4,759
Views
20
CrossRef citations to date
0
Altmetric
Original Research

T cells drive negative feedback mechanisms in cancer associated fibroblasts, promoting expression of co-inhibitory ligands, CD73 and IL-27 in non-small cell lung cancer

ORCID Icon, ORCID Icon, ORCID Icon, , ORCID Icon, , ORCID Icon, ORCID Icon, ORCID Icon, ORCID Icon, ORCID Icon & ORCID Icon show all
Article: 1940675 | Received 14 Mar 2021, Accepted 07 Jun 2021, Published online: 08 Jul 2021

References

  • Brown FD, Turley SJ. Fibroblastic reticular cells: organization and regulation of the T lymphocyte life cycle. J Immunol. 2015;194(4):1389–14. doi:10.4049/jimmunol.1402520.
  • Liu T, Han C, Wang S, Fang P, Ma Z, Xu L, Yin R. Cancer-associated fibroblasts: an emerging target of anti-cancer immunotherapy. J Hematol Oncol. 2019;12(1):86. doi:10.1186/s13045-019-0770-1.
  • Lakins MA, Ghorani E, Munir H, Martins CP, Shields JD. Cancer-associated fibroblasts induce antigen-specific deletion of CD8 + T Cells to protect tumour cells. Nat Commun. 2018;9(1):948. doi:10.1038/s41467-018-03347-0.
  • Costa A, Kieffer Y, Scholer-Dahirel A, Pelon F, Bourachot B, Cardon M, Sirven P, Magagna I, Fuhrmann L, Bernard C, et al. Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer. Cancer Cell. 2018;33:463–479 e410. doi:10.1016/j.ccell.2018.01.011.
  • Cremasco V, Astarita JL, Grauel AL, Keerthivasan S, MacIsaac K, Woodruff MC, Wu M, Spel L, Santoro S, Amoozgar Z, et al. FAP Delineates Heterogeneous and Functionally Divergent Stromal Cells in Immune-Excluded Breast Tumors. Cancer Immunol Res. 2018;6(12):1472–1485. doi:10.1158/2326-6066.CIR-18-0098.
  • Kraman M, Bambrough PJ, Arnold JN, Roberts EW, Magiera L, Jones JO, Gopinathan A, Tuveson DA, Fearon DT. Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha. Science. 2010;330(6005):827–830. doi:10.1126/science.1195300.
  • Phipps RP, Penney DP, Keng P, Quill H, Paxhia A, Derdak S, Felch ME. Characterization of two major populations of lung fibroblasts: distinguishing morphology and discordant display of Thy 1 and class II MHC. Am J Respir Cell Mol Biol. 1989;1(1):65–74. doi:10.1165/ajrcmb/1.1.65.
  • Yu M, Guo G, Huang L, Deng L, Chang C-S, Achyut BR, Canning M, Xu N, Arbab AS, Bollag RJ, et al. CD73 on cancer-associated fibroblasts enhanced by the A2B-mediated feedforward circuit enforces an immune checkpoint. Nat Commun. 2020;11(1):515. doi:10.1038/s41467-019-14060-x.
  • Thommen DS, Schreiner J, Muller P, Herzig P, Roller A, Belousov A, Umana P, Pisa P, Klein C, Bacac M, et al. Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors. Cancer Immunol Res. 2015;3(12):1344–1355. doi:10.1158/2326-6066.CIR-15-0097.
  • Simoni Y, Becht E, Fehlings M, Loh CY, Koo SL, Teng KWW, Yeong JPS, Nahar R, Zhang T, Kared H, et al. Bystander CD8(+) T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature. 2018;557:575–579. doi:10.1038/s41586-018-0130-2.
  • Canale FP, Ramello MC, Nunez N, Araujo Furlan CL, Bossio SN, Gorosito Serran M, Tosello Boari J, Del Castillo A, Ledesma M, Sedlik C, et al. CD39 Expression Defines Cell Exhaustion in Tumor-Infiltrating CD8 + T Cells. Cancer Res. 2018;78(1):115–128. doi:10.1158/0008-5472.CAN-16-2684.
  • Gupta PK, Godec J, Wolski D, Adland E, Yates K, Pauken KE, Cosgrove C, Ledderose C, Junger WG, Robson SC, et al. CD39 Expression Identifies Terminally Exhausted CD8+ T Cells. PLoS Pathog. 2015;11(10):e1005177. doi:10.1371/journal.ppat.1005177.
  • Ganesan A-P, Clarke J, Wood O, Garrido-Martin EM, Chee SJ, Mellows T, Samaniego-Castruita D, Singh D, Seumois G, Alzetani A, et al. Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer. Nat Immunol. 2017;18(8):940–950. doi:10.1038/ni.3775.
  • Thelen M, Lechner A, Wennhold K, von Bergwelt-Baildon M, Schlosser HA. CD39 Expression Defines Cell Exhaustion in Tumor-Infiltrating CD8 + T Cells—Letter. Cancer Res. 2018;78(17):5173–5174. doi:10.1158/0008-5472.CAN-18-0873.
  • van Duijn J, van Elsas M, Benne N, Depuydt M, Wezel A, Smeets H, Bot I, Jiskoot W, Kuiper J, Slutter B. CD39 identifies a microenvironment-specific anti-inflammatory CD8+ T-cell population in atherosclerotic lesions. Atherosclerosis. 2019;285:71–78. doi:10.1016/j.atherosclerosis.2019.04.217.
  • Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, Goodall CP, Blair TC, Fox BA, McDermott JE, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun. 2018;9(1):2724. doi:10.1038/s41467-018-05072-0.
  • Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen J-F, Enjyoji K, Linden J, Oukka M, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med. 2007;204(6):1257–1265. doi:10.1084/jem.20062512.
  • Raczkowski F, Rissiek A, Ricklefs I, Heiss K, Schumacher V, Wundenberg K, Haag F, Koch-Nolte F, Tolosa E, Mittrucker H-W. CD39 is upregulated during activation of mouse and human T cells and attenuates the immune response to Listeria monocytogenes. PLoS One. 2018;13(5):e0197151. doi:10.1371/journal.pone.0197151.
  • Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, Lieb DJ, Chen JH, Frederick DT, Barzily-Rokni M, et al. Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell. 2018;175(4):998–1013 e1020. doi:10.1016/j.cell.2018.10.038.
  • Parodi A, Battaglia F, Kalli F, Ferrera F, Conteduca G, Tardito S, Stringara S, Ivaldi F, Negrini S, Borgonovo G, et al. CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes. Cancer Immunol Immunother. 2013;62(5):851–862. doi:10.1007/s00262-013-1392-z.
  • Bastid J, Regairaz A, Bonnefoy N, Dejou C, Giustiniani J, Laheurte C, Cochaud S, Laprevotte E, Funck-Brentano E, Hemon P, et al. Inhibition of CD39 enzymatic function at the surface of tumor cells alleviates their immunosuppressive activity. Cancer Immunol Res. 2015;3(3):254–265. doi:10.1158/2326-6066.CIR-14-0018.
  • Gajewski TF, Schreiber H, Fu Y-X. Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol. 2013;14(10):1014–1022. doi:10.1038/ni.2703.
  • Borsellino G, Kleinewietfeld M, Di Mitri D, Sternjak A, Diamantini A, Giometto R, Hopner S, Centonze D, Bernardi G, Dell’Acqua ML, et al. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Blood. 2007;110(4):1225–1232. doi:10.1182/blood-2006-12-064527.
  • Gu J, Ni X, Pan X, Lu H, Lu Y, Zhao J, Guo Zheng S, Hippen KL, Wang X, Lu L. Human CD39hi regulatory T cells present stronger stability and function under inflammatory conditions. Cell Mol Immunol. 2017;14(6):521–528. doi:10.1038/cmi.2016.30.
  • Bai A, Moss A, Rothweiler S, Serena Longhi M, Wu Y, Junger WG, Robson SC. NADH oxidase-dependent CD39 expression by CD8+ T cells modulates interferon gamma responses via generation of adenosine. Nat Commun. 2015;6(1):8819. doi:10.1038/ncomms9819.
  • Park Y-J, Ryu H, Choi G, Kim B-S, Hwang ES, Kim HS, Chung Y. IL-27 confers a protumorigenic activity of regulatory T cells via CD39. Proc Natl Acad Sci U S A. 2019;116(8):3106–3111. doi:10.1073/pnas.1810254116.
  • Li J, Wang L, Chen X, Li L, Li Y, Ping Y, Huang L, Yue D, Zhang Z, Wang F, et al. CD39/CD73 upregulation on myeloid-derived suppressor cells via TGF-beta-mTOR-HIF-1 signaling in patients with non-small cell lung cancer. Oncoimmunology. 2017;6:e1320011. doi:10.1080/2162402X.2017.1320011.
  • Karakasheva TA, Lin EW, Tang Q, Qiao E, Waldron TJ, Soni M, Klein-Szanto AJ, Sahu V, Basu D, Ohashi S, et al. IL-6 Mediates Cross-Talk between Tumor Cells and Activated Fibroblasts in the Tumor Microenvironment. Cancer Res. 2018;78(17):4957–4970. doi:10.1158/0008-5472.CAN-17-2268.
  • Sun J, Dodd H, Moser EK, Sharma R, Braciale TJ. CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs. Nat Immunol. 2011;12(4):327–334. doi:10.1038/ni.1996.
  • Pot C, Jin H, Awasthi A, Liu SM, Lai C-Y, Madan R, Sharpe AH, Karp CL, Miaw S-C, Ho I-C, et al. Cutting edge: IL-27 induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells. J Immunol. 2009;183(2):797–801. doi:10.4049/jimmunol.0901233.
  • DeLong JH, O’Hara Hall A, Rausch M, Moodley D, Perry J, Park J, Phan AT, Beiting DP, Kedl RM, Hill JA, et al. IL-27 and TCR Stimulation Promote T Cell Expression of Multiple Inhibitory Receptors. Immunohorizons. 2019;3(1):13–25. doi:10.4049/immunohorizons.1800083.
  • Kündig TM, Bachmann MF, DiPaolo C, Simard JJ, Battegay M, Lother H, Gessner A, Kühlcke K, Ohashi PS, Hengartner H, et al. Fibroblasts as efficient antigen-presenting cells in lymphoid organs. Science. 1995;268(5215):1343–1347. doi:10.1126/science.7761853.
  • Feig C, Jones JO, Kraman M, Wells RJ, Deonarine A, Chan DS, Connell CM, Roberts EW, Zhao Q, Caballero OL, et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci U S A. 2013;110(50):20212–20217. doi:10.1073/pnas.1320318110.
  • Salmon H, Franciszkiewicz K, Damotte D, Dieu-Nosjean M-C, Validire P, Trautmann A, Mami-Chouaib F, Donnadieu E. Matrix architecture defines the preferential localization and migration of T cells into the stroma of human lung tumors. J Clin Invest. 2012;122(3):899–910. doi:10.1172/JCI45817.
  • Umetsu DT, Pober JS, Jabara HH, Fiers W, Yunis EJ, Burakoff SJ, Reiss CS, Geha RS. Human dermal fibroblasts present tetanus toxoid antigen to antigen-specific T cell clones. J Clin Invest. 1985;76(1):254–260. doi:10.1172/JCI111955.
  • Elyada E, Bolisetty M, Laise P, Flynn WF, Courtois ET, Burkhart RA, Teinor JA, Belleau P, Biffi G, Lucito MS, et al. Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts. Cancer Discov. 2019;9(8):1102–1123. doi:10.1158/2159-8290.CD-19-0094.
  • Clarke J, Panwar B, Madrigal A, Singh D, Gujar R, Wood O, Chee SJ, Eschweiler S, King EV, Awad AS, et al. Single-cell transcriptomic analysis of tissue-resident memory T cells in human lung cancer. J Exp Med. 2019;216(9):2128–2149. doi:10.1084/jem.20190249.
  • Thommen DS, Koelzer VH, Herzig P, Roller A, Trefny M, Dimeloe S, Kiialainen A, Hanhart J, Schill C, Hess C, et al. A transcriptionally and functionally distinct PD-1+ CD8+ T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade. Nat Med. 2018;24(7):994–1004. doi:10.1038/s41591-018-0057-z.
  • O’Brien SM, Klampatsa A, Thompson JC, Martinez MC, Hwang W-T, Rao AS, Standalick JE, Kim S, Cantu E, Litzky LA, et al. Function of Human Tumor-Infiltrating Lymphocytes in Early-Stage Non–Small Cell Lung Cancer. Cancer Immunol Res. 2019;7(6):896–909. doi:10.1158/2326-6066.CIR-18-0713.
  • Wang LC, Lo A, Scholler J, Sun J, Majumdar RS, Kapoor V, Antzis M, Cotner CE, Johnson LA, Durham AC, et al. Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity. Cancer Immunol Res. 2014;2:154–166. doi:10.1158/2326-6066.CIR-13-0027.
  • Gorchs L, Hellevik T, Bruun JA, Camilio KA, Al-Saad S, Stuge TB, Martinez-Zubiaurre I. Cancer-associated fibroblasts from lung tumors maintain their immunosuppressive abilities after high-dose irradiation. Front Oncol. 2015;5:87. doi:10.3389/fonc.2015.00087.
  • Nazareth MR, Broderick L, Simpson-Abelson MR, Kelleher RJ Jr., Yokota SJ, Bankert RB. Characterization of human lung tumor-associated fibroblasts and their ability to modulate the activation of tumor-associated T cells. J Immunol. 2007;178(9):5552–5562. doi:10.4049/jimmunol.178.9.5552.
  • Barnas JL, Simpson-Abelson MR, Brooks SP, Kelleher RJ Jr., Bankert RB. Reciprocal functional modulation of the activation of T lymphocytes and fibroblasts derived from human solid tumors. J Immunol. 2010;185(5):2681–2692. doi:10.4049/jimmunol.1000896.
  • Gorchs L, Fernandez Moro C, Bankhead P, Kern KP, Sadeak I, Meng Q, Rangelova E, Kaipe H. Human Pancreatic Carcinoma-Associated Fibroblasts Promote Expression of Co-inhibitory Markers on CD4+ and CD8+ T-Cells. Front Immunol. 2019;10:847. doi:10.3389/fimmu.2019.00847.
  • Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 2010;207(10):2187–2194. doi:10.1084/jem.20100643.
  • Mandai M, Hamanishi J, Abiko K, Matsumura N, Baba T, Konishi I. Dual Faces of IFNγ in Cancer Progression: a Role of PD-L1 Induction in the Determination of Pro- and Antitumor Immunity. Clin Cancer Res. 2016;22(10):2329–2334. doi:10.1158/1078-0432.CCR-16-0224.
  • Horton BL, Williams JB, Cabanov A, Spranger S, Gajewski TF. Intratumoral CD8 + T-cell Apoptosis Is a Major Component of T-cell Dysfunction and Impedes Antitumor Immunity. Cancer Immunol Res. 2018;6(1):14–24. doi:10.1158/2326-6066.CIR-17-0249.
  • AH D, CF N, DJ S. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of Activating Cytokines and Evidence for Independent Production the Journal of Immunology. 1988;141:2407–2412.
  • Sanceau J, Wijdenes J, Revel M, Wietzerbin J:IL-6. and IL-6 receptor modulation by IFN-gamma and tumor necrosis factor-alpha in human monocytic cell line (THP-1). Priming Effect of IFN-gamma J Immunol. 1991;147:2630–2637.
  • Ohmori Y, Schreiber RD, Hamilton TA. Synergy between Interferon-γ and Tumor Necrosis Factor-α in Transcriptional Activation Is Mediated by Cooperation between Signal Transducer and Activator of Transcription 1 and Nuclear Factor κB. J Biol Chem. 1997;272(23):14899–14907. doi:10.1074/jbc.272.23.14899.
  • Pechhold K, Patterson NB, Craighead N, Lee KP, June CH, Harlan DM. Inflammatory cytokines IFN-gamma plus TNF-alpha induce regulated expression of CD80 (B7-1) but not CD86 (B7-2) on murine fibroblasts. J Immunol. 2015;5:4921–4929.
  • Ju X, Zhang H, Zhou Z, Wang Q. Regulation of PD-L1 expression in cancer and clinical implications in immunotherapy. Am J Cancer Res. 2020;10:1–11.
  • Yoshida H, Hunter CA. The immunobiology of interleukin-27. Annu Rev Immunol. 2015;33(1):417–443. doi:10.1146/annurev-immunol-032414-112134.
  • Yoshimoto T, Morishima N, Mizoguchi I, Shimizu M, Nagai H, Oniki S, Oka M, Nishigori C, Mizuguchi J. Antiproliferative activity of IL-27 on melanoma. J Immunol. 2008;180(10):6527–6535. doi:10.4049/jimmunol.180.10.6527.
  • Cocco C, Giuliani N, Di Carlo E, Ognio E, Storti P, Abeltino M, Sorrentino C, Ponzoni M, Ribatti D, Airoldi I. Interleukin-27 acts as multifunctional antitumor agent in multiple myeloma. Clin Cancer Res. 2010;16(16):4188–4197. doi:10.1158/1078-0432.CCR-10-0173.
  • Morishima N, Owaki T, Asakawa M, Kamiya S, Mizuguchi J, Yoshimoto T. Augmentation of Effector CD8+ T Cell Generation with Enhanced Granzyme B Expression by IL-27. J Immunol. 2005;175(3):1686–1693. doi:10.4049/jimmunol.175.3.1686.
  • Schneider R, Yaneva T, Beauseigle D, El-Khoury L, Arbour N. IL-27 increases the proliferation and effector functions of human naïve CD8+ T lymphocytes and promotes their development into Tc1 cells. Eur J Immunol. 2011;41(1):47–59. doi:10.1002/eji.201040804.
  • Salcedo R, Stauffer JK, Lincoln E, Back TC, Hixon JA, Hahn C, Shafer-Weaver K, Malyguine A, Kastelein R, Wigginton JM. IL-27 Mediates Complete Regression of Orthotopic Primary and Metastatic Murine Neuroblastoma Tumors: role for CD8+ T Cells. J Immunol. 2004;173(12):7170–7182. doi:10.4049/jimmunol.173.12.7170.
  • Awasthi A, Carrier Y, Peron JP, Bettelli E, Kamanaka M, Flavell RA, Kuchroo VK, Oukka M, Weiner HL. A dominant function for interleukin 27 in generating interleukin 10–producing anti-inflammatory T cells. Nat Immunol. 2007;8(12):1380–1389. doi:10.1038/ni1541.
  • Hirahara K, Ghoreschi K, Yang X-P, Takahashi H, Laurence A, Vahedi G, Sciume G, Hall AO, Dupont CD, Francisco LM, et al. Interleukin-27 priming of T cells controls IL-17 production in trans via induction of the ligand PD-L1. Immunity. 2012;36(6):1017–1030. doi:10.1016/j.immuni.2012.03.024.
  • Zhu C, Sakuishi K, Xiao S, Sun Z, Zaghouani S, Gu G, Wang C, Tan DJ, Wu C, Rangachari M, et al. An IL-27/NFIL3 signalling axis drives Tim-3 and IL-10 expression and T-cell dysfunction. Nat Commun. 2015;6(1):6072. doi:10.1038/ncomms7072.
  • Huang Z, Zak J, Pratumchai I, Shaabani N, Vartabedian VF, Nguyen N, Wu T, Xiao C, Teijaro JR. IL-27 promotes the expansion of self-renewing CD8+ T cells in persistent viral infection. J Exp Med. 2019;216(8):1791–1808. doi:10.1084/jem.20190173.
  • Dong S, Zhang X, He Y, Xu F, Li D, Xu W, Wang H, Yin Y, Cao J. Synergy of IL-27 and TNF-α in Regulating CXCL10 Expression in Lung Fibroblasts. Am J Respir Cell Mol Biol. 2013;48(4):518–530. doi:10.1165/rcmb.2012-0340OC.
  • Wong CK, Chen DP, Tam LS, Li EK, Yin YB, Lam CW. Effects of inflammatory cytokine IL-27 on the activation of fibroblast-like synoviocytes in rheumatoid arthritis. Arthritis Res Ther. 2010;12(4):R129. doi:10.1186/ar3067.
  • Patel MV, Shen Z, Rossoll RM, Wira CR. IL-27 Expression and Responsiveness in Human Uterine Epithelial Cells and Fibroblasts In Vitro and the Role of Estradiol. J Interferon Cytokine Res. 2018;38(3):101–110. doi:10.1089/jir.2017.0038.
  • Cao W, Fang F, Gould T, Li X, Kim C, Gustafson C, Lambert S, Weyand CM, Goronzy JJ. Ecto-NTPDase CD39 is a negative checkpoint that inhibits follicular helper cell generation. J Clin Invest. 2020;130(7):3422–3436. doi:10.1172/JCI132417.
  • Fang F, Yu M, Cavanagh MM, Hutter Saunders J, Qi Q, Ye Z, Le Saux S, Sultan W, Turgano E, Dekker CL, et al. Expression of CD39 on Activated T Cells Impairs their Survival in Older Individuals. Cell Rep. 2016;14:1218–1231.
  • Young A, Ngiow SF, Barkauskas DS, Sult E, Hay C, Blake SJ, Huang Q, Liu J, Takeda K, Teng ML, et al. Co-inhibition of CD73 and A2AR Adenosine Signaling Improves Anti-tumor Immune Responses. Cancer Cell. 2016;30(3):391–403. doi:10.1016/j.ccell.2016.06.025.
  • Hilchey SP, Kobie JJ, Cochran MR, Secor-Socha S, Wang JC, Hyrien O, Burack WR, Mosmann TR, Quataert SA, Bernstein SH. Human Follicular Lymphoma CD39+-Infiltrating T Cells Contribute to Adenosine-Mediated T Cell Hyporesponsiveness. J Immunol. 2009;183(10):6157–6166. doi:10.4049/jimmunol.0900475.
  • Beavis PA, Henderson MA, Giuffrida L, Mills JK, Sek K, Cross RS, Davenport AJ, John LB, Mardiana S, Slaney CY, et al. Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy. J Clin Invest. 2017;127(3):929–941. doi:10.1172/JCI89455.
  • Mascanfroni ID, Takenaka MC, Yeste A, Patel B, Wu Y, Kenison JE, Siddiqui S, Basso AS, Otterbein LE, Pardoll DM, et al. Metabolic control of type 1 regulatory T cell differentiation by AHR and HIF1-α. Nat Med. 2015;21(6):638–646. doi:10.1038/nm.3868.