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CD164, a Novel Sialomucin on CD34 1 and Erythroid Subsets,
Is Located on Human Chromosome 6q21
By Suzanne M. Watt, Hans-Jörg Bühring, Irene Rappold, James Yi-Hsin Chan, Jane Lee-Prudhoe, Tania Jones,
Andrew C.W. Zannettino, Paul J. Simmons, Regis Doyonnas, Denise Sheer, and Lisa H. Butler
CD164 is a novel 80- to 90-kD mucin-like molecule expressed
by human CD341 hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in
hematopoiesis by facilitating the adhesion of CD341 cells to
bone marrow stroma and by negatively regulating CD341
hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes,
defined by the monoclonal antibodies (MoAbs), 103B2/9E10
and 105A5. In this report, we show that these MoAbs,
together with two other CD164 MoAbs, N6B6 and 67D2,
show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical
cord blood, and peripheral blood. Flow cytometric analyses
revealed that, on average, 63% to 82% of human bone
marrow and 55% to 93% of cord blood CD341 cells are
CD1641, with expression of the 105A5 epitope being more
variable than that of the other identified epitopes. Extensive
multiparameter flow cytometric analyses were performed
on cells expressing the 103B2/9E10 functional epitope. These
analyses showed that the majority (G90%) of CD341 human
bone marrow and cord blood cells that were CD38lo/2 or that
coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)1. This CD164 epitope was
generally detected on a significant proportion of CD341-
CD71lo/2 or CD341CD33lo/2 cells. In accord with our previous
in vitro progenitor assay data, these phenotypes suggest
that the CD164(103B2/9E10) epitope is expressed by a very
primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD341CD164(103B2/9E10)lo/2
cells in bone marrow are mainly CD191 B-cell precursors,
with the CD164(103B2/9E10) epitope subsequently appearing on CD34lo/2CD191 and CD34lo/2CD201 B cells in bone
marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34lo/2CD164(103B2/
9E10)1 subsets indicated that one of the most prominent
populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III
erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/
9E10) epitope is also found on developing myelomonocytic
cells in bone marrow, being downregulated on mature
neutrophils but maintained on monocytes in the peripheral
blood. We have extended these studies further by identifying
Pl artificial chromosome (PAC) clones containing the CD164
gene and have used these to localize the CD164 gene
specifically to human chromosome 6q21.
r 1998 by The American Society of Hematology.
T
cell-cell adhesion, involving interactions between their mucinlike domains and the N-terminal C-type lectin domains of their
appropriate L-, E-, or P-selectin ligands.4 These interactions
may be enhanced by cooperativity between mucin-like domains
and non-mucin motifs on the same molecule, thereby forming
HE SIALOMUCINS are thought to play two key, but
opposing, roles in vivo; the first as cytoprotective or
anti-adhesive agents, and the second as adhesion receptors.1-4
Despite their common functions, these mucins encompass a
heterogeneous group of secreted or membrane-associated proteins that vary in their apparent molecular weights from 50 to
3,000 kD and that share limited similarity/homology at the
amino acid and nucleotide levels. They were identified initially
on epithelial cells and, more recently, on endothelial cells and
leukocytes. The epithelial-associated mucins include the MUC-1
to MUC-8 molecules, whereas the endothelial/leukocyteassociated mucins encompass CD34, CD43, CD45RA, CD164,
GlyCAM-1, MAdCAM-1, TACTILE (CD96), and PSGL1(CD162).1,3-5 Because of their structural heterogeneity, mucins
have recently been defined by two criteria: (1) their high
regional content of proline, threonine, and/or serine residues
(20% to 55% of their amino acid compositions) and (2) their
dense local concentrations of O-linked carbohydrates that are
attached to these serine and threonine residues and that constitute one or more mucin-like domains.3,6 Such domains are a
primary feature of molecules, such as PSGL-1, CD43 and
CD164.5,7-9 However, for MAdCAM-1, CD34, and CD96, these
mucin-like domains are interspersed with or linked to Ig-like
structural motifs.3,10-13
For all the mucins, their high proline content and heavy
O-linked glycosylation predict an extended filamentous conformation, with the membrane linked forms protruding above the
glycocalyx. This provides an opportunity for their oligosaccharide sidechains and/or Ig-like domains to interact with ligands
on opposing cells, bacteria, or viruses.3 It is now known that
several of the leukocyte/endothelial-associated mucins mediate
Blood, Vol 92, No 3 (August 1), 1998: pp 849-866
Fom the MRC Molecular Haematology Unit, Institute of Molecular
Medicine, The John Radcliffe Hospital, Oxford, UK; Medizinische
Universitatsklinik II, University of Tübingen, Tübingen, Germany;
Human Cytogenetics Laboratory, Imperial Cancer Research Fund,
London, UK; and the Hanson Centre for Cancer Research, Matthew
Roberts Laboratory, Instiute of Medical and Veterinary Sciences,
Adelaide, South Australia.
Submitted November 18, 1997; accepted March 26, 1998.
Supported by the Medical Research Council (S.M.W., J.L.-P.) and
Leukaemia Research Fund (S.M.W.) of Great Britain; Smith-KlineBeecham (L.H.B.); INTAS/RFBR, PECO, and E.U. Concerted Action
and Biotech Grants on the Human Haematopoietic Stem Cell (S.M.W.);
the Deutsche Forschungsgemeinschaft (SFB 510, project A1 to H.J.B.)
and the fortune project 244 of the University of Tübingen (I.R.); grants
from the Anti-Cancer Foundation of the Universities of South Australia
and the National Health and Medical Research Council of Australia
(P.J.S.); the Imperial Cancer Research Fund (T.J., D.S.); and the
Taiwanese Government (J.Y.-H.C.).
Address reprint requests to Suzanne M. Watt, PhD, MRC Molecular
Haematology Unit, Institute of Molecular Medicine, The John Radcliffe
Hospital, Headington, Oxford OX3 9DS, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9203-0018$3.00/0
849
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850
part of an adhesion cascade. In such cases, the mucin receptor/
selectin ligand interactions would mediate the initial weak
tethering of leukocytes to endothelium that precedes stronger
integrin-mediated adhesion and subsequent transendothelial
migration required for leukocyte trafficking.4 Although mucin
receptors may be widely expressed, their function may differ on
different cell types or on the same cell type under different states
of activation. This functional diversity is dependent on the core
peptide of the mucin and on the cell-specific expression of
glycosyl transferases.1,4,14 These, in turn, regulate the structure
and presentation of the O-linked oligosaccharide sidechains,
membrane anchorage, signal transduction abilities, and/or the
trafficking of the mucin to the correct cellular domain.15-18
These alterations may then affect function.
Although a great deal of research has been directed toward
the expression and function of the mucins on mature leukocytes
and endothelial cells, there is still a paucity of information about
their expression and function on human CD341/hematopoietic
‘‘stem’’ and progenitor cells and on the associated stromal and
endothelial cells that constitute the immediate stem-cell microenvironment. Previous studies have identified three of the
mucin-like receptors, CD34, PSGL-1, and CD43, on primitive
human bone marrow hematopoietic progenitor cells and/or
associated microenvironmental stromal/endothelial cells.9,19-28
More recently, we have identified and cloned a fourth sialomucin, CD164, on human hematopoietic progenitor cells and bone
marrow stromal reticular cells.5,7 On such cells, the four
sialomucins have a variety of functions, with the specificity in
receptor/ligand interactions depending on the structural characteristics of the mucin-like receptor. These functions include
mediating,5,7,20,29,30 or regulating31,32 hematopoietic progenitor
cell adhesion and the negative regulation of their growth and/or
differentiation.5,7,24,25,33-35
In this report, we have characterized four monoclonal antibodies (MoAbs) against the CD164 molecule. These MoAbs each
recognize distinct epitopes on the CD164 molecule. We have
examined the expression of these epitopes on CD341 cells from
cord blood and bone marrow. Because we have recently shown
that antibody ligation of the 103B2/9E10 epitope can inhibit the
proliferation of single human CD341CD38lo/2 hematopoietic
progenitor cells in the presence of the cytokines, interleukin-3
(IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF),
and Steel factor in serum free medium and the adhesion of
CD341 cells to bone marrow stromal reticular cells,5,7 we also
analyzed the distribution of this epitope on subsets of human
bone marrow and cord blood cells and on their CD341 cell
precursors in some detail. Our results show that this epitope is
present on phenotypically very primitive CD341 progenitor
cells, as defined by their coexpression of such surface antigens
as AC133, Thy-1, FLT3 receptor, and c-kit receptor, but it
exhibits differential expression on maturing B cells. Furthermore, using the CD164 cDNA as a probe, we have isolated PAC
clones containing the CD164 gene and have used these to show,
by fluorescence in situ hybridization (FISH) analysis, that the
CD164 gene is localized to human chromosome 6q21.
WATT ET AL
MATERIALS AND METHODS
Cells and Cell Lines
Cell lines were cultured in RPMI 1640 or Iscove’s modified
Dulbecco’s medium (IMDM) containing 10% to 15% (vol/vol) fetal
calf serum (FCS; GIBCO-BRL, Paisley, Scotland). Heparinized human
umbilical cord blood, normal peripheral blood, and normal bone
marrow were drawn with informed consent of donors and with ethical
permission from the Department of Transplantation Sciences, Southmead Hospital, Bristol, from local institutes or from the Medical Clinic,
University of Tübingen, Tübingen, Germany.
Cell Isolation and Erythroid Cultures
Fresh human bone marrow, cord blood, and peripheral blood samples
were fractionated on Ficoll-Hypaque (1.077 g/mL; Sigma Chemical Co,
St Louis, MO). The light density cells were collected and any
contaminating erythrocytes lysed in 0.147 mol/L NH4Cl for 30 to 60
minutes at room temperature. CD341 cells were isolated using the
Miltenyi Biotech (Bergisch Gladbach, Germany) Mini-MACS CD34
stem cell isolation kit as specified by the manufacturer. Cells were
resuspended and cultured at 3,000 to 5,000 cells per mL in 24-well
Falcon 3047 tissue culture plates (Becton Dickinson, Sunnyvale, CA) in
defined StemBio-A.Erythro-CNRS serum-free media (StemBio-CNRS,
Villejuif, France) containing optimal concentrations of the cytokines,
IL-3, IL-6, Steel factor, and erythropoietin in a humidified incubator at
37°C in the presence of 4.5% (vol/vol) CO2 and 5% (vol/vol) O2 gas
mixtures. Cells were obtained on days 5, 6, 9, and 13; the nucleated cell
number determined; and cytospins phenotyped after centifugation onto
slides as described below. In some instances, the cells were stained with
antibodies and analyzed on the FACSCalibur (Becton Dickinson).
Peripheral blood granulocytes (neutrophils) were isolated from 1 mL of
Ficoll-hypaque–pelleted cells (density .1.077 g/mL) after a 20-second
erythrocyte lysis with 20 mL of distilled water. After mixing, 20 mL of
0.308 mol/L ice-cold NaCl was added and the cells washed twice before
staining.
Generation and Characterization of CD164 Antibodies
The CD164-specific MoAbs, 103B2/9E10 and 105A5, were generated5,7 after the immunization of mice with the erythromegakaryocytic
cell line, MOLM-1; antibody 67D2 by immunization with the breast
carcinoma cell line T47-D (obtained from the American Tissue Culture
Collection); and antibody N6B6 by immunization with the pre–B-cell
line Nalm-1 (obtained from the German Collection of Microorganisms
and Cell Cultures), according to previously described methods.36 The
103B2/9E10 and 105A5 MoAbs were used for expression cloning.
Comparative analysis revealed that both MoAbs recognized isolated
FDCP-1 transfectant cell lines expressing human CD164. A detailed
description of the isolation, sequencing, and generation of CD164
transfectant cell lines is described separately.7 In addition, the 67D2 and
N6B6 MoAbs were also found to specifically recognize murine FDCP-1
cells expressing human CD164 but not the parental FDCP-1 cells (see
Fig 1).
Antibodies and Antibody Conjugates
The isotypes of the MoAbs were determined by enzyme-linked
immunosorbent assay (ELISA) (Boehringer-Mannheim, Mannheim,
Germany). The murine 103B2/9E10 (IgG3 isotype), 105A5 (IgM
isotype), 67D2 (IgG1 isotype,) and N6B6 (IgG2a isotype) MoAbs to
human CD164 were used as culture supernatants or purified Ig
preparations. The mouse anti-human CD34 MoAb (clone 43A1; IgG3)
was generated by immunization with KG1A cells and then assigned to
the CD34 cluster as described by Greaves et al.37 For immunohistochemistry, mouse MoAbs to human CD3 (clone 3D4; IgG1), glycophorin A
(clone JC159; IgG1), glycophorin C (clone Ret40S; IgG1), and band III
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CD164 EXPRESSION AND CHROMOSOME LOCATION
(clone Q1/156; IgG1) were purchased from Dakopatts (Copenhagen,
Denmark) as culture supernatants or purified Ig fractions. For dual and
multicolor fluorescence-activated cell sorter (FACS) analysis, the
following mouse antibody conjugates, coupled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or PerCP were used: (1) CD3-PE
or -FITC (clone SK7; IgG1); CD4-FITC (clone SK31,2; IgG1); CD8FITC (clone SK1; IgG1); CD14-FITC (clone MOP9; IgG2b); CD20-PE
or -FITC (clone L27; IgG1); CD19-PE or -FITC (clone SJ25C1; IgG1);
HLA-DR-PE (clone L243; IgG2a); CD34-FITC, -PE, or -PerCP (clone
8G12; IgG1); CD38-PE (Hb-7; IgG1); and CD33-PE (clone p67.6; IgG1;
all from Becton-Dickinson, San Jose, CA); (2) CD117-PE (clone 95C3;
IgG1); CD65-FITC (clone 88H7; IgM); CD66b-FITC (clone 80H3;
IgG1); and anti-glycophorin A-PE (clone 11E4B7; IgG1; all from
Immunotech, Marseille, France); (3) CD90-PE (clone 5E10; IgG1;
Pharmingen, Hamburg, Germany); (4) CD71 (clone T56/14; IgG1;
Biotrend, Cologne, Germany); and (5) AC133-PE (clone AC133; IgG1;
Amcell Corporation, Sunnyvale, CA). The CD135-PE (FLT3; clone
SF1.340; mouse IgG1) was a kind gift from Drs O. Rosnet and A. van
Agthoven, Marseille, France.38 As comparative negative controls,
irrelevant antibodies (Dakopatts, Immunotech, or Becton Dickinson) of
the same isotypes and with equivalent fluorescent tags or phosphatebuffered saline (PBS) were used in place of primary antibodies. Texas
Red (TR)-, FITC-, or PE-conjugated isotype-specific secondary antibodies were purchased from Southern Biotechnology Associates Inc,
Birmingham, AL, and FITC (Fab8)2 goat or rabbit anti-mouse Ig from
Dianova, Hamburg, Germany or Dakopatts. All antibodies were used at
5 to 10 µg/mL per 107 cells/mL or at the concentrations recommended
by the manufacturer.
Immunofluorescence Staining for Flow Cytometric Analysis
and Cell Sorting
Single-color staining. Cells were blocked with 10% (vol/vol)
human AB serum (Behring, Marburg, Germany) or human gamma
globulin (30% [vol/vol] Fc blocking reagent; Mitenyi Biotech) for 10 to
20 minutes at 4°C and then labeled with saturating concentrations of
culture supernatants of the CD164 MoAbs, 103B2/9E10, 105A5, 67D2,
or N6B6 MoAbs and counterstained with PE-conjugated goat antimouse isotype-specific Ig or with FITC-F(ab8)2 goat or rabbit antimouse Ig. To indicate background staining, isotype-matched control
antibodies were used. For these and all subsequently described FACS
analyses, positivity was determined by the use of arbitrary gates set
against these negative isotype-matched controls.
Two-color staining of bone marrow and cord blood cells. In some
experiments (see Fig 3), bone marrow or cord blood cells were labeled
with the CD164 MoAbs, 103B2/9E10 (IgG3), 105A5 (IgM), or N6B6
(IgG2a) and FITC-CD34 (8G12; IgG1) and then counterstained with
PE-conjugated goat anti-mouse isotype-specific Ig. Alternatively, cells
were stained with 67D2 (IgG1) plus CD34 (43A1; IgG3) before
application of PE-conjugated anti-mouse IgG1 and FITC–anti-mouse
IgG3. Peripheral blood mononuclear cells (see Fig 9) were labeled with
103B2/9E10 or an irrelevant IgG32 control antibody, together with
CD4-FITC, CD8-FITC, CD3-FITC, CD20-FITC, or irrelevant FITC–
isotype-matched controls before counterstaining with PE-conjugated
anti-mouse IgG3.
Three-color staining of bone marrow and cord blood cells. Ficoll
separated bone marrow and cord blood mononuclear cells or purified
CD341 cells were labeled with the 103B2/9E10 MoAb and stained with
CD34-PerCP (clone 8G12) and with the PE-conjugates (as described in
Figs 4, 5, 8, and 10), followed by FITC–anti-mouse IgG3.
Flow cytometric analysis and cell sorting. Cells were analyzed on a
FACSCalibur using Cellquest software or analyzed and sorted on a
FACS-Vantage flow cytometer (all from Becton Dickinson). The
fluorescence of FITC, PE, and PerCP was excited with an argon ion
851
laser at 488 nm and detected at emission wavelengths of 530 nm, 570
nm, and 670 nm, respectively. Cell sorting of CD341CD164(103B2/
9E10 epitope)1 and CD342CD164(103B2/9E10 epitope)1 bone marrow cells was performed at 25 kHz using FITC-CD34 and the
103B2/9E10 MoAb followed by PE-conjugated anti-mouse IgG3.
Sorted fractions were cytocentrifuged and stained with May-GrunwaldGiemsa solution for morphological analysis.
Dual-Color Immunofluorescence of Cytospins
All incubations were performed at room temperature for 30 minutes.
After Fc receptor blockade as above, cytospins were incubated with
103B2/9E10, 105A5, or N6B6 MoAbs followed by FITC-conjugated
isotype-specific secondary antibodies (1:25 dilutions in PBS). After
washing in PBS, cells were incubated with CD3 (as a negative irrelevant
IgG1 control), anti–glycophorin A, anti–glycophorin C, or anti–band III
and developed with TR-conjugated goat anti-mouse IgG1 (1:50 dilution
in PBS). The slides were mounted in fluorescent mounting medium
(Dakopatts) containing 2% (wt/vol) 4,6-diamidine-2-phenylindole dihydrochloride (DAPI; Sigma) and viewed under an Olympus BX-60
fluorescence microscope (Olympus, London, UK).
Cross-Blocking Analysis of CD164-Specific MoAbs
For epitope mapping studies, MOLM-1 cells were incubated with
either 103B2/9E10, 105A5, 67D2, N6B6 (all different isotypes), or with
isotype-matched negative control antibodies all at concentrations of 5
µg/mL at 107 cells/mL for 30 minutes on ice. After washing, cells were
incubated either with the same MoAbs to indicate positive or negative
controls, respectively, or with test CD164 MoAbs that differed from the
blocking MoAbs. In the final step, cells were stained with PEconjugated anti-Ig that specifically identified the CD164 test MoAb and
analyzed on a FACSCalibur flow cytometer. The percent blocking was
calculated as follows: 100 2 [(Median Fluorescence of Cells Stained
With Test MoAb After Incubation with Blocking MoAb) 2 (Median
Fluorescence of Cells Stained With Isotype-Matched Negative Control
MoAb)] 4 [(Median Fluorescence of Cells Stained With Test MoAb After
Incubation With Negative Control MoAb) 2 (Median Fluorescence of
Cells Stained With Isotype-Matched Negative Control MoAb)] 3 100.
Sensitivity of CD164 Epitopes to Vibrio cholerae
Neuraminidase Treatment
Calu-1, KG1A, MOLM-,1 and BV-173 cells were incubated with 0.2
U/mL of neuraminidase from V. cholerae (Calbiochem, Heidelberg,
Germany) for 60 minutes at 37°C in 250 mL PBS. After washing with
ice-cold staining buffer, cells were blocked and labeled with each of the
CD164 MoAbs or the appropriate negative isotype-matched control
MoAb and counterstained with PE-conjugated isotype-matched anti-Ig.
Cells were analyzed on a FACSCalibur, and percent binding was
calculated as follows: [(Median Fluorescence of NeuraminidaseTreated Cells Stained With CD164 MoAb) 2 (Median Fluorescence of
Neuraminidase-Treated Cells Stained With the Isotype-Matched Negative Control MoAb)] 4 [(Median Fluorescence of Untreated Cells
Stained With CD164 MoAb) 2 (Median Fluorescence of Untreated
Cells Stained With the Isotype-Matched Negative Control MoAb)] 3
100.
CD164 cDNA and Probes
Two human CD164 cDNA clones (clone 105A5 and 103B2), isolated
from a retroviral cDNA library of human bone marrow stromal cells39,40
by expression cloning with the 105A5 and 103B2/9E10 MoAbs,5,7 and
subcloned into the pGEM-T vector (Promega, Southampton, UK) were
transformed into XL2 Blue-MRF8 bacteria (Stratagene, Cambridge,
UK). Large-scale DNA preparations of the two CD164 cDNA clones
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852
WATT ET AL
C), which contained CD164 cDNA sequence, including a region
spanning 1309 to 2487 bp of untranslated CD164 sequence in the
38UTR. The restriction enzymes were purchased from BoehringerMannheim and endonuclease digestions performed in the buffers
supplied with the enzymes and according to the manufacturer’s
instructions. Restriction fragments were isolated after electrophoresis
on 1.5% to 2% (wt/vol) NuSieve agarose (FMC Bioproducts, Rockland,
ME) in TAE buffer (40 mmol/L Tris-acetate buffer, pH 8.5, containing 2
mmol/L EDTA) and purification on Wizard PCR (polymerase chain
reaction) preps columns (Promega) as detailed by the manufacturer.
DNA concentrations were determined by agarose gel electrophoresis
against known phage DNA quantitation standards (GIBCO-BRL). For
labeling, 20 to 50 ng of each cDNA probe was labeled with 50 µCi
a-32P–dCTP (Amersham Int, High Wycombe, Bucks, UK) using the T7
Quickprime kit (Pharmacia, Uppsala, Sweden) according to the manufacturer’s protocol.
Fig 1. Binding of CD164 MoAbs to stable FDCP-1 cells expressing
CD164. FDCP-1 cells stably transfected with CD164 cDNA were
labeled with the N6B6, 67D2, 105A5, or 103B2/9E10 MoAbs or with
irrelevant first MoAbs of the same isotype. The reaction was developed with FITC-conjugated anti-mouse Ig antibody as detailed in
Materials and Methods, and the median fluorescence values were
determined after FACSCalibur analysis. The results of one of three
experiments are shown. The IgG negative control histogram contains
the second FITC antibody only. The median fluorescence intensity
values for the negative isotype matched controls were: no first
MoAb 5 3.4; IgG3 5 3.4; IgG2a 5 3.4; IgM 5 3.4; and IgG1 5 3.3. The
median fluorescence for each CD164 MoAb was: 103B2/9E10 5 45.32;
105A5 5 26.42; 67D2 5 138.24; and N6B6 5 121.37.
were purified on CsCl gradients,41 completely sequenced5,7 using
oligonucleotide primers, and used to probe genomic libraries. The
nucleotide and the predicted peptide sequences have been described
previously.7 Initial screening of a human PAC library was performed
with two CD164 cDNA probes, A and B, derived from the 105A5 cDNA
clone in the pGEMT vector (Promega). The Sac 1/Spe 1 probe A
fragment contained 1 to 1907 bp of cDNA sequence, where bp1
indicates the translational start site. This encompasses the whole
translated sequence plus part of the 38UTR. The Spe 1/Apa 1 probe B
fragment comprised 1908 to 2867 bp of 38UTR. Subsequent screening
of the human PAC subclones and of Southern blots was performed with
a 1.173 kb EcoRV/HindIII CD164 probe from the 105A5 cDNA (Probe
Isolation and Subcloning of PAC Clones
The human PAC library derived from normal human male genomic
DNA in the PCYPAC2N vector, containing 120,000 clones and grided
on to 7 Hybond N filters, was kindly provided by the HGMP Resource
Centre (Cambridge, UK) via Dr P. de Jong’s group. Filters were
hybridized with the a-32P–labeled human CD164 probes A and B as
described below. Five identified positive clones were obtained from the
HGMP Resource Centre and grown overnight in 23 TY broth
containing 25 µg/mL kanamycin. These were digested with EcoR1,
electrophoresed on a 0.7% (wt/vol) agarose gel, Southern blotted, and
probed with a-32P–labeled CD164 probe C or analyzed by PCR using
forward MGC-GP-F3 and reverse MGC-GP-B3 oligonucleotide primers (Oswell, Southampton, UK) and the PCR products sequenced as
described below. The MGC-GP-F3 (455-479) and MGC-GP-B3 (16011577) primers were 58-CCTCACAACCTGTGCGAAAGTCTAC-38
and 58-ACTCAAGACAGTCTGGTGG AAATCC-38, respectively. Two
positive clones (termed CD164 PAC1 and PAC5) were selected and
digested with Pst 1 or BglII according to the manufacturer’s instructions
and used directly for ligation to pCRScript vector. The pCRScript
(SK1) vector was digested with Pst 1 or BamH1 for 1 hour at 37°C in
the appropriate buffers before the addition of 1 µL of shrimp alkaline
phosphatase (Boehringer Mannheim) for 1 hour at 37°C. After heat
inactivation at 68°C for 15 minutes, the enzyme-digested vector was
Table 1. Epitope Mapping of CD164-Specific MoAbs
CD164 MoAb Reactivity With Soluble
Recombinant CD164 Domain Constructs*
% Inhibition of Test CD164 MoAb Binding
Test CD164 MoAb
103B2/9E10
105A5
N6B6
67D2
103B2/9E10
—
60.4
21.4
13.5
Blocking CD164 MoAb
105A5
N6B6
0
3.5
—
0
0
—
3.5
98.3
67D2
3.5
0
90.1
—
Exon 1
Exons 1 and 2
Exons 1, 2, and 3
1
1
2
2
1
1
2
2
1
1
1
1
MOLM-1 cells were stained with one of the unconjugated CD164-blocking MoAbs or with the appropriate isotype-matched negative control
MoAb before labeling with the second test CD164 MoAb, which differed from the blocking MoAb. After staining these cells with PE-conjugated
anti-Ig that specifically reacted with the CD164 test MoAb, they were analyzed on the FACSCalibur and their median fluorescence intensities
determined. Percent blocking was calculated from median fluorescence intensities as indicated in Materials and Methods.
*Soluble domain deletion constructs, derived from exons 1; 1 and 2; and 1, 2, and 3 were prepared after determination of the intron-exon
structure of the CD164 gene,42 subcloned into the pEFBos-Ig mu vector,43 and expressed in 293T cells as soluble recombinant proteins as detailed
elsewhere.45 The predicted peptide sequences for these soluble constructs are: Exon 1: DKNTTQHPNVTTLAPISNV TSAPVTSLPLVTTPAP; Exon 2:
ETCEGRNSCVSCFNVSVVNTTCFWIECK; Exon 3: DESYCSHNSTVSDCQVGNTTDFCS with exon 1 predicted to contain nine O-linked glycosylation
sites on the serine and threonine residues underlined. Potential N-linked glycosylation sites are indicated in bold lettering. The data represent a
summary of the results of CD164 MoAb binding to these soluble recombinant proteins as determined by ELISA assays.45
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CD164 EXPRESSION AND CHROMOSOME LOCATION
853
Table 2. Sensitivity of CD164 Epitopes to Sialidase
Cell Line
CD164 MoAb
Calu-1
KG1A
% CD164 MoAb Binding
103B2/9E10
93.8 6 5.6
74.8 6 8.7
105A5
18.8 6 1.1
060
N6B6
88.9 6 10.2
85.2 6 6.4
67D2
118.5 6 1.5
94.6 6 4.4
Calu-1 and KG1A cells were incubated with 0.2 U/mL of Vibrio
cholerae sialidase for 60 minutes at 37°C. Cells were then washed and
stained with each CD164 MoAb indicated or with an isotype-matched
negative control MoAb and counterstained with PE- or FITCconjugated isotype-matched anti-Ig. The median fluorescence for
each cell group was determined by analysis on a FACSCalibur, and the
percent binding determined as indicated in Materials and Methods.
Values are means 6 SD of three determinations.
precipitated on dry ice with 2 volumes of ethanol in the presence 0.3
mol/L sodium acetate buffer, pH 5.2. The resulting pellet was resuspended in double distilled water and used for ligations of the digested
PAC fragments. In brief, all ligation reagents used were derived from
the pMOS-Blue kit (Amersham). Fifty nanograms of enzyme-digested
and phosphatased pCRScript vector was mixed with 170 to 350 ng
enzyme-digested PAC clone, 1 µL 103 ligation buffer, 0.5 µL 100
mmol/L dithiothreitol (DTT), 0.5 µL 10 mmol/L adenosine triphosphate
(ATP), and 0.5 µL T4 DNA ligase (4 U/mL) in a final volume of 10 µL
and incubated at 15 to 20°C for 2 hours. The ligated samples were
transformed into XL2-Blue MRF8 competent bacteria, plated onto
L-broth agar plates containing 50 to 100 µg/mL ampicillin, and grown
overnight at 37°C. Lifts were made on Hybond N filters, and the filters
were probed with a-32P–labeled CD164 Probe C. Positive clones were
picked off each master plate, grown in 10 to 15 mL of L-broth
containing 50 to 100 µg/mL ampicillin, and characterized by restriction
enzyme digestion, Southern blotting, and probing with a-32P–labeled
CD164 Probe C or by PCR analysis using the MGC-GP-F3/MGC-
Fig 2. Differential expression of CD164 epitopes
on peripheral blood lymphocytes, monocytes, and
granulocytes. Ficoll separated cells were stained
with CD164-specific MoAbs, 103B2/9E10, 105A5,
67D2, and N6B6, selected for lymphocytes, monocytes, or granulocytes on the basis of forward and
side scatter parameters and analyzed on a FACSCalibur flow cytometer. Representative histograms from
one of three independent experiments are shown.
Median fluorescence values after subtraction of the
median fluorescence value of the negative isotypematched control for each population in this experiment are as follows: for the lymphocytes: N6B6 5
8.01; 67D2 5 6.49; 103B2/9E10 5 5.01; 105A5 5 1.37;
for the monocytes: N6B6 5 35.05; 67D2 5 58.51;
103B2/9E10 5 53.50; 105A5 5 2.09; for the granulocytes: N6B6 5 2.41; 67D2 5 4.41; 103B2/9E10 5 2.59;
105A5 5 0.31. Mature non-nucleated glycophorin-A1
erythrocytes did not stain with any of the CD164
MoAbs.
GP-B3 primers and sequencing of the PCR products. Using oligonucleotide primers derived from the cDNA sequence, the CD164 gene was
contained in a 23-kb region of the PAC clones. Sequencing of these
fragments has identified 5 exons that encode the isolated CD164 cDNA,
interspersed with introns of varying sizes and extending from the
translational start site (bp1) into the 38 UTR ( to position 2867 bp of the
cDNA).42 Exons 1; 1 to 2; and 1, 2, and 3 corresponding to the CD164
cDNA were subcloned into the pEFBos-Ig mu vector,43 expressed as
soluble recombinant proteins in 293T cells, and analyzed for reactivity
with the 4 CD164 MoAbs or with isotype-matched negative controls by
Elisa assays.44,45 A detailed description of the PAC clones, the genomic
structure of CD164, and the epitope mapping using soluble recombinant
CD164 protein domains are presented in separate papers.42,45
PCR Analysis of Somatic Cell Hybrids, PAC Clones,
and Human Genomic DNA
DNA extracts from human x hamster or human x mouse somatic
cell hybrids were kindly provided by the HGMP Resource Centre,
Cambridge, UK. These (100 ng) were analyzed by PCR using the
MGC-GP-F3 and MGC-GP-B3 primer pairs described above or the
MGC-GP-F4 (1566-1591) and MGC-GP-B4 (2069-2045) primer pairs,
58-GTACCTTGAAAGGA TTTCCACCAGAC-38 and 58-CAAGTGCGAAACTCAGCCACTATTG-38, respectively (all from Oswell, Southampton, UK) to the CD164 cDNA sequence. PCR analysis was also
performed on two human male and female genomic DNA preparations,
human genomic DNA provided with the hybrid panel, the BglII subclone of
CD164 PAC1, and the CD164 cDNA clones, 103B2/9E10 and 105A5. The
PCR on the somatic cell hybrid panel was performed with 100 ng of each
chromosome 1-22; chromosome X; chromosome Y DNA; plus mouse,
human, and hamster DNA controls using the Expand Long Template
System (GIBCO-BRL) and the following program: a hotstart of 95°C
for 2 minutes, followed by 30 cycles of 95°C for 1 minute for
denaturation, 65°C for 30 seconds for annealing, and 69°C for 4 minutes
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854
for extension; and a final extension cycle of 69°C for 10 minutes.
Essentially the same protocol was used for PCR analysis of the PAC
clones and subclones and the CD164 cDNAs. PCR analysis was,
however, performed on the human genomic DNA samples using the
Advantage Klentaq System (Clontech, Palo Alto, CA) and the following
PCR program used: a hotstart of 95°C for 2 minutes, followed by 5
cycles of 95°C for 30 seconds and 72°C for 3 minutes, 5 cycles of 95°C
for 30 seconds and 70°C for 3 minutes, 25 cycles of 95°C for 30 seconds
and 68°C for 3 minutes, and a final extension cycle of 68°C for 7
minutes. The PCR products (10 µL) were analyzed by 2% (wt/vol)
agarose gel electrophoresis in Tris borate EDTA (TBE) buffer using a
1-kb DNA ladder (GIBCO-BRL) as the molecular weight marker and
blotted onto Nytran-N Nylon membranes (Schleicher and Schuell,
Dassel, Germany). These membranes were prehybridized in 30 mL of
50% (wt/vol) formamide, 43 SSC, 0.001 mol/L EDTA, 0.05 mol/L
WATT ET AL
sodium phosphate buffer (pH 7.2), 8% (wt/vol) dextran sulfate, 100
µg/mL of sonicated Herring sperm DNA (Sigma), and 25 µg/mL yeast
tRNA (Sigma), 103 Denhardt’s solution46 and 50 µg human placental
DNA (Sigma) at 42°C for 1 to 18 hours before addition of the
a-32P–labeled CD164 probes A, B, or C for 18 to 48 hours at 42°C.
Filters were washed twice at 42°C in 23 SSC with 0.1% (wt/vol)
sodium dodecyl sulfate (SDS) for 30 minutes each, 3 times at 65°C in
0.23 SSC containing 0.1% (wt/vol) SDS for 45 minutes each, and then
exposed to Kodak X-OMat film (Eastman Kodak, Rochester, NY) with
intensifying screens at 270°C as described.46
Automatic Sequencing
Miniprep or CsCl gradient prepared DNA (200 µL) was denatured at
37°C for 30 minutes with 20 µL 2 mol/L NaOH and 2 mmol/L EDTA
Fig 3. Differential expression of CD164 epitopes
on CD341 bone marrow and cord blood cells from
normal donors. Mononuclear cells were stained with
CD164-specific MoAbs 103B2/9E10, 105A5, 67D2, and
N6B6 together with CD34 as indicated in Materials
and Methods and gated on forward and side scatter
(top dot plots) before analysis on a FACSCalibur flow
cytometer. In four independent bone marrow and
three independent cord blood analyses, 4.6 6 0.8%
and 1.2 6 0.1% of the scatter gated cells were CD341,
respectively. In the representative experiment shown,
median fluorescence values for the CD341CD1641
subsets of the human bone marrow (BM) gated cells
were: N6B6 5 138.24; 103B2/9E10 5 212.88; 105A5 5
45.32; 67D2 5 198.10 and for the CD342CD1641 gated
subsets were: N6B6 5 294.97; 103B2/9E10 5 184.34;
105A5 5 220.67; 67D2 5 697.83. Median fluorescence
values for the CD341CD1641 subsets of human cord
blood (CB) cells were: N6B6 5 148.55; 103B2/9E10 5
294.27; 105A5 5 37.86; 67D2 5 283.87 and for the
CD342CD1641 gated subsets were: N6B6 5 119.71;
103B2/9E10 5 69.78; 105A5 5 77.74; 67D2 5 273.84.
Cells were also labeled with CD34-FITC or the CD34
MoAb, 43A1, plus an anti–IgG3-FITC secondary antibody, together with isotype-matched irrelevant control MoAbs for each CD164 MoAb used plus antiisotype–specific PE-conjugated antibodies. Under
these conditions, 0.08% to 0.12% of cells occured in
the CD341 Ig Isotype2 gates, and 0.08% to 0.38% of
cells occured in the CD342 Ig Isotype2 gates.
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CD164 EXPRESSION AND CHROMOSOME LOCATION
and then ethanol precipitated. In some cases, the PCR products were
directly sequenced after PEG precipitation. The PCR products (4 µL) or
the DNA (0.5 µg) in 5 µL sterile double-distilled were added to 4 µL of
ABI Prism Ready Dye de-oxy Terminator mix (Applied Biosystems,
Perkin Elmer, Foster City, CA) with 0.025 µg M13 (Stratagene) or 3
pmol of CD164 forward or reverse primers (Oswell) in 2 to 3 µL sterile
double-distilled water, and the sequencing reaction was performed
according to the manufacturer’s protocol before analysis on an ABI 373
Automatic Sequencer (Applied Biosystems). The sequences were
analyzed using MacVector, Seqed, Assemblign, Analysis, and Sequencher software packages (Oxford Molecular, Oxford, UK), aligned
to each other and to the CD164 cDNAs and a contig generated.
FISH
Metaphase spreads were prepared from phytohemagglutininstimulated normal human lymphocytes by use of standard techniques.
Before hybridization, the slides were denatured in 70% (vol/vol)
formamide and 23 SSC at 73°C for 3 minutes, then washed in 23 SSC
and dehydrated through an ethanol series of cold 70% (wt/vol), 95%
(wt/vol), and absolute ethanol. The BglII and Pst 1 subcloned PAC
DNA in the pCRScript vector was biotinylated using the Bionick kit
(GIBCO-BRL). Two hundred nanograms labeled probe was mixed with
5 µg Cot-1 DNA (GIBCO-BRL), precipitated, resuspended in 11 µL
hybridization mix, denatured at 85°C for 5 minutes, and allowed to
preanneal at 37°C for 30 minutes. The probe was then applied to a
denatured slide and hybridized overnight. Slides were washed in 50%
(vol/vol) formamide, 23 SSC pH 7 at 42°C, followed by 13 SSC at
Fig 4. Triple-color analysis of primitive markers
on CD341CD164(103B2/9E10 epitope)1 bone marrow
cells. CD341 bone marrow cells were labeled with
103B2/9E10 plus CD34-PerCP and with each of the
antibody PE conjugates indicated before staining
with FITC-F(ab)2 goat anti-mouse IgG3. Plots are
two-color dot plot displays from a representative
experiment of CD34 purified cells gated on forward
and side scatter parameters (left top dot plot) and on
PerCP-CD341 fluorescence and side scatter (middle
dot plot). The arbitrary gates indicated on the dot
plots were determined with appropriate isotypematched negative controls. Examples of the IgG1/
IgG3 isotype negative controls (top right dot plot)
and of CD164(103B2/9E10 epitope)/anti–IgG3-FITC
staining plus labeling with a nonbinding PE-conjugated anti–glycophorin A conjugate (bottom right
dot plot) are indicated. Similar results were obtained
for the IgG1/IgG2a isotype negative controls.
855
60°C. Blocking solution (3% [wt/vol] BSA, 43 SSC and 0.1% [vol/vol]
Tween 20) was applied and slides incubated at 37°C for 30 minutes.
After incubation, avidin-FITC (diluted in 1% [wt/vol] BSA, 43 SSC,
0.1% [vol/vol] Tween 20) was applied and slides incubated at 37°C for
40 minutes. Slides were washed in 43 SSC, 0.1% (vol/vol) Tween 20 at
42°C and counterstained with 200 ng/mL DAPI, followed by 2 minutes
in 23 SSC. Slides were mounted in Citifluor and images captured
using a Photometrics KAF 1400-500 CCD camera (Photometrics,
Tuscan, AZ) attached to a Zeiss Axioskop (Zeiss, New York, NY)
epifluorescence microscope. Separate images of probe signals and
DAPI banding patterns were pseudocoloured and merged using SmartCapture software (Vysis Inc, Chicago, IL).
RESULTS
The MoAbs 103B2/9E10, 105A5, 67D2, and N6B6 Specifically
Detect CD164 and Recognize Distinct CD164 Epitopes
The predicted amino acid sequence of cDNA clones selected
by expression cloning of a human bone marrow stromal cell
cDNA library with the CD164 MoAbs, 103B2/9E10 and
105A5, were identical. The complete sequence is described
separately.7 Parental FDCP-1 cells or FDCP-1 cells expressing
these human CD164 cDNAs were stained with 67D2 and N6B6
MoAbs, as well as with 103B2/9E10 and 105A5, and analyzed
by flow cytometry. All four MoAbs selectively recognized
CD164 cDNA transfected, but not parental, FDCP-1 cells (Fig
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856
1). Our more recent data42 indicate that the identified CD164
cDNA is encoded by 5 separate exons, with the 103B2/9E10
and 105A5 MoAbs reacting with soluble recombinant protein
derived from exon 1. In contrast, the N6B6 and 67D2 MoAbs
do not react with soluble recombinant proteins derived from
exon 1 or exons 1 and 2, but bind to a recombinant protein
comprising the extracellular domains encoded by exons 1, 2 and
3, as summarized in Table 1. Detailed epitope analyses using
these and other soluble CD164 recombinant constructs are
described elsewhere.45 Reactivities of the four MoAbs with cell
surface–expressed epitopes of CD164 were analyzed in more
detail on a set of hematopoietic and nonhematopoietic cell lines
(data not shown). Because all four MoAbs reacted strongly with
the MOLM-1, Calu-1, KG1A, and BV-173 cell lines, these were
used in further experiments. Cross-blocking experiments were
performed to determine whether the 103B2/9E10 and 105A5 or
the N6B6 and 67D2 MoAbs recognized identical or different
epitopes on cell lines. As indicated in Table 1, prelabeling of
MOLM-1 with the 103B2/9E10 and 105A5 MoAbs did not
substantially block 67D2 or N6B6 binding, nor did 105A5
MoAb prelabeling prevent the 103B2/9E10 MoAb from binding. However, 67D2 almost completely blocked N6B6 staining,
and vice versa. Partial blocking of 105A5 was observed after
103B2/9E10 prelabeling, but not vice versa. This partial
inhibition might be caused by conformational changes of the
CD164 molecule potentially induced by the 103B2/9E10 MoAb.
These results support those obtained for exon mapping and
show a close association between the 103B2/9E10 and 105A5
epitopes and between the 67D2 and N6B6 epitopes.
WATT ET AL
103B2/9E10, 67D2, and N6B6, whereas staining with the
105A5 MoAb resulted only in faint signals near background
staining. Monocytes generally showed higher levels of CD164
MoAb binding than did lymphocytes. Median fluorescence
intensity (MFI) values for CD164 staining of monocytes were
Sensitivity of CD164 Binding to Sialidase Treatment
To further characterize CD164 epitopes, Calu-1 and KG1A
cells were treated with V. cholerae sialidase, an enzyme that
selectively hydolyzes N- or O-acyl-neuraminic acids, which are
a2,3-, a2,6- or a2,8-linked to galactose, hex, NAc, or N- or
O-acylated neuraminyl residues in oligosaccharides, before
staining with the CD164 MoAbs. Table 2 shows that the
epitopes detected by the 103B2/9E10, N6B6, and 67D2 MoAbs
are relatively resistant to desialylation compared with the
untreated positive controls, whereas binding of 105A5 is almost
completely abrogated. Preliminary results also indicate that the
105A5 MoAb (in contrast to the other three CD164 MoAbs)
does not bind to sialidase treated MOLM-1 and BV-173 cell
lines, with labeling being reduced to 7.8% and 0%, respectively,
of the untreated cells. These sialidase studies are in line with the
exon localization and cross-blocking studies in showing that,
although the epitopes recognized by the 103B2/9E10 and
105A5 MoAbs are closely associated, they are distinct from one
another and are different from those recognized by N6B6 and
67D2.
Differential Expression of CD164 Epitopes on Normal Adult
Peripheral Blood Cells and on Normal Bone Marrow and
Cord Blood CD341 Cells
Staining of Ficoll separated peripheral blood cells from
normal donors with the CD164 MoAbs 103B2/9E10, 105A5,
67D2, and N6B6 and the separation of cell subsets based on
forward and side scatter parameters revealed that peripheral
blood lymphocytes were weakly cell-surface positive with
Fig 5. CD342CD164(103B2/9E10 epitope)1 bone marrow and cord
blood cells contain nucleated erythroid cells. Ficoll separated bone
marrow or cord blood mononuclear cells were labeled with 103B2/
9E10 together with the PE-antibody conjugates indicated on the dot
plots before staining with FITC-F(ab)2 goat anti-mouse IgG3 and flow
cytometric analyses. Plots are two-color displays of cells gated on
the lymphoid/erythroid/blast cell gate in Fig 3. The arbitrary gates
shown on the dot plots were determined with negative control
isotype-matched irrelevant antibodies as described in Materials and
Methods.
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CD164 EXPRESSION AND CHROMOSOME LOCATION
28.9 6 6.8 for N6B6, 41.5 6 15.6 for 67D2, 22.3 6 27.1 for
103B2/9E10, and 7.5 6 4.8 for 105A5, after subtraction of the
MFI values for isotype-matched negative controls and where
the values are means 6 SD of three independent experiments.
This compares with MFI values for lymphocyte staining of
7.9 6 3.4 for N6B6, 8.3 6 2.7 for 67D2, 2.7 6 2.0 for
103B2/9E10, and 3.6 6 2.0 for 105A5. Mature non-nucleated
erythrocytes showed negligible staining with all four CD164
MoAbs (data not shown). Granulocytes were very weakly
stained by the CD164 MoAbs, except for 105A5, which was
completely negative. The relevant fluorescence profiles and
MFI values of a representative experiment are shown in Fig 2.
The expression of the four CD164 epitopes was significantly
higher on bone marrow and cord blood CD341 cells than on
peripheral blood mononuclear cells. When CD341 cells were
analyzed, staining was more consistent with the N6B6 and
67D2 MoAbs. In four independent experiments, 81.8 6 9.9%
Fig 6. Immunofluorescence staining with
CD164 and erythroid specific MoAbs of cord
blood CD341 cells cultured under erythroid
conditions. CD341 cells were isolated from
cord blood and cultured for 5 to 9 days under
the conditions described. Cultured cells were
cytocentrifuged, fixed in acetone, and stained
by dual immunofluorescence using DAPI as a
nuclear counterstain. Of interest are day-5
cultured cells stained in the Golgi region with
103B2/9E10 (A) and105A5 (B) antibodies.
These cells were negative with anti-glycophorin C (C). Similar staining was obtained for
day-9 cultured cells using 103B2/9E10 (D).
Also shown are day-9 cells labeled with an
IgG3 irrelevant isotype-matched negative control (E), anti–glycophorin C (F), anti–glycophorin A (G), and band III (H); and day-6
cultured cells labeled with 103B2/9E10 plus
FITC–anti-mouse IgG3 (I through K) and glycophorin C (I), glycophorin A (J), or band III (K)
plus Texas Red–anti-mouse IgG1. Arrow heads
indicate double-stained cells and arrows show
cells stained only with 103B2/9E10 (I through
K); (A through H: 40 3 original magnification; I
through K: 100 3 original magnification).
857
and 81.5 6 9.9% of CD341 bone marrow cells expressed these
respective epitopes. This is in contrast to more variable staining
with the 103B2/9E10 and 105A5 MoAbs with 70.2 6 26.3%
and 63.3 6 26.1% of CD341 cells labeling, respectively.
CD341 bone marrow cells expressing the 103B2/9E10 epitope
of CD164 exhibited the highest MFI values. Examples of dot
plots and MFI values for the CD341CD1641 cell subsets
defined with the different CD164 MoAbs are shown in Fig 3.
Three-color immunofluorescence analysis indicated that the
CD341CD164(103B2/9E10)1 bone marrow cells coexpressed
the N6B6 and 67D2 epitopes (data not shown). Some differential cell-surface staining with the CD164 MoAbs was also
observed on cord blood. When cord blood mononuclear cells
were dual-labeled with CD34 and the different CD164 MoAbs
(Fig 3), approximately 90% of the CD341 cells expressed the
103B2/9E10 (92.6 6 2.8%), 67D2 (88.7 61 0.5%), and N6B6
(89.1 6 8.9%) epitopes, whereas only 55.0 6 11.6% of these
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858
cells were 105A5 epitope1, where values are means 6SD of
three independent experiments. Again, the MFI values obtained
for the CD341CD1641 cord blood subset were higher when the
103B2/9E10 epitope was detected than for the other CD164
reactive epitopes (Fig 3).
The CD341CD1641 Subset Contains Phenotypically
Primitive Cells
We have recently shown that the 103B2/9E10 epitope is
expressed by clonogenic progenitors, pre–colony-forming unit–
granulocyte-macrophage (pre–CFU-GM), colony-forming unit–
granulocyte, erythroid, monocyte, megakaryocyte (CFUGEMM), burst-forming unit–erythroid (BFU-E), and
granulocyte-macrophage colony-forming cells (GM-CFC), and
that exogenous addition of the 103B2/9E10 MoAb to single
CD341CD38lo/2 cells cultured in serum-free media in the
presence of IL-3, IL-6, G-CSF, and Steel factor can inhibit the
proliferation of these cells by up to 65%. Within these cultures,
5 6 3% of 103B2/9E10 treated cells compared with 14 6 2% of
cells treated with an irrelevant IgG3 MoAb proliferated by day
3, whereas 23 6 1% compared with 50 6 6%, respectively, of
these same cells proliferated by day 10 when 100 to 200 single
cells were analyzed.7 The 103B2/9E10 MoAb also partially
inhibited the adhesion of CD341 cells to bone marrow stroma.7
To gain more insight into the phenotype of the most immature
CD341CD164(103B2/9E10 epitope)1 subset, CD341 bone
marrow and cord blood cells were stained with 103B2/9E10 and
with selected MoAbs that define primitive CD341 cell subgroups. Representative dot plots of human bone marrow–
purified CD341 cells stained with these markers are shown in
Fig 4. The most primitive CD341 cells are thought to occur in
the CD90(Thy1)1, CD117(c-kit)1, CD135(FTL-3)1, AC-1331,
CD38lo/2, CD33lo/2, and CD71lo/2 subsets. It was therefore of
interest that 49.9 6 10.9%, 88.7 6 6.4%, 94.8 6 3.8%, and
54.3 6 17% of the CD341 CD164(103B2/9E10 epitope) 1
bone marrow cells expressed CD90, CD117, AC-133, and
CD135, respectively, in four independent experiments. This
represented greater than 90% of those CD341 bone marrow
cells that were CD901 (96.9 6 2.2%), CD1171 (98.3 6 1.1%),
AC1331 (96.9 6 1.8%), and CD1351 (90.2 6 10.2%). The
CD341CD164(103B2/9E10)1 bone marrow cell subset also
contained both HLA-DR1 and HLA-DRlo/2 cells and the
CD38lo/2 subset (Fig 4). In these same studies, 10.3 6 4.3% and
25.9 6 16.7% of the CD341 bone marrow cells were CD71lo/2
and CD33lo/2, respectively. Of these, an average of 65% of the
CD71lo/2 and 89% of the CD33lo/2 expressed the CD164(103B2/
9E10) epitope. Similar results (data not shown) were obtained
with cord blood, with essentialy all CD341 cells that coexpressed CD90, CD117, AC133, and CD135 being
CD164(103B2/9E10 epitope)1. These data indicate that CD164
is present on a very primitive hematopoietic CD341 progenitor
cell subset in addition to early clonogenic cells.
CD164 Expression Is Maintained on Nucleated Erythroid Cell
Subsets, but Is Lost on Terminal Erythroid Differentiation
The localization of day-14 BFU-E within the CD341CD164(103B2/9E10 epitope)1 fraction of human bone marrow5 is
consistent with the finding that a proportion of CD341CD164(103B2/9E10 epitope)1 cells coexpress the CD33, CD117,
WATT ET AL
HLA-DR, and CD71 markers (Fig 4). Because the CD164/
103B2/9E10 epitope was present on the BFU-E but absent from
the mature erythrocytes in peripheral blood, and as the whole
CD342 erythroid lineage from the proerythroblast stage to the
erythroblast, normoblast, and mature non-nucleated erythrocyte
stages can be defined by the regulated expression of three
markers, CD71, CD117, and the erythroid specific marker
glycophorin A,47 we used these markers to determine at which
stage in the erythroid lineage this CD164 epitope was lost.
Within the CD342 erythroid lineage, CD117 and CD71 are
expressed on both the proerythroblasts and erythroblasts, with
Fig 7. Flow cytometric analysis of day-9 cultured cord blood
erythroid cells. Cord blood CD341 cells were cultured for 9 days under
erythroid conditions as described. These cells were labeled with
MoAbs to the erythroid-specific markers, glycophorin C (A through
C), glycophorin A (D through F), or band III (G through I) or with the
CD164(103B2/9E10) MoAb (J through L), followed by FITC anti-mouse
IgG. Cells were analyzed on a FACSCalibur on the basis of forward
and side light scatter parameters. Either the whole cell population
was analyzed (dark histograms A, D, G, J) or the cells were gated into
two subsets based, both with low side scatter and with low to
medium forward scatter (R1) or medium to high forward scatter (R2)
before fluorescence analysis (dark histograms B, E, H, K for R1 and C,
F, I, L for R2). Values adjacent to each histogram represent median
fluorescence values. Isotype-matched negative controls for A through
I were an irrelevant IgG1 mouse MoAb (light histograms in A through
C) or an irrelevant mouse IgG3 MoAb (light histograms in J through
L). Median fluorescence values for negative controls were A 5 9; B 5
9; C 5 6; J 5 9; K 5 9; L 5 8.
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CD164 EXPRESSION AND CHROMOSOME LOCATION
glycophorin A appearing on some erythroblasts, being strongly
expressed on the more differentiated normoblasts, and then
maintained to the mature erythrocyte stage. Although CD71 is
maintained on normoblasts and then lost from reticulocytes and
mature erythrocytes, CD117 expression is lost at the normoblast
stage.47 When human bone marrow cells were defined by the
erythroid/lymphoid/blast cell light scatter parameters shown in
Fig 5, 52.9 6 9.2% of these cells were CD342CD164(103B2/
9E10 epitope)1, where values are means 6SD of four independent evaluations. When these cells were examined for their
coexpression of erythroid associated markers, averages of 93%,
82%, and 8% of these cells were CD711, glycophorin-A1, and
CD1171, respectively, in two independent experiments (Fig 5).
In addition, a mean of 88% of the glycophorin A1 nucleated
erythroid cells within these bone marrow samples were 103B2/
9E101. Similar results with cord blood (Fig 5) confirmed that
this prominent CD342CD1641 cell population consists predominantly of nucleated erythroid cells, although most but not all
glycophorin A1 nucleated cells express the 103B2/9E10 epitope. This was confirmed in a separate experiment by morphological analysis of sorted CD34lo/2CD164(103B2/9E10 epitope)1 bone marrow cells, which revealed that a large proportion
of these cells belonged to the erythroid lineage (9% lymphocytes, 13% monocytes, 1% promyelocytes, 49% polychromatic
normoblasts, 10% basophilic normoblasts, 3% erythroblasts,
and 13% proerythroblasts).
To analyze at which stage of erythroid development the
CD164(103B2/9E10) epitope was expressed, CD34-enriched
human umbilical cord blood cells were expanded in liquid
culture under conditions known to generate erythroid cells.
Cells were collected at days 5, 6, 9, and 13 of culture and their
Fig 8. Bone marrow lymphoid cells differentially
express the CD164(103B2/9E10) epitope. Representative dot plots from one of two separate experiments
showing CD341 bone marrow cells and Ficoll separated bone marrow mononuclear cells labeled with
the 103B2/9E10 MoAb or with an IgG3 negative
control MoAb plus CD34-PerCP and with the PEconjugated MoAbs indicated before counterstaining
with FITC anti-mouse IgG3 and analysis on the FACSCalibur. In contrast to the more mature CD342CD191
and CD342CD201 B cells, the immature CD341CD191
B-cell subset failed to express the CD164(103B2/
9E10) epitope. The median fluorescence intensity for
CD164(103B2/9E10) staining of the CD342CD31 cells
was 9.08 after subtraction of the isotype-matched
negative control value, indicating that the CD342CD31
T-cell subset was weakly reactive with the 103B2/
9E10 MoAb.
859
total nucleated cell number determined. In two separate experiments, 100- to 200-fold and 400- to 600-fold expansions in cell
numbers were observed in these cultures at days 9 and 13,
respectively. Cells were then centrifuged onto slides for fixation
and staining with the CD164 MoAbs and with the erythroid
specific markers, glycophorin C, glycophorin A, and band III,
with glycophorin C being expressed on BFU-E and proerythroblasts before the appearance of the other markers on erythroblasts and normoblasts. At day 5, the cultured cells were mostly
glycophorin A, glycophorin C, or band III negative (.98.5%).
However, these cells stained strongly in the Golgi region for all
CD164 MoAbs (95% to 99%). By days 6 to 9 of culture, the
majority of cells were glycophorin C positive (80 and 90%; Fig
6) and a subset of these had begun to express glycophorin A
(42% and 61%) and band III (11% and 24%). Essentially all
cells in these day-6 to -9 cultures maintained their expression of
the different CD164 epitopes (.92%; Fig 6 and data not
shown). It was of interest to note, however, that in these cultures
only a very small proportion of these nucleated erythroid cells
that were exclusively glycophorin A or band III positive either
failed to express or expressed very low levels of the CD164
epitopes. The day-9 erythroid-cultured cells were also analyzed
on the FACSCalibur to compare the levels of cell surface
expression of the CD164 epitopes and those of the erythroid
specific markers. As indicated in Fig 7, the cultured erythroid
cells at day 9 could be divided into two subsets, both exhibiting
low side light scatter characteristics, but with either low to
medium (the R1 subset) or medium to high forward light scatter
parameters (the R2 subset). Both subsets expressed glycophorin
C in high amounts (histograms B and C). However, cells within
the R1 or lower forward scatter gate expressed higher levels of
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860
glycophorin A (histogram E) and band III (histogram H) than
those in the R2 or higher forward scatter gate (histograms F and
I, respectively). The R1 gated cells were essentially all glycophorin A positive, but could be separated into two subsets based on
positive or negative expression of band III (histogram H). This
suggested that cells within the lower forward scatter gate were
more mature, but not fully developed, nucleated erythroid
components. However, all subsets exhibited essentially the
same surface fluorescence intensities when stained with the
103B2/9E10, as indicated in a typical experiment in Fig 7
(histograms J to L). Thus, the 103B2/9E10 epitope is evident
before the expression of specific erythroid markers and is lost
only at the late stages of erythroid differentiation.
CD164 Is Differentially Expressed During B Lymphoid
and Myelomonocytic Development
It was of interest that the CD341CD164(103B2/9E10)lo/2 cell
population in bone marrow (Fig 8) consisted mainly of CD191
B-cell precursors, suggesting that the most immature B-cell
subset in bone marrow is CD1642. Developing B cells in bone
marrow, however, were CD164(103B2/9E10)1 as evidenced in
WATT ET AL
Fig 8, which further shows that 60% to 70% of CD342 B cells
expressing CD19 or CD20 surface molecules coexpress the
CD164(103B2/9E10) epitope. In contrast, the majority of
CD342 T cells, in bone marrow, staining with the CD3 marker
were found in the CD164(103B2/9E10)lo/2 cell gate (Fig 8). To
determine whether this CD164 epitope was maintained on
peripheral blood lymphocyte subsets, three individual Ficoll
separated peripheral blood samples were scatter gated on the
lymphoid gate and analyzed by two-color FACS analysis for the
B lymphoid marker, CD20, and for the T-cell markers, CD3,
CD4, and CD8 (Fig 9). The majority of CD31, CD41, CD81,
and CD201 cells fell within the CD164(103B2/9E10)lo/2 gate.
Analyses of the MFI indicated that for these three peripheral
blood samples the CD201 B cells showed lower levels of
CD164(103B2/9E10) expression (MFI 5 3.95 6 0.29) than the
CD31 T cell subset (MFI 5 6.69 6 0.44). In addition, within
the T-cell subset, the CD81 cells (MFI 5 4.11 6 0.33) showed
lower levels of CD164(103B2/9E10) epitope expression than
did the CD41 cells (MFI 5 8.47 6 0.12). Our other studies
(data not shown) examining the correlated expression of the
CD164(103B2/9E10) epitope and defined lymhoid surface
markers on cord blood cells indicated that the majority of cord
Fig 9. The expression of the CD164(103B2/9E10)
epitope on peripheral blood lymphoid subsets. Peripheral blood mononuclear cells were labeled with
the 103B2/9E10 MoAb or with an IgG3 negative
control MoAb plus the FITC-conjugated MoAbs indicated before counterstaining with PE-conjugated
anti-mouse IgG3. Lymphoid cells were gated on low
forward and low side scatter gates before analysis
on the FACSCalibur.
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CD164 EXPRESSION AND CHROMOSOME LOCATION
blood CD342CD191 and CD201 B cells were CD164(103B2/
9E10)1, whereas the CD342CD31 T cells were CD164(103B2/
9E10)lo/2. Together these results show that T cells in bone
marrow and peripheral blood are very weakly positive for the
103B2/9E10 epitope. However, the expression of this epitope
was differentially regulated on developing B cells, with the
most primitive bone marrow CD341CD191 B cells and the
most mature peripheral blood B cells being CD164(103B2/
9E10)lo/2, whereas a large proportion of maturing CD342CD191
or CD342CD201 B cells in bone marrow were CD164(103B2/
9E10)1.
In earlier experiments,7 we have shown that myeloid precursors, the pre–CFU-GM and GM-CFC, were CD164(103B2/
9E10)1, whereas this epitope continues to be expressed on
monocytes but is virtually absent from mature neutrophils in
peripheral blood (Fig 2). We therefore used three cell-surface
markers to analyze the expression of this CD164 epitope on
CD342 myelomonocytic cells in bone marrow. These were
CD14,48 which is expressed from the promonocyte to the
monocyte stages; the myelomonocytic marker, CD6549; and
CD66b,50 which exhibits high levels of expression on myelocytes and metamyelocytes and lower levels of expression on the
more primitive promyelocytes and early myelocytes and on the
more mature band forms and neutrophils in the neutrophilic
lineage. Figure 10 confirms that essentially all CD141 cells
within the monocytic lineage and all except the more mature
cells within the neutrophilic lineage are CD164(103B2/9E10)1.
861
PCR Analysis of Somatic Cell Hybrids Localizes CD164
to Chromosome 6
PCR analysis was performed using DNA from a set of
somatic cell hybrids (Tables 3 and 4), from the BglII subclone of
CD164 PAC1 and from three human genomic DNA samples.
For the human genomic DNA, the CD164 PAC1 subclone and
the hybrid containing human chromosome Xq13 translocated to
6p21, t(6,X)(p21,q13), single bands of approximately 1.1 kb
and 500 bp were obtained with the MGC24-Gp- F3/B3 and
F4/B4 primers, respectively, on ethidium bromide–stained gels.
These bands hybridized to the CD164 cDNA probe C. An
example of the PCR products generated with the MGC24-GpF4/B4 primers from the somatic cell hybrid panel is shown in
Fig 11A and B. These PCR products were not generated with the
hybrid DNA containing the X chromosome alone or in conjunction with chromosomes other than 6. This indicated that the
CD164 gene was very likely to be located on chromosome 6. A
sequence comparison of the products generated by PCR analysis of the hybrids revealed identical sequence for both the cDNA
clones, the BglII subclones of the CD164 PACs, and genomic
DNA from male or female peripheral blood leukocytes.
Identification of CD164 Containing PAC Clones
A PAC library containing 120,000 recombinants was screened
with full-length CD164 cDNA probes, and two positive clones
(termed CD164 PAC1 and PAC5) were analyzed in detail.
Restriction enzyme analyses of these clones, using EcoR1,
BglII, Pst 1, HindIII, and BamH1 revealed that the pattern of
bands on a 0.7% agarose gel stained with ethidium bromide was
similar but not exactly identical (data not shown). When these
gels were Southern blotted and hybridized with the 1.173 kb
EcoRV/HindIII CD164 probe C, however, the pattern of bands
observed for CD164 PAC1 was identical to that obtained with
CD164 PAC5. Southern blotting of human placental genomic
DNA digested with all five restricton enzymes and hybridized
with CD164 cDNA probe C produced the same banding pattern
(data not shown), indicating that the PAC clones had not
undergone gross rearrangements within the region of the
CD164 gene. A single hybridization band was observed with
each enzyme, the smallest being an EcoR1 fragment of approximately 2.2 kb, whereas the largest fragment of greater than or
equal to 18 kb was obtained after BamH1 restriction digestion.
When the blot was reprobed with the 58 end of the cDNA, two
additonal BamH1 fragments of 2 and 3 kb were identified.
These three BamH1 fragments cover the entire CD164 gene and
span approximately 23 kb of DNA. Complete sequence analysis
has been obtained from these BamH1 fragments derived from
the CD164 PACs after subcloning into pCRScript and these
contain a sequence that is identical to the CD164 cDNA from 1
to 2867 bp, but they are interspersed with intronic sequences of
varying sizes.42
Fig 10. The CD164(103B2/9E10) epitope is expressed on myelomonocytic cells in bone marrow. Representative dot plots from one of
two separate experiments showing Ficoll separated bone marrow
mononuclear cells labeled with the 103B2/9E10 MoAb or with an IgG3
negative control MoAb plus CD34-PerCP and with the FITC-conjugated MoAbs indicated before counterstaining with PE anti-mouse
IgG3 and analysis on the FACSCalibur. Gates were set to exclude the
CD341 cell population before myelomonocytic cell analysis.
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862
WATT ET AL
Table 3. Monochromosomal Somatic Cell Hybrid DNA Panel From the UK HGMP Resource Centre
Hybrid
GM07299
GM10826B
GM10253
HHW416
GM10114
MCP6BRA
CLONE21E
C4A
GM10611
762-8a
JICL4
1aA96021
289
GM10479
HORLI
2806H7
PCTBA1.8
DL18TS
GM10612
GM10478
THYB1.3
PgME25NU
HORL9X
853
Chromosome
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
/
2
2
2
2
5
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
6
2
2
2
2
2
/
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
7
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
2
2
2
2
2
2
2
1
2
2
2
2
/
2
2
2
2
2
2
/
2
2
2
2
9
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
10
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
11
2
2
2
2
2
2
2
2
2
2
1
2
/
2
/
2
2
2
2
2
2
2
2
2
12
2
2
2
2
2
2
2
2
2
2
2
1
/
2
2
2
2
2
2
2
2
2
2
2
13
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
14
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
15
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
16
2
2
2
2
2
2
2
2
2
2
2
2
2
/
2
1
2
2
2
2
2
2
2
2
17
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
18
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
19
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
20
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
21
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
1
2
2
2
22
2
2
2
2
2
2
2
/
2
2
2
2
2
2
2
2
2
2
2
/
2
1
2
2
X
1
2
2
2
2
/
2
2
2
2
2
1
2
2
/
2
2
2
2
1
1
/
1
2
Y
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
1
Hybrid: GM07299, 1 and X; MCP6BRA, Xqter-Xq13:6p21-6qter; C4a, 8 and fragment of 22; 762-8a, 10 and Y; 1aA9602 1 ve, 12, 21 and X; 289, 13
plus fragments of 8, 11, and 12; GM10479, 14 plus part of 16 (probably 16p13.3-16q22.1); HORLI, 15, 11q, part of Xp and proximal Xq; GM10478, 20,
4 (part), 8 (part), 22q, and X; GM10612, 9p stronger than 9q, only 10% of cells with whole 9; PGME25NU, 22, part of Xp.
1 Indicates presence of chromosome; 2 indicates absence of chromosome; / indicates chromosome translocation, extra chromosomes, or
other modifications.
FISH Localizes CD164 to Chromosome 6q21
Sixty metaphase spreads were examined for signal using each
of the BglII subclones of CD164 PACS 1 and 5. Probe efficiency
was approximately 60%, with signal being observed on both
chromatids of chromosome 6 on band 6q21 only (Fig 11C).
DISCUSSION
The interaction of hematopoietic progenitor cells with stromal/
endothelial cells in their immediate microenvironment is thought
to be of major importance in the regulation of hematopoietic
stem cell self-renewal, quiescence, commitment, and migration.
These interactions involve cooperation between adhesion receptors, their cognate ligands, and cytokines. The adhesion receptors/
ligands implicated in these processes belong to several families
of adhesion molecules. These include the integrins, selectins, Ig
superfamily, and sialomucins. Recently, mucin-like receptors or
sialomucins, in particular, have assumed some importance in
hematopoiesis, with four of these receptors, CD34, PSGL-1,
CD43, and CD164, having been identified on primitive hematopoietic precursor cells and/or their associated stromal/endothelial elements.5,7,19-27,51 Our own studies and those of other
researchers indicate that these receptors are involved in mediating or regulating hematopoietic precursor cell adhesion in vitro
and/or the regulation of hematopoietic progenitor cell proliferation.5,7,20,31,32
In this report, our studies have concentrated on the CD164
molecule. To this end, we have characterized the reactivity of
the first four MoAbs, 103B2/9E10, 105A5, 67D2, and N6B6, to
this sialomucin. Each of these antibodies shows distinct patterns
of reactivity on CD341 hematopoietic progenitor cells, labeling
on average 55% to 93% of these cells. When compared with
other CD164 epitopes, the 103B2/9E10 epitope is more highly
expressed on CD341 cells from both cord blood and bone
marrow, whereas the 105A5 epitope exhibits the most variable
level of expression. Our recent studies suggest that this may
reflect glycosylation differences in the 105A5 and 103B2/9E10
epitopes on different CD341 subsets. We have cloned the
CD164 cDNA5,7 and have shown that this gene encodes an 80 to
90 kD heavily O-glycosylated protein that is able to form
homodimers with an apparent molecular weight of 160 to 180
kD. Our recent studies have also shown that the CD164 receptor
mediates the adhesion of CD341 hematopoietic progenitor cells
to bone marrow stroma, since this adhesion can be partially
blocked by the 103B2/9E10 MoAb.5,7 In addition, two-color
phenotypic selection, based on expression of CD34 and the
CD164(103B2/9E10) epitope, revealed that essentially all the
committed myeloid and erythroid progenitors and their immature precursors were present in this CD1641 population and
correspondingly absent from the CD341CD1642 subset. In
contrast, mature peripheral blood cells showed only low or
negligible levels of CD164 expression. In view of these results,
we have characterized, in more detail, the expression of the
103B2/9E10 epitope on CD341 and CD342 subsets of cord
blood and bone marrow cells. Multiparameter flow cytometric
analyses revealed that the majority (.90%) of CD341 human
bone marrow and cord blood cells that expressed CD90(Thy-1),
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CD164 EXPRESSION AND CHROMOSOME LOCATION
863
Table 4. Somatic Cell Hybrid DNA Panel Positive for CD164
Hybrid
GM07299
GM10826B
GM10253
HHW416
GM10114
MCP6BRA
CLONE21E
C4A
GM10611
762-8a
JICL4
1aA96021
289
GM10479
HORLI
2806H7
PCTBA1.8
DL18TS
GM10612
GM10478
THYB1.3
PgME25NU
HORL9X
853
Chromosome
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
6
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
7
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
9
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
10
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
11
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
12
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
13
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
14
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
15
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
16
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
17
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
18
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
19
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
20
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
21
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
22
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
X
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Y
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
DNA samples were PCR amplified using CD164 primers as indicated in Materials and Methods and analyzed on 2% agarose gels. Positive bands
of the correct size were obtained with the chromosome 6 DNA sample only. These products were Southern blotted and probed with CD164 cDNA
Probe C or sequenced and were shown to have sequence identifical to CD164 cDNA. 2, no PCR product; 1, PCR product of the appropriate size.
Human, mouse, and hamster genomic DNA samples were also analyzed, but only the human DNA proved positive for CD164 under the conditions
used.
CD117(c-kit), AC-133, and CD135(FLT3) also coexpressed the
103B2/9E10 epitope. Because human AC-1331 cells have the
capacity for hematopoietic engraftment in fetal sheep,52 these
results suggest that the 103B2/9E10 epitope is present on a very
A
B
primitive subset of CD341 cells.52,53 Similar analyses of the
CD34lo/2CD164(103B2/9E10 epitope)1 subsets have also indicated that one of the most prominent populations consists of
nucleated erythroid cells, with the appearance of this CD164
C
Fig 11. CD164 is located on human chromosome 6q21. Somatic cell hybrids shown in Tables 3 and 4 were analyzed by PCR for the presence of
the CD164 gene. (A) Ethidium bromide–stained gel of PCR products using the MGC24-Gp-F4/B4 primer pairs. This gel was Southern blotted and
probed with CD164 cDNA probe C as indicated in (B). Partial metaphase spreads (C) showing localization of the Bgl II subclone of CD164 PAC1 to
human chromosome band 6q21 (arrowed and shown on the idiogram).
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
864
epitope preceding the expression of the erythroid markers,
glycophorin C, glycophorin A, and band III. This is consistent
with our previous studies5,7 showing that the 103B2/9E10 and
105A5 epitopes of CD164 are present on the majority of day-14
CFU-GEMM and BFU-E. Of added interest was the finding that
the expression of the 103B2/9E10 epitope was differentially
regulated during B-cell development, because the majority of
CD342 B cells in bone marrow expressed the 103B2/9E10
epitope, whereas both mature B cells from peripheral blood and
the most immature CD341CD191 B-cell precursors were
CD164(103B2/9E10 epitope)lo/2.
Our recent studies45 indicate that the 103B2/9E10 and 105A5
epitopes both localize to the N-terminal domain of the CD164
molecule encoded by exon 1. Although the epitope recognized
by the 103B2/9E10 antibody was not affected by sialidase
treatment, that detected by 105A5 was completely abrogated by
CD164 desialylation. This indicates that these epitopes are
spatially distinct from one another and implies that the epitope
required for CD164-mediated cell adhesion is not sialylated.
Further to these finding, we have shown by cross-blocking
experiments that the binding of the 105A5 MoAb was partially
inhibited by prelabeling the cells with the 103B2/9E10 MoAb,
but not vice versa. This partial inhibition might be due to the
induction, by the 103B2/9E10 antibody, of a conformational
change in the CD164 molecule that results in masking the
105A5 epitope. It therefore seems possible that these two
epitopes may cooperate or compete with one another in the
initial adhesion of CD341 cells to stromal cells, with expression
of the 105A5 epitope serving to modulate the function of the
CD164(103B2/9E10) epitope. Further studies are in progress to
fully characterize the epitopes recognized by these different
CD164 MoAb. However, it is possible that the different patterns
of expression of the CD164 epitopes on CD341 cells reflect
differences in glycosylation patterns, carbohydrate modifications, or splice variant expression among different precursor
cells within the hematopoietic progenitor cell pool.
One major unanswered question in hematopoiesis involves
the mechanisms by which CD341 hematopoietic progenitor
cells identify particular microenvironmental niches. It is thought
that the lodgement of such cells in these niches is required for
the maintenance of their quiescent state or for their self-renewal
or commitment to particular hematopoietic lineages. It is
possible that the sialomucins as a family may act to regulate
hematopoietic proliferation or differentiation. Evidence for this
comes from both in vitro and in vivo studies. For instance,
transfection of the long form of CD34 into M1 cells inhibits
their differentiation, whereas transfection of the short isoform
has no effect.33 In other studies, ligation of the CD43 engenders
an apoptotic response in more mature cells within the
CD34hiLin2 subset but not in the more immature stem or severe
combined immunodeficiency disease (SCID) repopulating
cells.24,25 Our recent experiments suggest that ligation of
CD164 with both the 103B2/9E10 and 105A5 MoAbs can
inhibit the clonogenic growth of CD341 CD38lo/2 cells.7 An
alternative way of determining the potential importance of these
mucin-like molecules is to generate knock-out mice lacking the
gene of interest or to create transgenic mice overexpressing a
particular sialomucin in a specified subset of hematopoietic
cells and then to examine their effects on hematopoietic
WATT ET AL
development. Although studies are limited, CD34-null embryonic stem (ES) cells and mice have been generated.35 In the
mutant ES cells, erythroid and myeloid development is delayed
during embryoid body formation, but it can be restored by the
introduction of either a full-length or cytoplasmically truncated
CD34 gene into these cells. In the CD34-null mice, the number
of clonogenic progenitor cells is reduced in the yolk sac, fetal
liver, adult bone marrow, and adult spleen, and cytokineinduced expansion of bone marrow hematopoietic progenitors
ex vivo is inhibited. These studies suggest that cytokines may
cooperate with adhesion receptors to induce cell proliferation
and/or differentiation by presenting cytokines on the CD34
molecule,54 by interaction between CD34 and cytokine receptors expressed on the same cell, or by engagement of the CD34
molecule on one cell with its cognate ligand on an opposing
cell. Such an adhesion process might precede or be required for
interaction with membrane associated growth factors on accessory cells. It will be of interest to see if CD164 possesses similar
functions or if signaling via CD164 is required for the regulation of hematopoietic cell proliferation or differentiation.
In the final section of this report, we have identified PAC
clones containing genomic fragments of CD164 and have used
these to localize the CD164 gene to human chromosome 6q21.
These PAC clones have now been fully sequenced and the
genomic structure of CD164 determined.42 These constructs,
together with the murine CD164 gene, when cloned, will
provide important reagents for identifying elements that regulate CD164 gene expression in hematopoietic progenitor cells
and for generating transgenic and knock-out mice for the
analysis of the function of CD164 in vivo. In addition, the PAC
clones containing CD164 may help to identify other genes on
chromosome 6q21 that are related to CD164 or that have similar
functions to CD164 in regulating hematopoietic progenitor cell
proliferation and differentiation.
ACKNOWLEDGMENT
The authors thank Professor Sir D.J. Weatherall and Professor L.
Kanz for their support, Dr D Buck for the AC-133 MoAb, and Heike
Hanisch and Doris Schweigert for excellent technical assistance.
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1998 92: 849-866
CD164, a Novel Sialomucin on CD34+ and Erythroid Subsets, Is Located on
Human Chromosome 6q21
Suzanne M. Watt, Hans-Jörg Bühring, Irene Rappold, James Yi-Hsin Chan, Jane Lee-Prudhoe, Tania
Jones, Andrew C.W. Zannettino, Paul J. Simmons, Regis Doyonnas, Denise Sheer and Lisa H. Butler
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