Adenosine signaling via the A2a receptor (A2aR) is emerging as an important checkpoint of immune responses. The presence of adenosine in the inflammatory milieu or generated by the CD39/CD73 axis on tissues or T regulatory cells serves to regulate immune responses. By nature of the specialized metabolism of cancer cells, adenosine levels are increased in the tumor microenvironment and contribute to tumor immune evasion. To this end, small molecule inhibitors of the A2aR are being pursued clinically to enhance immunotherapy. Herein, we demonstrate the ability of the novel A2aR antagonist, CPI-444, to dramatically enhance immunologic responses in models of checkpoint therapy and ACT in cancer. Furthermore, we demonstrate that A2aR blockade with CPI-444 decreases expression of multiple checkpoint pathways, including PD-1 and LAG-3, on both CD8+ effector T cells (Teff) and FoxP3+ CD4+ regulatory T cells (Tregs). Interestingly, our studies demonstrate that A2aR blockade likely has its most profound effects during Teff cell activation, significantly decreasing PD-1 and LAG-3 expression at the draining lymph nodes of tumor bearing mice. In contrast to previous reports using A2aR knockout models, pharmacologic blockade with CPI-444 did not impede CD8 T cell persistence or memory recall. Overall these findings not only redefine our understanding of the mechanisms by which adenosine inhibits immunity but also have important implications for the design of novel immunotherapy regimens.
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Adenosine A2a receptor
American type culture collection
OVA-expressing B16 murine melanoma
Tumor-draining lymph nodes
Inducible nitric oxide synthase
OVA-expressing Listeria monocytogenes
Non-draining lymph nodes
CD8+ effector T cell
OVA class-I tetramer+
Ohta A, Sitkovsky M (2001) Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature 414:916–920
Ohta A, Gorelik E, Prasad SJ, Ronchese F, Lukashev D, Wong MK, Huang X, Caldwell S, Liu K, Smith P, Chen JF, Jackson EK, Apasov S, Abrams S, Sitkovsky M (2006) A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci USA 103:13132–13137
Allard B, Pommey S, Smyth MJ, Stagg J (2013) Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs. Clin Cancer Res 19:5626–5635
Beavis PA, Milenkovski N, Henderson MA, John LB, Allard B, Loi S, Kershaw MH, Stagg J, Darcy PK (2015) Adenosine receptor 2A blockade increases the efficacy of anti-PD-1 through enhanced anti-tumor T cell responses. Cancer Immunol Res 3:506–517
Waickman AT, Alme A, Senaldi L, Zarek PE, Horton M, Powell JD (2012) Enhancement of tumor immunotherapy by deletion of the A2A adenosine receptor. Cancer Immunol Immunother 61:917–926
Stagg J, Divisekera U, McLaughlin N, Sharkey J, Pommey S, Denoyer D, Dwyer KM, Smyth MJ (2010) Anti-CD73 antibody therapy inhibits breast tumor growth and metastasis. Proc Natl Acad Sci USA 107:1547–1552
Loi S, Pommey S, Haibe-Kains B, Beavis PA, Darcy PK, Smyth MJ, Stagg J (2013) CD73 promotes anthracycline resistance and poor prognosis in triple negative breast cancer. Proc Natl Acad Sci USA 110:11091–11096
Beavis PA, Divisekera U, Paget C, Chow MT, John LB, Devaud C, Dwyer K, Stagg J, Smyth MJ, Darcy PK (2013) Blockade of A2A receptors potently suppresses the metastasis of CD73 + tumors. Proc Natl Acad Sci USA 110:14711–14716
Mittal D, Young A, Stannard K, Yong M, Teng MW, Allard B, Stagg J, Smyth MJ (2014) Antimetastatic effects of blocking PD-1 and the adenosine A2A receptor. Cancer Res 74:3652–3658
Zarek PE, Huang CT, Lutz ER, Kowalski J, Horton MR, Linden J, Drake CG, Powell JD (2008) A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells. Blood 111:251–259
Parks SK, Cormerais Y, Pouysségur J (2017) Hypoxia and cellular metabolism in tumour pathophysiology. J Physiol (Lond) 595:2439–2450
Kishton RJ, Sukumar M, Restifo NP (2017) Metabolic regulation of T cell longevity and function in tumor immunotherapy. Cell Metab 26:94–109
Blay J, White TD, Hoskin DW (1997) The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine. Cancer Res 57:2602–2605
Dubyak GR, el-Moatassim C (1993) Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol 265:C577–C606
Robeva AS, Woodard RL, Jin X, Gao Z, Bhattacharya S, Taylor HE, Rosin DL, Linden J (1996) Molecular characterization of recombinant human adenosine receptors. Drug Dev Res 39:243–252
Apasov S, Koshiba M, Redegeld F, Sitkovsky MV (1995) Role of extracellular ATP and P1 and P2 classes of purinergic receptors in T-cell development and cytotoxic T lymphocyte effector functions. Immunol Rev 146:5–19
Apasov SG, Koshiba M, Chused TM, Sitkovsky MV (1997) Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor. J Immunol 158:5095–5105
Filippini A, Taffs RE, Agui T, Sitkovsky MV (1990) Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP. J Biol Chem 265:334–340
Resta R, Yamashita Y, Thompson LF (1998) Ecto-enzyme and signaling functions of lymphocyte CD73. Immunol Rev 161:95–109
Emens L, Powderly J, Fong L, Brody J, Forde P, Hellmann M, Hughes B, Kummar S, Loi S, Luke J, Mahadevan D, Markman B, McCaffery I, Miller R, Laport G (2017) CPI-444, an oral adenosine A2a receptor (A2aR) antagonist, demonstrates clinical activity in patients with advanced solid tumors. AACR Annual Meeting 2017. Cancer Res 77:Abstract CT119
Willingham S, Ho P, Leone R, Piccione E, Choy C, Hotson A, Buggy J, Powell J, Miller R (2016) The adenosine A2A receptor antagonist CPI-444 blocks adenosine-mediated T-cell suppression and exhibits antitumor activity alone and in combination with anti-PD-1 and anti-PD-L1. AACR Annual Meeting 2017. Cancer Res 76:Abstract 2337
Cekic C, Sag D, Day YJ, Linden J (2013) Extracellular adenosine regulates naive T cell development and peripheral maintenance. J Exp Med 210:2693–2706
Cekic C, Linden J (2014) Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment. Cancer Res 74:7239–7249
Ngiow SF, Young A, Jacquelot N, Yamazaki T, Enot D, Zitvogel L, Smyth MJ (2015) A threshold level of intratumor CD8+ T-cell PD1 expression dictates therapeutic response to anti-PD1. Cancer Res 75:3800–3811
Chaudhary B, Elkord E (2016) Regulatory T Cells in the tumor microenvironment and cancer progression: role and therapeutic targeting. Vaccines (Basel). https://doi.org/10.3390/vaccines4030028
Huang CT, Workman CJ, Flies D, Pan X, Marson AL, Zhou G, Hipkiss EL, Ravi S, Kowalski J, Levitsky HI, Powell JD, Pardoll DM, Drake CG, Vignali DA (2004) Role of LAG-3 in regulatory T cells. Immunity 21:503–513
Ohta A (2016) A metabolic immune checkpoint: adenosine in tumor microenvironment. Front Immunol 7:109
Smyth MJ, Ngiow SF, Ribas A, Teng MWL (2016) Combination cancer immunotherapies tailored to the tumour microenvironment. Nat Rev Clin Oncol 13:143–158
Jiang S, Yan W (2016) T-cell immunometabolism against cancer. Cancer Lett 382:255–258
Mockler MB, Conroy MJ, Lysaght J (2014) Targeting T cell immunometabolism for cancer immunotherapy; understanding the impact of the tumor microenvironment. Front Oncol 4:107
Siska PJ, Rathmell JC (2015) T cell metabolic fitness in antitumor immunity. Trends Immunol 36:257–264
Curran MA, Montalvo W, Yagita H, Allison JP (2010) PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci USA 107:4275–4280
Koyama S, Akbay EA, Li YY, Herter-Sprie G, Buczkowski KA, Richards WG, Gandhi L, Redig AJ, Rodig SJ, Asahina H, Jones RE, Kulkarni MM, Kuraguchi M, Palakurthi S, Fecci PE, Johnson BE, Janne PA, Engelman JA, Gangadharan SP, Costa DB, Freeman GJ, Bueno R, Hodi FS, Dranoff G, Wong K, Hammerman PS (2016) Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun 7:10501
Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, Konieczny BT, Daugherty CZ, Koenig L, Yu K, Sica GL, Sharpe AH, Freeman GJ, Blazar BR, Turka LA, Owonikoko TK, Pillai RN, Ramalingam SS, Araki K, Ahmed R (2017) Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science 355:1423–1427
Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, Sasmal DK, Huang J, Kim JM, Mellman I, Vale RD (2017) T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science 355:1428–1433
Chamoto K, Chowdhury PS, Kumar A, Sonomura K, Matsuda F, Fagarasan S, Honjo T (2017) Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity. Proc Natl Acad Sci USA 114:E770
Vijayan D, Young A, Teng MWL, Smyth MJ (2017) Targeting immunosuppressive adenosine in cancer. Nat Rev Cancer 17:709–724
Smyth LA, Ratnasothy K, Tsang JYS, Boardman D, Warley A, Lechler R, Lombardi G (2013) CD73 expression on extracellular vesicles derived from CD4+ CD25+ Foxp3+ T cells contributes to their regulatory function. Eur J Immunol 43:2430–2440
Abbott RK, Thayer M, Labuda J, Silva M, Philbrook P, Cain DW, Kojima H, Hatfield S, Sethumadhavan S, Ohta A, Reinherz EL, Kelsoe G, Sitkovsky M (2016) Germinal center hypoxia potentiates immunoglobulin class switch recombination. J Immunol 197:4014–4020
Ohta A, Diwanji R, Kini R, Subramanian M, Ohta A, Sitkovsky M (2011) In vivo T cell activation in lymphoid tissues is inhibited in the oxygen-poor microenvironment. Front Immunol 2:27
Martin C, Leone M, Viviand X, Ayem ML, Guieu R (2000) High adenosine plasma concentration as a prognostic index for outcome in patients with septic shock. Crit Care Med 28:3198–3202
Funaya H, Kitakaze M, Node K, Minamino T, Komamura K, Hori M (1997) Plasma adenosine levels increase in patients with chronic heart failure. Circulation 95:1363–1365
Vallon V, Miracle C, Thomson S (2008) Adenosine and kidney function: potential implications in patients with heart failure. Eur J Heart Fail 10:176–187
Ramakers BPC, Riksen NP, Broek PHH, Franke B, Peters WHM, Hoeven JG, Smits P, Pickkers P (2011) Circulating adenosine increases during human experimental endotoxemia but blockade of its receptor does not influence the immune response and subsequent organ injury. Crit Care 15:R3
Angelin A, Gil-de-Gómez L, Dahiya S, Jiao J, Guo L, Levine MH, Wang Z, Quinn WJ, Kopinski PK, Wang L, Akimova T, Liu Y, Bhatti TR, Han R, Laskin BL, Baur JA, Blair IA, Wallace DC, Hancock WW, Beier UH (2017) Foxp3 reprograms T cell metabolism to function in low-glucose, high-lactate environments. Cell Metab 25:1282–1293.e7
Sitkovsky MV (2009) T regulatory cells: hypoxia-adenosinergic suppression and re-direction of the immune response. Trends Immunol 30:102–108
Hatfield SM, Kjaergaard J, Lukashev D, Schreiber TH, Belikoff B, Abbott R, Sethumadhavan S, Philbrook P, Ko K, Cannici R, Thayer M, Rodig S, Kutok JL, Jackson EK, Karger B, Podack ER, Ohta A, Sitkovsky MV (2015) Immunological mechanisms of the antitumor effects of supplemental oxygenation. Sci Transl Med 7:277ra30
Whiteside TL (2014) Induced regulatory T cells in inhibitory microenvironments created by cancer. Expert Opin Biol Ther 14:1411–1425
Beavis PA, Henderson MA, Giuffrida L, Mills JK, Sek K, Cross RS, Davenport AJ, John LB, Mardiana S, Slaney CY, Johnstone RW, Trapani JA, Stagg J, Loi S, Kats L, Gyorki D, Kershaw MH, Darcy PK (2017) Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy. J Clin Invest 127:929–941
Sitkovsky MV, Ohta A (2005) The ‘danger’ sensors that STOP the immune response: the A2 adenosine receptors? Trends Immunol 26:299–304
We thank members of the Powell lab, especially Chirag Patel, for critical discussion of the manuscript; Corvus pharmaceuticals for their generous gift of CPI-444; and Aduro Biotech for their generous gift of LM-OVA.
This work was supported in part by funds from the Bloomberg~Kimmel Institute for Cancer Immunotherapy. In addition, CPI-444 and unrestricted research funds were provided by Corvus.
Conflict of interest
Jonathan D. Powell has been a paid consultant for Corvus and has equity in the company. All other authors declare that they have no conflicts of interest.
Ethical approval and ethical standards
All applicable international and national guidelines for the care of animals were followed. All mouse procedures approved by Johns Hopkins University Institutional Animal Care and Use Committee (Protocol #M016M103, approved 4/1/2016).
C57BL/6 obtained from Charles River Laboratories (MC38 experiments) or Jackson Laboratories (ACT; 000664). OT-I and CD90.1, BALB/c mice obtained from The Jackson Laboratory. Male or female mice were used for each experiment; mice were sex and age matched accordingly.
Cell line authentication
MC38 cells were donated by CORVUS pharmaceuticals. The identity and specific pathogen free status of these cells was validated by microsatellite genotype analysis (IDEXX Bioresearch). B16-OVA melanoma cells were a gift from Hyam Levitsky. All other tumor cell lines used were obtained from the ATCC. All cell lines were mycoplasma free via ELISA-based assays performed every 6 months.
Some of the data contained herein had been presented as an abstract and oral presentation. Proceedings of the 107th Annual Meeting of the American Association for Cancer Research (AACR); 2016 April 16–20; New Orleans, LA. Cancer Res 2016; 76: (Abstract 4364).
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Leone, R.D., Sun, IM., Oh, MH. et al. Inhibition of the adenosine A2a receptor modulates expression of T cell coinhibitory receptors and improves effector function for enhanced checkpoint blockade and ACT in murine cancer models. Cancer Immunol Immunother 67, 1271–1284 (2018). https://doi.org/10.1007/s00262-018-2186-0
- Immune checkpoint