CD164 Monoclonal Antibodies That Block Hemopoietic Progenitor Cell Adhesion and Proliferation Interact with the First Mucin Domain of the CD164 Receptor This information is current as of September 28, 2021. Regis Doyonnas, James Yi-Hsin Chan, Lisa H. Butler, Irene Rappold, Jane E. Lee-Prudhoe, Andrew C. W. Zannettino, Paul J. Simmons, Hans-Jörg Bühring, Jean-Pierre Levesque and Suzanne M. Watt J Immunol 2000; 165:840-851; ; Downloaded from doi: 10.4049/jimmunol.165.2.840 http://www.jimmunol.org/content/165/2/840 http://www.jimmunol.org/ References This article cites 41 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/165/2/840.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD164 Monoclonal Antibodies That Block Hemopoietic Progenitor Cell Adhesion and Proliferation Interact with the First Mucin Domain of the CD164 Receptor1 Regis Doyonnas,* James Yi-Hsin Chan,* Lisa H. Butler,* Irene Rappold,* Jane E. Lee-Prudhoe,* Andrew C. W. Zannettino,† Paul J. Simmons,‡ Hans-Jo¨rg Bu¨hring,§ Jean-Pierre Levesque,‡ and Suzanne M. Watt2§ The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34؉ cell subsets in bone marrow and as a potent negative regulator of CD34؉ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble Downloaded from recombinant chimeric proteins encoded by exons 1–6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; http://www.jimmunol.org/ the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function. The Journal of Immunology, 2000, 165: 840–851. ecently, we have identified and cloned human CD164, a al., manuscript in preparation) and prevents the recruitment of novel 80- to 100-kDa type 1 transmembrane sialomucin CD34ϩCD38low/Ϫ cells into cycle in response to the cytokines, ϩ 3 R that is highly expressed on primitive CD34 hemopoietic IL-3, IL-6, stem cell factor (SCF), and G-CSF (1). All these re- by guest on September 28, 2021 progenitor cells (1–4) (J.Y.-H. Chan et al., manuscript in prepa- sults suggest that CD164 may act as a potent negative signaling ration). Analyses of transfectants expressing human CD164 have molecule for hemopoietic progenitor cell proliferation. The CD164 allowed the identification of at least four mAbs, 103B2/9E10, Ag, when analyzed with any of the four CD164 mAbs described 105A5, N6B6, and 67D2, that specifically recognize this sialomu- above, is expressed during ontogeny on CD34ϩ intra-aortic cell cin (1–4). Of these, the interaction of the 103B2/9E10 mAb with clusters in human wk 4–5 embryos and has been shown to be the CD164 receptor inhibits the adhesion of CD34ϩ cells to bone expressed on primitive human hemopoietic progenitors from fetal marrow stromal cells in vitro (1). Interestingly, similar interactions liver, cord blood, and bone marrow (4). The highest cell surface with the 103B2/9E10 or 105A5 mAbs inhibit the proliferation and expression of CD164 epitopes on these cells occurs on the more differentiation of primitive CD34ϩ erythroid and granulocyte- primitive subset of CD34ϩ cells (CD34high, AC133high, monocyte progenitors in colony forming assays (J.Y.-H. Chan et CD38low). It is also expressed on the vast majority of the linϪCD34low/ϪCD38low/Ϫ cells with the capacity for long term repopulation of hemopoiesis in an in vivo fetal sheep model (4, 5). *Medical Research Council Molecular Haematology Unit, Institute of Molecular CD164 expression is maintained at a lower level on the surface of Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom; †Hanson all the committed myeloid and erythroid progenitors, with low or ‡ Centre for Cancer Research, Adelaide, Australia; Stem Cell Laboratory, Peter Mac- negligible levels of expression on mature peripheral blood neutro- Callum Cancer Institute, Melbourne, Australia; and §Medizinische Universita¨tsklinik II, University of Tubingen, Tubingen, Germany phils and erythrocytes (4). In contrast to their common distribution Received for publication December 15, 1999. Accepted for publication April pattern on hemopoietic progenitor cells, the CD164 epitopes de- 25, 2000. fined by the 105A5 and 103B2/9E10 mAbs are differentially and The costs of publication of this article were defrayed in part by the payment of page often reciprocally expressed on lymphoid cells, endothelia, postcap- charges. This article must therefore be hereby marked advertisement in accordance illary high endothelial venules, and basal/subcapsular epithelia in he- with 18 U.S.C. Section 1734 solely to indicate this fact. mopoietic and nonhemopoietic tissues, while the N6B6 and 67D2 1 This work was supported by the United Kingdom Medical Research Council, the ϩ ϩ United Kingdom Leukaemia Research Fund, SmithKline Beecham, INTAS/RFBR, mAbs react with both the 103B2/9E10 and 105A5 cell subsets (4). E. U. Biotech. Framework 4, and Taiwan Government Grants (to R.D., J.Y.-H.C., Differential epitope expression has also been described for other L.H.B., I.R., J.L.-P., and S.M.W.), National Health and Medical Research Council of members of the sialomucin adhesion receptor family, to which Australia (to A.C.W.Z., P.J.S., and J.-P.L.), and Deutsche Forschungsgemeinschaft SFB 510, Project A1 (to H.J.B.). 2 Address correspondence and reprint requests to Dr. Suzanne M. Watt, Medical 3 Abbreviations used in this paper: SCF, stem cell factor; GAG, glycosamino- Research Council Molecular Hematology Unit, Institute of Molecular Medicine, The glycan; PCLP, podocalyxin-like protein; PSGL-1, P-selectin glycoprotein ligand-1; John Radcliffe Hospital, Headington, Oxford, United Kingdom OX3 9DS. E-mail MAdCAM-1, mucosal addressin cell adhesion molecule-1; GlyCAM-1, glycosyla- address: [email protected] tion-dependent cell adhesion molecule-1. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 841 CD164 belongs. The expanding family of sialomucin receptors Human bone marrow stromal cells, CD34ϩ cell isolation, and includes CD34, PCLP, PSGL-1 (CD162), CD45RA, MAdCAM,-1, cell lines Sgp200, GlyCAM-1, and CD43 (reviewed in Refs. 6–10). These molecules are expressed on hemopoietic progenitor cells and/or on All human cell samples were obtained with patient permission and with associated stromal, macrophage, T lymphoid, and/or endothelial ethical consent of the institutions or hospitals concerned. Human bone marrow stromal cells were prepared and stained with the CD164 mAbs cells in hemopoietic microenvironments, where they function in using the immunofluorescence technique previously described (2, 4). Hu- regulating hemopoiesis, leukocyte trafficking, inflammatory re- man CD34ϩ cells (Ͼ90% purity) were purified from fresh cord samples sponses, or T cell activation. They are all serine and threonine rich, provided by Prof. J. Hows (Southmead Hospital, Bristol, U.K.), using the allowing the potential for extensive O-linked glycosylation. They Miltenyi Biotech (Bergish Gladbach, Germany) miniMACS CD34 stem cell isolation kit (2). The human KG1a, KG1B, THP-1, U937, CEM, are either secreted or transmembrane molecules with the ability to RPMI-1788, TF1, and 293T and the mouse MS.5 cell lines were cultured extend well above the glycocalyx to promote ligand interactions. as previously described (2, 4). The diversity of these sialomucin receptors is further enhanced by alternative splicing and by cell-specific glycosyltransferase-medi-