[en] The activities, ontogeny, and mechanisms of lineage expansion of eosinophils are less well resolved than those of other immune cells, despite the use of biological therapies targeting the eosinophilia-promoting cytokine interleukin (IL)-5 or its receptor, IL-5Rα. We combined single-cell proteomics and transcriptomics and generated transgenic IL-5Rα reporter mice to revisit eosinophilopoiesis. We reconciled human and murine eosinophilopoiesis and provided extensive cell-surface immunophenotyping and transcriptomes at different stages along the continuum of eosinophil maturation. We used these resources to show that IL-5 promoted eosinophil-lineage expansion via transit amplification, while its deletion or neutralization did not compromise eosinophil maturation. Informed from our resources, we also showed that interferon response factor-8, considered an essential promoter of myelopoiesis, was not intrinsically required for eosinophilopoiesis. This work hence provides resources, methods, and insights for understanding eosinophil ontogeny, the effects of current precision therapeutics, and the regulation of eosinophil development and numbers in health and disease.
Research Center/Unit :
GIGA I3-Cellular and Molecular Immunology - ULiège
Disciplines :
Immunology & infectious disease
Author, co-author :
Jorssen, Joseph ✱; Université de Liège - ULiège > GIGA > GIGA I3 - Cellular and Molecular Immunology
Van Hulst, Glenn ✱; Université de Liège - ULiège > Département des sciences fonctionnelles (DSF) > Biochimie et biologie moléculaire
Mollers, Kiréna ; Université de Liège - ULiège > Département des sciences fonctionnelles (DSF)
Pujol, Julien ; Université de Liège - ULiège > GIGA > GIGA I3 - Cellular and Molecular Immunology
Petrellis, Georgios ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH) > FARAH: Santé publique vétérinaire
Baptista, Antonio P; Laboratory of Immunoregulation and Mucosal Immunology, VIB-UGent Center for Inflammation Research, Ghent, Belgium, Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium
Schetters, Sjoerd; Laboratory of Immunoregulation and Mucosal Immunology, VIB-UGent Center for Inflammation Research, Ghent, Belgium, Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium
Baron, Frédéric ; Université de Liège - ULiège > Département des sciences cliniques
Caers, Jo ; Université de Liège - ULiège > Département des sciences cliniques > Hématologie
Lambrecht, Bart N; Laboratory of Immunoregulation and Mucosal Immunology, VIB-UGent Center for Inflammation Research, Ghent, Belgium, Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium, Department of Pulmonary Medicine, Erasmus MC, Rotterdam, the Netherlands
Dewals, Benjamin G ; Université de Liège - ULiège > Département des maladies infectieuses et parasitaires (DMI) > Immunologie vétérinaire
F.R.S.-FNRS - Fonds de la Recherche Scientifique FWO - Fonds Wetenschappelijk Onderzoek Vlaanderen Leon Fredericq Foundation
Funding text :
J.J. was supported by a PhD fellowship and C.J.D. and F. Baron are senior
research associates of the F.R.S.-FNRS (Fonds de la Recherche
Scientifique-Fonds National de la Recherche Scientifique, Belgium). This
work was supported by the Fonds Wetenschappelijk Onderzoek – Vlaanderen
(FWO) and the F.R.S.-FNRS under EOS projects number 30565447 (U-HEAD)
and number G0H1222N (BENEFICIARIES), by a research project grant
(T.0052.18 REGEOS) and a research credit grant (35278176) of the F.R.S.-
FNRS, by the Le´ on Fredericq Foundation (Liege University), and by Liege Uni-
versity.
Klion, A.D., Ackerman, S.J., Bochner, B.S., Contributions of Eosinophils to Human Health and Disease. Annu. Rev. Pathol. 15 (2020), 179–209, 10.1146/annurev-pathmechdis-012419-032756.
Jacobsen, E.A., Jackson, D.J., Heffler, E., Mathur, S.K., Bredenoord, A.J., Pavord, I.D., Akuthota, P., Roufosse, F., Rothenberg, M.E., Eosinophil Knockout Humans: Uncovering the Role of Eosinophils Through Eosinophil-Directed Biological Therapies. Annu. Rev. Immunol. 39 (2021), 719–757, 10.1146/annurev-immunol-093019-125918.
Mitre, E., Klion, A.D., Eosinophils and helminth infection: protective or pathogenic?. Semin. Immunopathol. 43 (2021), 363–381, 10.1007/s00281-021-00870-z.
Ignacio, A., Shah, K., Bernier-Latmani, J., Köller, Y., Coakley, G., Moyat, M., Hamelin, R., Armand, F., Wong, N.C., Ramay, H., et al. Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization. Immunity 55 (2022), 1250–1267.e12, 10.1016/j.immuni.2022.05.014.
Mesnil, C., Raulier, S., Paulissen, G., Xiao, X., Birrell, M.A., Pirottin, D., Janss, T., Starkl, P., Ramery, E., Henket, M., et al. Lung-resident eosinophils represent a distinct regulatory eosinophil subset. J. Clin. Invest. 126 (2016), 3279–3295, 10.1172/JCI85664.
Bohrer, A.C., Castro, E., Hu, Z., Queiroz, A.T.L., Tocheny, C.E., Assmann, M., Sakai, S., Nelson, C., Baker, P.J., Ma, H., et al. Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice. J. Exp. Med., 218, 2021, e20210469, 10.1084/jem.20210469.
Poznanski, S.M., Mukherjee, M., Zhao, N., Huang, C., Radford, K., Ashkar, A.A., Nair, P., Asthma exacerbations on benralizumab are largely non-eosinophilic. Allergy 76 (2021), 375–379, 10.1111/all.14514.
Gurtner, A., Borrelli, C., Gonzalez-Perez, I., Bach, K., Acar, I.E., Núñez, N.G., Crepaz, D., Handler, K., Vu, V.P., Lafzi, A., et al. Active eosinophils regulate host defence and immune responses in colitis. Nature 615 (2023), 151–157, 10.1038/s41586-022-05628-7.
Brigger, D., Riether, C., van Brummelen, R., Mosher, K.I., Shiu, A., Ding, Z., Zbären, N., Gasser, P., Guntern, P., Yousef, H., et al. Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age. Nat. Metab. 2 (2020), 688–702, 10.1038/s42255-020-0228-3.
Grisaru-Tal, S., Rothenberg, M.E., Munitz, A., Eosinophil-lymphocyte interactions in the tumor microenvironment and cancer immunotherapy. Nat. Immunol. 23 (2022), 1309–1316, 10.1038/s41590-022-01291-2.
Iwasaki, H., Mizuno, S., Mayfield, R., Shigematsu, H., Arinobu, Y., Seed, B., Gurish, M.F., Takatsu, K., Akashi, K., Identification of eosinophil lineage–committed progenitors in the murine bone marrow. J. Exp. Med. 201 (2005), 1891–1897, 10.1084/jem.20050548.
Mori, Y., Iwasaki, H., Kohno, K., Yoshimoto, G., Kikushige, Y., Okeda, A., Uike, N., Niiro, H., Takenaka, K., Nagafuji, K., et al. Identification of the human eosinophil lineage-committed progenitor: revision of phenotypic definition of the human common myeloid progenitor. J. Exp. Med. 206 (2009), 183–193, 10.1084/jem.20081756.
Tusi, B.K., Wolock, S.L., Weinreb, C., Hwang, Y., Hidalgo, D., Zilionis, R., Waisman, A., Huh, J.R., Klein, A.M., Socolovsky, M., Population snapshots predict early haematopoietic and erythroid hierarchies. Nature 555 (2018), 54–60, 10.1038/nature25741.
Weinreb, C., Rodriguez-Fraticelli, A., Camargo, F.D., Klein, A.M., Lineage tracing on transcriptional landscapes links state to fate during differentiation. Science, 367, 2020, eaaw3381, 10.1126/science.aaw3381.
Jacobsen, S.E.W., Nerlov, C., Haematopoiesis in the era of advanced single-cell technologies. Nat. Cell Biol. 21 (2019), 2–8, 10.1038/s41556-018-0227-8.
Drissen, R., Buza-Vidas, N., Woll, P., Thongjuea, S., Gambardella, A., Giustacchini, A., Mancini, E., Zriwil, A., Lutteropp, M., Grover, A., et al. Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing. Nat. Immunol. 17 (2016), 666–676, 10.1038/ni.3412.
Drissen, R., Thongjuea, S., Theilgaard-Mönch, K., Nerlov, C., Identification of two distinct pathways of human myelopoiesis. Sci. Immunol., 4, 2019, eaau7148, 10.1126/sciimmunol.aau7148.
Mack, E.A., Pear, W.S., Transcription factor and cytokine regulation of eosinophil lineage commitment. Curr. Opin. Hematol. 27 (2020), 27–33, 10.1097/MOH.0000000000000552.
Dougan, M., Dranoff, G., Dougan, S.K., GM-CSF, IL-3, and IL-5 Family of Cytokines: Regulators of Inflammation. Immunity 50 (2019), 796–811, 10.1016/j.immuni.2019.03.022.
Foster, P.S., Hogan, S.P., Ramsay, A.J., Matthaei, K.I., Young, I.G., Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J. Exp. Med. 183 (1996), 195–201, 10.1084/jem.183.1.195.
Kopf, M., Brombacher, F., Hodgkin, P.D., Ramsay, A.J., Milbourne, E.A., Dai, W.J., Ovington, K.S., Behm, C.A., Köhler, G., Young, I.G., et al. IL-5-Deficient Mice Have a Developmental Defect in CD5+ B-1 Cells and Lack Eosinophilia but Have Normal Antibody and Cytotoxic T Cell Responses. Immunity 4 (1996), 15–24, 10.1016/S1074-7613(00)80294-0.
Jarick, K.J., Topczewska, P.M., Jakob, M.O., Yano, H., Arifuzzaman, M., Gao, X., Boulekou, S., Stokic-Trtica, V., Leclère, P.S., Preußer, A., et al. Non-redundant functions of group 2 innate lymphoid cells. Nature 611 (2022), 794–800, 10.1038/s41586-022-05395-5.
Roufosse, F., Targeting the Interleukin-5 Pathway for Treatment of Eosinophilic Conditions Other than Asthma. Front. Med. (Lausanne), 5, 2018, 49, 10.3389/fmed.2018.00049.
Pavord, I.D., Menzies-Gow, A., Buhl, R., Chanez, P., Dransfield, M., Lugogo, N., Keene, O.N., Bradford, E.S., Yancey, S.W., Clinical Development of Mepolizumab for the Treatment of Severe Eosinophilic Asthma: On the Path to Personalized Medicine. J. Allergy Clin. Immunol. Pract. 9 (2021), 1121–1132.e7, 10.1016/j.jaip.2020.08.039.
Howell, I., Howell, A., Pavord, I.D., Type 2 inflammation and biological therapies in asthma: Targeted medicine taking flight. J. Exp. Med., 220, 2023, e20221212, 10.1084/jem.20221212.
FitzGerald, J.M., Bleecker, E.R., Nair, P., Korn, S., Ohta, K., Lommatzsch, M., Ferguson, G.T., Busse, W.W., Barker, P., Sproule, S., et al. Benralizumab, an anti-interleukin-5 receptor α monoclonal antibody, as add-on treatment for patients with severe, uncontrolled, eosinophilic asthma (CALIMA): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet 388 (2016), 2128–2141, 10.1016/S0140-6736(16)31322-8.
Castro, M., Corren, J., Pavord, I.D., Maspero, J., Wenzel, S., Rabe, K.F., Busse, W.W., Ford, L., Sher, L., FitzGerald, J.M., et al. Dupilumab Efficacy and Safety in Moderate-to-Severe Uncontrolled Asthma. N. Engl. J. Med. 378 (2018), 2486–2496, 10.1056/NEJMoa1804092.
Diver, S., Khalfaoui, L., Emson, C., Wenzel, S.E., Menzies-Gow, A., Wechsler, M.E., Johnston, J., Molfino, N., Parnes, J.R., Megally, A., et al. Effect of tezepelumab on airway inflammatory cells, remodelling, and hyperresponsiveness in patients with moderate-to-severe uncontrolled asthma (CASCADE): a double-blind, randomised, placebo-controlled, phase 2 trial. Lancet Respir. Med. 9 (2021), 1299–1312, 10.1016/S2213-2600(21)00226-5.
Wechsler, M.E., Ruddy, M.K., Pavord, I.D., Israel, E., Rabe, K.F., Ford, L.B., Maspero, J.F., Abdulai, R.M., Hu, C.-C., Martincova, R., et al. Efficacy and Safety of Itepekimab in Patients with Moderate-to-Severe Asthma. N. Engl. J. Med. 385 (2021), 1656–1668, 10.1056/NEJMoa2024257.
Laurenti, E., Göttgens, B., From haematopoietic stem cells to complex differentiation landscapes. Nature 553 (2018), 418–426, 10.1038/nature25022.
Becht, E., McInnes, L., Healy, J., Dutertre, C.-A., Kwok, I.W.H., Ng, L.G., Ginhoux, F., Newell, E.W., Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. 37 (2018), 38–44, 10.1038/nbt.4314.
Giladi, A., Paul, F., Herzog, Y., Lubling, Y., Weiner, A., Yofe, I., Jaitin, D., Cabezas-Wallscheid, N., Dress, R., Ginhoux, F., et al. Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis. Nat. Cell Biol. 20 (2018), 836–846, 10.1038/s41556-018-0121-4.
Dahlin, J.S., Hamey, F.K., Pijuan-Sala, B., Shepherd, M., Lau, W.W.Y., Nestorowa, S., Weinreb, C., Wolock, S., Hannah, R., Diamanti, E., et al. A single-cell hematopoietic landscape resolves 8 lineage trajectories and defects in Kit mutant mice. Blood 131 (2018), e1–e11, 10.1182/blood-2017-12-821413.
Bettigole, S.E., Lis, R., Adoro, S., Lee, A.-H., Spencer, L.A., Weller, P.F., Glimcher, L.H., The transcription factor XBP1 is selectively required for eosinophil differentiation. Nat. Immunol. 16 (2015), 829–837, 10.1038/ni.3225.
Bedi, R., Du, J., Sharma, A.K., Gomes, I., Ackerman, S.J., Human C/EBP-ϵ activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation. Blood 113 (2009), 317–327, 10.1182/blood-2008-02-139741.
Milanovic, M., Terszowski, G., Struck, D., Liesenfeld, O., Carstanjen, D., IFN Consensus Sequence Binding Protein (Icsbp) Is Critical for Eosinophil Development1. J. Immunol. 181 (2008), 5045–5053, 10.4049/jimmunol.181.7.5045.
Kwok, I., Becht, E., Xia, Y., Ng, M., Teh, Y.C., Tan, L., Evrard, M., Li, J.L.Y., Tran, H.T.N., Tan, Y., et al. Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor. Immunity 53 (2020), 303–318.e5, 10.1016/j.immuni.2020.06.005.
Becht, E., Tolstrup, D., Dutertre, C.-A., Morawski, P.A., Campbell, D.J., Ginhoux, F., Newell, E.W., Gottardo, R., Headley, M.B., High-throughput single-cell quantification of hundreds of proteins using conventional flow cytometry and machine learning. Sci. Adv., 7, 2021, eabg0505, 10.1126/sciadv.abg0505.
Kotas, M.E., Dion, J., Van Dyken, S., Ricardo-Gonzalez, R.R., Danel, C.J., Taillé, C., Mouthon, L., Locksley, R.M., Terrier, B., A role for IL-33–activated ILC2s in eosinophilic vasculitis. JCI Insight, 6, 2021, e143366, 10.1172/jci.insight.143366.
Bouchery, T., Volpe, B., Shah, K., Lebon, L., Filbey, K., LeGros, G., Harris, N., The Study of Host Immune Responses Elicited by the Model Murine Hookworms Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Curr. Protoc. Mouse Biol. 7 (2017), 236–286, 10.1002/cpmo.34.
Rolot, M., Dougall, A.M., Chetty, A., Javaux, J., Chen, T., Xiao, X., Machiels, B., Selkirk, M.E., Maizels, R.M., Hokke, C., et al. Helminth-induced IL-4 expands bystander memory CD8+ T cells for early control of viral infection. Nat. Commun., 9, 2018, 4516, 10.1038/s41467-018-06978-5.
Fulkerson, P.C., Schollaert, K.L., Bouffi, C., Rothenberg, M.E., IL-5 Triggers a Cooperative Cytokine Network That Promotes Eosinophil Precursor Maturation. J. Immunol. 193 (2014), 4043–4052, 10.4049/jimmunol.1400732.
Menzies-Gow, A., Flood-Page, P., Sehmi, R., Burman, J., Hamid, Q., Robinson, D.S., Kay, A.B., Denburg, J., Anti-IL-5 (mepolizumab) therapy induces bone marrow eosinophil maturational arrest and decreases eosinophil progenitors in the bronchial mucosa of atopic asthmatics. J. Allergy Clin. Immunol. 111 (2003), 714–719, 10.1067/mai.2003.1382.
Yoshida, T., Ikuta, K., Sugaya, H., Maki, K., Takagi, M., Kanazawa, H., Sunaga, S., Kinashi, T., Yoshimura, K., Miyazaki, J., et al. Defective B-1 Cell Development and Impaired Immunity against Angiostrongylus cantonensis in IL-5Rα-Deficient Mice. Immunity 4 (1996), 483–494, 10.1016/S1074-7613(00)80414-8.
Wang, W., Xu, Y., Wang, L., Zhu, Z., Aodeng, S., Chen, H., Cai, M., Huang, Z., Han, J., Wang, L., et al. Single-cell profiling identifies mechanisms of inflammatory heterogeneity in chronic rhinosinusitis. Nat. Immunol. 23 (2022), 1484–1494, 10.1038/s41590-022-01312-0.
Grisaru-Tal, S., Dulberg, S., Beck, L., Zhang, C., Itan, M., Hediyeh-Zadeh, S., Caldwell, J., Rozenberg, P., Dolitzky, A., Avlas, S., et al. Metastasis-Entrained Eosinophils Enhance Lymphocyte-Mediated Antitumor Immunity. Cancer Res. 81 (2021), 5555–5571, 10.1158/0008-5472.CAN-21-0839.
Stacy, N.I., Ackerman, S.J., A tribute to eosinophils from a comparative and evolutionary perspective. J. Allergy Clin. Immunol. 147 (2021), 1115–1116, 10.1016/j.jaci.2020.12.002.
Lee, J.J., Jacobsen, E.A., Ochkur, S.I., McGarry, M.P., Condjella, R.M., Doyle, A.D., Luo, H., Zellner, K.R., Protheroe, C.A., Willetts, L., et al. Human versus mouse eosinophils: “that which we call an eosinophil, by any other name would stain as red.”. J. Allergy Clin. Immunol. 130 (2012), 572–584, 10.1016/j.jaci.2012.07.025.
Wright, A.K.A., Weston, C., Rana, B.M.J., Brightling, C.E., Cousins, D.J., Human group 2 innate lymphoid cells do not express the IL-5 receptor. J. Allergy Clin. Immunol. 140 (2017), 1430–1433.e4, 10.1016/j.jaci.2017.04.025.
Lommatzsch, M., Marchewski, H., Schwefel, G., Stoll, P., Virchow, J.C., Bratke, K., Benralizumab strongly reduces blood basophils in severe eosinophilic asthma. Clin. Exp. Allergy 50 (2020), 1267–1269, 10.1111/cea.13720.
Willebrand, R., Voehringer, D., Regulation of eosinophil development and survival. Curr. Opin. Hematol. 24 (2017), 9–15.
Van Hulst, G., Jorssen, J., Jacobs, N., Henket, M., Louis, R., Schleich, F., Bureau, F., Desmet, C.J., Anti-IL-5 mepolizumab minimally influences residual blood eosinophils in severe asthma. Eur. Respir. J., 59, 2022, 2100935, 10.1183/13993003.00935-2021.
Li, H., Natarajan, A., Ezike, J., Barrasa, M.I., Le, Y., Feder, Z.A., Yang, H., Ma, C., Markoulaki, S., Lodish, H.F., Rate of Progression through a Continuum of Transit-Amplifying Progenitor Cell States Regulates Blood Cell Production. Dev. Cell 49 (2019), 118–129.e7, 10.1016/j.devcel.2019.01.026.
Hérault, A., Binnewies, M., Leong, S., Calero-Nieto, F.J., Zhang, S.Y., Kang, Y.-A., Wang, X., Pietras, E.M., Chu, S.H., Barry-Holson, K., et al. Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis. Nature 544 (2017), 53–58, 10.1038/nature21693.
Schultze, J.L., Mass, E., Schlitzer, A., Emerging Principles in Myelopoiesis at Homeostasis and during Infection and Inflammation. Immunity 50 (2019), 288–301, 10.1016/j.immuni.2019.01.019.
Hysenaj, L., de Laval, B., Arce-Gorvel, V., Bosilkovski, M., González-Espinoza, G., Debroas, G., Sieweke, M.H., Sarrazin, S., Gorvel, J.-P., CD150-dependent hematopoietic stem cell sensing of Brucella instructs myeloid commitment. J. Exp. Med., 220, 2023, e20210567, 10.1084/jem.20210567.
Wang, S.-W., Herriges, M.J., Hurley, K., Kotton, D.N., Klein, A.M., CoSpar identifies early cell fate biases from single-cell transcriptomic and lineage information. Nat. Biotechnol. 40 (2022), 1066–1074, 10.1038/s41587-022-01209-1.
Van Hulst, G., Bureau, F., Desmet, C.J., Eosinophils as Drivers of Severe Eosinophilic Asthma: Endotypes or Plasticity?. Int. J. Mol. Sci., 22, 2021, 10150, 10.3390/ijms221810150.
Sichien, D., Scott, C.L., Martens, L., Vanderkerken, M., Van Gassen, S., Plantinga, M., Joeris, T., De Prijck, S., Vanhoutte, L., Vanheerswynghels, M., et al. IRF8 Transcription Factor Controls Survival and Function of Terminally Differentiated Conventional and Plasmacytoid Dendritic Cells, Respectively. Immunity 45 (2016), 626–640, 10.1016/j.immuni.2016.08.013.
Rosu, A., El Hachem, N., Rapino, F., Rouault-Pierre, K., Jorssen, J., Somja, J., Ramery, E., Thiry, M., Nguyen, L., Jacquemyn, M., et al. Loss of tRNA-modifying enzyme Elp3 activates a p53-dependent antitumor checkpoint in hematopoiesis. J. Exp. Med., 218, 2021, e20200662, 10.1084/jem.20200662.
Traag, V.A., Waltman, L., van Eck, N.J., From Louvain to Leiden: guaranteeing well-connected communities. Sci. Rep., 9, 2019, 5233, 10.1038/s41598-019-41695-z.
Street, K., Risso, D., Fletcher, R.B., Das, D., Ngai, J., Yosef, N., Purdom, E., Dudoit, S., Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics, 19, 2018, 477, 10.1186/s12864-018-4772-0.
Love, M.I., Huber, W., Anders, S., Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol., 15, 2014, 550, 10.1186/s13059-014-0550-8.
Ge, S.X., Jung, D., Yao, R., ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics 36 (2020), 2628–2629, 10.1093/bioinformatics/btz931.
Hao, Y., Hao, S., Andersen-Nissen, E., Mauck, W.M. III, Zheng, S., Butler, A., Lee, M.J., Wilk, A.J., Darby, C., Zager, M., et al. Integrated analysis of multimodal single-cell data. Cell 184 (2021), 3573–3587.e29, 10.1016/j.cell.2021.04.048.
