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Role of TGF-β Signaling in Generation of CD39 +CD73+ Myeloid Cells in Tumors Sergey V. Ryzhov, Michael W. Pickup, Anna Chytil, Agnieszka E. Gorska, Qinkun Zhang, Philip Owens, Igor This information is current as Feoktistov, Harold L. Moses and Sergey V. Novitskiy of September 27, 2021. J Immunol 2014; 193:3155-3164; Prepublished online 15 August 2014; doi: 10.4049/jimmunol.1400578

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Role of TGF-b Signaling in Generation of CD39+CD73+ Myeloid Cells in Tumors

Sergey V. Ryzhov,* Michael W. Pickup,† Anna Chytil,† Agnieszka E. Gorska,† Qinkun Zhang,* Philip Owens,† Igor Feoktistov,* Harold L. Moses,† and Sergey V. Novitskiy†

There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-b type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-b signaling. In this study, we tested the hypothesis that TGF-b drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/ CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in Downloaded from the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-b receptor in mammary epithelium, an increased level of TGF-b protein was associated with further increase in number of CD39+CD73+ TDMMCs compared with MMTV-PyMT/TGFbRIIWT control tumors with intact TGF-b signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-b signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-b signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and http://www.jimmunol.org/ CD73, and was accompanied by increased inﬁltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-b signaling can directly induce the generation of CD39+CD73+ TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment. The Journal of Immunology, 2014, 193: 3155–3164.

D39 (ectonucleoside triphosphate diphosphohydrolase-1) effects of extracellular adenosine have been attributed to suppression hydrolyzes extracellular ATP and ADP into AMP, which of immune responses through A2A adenosine receptor–mediated by guest on September 27, 2021 C is then processed into adenosine by the CD73 (ecto-59- inhibition of T cell proliferation (7) and A2A/2B receptor–mediated nucleotidase). There is growing evidence that the cell-surface stimulation of tumor angiogenesis (8–10). Therefore, cell pop- CD39/CD73 enzyme pair plays an important role in regulating ulations present in tumors that express CD39 and CD73 including antitumor responses by catabolizing the tumor-suppressing ATP to regulatory T cells (11, 12), myeloid-derived suppressor cells the tumor-promoting adenosine. Antitumor effects of extracellular (MDSCs) (13), endothelial cells (14), and some types of cancer ATP are explained by its direct inhibition of tumor cell growth (1) cells (15, 16) or even exosomes (17), have attracted much attention and by acting as a natural adjuvant (2) or danger signal (3) that lately due to their involvement in ATP–CD39–CD73–adenosine activates the immune system (4–6). Conversely, tumor-promoting pathway, which can shift the balance from tumor-suppressive extracellular ATP toward tumor-promoting extracellular adenosine. However, studies on CD39 and CD73 expression so far have *Cardiovascular Division, Department of Medicine, Vanderbilt University, Nashville, TN 37232; and †Cancer Biology Department, Vanderbilt-Ingram Cancer Center, focused only on separate cell subpopulations without taking into Nashville, TN 37232 account other cells present in the microenvironments of tumors. Received for publication March 5, 2014. Accepted for publication July 17, 2014. The pleiotropic cytokine TGF-b is a part of the tumor micro- This work was supported by National Institutes of Health Grants R01HL095787, environment and plays a critical role in the regulation of tumor R01CA138923 (to I.F.), CA085492, and CA102162 (to H.L.M.), the T.J. Martell growth. High levels of TGF-b are present in many types of Foundation, the Vanderbilt-Ingram Cancer Center, and the Robert J. and Helen C. Kleberg Foundation. National Institutes of Health Grant CA068485 provided core tumors, including melanomas and carcinomas of the breast, colon, laboratory support. esophagus, stomach, liver, lung, pancreas, and prostate, as well as Address correspondence and reprint requests to Dr. Sergey V. Novitskiy or Sergey V. hematologic malignancies (18, 19). Through its pleiotropic effects Ryzhov, 2220 Pierce Avenue, PRB 612, Vanderbilt University, Nashville, TN 37232 on immune cells, TGF-b maintains a delicate balance between (S.V.N.) or 2220 Pierce Avenue, PRB 361, Vanderbilt University, Nashville, TN 37232 (S.V.R.). E-mail addresses: [email protected] (S.V.N.) or immunosuppression and activation of the immune system. For [email protected] (S.V.R.) example, genetic ablation of Smad4-dependent signaling in The online version of this article contains supplemental material. T lymphocytes resulted in spontaneous development of gastroin- Abbreviations used in this article: HPC, hematopoietic progenitor cell; Lin2, lineage testinal tumors (20), suggesting tumor-suppressive role of TGF-b negative; LLC, Lewis lung carcinoma; MDSC, myeloid-derived suppressor cell; signaling in T cells. However, T cell–speciﬁc TGF-b signaling can DMFI, difference in mean ﬂuorescence intensity; MHC II, MHC class II; MMTV, mouse mammary tumor virus; NECA, 59-N-ethylcarboxamido adenosine; PyMT, also contribute to suppression of antitumor immunity directly via polyoma middle T Ag; SRA, class A scavenger receptor; TDMMC, terminally dif- inhibition of generation and activity of cytotoxic T lymphocyte or ferentiated myeloid mononuclear cell; Tgfbr2, TGF-b type II receptor gene; VEGF, indirectly through induction of regulatory T cells (21). vascular endothelial growth factor; WT, wild-type. In myeloid cells, we have recently shown that genetic disruption Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 of the TGF-b type II receptor gene, Tgfbr2,inLysM-Cre/ www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400578 3156 TGF-b IN GENERATION OF CD39/CD73 TDMMC

Tgfbr2KO mice resulted in decreased implanted tumor growth, Adenosine generation assay suggesting protumorigenic effects of TGF-b signaling (22). In- The optimal number of myeloid cells (5 3 104) per assay was determined in deed, TGF-b has been shown to promote recruitment of MDSCs ancillary studies (Supplemental Fig. 1). Magnetically sorted CD11b+,Gr-1+, into tumors (23). MDSCs may contribute to immunosuppressive or Gr-12 myeloid cells were resuspended in 50 ml modified Tyrode’s buffer tumor networks through a variety of mechanisms including gen- (20 mM HEPES, 10 mM glucose, 5 mM KCI, 120 mM NaCI, and 2 mM eration of reactive oxygen species, secretion of TGF-b, depletion CaCI2 [pH 7.5]) containing 2 mM erythro-9-(2-hydroxy-3-nonyl) adenine (R&D Systems/Tocris Biosciences). The reaction was started with addition of L-arginine, cystine, and cysteine (24). However, in the tumor of 50 ml of the same buffer containing 20 mM[8-14C] ADP (American microenvironment, MDSCs rapidly mature into terminally dif- Radiolabeled Chemicals, St. Louis, MO). After a 10-min incubation period ferentiated myeloid cells that include neutrophils, macrophages, at 37˚C, the reaction was stopped with addition of TCA (5% final concen- 14 and dendritic cells (25). Differentiated cells of the myeloid lineage tration), and tubes were immediately placed on ice. Radioactive [8- C] adenosine, generated by CD39+CD73+ myeloid cells, was separated from represent a major component of the leukocyte infiltrate of many [8-14C] nucleotides on columns of acidic aluminum oxide (1.3 g/column) solid tumors. These cells are composed of multiple distinct sub- by elution with 4 ml 0.005 N hydrochloric acid as described previously (31). populations with pro- or antitumorigenic properties depending on [14C] radioactivity in eluents was measured with a liquid scintillation counter stimuli that triggered their differentiation (26–29). (LS6000IC; Beckman Coulter, Fullerton, CA), and adenosine concentrations In the current study, we report for the first time, to our knowledge, were calculated from calibration curves. + + + + + 2 that CD45 CD11b CD11c F4/80 MHC class II (MHC II) Gr-1 ATP breakdown assay terminally differentiated myeloid mononuclear cells (TDMMCs) Magnetically sorted CD11b+ cells from tumors were resuspended in represent a major cell subpopulation in tumors expressing high modified Tyrode’s buffer (20 mM HEPES, 10 mM glucose, 5 mM KCI, b 5 Downloaded from levels of both CD39 and CD73 and that TGF- acting on myeloid 120 mM NaCI, and 2 mM CaCI2 [pH 7.4]) at a concentration of 10 cells/ml cells can directly regulate the generation of CD39/CD73 TDMMCs, and incubated in the presence of 5 mM ATP for 10 min at 37˚C. Concen- thus contributing to the tumor-promoting effects of this pleiotropic trations of remaining unhydrolyzed ATP in supernatants were determined effector of tumor microenvironment. using an ATP Determination Kit (A22066, Life Technologies/Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Materials and Methods Whole-lung mounting

Mice and cell lines Mice were sacrificed by anesthetic overdose. Lungs were processed as http://www.jimmunol.org/ Mice with conditional deletion of the type II TGF-b receptor in myeloid cells described (32). The tumor nodules in lung were then counted. MyeKO (TGFbRII ) and normal mice (intact TGF-b signaling) as a control for Measurements of secreted vascular endothelial growth factor mice with conditional deletion of the type II TGF-b receptor in myeloid cells (TGFbRIIMyeWT), on a C57BL6 background, and MMTV–polyoma middle Magnetically sorted CD11b+, Gr-1+ or Gr-12 myeloid cells were resus- T Ag (PyMT)/TGFbRIIfloxed and mice with spontaneous tumor formation of pended in RPMI 1640 media at a concentration of 106 cells/ml and in- mammary gland and conditional deletion of the type II TGF-b receptor in cubated in the absence or presence of 10 mM59-N-ethylcarboxamido mammary epithelium (MMTV-PyMT/TGFbRIIKO), on an FVB background, adenosine (NECA; Sigma-Aldrich) for 6 h at 37˚C. Mouse vascular en- were established and maintained as described (22). To generate mice with dothelial growth factor (VEGF) concentrations in supernatants were spontaneous tumor formation of mammary gland and conditional deletion of quantified using the DuoSet ELISA Development Systems (R&D Systems) MyeKO the type II TGF-b receptor in myeloid cells (MMTV-PyMT/TGFbRII ) according to the manufacturer’s instructions. by guest on September 27, 2021 mice, we first crossed LysM-Cre mice (FVB background, kindly provided by Timothy Blackwell, Vanderbilt University, Nashville, TN) with MMTV- Statistical analysis PyMT mice and then MMTV-PyMT/TGFbRIIfloxed mice with MMTV- Data were analyzed using GraphPad Prism 5.02 software (GraphPad, San PyMT/LysM-Cre mice. The studies were approved by the Institutional An- 6 imal Care and Use Committee at Vanderbilt University Medical Center. Diego, CA) and presented as mean SEM. Comparisons between two Lewis lung carcinoma (LLC) cell line (CRL-1642) was obtained from groups were performed using two-tailed unpaired t tests. Multiple comparisons were performed using one-way ANOVAwith appropriate post hoc American Type Culture Collection (Manassas, VA) and maintained following , the manufacturer’s protocols. LLC cells (5 3 105 cells) were injected s.c. tests. A p value 0.05 was considered significant. into the right flank of mice. Results Flow cytometry analysis TDMMCs represent a major cell subpopulation characterized Single-cell suspension from explant of LLC tumor was prepared after colla- by high level of CD39 and CD73 expression in mouse genase I/hyaluronidase digestion for 1 h as described (13). Collagenase mammary carcinomas I/Dispase II solution was used to obtain cell suspension from MMTV-PyMT tumors (23). After treatment with FcR Blocking Reagent, cells (106 cells/ml) To identify tumor cell subpopulations contributing to the genera- were incubated with the relevant Abs for 25 min at 4˚C. If not stated otherwise, tion of adenosine, we compared the cell-surface expression of all Abs were obtained from eBioscience (San Diego, CA) and BioLegend (San CD39 (NTPDase-1) and CD73 (59-nucleotidase) in cell sub- Diego, CA). Data acquisition was performed on LSRII and FACSCalibur flow cytometers (BD Biosciences, Franklin Lakes, NJ), and the data were analyzed populations of tumors obtained from MMTV-PyMT mice on day with FlowJo software (Tree Star). Ag negativity was defined as having the 21 after tumor palpation with their expression on cells from nor- same fluorescence intensity as the isotype-matched control Ab. mal mammary gland tissue of naive FVB mice. Using simulta- neous flow cytometric analysis of several surface markers, we Generation of cells from bone marrow hematopoietic identified a number of phenotypically distinct subpopulations progenitors within CD45-positive immune cells, which consisted mainly of Bone marrow cells were harvested from the femurs and tibias of T lymphocytes and myeloid cells, and within CD45-negative cells, TGFbRIIMyeWT or TGFbRIIMyeKO mice. Hematopoietic progenitor cells 2 which were represented by fibroblast, epithelial, and endothelial (HPC; lineage negative [Lin ]) were isolated using lineage cell depletion kit and LS columns from Miltenyi Biotec (Auburn, CA) according to the cells. Gating strategies used to define cell subpopulations and their manufacturer’s instructions. Resulting cells were .50% CD117 positive as percentage in normal mammary glands or tumors are summarized assayed by flow cytometry. HPC were cultured at an initial concentration in Table I. Representative cytofluorimetric contour plots of CD39 4 of 5 3 10 cells/ml concentration in RPMI 1640 medium containing 10% and CD73 expression in different cell subpopulations from tumor FBS, 20 mM HEPES, 50 mM 2-ME, and 13 antibiotic-antimycotic solution (Sigma-Aldrich, St. Louis, MO) and supplemented with GM-CSF (20 ng/ml) and normal mammary gland tissues are shown in Fig. 1A. and IL-6 (10 ng/ml; both from R&D Systems, Minneapolis, MN) (30) for 3 Tumor growth resulted in an increase in the number of cells to 4 d under a humidified atmosphere of air/CO2 (19:1) at 37˚C. expressing the enzyme pair CD39 and CD73 known to be a major The Journal of Immunology 3157

Table I. Cell subpopulations in normal mammary glands and tumors extracted from MMTV-PyMT mice

Percent of CD45-Positive Cells Percent of CD45-Negative Cells

Normal MG MMTV-PyMT Tumor Normal MG MMTV-PyMT Tumor TDMMC (CD45+CD11b+CD11c+F4/80+MHC II+Gr12) 14.9 6 0.7 76.5 6 5.4 — — MDSC (CD45+CD11b+CD11c2F4/802MHC II2Gr1+) 2.1 6 0.09 9.2 6 1.7 — — T cells (CD45+CD3+) 73.3 6 3.8 11.7 6 0.6 — — Fibroblasts (CD452CD3262CD140a+) — — 81.7.0 6 6.8 11.4 6 1.3 Epithelial/cancer cells (CD452CD326+CD140a2) — — 8.9 6 1.5 75.9 6 6.9 Endothelial (CD452CD3262CD31+CD102+) — — 7.3 6 0.8 1.8 6 0.4 Data are presented as mean 6 SEM of ﬁve animals in each group. Mean values of percentage of CD45+ cells were 49.6 6 5.3 and 18.3 6 7.1% in normal MG and MMTV- PyMT tumors, respectively. Total cell numbers in cell suspensions from normal mammary glands (MG) and tumors were 1.1 6 0.3 3 107 and 9.8 6 2.6 3 107 cell/g of tissue, respectively. Single-cell suspensions obtained from normal MG or MMTV-PyMT tumors were initially gated as DAPI-negative population to exclude dead cells. Then, viable cells were adjusted to singlets using forward light scatter area/forward light scatter height dot plot. Viable singlet-cell population was then used to identify different cell subpopulation based on cell-surface markers indicated in the table. contributor to extracellular adenosine generation (33). Mean values receptor (SRA), also known as CD204, in CD39+CD73+ TDMMCs + + 6 + 2 of CD39 CD73 cell numbers were 19.2 6 4.5 and 2.7 6 0.8 3 10 / compared with CD39 CD73 TDMMCs (Fig. 2D, 2E). Thus, our Downloaded from gram of tissue in tumors and normal mammary glands, respectively in vivo experiments provided indirect evidence of a potential role for (p , 0.01; n = 5, unpaired t test). An increase in CD39+CD73+ cell TGF-b in regulation of CD39+CD73+ TDMMC accumulation in numbers was detected in every cell subpopulation examined, except tumor tissue. for ﬁbroblasts (Fig. 1B). The largest numbers of cells expressing TGF-b signaling promotes differentiation of MDSCs into CD39 and CD73 were found within the cell population with epi- CD39+CD73+ TDMMCs

thelial characteristics most likely represented by tumor cells and http://www.jimmunol.org/ within the myeloid cell population comprised of TDMMC and The tumor microenvironment directs MDSC differentiation toward MDSC subpopulations. Although the numbers of tumor and mye- terminally differentiated myeloid cells, characterized by protu- loid cells expressing CD39 and CD73 were comparable, the ex- morigenic properties (25). To determine whether TGF-b can di- + + pression levels of these ectonucleotidases on their surface, rectly affect differentiation of myeloid cells into CD39 CD73 - calculated as a difference in mean fluorescence intensity (DMFI), expressing TDMMCs, we performed in vitro experiments with differed greatly (Fig. 1C, 1D). CD11b+Gr1+ cells in normal TGF-b stimulation of MDSCs. 2 mammary glands expressed high levels of both CD39 and CD73 MDSCs were generated from Lin HPC as described (30). After on their surface, whereas the expression levels of these ectonu- day 3 in culture, .95% of cells expressed CD11b and Gr-1, the cleotidases on epithelial cells and TDMMCs were significantly cell-surface markers characteristic of the MDSC phenotype (36). by guest on September 27, 2021 lower. In the tumor microenvironment, however, the expression of The percentage of Gr-1–negative cells, which also expressed CD73 and CD39 on TDMMC dramatically increased by 2- and markers of terminally differentiated myeloid cells, including + 30-fold, respectively. In contrast, the expression levels of CD73 F4/80, MHC II, and CD11c, was ,2% of CD11b cells (data not and CD39 on epithelial cells were actually reduced by 20 and shown). Slow spontaneous differentiation of MDSCs and accu- + + + 2 80%, respectively. Although the expression of CD73 on MDSCs mulation of F4/80 MHC II CD11c Gr-1 TDMMCs was ob- was also increased, the expression of CD39 was decreased by served within the next 24 h (day 4) of incubation in the absence of 50%. Therefore, we conclude that, in the tumor microenviron- TGF-b. The expression of CD39 was detected on the cell surface ment, TDMMCs become a major cell subpopulation characterized of all TDMMCs. At least one-fourth of TDMMCs also expressed by high levels of CD39 and CD73 expression. CD73. Incubation of cells in the presence of TGF-b resulted in enhanced MDSC differentiation into TDMMCs and increased Elevated levels of TGF-b protein in the tumor are associated + + + + expression of CD73. Numbers of CD39 CD73 TDMMCs were with an increase in CD39 CD73 TDMMCs increased 2.9-fold in the presence of TGF-b compared with ve- We have recently reported that conditional deletion of Tgfbr2 in hicle (Fig. 3A, 3C). The potent and selective inhibitor of type I mammary epithelium of MMTV-PyMT/TGFbRIIKO mice results receptor activin receptor-like kinase 5 SB431542, at a concentra- in shortened tumor latency and a dramatic increase in the number tion of 2 mM, abrogated the effect of TGF-b in myeloid cells of metastases in the lungs (34). We have also reported that these (Fig. 3C). Accordingly, MDSC numbers were decreased in the aggressive tumors are characterized by a .2-fold increase in presence of TGF-b compared with vehicle, and the TGF-b in- TGF-b levels compared with MMTV-PyMT/TGFbRIIfloxed (35). hibitor SB431542 abrogated these induced effects (data not In this study, we used MMTV-PyMT/TGFbRIIKO and MMTV- shown). To determine whether the effect of TGF-b is mediated via PyMT/TGFbRIIfloxed mice to determine if changes in the tumor activation of TGFbRII, we performed experiments using Lin2 microenvironment, characterized by increased TGF-b levels, can HPC isolated from mice lacking Tgfbr2 in myeloid cells affect tumor-associated populations of CD39+CD73+-expressing (Tgfbr2MyeKO). Representative flow cytometric dot plots showing TDMMCs. Indeed, cytofluorimetric analysis revealed a 2.8-fold in- percentage of MDSCs and TDMMCs, including CD39+CD73+ crease in the number of CD39+CD73+ TDMMCs in tumors ex- TDMMCs, in the absence or presence of TGF-b are shown in tracted from MMTV-PyMT/TGFbRIIKO mice, compared with Fig. 3B. Conditional deletion of Tgfbr2 in myeloid cells isolated MMTV-PyMT/TGFbRIIfloxed controls (Fig. 2A, 2B). In contrast, we from TGFbRIIMyeKO mice resulted in a significant attenuation found no differences in the number of CD39+CD732 TDMMCs of CD39+CD73+ TDMMC accumulation (Fig. 3C), suggesting (Fig. 2C), tumor cells, fibroblasts, or endothelial cells (data not a functional importance of intact TGF-b signaling. The effect of shown). Of interest, analysis of other cell-surface markers on TGF-b was dose dependent with an EC50 of 80 pg/ml (Fig. 3D), TDMMCs revealed higher expression levels of the class A scavenger close to the reported affinity of TGF-b1 to TGF-b receptors (37). 3158 TGF-b IN GENERATION OF CD39/CD73 TDMMC Downloaded from http://www.jimmunol.org/

FIGURE 1. Analysis of the cell-surface expression of CD39 and CD73 in different cell subpopulations in normal pretumor mammary glands and tumors from MMTV-PyMT mice. (A) Representative flow cytometric contour plots showing percentage of CD39+CD73+ cells within different cell subpopulations in normal mammary gland (MG; top panel) and tumor (bottom panel) from MMTV-PyMT mice. (B) Graphical representation of data from flow cytometric analysis of CD39/CD73-expressing cells. Numbers of cells expressing CD39 and CD73 per gram of tissue were calculated from percentages of each cell by guest on September 27, 2021 subpopulation and corresponding percentages of CD39+CD73+ cells. Isotype-matched Abs were used to determine positivity or negativity for particular markers and set the location of gates. Variation in gates location is due to different levels of autofluorescence in each individual cell subpopulation. Data represent mean 6 SEM of five independent experiments with two mice each. Cell-surface expression of CD39 (C) and CD73 (D) in subpopulations of epithelial and myeloid cells obtained from normal MG (open bars) or tumor (closed bars). Bars represent the average DMFI (geometric MFI corresponded to isotype-matched Ab subtracted from geometric MFI of specific Ab) from five independent experiments with two mice each. Asterisks indicate the level of statistical significance: *p , 0.05, **p , 0.01, ***p , 0.001; unpaired two-tailed t test.

It should be noted that only CD73+, but not CD732 TDMMCs, cells. Myeloid cells lacking TGFbRII were also less efficient in generated in vitro from MDSCs in the presence of TGF-b, also ATP breakdown. As seen in Fig. 3I, incubation of these cells (104 expressed a high level of CD204 (Fig. 3E, 3F), highlighting the in 0.15 ml) in the presence of 5 mM ATP for 10 min decreased similarity between the in vivo and in vitro TDMMC phenotypes. ATP concentration in supernatant to 1.5 6 0.04 mM, whereas the Of interest, CD73high intermediate populations of cells with a same number of WT cells decreased extracellular ATP concen- Gr-1low/dim and (F4/80/MHC II/CD11c)low/dim phenotype had tration to 0.17 6 0.04 mM. Taken together, our results suggest that higher CD204 expression compared with corresponding CD73low TGF-b signaling is important for differentiation of MDSCs into intermediate populations (data not shown). Therefore, we con- CD39+CD73+TDMMCs, which are characterized by high clude that TGF-b, acting via TGFbRII, guides MDSC differen- adenosine-producing capacity. tiation into CD39+CD73+ terminally differentiated myeloid cells. Myeloid cell–specific deletion of Tgfbr2 is associated with To compare adenosine-generating capacities of TDMMCs and + + MDSCs, we prepared myeloid cells from Lin2 HPCs in the decreased accumulation of CD39 CD73 TDMMCs and presence of TGF-b as described above and magnetically separated reduced tumor growth and lung metastasis them into Gr-12 and Gr-1+ cell populations. Cells (5 3 104 in 0.1 ml) To evaluate the effect of Tgfbr2 deletion on differentiation of were incubated in the presence of 10 mM ADP for 10 min. Fig. 3G MDSCs into CD39+CD73+ TDMMCs within the tumor microen- shows that adenosine generation by Gr-12 cell population containing vironment, we first analyzed the number of these cells in LLC tumors TDMMCs was .1.5 times higher compared with Gr-1+ cells, which grown in mice lacking Tgfbr2 in myeloid cells (TGFbRIIMyeKO)or are represented mostly by MDSCs. We then compared adenosine- TGFbRIIMyeWT control animals. Flow cytometric examination of generating capacities of myeloid (CD11b+) cells isolated from single-cell suspensions revealed that there was no difference in the LLC tumors grown in mice lacking Tgfbr2 in myeloid cells proportion of tumor-infiltrating CD45+ immune cells between (TGFbRIIMyeKO) or TGFbRIIMyeWT control animals. As seen in TGFbRIIMyeKO and control TGFbRIIMyeWT mice (31.2 6 5.8 and Fig. 3H, adenosine generation by myeloid cells lacking TGFbRII 34.7 6 7.4%, respectively; p . 0.05; n = 5, unpaired t test). We next was more than four times lower compared with wild-type (WT) analyzed TDMMC and MDSC subpopulations within CD11b- The Journal of Immunology 3159 Downloaded from http://www.jimmunol.org/

+ + KO

FIGURE 2. Analysis of CD39 CD73 TDMMC populations in tumors from MMTV-PyMT/TGFRII and MMTV-PyMT mice. (A) Representative dot by guest on September 27, 2021 plots showing percentage of CD39+CD73+ cells within TDMMC subpopulation in tumors extracted from MMTV-PyMT mice lacking Tgfbr2 expression in mammary epithelial cells (TGFbRIIKO, right panel) or MMTV-PyMT/TGFbRIIfloxed control (left panel) animals on day 28 after tumor palpation. Graphic representation of data from flow cytometric analysis of CD73+ (B) and CD732 (C) TDMMCs in tumors extracted from MMTV-PyMT/TGFbRIIKO (closed bars) mice or tumors from MMTV-PyMT/TGFbRIIfloxed (open bars) control animals. Data represent mean 6 SEM from three independent experiments with two mice each. (D) Representative cytofluorimetric histograms demonstrating the expression of cell markers of mature myeloid cells in subsets of CD39+CD732 and CD39+CD73+ TDMMCs. Gray-shaded histograms correspond to the expression of Ags on cell surface of CD39+CD732 TDMMCs, white histograms represent the expression on CD39+CD73+ TDMMCs, and black histograms represent isotype-matched Abs. (E) Graphical representation of mature myeloid cell markers expression on CD39+CD732 (open bars) and CD39+CD73+ (closed bars) TDMMCs. Data are expressed as DMFI and presented as mean 6 SEM from five independent experiments with two mice each. *p , 0.05 TGFbRIIKO versus TGFbRIIfloxed; unpaired two- tailed t test. positive immune cells. Fig. 4A depicts representative flow cyto- latency between MMTV-PyMT/TGFbRIIMyeKO and MMTV-PyMT/ metric dot plots showing percentage of tumor myeloid cell sub- TGFbRIIMyeWT mice (Fig. 4C). However, tumor weight was signifi- populations and the expression of CD39 and CD73. We found that cantly reduced, and the average number of lung metastasis was de- the conditional deletion of Tgfbr2 in myeloid cells resulted in in- creasedinmicewithablatedTgfbr2 in myeloid cells, compared with creased numbers of MDSCs and reduced numbers of TDMMCs control (Fig. 4D, 4E). Total cell numbers in single-cell suspensions (Fig. 4B). The number of CD39+CD73+ TDMMCs was decreased obtained from MMTV-PyMT/TGFbRIIMyeKO tumors were lower than approximately by one-third in TGFbRIIMyeKO mice compared with those in MMTV-PyMT/TGFbRIIMyeWT mice (43.6 6 5.5 and 97.7 6 TGFbRIIMyeWT animals. Importantly, tumor growth was signifi- 11.3 3 106 cell/tumor, respectively; p , 0.01; n = 5, unpaired t test), cantly reduced in mice lacking Tgfbr2 in myeloid cells compared reflecting attenuation of tumor growth in animals lacking Tgfbr2. with control animals (22), implicating the tumor-promoting role for However, the percentage of CD45+ cells was comparable between CD39+CD73+ TDMMCs in an LLC isograft model. tumors obtained either from MMTV-PyMT/TGFbRIIMyeKO or To investigate effects of Tgfbr2 deletion in myeloid cells on differ- MMTV-PyMT/TGFbRIIMyeWT mice with respective mean values ent aspects of multistage breast carcinogenesis, we took an advantage of 25.3 6 5.4 and 29.1 6 7.1% (p . 0.05; n = 5, unpaired t test). of the mouse line established in our laboratory with spontaneous Representative cytofluorometric dot plots showing the percentage mammary gland tumorigenesis, in which Tgfbr2 gene was specifically of MDSCs and TDMMCs within CD45+ cell population are shown deleted in myeloid cells (MMTV-PyMT/TGFbRIIMyeKO). The ad- in Fig. 4F and 4G. Flow cytometric analysis revealed that the vantage of MMTV-PyMT/TGFbRIIMyeKO/WT model is its close sim- number of MDSCs was significantly increased in MMTV-PyMT/ ilarity to human breast cancer characterized by the development of TGFbRIIMyeKO compared with control (Fig. 4H). The higher metastatic lesions in the lungs. No difference was observed in tumor number of MDSCs were reciprocated by decreased numbers of 3160 TGF-b IN GENERATION OF CD39/CD73 TDMMC Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. TGFb/TGFbRII signaling promotes differentiation of MDSCs into CD39+CD73+TDMMCs. Lin2 HPCs were isolated from bone marrow of TGFbRIIMyeWT (A) or TGFbRIIMyeKO (B) mice. Representative flow cytometric dot plots showing percentage of TDMMCs (CD11b+F4/80+MHC II+ CD11c+Gr12) and MDSCs (CD11b+F4/802MHC II2CD11c2Gr1+) and percentage of CD73+ cells within the population of TDMMCs in the absence or presence of 1 ng/ml of TGF-b.(C) Graphical representation of data from flow cytometric analysis of CD39+CD73+TDMMC accumulation expressed as cell numbers per well in the absence (open bars) or presence of 1 ng/ml TGF-b (black bars) alone or in combination with 2 mM SB431542 (hatched bars). (D) Dose-dependent curve for TGF-b to induce an increase in numbers (per well) of CD39+CD73+TDMMCs generated from TGFbRIIMyeWT Lin2 HPCs. (C and D) For details, please see Materials and Methods. The data are expressed as means and SEM of three independent experiments. Flow cytometric histograms showing expression of CD204 on cell surface of CD39+CD732 (gray shaded) and CD39+CD73+ (white) TDMMCs (E) and graphical representation of flow cytometric data (F). Data represent mean 6 SEM from three independent experiments. (G) Adenosine (Ado) production by Gr-12 and Gr-1+ populations of myeloid cells derived from WT (Lin2) HPCs in vitro. Data are presented as mean 6 SEM of adenosine concentrations; n =6.(H) Adenosine (Ado) production by myeloid (CD11b+) cells isolated from LLC tumors grown in TGFbRIIMyeWT (WT) or TGFbRIIMyeKO (KO) mice. Data are presented as mean 6 SEM of adenosine concentrations; n = 4 animals in each group. (I) ATP breakdown by myeloid (CD11b+) cell isolated LLC tumors grown in TGFbRIIMyeWT (WT) or TGFbRIIMyeKO (KO). Data are presented as mean 6 SEM of ATP concentrations remaining in media; n = 5. Statistical significance was calculated by unpaired two-tailed t test (F–I) or one-way ANOVAwith Bonferroni (C) or Dunnett (D) post hoc tests. *p , 0.05, **p , 0.01, ***p , 0.001.

TDMMCs including CD39+CD73+TDMMCs, indicating impaired seen in tumor microenvironment (11, 12), which in turn can differentiation of MDSCs into CD39+CD73+TDMMCs in animals promote angiogenesis (10) and the tumor-associated immuno- lacking myeloid cell–speciﬁc TGF-bRII signaling. suppression (13, 38, 39). In agreement with this concept, we observed a lower density of CD31-positive blood vessels in tumors Myeloid cell–speciﬁc deletion of Tgfbr2 is associated with extracted from MMTV-PyMT/TGFbRIIMyeKO mice compared with decreased tumor angiogenesis and immunosuppression tumors from MMTV-PyMT/TGFbRIIMyeWT (Fig. 5A, 5B). To Immune cells expressing high levels of CD39 and CD73 have been determine if myeloid cells could be a source of angiogenic factors, suggested to contribute to an increase of extracellular adenosine we magnetically separated WT bone marrow–derived myeloid The Journal of Immunology 3161 Downloaded from http://www.jimmunol.org/

FIGURE 4. Myeloid cell–speciﬁc deletion of Tgfbr2 is associated with decreased accumulation of CD39+CD73+ TDMMCs and reduced tumor growth and lung metastasis. (A) Single-cell suspensions were prepared from LLC tumors (3 wk) extracted from TGFbRIIMyeWT (left panel) and TGFbRIIMyeKO (right panel) mice. Representative FACS dot plots showing percentage of TDMMCs (left upper quadrant) and MDSCs (right lower quadrant). Subpop- ulation of TDMMCs was gated and analyzed for expression of CD39 and CD73. (B) Graphical representation of data from ﬂow cytometric analysis of + + MyeWT MyeKO C MDSCs, TDMMCs, and CD39 CD73 TDMMCs in tumors extracted from TGFbRII (open bars) and TGFbRII (closed bars) mice. ( ) by guest on September 27, 2021 Mammary tumor onset in MMTV-PyMT/TGFbRIIMyeWT and MMTV-PyMT/TGFbRIIMyeKO mice. Age of onset is the time that a palpable mammary tumor

first appears. T50 denotes the age at which 50% of mice first possess a tumor, and n is the number of mice examined. (D) Weight of tumor tissue in MMTV- PyMT/TGFbRIIMyeWT and MMTV-PyMT/TGFbRIIMyeKO mice on day 28 after tumor palpation. The total weight of tumors from all 10 mammary glands is indicated. (E) Number of metastatic foci in lungs. Representative cytofluorometric dot plots demonstrating percentage of MDSCs, TDMMCs, and CD39+ CD73+TDMMCs in tumors extracted from MMTV-PyMT/TGFbRIIMyeWT (F) and MMTV-PyMT/TGFbRIIMyeKO (G) mice. (H) Graphical representation of data from FACS analysis of MDSCs and TDMMCs in MMTV-PyMT/TGFbRIIMyeWT and MMTV-PyMT/TGFbRIIMyeKO mice on day 28 after tumor palpation. Data represent mean 6 SEM three independent experiments with two mice each. *p , 0.05; unpaired two-tailed t test. cells into Gr-12 and Gr-1+ cell populations and measured VEGF had no significant effect on VEGF secretion from TGFbRIIMyeKO secretion from these cells in response to their stimulation with the myeloid cells. nonhydrolyzed adenosine analog NECA (1025 M). Fig. 5C shows We have also found that the percentage of CD3e+ T lymphocytes that even in the absence of NECA, Gr-12 cells tended to produce was significantly increased in mammary tumors obtained from mice higher levels of VEGF compared with Gr-1+ cells, though the dif- lacking Tgfbr2 gene expression in myeloid cells compared with ference between basal VEGF levels did not reach statistical sig- control animals (Fig. 5E). Accordingly, the absolute number of nificance. Stimulation of Gr-12 cells with NECA significantly CD3e+ T lymphocytes, and particularly CD3e+CD8+ T lymphocytes, increased their VEGF secretion by ∼3-fold, whereas NECA had no was also significantly higher in tumors obtained from MMTV- significant effect on VEGF secretion from Gr-1+ cells. Therefore, PyMT/TGFbRIIMyeKO mice compared with MMTV-PyMT/ our data suggest that in contrast to MDSCs (Gr-1+), TDMMCs are TGFbRIIMyeWT control animals (Fig. 5F). Although no difference in capable not only of generating high levels of adenosine but also total numbers of CD3e+CD4+ T lymphocytes was seen between responding to stimulation of their adenosine receptors with a sig- tumors obtained from TGFbRIIMyeKO and TGFbRIIMyeWT (Fig. 5F), nificant increase in VEGF secretion. we found that the proportion of activated CD154+CD3e+CD4+ Next, we compared VEGF secretion from myeloid (CD11b+) cells T lymphocytes (40) was 2.5-fold higher in tumors obtained from isolated from LLC tumors grown in mice lacking Tgfbr2 in myeloid mice lacking TGF-bRII in myeloid cells compared with control cells TGFbRIIMyeKO or TGFbRIIMyeWT control animals. As seen in animals (Fig. 5G). Similarly, the proportion of activated cytotoxic Fig. 5D, tumor-derived WT myeloid cells tended to produce higher CD107a+CD3e+CD8+ T lymphocytes (41) was 2-fold higher in levels of VEGF compared with myeloid cells lacking TGF-bRII, tumors obtained from TGFbRIIMyeKO mice compared with control though the difference between basal VEGF levels did not reach sta- animals. Remarkably, the proportion of IFN-g–producing CD3e+ tistical significance. Stimulation of WT myeloid cells with NECA CD8+ T lymphocytes was 3-fold higher in tumors obtained from significantly increased their VEGF secretion by 2-fold, whereas NECA MMTV-PyMT/TGFbRIIMyeKO mice (Fig. 5G). Taken together, our 3162 TGF-b IN GENERATION OF CD39/CD73 TDMMC Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Myeloid cell–specific deletion of Tgfbr2 leads to decreased tumor angiogenesis and immunosuppression. (A) Double immunofluorescence microscopy of frozen tumor sections stained with Abs against CD31 (green) and cytokeratin 8 (K8; red). Scale bar, 100 mm. (B) Graphical representation of vessel density: CD31-positive vessels were quantified within the tumor epithelium and given as fraction of cytokeratin-–positive area. Quantification of immunofluorescence was performed with ImageJ software (National Institutes of Health). Bars represent mean 6 SEM measurements in five fields per tumor of five animals. (C) VEGF secretion by Gr-12 and Gr-1+ populations of myeloid cells derived from WT (Lin2) HPCs in the absence (open bars) or presence (closed bars) of NECA. Data are presented as mean 6 SEM of VEGF concentrations; n =4.(D) VEGF secretion by myeloid (CD11b+) cells isolated from LLC tumors grown in TGFbRIIMyeWT (WT) or TGFbRIIMyeKO (KO) mice in the absence (open bars) or presence (closed bars) of NECA. Data are presented as mean 6 SEM of VEGF concentrations; n = 4 animals in each group. (E) Percentage of CD3-positive T lymphocytes in tumors extracted from MMTV-PyMT/TGFbRIIMyeWT (open bars) and MMTV-PyMT/TGFbRIIMyeKO (closed bars) mice. Data represent mean 6 SEM from five tumors. (F) Total numbers of CD3+ T lymphocytes including numbers of CD3+CD4+ and CD3+CD8+ T lymphocytes in tumors extracted from MMTV- PyMT/TGFbRIIMyeWT (open bars) and MMTV-PyMT/TGFbRIIMyeKO (closed bars) mice. Data represent mean 6 SEM from three tumors. (G)Rep- resentative cytofluorographic outlier contour plots of CD154 and CD69 expression on CD3+CD4+ T lymphocytes (top panel), CD137 and CD107a expression on CD3+CD8+ T lymphocytes (middle panel), and intracellular IFN-g staining of CD3+CD8+ T lymphocytes (bottom panel) in tumors extracted from MMTV-PyMT/TGFbRIIMyeWT (left panels) and MMTV-PyMT/TGFbRIIMyeKO (right panels) mice. Asterisks indicate the level of statistical significance calculated by unpaired two-tailed t test (B, E, F) or one-way ANOVA with Bonferroni (C, D) post hoc test. *p , 0.05, **p , 0.01, ***p , 0.001. results suggest that TGF-b signaling is important for generation of Discussion CD39+CD73+ TDMMCs capable to increase extracellular adenosine In the current study, we adopted a systematic approach to simul- in tumor microenvironment, which in turn can promote angiogenesis taneously analyze CD39 and CD73 expression on various cell and the tumor-associated immunosuppression. populations present in tumors from MMTV-PyMT mice, a clini- The Journal of Immunology 3163 cally relevant spontaneous breast tumor model. We discovered that, esis not only through the production of high levels of extracellular whereas TDMMCs represent a minor cell subpopulation expressing adenosine but also through autocrine adenosine-dependent up- relatively low levels of CD39 and CD73 in normal mammary gland regulation of their VEGF secretion. The latter is in agreement with tissue, they become a major cell subpopulation expressing high our previous data on the significant role of A2B adenosine re- levels of both CD39 and CD73 in the tumor microenvironment. In ceptor–dependent VEGF secretion from immune cells in tumor contrast to normal mammary gland, in which CD140a+ fibroblasts angiogenesis (10). are the major cell population, characterized by CD39 and CD73 Our study clearly demonstrated that the loss of TGF-b signaling surface expression, the majority of cells expressing high levels of in myeloid cells led not only to a decrease in number of TDMMC both CD39 and CD73 in tumors were represented by myeloid expressing the adenosine-generating enzymes CD39 and CD73 cells. We and others have previously reported that the entire subset but also to increased tumoral T lymphocyte infiltration and their of Gr-1high granulocytic MDSCs is characterized by high ex- activation status, reduced VEGF levels and density of blood ves- pression of CD73. In contrast, only a minor fraction of Gr-12/low sels, and slow progression of both LLC and mammary carcino- myeloid cells expressed CD73 when generated in vitro from Lin2 mas. In agreement with our findings, Pang et al. (46) have recently HPC (13) or in melanoma and pancreatic adenocarcinoma mouse reported that the conditional deletion of Tgfbr2 gene expression in tumor models (42). In agreement with these data, we have found myeloid cells decreased tumor metastasis in implanted tumor that the majority of MDSCs and a lesser fraction of TDMMCs models. The authors explained this phenomenon by impaired expressed CD39 and CD73 in tumors from MMTV-PyMT mice. production of type II cytokines. We and others have previously However, due to significant accumulation of TDMMCs in breast shown that adenosine is a potent inducer of IL-4, IL-5, IL-6, IL- tumors, these cells represented a major subpopulation with high 10, IL-13, and arginase in immune cells (9, 10, 47, 48). Therefore, Downloaded from expression levels of both CD39 and CD73. Changes in the tumor it is possible that the effects observed by Pang et al. (46) can be microenvironment associated with elevated TGF-b resulted in explained by reduced contribution of adenosine in stimulation of a further increase in number of CD39+CD73+ TDMMCs. There- type II cytokines in their model. fore, we hypothesized that TGF-b acting on myeloid cells can Of interest, we found that the TGF-b signaling in myeloid cells is directly regulate generation of CD39+CD73+ TDMMCs, thus also involved in regulation of the expression of SRA/CD204.

contributing to the tumor-promoting effects of this pleiotropic Analysis of cell markers revealed an association between the ex- http://www.jimmunol.org/ effector of tumor microenvironment. pression of CD73 and scavenger receptor SRA/CD204 on TDMMCs TGF-b has been characterized as both a tumor-promoting and in tumors from MMTV-PyMT mice. Furthermore, CD39+CD73+, tumor-suppressing growth factor depending on the phase of tumor but not CD39+CD732, TDMMCs, generated in the presence of development, local microenvironmental milieu, and cell type (43). TGF-b in vitro, also were characterized by high levels of CD204 We have previously shown that the deletion of Tgfbr2 expression expression. We have previously shown that upregulation of CD204 in myeloid cells is associated with decreased tumor growth, sug- increases lipid accumulation in dendritic cells, leading to their re- gesting a protumorigenic effect of TGF-b/TGF-bRII signaling duced capacity to process Ags (49). Recent studies have reported (22). It has been suggested that this protumorigenic effect may be a strong correlation between numbers of CD204+ tumor-associated associated with accumulation of MDSCs (35). In the tumor mi- macrophages and tumor aggressiveness, suggesting a tumor- by guest on September 27, 2021 croenvironment, however, MDSCs rapidly maturate into termi- promoting role of these macrophages (50–52). Therefore, it would nally differentiated myeloid cells (25). In the current study, we be interesting in the future to determine whether the protumorigenic demonstrated the direct effect of TGF-b on differentiation of effect of CD204-positive macrophages is mediated, at least in part, myeloid cells into CD39+CD73+ TDMMCs in vitro. Using genetic via action of CD39+ and CD73+. and pharmacological approaches, we found that TGF-b signaling skews maturation of MDSCs into TDMMCs expressing high Disclosures levels of CD39 and CD73. 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M  4

Ado, 2

1 10 100 1000 cells x 103/well

Supplementary Figure 1. Cell concentration-dependent generation of adenosine.

Total population of myeloid cells derived in vitro from WT (Lin-) HPCs was serially diluted in modified Tyrode’s buffer containing 2 μM erythro-9-(2-hydroxy-3-nonyl) adenine. Adenosine generation from 10 μM of [8-14C] adenosine 5’-diphosphate (ADP) was measured as described in the Materials and Methods section.