CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-CD100 (n = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

Biomedical Research Networking Centre Consortium of Oncology Instituto de Salud Carlos 3 Madrid Spain

CLIP Childhood Leukaemia Investigation Prague Department of Paediatric Haematology and Oncology Charles University Prague Czechia

Department of Biomedical Sciences University of Barcelona Barcelona Spain

Department of Immunology and Pathology Monash University and the Alfred Hospital Melbourne VIC Australia

Department of Immunology Erasmus MC University Medical Center Rotterdam Netherlands

Department of Medicine Cancer Research Centre Institute of Biomedical Research of Salamanca University of Salamanca Salamanca Spain

Department of Paediatric Haematology and Oncology University Hospital Motol Prague Czechia

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Fagerberg L, Jonasson K, von Heijne G, Uhlen M, Berglund L. Prediction of the human membrane proteome. Proteomics. (2010) 10:1141–9. 10.1002/pmic.200900258 PubMed DOI

Bausch-Fluck D, Goldmann U, Muller S, van Oostrum M, Muller M, Schubert OT, et al. . The in silico human surfaceome. Proc Natl Acad Sci USA. (2018) 115:E10988–97. 10.1073/pnas.1808790115 PubMed DOI PMC

Bausch-Fluck D, Hofmann A, Bock T, Frei AP, Cerciello F, Jacobs A, et al. . A mass spectrometric-derived cell surface protein atlas. PLoS ONE. (2015) 10:e0121314. 10.1371/journal.pone.0121314 PubMed DOI PMC

Diaz-Ramos MC, Engel P, Bastos R. Towards a comprehensive human cell-surface immunome database. Immunol Lett. (2011) 134:183–7. 10.1016/j.imlet.2010.09.016 PubMed DOI

Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. (1975) 256:495–7. 10.1038/256495a0 PubMed DOI

Julius MH, Masuda T, Herzenberg LA. Demonstration that antigen-binding cells are precursors of antibody-producing cells after purification with a fluorescence-activated cell sorter. Proc Natl Acad Sci USA. (1972) 69:1934–8. 10.1073/pnas.69.7.1934 PubMed DOI PMC

Clark G, Stockinger H, Balderas R, van Zelm MC, Zola H, Hart D, et al. . Nomenclature of CD molecules from the Tenth Human Leucocyte Differentiation Antigen Workshop. Clin Transl Immunol. (2016) 5:e57. 10.1038/cti.2015.38 PubMed DOI PMC

Engel P, Boumsell L, Balderas R, Bensussan A, Gattei V, Horejsi V, et al. . CD nomenclature 2015: human leukocyte differentiation antigen workshops as a driving force in immunology. J Immunol. (2015) 195:4555–63. 10.4049/jimmunol.1502033 PubMed DOI

Iwai Y, Hamanishi J, Chamoto K, Honjo T. Cancer immunotherapies targeting the PD-1 signaling pathway. J Biomed Sci. (2017) 24:26. 10.1186/s12929-017-0329-9 PubMed DOI PMC

Rosman Z, Shoenfeld Y, Zandman-Goddard G. Biologic therapy for autoimmune diseases: an update. BMC Med. (2013) 11:88. 10.1186/1741-7015-11-88 PubMed DOI PMC

Horvath C, Andrews L, Baumann A, Black L, Blanset D, Cavagnaro J, et al. . Storm forecasting: additional lessons from the CD28 superagonist TGN1412 trial. Nat Rev Immunol. (2012) 12:740; author reply 740. 10.1038/nri3192-c1 PubMed DOI

Barnkob MS, Simon C, Olsen LR. Characterizing the human hematopoietic CDome. Front Genet. (2014) 5:331. 10.3389/fgene.2014.00331 PubMed DOI PMC

Andersen MN, Al-Karradi SN, Kragstrup TW, Hokland M. Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages. Cytometry A. (2016) 89:1001–9. 10.1002/cyto.a.22995 PubMed DOI

Kalina T, Flores-Montero J, van der Velden VH, Martin-Ayuso M, Bottcher S, Ritgen M, et al. . EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia. (2012) 26:1986–2010. 10.1038/leu.2012.122 PubMed DOI PMC

Kalina T, Flores-Montero J, Lecrevisse Q, Pedreira CE, van der Velden VH, Novakova M, et al. . Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds. Cytometry A. (2015) 87:145–56. 10.1002/cyto.a.22581 PubMed DOI

R Development Core Team . R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria: (2014).

Caglar MU, Teufel AI, Wilke CO. Sicegar: R package for sigmoidal and double-sigmoidal curve fitting. PeerJ. (2018) 6:e4251. 10.7717/peerj.4251 PubMed DOI PMC

Christopoulos DT. A practical guide for using Unit Invariant Knee (UIK) as elbow point estimation in multivariate analysis. ResearchGate. (2014). 10.13140/RG.2.2.10201.31846 DOI

Murtagh F, Legendre P. Ward's hierarchical agglomerative clustering method: which algorithms implement ward's criterion? J Classif. (2014) 31:274–95. 10.1007/s00357-014-9161-z DOI

Bernard A, Dausset J, Milstein C, Schlossmann SF. editors. Leucocyte Typing: Human Leucocyte Differentiation Antigens Detected by Monoclonal Antibodies. New York, NY: Springer-Verlag; (1984).

Reinherz EL, Haynes BF, Nadler LM, Bernstein ID. editors. Leukocyte Typing II. New York, NY: Springer-Verlag; (1986). 10.1007/978-1-4612-4850-7 DOI

McMichael AJ, Beverley PCL, Cobbold S, Crumpton MJ, Gilks W, Gotch FM, et al. editors. Leucocyte Typing III: White Cell Differentiation Antigens. Oxford: Oxford University Press; (1987).

Knapp W, Dörken B, Gilks WR, Rieber EP, Schimdt RE, Stein H, et al. editors. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: Oxford University Press; (1989).

Schlossman SF, Boumsell L, Gilks W, Harlan JM, Kishimoto T, Morimoto C, et al. editors. Leucocyte Typing V: White Cell Differentiation Antigens. Oxford: Oxford University Press; (1995).

Clark GJ, Kupresanin F, Fromm PD, Ju X, Muusers L, Silveira PA, et al. . New insights into the phenotype of human dendritic cell populations. Clin Transl Immunol. (2016) 5:e61. 10.1038/cti.2015.40 PubMed DOI PMC

van der Burg M, Kalina T, Perez-Andres M, Vlkova M, Lopez-Granados E, Blanco E, et al. . The EuroFlow PID orientation tube for flow cytometric diagnostic screening of primary immunodeficiencies of the lymphoid system. Front Immunol. (2019) 10:246. 10.3389/fimmu.2019.00246 PubMed DOI PMC

van Zelm MC, Szczepanski T, van der Burg M, van Dongen JJ. Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion. J Exp Med. (2007) 204:645–55. 10.1084/jem.20060964 PubMed DOI PMC

Perez-Andres M, Paiva B, Nieto WG, Caraux A, Schmitz A, Almeida J, et al. . Human peripheral blood B-cell compartments: a crossroad in B-cell traffic. Cytometry B Clin Cytom. (2010) 78 (Suppl 1):S47–60. 10.1002/cyto.b.20547 PubMed DOI

Stepanek O, Kalina T, Draber P, Skopcova T, Svojgr K, Angelisova P, et al. . Regulation of Src family kinases involved in T cell receptor signaling by protein-tyrosine phosphatase CD148. J Biol Chem. (2011) 286:22101–12. 10.1074/jbc.M110.196733 PubMed DOI PMC

Dik WA, Pike-Overzet K, Weerkamp F, de Ridder D, de Haas EF, Baert MR, et al. . New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. J Exp Med. (2005) 201:1715–23. 10.1084/jem.20042524 PubMed DOI PMC

Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, et al. . Nomenclature of monocytes and dendritic cells in blood. Blood. (2010) 116:e74–80. 10.1182/blood-2010-02-258558 PubMed DOI

Ziegler-Heitbrock L, Hofer TP. Toward a refined definition of monocyte subsets. Front Immunol. (2013) 4:23. 10.3389/fimmu.2013.00023 PubMed DOI PMC

Clark GJ, Silveira PA, Hogarth PM, Hart DNJ. The cell surface phenotype of human dendritic cells. Semin Cell Dev Biol. (2018) 86:3–14. 10.1016/j.semcdb.2018.02.013 PubMed DOI

Piccioli D, Tavarini S, Borgogni E, Steri V, Nuti S, Sammicheli C, et al. . Functional specialization of human circulating CD16 and CD1c myeloid dendritic-cell subsets. Blood. (2007) 109:5371–9. 10.1182/blood-2006-08-038422 PubMed DOI

Hernandez-Campo PM, Almeida J, Sanchez ML, Malvezzi M, Orfao A. Normal patterns of expression of glycosylphosphatidylinositol-anchored proteins on different subsets of peripheral blood cells: a frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry B Clin Cytom. (2006) 70:71–81. 10.1002/cyto.b.20087 PubMed DOI

Larbi A, Fulop T. From “truly naive” to “exhausted senescent” T cells: when markers predict functionality. Cytometry A. (2014) 85:25–35. 10.1002/cyto.a.22351 PubMed DOI

Kared H, Martelli S, Ng TP, Pender SL, Larbi A. CD57 in human natural killer cells and T-lymphocytes. Cancer Immunol Immunother. (2016) 65:441–52. 10.1007/s00262-016-1803-z PubMed DOI PMC

Lugli E, Dominguez MH, Gattinoni L, Chattopadhyay PK, Bolton DL, Song K, et al. . Superior T memory stem cell persistence supports long-lived T cell memory. J Clin Invest. (2013) 123:594–9. 10.1172/JCI66327 PubMed DOI PMC

Mahnke YD, Brodie TM, Sallusto F, Roederer M, Lugli E. The who's who of T-cell differentiation: human memory T-cell subsets. Eur J Immunol. (2013) 43:2797–809. 10.1002/eji.201343751 PubMed DOI

Hamann D, Baars PA, Rep MH, Hooibrink B, Kerkhof-Garde SR, Klein MR, et al. . Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med. (1997) 186:1407–18. 10.1084/jem.186.9.1407 PubMed DOI PMC

Romero X, Benitez D, March S, Vilella R, Miralpeix M, Engel P. Differential expression of SAP and EAT-2-binding leukocyte cell-surface molecules CD84, CD150 (SLAM), CD229 (Ly9) and CD244 (2B4). Tissue Antigens. (2004) 64:132–44. 10.1111/j.1399-0039.2004.00247.x PubMed DOI

Res P, Blom B, Hori T, Weijer K, Spits H. Downregulation of CD1 marks acquisition of functional maturation of human thymocytes and defines a control point in late stages of human T cell development. J Exp Med. (1997) 185:141–51. 10.1084/jem.185.1.141 PubMed DOI PMC

Berkowska MA, Schickel JN, Grosserichter-Wagener C, de Ridder D, Ng YS, van Dongen JJ, et al. . Circulating human CD27-IgA+ memory B cells recognize bacteria with polyreactive Igs. J Immunol. (2015) 195:1417–26. 10.4049/jimmunol.1402708 PubMed DOI PMC

Jacobsen M, Schweer D, Ziegler A, Gaber R, Schock S, Schwinzer R, et al. . A point mutation in PTPRC is associated with the development of multiple sclerosis. Nat Genet. (2000) 26:495–9. 10.1038/82659 PubMed DOI

Lopez-Cabrera M, Santis AG, Fernandez-Ruiz E, Blacher R, Esch F, Sanchez-Mateos P, et al. . Molecular cloning, expression, and chromosomal localization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the C-type animal lectin superfamily of signal-transmitting receptors. J Exp Med.(1993) 178:537–47. 10.1084/jem.178.2.537 PubMed DOI PMC

Roederer M, Quaye L, Mangino M, Beddall MH, Mahnke Y, Chattopadhyay P, et al. . The genetic architecture of the human immune system: a bioresource for autoimmunity and disease pathogenesis. Cell. (2015) 161:387–403. 10.1016/j.cell.2015.02.046 PubMed DOI PMC

Cossarizza A, Chang HD, Radbruch A, Akdis M, Andra I, Annunziato F, et al. . Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol. (2017) 47:1584–797. 10.1002/eji.201646632 PubMed DOI PMC

Davis KA, Abrams B, Iyer SB, Hoffman RA, Bishop JE. Determination of CD4 antigen density on cells: role of antibody valency, avidity, clones, and conjugation. Cytometry. (1998) 33:197–205. 10.1002/(SICI)1097-0320(19981001)33:2<197::AID-CYTO14>3.0.CO;2-P PubMed DOI

Wang L, Abbasi F, Gaigalas AK, Hoffman RA, Flagler D, Marti GE. Discrepancy in measuring CD4 expression on T-lymphocytes using fluorescein conjugates in comparison with unimolar CD4-phycoerythrin conjugates. Cytometry B Clin Cytom. (2007) 72:442–9. 10.1002/cyto.b.20354 PubMed DOI

Sikorski K, Mehta A, Inngjerdingen M, Thakor F, Kling S, Kalina T, et al. . A high-throughput pipeline for validation of antibodies. Nat Methods. (2018) 15:909–12. 10.1038/s41592-018-0179-8 PubMed DOI

Uhlen M, Bandrowski A, Carr S, Edwards A, Ellenberg J, Lundberg E, et al. . A proposal for validation of antibodies. Nat Methods. (2016) 13:823–7. 10.1038/nmeth.3995 PubMed DOI PMC

Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative