biotec) according to the manufacturer’s instructions. Gr-1–enriched MDSCs were sorted by FACS as Gene expression profiling and data analysis followed: PMN-MDSCs, Gr-1+CD11b+F4/8027-aminoactinomycin D2; and MO-MDSCs, Gr-1+CD11b+F4/80+7-aminoactinomycin D2. Purities MDSC subsets were sorted by flow cytometry from peripheral blood of of MDSC subsets were .96%. PBS-injected naive mice (pooled, n = 30) or from blood and tumor of RMA-S–injected mice (pooled, n = 25) 19 d after tumor cell inoculation. In vivo BrdU staining The purity of sorted cells (PMN-MDSCs, Gr-1+CD11b+F4/802, and MO- MDSCs, Gr-1+CD11b+F4/80+)was$98%. RNA was extracted from sorted RMA-S tumor–bearing or naive mice were injected with 2 mg BrdU i.p. on MDSC subsets using the RNeasy Mini Kit (Qiagen). Obtained RNA was day 18 after tumor cell inoculation. After 1 h, mice were sacrificed and treated with DNase using TURBO DNA-free kit (Ambion) and precipitated bone marrow, blood, spleen, and tumor suspensions were prepared. BrdU with linear polyacrylamide. Gene expression was determined using the incorporation in MDSCs was analyzed using the BD FITC BrdU Flow Kit Mouse Ref-8 v2 array from Illumina. Three samples generated in three (BD Biosciences) according to the manufacturer’s instructions. independent experiments per group were analyzed. cDNA synthesis and Chemokine application in vivo hybridization to arrays were performed according to the manufacturer’s instructions. Raw microarray data were processed using BeadStudio v9. A total of 1 mg rCCL3, CCL4, or CCL5 (PeproTech) were coinjected with Briefly, raw values were quantile normalized, background subtracted, and the tumor cells. Chemokines (1 mg/mouse) were reinjected intratumorally log2-transformed. Statistical and cluster analyses were performed using every third day. A total of 10 mg/mouse Met-RANTES (kindly provided by software package MeV v4.6 (MultiExperiment Viewer) from TM4 Micro- Amanda Proudfoot; Merck Serono Geneva Research Center) was injected array Software Suite. Statistical analysis of microarrays was used to identify i.p. daily for 3 wk. significantly differentially expressed genes between defined groups. A false 5604 Treg RECRUITMENT BY TUMOR-INFILTRATING MO-MDSCs discovery rate of 5% was used as a cutoff for determination of significance MDSCs from solid tumors and ROS production by PMN-MDSCs, (Gene Expression Omnibus accession number GSE41620; http://www.ncbi. indicative of the suppressive function of tumor-infiltrating MDSC nlm.nih.gov/geo/query/acc.cgi?acc=GSE41620). subsets. Statistical analysis Gene expression profiling reveals high chemokine expression in Differences between groups were calculated by unpaired Student t test or tumor-infiltrating MO-MDSCs by two-way ANOVA. Values of p , 0.05 were considered to be statisti- cally significant. To further characterize tumor-infiltrating MDSC subpopulations, we performed genomewide expression profiling using Illumina Results microarray chips (Illumina). PMN- and MO-MDSCs were isolated MDSC subsets accumulate in peripheral blood, spleen, and from peripheral blood of naive C57BL/6 mice and from blood and tumor tissue of RMA-S lymphoma–bearing mice tumor of RMA-S tumor–bearing mice when the tumor diameter reached 1.5 cm (day 19 after tumor cell inoculation). To obtain The aim of our study was to elucidate the molecular signature and highly purified MDSC subsets ($ 98%), cells were sorted by flow function of MDSC subsets in the tumor tissue of mice. As a first step, cytometry. mRNA samples obtained in three independently per- numbers of MDSC subsets were determined in mice after s.c. in- formed experiments were analyzed. Pan-genomic microarray analy- oculation of RMA-S lymphoma cells. Within 3 wk, progressive sis revealed a cluster of .400 genes that were explicitly upregulated tumor growth was accompanied by an expansion of Gr-1+ cells and + + in MO-MDSCs from tumor tissue compared with all other samples Gr-1 CD11b cells in peripheral blood and spleen (Fig. 1A). (Supplemental Table I). Moreover, 250 genes were significantly Higher amounts of CD11b+ cells were observed in spleen of tumor- + upregulated in tumor-infiltrating MO-MDSCs compared with MO- Downloaded from bearing mice. Tumor-infiltrating cells consisted mainly of Gr-1 MDSCs from blood of tumor-bearing mice (Supplemental Table and CD11b+ that comprised Gr-1+CD11b+ cells and Gr-12CD11b+ + II). Among the highly differentially expressed genes (fold change F4/80 cells that were previously described as tumor-associated .5), we detected several members of the chemokine family, in- macrophages (3). In addition, lower percentages of CD4+ Tcells, + cluding the chemokines Ccl7, Ccl24, Cxcl16, Cxcl9, Cxcl2, Ccl3, Tregs, CD8 T cells, and NK cells were present in the tumor tissue and Ccl4 (Fig. 3A, 3B). In the tumor-infiltrating PMN-MDSC (26). Two MDSC subsets, Gr-1+CD11b+F4/802 and Gr-1+CD11b+ + subset, only CCL3 was significantly increased. Because the genes http://www.jimmunol.org/ F4/80 cells, exhibited distinct morphological properties (Fig. 1B). Ccl3 and Ccl4, encoding the CCR5 ligands CCL3 (MIP-1a) and Gr-1+CD11b+F4/802 MDSCs from blood and tumor were poly- + + + CCL4 (MIP-1b), showed the strongest upregulation, protein levels morphonuclear (PMN-MDSC), whereas Gr-1 CD11 F4/80 MDSCs of all CCR5 ligands were analyzed. Indeed, tumor-infiltrating MO- displayed mononuclear features (MO-MDSC). Of note, PMN- MDSCs produced significantly higher levels of CCL3, CCL4, and MDSCs from blood and tumor expressed Ly6G and low levels 2 high CCL5 compared with all other subsets (Fig. 3C). The RMA-S cell of Ly6C, whereas MO-MDSCs displayed a Ly6G Ly6C phe- line neither produced any of these chemokines nor expressed CCR5 notype (data not shown). To determine the kinetics of PMN- and (data not shown). Moreover, tumor-infiltrating MO-MDSCs from MO-MDSC accumulation, total cell numbers of these subsets were B16 melanoma were the most potent producers of CCL3, CCL4, assessed in blood and tumors at indicated time points (Fig. 1C). and CCL5 among PMN- and MO-MDSC subsets isolated from by guest on September 26, 2021 In blood of tumor-bearing mice, both subpopulations accumulated blood and tumor (Fig. 3D). Finally, we analyzed MDSC subsets in during tumor progression, but the frequency of PMN-MDSCs was a ret transgenic mouse model of spontaneous skin melanoma. In significantly higher. In tumor tissue, PMN- and MO-MDSCs ac- this mouse model, after a short latency, ∼25% mice develop mac- cumulated with comparable kinetics, and similar cell numbers were roscopic skin melanoma with disseminated metastases in lymph detected. To address the generation/expansion of tumor-infiltrating nodes, bone marrow, lung, liver, and brain (25, 27). As shown in MDSCs, we analyzed BrdU incorporation in MDSC subsets from + Fig. 3E, highly elevated levels of CCL3, CCL4, and CCL5 were blood, spleen, bone marrow, and the tumor tissue. BrdU PMN- detected in MO-MDSCs from the peripheral blood of mice carrying MDSCs were only detected in the bone marrow at low percentages + skin melanoma as compared with nontransgenic littermates. Lower (Fig. 1D). High percentages of BrdU MO-MDSCs were mainly amounts of CCL3, CCL4, and CCL5 were detectable within the detected in the bone marrow regardless of whether naive or tumor- tumor-infiltrated MO-MDSCs. bearing mice were analyzed. In the tumor tissue, only 4% MO- MDSCs were labeled with BrdU, suggesting that their accumu- Chemoattraction of Tregs to the tumor tissue by CCL4 and lation in the tumor tissue was mainly due to their recruitment from CCL5 the bone marrow and to a lesser extent to proliferation at the tumor So far, our results indicate that CCL3, CCL4, and CCL5 were the site. most abundant chemokines produced by tumor-infiltrating MO- MDSCs. The only chemokine receptor shared by all of the three Suppressive factors are produced by tumor-infiltrating MDSC chemokines CCL3, CCL4, and CCL5 is CCR5. CCL3 and CCL5 subsets also bind to other receptors. To determine the cell population that To evaluate factors involved in suppression by tumor-infiltrating might be recruited to the tumor tissue via CCR5, immune cells from MDSC subpopulations, PMN- and MO-MDSCs were sorted from blood, spleen, and tumor were stained for CCR5 surface expres- blood and tumor of RMA-S–injected mice. MO-MDSC subsets sion. On day 17 after tumor cell inoculation, Tregs from peripheral displayed significantly higher arginase-1 and NO production as blood and spleen of tumor-bearing mice and from tumor tissue compared with their granulocytic counterparts (Fig. 2A 2B). displayed elevated levels of CCR5, whereas expression of CCR5 Tumor-infiltrating MO-MDSCs showed dramatically (28-fold) was hardly detectable on conventional CD4+Foxp32 T cells (Fig. higher levels of arginase-1 and increased levels (3-fold) of NO 4A, B). CD8+ T cells from blood and spleen expressed CCR5 at compared with blood MO-MDSCs. Furthermore, the PMN-MDSC low levels and expression of CCR5 was increased on tumor- subsets produced highly elevated levels of ROS compared with infiltrating CD8+ T cells. MO-MDSCs (Fig. 2C). Highest amounts of ROS were detected in Because Tregs exhibited high surface expression of CCR5, we PMN-MDSCs from blood. Overall, our results demonstrate high investigated whether CCL3, CCL4, or CCL5 attracts Tregs to the arginase-1 and inducible NO synthase (iNOS) activity by MO- tumor tissue in vivo. Upon intratumoral injection of recombinant The Journal of Immunology 5605 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. PMN-MDSCs and MO-MDSCs accumulate in blood, spleen, and tumor tissue. (A) C57BL/6 mice were s.c. injected with RMA-S tumor cells, and peripheral blood, splenocytes, and tumor cell suspensions were stained with anti–Gr-1 and CD11b mAbs on day 19 after tumor cell inoculation. Representative dot plots are shown. Graphs show the mean (n =4)6 SEM among living cells. (B) Cytospins from FACS-sorted PMN-MDSCs and MO- MDSCs from blood and tumor were prepared, and H&E staining was performed. Images were acquired with 3200 original magnification. (C) Absolute cell numbers of PMN- and MO-MDSCs were determined in blood and tumor at the indicated time points after tumor cell inoculation. Graph indicates the mean (n =3)6 SEM. (D) RMA-S tumor–bearing or naive mice were injected i.p. with BrdU on day 18 after tumor cell inoculation. After 1 h, BrdU uptake by PMN- and MO-MDSCs from bone marrow, blood, spleen, and tumor was analyzed by flow cytometry. Graph indicates the mean (n =3)6 SEM. Data (A– D) are representative of two independent experiments. chemokines, we observed a significant increase of tumor-infiltrating Tumor-infiltrating MO-MDSCs recruit Tregs via CCR5 + Tregs among CD4 T cells after CCL4 and CCL5 (Fig. 4C). The To validate the chemoattractive potential of tumor-infiltrating injection of CCL3 did not significantly alter the percentage of MDSC subsets, Treg migration toward tumor-infiltrating MDSCs Tregs in the tumor tissue. Taken together, these results demon- was analyzed in vitro. As shown in Fig. 5A, high numbers of Tregs strate that the CCR5 ligands CCL4 and CCL5 promote the ac- migrated toward MO-MDSCs isolated from tumor tissue compared cumulation of Tregs within the tumor tissue. with PMN-MDSCs. Importantly, Tregs from CCR5 knockout (KO) 5606 Treg RECRUITMENT BY TUMOR-INFILTRATING MO-MDSCs

shown). The lower percentages of Treg correlated with reduced RMA-S tumor growth in CCR5 KO mice (Fig. 5C). In a next step, we analyzed mice bearing B16 melanoma. In B16 tumors, Treg levels in CCR5 KO mice were significantly reduced accompanied by an increased CD8/Treg ratio (Fig. 5D). In this tumor model, no differences in the CD4/Treg ratio between CCR5 KO and wt mice were detected. Similar to the RMA-S tumor model, tumor growth of B16 in CCR5 KO mice was significantly reduced compared with wt mice (Fig. 5E). Finally, we investigated the effect of Met-RANTES, a CCL5 analog that inhibits agonist-induced activities, in wt mice after inoculation of different doses (1 3 106 or 0.5 3 106) of RMA-S tumor cells. In mice that received Met-RANTES, RMA-S tumor growth was significantly delayed compared with PBS-injected control mice regardless of whether 1 3 106 or 0.5 3 106 RMA-S cells were injected (Fig. 5F). Overall, our data indicate that CCR5 deficiency and CCR5 blockade result in delayed tumor growth.

Discussion Downloaded from Tumors use a variety of suppressive strategies to escape from antitumor immune responses. One common feature of tumor progression is the deregulation of myelopoiesis and the expansion of MDSCs. MDSCs are a heterogeneous population comprising monocytic and granulocytic cells. The characterization of MDSC

subsets present in the tumor tissue is crucial to understand their http://www.jimmunol.org/ involvement in tumor-associated immune suppression. Thus, in our study, we investigated the molecular signature, phenotype, and function of the monocytic and granulocytic MDSC subsets isolated from tumors of RMA-S lymphoma–bearing mice compared with MDSCs from blood of tumor-bearing or naive mice. FIGURE 2. Suppressive factors are produced by tumor-infiltrating + + MDSC subsets. (A) MDSC subpopulations were sorted from blood and A previous study described that Gr-1 CD11b MDSCs infil- tumor of RMA-S–injected mice (pooled, n = 30) on day 19 after tumor cell trating 3LL tumors were composed primarily of macrophages and inoculation, and arginase-1 activity was measured. Data were obtained neutrophils (29). Moreover, Movahedi et al. (4) demonstrated that from two independent experiments. (B) FACS-sorted populations of mouse splenic Gr-1+CD11b+ MDSCs comprised an Ly6G+SSChigh by guest on September 26, 2021 MDSCs (pooled, n = 20) were stimulated with 1 mg/ml LPS for 24 h, polymorphonuclear (PMN) and an Ly6G2SSClow mononuclear supernatants were collected, and the nitrite/NO concentration was mea- (MO) subset (4). MO-MDSCs expressed the macrophage marker sured. (C) MDSCs from blood and tumor cell suspensions were pregated + + 2 + + + F4/80 that was absent on PMN-MDSCs. In concordance with on Gr-1 CD11b F4/80 (PMN) and Gr-1 CD11b F4/80 (MO), and ROS previous reports (4, 5, 30) we observed that PMN-MDSCs from production was determined by flow cytometry. The graph depicts mean blood and tumor expressed Ly6G, low levels of Ly6C, whereas fluorescence intensity (MFI) of DCFDA after 30 min of PMA stimulation. 2 high Values represent the mean (n =3)6 SEM. Data are representative of two MO-MDSCs displayed a Ly6G Ly6C phenotype. Both pop- (B) and three (C) independent experiments. *p , 0.05, **p , 0.01, ***p , ulations expressed CD124 [(31), data not shown]. In our study, we + + 2 + 0.001. ND, Not detected. characterized Gr-1 CD11b F4/80 PMN-MDSC and Gr-1 CD11b+F4/80+ MO-MDSC subsets in blood and tumor tissue. Our results reveal that both subpopulations increased in numbers mice almost completely lost the ability to migrate toward che- during tumor growth in blood, spleens, and tumors. PMN-MDSCs mokines secreted by MO-MDSCs (Fig. 5A). Of note, similar de- represented the most prominent subset in the blood, whereas ac- velopment of Tregs in CCR5-deficient and wt mice was described cumulation of MO-MDSCs was more pronounced in the tumor (28). tissue. Our experiments, addressing the proliferation of the two MDSC CCR5 deficiency and CCR5 blockade result in delayed tumor subpopulations by BrdU labeling, revealed a high turnover rate of growth MO-MDSCs in the bone marrow and only low percentages of To analyze the effect of CCR5 deficiency in vivo, the amounts of BrdU+ MO-MDSCs in spleen and tumor. These data suggest that tumor-infiltrating conventional CD4+Foxp32, CD8+ T cells, and MO-MDSCs expand in the bone marrow and migrate through the Tregs from wt or CCR5-deficient mice were assessed. Analysis blood stream to spleen and tumor, where they proliferate at low of lymphocyte populations revealed significantly reduced levels levels. Comparatively low percentages of BrdU+ PMN-MDSCs of Tregs in the tumor tissue of RMA-S tumor–bearing CCR5- were detected in the bone marrow but not in the peripheral deficient mice (Fig. 5B). Furthermore, higher ratios of CD4/ blood, spleen, or tumors, suggesting that these cells might migrate Tregs and CD8/Tregs were detected in tumors of CCR5 KO mice from the bone marrow to the tumor tissue and/or might arise from compared with wt mice. Decreased Treg numbers were also precursors other than Gr-1+CD11b+F4/802. These results are in detected at late stage tumors (day 19) from CCR5 KO mice (data line with a recent study addressing the turnover of MDSC sub- not shown). Although CCR5 was expressed by MO-MDSC from populations in the 3LL tumor model (29). blood and tumor, similar amounts of tumor-infiltrating MO- Several tumor-derived factors such as GM-CSF (32), Cox-2 MDSC were observed in CCR5 KO and wt mice (data not (33), S100A8/A9 (34), IL-1b (35), or IFN-g (4) mediate expan- The Journal of Immunology 5607 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Tumor-infiltrating MO-MDSCs express high levels of chemokines including the CCR5 ligands CCL3, CCL4, and CCL5. (A) Differentially expressed genes of the chemokine family were identified by microarray analysis on the different subsets of MDSCs. The heat map represents selected members of the chemokine family significantly upregulated in the tumor-infiltrating MO-MDSC subset. Expression is displayed as log2 intensity value generated from normalized microarray data. (B) Log2 intensity values of chemokine gene expression for Ccl3, Ccl4, Cxcl2, Cxcl9, Ccl7, and Ccl24 in different MDSC subsets are depicted. (C) MDSC subsets were FACS-sorted 19 d after RMA-S cell inoculation (pooled, n = 15) and cultured in vitro for 24 h. Supernatants were analyzed for chemokine production by Bioplex (Bio-Rad). Data are representative of three independent experiments. (D) B16 tumor cells were s.c. injected into mice. MDSC subsets from blood and tumor (pooled, n = 15) were purified on day 17 after tumor cell inoculation, and chemokine production was measured as described in (C). (E) MDSC subsets from the peripheral blood and skin tumors of ret transgenic mice (pooled n = 20) were purified, and the chemokine production was measured as described in (C). Graphs (B–E) represent the mean 6 SEM of cultures set up in triplicates. MO, MO-MDSC; PMN, PMN-MDSC. sion and activation of MDSC subsets and induce their suppressive duced iNOS expression was further increased upon addition of activity (36). Our data indicate that MDSC subpopulations from IL-13 (38). Thus, it is likely that also other more physiological blood and tumors differ in their capacity to produce factors in- stimuli such as IFN-g/IL-13 would induce NO from tumor- volved in T cell suppression. PMN-MDSCs exhibited high levels infiltrating MO-MDSCs. Corzo et al. (39) demonstrated rapid of ROS in blood, lower levels in the tumor tissue, and negligible upregulation of arginase-1 and iNOS in i.p. CD11b+Gr-1+ MDSCs arginase-1 or iNOS activity. In contrast, tumor-infiltrating MO- in a tumor model of EL-4 lymphoma growing in the peritoneum. MDSCs displayed increased amounts of arginase-1 and low lev- The upregulation of iNOS and arginase-1 and the decreased levels els of ROS. In our study, MDSC subsets were stimulated with LPS of ROS were mediated by the hypoxia-inducible factor-1a. for NO production. Only tumor-infiltrating MO-MDSC produced Moreover, other transcription factors like STAT1, STAT6, or C/ high amounts of NO. In previous studies, similar levels of NO EBPb were reported to be involved in the regulation of MDSC were induced upon stimulation with IFN-g (37). The IFN-g–in- activity (40–42). The transcription factors mediating the sup- 5608 Treg RECRUITMENT BY TUMOR-INFILTRATING MO-MDSCs Downloaded from http://www.jimmunol.org/

FIGURE 4. CCR5 ligands, CCL4 and CCL5, increase Treg numbers in the tumor tissue. (A) Immune cells from blood, spleen, and tumor (n = 3) were stained with anti-CCR5 mAb 19 d after tumor cell inoculation. Cells were pregated as follows: Tregs, CD4+Foxp3+CD25+; CD8, CD8+CD3+CD42; conventional CD4, CD4+CD3+CD82Foxp32. Representative histograms are shown (left panel). (B) Graph (right panel) shows the mean fluorescence intensity (MFI) (n =3)6 SEM and the mean percentage (n =3)6 SEM of CCR5+ cells. (C) C57BL/6 mice were s.c. injected with RMA-S cells and intratumorally with 1 mg rCCL3, CCL4, CCL5, or PBS every third day. Percentages of tumor-infiltrating Tregs within total CD4+ T cells were assessed on day 13 after tumor cell inoculation. Graph represents the mean (n =5)6 SEM. Data are representative of two (A, B) and three (C) independent experiments. by guest on September 26, 2021 *p , 0.05. pressive activity of MDSCs in our model are currently under in- CCL5–CCR5 interactions have already been linked to the tumor vestigation. microenvironment (45). However, several aspects of CCR5 and its Increasing evidence exists that chemokines recruiting inflam- ligands in cancer have remained unclear. In concordance with our matory cells to the tumor tissue are produced not only by cancer results indicating a protumoral role of CCR5/CCR5 ligands in cells, but also by tumor-infiltrating cells (43). In our study, global cancer, CCL5 secretion by mesenchymal stem cells enhanced the gene expression profiling of MDSC subsets from blood and tumor metastatic potential of breast cancer (46). Ng-Cashin et al. (47) revealed distinct chemokine patterns. Purified MO-MDSCs isolated demonstrated that the deficiency of CCR5 or its ligand CCL3 from the tumor tissue of RMA-S lymphoma– or B16 melanoma– resulted in delayed or accelerated melanoma growth depending on bearing mice secreted high amounts of chemokines including the tumor load. Furthermore, in a mouse model of pancreatic CCL3, CCL4, and CCL5. To address the chemokine production by adenocarcinoma, knockdown of CCL5 production in tumors MO-MDSCs in a spontaneous autochthonous mouse model, skin resulted in the delayed tumor growth (20). In addition, CCL5/ cancer–bearing ret transgenic mice were analyzed. Highly increased CCR5 blockade improved the efficiency of immunochemother- levels of CCL3, CCL4, and CCL5 were found in MO-MDSCs from apy (48). In our models, tumor-infiltrating CD8+ T cells expressed blood. Lower levels of these chemokines were detected in MO- CCR5 but their accumulation in tumors in CCR5-deficient mice MDSCs from tumors. It is likely that in this model disseminated was higher compared with wt mice, indicating that CCR5 ex- melanoma metastases also affected MO-MDSCs in the blood. Of pression was not required for their tumor infiltration. In contrast, note, other myeloid cells present in the tumors such as Gr-12 tumor- CCR5-deficient mice exhibited lower numbers of CD4+ and CD8+ associated macrophages also produced certain chemokines (data not T cells and similar amounts of Tregs infiltrating pancreatic car- shown). The chemokine production by non-MDSC myeloid subsets cinoma compared with wt mice (49). The diverging tumor- and their function in the tumor microenvironment will be addressed promoting versus tumor-delaying results described for CCR5/ in future studies. Previously, it has been reported that MDSCs can CCR5 ligands might be explained by the use of different tumor produce certain chemokines (44), but the chemokine production by models. We believe that the differential ability of tumor cells to MDSC subsets from different organs and the impact of MDSC- produce certain chemokines by themselves, the immunogenicity derived chemokines on the recruitment of immune cells to the of tumor cells, and the types and amounts of tumor-infiltrating tumor tissue has not been addressed so far. In this study, we show immune cells greatly influence the contribution of the CCR5/ for the first time, to our knowledge, that chemokine secretion by CCR5 ligand axis to the outcome of antitumor responses. tumor-infiltrating MO-MDSCs results in direct Treg recruitment Previously, we have shown that depletion of Tregs in RMA-S dependent on CCR5. lymphomas increased antitumor immunity mediated by NK and The Journal of Immunology 5609 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Tumor-infiltrating MO-MDSCs attract Tregs in a CCR5-dependent manner. (A) Tumor-infiltrating MDSC subsets were purified by FACS on day 19 after tumor cell inoculation (pooled, n = 30) and 5 3 105 (left panel) and 4 3 105 cells (right panel) were cultured for 18 h in the lower chamber. Preactivated Tregs from naive C57BL/6 wt (pooled, n = 6) or CCR5 KO mice (pooled, n = 6) were added to the upper chamber, and the percentage of migrated Tregs was assessed after 5 h. Values represent the mean (left panel, n =3;right panel, n =2)6 SEM. (B) Amounts of immune cells in the tumor tissues of wt or CCR5 KO mice were analyzed on day 11 after RMA-S tumor cell inoculation. Percentages of Tregs within total CD4+ T cells and the CD4/ Treg and CD8/Treg ratios are depicted. (C) wt and CCR5-deficient mice were s.c. injected with RMA-S cells. Tumor growth was assessed for 19 d. (D) Amounts of immune cells in the tumor tissues of wt or CCR5 KO mice were analyzed on day 14 after B16 tumor cell inoculation. Percentages of Tregs within total CD45+ cells and the CD4/Treg and CD8/Treg ratios are depicted. (E) wt and CCR5-deficient mice were s.c. injected with B16 cells. Tumor growth was assessed for 14 d. (F) C57BL/6 mice were s.c. injected with RMA-S cells and i.p. with 10 mg Met-RANTES or PBS every day. Tumor growth was determined as indicated. Data are representative of three independent experiments. Values (B–F) represent the mean (B, n =4;C, n =5;D, E, n = 12; F, n =9)6 SEM. *p , 0.05, **p , 0.01.

T cells, supporting the suppressive role of Tregs in our model (26). in RMA-S tumors and not in B16 tumors from CCR5 KO mice. It Because Tregs are able to suppress T cells at the tumor site (11) is likely that in B16 tumors, the composition of CD4+ T cells and their accumulation in tumors correlates with bad prognosis within the tumor tissue differs from RMA-S tumors due to the (16), the identification of factors involved in Treg accumulation in different tumor microenvironment. At this point, we cannot for- tumors is of high clinical relevance. Our results demonstrate that mally exclude that in addition to CCR5, other mechanisms are also tumor-infiltrating MO-MDSCs directly mediate the migration of directly involved in recruitment of Tregs to tumors in vivo. Tregs via CCR5 in vitro. In vivo, deficiency of CCR5 resulted in Collectively, we demonstrate for the first time, to our knowledge, reduced Treg numbers in the tumor tissue in RMA-S and B16 that the monocytic subset of tumor-infiltrating MDSCs is a potent tumor–bearing mice. Increased CD4/Treg ratios were only detected producer of chemokines resulting in CCR5-dependent Treg re- 5610 Treg RECRUITMENT BY TUMOR-INFILTRATING MO-MDSCs cruitment favoring lymphoma growth. These findings illustrate an suppression and T regulatory cell activation mediated by myeloid-derived suppressor cells in cancer. Cancer Res. 70: 99–108. important role of tumor-infiltrating MO-MDSCs in Treg recruit- 22. Yang, R., Z. Cai, Y. Zhang, W. H. Yutzy, IV, K. F. Roby, and R. B. Roden. 2006. ment, defining a novel aspect of the suppressive network of CD80 in immune suppression by mouse ovarian carcinoma-associated Gr-1 MDSCs and Tregs within the tumor microenvironment. Because +CD11b+ myeloid cells. Cancer Res. 66: 6807–6815. 23. Serafini, P., S. Mgebroff, K. Noonan, and I. Borrello. 2008. Myeloid-derived in addition to CCR5, other chemokine receptors (19, 50) have also suppressor cells promote cross-tolerance in B-cell lymphoma by expanding been implicated in the recruitment of Treg to tumor tissue, a regulatory T cells. Cancer Res. 68: 5439–5449. combination of chemokine receptor antagonists, including an in- 24. Dilek, N., N. Poirier, C. Usal, B. Martinet, G. Blancho, and B. Vanhove. 2012. Control of transplant tolerance and intragraft regulatory T cell localization by hibitor for CCR5, should be considered for the development of myeloid-derived suppressor cells and CCL5. J. Immunol. 188: 4209–4216. innovative immunotherapeutic strategies against cancer. 25. Kato, M., M. Takahashi, A. A. Akhand, W. Liu, Y. Dai, S. Shimizu, T. Iwamoto, H. Suzuki, and I. Nakashima. 1998. Transgenic mouse model for skin malignant melanoma. Oncogene 17: 1885–1888. Acknowledgments 26. Galani, I. E., M. Wendel, A. Stojanovic, M. Jesiak, M. M. Mu¨ller, C. Schellack, We thank Dr. Amanda Proudfoot and Dr. Peter Nelson for providing Met- E. Suri-Payer, and A. Cerwenka. 2010. Regulatory T cells control macrophage accumulation and activation in lymphoma. Int. J. Cancer 127: 1131–1140. RANTES and Dr. Alexander Ro¨lle and Dr. Sonja Textor for critical review 27. Umansky, V., O. Abschuetz, W. Osen, M. Ramacher, F. Zhao, M. Kato, and of the manuscript. D. Schadendorf. 2008. Melanoma-specific memory T cells are functionally ac- tive in Ret transgenic mice without macroscopic tumors. Cancer Res. 68: 9451– 9458. Disclosures 28. Yurchenko, E., M. Tritt, V. Hay, E. M. Shevach, Y. Belkaid, and C. A. Piccirillo. The authors have no financial conflicts of interest. 2006. CCR5-dependent homing of naturally occurring CD4+ regulatory T cells to sites of Leishmania major infection favors pathogen persistence. J. Exp. Med. 203: 2451–2460. 29. Sawanobori, Y., S. Ueha, M. Kurachi, T. Shimaoka, J. E. Talmadge, J. Abe, Downloaded from References Y. Shono, M. Kitabatake, K. Kakimi, N. Mukaida, and K. Matsushima. 2008. 1. Rabinovich, G. A., D. Gabrilovich, and E. M. Sotomayor. 2007. Immunosup- Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in pressive strategies that are mediated by tumor cells. Annu. Rev. Immunol. 25: tumor-bearing mice. Blood 111: 5457–5466. 267–296. 30. Huang, B., P. Y. Pan, Q. Li, A. I. Sato, D. E. Levy, J. Bromberg, C. M. Divino, 2. Sica, A., and V. Bronte. 2007. Altered macrophage differentiation and immune and S. H. Chen. 2006. Gr-1+CD115+ immature myeloid suppressor cells me- dysfunction in tumor development. J. Clin. Invest. 117: 1155–1166. diate the development of tumor-induced T regulatory cells and T-cell anergy in 3. Gabrilovich, D. I., and S. Nagaraj. 2009. Myeloid-derived suppressor cells as tumor-bearing host. Cancer Res. 66: 1123–1131.

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Original row SYMBOL ProbeID Fold change(Unlogged) 19916 Osm 106510484 4,02 43509 Cope 6620279 1,72 32260 F10 3830450 4,41 37779 Tcerg1 5220746 1,13 12873 G630014P10Rik 104150037 1,15 32314 Nol6 3840021 1,13 38953 Adamts20 5570059 1,16 15503 Stab1 105080047 3,08 32361 Cd274 3840180 3,00 38944 Impg1 5570025 1,14 13935 D230015O06Rik 104570039 1,15 30498 Clic4 3290368 1,28 5402 LOC384907 101850435 1,10 2723 scl0001576.1_111 100870121 1,25 31650 BC004044 3710195 1,25 18415 6720403M19Rik 106110204 1,43 45110 Adssl1 7000288 1,60 18398 Arvcf 106110131 1,17 16004 C630024B01Rik 105270603 1,54 39036 4921509E07Rik 5570465 1,13 46070 Bcl2l11 780044 1,49 28238 Rab20 2640601 1,35 28342 Selenbp1 2650372 2,65 12013 A730052K04Rik 103940164 1,14 16315 5430416B10Rik 105360092 1,34 19196 2610029J22Rik 106350711 1,33 41761 Stx4a 6270632 1,43 37584 0610031J06Rik 520397 1,35 40245 Pou2f1 5910056 1,14 15845 E130318A13Rik 105220451 1,47 33354 Ramp3 4060215 1,17 14065 B020006M18Rik 104590066 1,14 30383 2410042D21Rik 3190500 1,11 35418 Mosc1 4590452 1,17 44720 Tcirg1 6900619 2,30 31243 Hist1h4d 3520398 2,60 39879 Actr10 5860458 1,14 40695 H2afy3 6040433 1,17 46262 Fgf11 840292 1,15 38978 4930583H14Rik 5570142 2,09 46456 Pitx2 870537 1,12 46121 4930431B09Rik 780301 2,02 40875 Pla1a 6100204 1,25 13620 F630001K14Rik 104480452 2,94 38322 Hnrpdl 5360471 1,69 42819 H2‐DMb1 6450593 3,79 35673 Egr1 4610347 2,44 38089 Dcn 5340026 1,17 8340 LOC634448 102650528 1,13 29356 Cd40 2940390 2,25 45599 Osr2 70711 1,20 36290 B3gnt5 4780528 1,28 26064 Appl2 2120280 2,21 36773 Necap1 4920750 1,58 42947 Saa3 6510390 3,88 12172 3110027N22Rik 103990390 1,58 2529 LOC383885 100780546 1,16 38318 Tgm2 5360452 3,83 40508 Il1rl1 6020347 1,18 35306 Snx30 4570632 2,04 14264 Hspa1b 104610450 1,27 7914 LOC382258 102570195 1,18 20388 LOC381188 106620181 1,15 21113 BC049349 106860044 1,09 27200 H2‐Ab1 2370433 5,46 36167 Socs2 4760692 1,51 16064 Mterfd3 105290152 1,16 44124 Mapk6 6760520 2,02 25714 Vim 20431 2,11 30648 Snx2 3360364 1,66 35147 LOC640441 4560494 1,28 3357 Ern1 101170717 1,16 24826 Erg 1770739 1,19 29697 Ms4a6d 3120035 2,00 36706 Gzmd 4920368 1,30 31597 Olfr1118 3710021 1,14 15962 D030016M11Rik 105270341 1,16 28561 Dusp4 2690044 1,80 46191 Fcgr2b 780750 3,66 5299 4932443E23Rik 101780672 1,13 17057 Tens1 105670671 2,68 38117 LOC671462 5340128 1,63 39200 Ifng 5670592 1,39 29162 LOC667266 2900121 1,14 41130 Slc27a1 6110093 1,53 37556 Mfap4 520181 1,13 9397 E130203E12Rik 102970746 1,15 41030 4930589M24Rik 610315 1,14 27423 Snx2 2470068 1,44 19316 3010031K01Rik 106370605 1,25 27495 Rsc1a1 2470397 1,65 34004 Lmna 4210020 2,63 25533 Tnnc1 1990575 1,15 15872 Myo5a 105220632 1,14 39560 Renbp 5720332 3,34 11388 E130319N12Rik 103800332 1,13 34238 LOC641240 4230427 5,48 6038 A630059F13Rik 102060333 1,23 28999 1700123K08Rik 2850025 1,13 38745 Bcl2l1 5420484 1,24 2038 Osm 100630577 2,76 21594 4930563C04Rik 106980537 1,17 30483 Igf1 3290280 1,16 2492 A830081I21Rik 100780332 1,25 11989 D530043N20Rik 103940079 1,38 21821 7330410H16Rik 107040288 1,19 28737 Cpne9 2760110 1,12 37601 Lmna 520471 2,41 27761 Apba3 2510348 2,20 27870 LOC675179 2570133 1,15 23825 Lmna 1500075 3,02 34453 Eif2b2 4290048 1,89 41831 Sfn 6290301 1,16 22776 Clic4 1230025 2,44 21430 Grasp 106940324 1,40 39424 Pde6c 5700292 1,15 32104 Tmcc3 380398 1,14 42177 Hipk2 6350647 1,38 14375 Cspg2 104670333 1,19 37092 Ccrk 5080112 2,46 28312 Il17rd 2650156 1,10 19529 LOC633961 106400324 1,15 30929 1600014C23Rik 3440239 1,15 44486 Lag3 6860112 1,46 12760 Tatdn2 104120072 2,00 44621 E130112H22Rik 6900068 1,16 43169 Pmp22 6550072 1,13 39750 Smurf1 580647 1,18 34700 Foxo3a 4480451 1,15 18038 2010013I23Rik 106020278 1,49 32831 Disc1 3940487 1,37 1978 Slco2a1 100630176 1,20 44334 Mgat4b 6840100 1,21 25446 Gzme 1990112 1,24 8244 Tanc2 102640739 1,13 44941 Icam1 6980138 2,65 36872 Ccrn4l 50438 2,18 26249 Cables1 2190504 1,24 39404 Bcl2a1b 5700138 3,08 29073 Tgif1 2850403 1,63 13391 D630011N09Rik 104230707 1,19 14575 Camk2d 104760021 1,50 27769 Tnfrsf9 2510400 1,28 19026 Cul1 106290411 1,72 38524 Zbtb43 540022 1,14 44362 Dusp3 6840215 1,41 1594 Il1a 100540167 1,29 26168 Nfkbie 2190086 1,33 17385 Scamp1 105720369 2,11 44105 Hspb1 6760435 1,45 41509 Pcqap 6200273 1,54 31989 Gipc2 3800463 1,33 28445 Spon2 2680136 1,13 25904 Tpi1 2100154 2,76 41044 Cxcl2 610398 3,79 24477 Ctsz 1690364 2,53 45877 Fabp5 730452 1,21 39907 Ube3a 5860609 1,19 27132 Bcl2a1b 2370092 3,53 36410 Paox 4810379 1,24 23696 Ccrk 1450161 2,12 9192 PEBP2aB 102940156 1,31 29904 Dpep2 3130309 4,69 25496 Ddb2 1990369 2,06 41065 Etv6 610524 1,54 83 LOC546212 100050398 1,17 405 2900084C01Rik 100110079 1,30 39059 Cd14 5570600 4,07 43678 Plekhm2 6650438 1,79 8345 LOC672207 102650563 1,94 38505 Stab1 5390707 2,22 44791 St6galnac4 6940161 1,41 30360 Dpep2 3190364 4,03 40458 Folr2 6020075 1,25 38543 Pgm2 540092 2,40 44719 9330175E14Rik 6900609 1,12 29451 Gab1 2970156 3,21 46557 LOC674367 940253 1,13 41041 St7 610373 1,73 14747 Acp2 104780162 1,93 4535 6330414G02Rik 101580021 1,46 41896 Cntn3 6290725 1,14 38292 Slc9a7 5360301 1,25 18958 Frap1 106290044 1,26 45290 Kcnk4 7040064 1,22 21500 E030040L08Rik 106980026 1,60 24584 Adfp 1740102 3,12 40302 LOC675521 5910341 2,95 1317 Rbm11 100510112 1,16 24849 Plekha1 1780059 1,29 25053 Ikbke 1850332 2,24 5119 2610207I05Rik 101770341 1,18 4874 Chk 101690441 1,20 34117 Slc17a6 4210576 1,51 31071 Ddhd1 3450241 2,35 29437 Clcn7 2970095 1,97 13672 Zfhx1b 104540040 1,90 34660 Srxn1 4480239 1,18 15389 Traf1 105050288 2,58 31075 Errfi1 3450273 2,24 30424 Cspg2 3290017 1,57 38358 LOC623133 5360707 1,10 42035 Pfkfb3 630706 2,49 43173 Clec4n 6550086 3,84 43634 P2rx4 6650162 2,68 26832 H2‐Eb1 2350079 5,93 37768 Serpina3f 5220692 1,50 10684 LOC625098 103440671 1,49 31006 Traf1 3440735 1,86 8735 Depdc1a 102760538 1,16 23965 Nab2 1570014 1,41 41590 Gcap27 6200736 1,98 34443 Ednrb 4280717 3,16 23866 Sh2b2 1500278 1,86 3351 D630023B12Rik 101170673 1,22 40168 Rog 5900397 1,50 35422 2810405K02Rik 4590484 1,83 25609 Hbegf 2030156 1,26 41029 1700096C12Rik 610309 1,33 38826 Rbpj 5550136 1,27 43379 Cndp2 6590333 2,29 43711 Tnf 6650603 3,06 40228 2810055G22Rik 5910017 1,22 44219 Ngfrap1 6770195 1,79 28655 Cxcl1 2690537 4,31 43744 Crem 6660041 1,51 34476 Rab15 430070 2,96 1220 Dock4 100460242 1,32 19507 BC037006 106400170 1,36 27707 Tbc1d1 2510072 1,31 37672 Pdcd1lg2 5220114 1,31 36338 H2‐Ab1 4810050 7,25 36138 Mid1 4760458 1,42 29890 Srxn1 3130195 1,31 45613 Stac2 7100022 1,59 36384 Nr1h3 4810215 4,53 36474 Mir16 4850014 1,66 33177 Cdkn1a 4050088 5,48 39047 Hist1h4c 5570528 1,73 31083 Snx8 3450315 2,70 5870 Tmem65 102030086 1,61 46511 Ecm1 940068 3,00 43942 Slc39a14 670300 1,27 41498 Nt5dc2 6200176 3,60 15349 A730085F06Rik 105050068 1,40 20726 Mitf 106760093 1,31 5448 Serpine2 101940008 1,74 32452 Tes 3840736 1,37 28323 C2ta 2650242 3,89 44574 Havcr2 6860592 3,74 17248 Hspa1b 105700270 1,41 39709 Nfkbie 580390 2,38 19919 B230319K02Rik 106510504 1,27 41343 P2rx5 6130435 1,32 29936 Car5b 3130465 1,23 33067 Pmp22 4010239 1,19 33492 Prg2 4070176 5,28 42598 Cdkn1a 6400706 8,08 41078 Serpina3g 610600 6,23 44555 Fabp3 6860452 1,70 36635 Areg 4920025 1,27 27348 Cxcl10 2450408 1,26 40244 Cspg2 5910053 1,57 28782 Rassf1 2760364 1,51 27138 Lrp12 2370112 2,82 38202 Ces2 5340609 1,19 27740 Spag9 2510180 1,27 42040 Srxn1 630731 1,63 41484 Traf6 6200132 1,42 29308 Zfand2a 2940112 2,32 29917 Igf1 3130377 1,31 34470 Ccl4 430047 10,23 40933 P2ry14 6100497 2,93 30994 1700113I22Rik 3440647 1,30 20854 Il1rn 106770079 2,97 28874 Ccl3 2810092 10,12 7765 scl0001259.1_60 102510131 1,42 15184 Nr4a2 104920411 1,22 3761 Hs3st3b1 101340047 2,21 44584 Socs7 6860671 1,22 31463 Il1a 3610056 1,37 23313 Mir16 1340471 2,22 8447 Synpo 102680377 1,48 15972 A630076G18Rik 105270390 1,27 24687 Hmox1 1740687 2,64 28347 Pcgf5 2650403 1,59 44253 Cox7a1 6770427 1,48 43899 Dnase1l3 670086 1,54 4348 Hipk2 101500403 1,40 45420 Serpinb2 70487 1,21 20670 Sh3pxd2b 106660487 2,91 32554 Tff1 3850452 1,66 25550 Slc39a14 1990711 1,25 23111 2310016C08Rik 130152 6,04 25122 Scamp1 1940020 1,27 46243 Jun 840170 3,60 36141 Ccrl2 4760471 6,96 31511 Lgmn 3610301 9,11 1487 Slc6a6 100520301 1,47 37304 Cttnbp2nl 510451 2,46 40463 Pdgfa 6020095 1,53 33770 Cd83 4150242 2,23 36162 4833422F24Rik 4760609 4,37 39354 Slc7a8 5690672 3,41 39041 Insl6 5570497 2,49 29182 Car13 2900184 1,72 17553 Cxcl9 105860594 3,22 14411 A430084P05Rik 104670551 3,40 39811 Rhov 5860100 2,23 28577 Fndc3a 2690097 1,96 7103 Spint1 102360088 7,76 1225 Ifrd1 100460286 1,38 46082 Rgs16 780091 1,85 34524 Thbs1 430288 1,56 32312 Cd276 3840019 1,60 37775 Gja1 5220731 2,06 14577 Raet1d 104760025 1,29 45384 Bhlhb2 7040603 2,45 5347 2210008N01Rik 101850102 1,44 38437 Ecm1 5390309 1,37 41948 Car5b 630176 1,33 15240 Vat1 1050040 2,54 4052 Aps‐pending 101410239 1,47 32533 Hyal1 3850341 1,37 19134 Hak‐pending 106350253 1,31 30816 Cd86 3390471 1,73 39287 Casp6 5690239 4,12 36857 Ccl4 50368 9,58 31975 Egr2 3800403 1,60 44453 Dnmt3l 6860017 2,79 45480 Eps8 7050204 1,72 14509 Slc23a1 104730368 1,37 35738 Pdpn 4640280 2,50 32807 Slc6a8 3940341 1,56 30803 Gpnmb 3390403 1,40 46146 Mtss1 780435 1,59 39634 Adamtsl5 5720750 1,35 8846 3830421F03Rik 102810433 1,51 25951 Pvr 2100450 1,46 7185 9430049I24Rik 102360576 1,66 41083 Fgd6 610632 1,33 36919 Prr15 5050133 1,50 35334 Pla1a 4590040 1,41 36648 Mmp12 4920070 1,33 35082 Cstb 4560114 2,67 40183 BC025833 5900465 1,41 34555 Ralgds 430463 1,36 37223 Pld3 510041 3,15 21748 E230029F23Rik 107000673 5,29 40581 Nr4a2 60273 1,44 38144 Dok2 5340273 5,09 42873 Plau 6510047 2,55 30323 Il4i1 3190161 2,64 40887 Hebp1 6100279 2,44 15657 Hspa1a 105130121 4,13 46266 Cish 840315 7,19 35208 Tmem49 4570088 2,58 36038 Ccl2 4760019 2,38 23113 Vdr 130156 1,83 24875 Gpnmb 1780138 15,86 40897 X99384 6100347 1,80 1739 Ear7 100580300 2,27 24088 Cxcl9 1570673 13,80 3882 Hspa1b 101400026 12,41 33178 Slc7a2 4050091 1,62 42617 Mertk 6420056 2,80 14576 Hspa1a 104760022 11,61 25233 Atf3 1940546 6,75 36445 Pgf 4810593 1,42 30610 Pim3 3360138 2,70 33373 Rgs1 4060347 2,38 5150 LOC381140 101770541 4,88 45807 Mrc1 730097 9,70 35015 Dnahc2 4540563 2,05 30294 Spint1 3190059 2,73 34956 Rgs1 4540181 8,96 24713 Edn1 1770047 1,62 26144 Ccl12 2120746 6,39 127 Il1rl2 100050707 1,67 2960 Timp1 1010326 2,80 30533 Dmkn 3290575 10,52 41615 Dscr1 6220039 1,59 37269 Cxcl16 510278 6,85 23030 C330008K14Rik 1240576 2,17 9773 Dab2 103130670 1,84 8407 A930007I19Rik 102680129 2,56 33663 Emp1 4120438 14,46 40296 C1qb 5910292 10,16 45162 Ccl12 7000592 7,19 39574 Snx24 5720398 3,21 35095 Gpnmb 4560156 6,16 27537 Spp1 2470609 13,67 35893 Gdf15 4730017 3,67 25286 Ccr5 1980072 2,53 23834 Rgl1 1500097 7,28 37308 Mmp13 510471 8,75 46009 Rai14 770288 3,35 42904 Anpep 6510138 2,53 38701 Rab3il1 5420176 2,38 38502 C1qa 5390687 3,24 40588 Dab2 60309 7,86 37149 F7 5080411 4,46 39400 C1qc 5700131 5,35 43897 Mmp14 670079 23,59 28368 Ccl7 2650519 43,08 28805 Flrt3 2760497 4,07 27440 Sphk1 2470113 5,05 43911 Ccl24 670129 25,75 42285 Sdc4 6370411 7,07 46329 Arg1 840707 23,34

Genes significantly upregulated in MDSCs (RMA‐S tumor) compared to all other MDSC subsets (naïve blood and RMA‐S blood). False discovery rate (FDR) of 5 % was utilized as a cutoff for determination of significance.

Supplementary Table 2

Original row SYMBOL ProbeID Fold change(Unlogged) 26131 Speer4b 2120673 1.15 33178 Slc7a2 4050091 1.53 37799 Msi2 5270039 2.02 14576 Hspa1a 104760022 11.60 3819 AI324046 101340390 3.55 2834 5830436K05RIK 100940048 1.49 39766 Olfr900 580739 1.18 44584 Socs7 6860671 1.29 19888 F730045P10Rik 106510315 1.81 46281 Hist1h3i 840398 2.68 26832 H2‐Eb1 2350079 4.99 29364 Birc2 2940435 1.23 37601 Lmna 520471 2.49 41083 Fgd6 610632 1.30 8345 LOC672207 102650563 2.23 23113 Vdr 130156 1.59 37223 Pld3 510041 4.15 42120 B930007L02Rik 6350286 1.96 44574 Havcr2 6860592 2.53 42166 Impact 6350551 1.85 29657 Tjp3 3060551 1.22 7765 scl0001259.1_60 102510131 1.42 2738 4930401A07Rik 100870168 1.18 2038 Osm 100630577 5.10 29525 Sh2d2a 2970594 7.66 23594 Zap70 1410494 4.87 1201 6720411P22Rik 100460128 1.21 34299 Yif1b 4260307 2.62 37269 Cxcl16 510278 5.67 40581 Nr4a2 60273 1.52 11581 E430031D18Rik 103830750 2.57 30612 Lck 3360142 8.07 26911 Thop1 2350450 1.49 31195 Mxd1 3520132 1.44 17665 Myo1e 105890411 6.46 36706 Gzmd 4920368 1.45 14160 Ptafr 104590601 1.46 3845 A130082M07Rik 101340537 11.18 7600 Ptafr 102470687 1.98 13324 9130227C08Rik 104230239 4.34 35208 Tmem49 4570088 2.54 2960 Timp1 1010326 2.73 7041 A130014O09Rik 102350475 1.94 28276 Aqp9 2650047 2.17 40184 Zfp219 5900471 1.73 29257 Gadd45a 2900717 2.48 3076 Ptpn2 101050619 1.57 6213 9626962_229 102100670 28.59 2679 9626100_15 100840706 26.92 39296 Scd2 5690309 4.51 30441 4932417H02Rik 3290066 1.22 43711 Tnf 6650603 5.25 34215 Insl5 4230300 14.84 34165 Junb 4230048 2.14 44460 Trem1 6860035 1.83 41887 Aldoa 6290672 1.93 10718 Gm525 103450064 11.41 23825 Lmna 1500075 3.46 38701 Rab3il1 5420176 2.12 35015 Dnahc2 4540563 1.78 43660 Maged1 6650332 2.35 2232 2310036D04Rik 100730161 1.29 39709 Nfkbie 580390 2.33 8301 9626953_200 102650253 29.47 38122 Slc25a33 5340138 2.51 31461 Tcrb‐V8.2 3610048 13.33 40275 Thy1 5910162 14.82 27707 Tbc1d1 2510072 1.38 23125 Cox6a2 130215 20.72 44308 Rn18s 6840019 7.79 35673 Egr1 4610347 5.42 36776 Smox 50019 3.97 41615 Dscr1 6220039 1.45 43173 Clec4n 6550086 4.96 44630 Hist1h4i 6900092 3.75 16194 Ilf3 105340132 1.35 42441 Il7r 6380500 2.62 35156 Rnf126 4560563 1.29 46121 4930431B09Rik 780301 3.44 30529 Mc3r 3290538 1.25 4255 scl0003040.1_5 101450735 1.43 33067 Pmp22 4010239 1.17 33663 Emp1 4120438 7.57 41890 AI324046 6290685 4.24 24088 Cxcl9 1570673 6.73 27769 Tnfrsf9 2510400 1.38 39811 Rhov 5860100 2.52 11892 9626100_224 103870242 19.94 46070 Bcl2l11 780044 1.82 19429 9626958_317 106380519 23.05 44124 Mapk6 6760520 1.72 32475 Dot1l 3850068 1.78 33462 LOC432466 4070082 3.25 23634 Phc2 1430433 1.29 41948 Car5b 630176 1.26 38502 C1qa 5390687 3.02 37149 F7 5080411 3.66 38543 Pgm2 540092 2.93 14662 4833427B12Rik 104760465 1.78 25951 Pvr 2100450 1.45 24713 Edn1 1770047 1.59 43822 Ndg2 6660400 3.90 32533 Hyal1 3850341 1.36 37745 Atp6v1c1 5220528 1.72 12770 9626096_327 104120113 25.38 26265 Ldha 2190594 2.35 16457 LOC382127 105390148 9.67 22869 Fpgs 1230465 1.39 37308 Mmp13 510471 9.63 30533 Dmkn 3290575 4.70 28442 Kcnn4 2680129 4.52 38745 Bcl2l1 5420484 1.38 39400 C1qc 5700131 4.17 25235 Spc24 1940563 1.46 25977 Slc2a1 2100609 3.50 36110 Cpeb2 4760338 1.79 42598 Cdkn1a 6400706 7.99 40628 Yif1b 6040088 2.18 2063 Irg1 100650707 5.00 28655 Cxcl1 2690537 9.02 17058 Rn18s 105670673 12.33 42173 Myd116 6350601 3.14 43177 Retnlg 6550093 21.28 31336 Igh‐1a 360113 7.37 14728 9626965_344 104780093 13.66 39015 Cd6 5570368 1.90 27537 Spp1 2470609 16.75 25706 Clec4d 2030717 3.25 39219 Arnt 5670711 1.37 5347 2210008N01Rik 101850102 1.43 38666 Uck2 5420048 3.14 29032 Slc7a11 2850138 2.87 39785 Ier3 5860021 5.39 26709 Gys1 2340142 1.83 27729 Gadd45g 2510142 7.10 40588 Dab2 60309 7.21 11775 Ybx3 103850315 3.33 41106 Cnn3 6110020 3.69 2492 A830081I21Rik 100780332 1.41 40296 C1qb 5910292 5.39 42906 Hk3 6510142 1.56 38318 Tgm2 5360452 4.75 35738 Pdpn 4640280 3.06 42237 Erdr1 6370142 2.29 29372 Got1 2940465 1.50 20854 Il1rn 106770079 4.69 27162 Dusp2 2370184 4.81 38144 Dok2 5340273 4.52 41484 Traf6 6200132 1.60 35426 Cxcr4 4590519 2.30 40897 X99384 6100347 1.96 12644 Egln3 104070100 2.59 40366 Grasp 5910735 1.91 22736 Gpr68 1190537 2.73 40802 Abcg1 60692 3.62 23030 C330008K14Rik 1240576 2.32 29348 Vegfa 2940338 3.12 43678 Plekhm2 6650438 1.87 33177 Cdkn1a 4050088 3.94 10522 5primeMoMuSV_LTR 103390408 22.14 2281 Irg1 100730494 4.03 28191 Il1b 2640364 11.50 46146 Mtss1 780435 1.74 29182 Car13 2900184 1.50 46136 Ddit3 780373 4.53 40961 9530058B02Rik 6100670 2.75 26719 Unc45a 2340180 1.61 25694 Zfp36 2030605 3.99 29308 Zfand2a 2940112 3.11 29701 Dlg7 3120041 1.26 44778 Odf2 6940121 1.40 43834 Cd63 6660451 22.75 45423 Pkm2 70500 1.68 15389 Traf1 105050288 3.15 45807 Mrc1 730097 10.94 46009 Rai14 770288 2.98 12616 Dusp5 104060746 3.03 42035 Pfkfb3 630706 4.64 31666 LOC625421 3710332 9.88 9465 GIG2 103060348 2.58 8407 A930007I19Rik 102680129 2.52 41044 Cxcl2 610398 11.25 39574 Snx24 5720398 3.08 28234 Plk3 2640592 6.01 10067 BC022593 103190048 1.43 33373 Rgs1 4060347 2.49 46239 Galk1 840162 3.14 32450 Ehd1 3840731 3.33 25233 Atf3 1940546 8.40 28561 Dusp4 2690044 2.33 26238 Hist1h4i 2190450 2.90 9773 Dab2 103130670 1.82 46082 Rgs16 780091 2.09 1487 Slc6a6 100520301 1.68 21430 Grasp 106940324 1.57 28368 Ccl7 2650519 43.80 25778 Ffar2 2060280 3.24 27260 Phlda1 2450020 4.92 22785 Axud1 1230053 4.94 31457 Uck2 3610040 4.25 28874 Ccl3 2810092 43.94 30610 Pim3 3360138 2.73 35082 Cstb 4560114 1.95 1199 E030034J16Rik 100460114 1.86 39059 Cd14 5570600 7.37 32361 Cd274 3840180 6.48 27338 Fkbp1a 2450368 1.77 39041 Insl6 5570497 2.70 46243 Jun 840170 4.99 34639 1190002H23Rik 4480128 7.26 36445 Pgf 4810593 1.36 42599 A230097K15Rik 6400707 2.46 42904 Anpep 6510138 2.33 46266 Cish 840315 5.37 42741 Plk2 6450152 2.71 44489 Dusp1 6860121 3.40 64 LOC385068 100050286 7.17 35257 AA467197 4570343 10.44 38978 4930583H14Rik 5570142 3.11 3008 9530018I07Rik 101050161 2.38 42212 Nkg7 6370059 5.17 23834 Rgl1 1500097 6.49 38887 Gpr109a 5550471 31.96 34829 Adam8 450347 4.87 21748 E230029F23Rik 107000673 7.08 18671 A530045L16Rik 106180537 2.62 34956 Rgs1 4540181 11.64 39354 Slc7a8 5690672 2.76 36141 Ccrl2 4760471 19.03 43897 Mmp14 670079 13.49 43744 Crem 6660041 1.66 28805 Flrt3 2760497 4.17 35893 Gdf15 4730017 4.03 43911 Ccl24 670129 23.43 38505 Stab1 5390707 3.35 25904 Tpi1 2100154 3.44 5150 LOC381140 101770541 6.25 15588 LOC673501 105080494 22.06 23111 2310016C08Rik 130152 11.55 19916 Osm 106510484 30.79 14411 A430084P05Rik 104670551 4.32 24687 Hmox1 1740687 3.10 5784 Basp1 101990309 28.64 39207 Adam8 5670609 5.28 46329 Arg1 840707 23.26 36857 Ccl4 50368 26.19 27440 Sphk1 2470113 5.46 34470 Ccl4 430047 62.05 42285 Sdc4 6370411 6.89

Genes significantly upregulated in MO‐MDSCs (RMA‐S tumor) compared to MO‐MDSCs (RMA‐S blood). False discovery rate (FDR) of 5 % was utilized as a cutoff for determination of significance.