Suppression of Mll1-Complex by Stat3/Cebp −β Induced miR-21a/21b/181b Maintains the Accumulation, Homeostasis, and Immunosuppressive Function of This information is current as Polymorphonuclear Myeloid-Derived of September 25, 2021. Suppressor Cells Zhiqian Zhang, Xu Huang, Enlin Wang, Yugang Huang and Rongcun Yang Downloaded from J Immunol published online 1 May 2020 http://www.jimmunol.org/content/early/2020/04/30/jimmun ol.2000230 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2020/04/30/jimmunol.200023 Material 0.DCSupplemental

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 1, 2020, doi:10.4049/jimmunol.2000230 The Journal of Immunology

Suppression of Mll1-Complex by Stat3/Cebpb–Induced miR-21a/21b/181b Maintains the Accumulation, Homeostasis, and Immunosuppressive Function of Polymorphonuclear Myeloid-Derived Suppressor Cells

Zhiqian Zhang,*,† Xu Huang,‡ Enlin Wang,* Yugang Huang,* and Rongcun Yang*,x,{

Mixed-lineage leukemia 1 (MLL1), which exerts its H3K4 methyltransferase activity by interacting with WDR5, ASH2L, and RBBP5, plays a pivotal role in regulating hematopoietic stem cell homeostasis. Disrupting the integrity of MLL1-complex has been reported to be associated with acute leukemia. However, the exact role of MLL1-complex in myeloid cells is unknown. In this study, microarray analysis revealed that the core components of the Mll1-complex, Wdr5, Ash2l, and Mll1, were concurrently downregulated by tumor-secreted factors as well as GM-CSF + IL-6 during the accumulation and activation of murine myeloid- Downloaded from derived suppressor cells (MDSCs). These changes were further validated by quantitative RT-PCR and Western blotting both in vitro and in vivo. The expression levels of WDR5 and ASH2L were also significantly decreased in bone marrow MDSCs of lung cancer patients compared with that of healthy controls. Functionally, ectopic expression of Wdr5, Ash2l, and Mll1 (C terminus) reversed the accumulation and function of GM-CSF + IL-6–induced as well as tumor-cocultured polymorphonuclear MDSCs (PMN-MDSCs) by promoting them to differentiate into mature neutrophil-like cells. Mechanistically, GM-CSF + IL-6–activated http://www.jimmunol.org/ Stat3 and Cebpb synergistically induced the expression of miR-21a, miR-21b, and miR-181b, and thus inhibited the expression of Wdr5, Ash2l, and Mll1 by targeting to their 39 untranslated regions, respectively. Furthermore, knockdown of these microRNAs also suppressed the expansion and function of GM-CSF + IL-6–induced PMN-MDSCs. Taken together, our findings indicate that the Stat3/Cebpb–miR-21a/b/181b–Mll1-complex axis may play a critical role in PMN-MDSC expansion, activation, and differ- entiation, and this axis may provide an effectively immunological therapeutic approach for patients with cancer or other immu- nological diseases. The Journal of Immunology, 2020, 204: 000–000.

s a highly heterozygous group of bone marrow–derived been shown to exert immunosuppressive ability in tumor and

immature myeloid cells, myeloid-derived suppressor inflammatory microenvironments, particularly the latter (5). by guest on September 25, 2021 A cells (MDSCs) have been intensively studied in a wide Multiple extracellular factors such as GM-CSF, IL-6, TNF-a, range of pathological conditions, particularly in tumor and immu- vascular endothelial growth factor, IFN-g,IL-1b,TGF-b,and nological diseases (1, 2). In tumor and inflammatory microenviron- PGE2 can accelerate MDSC expansion and activation (6–8). ments, MDSCs act as one type of key tumor immunosuppressors by Meanwhile, several transcription factors such as CEBP a and b secreting various immune regulatory and interacting with (CEBPa and CEBPb) (9, 10), STAT family members (STAT1, other immune cells (3). MDSCs were first phenotypically identified STAT3, STAT4, STAT5, STAT6) (11), and CEBP-homologous as Gr1+CD11b+ cells in tumor-bearing mice (3). There are two main (CHOP) (12) have been demonstrated as critical in- subpopulations of MDSCs: monocytic MDSCs (Mo-MDSCs; ducers of MDSCs. The activation of MDSCs results in increased CD11b+Ly6ChiLy6G2) and polymorphonuclear MDSCs (PMN- enzymatic activity of arginase-1 (Arg1) and NO synthase 2 MDSCs; CD11b+Ly6ClowLy6G+) (4). In , Mo-MDSCs (iNOS), along with elevated production of reactive oxygen are defined as HLA2DR2CD14+CD33+CD11b+ and PMN- species (ROS) and NO, which are responsible for the immu- MDSCs are characterized as HLA2DR2CD15+CD33+CD11b+ nosuppressive effect of MDSCs (2). MDSCs promote tumor (4). Functionally, both Mo-MDSCs and PMN-MDSCs have growth and progression via a variety of mechanisms, including

*State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin Address correspondence and reprint requests to Prof. Zhiqian Zhang or Prof. Rongcun 300071, China; †School of Medicine, Southern University of Science and Technol- Yang, Southern University of Science and Technology, 1088 Xueyuan Road, Shenzhen ogy, Shenzhen 518055, Guangdong, China; ‡Department of Radiation Oncology, 518055, Guangdong, China (Z.Z.) or State Key Laboratory of Medicinal Chem- Harbin Medical University Cancer Hospital, Harbin 150040, Heilongjiang, China; ical Biology, Nankai University, Tianjin 300071, China (R.Y.). E-mail addresses: xKey Laboratory of Bioactive Materials Ministry of Education, Nankai University, [email protected] (Z.Z.) or [email protected] (R.Y.) Tianjin 300071, China; and {Department of Immunology, School of Medicine, Nan- The online version of this article contains supplemental material. kai University, Tianjin 300071, China Abbreviations used in this article: Arg1, arginase-1; BMC, bone marrow cell; BMDM, ORCID: 0000-0003-1226-4807 (Z.Z.). bone marrow–derived ; BM-MDSC, bone marrow MDSC; ChIP, chromatin Received for publication March 3, 2020. Accepted for publication April 13, 2020. immunoprecipitation; CHOP, CEBP-homologous protein; Co-IP, coimmunoprecipitation; CST, Cell Signaling Technology; H3K4, histone H3 at lysine 4; H3K4me3, H3K4 This work was supported partially by China Postdoctoral Science Foundation Grant trimethylation; iNOS, NO synthase 2; MDSC, myeloid-derived suppressor cell; miRNA, 2015M581292 and National Natural Science Foundation of China Grant 31470876 microRNA; MLL1, mixed-lineage leukemia 1; Mo-MDSC, monocytic MDSC; NC, and was self-financed partially by Z.Z. negative control; PMN-MDSC, polymorphonuclear MDSC; P/S, penicillin-streptomycin; Z.Z. and R.Y. designed the study. Z.Z. wrote, revised, and finalized the paper. Z.Z., ROS, reactive oxygen species; RT-qPCR, quantitative RT-PCR; siRNA, small interfering X.H., E.W., and Y.H. performed the experiments. Z.Z. analyzed the experimental RNA; 39-UTR, 39 untranslated region. results. Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000230 2 STAT3/CEBPb–miR-21a/21b/181b–MLL1-COMPLEX REGULATES PMN-MDSCs inhibition of functional T cells and NK cells, induction of other Materials and Methods immune-suppressive cell populations such as regulatory T cells, Cell culture and mice secretion of immunosuppressive cytokines, and production of ID8 murine ovarian carcinoma was provided by Dr. Katherine F. Roby from angiogenic factors (2). However, the molecular mechanisms the University of Texas, Austin, TX. 293T and murine melanoma B16 underlying accumulation, differentiation and function of MDSCs cells were obtained from the American Type Culture Collection (Manassas, have not yet been fully elucidated. VA). ID8 and B16 cells were cultured in RPMI 1640 (Thermo Fisher Sci- As one of the main research topics in the field of epigenetic entific, Waltham, MA), supplemented with 10% FBS (HyClone, Logan, UT) regulation, histone methylation plays a key role in a number of and 1% penicillin-streptomycin (P/S; Beyotime, Shanghai, China) at 37˚C in a humidified 5% CO2 atmosphere. 293T cells were cultured with DMEM biological processes. Abnormal histone methylation caused by (Thermo Fisher Scientific) with 10% FBS and 1% P/S. Female C57BL/6 and histone methylase disfunction usually leads to the occurrence BALB/c female mice (age, 5–6 wk) were purchased from the Beijing Vital and development of multiple genetic diseases or types of cancer River Laboratory Animal Technology (Beijing, China) and maintained in a (13, 14). As a critical member of the histone methyltransferases specific pathogen-free and controlled environment. family, mixed-lineage leukemia 1 (MLL1) is required for the Induction of MDSCs in vitro regulation of critical involved in vertebrate development and leukemogenesis (15, 16). The proper histone H3 at lysine 4 Fresh murine BMCs were prepared from 5- to 6-wk-old BALB/c female mouse femurs by depletion of RBCs with RBC Lysis Buffer (Beyotime) and (H3K4) trimethylation (H3K4me3) and transcriptional activity elimination of B/T lymphocytes by CD19/CD4/CD8 MicroBeads (Miltenyi of MLL1 depends on a core subcomplex consisting of MLL1, Biotec, Bergisch Gladbach, Germany). The remaining cells were cultured WDR5, RBBP5, and ASH2L (15). MLL1 is frequently dis- with an ultra-low attachment plate (Corning Costar, Corning, NY) in rupted by chromosomal translocation in acute leukemias (15). DMEM culture medium supplemented with 5% charcoal-stripped FBS Downloaded from Targeting the MLL1 enzymatic activity or disrupting the in- (Thermo Fisher Scientific), 40 ng/ml of GM-CSF (PeproTech, Rocky Hill, NJ), and 40 ng/ml of IL-6 (PeproTech) for 4 d to allow induction of MDSCs. teraction among MLL1 and other components has been pro- For the tumor-secreted factor-induced model, 5 3 104 ID8 cells or 1 3 105 posed as a potential therapeutic target for acute leukemia 4T1 cells (upper chamber) were cocultured with 2 3 106 BMCs (C57BL/6 treatment (17). Nevertheless, the exact role of MLL1-complex or BALB/c; lower chamber; depletion of RBC, B and T cells; cultured in in the myeloid cell differentiation and function is not clear. DMEM medium with 5% charcoal-stripped FBS) in a 24-transwell ultra- low attachment plates (pore size, 8 mm; BD Biosciences, San Jose, CA) to In contrast, emerging data implicate that noncoding RNAs induce functional MDSCs for 4 d. http://www.jimmunol.org/ play a vital role in the expansion and function of MDSCs (18–23). For instance, Li et al. (24) have observed that miR-155 MDSC, PMN-MDSC, and Mo-MDSC sorting and miR-21 are upregulated during the induction of MDSC by For FACS sorting of high-purity of Gr1+CD11b+ cells from BMCs and spleen GM-CSF + IL-6 in bone marrow cells (BMCs) and these two of C57BL/6 mice and CD11b+Ly6ChiLy6G2 Mo-MDSCs and CD11b+Ly6- microRNAs (miRNAs) show a synergistic effect on MDSCs via Clow/2Ly6G+ PMN-MDSCs from BMCs, indicated fluorochrome-conjugated suppressing the expression of SHIP-1 and PTEN, respectively. In Abs were added to the single-cell suspension, incubated at 4˚C for 15 min and late/chronic sepsis, the transcription factors STAT3 and CEBPb centrifuged at 1600 rpm for 5 min. The supernatant was discarded and then washed again, and the cell concentration was adjusted to 1 3 107/ml. The coordinately induce the expression of miR-21 and miR-181b to total MDSCs, PMN-MDSCs, and Mo-MDSCs were selected using a BD generate Gr1+CD11b+ cells and enhance its immunosuppressive FACSAria device (BD Biosciences). by guest on September 25, 2021 ability (23, 25). In the current study, to identify the differentially expressed Transfection of miRNA mimics and inhibitors genes during MDSC expansion and activation, an Affymetrix miR-21a mimic, miR-21a inhibitor, miR-21b mimic, miR-21b inhibitor, microarray was used to profile differentially expressed mRNAs miR-181b mimic, miR-181b inhibitor, negative control (NC) mimic, and in activated bone marrow MDSCs (BM-MDSCs) and control NC inhibitor were purchased from RiboBio (Guangzhou, China). Oligo- nucleotide transfection was performed using Lipofectamine RNAiMAX BMCs. It was revealed that the expression levels of Mll1, Wdr5, Transfection Reagent (Thermo Fisher Scientific) according to the manu- and Ash2l were concurrently downregulated by tumor-secreted facturer’s protocol. To determine the effect of miR-21a, miR-21b, or miR- factors as well as GM-CSF + IL-6 during MDSC accumulation 181b inhibition on GM-CSF + IL-6–induced MDSC and PMN-MDSC and activation. In the in vitro–induced models and in vivo tumor expansion, fresh BMCs were transfected with 50 nM of miRNA inhibi- models, these changes were further validated by quantitative tor and were then incubated with 40 ng/ml GM-CSF and 40 ng/ml IL-6 for 4 d. To detect the regulatory role of miR-21a, miR-21b, and miR-181b in RT-PCR (RT-qPCR) and Western blot analysis. Clinically, the the expression of Wdr5, Ash2l, and Mll1, fresh isolated PMN-MDSCs expression of WDR5 and ASH2L were also significantly de- were transfected with corresponding miRNA mimics (20, 50, and 100 nM, creased in BM-MDSCs of patients with lung cancer when respectively) for 2 d. To determine the transfection efficiency and toxicity of compared with that of healthy controls. Notably, rescue of miRNA mimics in PMN-MDSC, we transfected with freshly isolated PMN-MDSCs with 0, 20, 50, and 100 nM FAM-labeled miRNA mimics Wdr5, Ash2l, and MLL1 (C terminus) expression attenuated the control (synthesized by Sangon Biotech [Shanghai, China]). After 2 d, cells accumulation and immunosuppressive function of PMN-MDSCs were captured under a fluorescence microscope (BX53; OlymPus, Tokyo, via promoting them to differentiate into mature neutrophil-like Japan) and then collected for transfection efficiency analysis (green positive) and cells. Furthermore, it was revealed that miR-21a, miR-21b, apoptosis analysis (Annexin V-FITC/propidium iodide–double staining [Beyo- and miR-181b, which were induced by activated Stat3 and time]) using a BD FACSAria device (BD Biosciences, Franklin Lakes, NJ). Cebpb, suppressed the expression of Wdr5, Ash2l, and Mll1 Apoptosis assay in PMN-MDSCs by binding their 39 untranslated regions Treated cells were washed with PBS three times and resuspended with (39-UTRs), respectively. In addition, inhibition of these miR- 100 mlof13 Annexin V binding buffer (Beyotime). Then, cells were NAs also decreased the population and immunosuppressive incubated with 5 ml of FITC-Annexin V and propidium iodide for 30 min function of GM-CSF + IL-6–activated PMN-MDSCs. In con- at room temperature in the dark and then washed with 1 ml of 13 Annexin V clusion, the results of the current study demonstrate that the binding buffer three times. After that, cells were resuspended with 500 ml of PBS, and apoptotic cells were determined using a flow cytometry ma- downregulation of Mll1-complex members by Stat3/Cebpb– chine (BD FACSAria; BD Biosciences). induced miR-21a/b and miR-181b affects the differentiation and function of PMN-MDSCs, which may provide new op- In vivo mouse model portunities for therapeutic development toward immunological A total of six mice (C57BL/6, female, aged 5–6 wk) were s.c. injected diseases, including cancers. with 5 3 106 B16 melanoma tumor cells. BMCs transfected with control The Journal of Immunology 3 lentivirus, Wdr5/Ash2l/Mll1c lentivirus, control antagomir, miR-21a Microarray analysis antagomir, miR-21b antagomir, or miR-181b antagomir were induced by GM-CSF + IL-6 for 4 d and PMN-MDSCs were isolated by FACS The microarray assay was performed by the service provider KangCheng sorting. Purified PMN-MDSCs were injected into the tumors of mice on Biotechnology (Shanghai, China). Briefly, for each sample, 5 mgoftotalRNA days 1, 3, 6, and 8 (2 3 106 cells per mouse) after palpable tumors were was used to obtain biotin-labeled cRNA, which was then purified using an formed. After 16 d, the mice were euthanized and the tissues of tumor, Affymetrix GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). femurs, and spleen were isolated for the indicated studies. The animal Purified cRNA samples were heated at 94˚C for 30 min and cooled on ice experiment was approved by the Ethics Committee of Tianjin International using the buffer containing 200 mM tris-acetate (pH 8.1), 500 mM potassium Joint Academy of Biomedicine. acetate, and 150 mM magnesium acetate for fragmentation. Fragmented cRNA from each sample was hybridized to the Mouse Genome 430 2.0 Array Clinical specimens according to the manufacturer’s protocol (Affymetrix). The arrays were then scanned and processed using a GeneChip Scanner 3000 (Affymetrix). The BMCs were isolated from 11 patients with lung cancer and 16 heathy controls result was further processed and analyzed using the GeneChip Operating from Harbin Medical University Cancer Hospital (Harbin, Heilongjiang Software 1.4 (Affymetrix). The microarray result has been submitted to the China), which was approved by the Medical Ethics Committee of Harbin Expression Omnibus (GSE92303, https://www.ncbi.nlm.nih.gov/geo/ Medical University Cancer Hospital. Informed consent was obtained from query/acc.cgi?acc=GSE92303). all patients and healthy controls. After depletion of RBCs, BM-MDSCs (CD11b+CD33+HLA-DR2) were isolated by FACS sorting, and Western Western blot analysis blotting was performed to detect the protein expression of WDR5, ASH2L, Western blot analysis was performed as previously described (27). Rabbit MLL1, and RBBP5. anti-WDR5 (catalog no. ab178410), rabbit anti-ASH2L (catalog no. Flow cytometry analysis of MDSCs and PMN-MDSCs ab240191), rabbit anti-RBBP5 (catalog no. ab52084), rabbit anti-CEBPb (catalog no. ab32358), rabbit anti-LAMP2 (catalog no. ab203224), and After washing with 13 PBS three times, 1 3 106 cells were blocked with mouse anti–b-ACTIN (catalog no. ab6276) Abs were purchased from Downloaded from 13 PBS with 1% FBS at 4˚C for 30 min. A total of 5 ml of indicated Abcam (Cambridge, MA). Rabbit anti–p-STAT3 (Tyr705) (catalog no. 9145), fluorochrome-conjugated Abs (Gr1-FITC, catalog no. 108405; BioLegend, rabbit anti-STAT3 (catalog no. 9139), rabbit anti-CHOP (catalog no. 5554), San Diego, CA; CD11b-PE, catalog no. 101207; BioLegend; Ly6C-FITC, rabbit anti-CEBPa (catalog no. 2295), rabbit anti–Arg1 (catalog no. 93668), catalog no. 128005; BioLegend; Ly6G-allophycocyanin, catalog no. mouse anti-MLL1 C terminus (catalog no. 14197), and rabbit anti-iNOS 127613; BioLegend) were added and incubated in another 30 min at 4˚C. (catalog no. 13120) were purchased from Cell Signaling Technology Flow cytometry was conducted using a BD FACSCalibur device (BD (CST; Danvers, MA). The HRP-conjugated secondary Abs were pur-

Biosciences, San Jose, CA) and analyzed with FlowJo software (version chased from Zhongshan Golden Bridge Biotechnology (catalog no. http://www.jimmunol.org/ 10.6; Tree Star, Ashland, OR). ZDR5306 [goat anti-rabbit] or ZDR5307 [goat anti-mouse]; Beijing, China). Transfection of small interfering RNAs Lentivirus production Small interfering RNA (siRNA) transfection was performed as previously described (26). The sequences of siRNA oligonucleotides are as follows: The CDS region of Wdr5 and Ash2l were cloned from fresh BMCs of NC siRNA, 59-CCUACGCCACCAAUUUCGU-39; Stat3 siRNA-1, 59- BALB/c mice. The C terminus of MLL1 (7867–11,901 nt) was synthesized CAACAUGUCAUUUGCUGAA-39; Stat3 siRNA-2, 59-CGUUUGACAU- by Sangon Biotech. These cDNAs were inserted into the pSin4-EF2-IRES- GGAUCUGACAA-39;Cebpb siRNA-1, 59-GACAAGCUGAGCGACGAGUA- Puro vector. Lentiviruses expressing Wdr5, Ash2l, and MLL1 C terminus 39;Cebpb siRNA-2, 59-CACAAGGUGCUGGAGCUGA-39; Chop siRNA-1, were produced by transfecting the respective plasmids in 293T cells fol- 59-GCGUCCCUAGCUUGGCUGA-39; and Chop siRNA-2, 59-CACAG- lowing the protocols described on the Addgene website (http://www. by guest on September 25, 2021 CUAGCUGAAGAGAA-39. addgene.org/lentiviral/protocols-resources/). A total of 100 ml of viruses were added into the culture medium for 48 h and then the expression of RT-qPCR Wdr5, Ash2l, and MLL1 C terminus were determined via Western blot- ting. Equal amount of empty lentivirus was infected as the control. For mRNA detection, total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA) and was reverse transcribed to cDNA using a PrimeScript II First Strand cDNA Synthesis Kit (Takara, Dalian, China) Bone marrow–derived macrophage isolation and polarization according to the manufacturer’s protocol. The cDNA product was quan- C57BL/6 BMCs were cultured with DMEM containing 10% FBS supple- tified by Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) mented with 1% P/S and 10 ng/ml rM-CSF (Peprotech). The culture medium through an ABI 7500 Real-Time PCR System (Applied Biosystems, was replaced with fresh medium (DMEM + 10% FBS + 1% P/S + 10 ng/ml Foster City, CA). Relative was calculated using the GM-CSF) every other day. Isolated PMN-MDSCs generated in vitro were 2DDCycle threshold 2 method. b-actin was used as an internal control. The cocultured with 1 3 105 bone marrow–derived (BMDMs) (1:1) primers used in the current study are as follows: Arg1-forward: 59- in a Transwell chamber. After 12 h, PMN-MDSCs were removed and the CTCCAAGCCAAAGTCCTTAGAG-39, Arg1-reverse: 59-AGGAGCTGT- BMDMs were stimulated with LPS (200 ng/ml; Sigma-Aldrich, St. Louis, CATTAGGGACATC-39; iNOS-forward: 59-CCAAGCCCTCACCTACTTCC- MO) and recombinant murine IFN-g (10 ng/ml; Peprotech). After 24 h, 39, iNOS-reverse, 59-CTCTGAGGGCTGACACAAGG-39; Wdr5-forward: 59- BMDMs were harvested and the expression of CD86 (CD86-FITC, catalog GACCTACAGCCCTACTCCCA-39, Wdr5-reverse: 59-CACACCCAGTCCA- no. 105005; BioLegend) was determined by flow cytometry. The super- TACCCAC-39; Ash2l-forward: 59-CCGAAAGTGGGGATGCAAACT-39, natant was collected for ELISA. Ash2l-reverse: 59-GTCAGCGGTGAACCATTTTGT-39; Mll1-forward: 59- AAGATGCCTGGAAGTCACTG-39, Mll1-reverse: 59-GCTCAATCAGA- ELISA AACACAACGG-39; Rbbp5-forward: 59-TGGACAGAACTACCCAGAG- GA-39, Rbbp5-reverse: 59-CCATCGTTACAGCCAACAGC-39; Lamp2-forward: The levels of supernatant IL-6 and IL-12 were determined by the mouse 59-TGCTTTCTGTGTCTAGAGCGT-39, Lamp2-reverse: 59-ATGGGCA- IL-6 and IL-12 kits (catalog nos. ab222503 and ab100699; Abcam) CAAGGAAGTTGTC-39; Stat3-forward: 59-AATGGAAATTGCCCG- according to the manufacturer’s protocols. GATCG-39, Stat3-reverse: 59-TCCTGAAGATGCTGCTCCAA-39; Cebpb- Detection of ROS content forward: 59-CAAGCTGAGCGACGAGTACA-39, Cebpb-reverse: 59-AG- CTGCTCCACCTTCTTCTG-39; Chop-forward: 59-CCACCACACCT- The content of ROS in activated PMN-MDSCs was detected by using CM- GAAAGCAGAA-39, Chop-reverse: 59-AGGTGAAAGGCAGGGACTCA- H2DCFDA (Thermo Fisher Scientific) followed by flow cytometric analysis 3 9;andb-actin–forward: 59-GGTGGGAATGGGTCAGAAGG-39, b-actin– as previously described (28). reverse: 59-GTTGGCCTTAGGGTTCAGGG-39. For miRNA detection, the cDNA products of miR-21a, miR-21b, and miR-181b were transcribed Detection of arginase activity and amplified using Bulge-Loop mmu-mir-21a Primer Set (catalog no. The arginase activity was determined using an Arginase Activity Assay Kit MQPS8004894; RiboBio), Bulge-Loop mmu-mir-21b Primer Set (catalog (Sigma-Aldrich) according to the manufacturer’s protocol. no. MQPS MQPS0002616-1; RiboBio), and miDETECT A Track hsa-miR- 181b-5p Primer Set (catalog no. miRA100516-1; RiboBio), respectively, T cell proliferation assay according to the manufacturer’s protocol. The small nuclear RNA U6 was used as an internal control. The primer set of U6 was also purchased from For the T cell proliferation assay, CD4+ T and CD8+ T cells were isolated in RiboBio (catalog no. MQPS0000002-1). splenocytes of healthy C57/BL6 mice by FACS sorting and incubating with 4 STAT3/CEBPb–miR-21a/21b/181b–MLL1-COMPLEX REGULATES PMN-MDSCs

5 mM of CFSE (Sigma-Aldrich) and cocultured with activated PMN-MDSCs Statistical analysis at ratios of 2:1 and 4:1 in DMEM medium with 5% FBS (HyClone) in culture 6 plates precoated with anti-CD3 (catalog no. 100201; BioLegend) and CD28 All values are presented as the mean SEM. Significant differences be- mAbs (catalog no. 102101; BioLegend). After coculture for 3 d, the pro- tween the two groups were analyzed by using unpaired Student t test. For liferation rate was determined by flow cytometry. multiple groups, significance was evaluated by one-way ANOVA analysis. A p , 0.05 was considered to indicate a statistically significant difference. Luciferase reporter assay Results The whole 39-UTRs of Wdr5 and Ash2l were amplified from fresh BMCs Expression of Wdr5, Ash2l, and Mll1 are concurrently of BALB/c mice. The 39-UTR fragment (3123–4547 nt) of MLL1 con- taining three miR-181b binding sites were synthesized by Sangon Biotech. downregulated in activated MDSCs both in vitro and in vivo These products were inserted in the Sgf I and Pme I restriction sites of the Initially, the current study established in vitro MDSC induction reporter plasmid psiCHECK-2 (Promega, Madison, WI). For construction of Wdr5, Ash2l, and Mll1 39-UTR reporter gene plasmids with a mutant models by coculturing RBC, B cell–, and T cell–depleted BMCs of miR-21a, miR-21b, or miR-181b binding site, the Site-Directed Muta- BALB/c or C57BL/6 mice with ID8 ovarian carcinoma cells for 2 genesis System (Beyotime) was used according to the manufacturer’s or 4 d, coculturing BM-MDSCs with 4T1 breast cancer cells for protocol. The primers used for cloning and site-directed mutagenesis are as 4 d, or stimulating BM-MDSCs by GM-CSF (40 ng/ml) and IL-6 follows: Wdr5-39-UTR-WT-forward: 59-GACCGCGATCGCGTCCTGG- (40 ng/ml) for 4 d. As shown in Fig. 1A, 1B and Supplemental CTCCATGGGAGAC-39, Wdr5-39-UTR-WT-reverse: 59-CTTAGTTTAA- ACCATAAATCTACAACAGAG-39; Ash2l-39-UTR-WT-forward: 59-GA- Fig. 1A, 1B, 2A, 2B, Gr1 and CD11b double–staining was per- CCGCGATCGCCCAGTCCTTGCTTCTGGTG-39, Ash2l-39-UTR-WT-reverse: formed to identify murine MDSCs and the results demonstrated 59-CTTAGTTTAAACGCTGTCCTCAGATACTCCAG-39;Mll1-39-UTR-WT- that both ID8/4T1–secreted factors and GM-CSF + IL-6 promoted forward: 59-GACCGCGATCGCCACAATTAAGGAGGAAGCC-39, Mll1- the accumulation of MDSCs. Furthermore, compared with fresh Downloaded from 39-UTR-WT-reverse: 59-CTTAGTTTAAACTGCAGCAATAAACTTGACATG- 39; Wdr5-39-UTR-Mut-forward: 59-ACTGGTATCACTCAGATTCGAA- BMCs, activated BM-MDSCs expressed higher mRNA (Fig. 1C) CACACACTGTAATA-39, Wdr5-39-UTR-Mut-reverse: 59-TATTACAGT- and protein levels (Fig. 1E, Supplemental Fig. 1C) of Arg1 and GTGTGTTCGAATCTGAGTGATACCAGT-39; Ash2l-39-UTR-Mut-forward: iNOS, two immunosuppressive mediators produced by expanded 59-GAAGCTAGTGGGTTCTAATTTGAATAATTGTGAAAGG-39,Ash2l-39- MDSCs. Moreover, the essential transcription factors Cebpb, 9 UTR-Mut-reverse: 5 -CCTTTCACAATTATTCAAATTAGAACCCACTAG- Chop, and p-Stat3 were also induced by ID8-secreted factors and CTTC-39; Mll1-39-UTR-Mut1-forward: 59-CACAAAAAAATCTTTT- AATCTTACAATCTTTCTAAAGGACTG-39, Mll1-39-UTR-Mut1-reverse: GM-CSF + IL-6 in BM-MDSCs (Fig. 1D, 1E). These data suggest http://www.jimmunol.org/ 59-CAGTCCTTTAGAAAGATTGTAAGATTAAAAGATTTTTTTGTG-39; functional BM-MDSCs were successfully induced by tumor-secreted Mll1-39-UTR-Mut2-forward: 59-GTCTACTTCCGGTTATCTTACATGG- factors as well as GM-CSF + IL-6. To identify the differentially GGTCACCACCTG-39, Mll1-39-UTR-Mut2-reverse: 59-CAGGTGGTGA- expressed genes potentially involved in MDSC expansion and acti- CCCCATGTAAGATAACCGGAAGTAGAC-39; and Mll1-39-UTR-Mut3- forward: 59-GAAAGCTCTCTACGAAAGACTCTTACAAAAAGTAAA- vation, the current study applied an Affymetrix microarray to detect AAGTGTACATAG-39, Mll1-39-UTR-Mut3-reverse: 59-CTATGTACACT- the changes of mRNA profiles during MDSC activation induced by TTTTACTTTTTGTAAGAGTCTTTCGTAGAGAGCTTTC-39. The luciferase ID8-secreted factors for 4 d. Notably, among the differentially report assay was performed in 293T cells as previously described (26). expressed genes, the core components of Wdr5-Mll1 complex, in- Phagocytosis assay cluding Wdr5, Ash2l, and Mll1 were concurrently decreased in ac- tivated BM-MDSCs, compared with untreated BMCs (Fig. 1F), by guest on September 25, 2021 Activated PMN-MDSCs were isolated and suspended into a 96-well plate 4 although the expression of another core member of Wdr5/Mll1 with 100 ml of RPMI 1640 medium (HyClone) (5 3 10 per well) and incubated with 20 ml of FITC-labeled latex beads (2 mm; Sigma-Aldrich) complex, Rbbp5, showed no significant change (Fig. 1F). The re- at 37˚C for 1 h. Afterwards, cells were harvested and analyzed using flow sults were validated by RT-qPCR (Fig. 1G) and Western blot analysis cytometry. (Fig. 1H, Supplemental Fig. 1D). These findings suggest the Wdr5/ Coimmunoprecipitation assays Mll1 complex may be destroyed during MDSC activation. To address whether the observation that Wdr5, Ash2l, and Mll1 The coimmunoprecipitation (Co-IP) assays were performed as previously downregulation in activated MDSCs in vitro could be extrapolated described (29). Briefly, total proteinwasextractedfromisolatedPMN- 3 6 MDSCs using immunoprecipitation lysis buffer (Beyotime). Of protein to MDSCs expanded by tumors in vivo, 5 10 B16 melanoma extract, 20% was set apart as input. Co-IP was performed by incubating cells were s.c. injected into C57/BL6 mice for 16 d. A significant extract with Pierce Protein G Magnetic Beads (Thermo Fisher Scientific) increase in the proportion of Gr1+CD11b+ MDSCs in the BMCs conjugated with Abs against RBBP5 (catalog no. ab52084; Abcam), and splenocytes was observed (Fig. 1I, 1J). As expected, signifi- b CEBP (catalog no. ab32358; Abcam), STAT3 (catalog no. 9139; CST), or cant downregulation of Wdr5, Ash2l, and Mll1 and elevation of rabbit IgG (catalog no. ab172730; Abcam) overnight at 4˚C. The beads were washed with immunoprecipitation lysis buffer three times and eluted Cebpb, Chop, and p-Stat3 were observed in the BMCs (Fig. 1K, with 100 mlof13 SDS-PAGE sample loading buffer. After heating at 95˚C 1M) and splenocytes (Fig. 1L, 1N) of tumor-bearing mice com- for 10 min, 20 ml of samples were then subjected to electrophoresis and pared with that in BMCs and splenocytes of normal control mice. Western blotting for detection of Rbbp5, Wdr5, Ash2l, Mll1, Cebpb, p-Stat3, To confirm that the change of these genes was indeed attributed to Stat3, or Chop. MDSC activation, not to the differentiation of other cells, Chromatin immunoprecipitation Gr1+CD11b+ cells were sorted from the BMCs and splenocytes by FACS sorting. The results revealed that the majority of sorted cells Chromatin immunoprecipitation (ChIP) assay was performed using Sim- + + pleChIP Enzymatic Chromatin IP Kit (CST) following the manufacturer’s were Gr1 CD11b cells (.90%) (Fig. 1I, 1J). As expected, similar instruction. Abs against H3K4me3 (catalog no. 9751; CST), CEBPb observation was seen in sorted cells in which both the mRNA and (catalog no. ab15050; Abcam), and STAT3 (catalog no. 9139; CST) were protein levels of Wdr5, Ash2l, and Mll1 were significantly lower in used for immunoprecipitation. Quantitative analysis of ChIP-derived DNA sorted MDSCs derived from BMCs (Fig. 1K, 1M) and splenocytes was performed by semiquantitative PCR and/or real-time quantitative PCR. The assays were performed in triplicate. Primers are as follows: (Fig. 1L, 1N) of B16-bearing mice than control-sorted MDSCs. Lamp2-forward: 59-TCCCCATCAAGGAAAGAGAGT-39, Lamp2-reverse: Furthermore, the protein levels of Cebpb, Chop, and p-Stat3 were 59-TGGATAGGGAAGCTAGGGGG-39; miR-21a-forward: 59-ATTGGTC- also significantly higher in sorted BM-MDSCs and spleen MDSCs TATGTCTAGTGGC-39, miR-21a-reverse: 59-GTGATGGTGCACGTGT- in B16-bearing mice than control-sorted MDSCs (Fig. 1M, 1N). TAAC-39; miR-21b-forward: 59-GTCTGTCTGGTCAAAGAAGTC-39, miR-21b-reverse: 59-CTGCCTGAGGACCCTAACAG-39; and miR-181b- To determine whether the changes of WDR5, ASH2L, MLL1, forward: 59-TGATAGTGCAGTGGTCAAGC-39, miR-181b-reverse: 59- and RBBP5 also occurs in clinical samples. BMCs were isolated CTTCCTTTGAGATAGCTTTCC-39. from 11 patients with lung cancer and 16 heathy controls. Following The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Differential expression of Wdr5, Ash2l, Mll1, and Rbbp5 in naive MDSCs and activated MDSCs in vitro and in vivo. (A and B) ID8-secreted factors or GM-CSF + IL-6–induced MDSC accumulation in BMCs isolated from BALB/c mice. (A) One representative of three independent experiments. (B) The average MDSC percentage of three independent experiments. (C and D) RT-qPCR to determine the mRNA expression of Arg1 (C), iNOS (C), Cebpb (D), and Chop (D) in ID8-secreted factors or GM-CSF + IL-6–induced BM-MDSCs compared with fresh (Figure legend continues) 6 STAT3/CEBPb–miR-21a/21b/181b–MLL1-COMPLEX REGULATES PMN-MDSCs Downloaded from http://www.jimmunol.org/

A

FIGURE 2. WDR5 and ASH2L expression levels are decreased in BM-MDSCs of lung cancer patients compared with that of healthy controls. ( ) BMCs by guest on September 25, 2021 were isolated from 11 lung cancer patients and 16 heathy controls. After depletion of RBCs, BM-MDSCs were isolated by FACS sorting, and Western blotting was performed to detect the protein expression of WDR5, ASH2L, MLL1, and RBBP5. Data are representative of two independent experiments. (B) The relative protein levels of WDR5, ASH2L, MLL1, and RBBP5 were quantified by ImageJ. Data are presented as means 6 SEM. *p , 0.05. depletion of RBCs, CD11b+CD33+HLA-DR2 BM-MDSCs were PMN-MDSCs. The present study next determined the expression isolated by FACS sorting, and Western blotting were performed to and function of Wdr5, Ash2l, and Mll1 in these two subpopula- detect the protein expression levels of WDR5, ASH2L, MLL1, and tions of MDSCs. As shown in Fig. 3A, 3B and Supplemental Fig. RBBP5. The results demonstrated that the expression levels of WDR5 2C, 2D, both Mo-MDSCs and PMN-MDSCs were effectively and ASH2L were also significantly decreased in the BM-MDSCs of induced in BMCs by GM-CSF + IL-6 as well as ID8-secreted patients with lung cancer compared with that of healthy controls factors. Then, Mo-MDSCs and PMN-MDSCs were sorted in (Fig. 2A, 2B). The expression of MLL1 also showed a declining trend, fresh BMCs and GM-CSF + IL-6–treated BMCs. RT-qPCR (Fig. although it was not significant (Fig. 2A, 2B). 3C) and Western blotting (Fig. 3D, Supplemental Fig. 2E) results demonstrated the downregulation of Wdr5, Ash2l, and Mll1 only Downregulation of Wdr5, Ash2l, and MLL1 contributes to the occurred in GM-CSF + IL-6–induced PMN-MDSCs, compared accumulation and immunosuppressive ability of PMN-MDSCs with fresh-sorted PMN-MDSCs. in vitro Afterwards, to characterize the effect of Wdr5, Ash2l, and Mll1 As aforementioned, MDSCs mainly consist of two distinct subsets: in MDSC induction, the current study constructed lentiviral vectors CD11b+Ly6ChiLy6G2 Mo-MDSCs and CD11b+Ly6ClowLy6G+ containing Wdr5, Ash2l, or the C terminus of Mll1 (as the full-

BMCs (n = 3 independent experiments). (E) Determination of the protein levels of Arg1, iNOS, p-Stat3, Stat3, Cebpb, Cebpa, and Chop in ID8-secreted factors or GM-CSF + IL-6–induced BM-MDSCs compared with fresh BMCs. (F) The mRNA expression profiles between fresh BMCs and BM-MDSCs induced by ID8-secreted factors for 4 d were analyzed using Affymetrix microarray. The relative expression of the core factors of the Mll1-complex, Wdr5, Ash2l, Mll1, and Rbbp5, and MDSC-associated transcriptional factors, Chop and Cebpb, were shown. (G and H) Wdr5, Ash2l, Mll1, and Rbbp5 expression between fresh BMCs and BM-MDSCs induced by ID8-secreted factors or GM-CSF + IL-6 were measured by RT-qPCR [(G) n = 3 independent experi- ments] and Western blot analysis (H). (I and J) Gr1+CD11b+ MDSCs can be effectively sorted from the BMC (I) and spleen (J) of C57/BL6 control mice and B16 tumor–bearing mice 16 d after B16 cell injection. (K and L) RT-qPCR to detect Wdr5, Ash2l, Mll1, and Rbbp5 expression in BM-MDSCs (K) and spleen MDSCs (L) before and after sorting (n = 3 independent experiments). (M and N) Comparison of p-Stat3, Stat3, Cebpb, Chop, Wdr5, Ash2l, Mll1, and Rbbp5 protein levels by Western blot analysis in BM-MDSCs (M) and spleen MDSCs (N) before and after sorting. All data presented in bar graphs are shown as means 6 SEM. (E, H, I, J, M, and N) Data are representative of at least two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Overexpression of Wdr5 + Ash2l + Mll1c attenuates the accumulation and activities of PMN-MDSCs in vitro. (A and B) Representative images (A) and statistical analysis results [(B) n = 3 independent experiments] to show the proportion changes of CD11b+Ly6C+ and CD11b+Ly6G+ subsets in BM-MDSC with/without GM-CSF + IL-6 treatment. (C and D) RT-qPCR [(C) n = 3 independent experiments] and Western blotting (D) to determine the expression levels of Wdr5, Ash2l, Mll1, and Rbbp5 in sorted PMN-MDSCs and sorted Mo-MDSCs with/without GM-CSF + IL-6 treatment. (E) Fresh BMCs isolated from BALB/c mice were infected with Wdr5, Ash2l, and Mll1c lentivirus and then stimulated with (Figure legend continues) 8 STAT3/CEBPb–miR-21a/21b/181b–MLL1-COMPLEX REGULATES PMN-MDSCs length of Mll1 is too large to clone, the C terminus of Mll1 [aa PMN-MDSCs isolated from GM-CSF + IL-6–treated BMCs with 2623–3967] was constructed, which contains the binding sites for Wdr5 + Ash2l + Mll1c overexpression showed a markedly decreased Wdr5, Ash2l, and Mll1 and functional TAD and SET domains). ability to suppress LPS + IFN-g–induced CD86 expression and IL-6 Significantly higher expression levels of Wdr5, Ash2l, and Mll1c and IL-12 secretion in BMDMs (Fig. 4D–G). Collectively, these data were observed postinfection of these lentiviruses in MDSCs suggest Wdr5 + Ash2l + Mll1c overexpression attenuates the im- (Fig. 3E). Next, the current study determined whether restoration munosuppressive function of PMN-MDSCs. c of Wdr5, Ash2l, and Mll1 expression could influence the ex- c pansion of MDSCs. As shown in Fig. 3F, 3G and Supplemental Ectopic expression of Wdr5 + Ash2l + Mll1 promotes Fig. 2A, 2B, the flow cytometry results showed that over- PMN-MDSCs to differentiate into mature neutrophil-like expression of Wdr5, Ash2l, and Mll1c together impaired the cells in vitro induction of MDSC from BMCs by GM-CSF + IL-6 or ID8- Previous studies have demonstrated that PMN-MDSCs are com- secreted factors. Notably, ectopic expression of Wdr5, Ash2l, prised of pathologically activated precursors of neutrophils with and Mll1c only could suppress GM-CSF + IL-6 or ID8-secreted vastly different functions (33, 34). Therefore, the current study factor–induced accumulation of PMN-MDSCs, not Mo-MDSCs assessed whether Wdr5 + Ash2l + Mll1c overexpression decreased (Fig. 3H, 3I, Supplemental Fig. 2C, 2D). the immunosuppressive function of PMN-MDSCs via facilitating It is known that MDSCs exert its immunosuppressive function them to differentiate into mature neutrophil-like cells. One of via production of several enzymatic mediators, including Arg1 the classical characteristics of neutrophils is phagocytosis (33). In and iNOS. Therefore, the current study determined the expression the current study, the phagocytic activity of PMN-MDSCs isolated and activities of Arg and iNOS in PMN-MDSCs isolated from from GM-CSF + IL-6–treated or ID8-cocultured BMCs with/ Downloaded from GM-CSF + IL-6–treated BMCs with/without Wdr5 + Ash2l + Mll1c without Wdr5 + Ash2l + Mll1c overexpression was assessed overexpression. As expected, GM-CSF + IL-6 treatment markedly with latex beads. Wdr5 + Ash2l + Mll1c overexpression in acti- promoted the mRNA (Fig. 3J, 3K) and protein (Fig. 3L) levels of vated PMN-MDSCs increased their phagocytic activity (Fig. 5A, Arg1 and iNOS, as well as the activity of arginase (Fig. 3M) and 5B, Supplemental Fig. 2H, 2I), as well as the expression of the production of ROS (Fig. 3N, 3O) in PMN-MDSCs. However, phagocytosis marker LAMP2 (Fig. 5C, 5D). To further examine c overexpression of Wdr5 + Ash2l + Mll1 in these PMN-MDSCs how the MLL1-complex modulates PMN-MDSC maturation, the http://www.jimmunol.org/ decreased the expression of Arg1 and iNOS (Fig. 3J–L), decreased present study first detected the binding intensity of Wdr5, Ash2l, the activity of arginase (Fig. 3M) and suppressed the production of and MLL1 with Rbbp5 in PMN-MDSCs with/without Wdr5 + ROS (Fig. 3N, 3O). Ash2l + Mll1c overexpression. As shown in Fig. 5E and 5F, ec- To gain a more mechanistic insight into the potential role of topic expression of Wdr5 + Ash2l + Mll1c restored the binding Wdr5/Ash2l/Mll1c in PMN-MDSCs, CFSE-labeled CD4+ or intensity of Wdr5 and Ash2l with Rbbp5 in PMN-MDSCs. The CD8+ T cells sorted from spleen of healthy mice were cocultured Co-IP results indicated the Mll1c fragment also interacted with with/without PMN-MDSC cells (2:1 or 4:1 ratio) isolated from Rbbp5 in activated PMN-MDSCs (Fig. 5F). Furthermore, it was GM-CSF/IL-6–treated BMCs with/without Wdr5 + Ash2l + Mll1c revealed that Wdr5 + Ash2l + Mll1c overexpression enhanced overexpression in culture plates precoated with anti-CD3 and trimethylation of H3K4 in LAMP2 promoter region (Fig. 5G, by guest on September 25, 2021 CD28 mAbs for 3 d. PMN-MDSCs generated from wild-type GM- 5H). These findings suggest that MLL1-complex may promote CSF + IL-6–treated BMCs efficiently suppressed CD4+ and CD8+ LAMP2 expression by increasing H3K4me3 in its promoter T cell proliferation (Fig. 4A–C). In contrast, PMN-MDSCs gen- region. In addition, Wdr5 + Ash2l + Mll1c overexpression also erated from GM-CSF + IL-6–treated BMCs with Wdr5 + Ash2l + facilitated TNF-a secretion in GM-CSF + IL-6–treated PMN- Mll1c overexpression displayed considerably lower suppressive MDSCs (Fig. 5I, 5J). Taken together, these results support the activity on CD4+ and CD8+ T cell proliferation (Fig. 4A–C). hypothesis that the MLL1-complex can promote the matura- Similar results were observed in ID8-cocultured PMN-MDSCs tion of PMN-MDSCs, and its disruption is indispensable (Supplemental Fig. 2F, 2G). Additionally, MDSCs can suppress for the immature status and immunosuppressive function of the polarization of M1 macrophages (30–32). In vitro studies were PMN-MDSCs. c also performed to examine the effect of Wdr5/Ash2l/Mll1 over- c expressed PMN-MDSCs on BMDM polarization. BMDMs were Wdr5 + Ash2l + Mll1 overexpression attenuates the cocultured with/without PMN-MDSCs isolated from GM-CSF/IL- tumor-promoting function of activated PMN-MDSCs in vivo 6–treated BMCs with/without Wdr5, Ash2l, and Mll1c over- To determine the function of MLL1-complex in PMN-MDSCs expression for 24 h, then stimulated with LPS and IFN-g for 24 h. in vivo, a mouse B16 tumor model was established with intra- Strikingly, PMN-MDSCs generated from wild-type GM-CSF/IL-6– tumorous injection of activated PMN-MDSCs with/without Wdr5 + treated BMCs exert a significantly inhibitory effect on LPS/IFN-g– Ash2l + Mll1c overexpression. As shown in Fig. 6A–C, tumors induced the expression of M1 macrophage surface marker CD86 and injected with activated PMN-MDSCs with Wdr5 + Ash2l + Mll1c the secretion of IL-6 and IL-12 in BMDMs (Fig. 4D–G). However, overexpression grew markedly slower than those tumors injected

GM-CSF + IL-6 for 4 d. Western blotting to detect the expression levels of Wdr5, Ash2l, Mll1, and Rbbp5 in BM-MDSCs. (F and G) Dynamic levels of Gr1+CD11b+ MDSCs detected by flow cytometry to determine the effect of Wdr5 + Ash2l + Mll1c overexpression on GM-CSF + IL-6–induced MDSC expansion. One representative of three independent experiments is shown in (F). (G) represents the statistical analysis results. (H and I) Flow cytometry to examine CD11b+Ly6C+ and CD11b+Ly6G+ subsets in GM-CSF + IL-6–treated BM-MDSC with/without Wdr5 + Ash2l + Mll1c overexpression (n =3 independent experiments). (J–L) Fresh BMCs isolated from BALB/c mice were infected with Wdr5, Ash2l, and Mll1c lentivirus and then stimulated with GM-CSF + IL-6 for 4 d. Afterwards, the mRNA [(J and K) n = 3 independent experiments] and protein (L) levels of Arg1 and iNOS were determined in sorted PMN-MDSCs via Western blot analysis. (M) Arginase activities were detected in the cell lysate described in (J)(n =3 independent experiments). (N and O) Intracellular ROS levels were measured by flow cytometry analysis using DCFH-DA (n = 3 independent experiments). All data presented in bar graphs are shown as means 6 SEM. (D, E,andL) Data are representative of at least two independent experiments. **p , 0.01, ***p , 0.001. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Overexpression of Wdr5 + Ash2l + Mll1c inhibits the immunosuppressive function of PMN-MDSCs in vitro. (A–C) GM-CSF + IL-6– activated PMN-MDSCs, as mentioned in (A), were purified and cocultured with splenic CD4+ or CD8+ T cells (1:2 or 1:4) in the presence of anti-CD3/CD28 stimulation. CFSE assay was performed to detect the proliferation rate of CD4+ or CD8+ T cells. The bar graphs in (B) and (C) represent the proliferation index of CD4+ or CD8+ T cells (n = 3 independent experiments). (D and E) Fresh BMCs isolated from BALB/c mice were infected with Wdr5/Ash2l/Mll1c lentivirus and then stimulated with GM-CSF + IL-6 for 4 d. Then, the purified PMN-MDSCs were cocultured with BMDMs in the presence of LPS (200 ng/ml) and IFN-g (10 ng/ml) for 24 h. The surface functional marker CD86 was determined by flow cytometry. (D) One representative of three independent experiments is presented. (E) The average percentage CD86+ cells of three independent experiments was analyzed. (F and G) Levels of IL-6 (F) and IL-12 (G) in supernatant of BMDMs were determined by ELISA (n = 3 independent experiments). All data presented in bar graphs are shown as means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. with control activated PMN-MDSCs. Furthermore, FACS analysis GM-CSF + IL-6–induced miR-21a, miR-21b, and miR-181b of tumor-infiltrating lymphocytes showed that the proportion of suppressed the expression of Wdr5, Ash2l, and Mll1 by both CD4+ and CD8+ T cells were increased in tumors injected with targeting to their 39-UTRs, respectively, and modulates the activated PMN-MDSCs overexpressed Wdr5 + Ash2l + Mll1c accumulation and function of PMN-MDSCs c (Fig. 6D, 6E). In addition, overexpression of Wdr5 + Ash2l + Mll1 Multiple studies over the past decades have revealed the funda- in activated PMN-MDSCs also enhanced CD4+IFN-g+ Th1 and mental role of miRNAs in the differentiation and function of CD8+IFN-g+ CTL cells in B16 tumor (Fig. 6D, 6E). MDSCs (35, 36). To define regulators that could modulate the 10 STAT3/CEBPb–miR-21a/21b/181b–MLL1-COMPLEX REGULATES PMN-MDSCs Downloaded from http://www.jimmunol.org/

FIGURE 5. Overexpression of Wdr5 + Ash2l + Mll1c facilitates activated PMN-MDSCs differentiating into mature neutrophil-like cells. (A and B) by guest on September 25, 2021 GM-CSF + IL-6–activated PMN-MDSCs with/without Wdr5 + Ash2l + Mll1c overexpression were purified and subjected to phagocytosis assay. (A) One representative of three independent experiments. (B) The average percentages of phagocytic cells of three independent experiments. (C and D) RT-qPCR (n = 3 independent experiments) and Western blotting to detect the expression levels of LAMP2 in activated PMN-MDSCs with/without Wdr5 + Ash2l + Mll1c overexpression. (E and F) Co-IP of endogenous Rbbp5 with Wdr5, Ash2l, and Mll1/Mll1c.(E) Fresh BMCs were treated with vehicle or GM-CSF + IL-6 for 4 d and PMN-MDSCs were purified. (F) BMCs with/without Wdr5 + Ash2l + Mll1c overexpression were treated with GM-CSF + IL-6 for 4 d, and PMN-MDSCs were purified. Cell lysates were subjected to immunoprecipitation using rabbit anti-Rbbp5 Ab or rabbit IgG. The immunoprecipitants were separated and blotted with Wdr5, Ash2l, Mll1/Mll1c,andRbpp5Abs.(G and H) ChIP was performed with Abs against H3K4me3 to determine the enrichment of methylated histones at the promoter region of LAMP2 in cell samples described in (A). Enriched DNA were detected by semiquantitative RT-PCR (G)andRT-qPCR[(H) n = 3 independent experiments], respectively. (I and J) Flow cytometry to detect the secretion of TNF-a in cell samples described in (A). All data presented in bar graphs are shown as means 6 SEM. (D–G) Data are representative of at least two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. expression of Wdr5, Ash2l, and Mll1 in MDSCs, the current wild-type constructs, respectively (Fig. 7C–E). Afterwards, the study looked through the miRDB Database (http://mirdb.org/) wild-type constructs were mutated as depicted in Fig. 7A and also and identified miRNAs with putative binding sites in the 39- transfected into 293T cells along with miRNA mimic, miR-21a, UTRsofWdr5,Ash2l,andMll1.Notably,itwasrevealedthat miR-21b, or miR-181b mimics. The results showed that muta- miR-21 and miR-181b, which have been reported to be syner- tion of the binding sties abolished the effects of miR-21a, miR- gistically induced by STAT3-CEBPb–transcriptional complex 21b, or miR-181b on the luciferase activity of Wdr5, Ash2l, or in sepsis-associated MDSCs (23, 25), have potential binding Mll1 39-UTR plasmids, respectively (Fig. 7C–E). Notably, the sites within the 39-UTRs of Wdr5, Ash2l, and Mll1, respectively results revealed that ectopic expression of miR-21a, miR-21b, and (Fig. 7A). To determine whether the 39-UTRs of Wdr5, Ash2l, miR-181b with transfection of corresponding miRNA mimics and Mll1 are targeted by miR-21a, miR-21b, or miR-181b di- (Fig. 7F–H) significantly inhibited the protein levels of Wdr5, rectly, the 39-UTR fragments of Wdr5, Ash2l, and Mll1 con- Ash2l, and Mll1 in isolated PMN-MDSCs from BMCs, respec- taining the miR-21a, miR-21b, or miR-181b binding site(s) tively (Fig. 7I–K). To determine the transfection efficiency and were cloned into the psiCHECK2 dual luciferase reporter cytotoxicity of miRNA mimics in PMN-MDSC, we transfected plasmid and were transfected into 293T cells along with control with freshly isolated PMN-MDSCs with 0, 20, 50, and 100 nM of miRNA, miR-21a, miR-21b, or miR-181b mimics (Fig. 7B). FAM-labeled miRNA mimics control. The results revealed that The results of the luciferase assay demonstrated that enhanced miRNA mimic was successfully transfected (Supplemental Fig. expression of miR-21a, miR-21b, or miR-181b dramatically 3A, 3B) into PMN-MDSCs with low cytotoxicity (Supplemental decreased the luciferase activity of Wdr5, Ash2l, or Mll1 39-UTR Fig. 3C). The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 6. Activated PMN-MDSCs with Wdr5 + Ash2l + Mll1c overexpression decrease B16 tumor growth in vivo compared with control-activated PMN-MDSCs. (A–C) Tumor size (A), growth curve (B), and tumor weight (C) in C57/BL6 mice bearing B16 tumors (n = 5 or 6 per group) intratumorously injected with GM-CSF + IL-6–activated PMN-MDSCs with/without Wdr5 + Ash2l + Mll1c overexpression. (D and E) The proportion of total T, CD4+IFN- g+ Th1 and CD8+IFN-g+ CTL cells from tumor tissues were analyzed by flow cytometry (n = 5 or 6 per group). All data were shown as means 6 SEM.

**p , 0.01, ***p , 0.001. by guest on September 25, 2021

To address whether the downregulation of Wdr5, Ash2l, and synergistically suppressed the expression of Wdr5, Ash2l, and Mll1 could be, at least partially, attributed to upregulation of miR- Mll1 by inducing the transcription of miR-21a, miR-21b, and 21a, miR-21b, and miR-181b in activated BM-MDSCs and PMN- miR-181b, respectively. The Western blotting results indicated MDSCs, the current study then detected the expression of these that silence of Cebpb enhanced the protein levels of Wdr5, Ash2l, miRNAs in BM-MDSCs with/without GM-CSF + IL-6 treatment. and Mll1, whereas silence of Stat3 only enhanced the protein As expected, all the expression levels of miR-21a, miR-21b, and levels of Wdr5 in activated PMN-MDSCs (Fig. 9A). Notably, miR-181b were upregulated in GM-CSF + IL-6–induced BM- when both Stat3 and Cebpb were knocked down, both the mRNA MDSCs compared with fresh BMCs (Fig. 8A). Inhibition of and protein levels of Wdr5, Ash2l, and Mll1 were significantly these miRNAs (Fig. 8A) restored the expression of Wdr5, Ash2l, increased in activated PMN-MDSCs (Fig. 9B, 9C). Furthermore, and Mll1, respectively, in GM-CSF + IL-6–treated BM-MDSCs Co-IP experiments proved that Cebpb physically interacted with (Fig. 8B). Functionally, suppression of either miR-21a, miR-21b, p-STAT3 (Tyr705) in activated PMN-MDSCs (Fig. 9D, 9E). The or miR-181b attenuated GM-CSF + IL-6–induced BM-MDSC results also confirmed that silencing of Stat3 and/or Cebpb sig- expansion (Fig. 8C, 8D) as well as the proportion of PMN-MDSCs nificantly suppressed the expression of miR-21a, miR-21b, and (Fig. 8E, 8F) in activated BMCs. In vivo tumor experiment also miR-181b in activated PMN-MDSCs (Fig. 9F). The present study revealed that tumors injected with GM-CSF + IL-6–treated PMN- also performed a ChIP assay to determine the binding potential of MDSCs grew significantly faster than those injected with fresh p-Stat3 and Cebpb on the promoter regions of miR-21a, miR-21b, PMN-MDSCs (Fig. 8G–I). However, inhibition of either miR-21a, and miR-181b. In PMN-MDSCs isolated from fresh BMCs, no miR-21b, or miR-181b in activated PMN-MDSCs could slow down binding bands of p-Stat3 or Cebpb could be detected. Neverthe- the tumor growth rate (Fig. 8G–I). Taken together, the findings of less, both p-Stat3 and Cebpb showed strong binding activities on Figs. 7, 8 suggest that miR-21a, miR-21b, and miR-181b may fa- the promoter regions of these miRNAs in activated PMN-MDSCs cilitate PMN-MDSC expansion and activation via downregulating (Fig. 9G). Wdr5, Ash2l, and Mll1, respectively. In conclusion, the results of the current study reveal a critical regulatory axis in PMN-MDSC expansion, activation, and matu- b p-Stat3 and Cebp synergistically promote the transcription of ration, in which tumor environment–associated factors such as miR-21a, miR-21b, and miR-181b and suppress the expression GM-CSF and IL-6 activate transcription factors STAT3 and of Wdr5, Ash2l, and Mll1 in activated PMN-MDSCs CEBPb, and thus enhance the transcription of miR-21a, miR-21b, It has been reported that Stat3 and CEBPb coordinatively induce and miR-181b. Increased levels of these miRNAs suppressed the miR-21 and miR-181b expression in sepsis MDSCs (25). There- expression of Wdr5, Ash2l, and Mll1 to disrupt the Mll1-complex. fore, the current study then assessed whether p-Stat3 and Cebpb Disruption of MLL1-complex is beneficial to PMN-MDSCs to 12 STAT3/CEBPb–miR-21a/21b/181b–MLL1-COMPLEX REGULATES PMN-MDSCs Downloaded from http://www.jimmunol.org/

FIGURE 7. miR-21a, miR-21b, and miR-181b suppress the expression of Wdr5, Ash2l, and Mll1 by targeting their 39-UTRs respectively in PMN- by guest on September 25, 2021 MDSCs. (A) Schematic representation of the target regions (wild-type and mutant) of Wdr5, Ash2l, and Mll1 39-UTRs for miR-21a, miR-21b, and miR-181b, respectively. (B) Workflow to show the procedure of Luciferase reporter assays. (C–E) Luciferase reporter assays to assess the effect of miR-21a, miR-21b, and miR-181b on Wdr5 (C), Ash2l (D), and Mll1 (E), respectively. Each group was performed in six biological replicates. The Renilla luciferase activities were normalized to the firefly luciferase activity (Renilla/firefly). All data were shown as means 6 SEM of one representative of three independent experiments. The *, **, and *** values represent control mimic versus miR-21a, miR-21b, or miR-181b mimic. The # value represents Mll1-WT versus Mll1-Mut1, Mll1-Mut2, or Mll1-Mut3. (F–H) Semiquantitative RT-PCR to detect the expression of miR-21a, miR-21b, and miR-181b in PMN-MDSCs transfected with corresponding miRNA mimics. (I–K) The protein levels of Wdr5, Ash2l, and Mll1 were detected by Western blot analysis in PMN-MDSCs transfected with miR-21a, miR-21b, and miR-181b mimics (20, 50, or 100 nM), respectively. (F–K) Data are representative of at least two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, #p , 0.05. maintain its immature status and immunosuppressive function p-Stat3– and CEBPb-induced miR-21a, miR-21b, and miR-181b (Fig. 10). This axis may provide an effective immunological play an inhibitory role in the expression of Wdr5, Ash2l, and therapeutic approach for patients with tumor or other immune- Mll1, respectively. Taken together, the results of the current study associated diseases. have demonstrated a key role of Stat3/Cebpb–miR-21a/b/181b– Mll1-complex in regulating the differentiation and function of Discussion PMN-MDSCs. MDSCs are a heterogeneous population of myeloid cells with The results of protein expression of WDR5, ASH2L, MLL1, and immature and undifferentiated phenotype. Accumulating evidence RBBP5 in BM-MDSCs of patients with lung cancer and that of has revealed that MDSCs exert its immunosuppressive ability in healthy controls showed that the expression of WDR5 and ASH2L numerous pathological conditions, such as different types of cancer were also significantly decreased in BM-MDSCs of patients with and inflammatory diseases. Inhibition of MDSC function may be an lung cancer compared with that of healthy controls. The expression effective therapeutic strategy for these diseases (37–39). However, of MLL1 also showed a declining trend, although it was not sig- the intrinsic mechanisms underlying MDSC expansion, differentia- nificant, which suggest more clinical samples should be used. tion, and activation urgently requires further investigation. The Over the past few decades, the mechanisms by which the MLL1- present study revealed that the core members of the Mll1-complex, complex modulates leukemogenesis have been deeply investigated. Wdr5, Ash2l, and Mll1 were concurrently suppressed by GM-CSF However, its function in MDSCs remains unknown. To the best of andIL-6inPMN-MDSCs.DownregulationofWdr5,Ash2l,and our knowledge, these data are the first to demonstrate the critical Mll1 impaired the formation of the Mll1-complex and is in- role of MLL1-complex in MDSCs. As a pivotal member of the dispensable for maintaining the immature phenotype and im- histone methyltransferases family, MLL1 encodes an enzyme munosuppressive function of activated PMN-MDSCs. Furthermore, catalyzing the mono-, di-, and trimethylation of H3K4 to control The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 8. Inhibition of miR-21a, miR-21b, and miR-181b attenuates the accumulation and immunosuppressive function of PMN-MDSCs. (A)RT- qPCR to detect the expression of miR-21a, miR-21b, and miR-181b in GM-CSF + IL-6–treated BM-MDSCs with/without miR-21a, miR-21b, or miR-181b inhibition (n = 3 independent experiments). (B) Western blot analysis to determine the protein levels of Wdr5, Ash2l, and Mll1 in GM-CSF + IL-6–treated BM-MDSCs with/without miR-21a, miR-21b, or miR-181b inhibition. Data are representative of two independent experiments. (C and D) Flow cytometry to determine the effect of transfection of control miRNA, miR-21a, miR-21b, or miR-181b (50 nM each) inhibitor on the accumulation of BM-MDSCs under the treatment of GM-CSF + IL-6 (40 ng/ml each for 4 d). The average percentage of MDSCs of three independent experiments was shown in (D). (E and F) Flow cytometry to examine CD11b+Ly6C+ and CD11b+Ly6G+ subsets in cells depicted in (A)(n = 3 independent experiments). (G–I) B16 tumor size (G), growth curve (H), and tumor weight (I) are presented. C57/BL6 mice bearing B16 tumors (n = 6) were intratumorously injected with activated PMN-MDSCs transfected with control miRNA, miR-21a, miR-21b, or miR-181b (50 nM each) inhibitor, respectively (n = 5 or 6 per group). All data were shown as means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. gene transcription. Alteration of this process often causes changes 41). In the current study, it was revealed that overexpression of in the gene expression patterns involved in stem cell maintenance Wdr5 + Ash2l + Mll1C in murine PMN-MDSCs suppressed Arg1 and development. The present study hypothesized that disrup- and iNOS and elevated the secretion of TNF-a. However, tion of the Mll1-complex may influence the genes associated H3K4me3 was not detectable in their promoter regions, which with PMN-MDSC differentiation and function. In fact, the current suggests that these genes may be regulated by Mll1-complex in- study indeed proved that the Mll1-complex enhanced the directly. Future studies will address this problem. In contrast, transcription and expression of phagocytosis-associated genes other genes and cytokines regulated by the MLL1-complex in LAMP2 by enhancing H3K4me3 in its promoter region, and thus PMN-MDSCs also require investigation. Future studies will be facilitated PMN-MDSC maturation. In contrast, several critical focused on the epigenetic events controlled by MLL1-complex on cytokines and transcription factors involved in MDSC expansion the whole genome during PMN-MDSC accumulation and activa- and activation have been reported to be regulated by MLL1 in tion, particularly on the histone methylation profiles. other immune cells. For instance, deletion of MLL1 in T cells and One of the interesting findings in the current study is that we macrophages impairs the production of lineage-specific cytokines, found that overexpression of Wdr5 + Ash2l + Mll1C might promote such as TNF-a, IFN-g, IL-1b, and IL-4, concurrently with the PMN-MDSCs differentiating into mature neutrophil-like cells. In dysregulated transcriptional activities of NF-kB and GATA3 (40, our future study, it is interesting to determine whether these 14 STAT3/CEBPb–miR-21a/21b/181b–MLL1-COMPLEX REGULATES PMN-MDSCs Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 9. Cebpb couples with p-Stat3 promote the transcription of miR-21a, miR-21b, and miR-181b and suppressed the expression of Wdr5, Ash2l, and Mll1 in activated PMN-MDSCs. (A) PMN-MDSCs isolated from GM-CSF + IL-6–treated BMCs were transfected with two distinct siRNAs targeting Stat3, Cebpb, and Chop, respectively. Western blot analysis to determine the protein levels of Wdr5, Ash2l, Mll1, Rbbp5, Cebpb, p-Stat3, Stat3, and Chop. (B and C) mRNA [(B) n = 3 independent experiments] and protein (C) expression of Stat3, Cebpb, Wdr5, Ash2l, Mll1, and Rbbp5 were determined by RT- qPCR and Western blotting, respectively, in activated PMN-MDSCs transfected with Stat3-siRNA1, Cebpb-siRNA1, and Stat3-siRNA1 + Cebpb-siRNA1. (D and E) Co-IP to determine the physical interaction between Cebpb and p-Stat3 in naive and activated PMN-MDSCs. (F) RT-qPCR to determine the expression of miR-21a, miR-21b, and miR-181b in activated PMN-MDSCs transfected with Stat3-siRNA1, Cebpb-siRNA1, and Stat3-siRNA1 + Cebpb- siRNA1 (n = 3 independent experiments). (G) ChIP analysis for the recruitment of Cebpb and p-Stat3 on to the promoter regions of miR-21a, miR-21b, and miR-181b naive and activated PMN-MDSCs. All data presented in bar graphs are shown as means 6 SEM. (A, C, D, E, and G) Data are representative of at least two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. mature neutrophil-like cells exert their phagocytic ability to erase Another interesting finding in the current study is that the tumor cells directly or present tumor-associated Ags to T cells in components of Mll1-complex, Wdr5, Mll1, and Ash2l are con- tumor microenvironment to activate tumor immune response. currently downregulated in MDSC expansion and activation. The The Journal of Immunology 15 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 10. Schematic view to present the suppressive role of the Cebpb/p-Stat3–miR-21a/b/181b–Mll1-complex axis in murine PMN-MDSC ex- pansion, differentiation, and activation. In the tumor environment, several factors such as GM-CSF and IL-6 activate transcription factors STAT3 and CEBPb, and thus enhance the transcription of miR-21a, miR-21b, and miR-181b. Increased levels of these miRNAs suppressed the expression of Wdr5, Ash2l, and Mll1 to disrupt the Mll1-complex. Disruption of MLL1-complex is beneficial to PMN-MDSCs to maintain its immature status and immu- nosuppressive function. mechanism underlying this phenomenon is worth investigating. There are several limitations to the current study. As afore- Numerous studies have shown that a wide range of miRNAs im- mentioned, first, the downstream effectors of Mll1-complex plicate in MDSC generation and differentiation. For example, as a during MDSC expansion and activation should be further in- downstream target of Stat3 and Cebpb, miR-181b couples with vestigated. Second, the upstream regulators of Wdr5, Mll1, and miR-21 to generate MDSCs and enhances its immunosuppressive Ash2l need to be further identified. Third, the dynamic changes ability in early and late sepsis (23, 25). Notably, by using online of interactions among Wdr5, Mll1, Ash2l, and other transcrip- software miRDB, the current study revealed that the 39-UTRs of tional factors should be deeply investigated during the differ- Wdr5, Ash2l, and Mll1 have potential binding sites for miR-21a, entiation and maturation of PMN-MDSCs. In addition, clinical miR-21b, and miR-181b, respectively. The present study also studies for more specimens are lacking for the study. Future demonstrated that suppression of miR-21a, miR-21b, and miR-181b studies will focus more on the role of the Mll1-complex in PMN- could reverse GM-CSF + IL-6–induced PMN-MDSC accumulation, MDSCsofcancerpatients. similar to the effect of Wdr5 + Ash2l + Mll1c overexpression. In conclusion, the results of the current study reveal the Meanwhile, luciferase assay proved that miR-21a, miR-21b, and suppressive role of Mll1-complex in murine MDSCs and its miR-181b directedly targeted to the 39-UTRs of Wdr5, Ash2l, and upstream regulators, as well as downstream effectors. It is be- Mll1, respectively. Furthermore, overexpression of miR-21a, miR- lieved that the work described in the current study not only 21b, and miR-181b indeed inhibited the expression of Wdr5, provides a novel role for MLL1-dependent H3K4me3 chromatin Ash2l, and Mll1, respectively. Even so, other factors involved in remodeling in immunosuppressive ability of MDSCs, but also regulating Wdr5, Ash2l, and Mll1 expression in MDSCs also need demonstrates an effective therapeutic target for inflammatory to be identified. diseases and cancer. 16 STAT3/CEBPb–miR-21a/21b/181b–MLL1-COMPLEX REGULATES PMN-MDSCs

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