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`HEMATOPOIESIS
`
`CD99 expressed on human mobilized peripheral blood CD34⫹ cells is involved
`in transendothelial migration
`Anne-Marie Imbert, Ghania Belaaloui, Florence Bardin, Cecile Tonnelle, Marc Lopez, and Christian Chabannon
`
`Hematopoietic progenitor cell trafficking
`is an important phenomenon throughout
`life. It is thought to occur in sequential
`steps, similar to what has been described
`for mature leukocytes. Molecular actors
`have been identified for each step of
`leukocyte migration; recently, CD99 was
`shown to play a part during transendothe-
`lial migration. We explored the expres-
`sion and role of CD99 on human hemato-
`poietic progenitors. We demonstrate that
`(1) CD34ⴙ cells express CD99, albeit with
`Introduction
`
`various intensities; (2) subsets of CD34ⴙ
`cells with high or low levels of CD99
`expression produce different numbers of
`erythroid, natural killer (NK), or dendritic
`cells in the in vitro differentiation assays;
`(3) the level of CD99 expression is related
`to the ability to differentiate toward B cells;
`(4) CD34ⴙ cells that migrate through an
`endothelial monolayer in response to
`SDF-1␣ and SCF display the highest level
`of CD99 expression; (5) binding of a neu-
`tralizing antibody to CD99 partially inhib-
`
`its transendothelial migration of CD34ⴙ
`progenitors in an in vitro assay; and
`(6) binding of a neutralizing antibody to
`CD99 reduces homing of CD34ⴙ progeni-
`tors xenotransplanted in NOD-SCID mice.
`We conclude that expression of CD99 on
`human CD34ⴙ progenitors has functional
`significance and that CD99 may be in-
`volved in transendothelial migration of
`progenitors. (Blood. 2006;108:2578-2586)
`
`© 2006 by The American Society of Hematology
`
`Hematopoiesis is a tightly regulated process during which mature
`and functional cells that belong to different lineages are continu-
`ously renewed through differentiation from a small pool of
`ancestral cells termed “stem cells.” Throughout adult life, low
`numbers of stem and progenitor cells circulate in the peripheral
`blood. Myeloablative chemotherapy or administration of hemato-
`poietic growth factors lead to transient increases in the number of
`circulating stem cells to therapeutically useful values allowing
`collection with apheresis procedures, a process termed “mobiliza-
`tion.” On the other hand, these cells have the property to engraft to
`the bone marrow (BM) and to fully reconstitute the hematopoietic
`system, a process referred to as “homing.” Thus, mechanisms
`controlling stem cell motility (migration, homing, retention, and
`release) play a critical role in the regulation of hematopoiesis.
`Although the migration of leukocytes to the inflammatory sites
`is well documented, the different factors controlling the migration
`of hematopoietic stem cells are still poorly understood. Studies
`have provided evidence that the chemokine stromal cell-derived
`factor-1␣ (SDF-1␣; CXCL12) and its receptor CXCR4 are in-
`volved in the control of the migration of hematopoietic stem
`cells.1-5 Mice with gene inactivation at the CXCR4 or SDF-1␣ loci
`die shortly after birth, with severe defects in stem cell traffick-
`ing and failure to establish hematopoiesis to the BM.6-9 However,
`
`CXCR4⫺/⫺ fetal liver cells can successfully engraft in the BM of
`wild-type mice.10,11 These results suggest that human hematopoi-
`etic stem cell
`trafficking is only partially dependent on the
`SDF-1␣/CXCR4
`interactions
`and
`that
`other molecular
`mediators exist.
`Hematopoietic stem cell trafficking involves interactions with
`BM endothelial cells that
`line vessels and marrow sinusoids.
`Transmigration of hematopoietic progenitors through BM endothe-
`lium is thought to occur in sequential steps, similar to what has
`been described for leukocytes migrating to the inflammatory
`sites.12 The initial tethering and rolling steps mediated by selectins
`and their ligands result in the slowdown of hematopoietic cells that
`escape the bloodstream. Tight adhesion is mostly mediated by
`␤ integrins and immunoglobulin superfamily members.13,14 Trans-
`migration itself is the last step, and molecular actors involved at
`this stage have recently begun to be identified. The complexity of
`this process is exemplified by the description of transcytosis as an
`alternative to interendothelial cell transmigration.15,16
`Different molecules present at interendothelial junctions are
`candidates to be actors in transendothelial migration. Antibodies
`against some of these molecules modulate leukocyte extravasation.
`This is the case for CD31 (PECAM-1),17 CD99,18 Jam-A,19 and
`Jam-C.20 More recently, the role of CD155 (PVR), a member of the
`
`From the Centre de The´rapie Cellulaire et Ge´nique, Institut Paoli-Calmettes;
`Centre Re´gional de Lutte Contre le Cancer Provence-Alpes-Coˆte d’Azur; and
`Equipe d’He´matopoı¨e`se Mole´culaire et Fonctionnelle, Unite´ Mixte de
`Recherche (UMR) 599 Institut National de la Sante´ et de la Recherche
`Me´dicale (Inserm)–Centre Re´gional de Lutte contre le Cancer (CRLCC)–
`Universite´ de la Me´diterrane´e, Centre de Recherche en Cance´rologie de
`Marseille, France.
`
`Submitted December 1, 2005; accepted June 15, 2006. Prepublished online as
`Blood First Edition Paper, July 6, 2006; DOI 10.1182/blood-2005-12-010827.
`
`from the
`in part, by Institut Paoli-Calmettes and by a grant
`Supported,
`European Economic Community (EEC) (EUROCORD III program; C.C.). G.B.
`is the recipient of a grant from a joint research training program established by
`the French and Algerian governments.
`
`Presented, in part, at the First European School of Hematology (ESH)–
`European Group for Blood and Marrow Transplantation (EBMT)–
`EUROCORD Euroconference on stem cell research in Cascaı¨s, Portugal,
`April 15, 2005.61
`
`Reprints: Anne-Marie Imbert, Centre de The´rapie Cellulaire et Ge´nique,
`Institut Paoli-Calmettes, Centre Re´gional de Lutte Contre le Cancer Provence-
`Alpes-Coˆte d’Azur, 232, boulevard Sainte Marguerite, 13273 Marseille Cedex
`9, France; e-mail: imbertam@marseille.fnclcc.fr or thercell@marseille.fnclcc.fr.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
`
`© 2006 by The American Society of Hematology
`
`2578
`
`BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
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`BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
`
`CD99 EXPRESSION ON HUMAN CD34
`
`2579
`
`nectin family, and its ligand DNAM-1, in monocyte transmigration
`through human umbilical vein endothelial cells (HUVECs) has
`been evidenced, using either blocking monoclonal antibodies
`(mAbs) or soluble forms of these molecules.21,22 Among these
`molecules, a role in the transmigration of hematopoietic progeni-
`tors has only been documented for PECAM-1.19,20,22-25
`The role of CD99 in the migration of monocytes has recently
`been described18 and occurs downstream of PECAM-1 blockade.
`CD99 is a highly O-glycosylated type I transmembrane protein of
`32 kDa encoded by the MIC2 gene that has no apparent structural
`homology with most molecules involved in cell-to-cell interac-
`tions.26 CD99 is involved in multiple cellular events such as
`E-rosetting for T lymphocytes,27 T-cell adhesion,28-30 apoptosis of
`immature thymocytes31 and Ewing sarcoma cells in a caspase-
`independent way,32 up-regulation of T-cell receptor,33 and major
`histocompatibility complex molecules.34,35 The CD99 gene en-
`codes 2 different proteins produced by alternative splicing,36 the
`short form harboring a deletion in the cytoplasmic domain.
`Differential expression of these 2 forms is associated with distinct
`behaviors for phenomena such as CD99-induced cell adhesion or
`T-cell death.36,37 The short CD99 isoform is a negative regulator of
`neural differentiation38 and is also involved in the induction of
`motility and invasiveness of human breast cancer cells.39
`Although the expression of CD99 on hematopoietic cells,
`especially at
`the early stage of B-cell, T-cell, or neutrophil
`differentiation has been reported,40 little is known about CD99
`expression and function in the hematopoietic progenitor compart-
`ment. Here, we investigated the expression of CD99 on human
`CD34⫹ hematopoietic progenitors and its relation to their prolifera-
`tive and differentiation abilities. Further, we investigated the role of
`CD99 in trafficking of CD34⫹ cells, using an in vitro model of
`transendothelial migration and homing of xenotransplanted human
`CD34⫹ cells in NOD-SCID mice.
`
`Materials and methods
`
`Primary cells and cell lines
`
`Following informed consent in accordance with the Declaration of Hel-
`sinki, apheresis samples were obtained from patients with poor-prognosis
`cancers, who underwent mobilization with recombinant human granulocyte
`colony-stimulating factor (rhG-CSF; Neupogen, Amgen, Thousand Oaks,
`CA, or Granocyte, Chugai, Paris, France), alone or in association with
`chemotherapy. Approval for these studies was obtained from the Institut
`Paoli-Calmettes institutional review board (Comite d’Orientation Strate-
`gique). Mononuclear cells were enriched by density gradient separation
`over Lymphoprep (d ⫽ 1.077 g/mL; AbCys, Paris, France). CD34⫹ and
`CD14⫹cells were immunoselected, using the magnetic-activated cell sort-
`ing (MACS) technique (Miltenyi Biotec, Bergisch-Gladbach, Germany).
`The average CD34⫹ cell content of the enriched fractions determined by
`fluorescence-activated cell sorting (FACS) analysis with a PE-conjugated
`anti-CD34 mAb was 94.7% (⫾ 0.9%).
`Cord blood (CB) samples were obtained from mothers after they
`provided informed consent and had normal deliveries. BM samples were
`obtained from healthy donors undergoing marrow collection for allogeneic
`transplantation, following informed consent.
`The human bone marrow endothelial cell (HBMEC) line was a gift of B.
`Weksler (Cornell University, New York, NY). This SV-40 immortalized cell
`line is derived from adult human HBMECs and retains characteristics of
`primary cells.41 HBMECs were cultured in flasks coated with 0.2% gelatin
`(Sigma-Aldrich, St Louis, MO), in DMEM containing 1% glucose (Cam-
`brex, Verviers, Belgium) supplemented with 5% (vol/vol) heat-inactivated
`fetal calf serum (FCS; Invitrogen, Paisley, United Kingdom), 10 mM
`HEPES (Invitrogen), 3 mM glutamine (Invitrogen), 1 ␮g/mL folate
`
`(Sigma-Aldrich), 100 U/mL penicillin, and 100 ␮g/mL streptomycin
`(Invitrogen).
`Nalm6 cells and MS-5 cells were maintained in RPMI 1640 (Cambrex)
`or in DMEM (Cambrex), respectively, supplemented with 10% (vol/vol)
`heat-inactivated FCS, 100 U/mL penicillin, and 100 ␮g/mL streptomycin.
`Cells were incubated at 37°C in 5% CO2 in a humidified atmosphere.
`
`Antibodies and reagents
`
`Anti-CD99 mAbs clones 12E7 and O662 were a gift from A. Bernard
`(UMR 576 Inserm, Nice, France); the hec2 anti-CD99 mAb was a gift from
`W. A. Muller (Cornell University, New York, NY). Anti-CD31 (PECAM-1,
`clone CLB/HEC75) was purchased from Sanquin (Amsterdam, The Nether-
`lands). Anti-CD106 (VCAM-1, clone 1G11) was purchased from Beckman
`Coulter (Miami, FL). The anti-CD155 mAb (PVR, clone PV.404) was
`previously described.42 The following mAbs and their isotypic controls
`were purchased from Beckman Coulter: phycoerythrin (PE)–conjugated
`anti-CD1a, PE-conjugated anti-CD3, fluorescein isothiocyanate (FITC)–
`conjugated anti-CD10, PE-conjugated anti-CD14, FITC-conjugated anti-
`CD16, PE-conjugated anti-CD19, PE-conjugated anti-CD33, FITC-
`conjugated anti-CD36, PE-cyanin 5.1 (PC5)–conjugated anti-CD34,
`PE- and PC5-conjugated anti-CD45, FITC-conjugated anti-CD80, FITC-
`conjugated anti-CD83, PE-conjugated anti-CD111 (nectin1), PE-conju-
`gated anti-CD117 (c-Kit). Allophycocyanin (APC)–conjugated anti-CD3,
`PE-conjugated anti-CD34, PE-conjugated anti-CD56, PE-conjugated anti-
`CD71, FITC-conjugated anti-CD86, PE-conjugated anti-CDw90 (Thy1),
`FITC- and PE-conjugated anti-CD99, PE-conjugated anti-CD235a (glyco-
`phorin A [GPA]) were from Becton Dickinson (San Jose, CA); FITC-
`conjugated anti-CD105 and PE-conjugated anti-CD184 (CXCR4) were
`from R&D Systems (Minneapolis, MN).
`SDF-1␣, monocyte chemotactic protein-1 (MCP-1), IL-15, and TNF-␣
`were purchased from R&D Systems. SCF, IL-3, and IL-6 were a gift of
`Amgen. Other cytokines used in this study were granulocyte-macrophage
`colony-stimulating factor (GM-CSF; Leucomax, Novartis, Rueil-Malmai-
`son, France), G-CSF (Neupogen, Amgen), and erythropoietin (Epo; Eprex,
`Janssen-Cilag, Issy-les-Moulineaux, France).
`
`Flow cytometry studies and cell sorting
`
`Flow cytometry analyses were conducted with a FACSCalibur (Becton
`Dickinson Immunocytometry Systems [BDIS], San Jose, CA). Cells were
`incubated with direct-labeled antibodies for 30 minutes on ice. Isotype
`control antibodies were used to exclude false-positive cells. Dead cells were
`gated out by staining with Topro3 (Molecular Probes, Eugene, OR).
`Cell sorting was performed on a FACSAria flow cytometer (BDIS).
`Briefly, CD34⫹ cells were stained with a FITC-conjugated anti-CD99 and a
`PC5-conjugated anti-CD34 as described and sorted into CD34⫹/CD99low
`and CD34⫹/CD99high, each fraction representing approximately 20% of the
`total CD34⫹ cell population.
`
`Aldehyde dehydrogenase assay
`
`Intracellular levels of aldehyde dehydrogenase (ALDH) in CD34⫹ cells
`were assessed by staining with Aldefluor reagent (StemCell Technologies,
`Vancouver, BC, Canada). ALDH substrate (0.6 ␮g) was added to 106
`CD34⫹ cells suspended in 1 mL assay buffer. Then, 0.5 mL of these cells
`was immediately transferred to a control tube containing 5 ␮L 1.5 mM
`diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. Cells were
`then incubated for 30 minutes at 37°C to allow the conversion of the ALDH
`substrate into a fluorescent product. Cells were then costained with
`PE-conjugated anti-CD99 and PC5-conjugated anti-CD34 and analyzed by
`flow cytometry.
`
`RNA extraction and quantitative real-time polymerase chain
`reaction (RQ-PCR)
`
`Total RNA was isolated from 5 ⫻ 105 cells using an RNA extraction kit
`(Macherey-Nagel, Hoerdt, France). Total RNA was denatured at 65°C for
`10 minutes and reverse transcribed using Superscript II reverse transcrip-
`tase (Invitrogen).
`
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`2580
`
`IMBERT et al
`
`BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
`
`For detection of the 2 CD99 isoforms that differ by their intracytoplas-
`mic tail, 2 sets of previously published oligonucleotides38 were used, and
`real-time polymerase chain reaction (PCR) was carried out using the Light
`Cycler 2.0 instrument and the software version 4.0 (Roche Applied Science,
`Mannheim, Germany). The 20-␮L reaction mixture contained 4 ␮L of 5 ⫻
`Master Mix (Roche Applied Science), 0.5 ␮M of each primer, and 1 ␮L
`cDNA sample. After initial incubation at 95° for 10 minutes, 60 cycles were
`carried out (10 seconds at 95°C, 10 seconds at 60°C for the long form, or
`1 second at 60°C for the spliced variant and the control GAPDH, and 20
`seconds at 72°C). For quantification, a standard curve was obtained from
`serial dilutions of a cDNA control and the relative expressions of the 2
`isoforms were normalized to GAPDH expression.
`
`Differentiation assays
`
`Clonogenic cultures. Two hundred fifty CD34⫹/CD99high or CD34⫹/
`CD99low cells were seeded in triplicate in 0.5 mL semisolid medium
`(Methocult 4230, StemCell Technologies) supplemented with rhIL-3,
`rhIL-6, rhGM-CSF, rhG-CSF (10 ng/mL each), rhSCF (100 ng/mL),
`and rhEpo (2 U/mL). Colonies were scored after 14 days of culture at 37°C,
`5% CO2.43
`For high proliferative potential (HPP)–colony-forming cells (HPP-
`CFCs) enumeration, double-layer agarose (Caltag Laboratories, Burlin-
`game, CA) cultures supplemented with rhIL-3, rhIL-6, rhG-CSF, rhGM-
`CSF, rhSCF, and rhFlt3-L (20 ng/mL each) were seeded at 1000 cells/well,
`as previously described.44 Colonies were enumerated on day 21. HPP-CFCs
`were defined as progenitors giving rise to dense colonies more than 1 mm in
`diameter, with a dense core.
`Limiting dilution analysis of cobblestone area colony-forming cells
`(CAFCs). Monolayers of MS-5 stromal cell
`line were established in
`96-well plates and allowed to reach confluence. Serial dilutions of
`CD34⫹/CD99low or CD34⫹/CD99high cells were then seeded on the
`monolayers in IMDM medium supplemented with 10% FCS without any
`human recombinant cytokine. CAFCs were scored at week 5.45,46
`Erythroid cell differentiation assay. The technique was adapted from a
`previously published report.47 CD34⫹/CD99low or CD34⫹/CD99high cells
`(104/mL) were cultured in IMDM supplemented with 20% BIT (StemCell
`Technologies), 900 ng/mL ferrous sulfate, and 90 ng/mL ferric nitrate
`(Sigma-Aldrich). From days 0 to 8, 10⫺6 M hydrocortisone (Sigma-
`Aldrich), 100 ng/mL SCF, 5 ng/mL IL-3, and 3 U/mL Epo were added to the
`cultures. On day 4, cultures were diluted in 4 volumes of the same medium.
`On day 9, cells were suspended at 3 ⫻ 105 cells/mL and cocultured on
`MS-5 layer in medium supplemented with Epo. Analysis of specific
`markers was performed at day 12.
`B- and NK cell differentiation assays. Layers were established in
`96-well plates with the murine stromal cell line MS-5. Then, 10 000
`CD34⫹/CD99low or CD34⫹/CD99high cells were added to the layers. B-cell
`differentiation was assessed after 5 weeks of culture without any human
`recombinant cytokine in IMDM supplemented with 5% FCS and 5% human
`AB serum (Institut Jacques Boy, Reims, France). In separate experiments,
`20 ng/mL IL-15 and 50 ng/mL SCF were added to the cultures to favor NK
`cell differentiation in IMDM supplemented with 15% FCS. Specific
`markers were analyzed after 5 weeks.
`Dendritic cell differentiation assays. CD34⫹/CD99low or CD34⫹/
`CD99high cells (10 000/mL) were cultured in 24-well plates in RPMI 1640
`medium (Cambrex) supplemented with 10% FCS, 20 ng/mL SCF, 100
`ng/mL GM-CSF, 2.5 ng/mL TNF-␣, and 20 ng/mL Flt3-L. At day 7,
`cultures were diluted in the same medium at the concentration of 5 ⫻ 105
`cells/mL. Specific markers were analyzed after 14 days.
`Cell cycle analyses. The percentages of cells in G0/G1, S, and G2/M
`phases were evaluated by staining living cells with propidium iodide. A
`total of 5 ⫻ 104 to 105 cells were fixed in 70% ethyl alcohol (Carlo Erba
`Reagenti, Milan, Italy) for 15 minutes, then treated with 200 to 300 ␮L of a
`10-␮g/mL DNase-free RNase solution (Roche Diagnostics, Indianapolis,
`IN) for 15 minutes at 37°C. An equal volume of a 50-␮g/mL propidium
`iodide solution was added, and cells were incubated for 1 hour at 37°C
`before flow cytometry analysis.46
`Detection of apoptotic cells. Apoptosis was determined by monitoring
`phosphatidyl serine exposure at the outer cell membrane, using an assay
`
`based on binding of annexin V-Cy5 (Molecular Probes); Sytox was used as
`a viability probe. Apoptosis was evaluated after 5 hours in the absence of
`cytokines and after 24 hours of culture in a medium supplemented with
`IL-3, IL-6 (10 ng/mL each), and SCF (100 ng/mL).
`
`Adhesion assay
`
`Thirty-thousand HBMECs were established in 96-well plates and allowed
`to grow to confluence for 72 hours; confluent cells were incubated for
`1 hour at 37°C with the relevant blocking mAbs. Simultaneously, mobilized
`peripheral blood (mPB) CD34⫹ cells in IMDM (Cambrex) supplemented
`with 20% BIT (StemCell Technologies) were incubated with various mAbs at
`4°C. An anti-CD34 mAb was used as control. Five to 10 ⫻ 104 mAb-treated
`CD34⫹ progenitors were cocultured with mAb-treated HBMECs for 1 hour at
`37°C. Wells were then filled with culture medium, covered with a plastic film,
`placed upside down, and centrifuged at 50 g for 5 minutes to eliminate
`nonadherent cells. Adherent cells were then detached with 5 mM EDTA,
`incubated with an anti-CD45 mAb (to exclude HBMECs), and the addition of a
`known number of calcein-AM–labeled (Molecular Probes) Nalm6 cells allowed
`for the estimation of their number by flow cytometry.
`
`Transendothelial migration studies
`
`Migration assays were performed in transwell culture inserts (Costar,
`Cambridge, MA) of 6.5 mm diameter and 5-␮m pore filters, coated with
`0.2% gelatin (Sigma-Aldrich). HBMECs were plated at 30 000 cells/
`transwell and allowed to grow to confluence for 72 hours. Before migration
`assays, the monolayers were washed once with assay medium (IMDM
`supplemented with 20% BIT). Then, 100 000 CD34⫹ cells or 500 000
`mononuclear cells in 0.1 mL were seeded in the upper compartment, and
`0.6 mL of assay medium was added to the lower chamber. In blocking
`experiments, the hematopoietic and the endothelial cells were preincubated
`with mAbs (20 ␮g/mL) for 30 minutes at 37°C for the layer or at 4°C for the
`hematopoietic cells. The mAbs were present during the migration assay.
`Cells were allowed to migrate for 4 hours in response to SDF-1␣
`(100 ng/mL) for CD34⫹ cells or to MCP-1 (200 ng/mL) for monocytes. In
`some experiments, SCF (100 ng/mL) was added to SDF-1␣. Before
`collecting the transmigrated cells, a fixed number of control Nalm6 cells
`labeled with calcein-AM was added to the lower compartment; cells were
`then collected and labeled with a PE-conjugated anti-CD45 mAb (for
`CD34⫹ cells) or a PE-conjugated anti-CD14 (for mononuclear cells) to
`eliminate contaminating HBMECs. After FACS analysis, the ratio between
`PE-labeled cells and calcein-labeled Nalm6 cells allowed us to determine
`the number of cells that had migrated from the upper to the lower chamber.
`
`Homing of CD34ⴙ progenitors in NOD-SCID animals
`
`The model has been adapted from a previously published report.48 Briefly,
`NOD-SCID animals were bred in our animal facility under germ-free
`conditions. Following sublethal total body irradiation at 350 cGy, 2 ⫻ 106
`human mPB CD34⫹ progenitors were injected in the tail vein after
`preincubation with Immu133 (anti-CD34 mAb), 12E7 (anti-CD99 mAb), or
`IgG1 (20 ␮g/mL). Animals were killed 18 to 24 hours after injection. BM
`cells were collected from femurs and tibias. Mononuclear cells were
`separated by density gradient and analyzed for the presence of human cells
`by using a human-specific anti-CD45 mAb; cells from nontransplanted
`mice were used as negative controls; human mononuclear cells were used
`as positive controls to establish the threshold for CD45 positivity; dead cells
`were excluded with Topro3 staining. Homing efficiency was determined by
`the percentage of CD45⫹ (human) cells among total number of recipient
`mononuclear BM cells. Only experiments in which at least 0.1% human
`cells were detectable in the BM of all control animals were kept for further
`analyses and interpretation.
`
`Statistical analyses
`
`Data are presented as the mean ⫾ SEM. Differences were tested by using the
`nonparametric Wilcoxon signed rank test for paired observations (differentiation
`assays) or the Mann-Whitney test in case of independent populations (migration
`experiments). P values below .05 were considered significant.
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`BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
`
`CD99 EXPRESSION ON HUMAN CD34
`
`2581
`
`fluorescence intensity (MFI) for CD99, each of these 2 subsets
`representing approximately 20% of the total CD34⫹ cell popula-
`tion. We next studied the relative expression of CD99 and several
`antigens within the CD34⫹ cell population. As shown in Figure 2A,
`the higher expression level for CD99 is associated with a lower
`level of expression of CD38 and nectin1, and on the other hand,
`with a higher level of expression of CD90 (Thy1), CD105 (a
`component of the TGF-␤ receptor), and CD133 (AC133), suggest-
`ing that the CD34⫹/CD99high subset is enriched with the most
`immature progenitors. Only rare cells appeared to be ABCG2⫹, an
`ABC transporter expressed on early hematopoietic progenitors49;
`these ABCG2⫹ cells expressed CD99 at higher levels than the
`majority of CD34⫹ cells. The coexpression of CD99 with c-Kit and
`CXCR4, the receptors of the chemoattractants SCF and SDF-1␣,
`respectively, used in this study, indicates that c-Kit and CXCR4
`expression is higher in the CD34⫹/CD99low subset. Finally, the
`Aldefluor assay demonstrated that the CD34⫹/CD99high cell subset
`possessed higher levels (4 ⫾ 0.9-fold, n ⫽ 4) of ALDH activity
`than its CD34⫹/CD99low counterpart; a representative experiment
`is shown in Figure 2B.
`
`Clonogenic progenitors and CAFCs are present at different
`frequencies in the mPB CD34ⴙ/CD99high and CD34ⴙ/CD99low
`cell subsets
`
`To further explore whether the level of CD99 expression is
`associated with different functional abilities within CD34⫹ cells,
`we sorted the CD34⫹/CD99low and CD34⫹/CD99high cell popula-
`tions (Figure 1B).
`As shown in Figure 3A-B, the CD34⫹/CD99high subset contains
`the majority of granulocyte-macrophage colony-forming units
`(CFU-GMs) and HPP-CFCs, whereas erythroid clonogenic progeni-
`tors are mostly present in the CD34⫹/CD99low subset. The fre-
`quency of more immature clonogenic progenitors (granulocyte,
`erythrocyte, megakaryocyte, macrophage colony-forming units
`
`0.76 ± 0.08
`
`1.34 ± 0.14
`
`1.84 ± 0.35
`
`0.53 ± 0.07
`
`Nectin 1
`
`ABCG2
`
`0.66 ± 0.12
`
`CD105
`
`CXCR4
`
`CD99
`
`2.91 ± 0.04
`
`Thy1
`
`AC133
`
`0.47 ± 0.04
`
`lortnoc BAED
`
`sllec +43DC
`
`99DC+43DC
`
`i
`
`ii
`
`low cells
`iii
`
`99DC+43DC
`
`high cells
`iv
`
`A
`
`CD38
`
`cKit
`
`SSCB
`
`ALDH activity
`
`Figure 2. Coexpression of different markers with CD99 within the mPB CD34ⴙ
`cell subset. (A) CD99 expression levels increase simultaneously with Thy1, CD105,
`and AC133. Conversely, levels of CD38, nectin1 (CD111), c-Kit, and CXCR4 tend to
`decrease, whereas levels of CD99 increase. The rare ABCG2⫹ cells express high
`levels of CD99. For each antigen, values in the dot plots represent the ratio of MFI for
`this specific marker in the CD34⫹/CD99high population versus the CD34⫹/CD99low
`population, as defined in Figure 1B (data are mean ⫾ SEM of 9 experiments). Due to
`the low percentage of ABCG2⫹ cells, this value was not computed for this marker. (B)
`A representative experiment showing detection of ALDH activity in total CD34⫹ cells
`and gated CD34⫹/CD99low and CD34⫹/CD99high cells. (i) Aldefluor-stained CD34⫹
`cells incubated with DEAB, a specific ALDH inhibitor; (ii) total CD34⫹ cells incubated
`with Aldefluor, analysis of Aldefluor-positive cells gated on (iii) CD34⫹/CD99low cells
`and (iv) CD34⫹/CD99high cells.
`
`T1
`T2
`GAPDH
`
`T1
`T2
`GAPDH
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`HBMEC
`HBMEC
`
`mPB CD34+
`monocytes
`monocytes mPB CD34+
`
`BM CD34+
`BM CD34+
`
`CB CD34+
`CB CD34+
`
`CD99low
`
`CD99high
`
`A
`
`CD99+ cells (%)
`
`B
`
`CD34 PC5
`
`CD99 FITC
`
`Figure 1. Expression of CD99. (A) Expression of CD99 on the HBMEC line, on
`human CD14⫹ mobilized peripheral blood (mPB) monocytes, and on mPB CD34⫹
`cells (n ⫽ 14), on bone marrow CD34⫹ cells (BM, n ⫽ 5) and cord blood CD34⫹ cells
`(CB, n ⫽ 4). Error bars represent SEM. The detection of the 2 isoforms, T1 (the
`full-length form) and T2 (the truncated form), shows the relatively low level of the T2
`isoform in mPB CD34⫹ progenitors when compared with mPB CD14⫹ monocytes (a
`representative illustration of gel electrophoresis of RQ-PCR products for CD14⫹ and
`CD34⫹ cells immunoselected from the same apheresis sample). (B) Representative
`flow cytometric detection of CD34 and CD99 expression on immunoselected CD34⫹
`progenitors obtained from an adult patient undergoing apheresis.
`
`Results
`
`CD99 is expressed at varying levels on CD34ⴙ cells
`from different sources
`
`Most CD34⫹ cells appear to express CD99, although the level of
`expression varies depending on cell source: 76.5% ⫾ 8.5% (n ⫽ 5)
`of BM CD34⫹ cells express CD99, 61.9% ⫾ 6.9% (n ⫽ 14) of
`adult mPB CD34⫹ progenitors express CD99, and 38% ⫾ 14.5%
`(n ⫽ 4) of CB CD34⫹ cells express CD99 (n ⫽ 4; Figure 1A). As
`already described, CD99 is also expressed on monocytes.18 The
`HBMEC line expresses CD99, similar to what has been shown with
`HUVECs18; pretreatment of HBMECs with TNF-␣ did not change
`the level of CD99 expression (data not shown).
`Because 2 isoforms of CD99 that differ by their intracytoplas-
`mic tail have been described, we used RQ-PCR and specific
`oligonucleotides38 to study their relative expression in human
`monocytes and CD34⫹ progenitors. Figure 1A shows a representa-
`tive illustration, where the short and truncated (T2) isoform
`expressed in monocytes is barely detectable in CD34⫹ cells; this
`result was reproduced in 7 paired experiments with monocytes and
`progenitors from the same individuals.
`The expression pattern of CD99 within the CD34⫹ mPB cells
`does not discriminate a clearly positive population but suggests a
`continuum of expression (Figure 1B), the highest expression of
`CD99 being associated with the highest expression of CD34. In
`further experiments, we defined CD34⫹/CD99low and CD34⫹/
`CD99high cells as CD34⫹ cells with the lowest and highest mean
`
`Sanquin EX2005
`Forty Seven v. Stichting Sanquin Bloedvoorziening
`IPR2016-01529
`
`

`
`From
`
`by guest
`www.bloodjournal.org
`
`
`on October 26, 2016.
`
`For personal use only.
`
`2582
`
`IMBERT et al
`
`BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
`
`Figure 3. Detection of clonogenic progenitors. (A-B) Detection of
`clonogenic progenitors from CD34⫹/CD99low (䡺) and CD34⫹/CD99high (f)
`cells sorted from different apheresis samples (n ⫽ 6). (A) Detection of
`committed progenitors (BFU-Es and CFU-GMs). (B) Detection of imma-
`ture clonogenic progenitors (CFU-GEMMs and HPP-CFCs). (A-B) Error
`bars represent SEM. (C) Scoring of CAFCs in limiting dilution analyses of
`CD34⫹/CD99low (䡺) and CD34⫹/CD99high (⽧) cells (n ⫽ 3). Data are from
`a representative experiment.
`
`5
`10
`Input cells at day 0 (x 103)
`
`15
`
`100
`
`10
`
`% negative wellsC
`
`1
`
`0
`
`0.50
`
`0.45
`
`0.40
`
`0.35
`
`0.30
`
`0.25
`
`0.20
`
`0.15
`
`0.10
`
`0.05
`
`0.00
`
`Clonogenicity (%)B
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`A
`
`Clonogenicity (%)
`
`CFU-GM
`
`BFU-E
`
`CFU-GEMM HPP-CFC
`
`[CFU-GEMMs] and HPP-CFCs) is significantly higher in the
`CD34⫹/CD99high subset. Figure 3C provides an estimate of CAFC
`frequency in the 2 tested cell subpopulations; CAFCs are mostly
`present within the CD34⫹/CD99high subset (approximately 1/3,000
`cells versus 1/50 000 for CD34⫹/CD99low cells).
`
`mPB CD34ⴙ/CD99high and CD34ⴙ/CD99low cells respond
`differently to cytokine combinations that induce erythroid, NK,
`B, or dendritic cell differentiation in the in vitro cultures
`
`In 3 of the 4 tested differentiation conditions, CD34⫹/CD99high and
`CD34⫹/CD99low cells differed only by their expansion ability;
`differentiated cells shared a similar phenotype. When cultured with
`cytokine conditions that favor erythroid differentiation, CD34⫹/
`CD99low cells produced higher numbers of CD36⫹, CD71⫹, or
`GPA⫹ cells than their CD34⫹/CD99high counterparts (Figure 4A).
`Conversely, CD34⫹/CD99high cells expanded to a higher degree
`toward the NK and dendritic lineages as indicated by the increased
`number of cells expressing CD16 and CD56 (NK lineage, Figure
`4B), or CD1a, CD80, CD83, and CD86 (dendritic lineage, Figure
`4C), leading to a greater output of differentiated cells. By contrast,
`coculture with the MS-5 cell line demonstrated that B-cell progeni-
`in the CD34⫹/CD99high cell subset,
`tors were mostly present
`suggesting a difference in differentiation potential in addition to
`different proliferative abilities (Figure 4D).
`These differences cannot be accounted for by a difference in cell
`cycling activity; both cell subsets had low and comparable
`
`percentages of cells in G2/M phase, immediately after cell sorting
`(0.6% ⫾ 0.09%; n ⫽ 4).
`
`A blocking mAb to CD99 inhibits the transmigration of human
`mPB CD34ⴙ progenitors through HBMECs in response
`to SDF-1␣ with or without SCF
`
`Before testing different anti-CD99 mAbs for their ability to
`interfere in hematopoietic progenitor–endothelial cell interactions,
`we checked whether they induced apoptosis of mPB CD34⫹ cells,
`because induction of apoptosis in T cells was described with the
`O662 mAb.50 Figure 5 shows that none of the 3 12E7, O662, and
`Hec2 mAbs induced apoptosis after 5 hours (in conditions mimick-
`ing those used for transmigration assays); the 12E7 mAb appar-
`ently protects CD34⫹ cells from apoptosis, because the percentage
`of annexin V⫹ cells tended to be lower than in the control
`conditions. After 24 hours in serum-free medium with IL-3, IL-6,
`and SCF, an approximately 3-fold increase in annexin V⫹ cells
`was detected when mPB CD34⫹ cells were exposed to the O662
`mAb, whereas no increase was induced by exposure to the 12E7 or
`Hec2 mAb.
`Next, we checked whether CD99 is involved in hematopoietic
`progenitor–endothelial cell adhesion. As expected,4,51 a blocking
`mAb against CD106/VCAM-1 decreased