• Sonuç bulunamadı

The expression of adenosine A2B receptor on antigen-presenting Cells suppresses CD8+ T-cell responses and promotes tumor growth

N/A
N/A
Protected

Academic year: 2021

Share "The expression of adenosine A2B receptor on antigen-presenting Cells suppresses CD8+ T-cell responses and promotes tumor growth"

Copied!
12
0
0

Yükleniyor.... (view fulltext now)

Tam metin

(1)

The Expression of Adenosine A2B Receptor on

Antigen-Presenting Cells Suppresses CD8

þ

T-cell

Responses and Promotes Tumor Growth

Siqi Chen

1

, Imran Akdemir

2

, Jie Fan

1

, Joel Linden

3

, Bin Zhang

1

, and Caglar Cekic

2,3

ABSTRACT

Accumulating evidence suggests that inhibiting adenosine-generating ecto-enzymes (CD39 and CD73) and/or adenosine A2A or A2B receptors (R) stimulates antitumor immunity and limits tumor progression. Although activating A2ARs or A2BRs causes similar immunosuppressive and protumoral functions, few studies have investigated the distinct role of A2BR in cancer. Here, we showed that A2BR expression by hematopoietic cells was primarily responsible for promoting tumor growth. Deletion of A2BR profoundly enhanced anticancer T-cell immunity. Although T-cell A2BR plays an insignificant role for A2BR-mediated immu-nosuppression and tumor promotion, A2BR deficiency in tumor-bearing mice caused increased infiltration of myeloid and CD103þ dendritic cells, which was associated with more effective

cross-priming of adoptively transferred tumor antigen–specific CD8þT cells. A2BR deletion also intrinsically favored accumulation of myeloid and CD11bdimantigen-presenting cells (APC) in the tumor microenvironment. Both myeloid-specific or CD11c-specific con-ditional deletion of A2BR delayed primary tumor growth. Myeloid, but not CD11c-specific conditional, depletion delayed lung metas-tasis. Pharmacologic blockade of A2BR improved the antitumor effect of adoptive T-cell therapy. Overall, these results suggested that A2BR expression on myeloid cells and APCs indirectly suppressed CD8þT-cell responses and promoted metastasis. These data pro-vide a strong rationale to combine A2BR inhibition with T-cell– based immunotherapy for the treatment of tumor growth and metastasis.

Introduction

Adenosine is present in the extracellular space and functions as a signaling molecule by engaging four different adenosine receptor subtypes; A1, A2AR, A2BR, and A3 (1). Among these, A2AR and A2BR are implicated in the signaling that resolves inflammation and promotes tissue repair (2). Adenosine-mediated tissue-healing responses can help tumors escape immune recognition and dissem-inate to other tissues (3–5). Therefore, identifying the roles of aden-osine receptor subtypes in different subsets of immune cells is needed for developing rational strategies targeting adenosine signaling as a potential cancer therapy.

A2BRs are expressed in both immune and nonimmune cells and have pleotropic protumoral effects. A2BR expression in tumor cells is implicated in decreased adherence, increased survival, and increased metastasis in both immune-deficient and proficient settings (4, 6–8). A2BR is also expressed by immune cells (9, 10), and A2BR expression is particularly increased in antigen-presenting cells (APC) upon

activation (11). Targeting A2BR by genetic deletion or by pharmaco-logic blockade slowed the growth of several syngeneic tumors by activation of T cells (4). In addition, promotion of tumor growth by A2BR signaling associates with expansion or activation of suppressive myeloid cells (12–14) and transfer of myeloid suppressor cells reverses the enhanced adaptive immune responses during A2BR blockade (12). However, it is not known if cell-intrinsic A2BR signaling by T cells versus APCs plays a major role in increasing tumor growth. Also, the impact of activating A2BRs on myeloid cells and APCs on lung colonization of tumors remains elusive.

Here, we confirmed that A2BR deletion delayed growth of a number of syngeneic ectopic solid tumors in a T-cell–dependent manner. Experiments utilizing adoptive transfer of wild-type (WT) versus A2BR/T cells to tumor bearing hosts or growth of tumors in mice reconstituted with a mixture of WT and A2BR/bone marrow (BM) cells demonstrated that T-cell A2BRs had a very limited role in A2BR-mediated suppression of tumor-associated T cells. Adoptively trans-ferred tumor antigen-specific CD8þT cells were more cross-primed in tumor bearing A2BR knockout (KO) hosts than that of WT hosts. A2BR deletion intrinsically favored accumulation and activation of myeloid APCs and Gr1þMDSCs in the tumor microenvironment. Both CD11c and myeloid deletion of A2BR delayed growth. However, lung coloni-zation of tumors was only inhibited by myeloid deletion of A2BR, suggesting monocytes and other Gr1þcells were important for A2BR-mediated promotion of lung dissemination. Finally, acute pharmaco-logic blockade of A2BR before adoptive transfer of tumor antigen– specific T cells improved antitumor efficacy compared with control or the single treatments. Thus, thesefindings suggested clinical potential for A2BR blockade to improve current cancer immunotherapies.

Materials and Methods

Mice and reagents

Animal experiments were approved by the Animal Care and Use Committee of the La Jolla Institute for Allergy & Immunology

1Robert H. Lurie Comprehensive Cancer Center, Department of

Medicine-Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, Illinois.2Department of Molecular Biology and Genetics,

Bilkent University, Ankara, Turkey.3Division of Inflammation Biology, La Jolla

Institute for Immunology, La Jolla, California.

Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Authors: Caglar Cekic, Department of Molecular Biology and Genet-ics, Bilkent University, Ankara, Turkey, 06800. E-mail: caglar.cekic@bilkent.edu.tr; and Bin Zhang, Robert H. Lurie Comprehensive Cancer Center, Department of Medicine-Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611. E-mail: bin.zhang@northwestern.edu Cancer Immunol Res 2020;8:1064–74

doi: 10.1158/2326-6066.CIR-19-0833

(2)

(La Jolla, CA) and the institutional animal use committees of Northwestern University (Chicago, IL) and Bilkent University (Ankara, Turkey). B16F10 cells stably expressing luciferase (B16-Luc) were obtained from Caliper Life Sciences and cultured in R5F (RPMI1640 medium containing 10% heat-inactivated FBS, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 50 U/mL

penicillin, 50 mg/mL streptomycin). Cell lines from Caliper Life Sciences were tested for being pathogen free. Dr. D. Theodorescu at University of Colorado (Aurora, CO) kindly provided us with the mouse urethelial carcinoma cell line MB49 originally produced by Dr. T. Ratliff of Purdue University (West Lafayette, IN). MB49 cells were confirmed to be mouse origin and tested negative for evidence of cross-species contamination and pathogen contamination by IDEXX BioResearch. All cell lines were passaged less than 10 times after initial revival from frozen stocks. Murine Lewis lung carci-noma (LLC1) cells were purchased from ATCC (CRL-1642). LLC1 cells expressing OVA (LLC1-OVA) were generated as described previously (15). For all experiments, tumor cells were recovered from frozen aliquots and cultured for 1 to 2 weeks prior to inoculation of mice. All the cell lines were routinely tested for Mycoplasma infections by culture and DNA stain and maintained in complete medium composed of RPMI1640 with 5% FBS, but have not been reauthenticated in the past year. Six-week-old C57BL/6 Rag1/, CD45.1 and CD90.1, LysMCre mice [B6.129P2-Lyz2tm1 (cre)Ifo/J), CD11c-Cre (B6.Cg-Tg(Itgax-cre)1-1Reiz/J] and Rag1/ mice were purchased from Jackson Laboratories. A2BR/mice were provided by Dr. Michael R. Blackburn (The University of Texas Health Science Center, Houston, TX) or by Katya Ravid of Boston University (Boston, MA). Dr. Hans Schreiber (University of Chicago, Chicago, IL) provided the 2C transgenic mice, SIYRYYGL peptides and MC38, B16-SIY cell lines. Adora2bf/f mice were generated as described previously (16) and crossed with LysMCre/þmice. The 10- to 12-week-old mice with adenosine receptor deletions used in this study were congenic to C57BL/6 and were created as described previously: A2AR/(17), A2BR/(18, 19). Antibodies used forflow cytometry are listed in Supplementary Table S1. Cytokines were used for the study is listed as Supplementary Table S2. All adenosine receptor agonists and antagonists were purchased from Tocris Bioscience (Supplementary Table S3).

Tumor challenge and treatments

B16-SIY, B16-Luc, LLC1, LLC1-OVA, or MC38 cells (1 106) and

MB49 cells (1 105) in 100 mL of PBS were injected subcutaneously. For A2BR blockade in vivo, 10 days after tumor cell injection, mice were injected intraperitoneally by MRS1754 in 100 mL of 0.1% DMSO (2 mg/kg) once daily. The tumor volume was determined by calipers at 2- to 3-day intervals. Tumor volumes were measured along 3 orthog-onal axes (a, b, and c) and calculated as (abc)/2.

BM reconstitution

BM chimeric mice were generated as described previously (20). Briefly, A2BR/KO mice and WT mice were exposed to 10 Gy total body irradiation using a Cesium-137 Mark I irradiator (J.L. Shepherd Inc, San Fernando, CA). BM cells from the femur and tibia of matched A2BR/mice and WT mice were harvested under sterile conditions. Irradiated recipient mice received 107BM cells in 100 mL of PBS intravenously. Mice were housed for 8 weeks after BM transplantation before experimentation. BM reconstitution of Rag1/mice with BMs from WT and A2BR/mice was performed as described previous-ly (21). Briefly, mice 6–12 weeks of age were fasted for 24 hours and then lethally irradiated (2 450 Rads for Rag1/and 2 500 Rads for

C57BL/6 recipients) using RS2000 Biological irradiator (Rad Source Technologies Inc.). After the second radiation exposure, a 1:1 mixture of 5–10  106BM cells from donor mice (WT and A2BR/) in 100 mL

of PBS were injected intravenously through the retro-orbital venous sinus. Mice were treated with antibiotics starting 3 days before tumor implantation up until 2 weeks after radiation. We injected 105MB49

bladder carcinoma cells in 100 mL of PBS subcutaneously into the right flanks 10 weeks after reconstitution of the BM. Single-cell suspensions from spleens and tumors were analyzed by cytofluorometry to deter-mine the proportion of T cells, NK cells, and myeloid cells 7, 14 and 21 days after tumor inoculation as described below.

Flow cytometry

Tumor tissues were minced into small pieces and digested in Collagenase IV (1 mg/mL; Roche) and DNase I (20 mg/mL; Roche) at 37C for 30 minutes, andfiltered by sequential pressing through 100- and 40-mm cell strainers. After red blood cell (RBC) lysis using RBC lysis buffer (BioLegend, catalog no. 420301) according to the manufacturer's instructions, cells were washed and resuspended in R10F and counted in a Z2-Coulter particle counter (Beckman Coul-ter). For BM-derived macrophages (BM-DM), cells were detached with Accutase solution (Innovative Cell Technologies, catalog no. AT104) 6 hours after 100 ng/mL LPS (Invivogen, catalog no. tlrl-smlps) treatment in the presence or absence of 1 mmol/L NECA (Tocris Bioscience, catalog no. 1691). Single-cell suspensions were preincu-bated for 10 minutes in 100 mL FACS buffer with antibody to block Fc receptors. Each sample tube received 100 mLfluorescently labeled antibody mixture (list of antibodies are indicated in Supplementary Table S1) and was incubated for 30 minutes at 4C in the dark. For intracellular cytokine staining, tumor samples were restimulated with 10 ng/mL PMA, 100 ng/mL ionomycin (Sigma-Aldrich), and Golgi Plug (eBioscience) for 5 hours at 37C. Cells werefixed and permea-bilized after surface staining and incubated for 25 minutes at 4C in 100 mL permeabilization/washing buffer (BioLegend, catalog no. 421002) containing 1:100 anti–IFNg for tumor samples or anti-TNFa and anti-IL10 for BM-DM samples. After a subsequent wash, cells were resuspended in 350 mL FACS buffer. Cells were analyzed using an LSR II equipped with four lasers and FACS Diva software (BD Biosciences). Live/deadfixable yellow (Invitrogen, catalog no. L34959) was used to exclude dead cells according to the manufacturer's instructions before analysis. Flow cytometry data were analyzed using FlowJo software (9.0.1 version; Tree Star) or Novocyte software (1.4.1. version; Acea Biosciences). Gating strategy for all the tested subpopulations and activation markers are listed in Supplementary Fig. S1.

Metastasis

For metastasis analysis, 3 105 B16-Luc in 100 mL PBS were injected intravenously into the tail vain and luciferase activity was measured one and two weeks after the injection of cancer cells by injection of 1 mgD-Luciferin (Caliper Life Sciences) in 100 mL PBS followed by IVIS Imaging (Caliper Life Sciences). Images were taken within 10 minutes of luciferase injection. Data were collected for 1 minute and represented as photons/second. After measuring luciferase activity, lungs were removed, and pictures from representative lungs were taken to observe metastatic dark spots. Lungs were also weighed to validate changes in overall metastatic tumor burden per group in LysMCre/þA2BRf/fversus littermate controls.

ELISA

IL12 ELISA was performed after exposing LPS-stimulated BM-derived dendritic cells (BM-DC) to various adenosine receptor APC Expression of Adenosine A2BR Controls Tumor Growth

(3)

antagonists (listed below and in Supplementary Table S3) in the presence of NECA or to various receptor subtype–specific adenosine receptor agonists. For cytokine detection IL12p40 ELISA kit (BioLe-gend, catalog no. 431601) was used by following manufacturer's instructions. For ELISA experiment, 100 nmol/L of following receptor subtype–specific agonists and antagonists from Tocris Biosciences were used: A1R agonist, 20-MeCCPA; A2AR agonist, CGS 21680 hydrochloride; A2BR partial agonist, BAY 60-6583; A3R agonist, 2-Cl-IB-MECA; A1R Antagonist, PSB36; A2AR Antagonist, SCH 58261; A2B Antagonist, PSB 603.

T-cell purification and adoptive transfer

Splenic CD8þ T cells from CD45.2þ 2C WT CD90.1þ or 2C A2BR/CD90.1mice were selected with a CD8þT Cell Enrichment Kit (StemCell Technologies, catalog no. 19853). For CFSE labeling, cells at 106per mL were stained with CellTrace CFSE staining solution

(Thermo Fisher Scientific, catalog no. C34554) for 20 minutes in a 37C water bath according to the manufacturer's instructions. A total of 2 106purified CFSE-labeled T cells in 100 mL of PBS were injected

intravenously into CD45.1þhosts through the retro-orbital venous sinus 1 day prior to B16 or B16-SIY injection. Three days later, the proliferation (CFSE dilution), intracellular IFNgþ production and activation (measured by expression of CD69 and CD44) of transferred T cells (gated on CD8þCD45.1CD45.2þ) in spleens were determined byflow cytometry. For adoptive T-cell therapy, splenic CD8þT cells were selected from 2C mice with a CD8þ T Cell Enrichment Kit (StemCell Technologies, catalog no. 19853) and stimulated with 0.5 mg/mL SIY peptides, 1 mg/mL anti-CD28, and 10 ng/mL IL2 in CR10, that is, RPMI1640 supplemented with 10% FCS (Life Technol-ogies, catalog no. 26140079), 100 IU/mL penicillin, 100 mg/mL strep-tomycin, 2 mmol/L L-glutamine, 25 mmol/L HEPES buffer, and nonessential amino acids for 3 days. Prestimulated T cells were injected intravenously at 5 106in 100 mL of PBS per mouse through the retro-orbital venous sinus into B16-SIY tumor–bearing mice on day 1 or day 13. To examine the role of A2BR on regulatory T cells (Treg), tumor-associated or splenic CD4þCD25hiTregs from WT or A2BR/were

sorted by BD Aria. For the colitis model as described previously (22), mouse splenocytes were stained with anti-CD45RB, anti-CD25, and anti-CD4, and sorted by BD Aria for CD4þCD45RBhi and

CD4þCD25hiT cells (considered as Tregs), respectively. A total of 4 105sorted CD4þCD45RBhicells in 100 mL of PBS were transferred intravenously into Rag1/mice with or without 105CD4þCD25hi

WT or A2BR/Tregs through the retro-orbital venous sinus. Mice developed clinical signs of colitis 3.5–4.5 weeks post transfer. Mice were observed daily and weighed weekly. To assess the clinical status of the recipient mice, aggregate clinical scores were assigned as described previously (23) on the day of injection, weekly thereafter, and at time of sacrifice. For histologic scores, colon tissue sections were stained with hematoxylin and eosin (H&E) as well as Alcian blue and periodic acid– Schiff solution. Colitis severity was graded semiquantitatively from 0 to 4 in a blinded fashion.

In vivo killing assay

Analysis of tumor antigen-specific effector CTL activity in vivo was performed as described previously (20). Briefly, SIY (SIYRYYGL) peptide-pulsed CFSE and OVA-I (SIINFEKL) peptide-pulsed CFSE low splenocytes were mixed at a ratio of 1:1, and a total of 2 107cells in 100 mL of PBS were injected intraperitoneally into recipient animals. Draining lymph nodes (DLN) and spleen were then harvested 24 hours after adoptive transfer, and CFSEfluorescence intensity was analyzed byflow cytometry. Gating on CFSEþcells, killing was calculated as

1-[(% SIY peptide-pulsed cells in B16-SIY tumor–bearing mice/% OVA-I–peptide-pulsed cells in B16-SIY tumor–bearing mice)/(% SIY peptide-pulsed cells in tumor-free mice/% OVA-I–peptide-pulsed cells in tumor-free mice)] and expressed as a percentage.

Thymidine incorporation assay

CD45 cell enrichment from tumor infiltrates was initially con-ducted. Single-cell suspensions of B16-SIY tumors from WT or A2BR/mice were stained with biotinylated anti-CD45, followed by streptavidin MACS beads, and sorted on an AutoMACS (Miltenyi Biotec). For the Treg suppression assay, the CD45þinfiltrating cells were further stained by anti-CD25 and anti-CD4 and sorted by BD Aria for CD4þCD25hiTregs. For the dendritic cell (DC)–mediated

T-cell proliferation assay, the CD45þinfiltrating cells were further stained by anti-CD11b and anti-CD11c and sorted by BD Aria for CD11bþCD11cþDCs. 2 1052C splenocytes per well were plated into 96-well bottom plates in CR10 medium as described above in the presence of 1 mg/mL SIY peptides with or without serially diluted sorted Tregs or DCs for 3 days. For both assays above, wells were pulsed with 1 mCi of [3H]thymidine (PerkinElmer) for 8–10 hours

and counted. 2C T-cell proliferation was defined as the mean CPM of the response of the antigen-stimulated cells divided by the mean of the response of cells cultured without antigen. Positive and negative controls were run on each plate.

BM-DM and BM-DC generation

BM-DCs were prepared as described previously (24). In brief, femurs and tibiae from 8- to 12-week-old mice were collected and flushed with sterile HBSS twice. The resulting BM cells were resus-pended in R10F (RPMI1640 medium containing 10% heat-inactivated FBS, 2 mmol/LL-glutamine, 1 mmol/L sodium pyruvate, 50 U/mL

penicillin, 50 mg/mL streptomycin) plus 50 mmol/L 2-ME, and 5 ng/ mL GM-CSF. A total of 2–3  106cells per bacteriological culture plate were cultured for 10 days, feeding cells on days 3 and 8 by adding 10 mL fresh medium, and on day 6 by replacing half of the culture medium. Nonadherent cells were collected on day 10 and verified to be at least 85%–95% CD11bþ/CD11cþ/MHC-IIþ/F4/80/Gr1byflow cytome-try before use in experiments.

BM-DMs were prepared as described previously (3). In brief, BM cells obtained as described above ere cultured overnight in standard tissue culture plates in the presence of 10 ng/mL M-CSF. Nonadherent cells from this initial culture were then transferred to low-attachment six-well plates (Corning Life Sciences) in 4 ml R5F containing 30% L929 conditioned medium and 10 ng/mL M-CSF for 7 days, adding 1.5 mL fresh medium on days 3 and 5. Cells were verified to be at least 90%–98% CD11bþ/CD11c/F4/80þ/Gr1byflow cytometry before

use in experiments. Statistical analysis

Data were analyzed by GraphPad Prism Software (Version 7.02). For datasets involving two groups Student t test, for datasets involving more than two groups one-way ANOVA and post hoc analysis and for samples involving more than one variable we performed two-way ANOVA and post hoc analysis. Significance is indicated as, P < 0.05;

, P < 0.01;, P < 0.001 and, P < 0.0001.

Results

Hematopoietic A2BR promoted tumor growth

Deletion or pharmacologic blockade of A2BR delays growth of syngeneic MB49 bladder carcinoma tumors in a hematopoietic cell–dependent manner (4). To extend the scope of these findings

(4)

using multiple syngeneic tumor models, A2BR/ mice were injected subcutaneously with parental or ovalbumin expressing lung (LLC1 and LLC1-OVA), colon adenocarcinoma (MC38), and melanoma (B16-SIY) syngeneic tumors. All these tumor cell lines showed slower tumor growth in A2BR/(KO) mice as compared with WT mice (Fig. 1A–D). To determine which A2BRþ(WT) host cell populations contributed to tumor protection, BM chi-meras (WT mice receiving KO BM, i.e., KO>WT; KO mice receiving WT BM, i.e., WT>KO, KO mice receiving KO BM, i.e., KO>KO, and WT mice receiving WT BM, i.e., WT>WT) were used to ablate the expression of A2BR selectively in hemato-poietic and nonhematohemato-poietic cells. We found that A2BR deletion only on hematopoietic cells effectively limited the growth of LLC1 (Fig. 1E) or B16-SIY (Fig. 1F), indicating the predominant impor-tance of A2BR expression on the hematopoietic compartment for tumor development.

Host A2BR expression impaired antitumor T-cell immunity We profiled immune infiltrates within the tumor microenviron-ment by flow cytometry. At 14 days postinoculation of B16-SIY tumors, there were no significant differences in the percentages of CD4þ, Treg (CD4þFoxp3þ), NK (CD49bþNK1.1þ), CD11bþCD11cþ cells, or granulocytic myeloid-derived suppressor cells (Gr1þCD11bþ) in the tumor infiltrates of B16-SIY tumors in A2BR/versus WT mice (Fig. 2A; Supplementary Fig. S2). In contrast, the frequencies of tumor-infiltrating total and tumor antigen–specific CD8þ T cells (Fig. 2A and B, respectively) increased in A2BR/mice as compared with WT mice. The frequency of infiltration of IFNg-secreting CD8þT cells was also increased in tumors isolated from A2BR/ hosts (Fig. 2C). To test whether these phenotypic differences translated

into a functional activity, we performed an in vivo cytotoxicity assay by transferring peptide pulsed splenocytes into WT or A2BR/hosts bearing B16-SIY tumors as depicted in Fig. 2D. Of note, the ability to kill antigen-pulsed target cells was significantly improved in DLNs of B16-SIY–bearing A2BR/ mice compared with WT mice (Fig. 2E). Our data demonstrated that host A2BR deficiency led to increased tumor antigen–specific effector CD8þ T-cell infiltration into the tumor microenvironment, contributing to delayed tumor growth.

A2BR expression on T cells played an insignificant role in the control of tumor growth

To evaluate the role of intrinsic A2BR expression on CD8þT-cell immunity, equal numbers of CFSE-labeled WT CD90.1þCD8þT cells and A2BR/CD90.2þCD8þT cells were cotransferred into CD45.1 transgenic mice one day before B16F10 or B16-SIY inoculation. Proliferation and IFNg secretion of transferred splenic A2BR/ CD8þT cells were comparable with those of cotransferred WT CD8þ T cells (Supplementary Fig. S3A and S3B). Adoptive transfer of A2BR/CD8þ T cells caused tumor inhibition in Rag1/mice similar to inhibition caused by WT CD8þ T cells. These results suggested an insignificant role of CD8þT-cell–intrinsic A2BR expres-sion for antitumor immunity (Supplementary Fig. S3C).

To determine whether A2BR expression influenced tumor-associated Treg function, we performed an in vitro suppression assay and an in vivo protection from colitis assay using A2BR/versus WT Tregs isolated from B16-SIY tumors or spleen, respectively. There were no significant differences in suppressive functions between A2BR/ and WT CD4þFoxp3þ Treg from B16-SIY–bearing mice (Supple-mentary Fig. S2). In line with this, WT and A2BR/Tregs showed Figure 1.

Expression of A2BR in hematopoietic cells promoted tumor growth. WT and A2BR-deficient (A2BR/) mice were inoculated subcutaneoulsy with 106LLC1 (A),

LLC1-OVA (B), MC38 (C), or B16-SIY (D) cells, and tumor growth was measured.,P < 0.05;,P < 0.01;,P < 0.001; n ¼ 5. Data are given as means  SEM. Data are representative of two independent experiments. WT and A2BR/chimeric mice were generated by BM reconstitution. Chimeric mice (5/group) were injected subcutaneously with 106LLC1 (E) or B16-SIY (F) cells, and tumor growth was measured.,

P < 0.05. Data are given as means  SEM. Data are representative of two independent experiments. For A–D, data were analyzed by Student t test; for E and F, data were analyzed by two-way ANOVA and post hoc Bonferroni test.

APC Expression of Adenosine A2BR Controls Tumor Growth

(5)

comparable ability to protect mice from colitis (Supplementary Fig. S4).

A2BR expression on tumor-infiltrating APCs mitigated antitumor CD8þT-cell immunity

To dissect the contribution of A2BR expression in APCs on antigen-specific T-cell priming, CFSE-labeled na€ve CD8þT cells were trans-ferred by intravenous injection into WT (A2BRþ/þ) or A2BR/mice prior to B16-SIY tumor challenge. Enhanced proliferation (CFSEdim) of transferred splenic CD8þT cells was found in A2BR/ hosts compared with WT hosts (Fig. 3A and B). There was also higher expression of activation markers (CD69 and CD44) and IFNg pro-duction in transferred CD8þT cells from spleens collected from B16-SIY–treated A2BR/mice compared with that of WT mice (Fig. 3A and B). These results suggested that A2BR-deficient APCs enhanced priming and activation of tumor antigen–specific A2BR-proficient CD8þT cells. More specifically, we observed that tumor-infiltrating myeloid APCs from A2BR/mice displayed elevated activation and maturation markers (CD86 and MHC II expression) as compared with that of WT mice (Fig. 3C). Sorted A2BR/infiltrating myeloid APCs had enhanced capacity to drive antigen-specific T-cell proliferation in a dose-dependent manner compared with that of WT APCs ex vivo (Fig. 3D). These data demonstrated that A2BR deficiency allowed

tumor-infiltrating myeloid APCs to more competently facilitate expansion and activation of tumor antigen–specific CD8þT cells.

We found that only the selective A2BR antagonist, PSB-603, blocked the suppression of IL12 from DCs stimulated with the nonselective adenosine agonist NECA and LPS (Fig. 3E). Conversely, IL12 secreted from myeloid DCs derived from BM were remarkably reduced by the treatment with the A2BR partial agonist, BAY-60-6583, but not by selective agonists for A1R, A2AR, or A3R (Fig. 3F). We found that NECA was only capable of reducing an LPS-induced TNFaþIL10 subpopulation of BM-DMs and increasing a TNFaIL10DC subpopulation when BM-DMs are proficient for A2BRs (Supplementary Fig. S5A). Compared with either WT or A2AR/BM-DM, A2BR/BM-DMs produced more TNFa and less of IL10 when stimulated with LPS (Supplementary Fig. S5B). Collectively, these results suggested that A2BR expression was impor-tant for polarization of myeloid APCs from tumor suppressor to tumor-promoting phenotypes in vivo and ex vivo.

Cell-intrinsic A2BR signaling in myeloid cells and APCs promoted tumor growth and lung metastasis

To investigate the cell-intrinsic role of A2BRs in hematopoietic cell differentiation in tumor-bearing mice, Rag1/hosts were recon-stituted with equal numbers of WT and A2BR/BM cells. The Figure 2.

Host A2BR impaired endogenous antitumor T-cell immunity. A, Percentages of CD4þTCRVbþ, CD8þTCRVbþ, Gr1þCD11bþ, CD49bþNK1.1þ, and CD11bþCD11cþcells in

tumor infiltrates of WT or A2BR/mice (n ¼ 5) collected 14 days after inoculation with B16-SIY tumor cells by flow cytometric analysis. The H-2Kb- SIYRYYGL-IgG dimer was used to track SIY-specific CD8þT cells from B16SIY tumor–bearing WT or A2BR/mice (n ¼ 5). Frequencies of dimerþCD8þcells (B) and IFNgþCD8þcells

(C) in response to SIYRYYGL peptide were measured byflow cytometry. D, Graphical representation of experimental approach in E. E, Percent killing of adoptively transferred SIYRYYGL peptide–pulsed splenocytes from parallel experiments involving B16-SIY–bearing WT versus A2BR/hosts (n ¼ 5).,P < 0.05;,P < 0.01. Data are representative of two independent experiments. For A–C and E, data were analyzed by Student t test.

(6)

proportion of A2BR/myeloid APCs, CD11bdimMHCIIþDCs and Gr-1þcells, but not T cells, in the tumor microenvironment increased compared with WT cells through 7–21 days posttumor inoculation (Fig. 4A). There was a slight but significant increase in the proportion of A2BR/NK cells (Fig. 4A). Consistent with the results shown in Fig. 3C, there was significantly higher expression of CD86 (Fig. 4B) and MHCII (Fig. 4C) in A2BR/infiltrating myeloid APCs than WT mDC on days 14 and 21. In contrast, the general activation status of both splenic and tumor-infiltrating CD4þ and CD8þ T cells was similar between A2BR/and WT as measured by CD69, CXCR3

expression and IFNg accumulation upon restimulation (Supplemen-tary Fig. S6A and S6B). We did not observe significant changes in activation markers such as CD44, CD69, CXCR3, CD11b, or CD107a in A2BR/NK cells (Supplementary Fig. S7). These results implicate a specific role for cell-intrinsic A2BR signaling in the differentiation and activation of myeloid cells within tumors.

To confirm cell-intrinsic effects of myeloid A2BR ablation on tumor progression, LysMCreþ/Adora2bf/fmice were generated for myeloid-selective A2BR deletion. Myeloid-myeloid-selective deletion of A2BR inhibited MB49 tumor growth (Fig. 4D) as compared with that of LysMCre/ Figure 3.

A2BR deficiency on tumor-infiltrating DCs promoted tumor- reactive CD8þT-cell immunity. A, CFSE-labeled 2C CD8þT cells were intravenously transferred into WT (A2BRþ/þ) or A2BR/mice (n ¼ 5) 1 day prior to subcutaneous challenge of B16-SIY cells. Three days later, the proliferation (CFSE dilution), IFNg production, and CD69/CD44 expression of transferred splenic T cells were measured byflow cytometry. B, The data were summarized and are shown as means  SEM (n ¼ 5). C, Expression of CD80, CD86, CD40, and MHC-II on tumor-infiltrating DCs (CD11bþCD11cþ) from B16-SIY–bearing WT or A2BR/mice. MFI, meanfluorescence intensity. D, Tumor-infiltrating DCs sorted from B16-SIY tumor–bearing WT or A2BR/mice were added at different ratios to 2C T cells for 3-day inculation, and 3[H] thymidine uptake was measured. Data are given as means SEM. Data are representative of two independent experiments. BM-DCs (3  105) were stimulated

with LPS. E, NECA was added to the culture in the presence or absence of antagonists specific for A1, A2A, and A2B receptors. F, Agonists specific for A1, A2A, A2B, and A3 adenosine receptors were added to the culture during stimulation. IL12 in the culture supernatants was tested by ELISA. Data are representative of two independent experiments. For B–D, data were analyzed by Student t test; for E and F, data were analyzed by one-way ANOVA and post hoc Tukey test (n.s., not significant;,P < 0.05;,P < 0.01;,P < 0.0001).

APC Expression of Adenosine A2BR Controls Tumor Growth

(7)

Figure 4.

Myeloid expression of A2BR intrinsically promoted tumor growth and dissemination. A, Rag/mice were reconstituted with mixed BMs from WT and A2BR/mice. Eight to 12 weeks later, mice received subcutaneous MB49 bladder carcinoma tumors. Tumors from different groups of mice were isolated 7, 14, and 21 days after tumor inoculation. Proportion of major immune cell populations is shown (n ¼ 4 per group for days 7 and 14, and n ¼ 5 per group for day 21). Activation markers for spleen- versus tumor-associated myeloid APCs from the tumor-bearing chimeric mice indicated in A were tested byflow cytometry 14 (left; B) or 21 (right; C) days after tumor growth (n ¼ 5 per group). Data are representative of two independent experiments. Data were analyzed by two-way ANOVA and post hoc Bonferroni test (n.s., not significant;,P < 0.05;,P < 0.01;,P < 0.001;,P < 0.0001). D, MB49 cells (105) were inoculated subcutaneously to the right

flanks of LysMCreþ/A2BRf/fmice or littermate controls. Tumor growth was measured by caliper.

N ≥ 11, pooled data from two independent experiments. E, B16-Luc cells (5 105) expressing luciferase enzyme were injected intravenously, and their localization in lung was measured noninvasively by IVIS imaging. F, Quantitation of E as

photons/second (p/s). Lungs were isolated 2 weeks after inoculation (G), and their weights were measured (H).N ≥ 6, pooled data from two independent experiments. For D and F, data were analyzed by two-way ANOVA andpost hoc Bonferroni test (n.s., not significant;,P < 0.01;,P < 0.001); for H, Student t test was performed (,P < 0.001).

(8)

littermate control mice. Likewise, deletion of A2BR on myeloid cells dramatically reduced lung metastases of B16F10 melanomas detected by luciferase activity and lung weight measurement (Fig. 4E–H). We next performed a detailed immuno-profiling of myeloid cells and DC populations using B16-SIY tumors. We observed that A2BR deletion particularly caused increased infiltration among the tumor-associated myeloid DCs and CD103þDCs (Fig. 5A). To test whether the role of DCs in tumor growth and dissemination we crossed Adora2bf/fmice with ItgaxCreþ/mice (CD11cCre). Deletion of A2BRs from DCs delayed growth B16F10-luciferase cells but failed to delay decrease lung dissemination (Fig. 5B–D) suggesting A2BR expression among DCs promoted tumor growth while A2BR expression of monocytes and/or Gr1þcells promoted lung dissemination.

Blockade of A2BR inhibited tumor growth and augmented the efficacy of adoptive T-cell therapy

Cell-based therapies are efficacious for treating leukemia but have failed to slow the growth solid tumors, possibly due to the suppressive tumor microenvironment (25). High adenosine in the TME can suppress tumor rejection by the immune system (1). Therefore, to extend ourfindings to a more clinically relevant setting, we evaluated the antitumor effect of pharmacologic A2BR blockade on adoptive T-cell therapy. In line with previously published results (20), transfer of SIY/anti-CD28 and IL2 prestimulated SIY-specific 2C T cells alone failed to control the growth of B16-SIY tumors (Fig. 6). Consistent with a role for host A2BRs in promoting tumor growth, treatment with the selective A2BR antagonist MRS-1754 caused tumor growth Figure 5.

A2BR expression in DCs promoted primary tumor growth but not experimental metastasis. A, Detailed immunophenotyping was performed using single-cell suspensions of B16-SIY tumors from WT or A2BR/mice. B, B16F10-luc cells (105) were inoculated subcutaneously to the right

flanks of CD11cCreþ/A2BRf/fmice or

littermate controls. Tumor growth was measured by caliper.N ¼ 9, pooled data from two independent experiments. C, B16-Luc cells (5  105) expressing luciferase enzyme were injected intravenously, and their localization in lung was measured by IVIS imaging (N ¼ 4). D, Quantitation of C as photons/second (p/s). For A, data were analyzed by Studentt test (,P < 0.05); for B–D, data were analyzed by two-way ANOVA and post hoc Bonferroni test (,P < 0.001).

APC Expression of Adenosine A2BR Controls Tumor Growth

(9)

inhibition in both B16-SIY (Fig. 6) and MC38 (Supplementary Fig. S8) tumor models as a monotherapy. Moreover, combining daily MRS1754 treatment with adoptive 2C T-cell therapy achieved syner-gistic antitumor efficacy (Fig. 6). Overall, our study suggested that APC expression of A2BR in the tumor microenvironment inhibits antigen-specific antitumor immune responses, thus can be exploited as a way to improve current strategies of cancer immunotherapy, includ-ing cell-based therapies.

Discussion

Adenosine A2B receptors are expressed in tumor cells and immune cells (4, 7, 11). Blockade or pharmacological deletion of A2BRs causes activation of both tumor-associated T cells and APCs in vivo (4). However, it was not clear which immune cell subtype have intrinsic A2BRs. Also, although tumor expression of A2BR is important for lung colonization of tumors (6, 7), thisfinding does not rule out a role for A2BRs on immune cells in contributing to this effect. Here, we provided evidence that cell-intrinsic signaling by A2BRs on APCs is involved in adenosine-mediated suppression of antitumor T-cell responses and for enhancing dissemination of tumors to the lung.

Activation of macrophages and DCs differentiated from bone marrow cells can be suppressed by A2BR signaling (10, 11). A2BR signaling in DCs strongly suppressed activation of T cells in vitro (11). Accordingly, in vivo models of inflammatory and infectious disease models suggest that A2BR signaling can suppress macrophage acti-vation and phagocytosis, potentially leading to increased bacterial pathogenesis but reduced sepsis (16, 26), suggesting a role for myeloid cell A2BRs as a tumor-promoting mechanism. Deletion of A2BR or pharmacologic targeting slows the growth of bladder carcinoma in a T-cell–dependent manner (4). However, it was not clear if myeloid cells and DCs intrinsically played a major role in producing this effect and whether similar mechanisms are at play in other tumors. Here, we addressed this question by extending the scope of previous

observa-tions showing antitumor effects of A2BR deletion in multiple synge-neic tumor models and defining the hematopoietic compartment and specific APCs as cellular targets of A2BRs in the tumor microenvi-ronment. A2BR expression is mostly observed in myeloid cells and myeloid DCs in PBMCs (ref. 27; https://www.proteinatlas.org/). Human DCs are susceptible to adenosine suppression ex vivo (28). Further investigation is required using human tumor samples tofind whether increased expression of A2BR particularly in tumor versus tumor-infiltrating APCs is associated with poor survival among these patients.

A2BR signaling promotes tumor metastasis (6, 7, 29, 30). A2BR blockade inhibited both spontaneous metastasis of primary tumors and lung colonization of tumors after systemic delivery. Mecha-nistically, A2BR expression in tumor cells decreases cell-to-cell contact and increases tumor cell survival through the ERK path-way (7, 8). Breast tumor cells express intrinsic A2B receptors that facilitate metastasis (6, 7). A2BR blockade or silencing CD73þin such tumors does not influence T cells or NKs (7). Our study suggests that A2BR expression in monocytes and/or Gr1þmyeloid cells but not DCs could also promote tumor colonization, especially at a later time point (week 2) after systemic delivery of the tumor cells; these data emphasized the importance of these subpopulations in regulation of tumor metastasis (31–33). One key difference between these studies and our study is the use of CD73þcells for tumor cell–intrinsic effects (6, 7, 30), and the use of A2BRlow,

CD73lowB16F10 cells to uncover antitumor immune effects,

respec-tively. Therefore, depending on the model, immune versus tumor-related effects of A2BR on metastasis may vary. Therefore, A2BRs on both tumor cells and immune cells, particularly myeloid cells, may be responsible for tumor invasiveness with varying degrees of influence depending on the CD73 status of the tumor cell. This concept requires additional investigation.

Adenosine targets both A2ARs and A2BRs to suppress immune responses (3–5, 20, 34, 35). Myeloid expression of adenosine A2ARs suppresses antitumoral T and NK-cell responses (3). Also, growth of certain syngeneic tumors such as MCA205 sarcomas or immunogenic CL8-1 melanomas are more sensitive to A2AR deletion than A2BR deletion (5, 36). Both A2ARs and A2BRs couple to Gs and increase cAMP to suppress immune responses (1). A2BRs also couples to Gq G proteins and has cAMP-independent effects. A2ARs are widely dis-tributed on immune cells (2), whereas A2BRs more preferentially expressed on myeloid immune cells (9). Therefore, A2AR and A2BR signaling can cause both overlapping and nonoverlapping effects in regulation of immune cell function causing differential effects in different tumor microenvironments. Another potential difference may be due to the affinities of these receptors for adenosine. A2ARs have higher affinity, and deletion of A2ARs can play important roles in homeostasis of immune cells systemically, while low-affinity A2BR signaling is preferentially involved in local pathologic reactions asso-ciated with high adenosine generation (37, 38). Therefore, tumors with high adenosine accumulation may be more sensitive to A2BR block-ade. Another possibility is based on the role of A2ARs in regulating the expression of A2BRs, suggesting that deletion of A2ARs may also reduce A2BR signaling indirectly (39). Future studies are needed to test if deletion or targeting both receptors especially in myeloid cells will have a synergistic or additive effect on tumor growth and dissemination.

In summary, our study identified cell-intrinsic A2BR expression on APCs as a suppressor of antitumor immune responses. There are distinct subtypes of APCs such as CD103þ DCs that are mainly responsible for antigen-cross presentation to CD8þ T cells and Figure 6.

Pharmacologic blockade of A2BR inhibited tumor growth and augmented the efficacy of adoptive T-cell therapy. WT mice (5/group) were subcutaneously challenged with 106B16-SIY cells. Ten days later, mice were treated with either MRS1754 or control vehicles daily, and 13 days after tumor challenge, mice were adoptively transferred with SIY/anti-CD28 and IL2 prestimulated 5  106 SIY-specific 2C T cells. Tumor volumes were measured at the indicated times. ,P < 0.01. Data are representative of two independent experiments. Data were analyzed by two-way ANOVA andpost hoc Bonferroni test.

(10)

myeloid DCs that activate CD4þ T cells and produce tolerogenic versus immunostimulatory cytokines such as IL10 versus IL12, respec-tively, to other immune cells including CD8þT cells depending on the microenvironment (40). Additional studies are needed to further dissect the roles of A2BRs in DC subtypes. Overall, our findings suggested that cell-based therapies such as adoptive cell therapy or DC vaccines, and immunotherapies can become more efficacious in combination A2BR inhibition.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Authors’ Contributions

Conception and design:S. Chen, B. Zhang, C. Cekic

Development of methodology:S. Chen, I. Akdemir, J. Fan, B. Zhang, C. Cekic Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.):S. Chen, J. Fan, J. Linden, B. Zhang, C. Cekic

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis):S. Chen, I. Akdemir, B. Zhang, C. Cekic

Writing, review, and/or revision of the manuscript:S. Chen, J. Linden, B. Zhang, C. Cekic

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases):S. Chen, I. Akdemir, B. Zhang, C. Cekic

Study supervision:B. Zhang, C. Cekic

Acknowledgments

The authors thank Long Wang and Lishi Sun (University of Texas Health Science Center at San Antonio) for their technical support with the in vivo mouse studies. The authors also thank Ali Can Savas, Merve Kayhan, and Altay Koyas (Bilkent Uni-versity, Ankara, Turkey) for their technical assistance with the in vitro studies. This research was supported in part by NIH grant CA149669, a Melanoma Research Alliance Pilot Award (to B. Zhang), Northwestern University Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Facility, and a Cancer Center Support Grant (NCI CA060553). This work is also supported in part by European Molecular Biology Organization (EMBO) Installation Grant 3297 and by 1001 - Scientific and Technological Research Projects Funding Program grant (215S729) from Scientific and Technological Research Council of Turkey (TUBITAK; to C. Cekic).

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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 23, 2019; revised March 30, 2020; accepted April 28, 2020; publishedfirst May 7, 2020.

References

1. Vijayan D, Young A, Teng MWL, Smyth MJ. Targeting immunosuppressive adenosine in cancer. Nat Rev Cancer 2017;17:709–24.

2. Cekic C, Linden J. Purinergic regulation of the immune system. Nat Rev Immunol 2016;16:177–92.

3. Cekic C, Day YJ, Sag D, Linden J. Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment. Cancer Res 2014;74:7250–9.

4. Cekic C, Sag D, Li Y, Theodorescu D, Strieter RM, Linden J. Adenosine A2B receptor blockade slows growth of bladder and breast tumors. J Immunol 2012; 188:198–205.

5. Ohta A, Gorelik E, Prasad SJ, Ronchese F, Lukashev D, Wong MK, et al. A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci U S A 2006;103:13132–7.

6. Desmet CJ, Gallenne T, Prieur A, Reyal F, Visser NL, Wittner BS, et al. Identification of a pharmacologically tractable Fra-1/ADORA2B axis promoting breast cancer metastasis. Proc Natl Acad Sci U S A 2013;110:5139–44. 7. Mittal D, Sinha D, Barkauskas D, Young A, Kalimutho M, Stannard K, et al.

Adenosine 2B receptor expression on cancer cells promotes metastasis. Cancer Res 2016;76:4372–82.

8. Ntantie E, Gonyo P, Lorimer EL, Hauser AD, Schuld N, McAllister D, et al. An adenosine-mediated signaling pathway suppresses prenylation of the GTPase Rap1B and promotes cell scattering. Sci Signal 2013;6:ra39.

9. Hasko G, Csoka B, Nemeth ZH, Vizi ES, Pacher P. A(2B) adenosine receptors in immunity and inflammation. Trends Immunol 2009;30:263–70.

10. Xaus J, Mirabet M, Lloberas J, Soler C, Lluis C, Franco R, et al. IFN-gamma up-regulates the A2B adenosine receptor expression in macrophages: a mechanism of macrophage deactivation. J Immunol 1999;162:3607–14.

11. Wilson JM, Ross WG, Agbai ON, Frazier R, Figler RA, Rieger J, et al. The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells. J Immunol 2009;182:4616–23.

12. Iannone R, Miele L, Maiolino P, Pinto A, Morello S. Blockade of A2b adenosine receptor reduces tumor growth and immune suppression mediated by myeloid-derived suppressor cells in a mouse model of melanoma. Neoplasia 2013;15: 1400–9.

13. Ryzhov S, Novitskiy SV, Goldstein AE, Biktasova A, Blackburn MR, Biaggioni I, et al. Adenosinergic regulation of the expansion and immunosuppressive activity of CD11bþGr1þ cells. J Immunol 2011;187:6120–9.

14. Sorrentino C, Miele L, Porta A, Pinto A, Morello S. Myeloid-derived suppressor cells contribute to A2B adenosine receptor-induced VEGF production and angiogenesis in a mouse melanoma model. Oncotarget 2015;6:27478–89. 15. Chen S, Wang L, Fan J, Ye C, Dominguez D, Zhang Y, et al. Host miR155

promotes tumor growth through a myeloid-derived suppressor cell-dependent mechanism. Cancer Res 2015;75:519–31.

16. Belikoff BG, Hatfield S, Georgiev P, Ohta A, Lukashev D, Buras JA, et al. A2B adenosine receptor blockade enhances macrophage-mediated bacterial phago-cytosis and improves polymicrobial sepsis survival in mice. J Immunol 2011;186: 2444–53.

17. Day YJ, Marshall MA, Huang L, McDuffie MJ, Okusa MD, Linden J. Protection from ischemic liver injury by activation of A2A adenosine receptors during reperfusion: inhibition of chemokine induction. Am J Physiol Gastrointest Liver Physiol 2004;286:G285–93.

18. Yang D, Zhang Y, Nguyen HG, Koupenova M, Chauhan AK, Makitalo M, et al. The A2B adenosine receptor protects against inflammation and excessive vascular adhesion. J Clin Invest 2006;116:1913–23.

19. Karmouty-Quintana H, Zhong H, Acero L, Weng T, Melicoff E, West JD, et al. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease. FASEB J 2012;26:2546–57.

20. Wang L, Fan J, Thompson LF, Zhang Y, Shin T, Curiel TJ, et al. CD73 has distinct roles in nonhematopoietic and hematopoietic cells to promote tumor growth in mice. J Clin Invest 2011;121:2371–82.

21. Cekic C, Sag D, Day YJ, Linden J. Extracellular adenosine regulates naive T cell development and peripheral maintenance. J Exp Med 2013;210:2693–706. 22. Mottet C, Uhlig HH, Powrie F. Cutting edge: cure of colitis by CD4þCD25þ

regulatory T cells. J Immunol 2003;170:3939–43.

23. Steinbach EC, Gipson GR, Sheikh SZ. Induction of murine intestinal in flam-mation by adoptive transfer of effector CD4þ CD45RB high T cells into immunodeficient mice. J Vis Exp 2015;(98):52533.

24. Cekic C, Casella CR, Eaves CA, Matsuzawa A, Ichijo H, Mitchell TC. Selective activation of the p38 MAPK pathway by synthetic monophosphoryl lipid A. J Biol Chem 2009;284:31982–91.

25. D'Aloia MM, Zizzari IG, Sacchetti B, Pierelli L, Alimandi M. CAR-T cells: the long and winding road to solid tumors. Cell Death Dis 2018;9:282. 26. Csoka B, Nemeth ZH, Rosenberger P, Eltzschig HK, Spolarics Z, Pacher P, et al.

A2B adenosine receptors protect against sepsis-induced mortality by dampening excessive inflammation. J Immunol 2010;185:542–50.

27. Ponten F, Jirstrom K, Uhlen M. The Human Protein Atlas–a tool for pathology. J Pathol 2008;216:387–93.

28. Kayhan M, Koyas A, Akdemir I, Savas AC, Cekic C. Adenosine receptor signaling targets both PKA and Epac pathways to polarize dendritic cells to a suppressive phenotype. J Immunol 2019;203:3247–55.

29. Lan J, Lu H, Samanta D, Salman S, Lu Y, Semenza GL. Hypoxia-inducible factor 1-dependent expression of adenosine receptor 2B promotes breast cancer stem cell enrichment. Proc Natl Acad Sci U S A 2018;115:E9640–E8.

30. Beavis PA, Divisekera U, Paget C, Chow MT, John LB, Devaud C, et al. Blockade of A2A receptors potently suppresses the metastasis of CD73þ tumors. Proc Natl Acad Sci U S A 2013;110:14711–6.

APC Expression of Adenosine A2BR Controls Tumor Growth

(11)

31. Butler KL, Clancy-Thompson E, Mullins DW. CXCR3(þ) monocytes/macro-phages are required for establishment of pulmonary metastases. Sci Rep 2017;7: 45593.

32. Young A, Ngiow SF, Barkauskas DS, Sult E, Hay C, Blake SJ, et al. Co-inhibition of CD73 and A2AR adenosine signaling improves anti-tumor immune responses. Cancer Cell 2016;30:391–403.

33. Hanna RN, Cekic C, Sag D, Tacke R, Thomas GD, Nowyhed H, et al. Patrolling monocytes control tumor metastasis to the lung. Science 2015;350:985–90. 34. Stagg J, Divisekera U, Duret H, Sparwasser T, Teng MW, Darcy PK, et al.

CD73-deficient mice have increased antitumor immunity and are resistant to exper-imental metastasis. Cancer Res 2011;71:2892–900.

35. Young A, Ngiow SF, Gao Y, Patch AM, Barkauskas DS, Messaoudene M, et al. A2AR adenosine signaling suppresses natural killer cell maturation in the tumor microenvironment. Cancer Res 2018;78:1003–16.

36. Kjaergaard J, Hatfield S, Jones G, Ohta A, Sitkovsky M. A2A adenosine receptor gene deletion or synthetic A2A antagonist liberate tumor-reactive CD8(þ) T cells from tumor-induced immunosuppression. J Immunol 2018;201:782–91. 37. Bruns RF, Lu GH, Pugsley TA. Characterization of the A2 adenosine receptor

labeled by [3H]NECA in rat striatal membranes. Mol Pharmacol 1986;29: 331–46.

38. Daly JW, Butts-Lamb P, Padgett W. Subclasses of adenosine receptors in the central nervous system: interaction with caffeine and related methylxanthines. Cell Mol Neurobiol 1983;3:69–80.

39. Moriyama K, Sitkovsky MV. Adenosine A2A receptor is involved in cell surface expression of A2B receptor. J Biol Chem 2010;285:39271–88.

40. Wculek SK, Cueto FJ, Mujal AM, Melero I, Krummel MF, Sancho D. Dendritic cells in cancer immunology and immunotherapy. Nat Rev Immu-nol 2020;20:7–24.

(12)

2020;8:1064-1074. Published OnlineFirst May 7, 2020.

Cancer Immunol Res

Siqi Chen, Imran Akdemir, Jie Fan, et al.

Growth

T-cell Responses and Promotes Tumor

+

Cells Suppresses CD8

The Expression of Adenosine A2B Receptor on Antigen-Presenting

Updated version

10.1158/2326-6066.CIR-19-0833

doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerimmunolres.aacrjournals.org/content/suppl/2020/05/07/2326-6066.CIR-19-0833.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerimmunolres.aacrjournals.org/content/8/8/1064.full#ref-list-1

This article cites 39 articles, 24 of which you can access for free at:

E-mail alerts

Sign up to receive free email-alerts

related to this article or journal.

Subscriptions

Reprints and

.

pubs@aacr.org

at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department

Permissions

Rightslink site.

Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.

http://cancerimmunolres.aacrjournals.org/content/8/8/1064

To request permission to re-use all or part of this article, use this link

Published OnlineFirst May 7, 2020; DOI: 10.1158/2326-6066.CIR-19-0833

Referanslar

Benzer Belgeler

2013 年國際口腔雷射應用醫學會(SOLA)世界年會假北醫大盛大舉行,來自歐 美亞等國近 200 名專業人士與會

神農本草經 陽湖孫星衍撰 原文 著本草者,代有明哲矣,而求道者必推本於神農,以

Ahidnamelere aykırı olarak yapılan bu hareketlerin Osmanlı devletine bildirilmesi sonucu, kanuna göre hareket edilmesini ve Fransa’nın kadim dost olması nedeniyle

[r]

3) Bir defter, bir kalem ve bir de eldiven aldım. Kasaya 200TL verdim. 4) Bir elbise ve bir gözlük aldım. Kasaya 200TL verdim. Kaç TL para üstü almalıyım?.... 2) Bir gözlük

In this study, the diagnosis and treatment process of a patient with an unrelated synchronous warthin tumor and basal cell adenoma combination in the ipsilateral parotid gland

Clinical findings of non-functional tumors, as in our case, are not relevant until the final stages and early diagnosis is difficult.. Key Words: Islet cell adenoma,

The tumors included in this group are Ewing’s sarcoma (peripheral neuroectodermal tumor), primitive neuroectodermal tumor (PNET), rhabdomyosarcoma, synovial sarcoma,