(2003) 17, 1137–1145 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Functional characterization of CD34+ cells that express low or high levels of the membrane antigen CD111 (nectin 1) G Belaaloui1, A-M Imbert1, F Bardin1, C Tonnelle1, P Dubreuil2, M Lopez2 and C Chabannon1

1Centre de The´rapie Cellulaire et Ge´nique, Institut Paoli-Calmettes, Centre Re´gional de Lutte Contre le Cancer Provence-Alpes- Coˆte d’Azur, Marseille, France; and 2Equipe d’He´matopoı¨ese Mole´culaire et Fonctionnelle, Inserm U119 : Cance´rologie Expe´rimentale, Marseille, France

Nectins are recently described adhesion molecules that are In some instances, the function of membrane antigens that are widely expressed on many tissues, including the hematopoietic otherwise used as ‘ markers’ is partially known. Some tissue. Nectin 1 (CD111) is expressed on a higher proportion of mobilized peripheral blood (mPB) than cord blood (CB) CD34+ of these molecules are receptors for cytokines: this is the case for cells, and of CD34+/CD38+ cells when compared with CD34+/ c- ligand or flt3 ligand. Others are molecules involved in cell- CD38À cells. We studied functional properties of human CB and to-cell or cell-to-matrix interactions, or adhesion molecules. mPB CD34+ cells that express low or high levels of CD111. This has been suspected, although not formally demonstrated, + dim CD34 /CD111 cells contain a higher proportion of cells in G0/ for CD34 expressed on hematopoietic progenitors. Other + bright + bright G1 phase than CD34 /CD111 cells. CD34 /CD111 cells adhesion molecules are clearly involved in the migration of contain more erythroid progenitors: CFU-E, than their counter- progenitors to (‘homing’) or from (‘mobilization’) the bone parts, which on the opposite contain more HPP-CFC. Limiting 8–10 11 dilution analyses demonstrate a higher frequency of immature marrow compartment: this is the case for VLA-4, PECAM-1 12 progenitors: cobblestone-area colony-forming cells, in CD34+/ and LFA-1. Chemokines and their receptors such as SDF-1/ CD111dim than in CD34+/CD111bright cells. In vitro differentiation CXCR48,13 and probably somatostatin/SSTR14 are also indis- assays demonstrate a higher frequency of B-, T- and dendritic- pensable partners in these phenomena. Within the marrow, + dim cell precursors, but less NK-cell precursors in CD34 /CD111 adhesion molecules may contribute to the proliferation, survival cells. Evaluation of engraftment in NOD-SCID mice shows that + and differentiation of hematopoietic stem and progenitor cells, SCID repopulating cells are more frequent among mPB CD34 / 15–21 CD111dim cells. Liquid culture of CD34+/CD111dim cells with through interactions with their environment. erythropoietin shows that CD111 expression increases simul- Nectins are a recently described family of molecules that are taneously with CD36, following CD71 and before structurally related to the immunoglobulin superfamily. Nectins expression. In conclusion, immature human hematopoietic were initially identified because of with the poliovirus progenitors express low levels of CD111 on their surface. 22,23 + receptor. Currently, four nectins have been described in During erythroid differentiation CD34 cells acquire higher 23–26 22,23 levels of the CD111 antigen. . Since their description, expression and func- Leukemia (2003) 17, 1137–1145. doi:10.1038/sj.leu.2402916 tion of human nectin 1 or CD111, and nectin 2 or CD112 have 24,27–29 Keywords: hematopoiesis; stem cells; adhesion moleculeserythroid been studied in a variety of models and tissues. Both differentiation molecules are widely expressed on numerous tissues of different embryonic origin, and appear to function as receptors for herpes simplex viruses.27,28 However, the physiological functions of Introduction these molecules have not been elucidated yet. The association between mutations in the nectin 1 gene and cleft lip/palate Hematopoiesis is a tightly regulated process during which 30 ectodermal dysplasia has been described. Nectin 1 is involved mature and functional cells that belong to different lineages, are 29 with nectin 3 in the formation of neurological synapses. continuously renewed through differentiation from a small pool Knock-out mice lacking nectin 2 have aberrant spermatozoa, of ancestral cells termed ‘stem cells’. Thus, identification of stem 31 and are sterile. The most plausible hypothesis is that nectins cells and understanding of mechanisms that control their play a role in cell–cell interactions and recognition. Recent data survival, proliferation and commitment are of the utmost suggest that nectins are involved in the tri-dimensional importance. For several decades, identification of stem cells organization of diverse tissues including epithelium and has relied on phenotype: expression (or lack of expression) of a , as they are expressed at the adherens junctions, number of membrane antigens characterize various stages of 26,32 on the lateral side of epithelial cells, and they are capable of mammalian hematopoiesis, including the earliest stages. One of 26,33 homophilic or heterophilic interactions. In addition, nectin the most useful tools for these approaches has been the 1 2 could play a role in interactions between hematopoietic and description of the CD34 membrane antigen , and the produc- 24 endothelial cells, as it is expressed on both cell populations. tion of numerous monoclonal (MoAb) directed at In the present report, we describe the expression of CD111 various epitopes of the molecule. However, functional assays + (nectin 1) on human cord blood (CB) and adult mobilized proved that the human CD34 cell population was hetero- + peripheral blood (mPB) CD34 cells. We next describe geneous, containing both early and late progenitors; thus, + functional properties of human CD34 cells that express low investigators worked at refining their phenotypic analyses by or high levels of CD111, using a variety of in vitro and in vivo subsetting the CD34+ cell population, using additional markers assays. such as CD38,2–4 CD90 (Thy1),5,6 or the absence of antigens that are expressed following commitment to hematolymphoid Patients and methods lineages (‘lineage –‘).7 Patients Correspondence: Dr C Chabannon, 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 Marguer- Following informed consent, apheresis samples were obtained ite, 13273 Marseille Cedex 9, France ; Fax : +33 04 91 22 36 59 from patients with a variety of poor-prognosis cancers, who Received 28 November 2002; accepted 27 January 2003 underwent mobilization with rhG-CSF (Neupogen, Amgen CD34+/CD111dim and CD34+/CD111bright cells G Belaaloui et al 1138 Thousand Oaks, CA, USA, or Granocyte, Chugai, Paris, France) equal volume of a 50 mg/ml propidium iodide solution was alone or in association with chemotherapy. Two samples were added, and cells were incubated for 1 h at 41C before flow obtained from healthy donors for allogeneic transplantation. CB cytometry analysis. samples were obtained after delivery from informed mothers. For all samples, mononuclear cells were enriched and red blood cells were depleted through density gradient separation Clonogenic cultures (d ¼ 1.077 g/ml, Nycomed, Oslo, Norway). Adherent cells were depleted, and CD34+ cells were immunoselected, using the The technique for the detection of granulomacrophagic pro- MACS technique according to the manufacturer’s recommenda- genitors (CFU-GM), erythroid progenitors (BFU-E and CFU-E) tions (Miltenyi Biotec, Bergisch-Gladbach, Germany). and early and highly proliferative progenitors (high-proliferative potential colony forming cells, HPP-CFC) has been described elsewhere.34–36. Briefly, for CFU detection, 250 cells were MoAb and flow cytometry studies seeded in triplicate in methyl-cellulose medium for 14 days in the presence of a combination of cytokines: IL3 (a kind gift from MoAb used for this study are listed in Table 1. All analyses were Amgen, Thousand Oaks, CA, USA), IL6 (Amgen), GM-CSF (Leucomaxs, Novartis, Rueil-Malmaison, France), G-CSF (Am- conducted with a FACScalibur (Becton-Dickinson Immunocy- s tometry Systems, BDIS, San Jose, CA, USA). Sorted cell gen) at 10 ng/ml, erythropoietin, Epo (Eprex , Janssen-Cilag, populations were obtained with a FACSVantage (BDIS) Issy-les-moulineaux, France) at 2 U/mL and SCF (Amgen) at equipped with a 448 nm argon laser. 100 ng/ml. Colonies of at least 50 nonhemoglobinized cells were scored as CFU-GM. CFU-E contained one or two colonies of hemoglobinized cells. BFU-E contained at least three colonies Cell cycle analyses of hemoglobinized cells. For HPP-CFC detection, 1000 cells were seeded in triplicate in an agar medium supplemented with The percentages of cells in G0/G1, S and G2/M phases were a combination of cytokines: IL3, IL6, GM-CSF, G-CSF and flt3-L evaluated for CD34+/CD111dim and CD34+/CD111bright cells, (R&D Systems, Minneapolis, MN, USA) at 20 ng/ml, and SCF at by staining living cells with propidium iodide. A total of 100 ng/ml. HPP-CFC were scored on day 21. 5 Â 104–105 cells were fixed in 70% ethyl alcohol (Carlo Erba Reagenti, Milan, Italy) for 15 min, then treated with 200–300 ml of a 10 mg/ml DNAse-free RNAse solution (Roche Diagnostics Limiting dilution analysis of Cobblestone-area Corporation, Indianapolis, IN, USA) for 15 min at 371C. An colony-forming cells (CAFC) The technique was adapted from a previously published report.37 Monolayers of the MS-5 stromal cell line were Table 1 MoAbs used in these experiments established in 96-well plates, and the cells were allowed to reach confluence.38 Serial dilutions of CD34+/CD111dim and Clone Recognized antigen Fluorochrome CD34+/CD111bright cells were then seeded on the monolayers. IMDM medium supplemented with 10% fetal calf serum (FCS, L01.1 CD71a FITC Stem Cell Technologies) was used for establishing monolayers, FA6.152 CD36b FITC and for culturing hematopoietic progenitors; no human recom- b 11E4B-7-6 (KC16) Glycophorin A FITC binant cytokine was added. CAFCs were scored at week 5. MOC-1 CD56c PE D3HL60.251 CD33b PE J4.119 CD19b PE b In vitro differentiation in erythroid cells RMO52 CD14 PE ALB 1 CD10b PE + dim R1.302.12 CD111 (Nectin 1)b PE CD34 /CD111 cells from either mPB or CB were cultured at R2.477.1 CD112 (Nectin 2)b PE a concentration of 2 Â 104 cells/ml, in serum-free medium: 581 CD34b PC-5 IMDM (BioWhitaker, Verviers, Belgique), supplemented with T16 CD38b FITC b 20% BIT (Stem Cell Technologies Vancouver, BC, Canada), and BL6 CD1a PE either with a combination of Epo (2 U/ml), IL3 and SCF (10 and MAB104 CD80b FITC HB15a CD83b FITC 100 ng/ml, respectively) to induce erythroid differentiation, or B70/B7-2 CD86a FITC with a combination of IL3, IL6, G-CSF and GM-CSF at a J.33 CD45b PC-5 concentration of 10 ng/ml for each, and SCF (100 ng/ml) to 25.3 CD11ab FITC induce granulomonocytic differentiation. Cultures were main- 2H5 CD15sa FITC 1 b tained for 15 days at a temperature of 37 C, 5% CO2 in a SAM1 CD49e FITC humidified atmosphere and the medium was changed at day 7. HP2/1 CD49db FITC 84H10 CD54/ICAN1b FITC Dreq56 CD62La FITC B-T1 CD102/ICAM2d FITC B- and NK-cell differentiation during coculture with the UCHT1 CD3b FITC murine stromal cell line MS-5 13B8.2 CD4b PE B9.11 CD8b PC5 CD34+/CD111dim and CD34+/CD111bright cells were cocultured b P2 CD41 PE with the murine stromal cell line MS-5, as previously 39,40 a reported; however, all exogenous cytokines were with- Becton Dickinson. 41 bBeckman Coulter. drawn to favor B-cell differentiation, in IMDM medium cDAKO A/S. supplemented with 5% FCS, 5% human male AB serum (Institut d À4 Southern Biotechnology Associates. Jacques Boy SA) and 10 M of b-mercaptoethanol (Sigma,

Leukemia CD34+/CD111dim and CD34+/CD111bright cells G Belaaloui et al 1139 Steinheim, Germany). In separate experiments, 20 ng/ml IL15 Results (R&D Systems) and 50 ng/ml SCF were added to the cultures to favor NK-cell differentiation in IMDM medium supplemented Phenotypic analyses of CD111 expression on human CB with 15% FCS. and adult mPB CD34+ cells

CD34+ cells obtained by immuno-selection from CB or Dendritic-cell differentiation apheresis samples (mean purity: 85711% for CB, n ¼ 31; 7 ¼ + dim + bright 80 22% for mPB, n 61) were simultaneously stained with a CD34 /CD111 and CD34 /CD111 cells were cultured in MoAb to CD34 and a MoAb to CD111. When compared to an 4 24-well plates at a concentration of 2 Â 10 cells/ml in RPMI- isotype-matched irrelevant control, the CD111 expression 1640 medium supplemented with 10% FCS, 20 ng/ml of SCF, pattern within the CD34+ cell subset does not discriminate a 100 ng/ml of GM-CSF, 2.5 ng/ml of TNFa (R&D Systems) and clearly positive population, but rather suggests a continuum of 20 ng/ml of flt3 ligand at 371C in a fully humidified incubator expression (Figure 1); for this reason, CD34+ cells will further be 42 with a 5% CO2 atmosphere during 14 days. The culture subset in CD34+/CD111bright and CD34+/CD111dim cells (see medium was changed at day 7. below). However, comparison of the CD34+ cell subpopulation from adult mPB and from CB shows that a higher percentage Fetal organotypic cultures of cells in the former population displays a fluorescence above the threshold set up with the control 39 (49.3721.6% vs 18.478.5%; Po0.001 with a Student t-test). Organotypic cultures were performed as previously described. Hence, the CD34+/CD111bright subset that accounts for approxi- In brief, thymus of 14–16 days old NOD-SCID mice embryos mately one-third of CD34+ cells represents cells with the highest were dissected. CB CD34+ cells were transferred to thymic lobes level of CD111 expression in samples from adult mPB, and all using the hanging drop method in Terazaki plates (in our hands, CD111+ cells in samples from CB origin. The CD34+/CD111dim mPB CD34+ cells did not differentiate towards the T-cell lineage subset was arbitrarily defined as the equivalent subset of CD34+ in these conditions). In total, 10 000–20 000 cells in 25 mlof cells that had the lower fluorescence for CD111 expression; culture medium were put in each well. A complete culture thus, a population exists with an intermediate level of medium containing 5 ng/ml of IL2 (Chiron, Suresnes, France), fluorescence for CD111: this population was not sorted (Figure 20 ng/ml of IL7 (R&D systems), and 50 ng/ml of SCF was used. 1b and c). After a 48 h incubation in a humidified incubator at 371C and Further, we studied CD111 expression on CD34+/CD38+ and 5% CO , lobes were transferred onto floating filters (Isopore 2 CD34+/CD38À cells, the latter being a minor subset (1.670.7% membrane, 25 mm diameter, 8 mm pore size; Millipore SA, St- for mPB, 6.673.2% for CB) enriched with the most Quentin-en-Yvelines, France) in a medium without cytokines, immature human progenitors. The percentages and mean and with the same atmospheric conditions. Cells were fluorescence intensities (MFI) for CD111 expression were lower recovered by mechanical disruption of thymic lobes after 30 in the CD34+/CD38À than in the CD34+/CD38+ subset, days, and used for cytometric analysis. both in cells originating from adult mPB (32726.9% vs 63716.8%, Po0.05, n ¼ 12; MFI: 31.3 vs 78.5) and from Detection of NOD-SCID repopulating cells CB (10718.4 vs 36730.5%, Po0.05, n ¼ 11; MFI: 34 vs 69.1). The technique has been described elsewhere,40 and reproduced It has been demonstrated that CD111 behaves as an adhesion from previously published reports,39,43,44 but was nevertheless molecule through hetero- and homotypic interactions with adapted to assay human adult peripheral blood progenitors. members of the nectin family. Thus, we studied with multicolor Briefly, NOD-SCID mice45 were obtained from Dr John Dick, flow cytometry the coexpression of other known adhesion + + + À Department of Molecular and Medical Genetics, Hospital for molecules on CD34 /CD111 and CD34 /CD111 cells. Sick Children, Toronto, Canada. Founders were bred at our Figure 2 demonstrates that the pattern of expression for CD11a animal facility. All animals were maintained in germ-free (LFA-1), CD15 s, CD54 (ICAM-1), CD62L (L-), CD49d conditions. Animals, 6–9 weeks old, were used for xenotrans- (VLA-4), CD49e (VLA-5) and CD102 differs within the two plantation. Recipients were prepared with total body gamma subsets. CD49e, CD49d and CD102 are statistically significantly + + + irradiation at a dose of 350 cGy. Within 24 h of irradiation, mice more expressed on CD34 /CD111 cells than on CD34 / À were injected with 0.9–4 Â 106 mPB CD34+/CD111dim cells or CD111 cells, although the differences are biologically small CD34+/CD111bright cells via the tail vein. After 10–12 weeks, for CD49d and CD102. The opposite is observed with CD54, animals were killed; bone marrow cells were recovered from CD62L, CD15 s and CD11a which are significantly more + À femurs and tibia by flushing, and the spleen was mechanically expressed on CD34 /CD111 cells. These observations are disrupted. Mononuclear cells were enriched and red blood cells valid for cells of mPB or CB origin. were depleted through density gradient separation and then analyzed by flow cytometry for the expression of human antigens. Cell cycle analyses

Owing to the relation between cell cycle activity and stem cell Statistics potential within the hematopoietic progenitor cell population,4 we next determined the percentage of cells in G0/G1 phase in Descriptive statistics, comparisons of paired values with the two cell subsets: CD34+/CD111bright and CD34+/CD111dim. nonparametric Wilcoxon’s test, and comparisons of unpaired Table 2 demonstrates that the proportion of cells that are not values with Student’s t-test were carried out by using the SPSS actively cycling (G0/G1 phase) is higher in the CD34+/ software for Windows (SPSS Inc., Chicago, IL, USA). A P-value CD111dim population than in its CD34+/CD111bright counter- of 0.05 was chosen for statistical significance. part. These observations are valid for mPB as well as CB cells.

Leukemia CD34+/CD111dim and CD34+/CD111bright cells G Belaaloui et al 1140

Figure 1 CD111 expression profile on CD34+ cells obtained by immunoselection from apheresis or CB samples. (a) CD111 expression profile on mPB CD34+ cells. (b) Sorting windows are indicated. CD34+/CD111dim cells and CD34+/CD111bright cells represent approximately one-third of all CD34+ cells for apheresis samples. (c) For CB samples, the CD34+/CD111bright subset contains all cells that display a fluorescence above the threshold set up with an irrelevant matched isotype control.

The frequency of clonogenic progenitors differs equivalent in the two subsets, for the three CB samples that between CD34+/CD111dim and CD34+/CD111bright we studied. cells

Overall, the frequency of committed clonogenic progenitors, as Evaluation of CD34+/CD111dim and CD34+/CD111bright estimated in methyl-cellulose assays, is higher in CD34+/ cell differentiation capacity into B, T, NK and dendritic CD111bright cells than in CD34+/CD111dim cells, although the cells difference appears to be statistically significant only for samples of mPB origin (Table 3). The higher frequency of mature Looking at the in vitro differentiation potential of the two erythroid progenitors (CFU-E) accounts for most of the differ- subsets, we cocultured the two sorted cell populations with the ence; the frequency of BFU-E being equivalent in both cell murine stromal cell line, MS-5, in the presence or absence of a subsets. In CB, the frequency of CFU-GM was significantly combination of IL-15 and SCF, to favor NK- or B-cell higher in the CD34+/CD111dim subpopulation. differentiation, respectively. NK and B cells were identified On the opposite, the frequency of more immature clonogenic with phenotypic features. After 5 weeks of culture, mPB CD34+/ progenitors – namely HPP-CFC – as assayed in agar cultures, CD111dim cells yielded more CD19+ but less CD56+ cells than was significantly higher in mPB or CB CD34+/CD111dim cells CD34+/CD111bright cells. CB CD34+/CD111bright also produced than in CD34+/CD111bright cells (Table 3). more CD56+ cells, while numbers of CD19+ cells that derived from both CB subpopulations appeared to be equivalent (Table 5). CD34+/CD111bright and CD34+/CD111dim were also cultured CAFC are more frequent in CD34+/CD111dim cells than in conditions that were previously demonstrated to favor CD34+/CD111bright cells dendritic-cell differentiation;42 these cells were identified as CD1a+ cells, and further studied for the expression of Cells that are able to reinitiate hematopoiesis in culture systems costimulatory molecules: CD80, CD83 and CD86. At day 14, with stromal cells, either long-term culture initiating Cells (LTC- the percentage of CD1a+ cells was higher in cultured CD34+/ IC) or CAFC, represent another index of stem cell activity. Using CD111dim cells than in CD34+/CD111bright cells, either from a limiting dilution assay with the murine stromal cell MS-5, we mPB or CB origin (Table 5). This higher percentage translated measured the frequency of CAFC37 in the two subpopulations. into a higher absolute number of dendritic cells produced from Despite intersample variability, Table 4 demonstrates the higher mPB CD34+/CD111dim cells. Within the CD1a+ subset, the frequency of CAFC in the CD34+/CD111dim population of mPB expression of CD80, CD83 and CD86 was comparable in cells origin; however, the frequency of CAFC appeared to be obtained from the two cultured populations.

Leukemia CD34+/CD111dim and CD34+/CD111bright cells G Belaaloui et al 1141 Table 3 Clonogenic potential of CD34+/CD111bright and CD34+/ CD111dim cells from adult mPB and CB

CD34+/ CD34+/ Colonies Origin CD111bright CD111dim

Total mPB* 76710 3476 CB 106711 83714 BFU-E/250 cells mPB 270.7 270.6 CB* 11771372 CFU-E/250 cells mPB* 4278572 CB* 57710 1874 CFU-GM/250 cells mPB 28772777 CB* 22733776 HPP-CFC/103 cells mPB* 270.4 872 CB* 6711672

*Po0.05: the Wilcoxon test for paired values was used. Results are means 7 s.e.m. of colonies numbers per 250 seeded cells (103 cells for HPP-CFC) in six to 12 experiments.

Table 4 Limiting dilution analyses of the frequency of CAFC in CD34+/CD111bright and CD34+/CD111dim cells

Figure 2 Expression of a variety of adhesion molecules following CAFC frequency CD111 expression. Expression of various adhesion molecules was + + + À determined on CD34 /CD111 cells and CD34 /CD111 cells from Cell population Exp 1 Exp 2 Exp 3 Exp.4 mPB or CB. Results are means 7s.e.m. of six to 12 experiments. *Po0.05 using a Wilcoxon’s test for paired samples. mPB CD34+/CD111bright 1/1600 1/460 1/10600 1/1200 CD34+/CD111dim 1/1100 1/310 1/4200 1/42 Table 2 Cell cycle analysis CB + bright + + CD34 /CD111 1/652 1/274 1/324 CD34 / CD34 / CD34+/CD111dim 1/216 1/235 1/304 Cell cycle phase Cell origin CD111bright (%) CD111dim (%) Cells from mPB (four experiments) or CB (three experiments) origin G0/G1 mPB* 88.972.6 98.770.2 were seeded on pre-established monolayers of the murine stromal cell CB* 8873.2 98.070.3 line MS-5 in limiting dilution conditions. For each cell population, six S mPB* 8.172.3 0.770.1 dilutions were prepared starting from 2000 to 2500 cells/well. CAFC CB* 8.172.2 0.870.1 frequency was calculated using the Poisson distribution. Owing to the G2/M mPB* 2.770.6 0.170.0 inter-individual variability and the low number of experiments we did CB* 2.370.5 0.370.1 not calculate averages and standard deviations; whatever the differences in absolute numbers in different samples, the numbers of A total of 5 Â 104–105 CD34+/CD111dim cells or an equal amount of CAFC are always higher in the CD34+/CD111dim cell subset. CD34+/CD111bright cells were stained with propidium iodide and the percentages of cells in G0/G1, S and G2/M phases were evaluated by flow cytometry. Results are mean 7 s.e.m. of six experiments. * Po0.05: the nonparametric Wilcoxon test for paired values was used. vein into the bloodstream of sublethally irradiated NOD-SCID animals. Mice were killed at designated time points after injection, and the presence of cells of human origin was Finally, for the assessment of our two population ability to assayed by flow cytometric identification of cells that stained differentiate into T lymphocytes, we seeded CB CD34+/ dim + bright positive for human CD45 or CD71 in the bone marrow and the CD111 and CD34 /CD111 into NOD-SCID 46 39 spleen. The presence of SCID repopulating cells (SRC) in CB thymic fragments, as previously described . After 14 days of + dim + bright CD34 /CD111 and CD34 /CD111 cells was not as- culture, the numbers of double positive CD3+/CD4+/CD8+ and sessed, because of low cell numbers. Human cells were the MFI for CD4 expression were higher in organotypic cultures + dim detected in both groups of animals. However, the percentage initiated with the CD34 /CD111 subset than in organotypic + + bright of CD45 cells was higher when mice were engrafted with cultures initiated with its CD34 /CD111 counterpart (three + dim CD34 /CD111 cells, than when mice were engrafted with separate experiments; Table 5 for percentages and data not + bright CD34 /CD111 cells (Table 6). Only small numbers of shown for MFI). À + CD45 /CD71 cells–a phenotype that identifies the erythroid progeny of SRC – were detected in hematopoietic organs of Evaluation of CD34+/CD111dim and CD34+/CD111bright engrafted animals, and these figures were similar for CD34+/ cell ability to repopulate hematopoietic organs CD111dim and CD34+/CD111bright cells. The distribution of in NOD/SCID mice CD45+ cells in myelomonocytic (CD13+/CD33+), megakaryo- cytic (CD41+) or B-lymphoid (CD19+/CD34À) cells was similar CD34+/CD111dim and CD34+/CD111bright cells sorted from in animals engrafted with CD34+/CD111dim or with CD34+/ apheresis samples were infused in equal numbers via the tail CD111bright cells (data not shown).

Leukemia CD34+/CD111dim and CD34+/CD111bright cells G Belaaloui et al 1142 Table 5 Evaluation of CD34+/CD111dim and CD34+/CD111bright cell differentiation capacity into B, T, NK and dendritic cells

Origin CD34+/CD111bright (%) CD34+/CD111dim (%) n

B lymphocytes (CD19+) mPB* 8.076.3 22.077.4 7 CB 34.4715.6 28.2711.0 6 NK cells (CD56+) mPB* 41.2716.7 23.978.8 8 CB* 87.178.8 48.9717.8 6 Dendritic cells (CD1a+) mPB* 11.873.0 50.6725.0 7 CB* 31.0712.0 52.3720.0 7 T lymphocytesa (CD3+/CD4+/CD8+) CB1 8.8 29.4 1 CB2 30.61 58.24 1 CB3 10.05 49.55 1

Culture conditions allowing B-, T-, NK-, and dendritic-cell differentiation from CD34+ progenitors are described in the Material and methods section. Results are average percentages 7s.e.m. of cells expressing CD19, CD56 or CD1a in n experiments, respectively. *Po0.05 (Wilcoxon’s test). aFor organotypic cultures allowing T differentiation, three experiments are shown: percentages are averages of CD3+/CD4+/CD8+ cells recovered from three to five thymic lobes.

Table 6 Engraftment of sublethally irradiated NOD-SCID mice with mPB CD34+/CD111dim and CD34+/CD111bright cells

BM Spleen Cell numbers Experiment (x106/mouse) CD111dim CD111bright (%) CD111dim CD111bright (%)

1 2.5 37.53 1.26 43.81 8.77 24.72 14.56 49.38 27.24 D 18.69 D 28.77 2 4 46.19 2.29 61.48 9.42 46.13 10.61 71.51 23.27 63.08 10.76 64.37 40.31 3 0.9 13.15 0.14 6.24 0.15 15.43 0.47 9.59 0.11 4 1.7 0.11 3.64 0.11 0.58 5.29 1.67 1.25 0.36 5.40 0.10 1.20 0.00

Adult mPB CD34+/CD111dim cells produced higher numbers of human CD45+ cells in the bone marrow and spleen of engrafted animals, than equal numbers of CD34+/CD111bright cells. D: the animal died before analysis.

In vitro differentiation in erythroid cells expression peaked and then decreased during the second week of culture. This pattern was similar for cells from mPB or CB The high frequency of CFU-E in the CD34+/CD111bright subset origin. suggested that CD111 may be highly expressed in the erythroid lineage, and prompted us to follow CD111 expression during in vitro differentiation. For this purpose, CD34+/CD111dim cells Discussion were sorted and cultured for 14 days in the presence of a cytokine combination known to favor erythroid differentiation, We demonstrate here that human CD34+ cells from CB or adult particularly as it contains Epo (SCF+IL3+Epo); as a control, the mPB origin are heterogeneous in terms of CD111 expression. same cells were cultured in parallel with another cytokine The profile of expression for CD111 appears to be different from combination that does not contain Epo, and rather favors the profile of expression for CD112 (PRR2 or nectin 2),24 a myelomonocytic differentiation (SCF + IL3 + IL6 + GM-CSF + G- structurally related surface antigen, and another member of this CSF). Figure 3 illustrates the kinetics of CD111 expression in one family of molecules; CD112 is found highly expressed on the representative experiment for each source of cells; while CD111 vast majority of bone marrow CD34+ cells, an observation that was upregulated in both culture systems, conditions allowing for is also valid for CB and mPB cells (data not shown). The pattern erythroid differentiation resulted in a higher percentage for for CD111 shows a lower level of expression, and does not CD111; in addition the MFI was also higher, suggesting higher subset CD34+ cells into a truly negative and a truly positive numbers of the molecules on the cell surface (157 for the population; it suggests that CD111 is expressed at various levels SCF+IL3+Epo condition vs 36.5 for the SCF+IL3+IL6+GM- on most CD34+ cells. CB and mPB CD34+ cells express CD111 CSF+G-CSF condition for mPB cells; 72 vs 46 for CB cells). with different intensities; therefore, the gating for the CD34+/ Upregulation of CD111 expression appeared to occur later than CD111bright cells may define slightly different populations when CD71, synchronously with CD36 and prior to the appearance of working with apheresis or CB samples, which may account for glycophorin A, a marker that is strictly specific of the erythroid some of the different results observed when this subpopulation lineage. Of note, CD111 was only transiently upregulated, as its was studied with functional assays (for example, the lack of

Leukemia CD34+/CD111dim and CD34+/CD111bright cells G Belaaloui et al 1143 Erythroid differentiation PB CB 120% 120% 100% 100% 80% 80% 60% 60% 40% 40% 20% 20% Percent Expression

Percent Expression 0% 0% 0 2 4 6 8 10 12 14 16 0 5 10 15 20 Days Days

CD34 CD111 CD36 CD71 Glycophorin A CD34 CD111 CD36 CD71 Glycophorin A

Myelomonocytic differentiation PB CB 120% 120% 100% 100% 80% 80% 60% 60% 40% 40% 20% 20% Percent Expression 0% Percent Expression 0% 0 5 10 15 20 0 5 10 15 20 Days Days

CD34 CD111 CD36 CD71 Glycophorin A CD34 CD111 CD36 CD71 Glycophorin A

Figure 3 CD34+/CD111dim cells from mPB samples or CB samples were cultured with two cytokines combinations allowing: erythroid differentiation (2 U/ml of Epo, 10 ng/ml of IL3 and 100 ng/ml of SCF) or myelomonocytic differentiation (IL3, IL6, G-CSF and GM-CSF at a concentration of 10 ng/ml and SCF at 100 ng/ml). Cultures were maintained for 15 days, and the expression of CD111, CD36, CD71, glycophorin A and CD34 on the cells was assessed at different time points.

difference for CAFC frequency in CB CD34+/CD111dim cells and suggests that cytokine combinations that induce erythroid CD34+/CD111bright cells). differentiation (and contain Epo) also induce a strong upregula- Nevertheless, all our observations suggest that CD111 is tion of CD111 expression on the cell surface. This is compatible expressed at low levels during early stages of human hemato- with the high numbers of CFU-E that were detected while poietic differentiation. First, CD34+/CD38À cells, a minor subset culturing CD34+/CD111bright cells in methyl-cellulose assays, of CD34+ cells known to be enriched for the most immature and with the increased expression of CD49d and CD49e on hematopoietic progenitors, express lower levels of CD111 than CD34+/CD111bright cells,49 as well as with the increased CD34+/CD38+ cells. Second, cell cycle analyses suggest a expression of CD11a on CD34+/CD111dim cells.50 The kinetics higher percentage of noncycling cells within the subpopulation of CD111 expression, compared with the kinetics of expression that express low levels of CD111. Third, functional assays that for other useful markers of the erythroid lineage (CD71, CD36 detect early progenitors (HPP-CFC, CAFC and SRC) suggest that and glycophorin A) suggests that CD111 may be highly the frequency of such progenitors is higher within the CD34+/ upregulated during the transition from the BFU-E stage, to the CD111dim cells than within the CD34+/CD111bright subset; CFU-E stage;50,51 during erythroid differentiation in vitro, however, the observation that HPP-CFC, CAFC and SRC can CD111 expression appears to peak after approximately 1 week also be detected, albeit at lower frequencies, within the CD34+/ of culture, to decrease later. These observations are consistent CD111bright subpopulation, along with the relative frequency of with a previous report that demonstrates CD111 expression on CD34+/CD111dim cells, suggests that low expression of CD111 mature (glycophorin A+) cells, with a higher percentage of will not constitute an appropriate ‘human ’ by glycophorin A+/CD111+ cells in the bone marrow than in itself. peripheral blood.52 In vitro differentiation assays suggest that B-, T- and dendritic- The role that CD111 may play during hematopoietic cell precursors are more frequent within the CD34+/CD111dim differentiation, and particularly during erythroid differentiation, cells than within the CD34+/CD111bright cells, as more CD19+ is unknown. E-cadherin provides an example of an adhesion cells, more CD3+/CD4+/CD8+ and more CD1a+ cells can be molecule, whose modulation can affect the ability of hemato- produced from CD34+/CD111dim cells than from equal numbers poietic progenitors to differentiate towards the erythroid lineage: of CD34+/CD111bright cells. Surprisingly, NK-cell precursors blocking antibodies directed to E-cadherin reduces the erythroid were detected at a higher frequency in the CD34+/CD111bright differentiation of human bone marrow mononucleated cells in subset; although a common origin has been reported for human vitro;53 in contrast, addition of a MoAb known to block CD111- T, B, NK and dendritic cells47, restriction in differentiation mediated heterophilic interactions,54 failed to block the capacities may occur sequentially48 rather than simultaneously, erythroid differentiation of human mPB CD34+ cells in vitro and could be associated with different patterns of CD111 (the pattern of glycophorin A upregulation on cultured cells did upregulation. Differentiation towards the nonlymphoid lineages not change), while we were able to reproduce inhibition with an

Leukemia CD34+/CD111dim and CD34+/CD111bright cells G Belaaloui et al 1144 anti-E-cadherin MoAb (data not shown). Elucidation of the role marrow and peripheral blood during G-CSF-enhanced recovery. of CD111 during eythroid differentiation will deserve further Exp Hematol 1995; 23: 1535–1542. investigation. 9 Craddock CF, Nakamoto B, Andrews RG, Priestley GV, Papayan- The function of nectin 1 and structurally related molecules nopoulou T. Antibodies to VLA4 mobilize long-term repopulating cells and augment cytokine-induced mobilization in remains elusive. Some members of the family act as virus 55 primates and mice. Blood 1997; 90: 4779–4788. receptors, and abnormal lead to defect in 10 Zanjani ED, Flake AW, Almeida-Porada G, Tran N, Papayanno- tissue organization.30,31 The localization of CD111 on the poulou T. Homing of human cells in the fetal sheep model: basolateral side of epithelial cells and endothelial cells, and modulation by antibodies activating or inhibiting very late associations of nectins with other membrane or cytoplasmic activation antigen-4-dependent function. Blood 1999; 94: 2515– molecules in supra-molecular complexes leads to speculate that 2522. 11 Yong KL, Watts M, Thomas NS, Sullivan A, Ings S, Linch DC. nectins may participate in the tri-dimensional organization of Transmigration of CD34+ cells across specialized and nonspecia- these tissues; however, the architecture of the hematopoietic lized endothelium requires prior activation by growth factors and tissue is poorly defined when compared to epitheliums, even is mediated by PECAM-1 (CD31). Blood 1998; 91: 1196–1205. though a stroma constituted of a variety of hematopoietic and 12 Pruijt JF, van Kooyk Y, Figdor CG, Lindley IJ, Willemze R, Fibbe nonhematopoietic cells embedded in an extracellular matrix WE. Anti-LFA-1 blocking antibodies prevent mobilization of exists, and is believed to play important roles in promoting the hematopoietic progenitor cells induced by interleukin-8. Blood 1998; 91: 4099–4105. survival, proliferation and differentiation of hematopoietic stem 13 Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, Byk T et al. and progenitor cells. Description of ‘anatomic’ structures has Dependence of human stem cell engraftment and repopulation 56,57 been limited to the ‘erythroblastic island’. Several adhesion of NOD/SCID mice on CXCR4. Science 1999; 283: molecules are likely to play a role in macrophage–erythroblast 845–848. cell interactions.58,59 Whether CD111 also plays a role in the 14 Oomen SP, van Hennik PB, Antonissen C, Lichtenauer-Kaligis EG, formation of these structures, and whether natural ligands for Hofland LJ, Lamberts SW, Lowenberg B, Touw IP. Somatostatin is a CD111 (nectins 3 and 4) are expressed on stromal cells, will selective chemoattractant for primitive (CD34(+)) hematopoietic progenitor cells. Exp Hematol 2002; 30: 116–125. deserve further attention, working with cells of marrow origin. 15 Miyake K, Medina KL, Hayashi S, Ono S, Hamaoka T, Kincade PW. Monoclonal antibodies to Pgp-1/CD44 block lympho- hemopoiesis in long-term bone marrow cultures. J Exp Med Acknowledgements 1990; 171: 477–488. 16 Moll J, Khaldoyanidi S, Sleeman JP, Achtnich M, Preuss I, Ponta H This work was supported in part by Institut Paoli-Calmettes, and et al. Two different functions for CD44 in human by a grant from the Comite´ Leuce´mie de la Fondation de France (# myelopoiesis. J Clin Invest 1998; 102: 1024–1034. 990038) to CC. GB is the recipient of a grant from a joint research 17 Khaldoyanidi S, Sikora L, Orlovskaya I, Matrosova V, Kozlov V, training program established by the French and Algerian govern- Sriramarao P. 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