Mast Cell–Dependent CD8 T-Cell Recruitment Mediates Immune

Published OnlineFirst January 30, 2018; DOI: 10.1158/2326-6066.CIR-17-0424

Research Article Cancer Immunology Research Mast Cell–Dependent CD8þ T-cell Recruitment Mediates Immune Surveillance of Intestinal Tumors in ApcMin/þ Mice Sobha R. Bodduluri1,2, Steven Mathis1,2, Paramahamsa Maturu1,2, Elangovan Krishnan1,2, Shuchismita R. Satpathy1,2, Paula M. Chilton2,3, Thomas C. Mitchell2,3, Sergio Lira4, Massimo Locati5,6, Alberto Mantovani5,7, Venkatakrishna R. Jala1,2, and Bodduluri Haribabu1,2

Abstract

The presence of mast cells in some human colorectal cancers is a CCR2 and CCR5 expression and were also efficient in antigen þ positive prognostic factor, but the basis for this association is presentation and activation of CD8 T cells. Mast cell–derived þ incompletely understood. Here, we found that mice with a leukotriene B4 (LTB4) was found to be required for CD8 T heterozygous mutation in the adenomatous polyposis coli gene lymphocyte recruitment, as mice lacking the LTB4 receptor þ – – – – þ (ApcMin/ ) displayed reduced intestinal tumor burdens and (ACKR2 / BLT1 / ApcMin/ ) were highly susceptible to intestinal increased survival in a chemokine decoy receptor, ACKR2-null tumor-induced mortality. Taken together, these data demonstrate background, which led to discovery of a critical role for mast cells that chemokine-mediated recruitment of mast cells is essential for –/– Min/þ þ in tumor defense. ACKR2 Apc tumors showed increased initiating LTB4/BLT1-regulated CD8 T-cell homing and genera- infiltration of mast cells, their survival advantage was lost in mast tion of effective antitumor immunity against intestinal tumors. – – – – þ cell–deficient ACKR2 / SA / ApcMin/ mice as the tumors grew We speculate that the pathway reported here underlies the positive rapidly, and adoptive transfer of mast cells restored control of prognostic significance of mast cells in selected human tumors. – – tumor growth. Mast cells from ACKR2 / mice showed elevated Cancer Immunol Res; 6(3); 332–47. 2018 AACR.

Introduction their antitumorigenic functions by direct interactions with tumor cells, the majority of their effector functions rely on Mast cells are critical mediators of allergic responses, path- releasing a wide variety of mediators and modulating the ogen recognition, and modulation of immune responses at immune response. Cytokines and chemokines produced by mucosal sites (1). The presence of mast cells in tumors has mast cells are known to have opposing roles in cancer progres- been correlated with either good or bad prognosis in several sion by recruiting immune cells to tumor environment (8). Mast studies of rodents and humans (2), highlighting the complexity cells also rapidly synthesize a wide range of lipid mediators, of effects mediated by mast cells. Tumor-associated mast cells such as leukotriene B (LTB ), leukotriene C (LTC ), and promote a variety of cancers by enhancing inflammation and 4 4 4 4 prostaglandin E2 (PGE2), that regulate a range of pathophys- angiogenesis (3, 4). However, in human colon cancers, mast cell iological activities, including leukocyte recruitment and smooth presence is correlated with better prognosis (5–7), but the muscle contraction (9). mechanisms remain unknown. Although mast cells can exert Chronic inflammation promotes a variety of cancers, but inflammation can also play an important role in tumor surveillance (10, 11). Atypical chemokine receptor 2 (Ackr2, 1 James Graham Brown Cancer Center, University of Louisville Health Sciences hereafter referred to as ACKR2, formerly known as D6) is a Center, Louisville, Kentucky. 2Department of Microbiology and Immunology, 3 "decoy" chemokine receptor that binds and internalizes a large University of Louisville Health Sciences Center, Louisville, Kentucky. Institute fl for Cellular Therapeutics, University of Louisville Health Sciences Center, cadre of in ammatory chemokines targeting them for degra- Louisville, Kentucky. 4Immunology Institute, Icahn School of Medicine at Mount dation, and, thus, limiting the inflammatory response (12). The Sinai, New York, New York. 5Humanitas Clinical and Research Center, University importance of ACKR2-mediated chemokine scavenging in the of Milan, Milan, Italy. 6University of Milan, Milan, Italy. 7Humanitas University, resolution of inflammation became evident in different animal Rozzano, Italy. models such as chemical-induced cutaneous inflammation Note: Supplementary data for this article are available at Cancer Immunology (13, 14), ovalbumin- or Mycobacterium-induced lung inflam- Research Online (http://cancerimmunolres.aacrjournals.org/). mation (15, 16), lipopolysaccharide (LPS)-induced placental Corresponding Authors: Bodduluri Haribabu, University of Louisville, 324 CTRB; inflammation (17), or methylcholanthrene/phorbol ester 505 S Hancock, Louisville, KY 40202. Phone: 502 852-7503; E-mail: (TPA)-induced skin cancer (18), as well as azoxymethane [email protected]; and Venkatakrishna R. Jala, Phone: 502 852-5523; (AOM)-induced dextran sodium sulfate (DSS)-promoted Fax: 502 852 2123; E-mail: [email protected] (AOM/DSS) colon tumor models (19). – – þ doi: 10.1158/2326-6066.CIR-17-0424 In this study, ACKR2 / mice were bred with ApcMin/ mice, 2018 American Association for Cancer Research. which develop spontaneous intestinal adenomas, in order to

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understand the link between chronic inflammation and tumor Immunohistochemistry (IHC) promotion. Contrary to the established role of ACKR2, the Cleaned small intestines were divided into 3 equal length – – þ ACKR2 / ApcMin/ mice showed decreased inflammation coin- sections (proximal, middle, and distal) and fixed with 10% ciding with decreased tumor burden and improved survival. neutral formalin for 16 hours followed by storage in 70% ethanol Using genetic, biochemical, and immunologic methods, we until embedded in paraffin. The sections were cut at 5-mm identified enhanced mast cell infiltration as the likely cause thickness and used for hematoxylin and eosin staining (H&E) – – þ of reduction in tumor burden in the ACKR2 / ApcMin/ mice. or immunohistochemical analysis. All images were scanned with We demonstrate that LTB4 produced by mast cells mediates an Aperio digital scanner (Leica Biosytems). þ CD8 T-cell recruitment and is essential for generating effective For mast cell IHC, formalin-fixed, paraffin-embedded (FFPE) antitumor immunity against intestinal tumor development. sections were heated at 90C in a 10 mmol/L Tris, 1 mmol/L EDTA (pH 9.0) solution followed by incubation for 1 hour at room temperature with rat monoclonal anti-mouse Mcpt-1 Materials and Methods (clone RF6; eBioscience) 1/200 dilution (2.5 ng/mL final con- Mice centration) using standard histochemical protocols and Vectas- – – ACKR2 / mice were obtained from Schering-Plough. To gen- tain ABC-AP staining kit (Vector Laboratories). To measure – – þ – – þ – – þ erate ACKR2 / ApcMin/ ,SA/ ApcMin/ , BLT1 / ApcMin/ , and tumor cell proliferation, mice were given 5'-bromo-2'-deoxyur- – – þ þ Rag2 / ApcMin/ strains, male C57BL/6-ApcMin/ mice (The Jack- idine (BrdUrd; 1 mg/mL) by intraperitoneal route and eutha- – – – – son Laboratory) were crossed individually with ACKR2 / ,SA / nized after 2 hours, followed by intestinal processing and (Sash mice/C57BL/6J-KitW-sh/W-sh; The Jackson Laboratory), paraffin embedding. IHC for BrdUrd was performed using a – – – – BLT1 / (20), and Rag2 / (Taconic Biosciences) mice, respective- BrdUrd in situ detection kit from BD Pharmigen, according to the ly. The following compound mouse strains were generated manufacturer's instructions. Cells containing BrdUrd-positive – – þ by breeding male ACKR2 / ApcMin/ mice individually with nuclei (brown nuclei) were manually counted by experienced – – – – female KitW-sh/W-sh, BLT1 / , or Rag2 / mice, respectively: lab personnel per 200 field. Apoptotic cells within tumors were – – – – þ – – – – þ ACKR2 / SA / ApcMin/ , ACKR2 / BLT1 / ApcMin/ , and determined by terminal deoxynucleotidyl transferase-mediated – – – – þ ACKR2 / Rag2 / ApcMin/ . Animals were housed under specific nick end-labeling (TUNEL) using the in situ Cell Death Detection þ pathogen-free conditions, and all mice carrying the ApcMin/ Kit, Fluorescein (Roche). Briefly, FFPE tissue sections were allele were maintained on breeder chow. The genotyping PCR dewaxed in xylene and alcohol. Epitope retrieval was performed þ for ApcMin/ and Rag2 was performed according to Jackson by heating to 95Cto100C for 30 minutes in sodium citrate Laboratories protocols and for the detection of ACKR2 and BLT1 buffer (pH 6.0) and exposed to the TUNEL reaction mixture, using previously described methods (refs. 13, 20; Supplementary according to the manufacturer's instructions. The TUNEL-pos- Table S1). All the experimental protocols were approved by the itive, green-fluorescent cells were captured using a Nikon Eclipse Institutional Animal Care and Use Committee (IACUC) at the TE300 fluorescence microscope and manually counted per field University of Louisville. at 200 magnification.

Survival of mice Immunofluorescence studies Min/þ þ/– Min/þ The overall survival of the mice (Apc , ACKR2 Apc , Distal intestine tissue was filled with OCT compound, Swiss –/– Min/þ –/– –/– Min/þ –/– ACKR2 Apc , ACKR2 SA Apc , and ACKR2 BL- rolled, mounted with OCT, and rapidly frozen by immersing in 2- –/– Min/þ T1 Apc ) was followed from birth to the endpoint of methylbutane (Sigma) chilled in dry ice. The OCT frozen tissue moribund or death. Significance of differences in survival was was cut into 6- to 7-mm size sections and fixed in cold acetone determined by the Mantel–Haenszel/log-rank test. (20C) for 10 minutes. After air drying for one hour, sections were washed with 1 PBS and blocked for 30 minutes at room Measurement of hematocrit temperature with 5% goat serum, followed by incubation with Animals were euthanized by CO2 asphyxiation, and 200 mLof primary rat anti-mouse CD8a (clone 53-6.7; BD Biosciences), blood was drawn from the heart and put into heparin-coated anti-mouse Mcpt-1 (clone RF6, eBioscience), or isotype control microvette (Sarstedt) tubes followed by analysis using the Hema- antibodies (at a 1/50 dilution) for 60 minutes at room temper- vet-950 Hematology System (Drew Scientific). ature in a humidified chamber. Slides were then washed with 1 Phosphate-buffered saline, followed by addition of goat anti- Analysis of adenomas rat IgG–Alexa Fluor 488 at a 1/500 dilution and incubated for Mice were sacrificed at 40 days or 110 days of age, and the entire 30 minutes. Sections were counterstained with DAPI-1, mounted intestinal tract was removed and flushed with 1 PBS using a using Vectashield mounting medium (Vector Laboratories) and blunt-end syringe to remove fecal material. Flushed small intes- visualized on Nikon A1R confocal microscope using appropriate þ tines were longitudinally opened and spread on filter paper using filters. The number of tumor-infiltrating CD8 T cells from at least forceps, immobilized by placing another filter paper on the top, 6 tumors in each genotype was counted, calculated per 100 mm2 and fixed in 10% neutral formalin for 16 hours followed by area, and plotted. transferring into 70% ethanol Polyps in proximal, middle, distal Bone marrow–derived mast cells (BMMC; 1 105) isolated, as small intestines, and colon were counted by experienced lab described in a later section, were washed and spread on superfrost personnel under a stereo microscope and classified by size: slides in 70 mL volume using a Shandon cytospin centrifuge. 0–1 mm, 1–2 mm, 2–3 mm, and >3 mm. The mean number of Cytospin preparations of BMMCs were stained indirectly using tumors/mouse SEM and the mean tumor diameter (mm) in the rat anti-CD117 (clone, 2B8; Santa Cruz Biotechnology) and group SEM were calculated for the small intestine and colon, biotin-labeled anti-FceR1 (clone MAR-1; eBioscience) antibodies separately. and visualized using anti-rat Green Alexa-488–labeled antibody

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(Invitrogen) and Red Alexa-647 Streptavidin (Jackson Immuno Western blot analysis of Mcpt-1 Research Laboratories). The immunofluorescence images were Size-matched intestinal tumors were dissected, snap-frozen in captured using a Nikon A1R confocal microscope using appro- liquid nitrogen, and stored at 80C. Tumors and normal distal priate filters. intestine tissues were homogenized in Bio-Rad lysis buffer containing 1 protease inhibitor cocktail (Roche) using a hand-held RNA Extraction, real-time PCR, and microarrays homogenizer (Omni International). Homogenates were incubat- The distal small intestine tissue from WT (C57BL/6) and ed for 1 hour at 4C with constant agitation, followed by centri- – – ACKR2 / mice, as well as size-matched intestinal tumors of fugation. Total cell extracts (50 mg of total protein as determined by – – þ þ ACKR2 / ApcMin/ and ApcMin/ mice (110 days age old) were Pierce BCA protein assay kit, Thermo Fisher Scientific) were sepa- dissected and frozen immediately in TRIzol reagent (Invitrogen) rated on 4% to 12% NuPAGE Bis-Tris precast gels and transferred at 80C. After quick thawing, tissue was homogenized using an onto nitrocellulose (Bio-Rad) membranes. Membrane blots were Omni GLH general homogenizer, RNA was extracted using the probed with rat anti-mouse Mcpt-1 (1 mg/mL; clone 285008; R&D RNA mini prep kit from Qiagen, followed by DNase treatment Systems), followed by peroxidase-coupled secondary anti-rat IgG (Ambion Inc and stored at 80 C until further usage. HRP and detected with Pierce ECL substrate. The same blot was RNA was extracted from BMMCs, macrophages, T cells, B cells, stripped with stripping buffer (Thermo Scientific) and reprobed and dendritic cells isolated as described in later sections and with b-actin-HRP antibody (Santa Cruz Biotechnology). frozen (80C) in TRIzol in a similar manner as above without homogenization. For BMMC microarray analysis, total RNA BMMC culture obtained from three cultures derived from independent mice BMMCs were generated from 4- to 6-week old female mice. for each genotype were analyzed on Bioanalyzer (Agillant- Bone marrow was flushed with RPMI from tibia and femur bones, 2100) and the integrity verified (RIN of 7.5 or above). The three gently mushed (pulped), filtered through 60-mm nylon mesh, and samples for each genotype were pooled and used immediately for centrifuged at 450 g for 5 minutes. The pellet was resuspended in microarray analysis. BMMC culture medium [DMEM containing 10% FCS, penicillin BMMCs microarray analysis was performed using the (100 units/mL), streptomycin (100 mg/mL), 2 mmol/L L-gluta- Affymetrix GeneChip Mouse Gene 1.0 ST Array, according to mine, and 50 mmol/L b-mercaptoethanol] supplemented with manufacturer's protocol at the microarray core facility, University recombinant mouse SCF (12.5 ng/mL; R&D Systems, #455-MC) of Louisville. The GeneChip-brand array is comprised of over and recombinant mouse IL3 (10 ng/mL; R&D Systems, #403-ML). 750,000 unique 25-mer oligonucleotide features constituting Cells were plated at a density of 1 106 cells/mL in a T-75 cm2 over 28,000 gene level probes with an average of 27 probes per flask. Nonadherent cells were transferred after 48 hours into fresh gene. Briefly, total RNA was amplified and labeled following the flasks without disturbing the adherent (fibroblast) cells. Flasks Affymetrix standard protocol for whole transcript expression were changed weekly or as needed to separate the nonadherent analysis and then hybridized to the Affymetrix Mouse Gene 1.0 mast cells from the contaminating adherent cells with BMMC ST arrays. The arrays were processed following the manufacturer- culture medium described above. Mast cells were visible after recommended wash and stain protocol on an Affymetrix FS-450 4 weeks of culture and propagated further by dividing once or fluidics station and scanned on an Affymetrix GeneChip 7G twice a week to a density of 0.5 106 to 1 106 cells/mL. For scanner using the Command Console 3.1. The resulting CEL files some experiments, mast cells were purified from non- were imported into Partek Genomics Suite 6.6, and transcripts homogenous cultures after 2 weeks on a MACS MS column were normalized on a gene level using RMA as the normalization (Miltenyi Biotec) using CD117 antibody positive selection, and background correction method. One-way ANOVAs were set according to the manufacturer's protocol. BMMCs were con- up to compare the genotypes (WT vs. ACKR2 / ) of interest. firmed by double-positive staining for mast cell–specific markers Pathway analysis was carried out by uploading data files into c-Kit and FceR1 (clones 2B8 and MAR-1, respectively; eBioscience) Ingenuity Pathway Analysis (IPA) software. by FACS analysis and used at least at 95% purity. The microarray for tumors was carried out using the whole mouse genome chip (Mouse 430 2.0 array; Affymetrix). Total RNA Chemotaxis – – obtained from three each of the following independent mice was Chemotaxis of BMMCs (from WT and ACKR2 / mice) was pooled. WT small intestine tissue; small intestine tumors from evaluated using 5-mm pore size Transwell filters (Corning Costar). þ þ ApcMin/ ; and ACKR2 / ApcMin/ mice. Experimental and sam- Briefly, cells were resuspended at a density of 5.0 106 cells/mL in ple preparation variations were standardized by applying the chemotaxis buffer (RPMI 1640 and 0.5% BSA). The lower cham- global scaling procedure to all absolute analysis data using ber was loaded with various concentrations of SCF (R&D Sys- constant global target intensity. The data were analyzed using tems), CCL2, and/or CCL5 (PeproTech Inc.) in a volume of 600 Affymetrix's MAS 5 algorithm for probe set summarization, mL, and 0.5 106 BMMCs were added to the upper chamber in followed by pairwise comparison. 100 mL BMMC medium. After 3 hours at 37 Cin5%CO2, the For all semiquantitative real-time PCRs (qPCR), cDNA was upper chamber was removed, and the cells in the lower chamber synthesized from 1 mg of total RNA using random hexamer were fixed in buffered 2% formaldehyde. The migrated cells were primers and Taqman reverse Transcription reagents counted using a FACS Canto (BD Biosciences) by running for fixed (N8080234, Applied Biosystems), followed by qPCR reaction time (fixed volume of cells) and analyzed using buffer only as a with SYBR-green master mix using PCR primers (Realtime Primers background control. LLC; listed in Supplementary Table S2). Real-time qPCR was performed in a Bio-Rad CFX96TM real-time PCR thermocycler. Calcium flux Data were normalized to b-actin, and the relative expression of BMMCs (4 106/2 mL) were loaded with 1 mmol/L INDO-1 target genes was calculated using the 2 DDCT method. (Molecular Probes) and the calcium response after stimulation

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– – with different concentrations of CCL2, CCL5, and SCF as indi- stored at 20C. WT or ACKR2 / BMMCs (2 106) were cated and measured using spectrofluorimeter. incubated with SIINFEKL for 3 hours in DMEM containing 10% FBS. Following incubation, BMMCs were washed 3 times Preparation of immune cells and resuspended in DMEM containing 10% FBS. For coculture þ þ þ CD8 T cells were isolated from the spleens and mesenteric experiments, 1 106 CD8 T cells or CFSE-labeled CD8 T cells lymph nodes of OT-1 mice (The Jackson Laboratory). Briefly, were mixed at different ratios with peptide-loaded BMMCs and spleen and lymph nodes were crushed and passed through 70-mm cultured for 48 or 72 hours to measure activation or proliferation, þ strainers (Corning), and the red blood cells were lysed with RBC respectively. CD8 T cell activation was measured by increased þ lysis buffer (BioLegend). After lysis, CD8 T cells were selected surface expression of CD69 (clone H1-2F3; eBioscience), CD44 þ using CD8a (Ly-2) microbeads (Miltenyi) on a MACS LS column, (clone IM-7), CD25 (clone PC61, BioLegend) by FACS. The CD8 þ according to the manufacturer's protocol. The purified CD8 T T cell proliferation was determined by FACS, following the CFSE þ cells were >95% positive for CD8 as analyzed by FACS. These cells dilution of CFSE-labeled CD8 T cells. were used for mast cell and T-cell coculture studies. – – – – þ For ACKR2 / Rag2 / ApcMin/ adoptive transfer experiments Flow cytometry (described in following sections), spleen cell suspensions Cells (1 to 5 105 cells) were resuspended in 50 mL FACS – – þ þ from ACKR2 / ApcMin/ mice were positively enriched for CD8 buffer (1 PBS containing 1% BSA), and FcR blocked (15 min- T cells using CD8a (Ly-2) microbeads, followed by purification utes at 4C) using rat anti-mouse CD16/CD32 (clone 2.4G2; þ on MACS LS columns. The CD8 T cell–negative fraction was BD Biosciences) antibody. Cells were then incubated on ice in þ used subsequently to isolate CD4 T cells using CD4 (L3T4) the dark with cell-specific fluorochrome-conjugated antibodies microbeads. listed in other sections for 40-minute labeling. Cells were wash- For CCR2 and CCR5 RNA expression analysis, T cells and B ed with cold 1 PBS, centrifuged at 400 g for 3 minutes, and cells were isolated from spleens and mesenteric lymph nodes fixed with 2% formaldehyde (Fisher Scientific). Cells were – – of WT and ACKR2 / mice using a Pan T-cell isolation kit (#130- acquired using a FACS Caliber or FACS Canto (BD Biosciences) 090-861) from Miltenyi and mouse B220 (clone RA3-6B2, BD and analyzed using FlowJo software v7.6.5). The cell viability Biosciences) positive selection on MACS MS columns. was measured using 0.5 mLoffixable viability Dye eFluor 660 Bone marrow–derived macrophages (BMDM) were generated (eBioscience) in 500 mL1 PBS. – – by isolating cells from femurs and tibia of C57BL/6 and ACKR2 / mice and culturing in DMEM containing 10% FCS, penicillin Intracellular IFNg staining (100 units/mL), streptomycin (100 mg/mL), 2 mmol/L T cells (1 106) were stimulated in 12-well plates for 5 hours L-glutamine, and 50 mmol/L b-mercaptoethanol) supplemented with 1 cell stimulation cocktail containing phorbol 12-myristate with recombinant M-CSF (100 ng/mL; catalog #576404, 13-acetate (PMA) and ionomycin (eBioscience) and then washed, BioLegend) for 7 to 8 days. Bone marrow–derived dendritic cells fixed in 1 buffer (# 00-8333, eBioscience), permeabilized (BMDC) were generated by culturing cells from bone marrow in (# 00-8222, eBioscience), and stained with rat anti-mouse IFNg RPMI containing 10% FCS, penicillin (100 units/mL), strepto- (clone XMG 1.2; BioLegend) according to eBioscience intracellu- mycin (100 mg/mL), 2 mmol/L L-glutamine, and 50 mmol/L lar staining instructions. b-mercaptoethanol in the presence of GM-CSF (40 ng/mL; catalog þ #415-ML, R&D Systems) and human Flt-3 (100 ng/mL; catalog Adoptive transfer of mast cells and CD8 T cells – – #308-FKN, R&D Systems) for 8 to 9 days. The purity of the BMMCs (1 107 in PBS) derived from ACKR2 / mice were – – – – þ immune cell population was determined by flow cytometry given intravenously (i.v.) to 30-day-old ACKR2 / SA / ApcMin/ using following antibodies: CD117 (clone 2B8, eBioscience), mice. Recipients and controls were analyzed for anemia and FceR1(clone MAR-1, eBioscience), MHCII (clone AMS-32.1, BD tumor development at 100 days age as described in other sections. – – – – þ Biosciences), CCR7 (clone 4B12, BioLegend), CD86 (clone GL-1 ACKR2 / Rag2 / ApcMin/ mice at 35 days of age were given 8.5 þ – – þ BD Biosciences), CD11b (clone M1/70, BD Biosciences), CD11c 105 CD8 T cells isolated from the spleens of ACKR2 / ApcMin/ þ þ (clone HL-3, BD Biosciences), F4/80 (clone BM8, eBioscience), mice or the same number of combined CD4 and CD8 T cells CD4 (clone GK1.5, eBioscience) and CD8 (clone 53-6.7), (1:1) in PBS. CD19 (clone MB-19-1, eBioscience), CD3 (clone 145-2C11, BD Biosciences), GR-1 (clone RB6-8C5 eBioscience, and CD45 (clone Leukotriene B4 measurement 30-F11, eBioscience). For measuring levels of LTB4, distal intestine tumors from – – þ – – – – þ – – ACKR2 / ApcMin/ , ACKR2 / SA / ApcMin/ , and ACKR2 / – – þ CFSE labeling BLT1 / ApcMin/ were resected into 500 mL of cold indomethacin The CFSE (Invitrogen) stock was freshly prepared at 10 mmol/L (10 mmol/L) containing 1 PBS and homogenized immediately þ in DMEM. The purified OT-1 CD8 T cells were washed with using a Omni GLH general homogenizer. The homogenates were DMEM and labeled with 2.5 mmol/L CFSE for 10 minutes at 37 C centrifuged at 14,000 g for 10 minutes, and the LTB4 levels in in the dark. The reaction was stopped quickly with DMEM con- the supernatant were quantified using a LTB4 EIA kit (Cayman taining 2% FCS, and cells were then centrifuged, washed, and Chemical). The LTB4 levels were normalized to the amount of resuspended in 2 mL DMEM containing 10% FBS. The cells were protein in the homogenates (measured by BCA protein assay, counted and diluted to 1 106 cells/mL for plating. Thermo Fisher Scientific).

BMMC and T-cell cocultures Statistical analysis The stock of OVA octapeptide SIINFEKL (ovalbumin 257–264; Standard Mann–Whitney U nonparametric, two-tailed test and Sigma) was prepared in 1 PBS at a 0.5 mg/mL concentration and two-tailed Student t test were used for comparisons between two

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– – þ experimental groups, with a P value of <0.05 considered from ACKR2 / ApcMin/ mice contained more apoptotic cells þ significant using Graph Pad Prism software (, P < 0.001; , compared with ApcMin/ mice indicating that immune surveil- P < 0.01; , P < 0.05). The significance of survival of animals was lance might play a role in controlling tumor growth (Fig. 1H). calculated using Kaplan–Meier survival curves by the Mantel– – – þ Haenszel/log-rank test. Increased mast cell infiltration in ACKR2 / ApcMin/ tumors In contrast to the proinflammatory phenotype observed with several ACKR2-null mouse models (13, 17, 23, 24), we found that Results – – þ ACKR2 / ApcMin/ mice displayed decreased inflammation and –/– Minþ Decreased mortality and tumorigenesis in ACKR2 Apc tumor burden. To examine the molecular mechanisims underly- – – þ mice ing this phenotype, gene expression profiles of ACKR2 / ApcMin/ þ In AOM/DSS-induced colon tumor models, genetic deficiency and ApcMin/ intestinal tumors were compared by microarray – – þ of ACKR2 accelerates tumor growth by enhancing chemokine- analysis. Tumors from ACKR2 / ApcMin/ were enriched in RNA mediated inflammation (19). In marked contrast, we found that transcripts for mast cell proteases (Mcpt) 1 and 2 relative to –/– Min/þ þ ACKR2 Apc mice displayed a significant decrease in mor- ApcMin/ tumors (Fig. 2A). This expression pattern was confirmed Min/þ bidity and mortality relative to Apc mice (Fig. 1A). The by qPCR (Fig. 2B) and Western blot analysis (Fig. 2C), and IHC –/– Min/þ average life expectancy of ACKR2 Apc at 208 days was a analysis revealed it was due to a significant increase in mast Min/þ – – þ third more than the 154 days observed for Apc mice. Het- cell infiltration of tumors in ACKR2 / ApcMin/ mice relative to þ/– Min/þ þ erozygous ACKR2 Apc mice also showed improved sur- ApcMin/ mice (Fig. 2D). The pattern appeared to be established vival with some mice approaching 300 days of age, whereas some early in disease, as small intestinal microadenomas analyzed from –/– Min/þ – – þ of the ACKR2 Apc mice were alive as long as 500 days 40-day-old ACKR2 / ApcMin/ mice were also enriched in mast þ (when the experiment was terminated). Intestinal adenomas cells relative to ApcMin/ mice (Supplementary Fig. S4). þ cause severe anemia in ApcMin/ mice, as seen here by their – – þ significant reduction in hematocrit values compared with WT Mast cells attenuate tumor burden in ACKR2 / ApcMin/ mice mice at 110 days age (Fig. 1B). Consistent with their improved To determine whether or not mast cells were responsible for –/– Min/þ – – þ survival, ACKR2 Apc displayed significantly increased protection from intestinal tumorigenesis, ACKR2 / ApcMin/ Min/þ hematocrit values compared with Apc mice, indicating mice were crossed with the mast cell deficient Kitw-sh/w-sh (SA) – – – – þ slower progression of the disease. No difference in hematocrit mice and tested for tumor burden. These ACKR2 / SA / ApcMin/ –/– values was observed between the WT mice and the ACKR2 mice died with average lifespans of 180 days, close to those of þ mice. ApcMin/ mice (Fig. 3A), indicating mast cells were required for the – – þ Tumor burden in the small intestine and colon was analyzed by survival advantage of ACKR2 / ApcMin/ mice. The hematocrit – – – – þ measuring tumor number and size in longitudinally opened values of ACKR2 / SA / ApcMin/ mice were significantly lower – – þ intestinal sections. In the small intestine, tumor number and size than ACKR2 / ApcMin/ mice, consistent with faster disease pro- –/– Min/þ were significantly reduced in ACKR2 Apc mice (Fig. 1C–F), gression in the absence of mast cells (Fig. 3B). Min/þ indicating slower disease progression compared with Apc . Although the total number of polyps in the small intestines of – – – – þ The frequency of polyps was similarly reduced in proximal, ACKR2 / SA / ApcMin/ mice was increased slightly, the increase –/– Min/þ – – þ middle and distal regions of ACKR2 Apc small intestines was not significantly different from ACKR2 / ApcMin/ mice (Fig. 1D). The gross appearance of longitudinally opened (Fig. 3C). However, analysis of the size distribution of polyps in distal small intestines (Fig. 1F) provides an image that clearly the small intestine revealed a significant increase that was restrict- –/– Min/þ illustrates the decrease in polyp numbers in ACKR2 Apc ed to larger tumors (1–2 mm, 2–3 mm, and 3 mm and above) in – – – – þ – – þ mice. In contrast, the number of tumors and the size frequency ACKR2 / SA / ApcMin/ relative to ACKR2 / ApcMin/ mice, of tumors found in the colons did not show significant despite comparable total number of tumors in both groups Min/þ –/– Min/þ differences between Apc and ACKR2 Apc mice (Sup- (Fig. 3C and D). H&E staining (Fig. 3E) of small intestinal sections plementary Fig. S1). Analysis of 40-day-old mice demonstrated revealed the presence of large tumors in the distal intestine of Min/þ – – – – þ a significant decrease in hematocrits only in Apc mice but ACKR2 / SA / ApcMin/ compared with mast cell sufficient mice. –/– Min/þ not in the ACKR2 Apc mice (Supplementary Fig. S2A). Tumors in the range of 0 to 1 mm were significantly less frequent –/– Min/þ – – – – þ ACKR2 Apc mice also showed significantly reduced in ACKR2 / SA / ApcMin/ mice, which suggests that tumor Min/þ intestinal polyps compared with Apc mice (Supplementary growth, and not tumor initiation, was affected most by the Fig. S2B). Histopathologic analysis of small intestines showed absence of mast cells. To further test for mast cell contributions Min/þ – – significant microadenomas in Apc mice at 40 days of age, to immune surveillance, BMMCs from ACKR2 / mice were –/– Min/þ – – – – þ whereas minimal disease was noted in ACKR2 Apc mice infused intravenously into 4-week-old ACKR2 / SA / ApcMin/ (Supplementary Fig. S2C). mice and analyzed for tumor burden at 110 days of age. The Min/þ – – – – þ In the Apc model, inflammation is known to promote hematocrit values of mast cell–transferred ACKR2 / SA / ApcMin/ intestinal tumor development (21, 22). Consistent with mice were significantly higher (Fig. 3F), with concomitant decreased tumor burden, analysis of mRNA from the reductions in tumor size and numbers (Fig. 3G and H). Hence, –/– Min/þ ACKR Apc tumors revealed that the expression of our genetic and cell-transfer experiments indicate mast cells – – inflammatory mediators COX-2, CXCL1, IL1b,andTNFa are responsible for slower tumor progression in ACKR2 / –/– Min/þ þ decreased in ACKR2 Apc mice compared with ApcMin/ mice. þ ApcMin/ mice (Supplementary Fig. S3). Tumor size reduction – – coincided with lower cell proliferation, as demonstrated by Sustained expression of CCR2 and CCR5 in ACKR2 / mast cells –/– Min/þ reduced BrdUrd incorporation in ACKR2 Apc tumors To explore the mechanisms behind the increased mast cell Min/þ – – þ compared with Apc tumors(Fig.1G).However,tumors abundance in ACKR2 / ApcMin/ polyps, we generated mast cells

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Figure 1. Decreased mortality and tumorigenesis in ACKR2–/–ApcMinþ mice. A, Kaplan–Meier survival plot analysis for ApcMin/þ (n ¼ 27; red), ACKR2þ/–ApcMin/þ (n ¼ 20; blue), and ACKR2–/–ApcMin/þ mice (n ¼ 30; green). Signiﬁcance determined by the Mantel–Haenszel/log-rank test (P < 0.0001). B, Hematocrit values determined in 110-day-old WT (N ¼ 11), ApcMin/þ (n ¼ 14), ACKR2/–(n ¼ 11), and ACKR2/–ApcMin/þ (n ¼ 14) mice. C, Total number of polyps in the small intestine quantiﬁed by stereoscopic microscopy in age-matched (105–110-day-old) ApcMin/þ (n ¼ 12) and ACKR2–/–ApcMin/þ (n ¼ 11) mice. D, Frequency of polyps in the proximal, middle, and distal regions, and (E) the size distribution of polyps in the small intestine for ApcMin/þ (n ¼ 12) and ACKR2–/–ApcMin/þ (n ¼ 11) mice. F, Representative images of longitudinally opened distal small intestines. FFPE distal intestine tumor sections were analyzed for (G) proliferation (BrdUrd incorporation) and (H) apoptotic cell death (TUNEL assay). Both BrdUrd-positive cells (brown) and apoptotic cells (green) were counted from 20 images of 5–6 tumors from 3 mice per genotype. Error bars, SEM. Scale bar, 100 mm. Statistical analysis performed using Mann–Whitney U test (, P < 0.05; , P < 0.01; , P < 0.001).

– – from the bone marrow of WT and ACKR2 / mice to compare their concentrations of CCL2 or CCL5, the ligands for CCR2 and – – properties. First, BMMCs positive for mast cell markers c-Kit and CCR5, respectively. ACKR2 / mast cells migrated efficiently, FceR1 (Supplementary Fig. S5A and S5B) were analyzed for showing the typical bell shape response curve (Fig. 4C and D). global changes in gene expression patterns using RNA microarray WT mast cells did not show chemotactic responses to either analyses. The expression of chemokine receptors CCR2 and CCR5 CCL2 or CCL5, but they migrated efficiently when tested with – – was upregulated in ACKR2 / mast cells (Fig. 4A and B). No the well-known mast cell chemotactic ligand, stem cell factor significant differences in expression of any other CC or CXC (SCF;Fig.4E).Thesignaling functions of CCR2 and CCR5 – – – – chemokine receptor were observed between the WT and ACKR2 / receptors on ACKR2 / mast cells were further tested in intra- – – mast cells (Supplementary Fig. S6). Because CCR2, CCR5, and cellular calcium flux assays. ACKR2 / mast cells released intra- other chemokine receptors, such as CXCR2 and BLT1, have been cellular calcium in a dose-dependent response to CCL2 implicated in mast cell homing to tissues (25–27), these results (Fig. 4F) and CCL5 (Fig. 4G). WT mast cells did not respond suggest that sustained elevated expression of CCR2 and/or CCR5 to either CCL2 or CCL5, even at the highest concentrations on mast cells might contribute to their increased presence in tested, 60 nmol/L or 10 nmol/L, respectively. However, both – – þ – – ACKR2 / ApcMin/ tumors. WT and ACKR2 / mast cells mobilized intracellular calcium – – To test for functional differences in migration, ACKR2 / equally well in response to 2,4-dinitrophenyl-human serum mast cells were tested in chemotaxis assays with increasing albumin (DNP-HSA)–mediated IgE-R stimulation, ensuring no

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Figure 2. Increased mast cell proteases in ACKR2–/–ApcMinþ mice. A, Fold changes in the expression of Mcpt-1 and Mcpt-2 in pooled samples of small intestinal tumors derived from three independent mice compared with WT small intestines. These were derived from pairwise comparisons between ACKR2–/–ApcMin/þ, ApcMin/þ mice small intestinal tumors and wild-type normal small intestine tissue. B, Relative fold changes of Mcpt-1 and Mcpt-2 mRNA determined in ACKR2–/–ApcMin/þ and ApcMin/þ tumors compared with wild type normal small intestine tissue by qPCR. Statistical analysis performed using unpaired t test – – þ – – þ (, P < 0.01). C, Western blot of distal intestines tissue lysates prepared from either normal tissue (WT, ACKR2 / ) or tumors (ApcMin/ , ACKR2 / ApcMin/ ) using anti-mouse Mcpt-1. The blot was stripped and reprobed for b-actin. The quantiﬁcation of band intensities was measured using Quantity One 4.0.3 software þ – – þ and expressed as a ratio of Mcpt-1/b-actin signals. D, Immunohistochemical staining of PPFE sections of 110-day-old ApcMin/ and ACKR2 / ApcMin/ mice for mucosal mast cells (red granules) with anti–Mcpt-1 and counterstained with hematoxicillin (blue). Scale bar, 100 mm.

– – þ inherent defects in the activation of WT BMMCs (Supplemen- tumor burden in ACKR2 / ApcMin/ is due to mast cell–mediated þ þ tary Fig. S7A and S7B). recruitment of CD8 T cells. Therefore, CD8 T cells were visu- þ It is known that the expression of CCR2 and BLT1 changes alized in the distal intestine tumors from 110-day-old ApcMin/ , – – þ – – – – þ during maturation of BMMCs, with high expression in mast cell ACKR2 / ApcMin/ , and ACKR2 / SA / ApcMin/ mice by immu- þ progenitors that is subsequently downregulated in mature mast nofluorescence. The CD8 T cell numbers were significantly – – – – þ þ cells (26). To examine if the ACKR2 / mast cells follow a similar increased in ACKR2 / ApcMin/ tumors compared with ApcMin/ pattern, we measured CCR2 and CCR5 mRNA at various time (Fig. 5A). We further analyzed sections from the same mice for points during the development of mast cells in culture (Fig. 4H the presence of mast cells by immunofluorescence. The – – þ and I). Transcripts for CCR2 and CCR5 were abundant early ACKR2 / ApcMin/ tumors displayed significantly more mast cells- þ – – – – during differentiation in both cell populations and decreased compared with ApcMin/ , and, as expected, the ACKR2 / SA / – – þ dramatically in WT cells thereafter, as expected. In the ACKR2 / ApcMin/ tumors were devoid of mast cells (Fig. 5B). This absence – – – – þ cell populations, the levels of CCR2 and CCR5 remained high of mast cells in ACKR2 / SA / ApcMin/ tumors was associated þ (>500-fold) relative to WT cells after 6 weeks of differentiation in with significantly reduced tumor infiltration by CD8 T cells culture. We next asked whether elevated expression of CCR2 and (Fig. 5A), consistent with a critical role for mast cells in their – – CCR5 in ACKR2 / mast cells is common to other leukocytes. recruitment. Analysis of total RNA from purified T cells, B cells, bone marrow– Mast cells are reported to facilitate immune surveillance by þ derived macrophages (BMDM), and dendritic cells (DC) from presenting tumor antigens to CD8 T cells and inducing their – – ACKR2 / mice all showed comparable levels of CCR2 and CCR5 proliferation and cytotoxic potential (30–32). To test the effi- – – expression (Supplementary Fig. S8). There was a slight increase in cacy of ACKR2 / mast cells in mediating T-cell activation, – – CCR2 and CCR5 expression in ACKR2 / BMDCs and in CCR5 coculture experiments were performed using BMMCs and – – þ expression in ACKR2 / B cells compared with WT. However, ovalbumin-specificCD8 T cells from OT-1 TCR transgenic – – these changes were relatively weak when compared with changes mice. Both WT and ACKR2 / mast cells were equally efficient in mast cells. in the uptake and presentation of ova peptide (Supplementary þ Fig. S9), as well as upregulation of the CD8 T-cell activation þ – – Mast cells are critical for CD8 T cell recruitment in markers CD69, CD44, and CD25 (Fig. 5C). ACKR2 / mast – – þ þ ACKR2 / ApcMin/ tumors cells induced CD8 T-cell proliferation and IFNg production þ þ Tumor antigen–specific CD8 T cells play a crucial role in tothesameextentasCD8 T cells activated by WT mast cells antitumor immunity (28, 29). We hypothesized that the reduced (Fig. 5D and E).

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Figure 3. Mast cells are critical for tumor protective phenotype of ACKR2–/–ApcMinþ mice. A, Overall survival of ACKR2–/–SA–/–ApcMin/þ (n ¼ 20; green), ApcMin/þ (n ¼ 27; red), and ACKR2–/–ApcMin/þ mice (n ¼ 30; blue) using the Kaplan–Meier method. Significance determined by the Mantel–Haenszel/log-rank test (P < 0.0001). B, Hematocrit values determined in 110-day-old ACKR2/–ApcMin/þ (n ¼ 14), ACKR2–/–SA–/–ApcMin/þ (n ¼ 9), and SA–/–ApcMin/þ (n ¼ 11) mice. C, Total number and (D) size distribution of polyps in the small intestine of ACKR2–/–ApcMin/þ(n ¼ 11), ACKR2–/–SA–/–ApcMin/þ(n ¼ 11), and SA–/–ApcMin/þ (n ¼ 12) mice. E, Representative images of H&E-stained cross-sections of distal intestines at indicated magnification. Scale bar, 1 mm. F–H, BMMCs (1 107) cultured from ACKR2–/– mice were adoptively transferred i.v. into 4-week-old ACKR2–/–SA–/–ApcMin/þ mice and analyzed for (F) hematocrit, (G) small intestinal polyp number, and (H) size at 100 days of age. Statistical analysis performed using two-tailed Mann–Whitney U test (, P < 0.05; , P < 0.01; , P < 0.001; NS, not significant).

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Figure 4. Increased CCR2 and CCR5 expression in ACKR2–/– BMMCs. A, Fold changes in the expression of the top 10 chemokines and chemokine receptors observed by microarray analysis of BMMCs from ACKR2–/– mice compared with WT BMMCs. Total RNA was isolated and pooled from BMMC cultures isolated from three independent mice for each genotype. B, mRNA of CCR2 and CCR5 in 10- week BMMC cultures. C–E, Chemotaxis assays were performed with WT and ACKR2–/– BMMCs for 3 hours with increasing concentrations of (C) CCL2, (D) CCL5, or (E) stem cell factor (SCF). Migrated cells counted by ﬂow cytometry. F and G, Agonist-mediated intracellular calcium release of Indo-1–loaded WT and ACKR2–/– BMMCs at the indicated concentrations of (F) CCL2 or (G) CCL5. The data are representative of 3 independent experiments performed. H and I, Total RNA from BMMCs at different time points (2, 4, 6 weeks) was isolated and mRNA of (H) CCR2 or (I) CCR5 was measured using real-time PCR.

þ CD8 T cells control tumor progression in of age revealed that although tumor frequencies were similar in – – þ – – þ – – – – þ ACKR2 / ApcMin/ mice ACKR2 / ApcMin/ and ACKR2 / Rag2 / ApcMin/ mice, those To investigate the role of the adaptive immune system in tumors grew to larger sizes in the absence of a functional adaptive – – þ intestinal tumor progression, the ACKR2 / ApcMin/ mice were immune system (Fig. 6B and C). To further examine the role of – – – – þ crossed onto the Rag2 / background and analyzed for anemia T cells, T cells from ACKR2 / ApcMin/ mice were adoptively – – – – – – – – þ and tumor burden. Hematocrit values in ACKR2 / Rag2 / transferred into ACKR2 / Rag2 / ApcMin/ mice. As shown in þ – – – – þ ApcMin/ mice showed a significant decrease compared Fig. 6D, the hematocrit values of ACKR2 / Rag2 / ApcMin/ mice – – þ þ with ACKR2 / ApcMin/ mice, indicating severe disease increased significantly with CD8 T-cell transfer compared with (Fig. 6A). Measurement of tumor numbers and size at 110 days the mice with sham transfer. There was also a significant decrease

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Figure 5. CD8þ T-cell infiltration correlates with mast cells' presence in ACKR2–/–ApcMin/þ tumors. The frozen distal intestine (110 days old) sections were stained with (A)CD8antibodyor(B) Mcpt-1 antibody. Tumor regions: white line; blue, DAPI; green: CD8 (A) or Mcpt-1 (B). Statistical analysis performed using two-tailed Mann–Whitney U test (, P < 0.01; , P < 0.001). Scale bar, 100 mm. C, BMMCs were pulse-loaded with 4 mmol/L SIINFEKEL, washed, andaddedtoMACSpurified OT-1 CD8þ T cells at different ratios, as indicated. Cell-surface expression of CD69, CD44, and CD25 on CD8þ T cells þ was analyzed by flow cytometry. D, BMMCs were pulsed with 4 mmol/L SIINFEKEL, washed, and added to MACS purified, CFSE-labeled OT-1 CD8 T cells at different ratios with 1 106 CD8þ T cells. Proliferation was analyzed by flow cytometry. The overlay in the 1:2 ratio shows the addition of the same number of mast cells without the peptide on their surface. E, WT and ACKR2–/– BMMC-mediated IFNg production in CD8þ T cells. SIINFEKL pulsed BMMCs were cocultured with MACS purified OT-1 CD8þ T cells at 1:2 ratio for 48 hours and then stimulated with PMA/ionomycin cocktail for 5 hours and analyzed for intracellular IFNg levels by flow cytometry.

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Figure 6. Tcell–mediated protection against tumor development in ACKR2–/–ApcMin/þ mice. Comparison of (A) hematocrit values, (B) small intestine polyp number, and (C) size distribution between ACKR2–/–ApcMin/þ (n ¼ 11) and ACKR2–/–Rag2–/–ApcMin/þ (n ¼ 12) mice. D–G, ACKR2–/–Rag2–/–ApcMin/þ mice at 35 days of age received either i.v. PBS (sham), 8.5 105 CD8þ T cells, or the same number of CD4þ and CD8þ T cells (1:1) isolated from ACKR2–/–ApcMin/þ mice. D, Hematocrit values, (E) small intestine polyp number, and (F) size distribution at 110 days age in adoptively transferred mice. G, Representative images of longitudinally opened distal small intestines of transferred and nontransferred ACKR2–/–Rag2–/–ApcMin/þ mice. Statistical analysis performed using Mann–Whitney U test (, P < 0.05; , P < 0.01; , P < 0.001; NS not signiﬁcant).

–/– –/– Min/þ in tumor number and size in ACKR2 Rag2 Apc mice to tumors (33). To examine whether mast cell–derived LTB4 is þ upon transfer compared with the sham mice (Fig. 6E and F). The responsible for recruitment of BLT1-expressing CD8 T cells to þ – – þ – – þ tumor protective effect of transferred CD8 T cells was clearly ACKR2 / ApcMin/ tumors, the ACKR2 / ApcMin/ mice were – – – – visible in the representative images of distal ileum sections crossedontotheBLT1/ background. The ACKR2 / – – þ (Fig. 6G). Thus, genetic and adoptive transfer experiments suggest BLT1 / ApcMin/ mice displayed accelerated disease and a sig- þ that CD8 T cells are both necessary and sufficient for tumor nificant decrease in survival (Fig. 7A and B). The tumor num- protection in this model. bers, as well as the size of the tumors, in the higher range – – – – þ To determine if T cells contribute to the enhanced presence of increased significantly in ACKR2 / BLT1 / ApcMin/ as com- – – þ – – þ mast cells in ACKR2 / ApcMin/ adenomas, cell lysates prepared pared with ACKR2 / ApcMin/ (Fig.7CandD).Frozensections – – – – þ – – from the distal intestine tumors of ACKR2 / Rag2 / ApcMin/ of distal intestine tumors from 110-day-old ACKR2 / – – þ þ mice were analyzed for Mcpt-1 (Supplementary Fig. S10). BLT1 / ApcMin/ mice were analyzed for mast cell and CD8 – – – – þ – – ACKR2 / Rag2 / ApcMin/ mice tumors showed similar expres- T cell recruitment by immunofluorescence. The ACKR2 / – – þ – – þ sion of Mcpt-1 as ACKR2 / ApcMin/ tumors, indicating that BLT1 / ApcMin/ tumors, despite elevated numbers of mast þ the absence of T cells did not alter mast cell recruitment in cells, showed significantly reduced CD8 Tcellinfiltration – – þ – – þ ACKR2 / ApcMin/ tumors. compared with ACKR2 / ApcMin/ tumors (Fig. 7E and F). Analysis of LTB4 levels in the adenomas showed negligible þ –/– –/– Min/þ LTB4–BLT1 axis mediates CD8 T-cell recruitment into LTB4 in mast cell–deficient ACKR2 SA Apc tumors – – þ ACKR2 / ApcMin/ tumors compared with mast cell–sufficient tumors (Fig. 7G). These þ Our findings from an implantable cervical cancer model show data together with reduced CD8 Tcellmigrationtomast þ – – – – þ that BLT1 expression on CD8 T cells is critical for their migration cell–deficient ACKR2 / SA / ApcMin/ tumors strongly suggest

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Figure 7. Antitumor activity in ACKR2–/–ApcMin/þ mice requires BLT1. A, Kaplan–Meier survival curves for ApcMin/þ (n ¼ 28), ACKR2–/–ApcMin/þ (n ¼ 28), and BLT1–/–ACKR2–/–ApcMin/þ (n ¼ 18) mice. B, Hematocrit values, (C) polyp number, and (D) size of polyps of ACKR2–/–ApcMin/þ and BLT1–/–ACKR2–/–ApcMin/þ mice. E, Number of Mcpt-1þ cells in the tumors of indicated mice quantified using confocal immunofluorescence images. F, Number of tumor-infiltrating CD8þ T cells

from at least 6 tumors in each genotype. G, LTB4 levels in tumor homogenates prepared from 5 to 6 distal intestine tumors. Data in E and F represent 3 different mice per genotype. Statistical analysis was performed using Mann–Whitney U test (, P < 0.05; , P < 0.01; , P < 0.001; NS, not signiﬁcant).

that mast cells produced LTB4 to promote effector T-cell recruit- In colorectal cancers, an absence of TLR signaling due to ment into intestinal adenomas. MyD88 deletion results in the reduction of adenomas in þ ApcMin/ mice, highlighting the critical role of inflammation Discussion in tumor development in this model (34). The current study showsthatdeletionofinflammatory chemokine scavenging – – þ þ The results presented here using ACKR2 / ApcMin/ mice in receptor ACKR2 in the context of ApcMin/ mice was protective, – – – – þ combination with the deletion of mast cells (SA / ), lymphocytes as the adenomas in ACKR2 / ApcMin/ mice were established –/– –/– (Rag2 ), and LTB4 receptors (BLT1 ) demonstrate that che- more slowly and displayed reduced inflammation and mokine-mediated mast cell recruitment initiates LTB4/BLT1- growth in tumor mass. This is in contrast to the well- þ regulated CD8 T-cell homing. This, in turn, leads to the gener- established role of ACKR2 in limiting inflammation demon- – – ation of effective antitumor immunity against intestinal tumor strated in several other models involving ACKR2 / mice, growth that slows progression of fatal disease in this spontaneous including colitis-associated colon cancers (18, 19). Thus, the tumor model. pathway by which oncogenesis is initiated appears to be a

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critical determinant of the consequences of elevated inflam- examine the basic mechanisms underlying the protective nature – – matory responses in ACKR2 / mice. of mucosal mast cells by focusing on their interactions with þ Among the many models that outline the protective role of effector CD8 T cells. ACKR2, the psoriasiform inflammation of the skin appears Mast cells are present in limited numbers in the mouse gut, but relevant to the current observations (13, 35). In the TPA-induced their numbers increase during infection and inflammatory con- psoriasis model, the characterization of the immune cell infiltrate ditions (43). Under reduced inflammatory conditions, the intes- – – – – þ in skin sections reveals that ACKR2 / mice recruit increasing tinal tumors from ACKR2 / ApcMin/ mice recruited more mast þ numbers of mast cells during the time course of disease progres- cells than ACKR2-sufficient ApcMin/ mice. Phenotypic and func- – – sion, whereas WT mice do not have any such increase in mast cell tional characterization of BMMCs revealed that ACKR2 / mast accumulation (13). The infiltration of macrophages and neutro- cells have high expression of functional CCR2 and CCR5, whereas phils into inflamed skin sections does not differ between WT and WT mast cells did not, providing a likely explanation of how mast – – – – ACKR2 / mice. Interestingly, more CD3-positive T cells were cells are selectively recruited into ACKR2 / tumors. Both CCR2 – – recruited into the treated ACKR2 / mice skin. Inhibiting either and CCR5, along with other chemoattractant receptors like mast cells or T cells reduced the TPA-induced inflammatory CXCR2 and BLT1, are implicated in mast cell homing to the – – pathology in ACKR2 / mice. As detailed in the current study, tissues (25–27). However, unlike CCR2 and CCR5, no change in the consequence of the similarly elevated mast cell and T-cell the expression of CXCR2 or BLT1 was detected. There are several þ recruitment to the site of intestinal tumors in ACKR2 / ApcMin/ potential explanations for why CCR2 and CCR5 are overexpressed – – mice appear to be in generating effective antitumor immunity. or sustained in ACKR2 / mast cells. Downregulation of CCR2 Clearly, the basis for the unexpected tumor protective pheno- mRNA has been demonstrated during development of B cells type appears to be dependent on mast cells because they are (44), DCs (45), and monocytes (46). CCR2 mRNA is down- – – þ significantly increased in ACKR2 / ApcMin/ adenomas. As appar- regulated in BMMCs after 2 weeks of culture, which appears to be a ent with the Mcpt-1 staining, mucosal mast cell numbers mechanism to confine them into tissues preventing them from increased significantly at the periphery, as well as in intratumoral emigrating (47). We also detected CCR2 and CCR5 mRNA – – þ regions, of ACKR2 / ApcMin/ adenomas. Several studies have expression in 2-week-old WT mast cell cultures and observed used mast cell–deficient strains, such as WBB6F1-Kitw/w-v and dramatic downregulation beyond 4 weeks of growth in culture. In – – C57BL/6-Kitw-sh/w-sh mice, to determine their role in cancer. contrast, the expression of these transcripts persisted in ACKR2 / However, the results obtained are variable, depending on the mast cells, suggesting that posttranscriptional processing might be – – tumor model used (2). Gounaris and colleagues reported infil- altered in the ACKR2 / mast cells. tration of mast cells into intestinal polyps in murine models of The dramatic upregulation of functional expression of CCR2 conditional b-catenin overexpression and truncation of the APC and CCR5 observed in cultured mast cells suggests that the – – þ (APCD468) gene (36). In lethally irradiated APCD468 mice, the enhanced mast cell infiltration observed in ACKR2 / ApcMin/ polyp number is reduced following the transfer of bone marrow tumors is likely mediated through the activity of either or both of from mast cell–deficient mice compared with WT mice, suggesting these chemokine receptors. Reciprocal control of ACKR2 and a protumor role for mast cells in polyp development. These results CCR2 in the recruitment of macrophages to developing lymphatic are in contrast to the observations of Sinnamon and colleagues in vessel surfaces has been demonstrated (48). It is suggested that in þ ApcMin/ mice, where mast cell infiltration in intestinal polyps was the absence ACKR2-scavenging function on lymphatic endothe- shown to negatively correlate with tumor growth (37). A similar lium, CCL2/CCR2-mediated macrophage recruitment is þ protective phenotype for mast cells has been observed in ApcMin/ enhanced. Likewise, Ly6Chigh monocytes increase in the spleen – – tumors lacking the receptor for glycation end products (RAGE), and blood of ACKR2 / mice in a CCR2-dependent manner with significant increases in mast cell number correlating with and exhibit enhanced immune suppressive activity (49). During 58% reduced polyp growth (38). The loss of survival advantage of LPS-induced inflammation, increased CCL2 accumulation in the – – þ – – the ACKR2 / ApcMin/ mice and the development of large-sized lymph nodes of ACKR2 / mice recruit high CCR2-expressing – – – – þ þ tumors in mast cell–deficient ACKR2 / SA / ApcMin/ mice CD11b Gr1high cells (50). Blocking CCR2 inhibits the migration reported here suggests a protective role for mast cells. The ability of other cellular flow and restores the migration of antigen – – of adoptively transferred ACKR2 / mast cells to reduce tumor presenting DCs. Graham and colleagues suggest that the draining growth further supports the notion that mast cells are protective in lymph node area becomes like a clogged sink in the absence intestinal adenoma development. Although the basis for the ACKR2, preventing the normal flow of leukocytes (51). However, protumor and antitumor activities observed in these studies our results showed a significantly different outcome, i.e., a more remains to be established, a few differences need consideration. efficient antitumor immune response in the absence of ACKR2. In In APCD468 mice, chymase-producing mucosal-type mast cells are the context of generating immunity to the altered self-antigens, detected in the intraepithelial regions of benign adenomas, and this clogged sink is what might be needed for unconventional tryptase-producing connective tissue-type mast cells are preferen- antigen presentation by antigen presenting cells such as mast cells. tially enriched in the stroma and invasive regions of adenocarci- Clearly, further work is needed to explore this possibility. nomas, indicating the plasticity of mast cell subtypes in a chang- It is clear from the immunofluorescence staining of tumors that þ ing tumor environment (39). Although APCD468 mice develop mast cell infiltration was correlated with CD8 T cell recruitment – – invasive intestinal tumors, only benign adenomas develop in in ACKR2 / tumors. Although T-cell cytokine-mediated mast cell þ ApcMin/ mice, and they die around the age of 150 days before hyperplasia is known to occur in the gut (52), the abundance of – – – – þ developing adenocarcinomas. It is possible that mast cells display mucosal mast cells in ACKR2 / Rag2 / ApcMin/ tumors clearly plasticity and switch between immune enhancing phenotypes suggests that mast cell recruitment in this model is independent of and immune suppressive phenotypes, depending on the tumor T-cell function. In contrast, we observed enhanced recruitment of þ – – þ microenvironment (40–42). Therefore, in this study, we sought to CD8 T cells in mast cell–enriched ACKR2 / ApcMin/ tumors and

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Mast Cell Mediated Immune Surveillance of Intestinal Cancer

Figure 8. A model for mast cell–mediated immune surveillance of intestinal tumors. Absence of ACKR2 in the background of ApcMin/þ results in mast cell accumulation in the small intestinal tumors and enhanced the survival of mice. Deletion of mast cells accelerates the tumor growth that is partially reversed by adoptively transferred mast cells. Deletion of lymphocytes in Rag2–/– mice also accelerates tumor growth in ACKR2–/–ApcMin/þ mice. Mast cell–produced þ LTB4 facilitates recruitment of effector CD8 T cells into adenomas curbing their growth.

their absence in mast cell–deficient tumors, indicating a critical mice outlines the importance of mast cell–mediated T-cell – – – – þ role for mast cells in T-cell recruitment. Because ACKR2 / responses in ACKR2 / ApcMin/ mice. –/– Min/þ Rag2 Apc mice displayed large tumors irrespective of mast Mast cell–derived LTB4 is known to mediate the recruitment of þ cell abundance, direct cytotoxicity, or phagocytic capabilities effector CD8 T cells to inflammatory sites via BLT1 (25, 55). – – of mast cells were likely not involved in controlling ACKR2 / Eicosanoids are one of the early mediators produced in the tumor þ ApcMin/ tumors. milieu by inflammatory cells such as mast cells, neutrophils, and Although the tumor-infiltrating mast cells might mediate pos- macrophages. Significantly, higher LTB4 is produced by human itive or negative outcomes for cancer patients, the presence of colon adenocarcinoma tissue (56), relative to the corresponding þ – – – – þ cytotoxic CD8 T cells within the core of the tumor or at the normal mucosal tissues. The data on ACKR2 / BLT1 / ApcMin/ invasive margin is usually correlated with good prognosis mice presented here highlight the importance of the LTB4/BLT1 (53, 54). Analysis by Galon and colleagues shows a clear associ- axis in mediating effector T-cell recruitment into intestinal ade- þ – – þ – – þ ation between high densities of CTLs and memory CD8 T cells nomas in ACKR2 / ApcMin/ mice. The ACKR2 / ApcMin/ mice þ (CD8 /CD45RO) and longer disease-free survival (28). The devoid of either mast cells or BLT1 develop large intestinal tumors – – – – þ þ development of large-size tumors in ACKR2 / Rag2 / ApcMin/ with reduced CD8 T-cell recruitment. Not only the survival – – þ – – without a change in the number of tumors clearly shows that T advantage of the ACKR2 / ApcMin/ mice was lost in ACKR2 / – – þ cell–mediated immune responses are critical in controlling tumor BLT1 / ApcMin/ mice, but the compound mice retained the þ – – þ growth in these mice. The ability of adoptively transferred CD8 T accelerated tumor development observed in BLT1 / ApcMin/ þ þ cells or CD4 /CD8 T cells to reinstate tumor protection demon- mice (57). The data showed that mast cells are the cellular origin þ strates that CD8 T cell–mediated immune response is essential in of LTB4 because elevated production was detected in – – þ – – þ ACKR2 / ApcMin/ mice. Although further studies are clearly ACKR2 / ApcMin/ adenomas, whereas mast cell–deficient þ needed to dissect the role and contribution of different CD4 tumors did not produce detectable LTB4. This is consistent with –/– –/– þ subsets, including Tregs, the use of SA and Rag2 compound the role of mast cell–produced LTB4 in CD8 T-cell migration

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Bodduluri et al.

reported in an airway hyperresponsiveness model, where adop- Authors' Contributions tive transfer of WT BMMCs into LTB4-deficient leukotriene A4 Conception and design: S.R. Bodduluri, E. Krishnan, T.C. Mitchell, V.R. Jala, –/– B. Haribabu hydrolase (LTA4H ) mice triggered LTB4 release in BAL fluid and restored AHR response (58). Development of methodology: S.R. Bodduluri, E. Krishnan, V.R. Jala, B. Haribabu A model summarizing our findings on the role of mast cell –/– Min/þ Acquisition of data (provided animals, acquired and managed patients, mediated immune surveillance in the ACKR2 Apc mice is provided facilities, etc.): S.R. Bodduluri, S. Mathis, P. Maturu, S.R. Satpathy, shown in Fig. 8. Elevated recruitment of mast cells resulted P.M. Chilton, S. Lira, V.R. Jala, B. Haribabu in reduced tumor burden and enhanced survival of the Analysis and interpretation of data (e.g., statistical analysis, biostati- – – þ þ ACKR2 / ApcMin/ mice relative to ApcMin/ mice. Accelerated stics, computational analysis): S.R. Bodduluri, S.R. Satpathy, P.M. Chilton, – – þ tumor growth in mast cell–deficient ACKR2 / ApcMin/ mice M. Locati, V.R. Jala, B. Haribabu Writing, review, and/or revision of the manuscript: S.R. Bodduluri, points to the importance of these cells in regulating adenoma P.M. Chilton, M. Locati, A. Mantovani, V.R. Jala, B. Haribabu progression. The lack of immune surveillance in either Administrative, technical, or material support (i.e., reporting or organizing –/– –/– Min/þ –/– –/– Min/þ ACKR2 Rag2 Apc or BLT1 ACKR2 Apc mice, data, constructing databases): V.R. Jala, B. Haribabu þ despite mast cell abundance, points to BLT1-mediated CD8 T Study supervision: V.R. Jala, B. Haribabu cell recruitment as a critical controller of adenoma growth in Other (performed research experiments): P. Maturu þ ApcMin/ mice. In selected human tumors, colorectal cancers in particular, high mast cell infiltration has been associated with Acknowledgments better prognosis. The mechanisms responsible for a tumor-pro- This work was supported by NIH grants CA-138623 (B. Haribabu) KLCRP (V.R. Jala) and James Graham Brown Cancer Center at U of L. Part of this tective function of mast cells and links to the known antitumor work was performed with assistance of the U of L Microarray Facility, which is function of infiltrating cells have remained elusive (5–7, 53, 54). supported by NCRR COBRE P20RR018733, KY-INBRE NCRR P20RR016481, Based on the results reported here, we surmise that interaction and the J.G. Brown Cancer Center at U of L. þ between mast cells and CD8 T cells with a pivotal role of The authors thank Dr. Nejat Egilmez for critical reading of the manuscript. chemokines and their receptors underlies these clinical We thank Michelle Smith and Becca Baby for expert technical assistance. observations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement Disclosure of Potential Conflicts of Interest in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. A. Mantovani is a consultant/advisory board member for Verily, Efranat, AbbVie, Compugen, Novartis, Pierre Fabre, and Roche. No potential conflicts of Received August 8, 2017; revised October 20, 2017; accepted January 19, interest were disclosed by the other authors. 2018; published OnlineFirst January 30, 2018.

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Mast Cell−Dependent CD8+ T-cell Recruitment Mediates Immune Surveillance of Intestinal Tumors in Apc Min/+ Mice

Sobha R. Bodduluri, Steven Mathis, Paramahamsa Maturu, et al.

Cancer Immunol Res 2018;6:332-347. Published OnlineFirst January 30, 2018.

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