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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 849-866
By
From 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.
CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human
CD34+ hematopoietic progenitor cells. Our previous
results suggest that this receptor may play a key role in hematopoiesis
by facilitating the adhesion of CD34+ cells to bone
marrow stroma and by negatively regulating CD34+
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 CD34+ cells are CD164+, 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 (>90%) of
CD34+ human bone marrow and cord blood cells that were
CD38lo/ © 1998 by The American Society of Hematology.
THE 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/leukocyte-associated mucins encompass CD34,
CD43, CD45RA, CD164, GlyCAM-1, MAdCAM-1, TACTILE (CD96), and
PSGL-1(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 cell-cell adhesion, involving interactions between their mucin-like 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 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 CD34+/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 CD34+ 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
CD34+CD38lo/ Cells and Cell Lines
Cell Isolation and Erythroid Cultures
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 (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); CD8-FITC (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 phosphate-buffered 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 (Fab )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 anti-mouse isotype-specific Ig or with
FITC-F(ab 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
IgG3 Three-color staining of bone marrow and cord blood cells. Ficoll separated bone marrow and cord blood mononuclear cells or purified CD34+ 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 laser at 488 nm and detected at emission wavelengths of 530 nm, 570 nm, and 670 nm, respectively. Cell sorting of
CD34+CD164(103B2/9E10 epitope)+ and
CD34 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 PE-conjugated anti-Ig that specifically identified the CD164 test MoAb and analyzed on a FACSCalibur flow cytometer. The percent blocking was calculated as follows: 100 [(Median
Fluorescence of Cells Stained With Test MoAb After Incubation with
Blocking MoAb) (Median Fluorescence of Cells Stained With
Isotype-Matched Negative Control MoAb)] / [(Median
Fluorescence of Cells Stained With Test MoAb After Incubation With Negative Control MoAb) (Median Fluorescence of
Cells Stained With Isotype-Matched Negative Control MoAb)] × 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 Neuraminidase-Treated Cells Stained With CD164 MoAb) (Median Fluorescence of
Neuraminidase-Treated Cells Stained With the Isotype-Matched Negative
Control MoAb)] / [(Median Fluorescence of Untreated Cells Stained
With CD164 MoAb) (Median Fluorescence of Untreated Cells
Stained With the Isotype-Matched Negative Control MoAb)] × 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-MRF
bacteria (Stratagene, Cambridge, UK). Large-scale DNA preparations of
the two CD164 cDNA clones 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 3UTR. The Spe
1/Apa 1 probe B fragment comprised 1908 to 2867 bp of 3UTR. 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
C), which contained CD164 cDNA sequence, including a region spanning
1309 to 2487 bp of untranslated CD164 sequence in the 3UTR. The
restriction enzymes were purchased from Boehringer-Mannheim 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  -32P-dCTP
(Amersham Int, High Wycombe, Bucks, UK) using the T7 Quickprime kit
(Pharmacia, Uppsala, Sweden) according to the manufacturer's protocol.
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-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 2× 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 -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 (1601-1577)
primers were 5-CCTCACAACCTGTGCGAAAGTCTAC-3 and
5-ACTCAAGACAGTCTGGTGG AAATCC-3, 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 (SK+) 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 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 10× 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 MRF 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
-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
-32P-labeled CD164 Probe C or by PCR analysis using the
MGC-GP-F3/MGC-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 3 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, 5-GTACCTTGAAAGGA
TTTCCACCAGAC-3 and 5-CAAGTGCGAAACTCAGCCACTATTG-3,
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 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, 4× SSC, 0.001 mol/L EDTA, 0.05 mol/L 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), 10× Denhardt's
solution46 and 50 µg human placental DNA (Sigma) at
42°C for 1 to 18 hours before addition of the
-32P-labeled CD164 probes A, B, or C for 18 to 48 hours
at 42°C. Filters were washed twice at 42°C in 2× SSC with
0.1% (wt/vol) sodium dodecyl sulfate (SDS) for 30 minutes each, 3 times at 65°C in 0.2× 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 70°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 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 phytohemagglutinin-stimulated normal human lymphocytes by use of standard techniques. Before hybridization, the slides were denatured in 70% (vol/vol) formamide and 2× SSC at 73°C for 3 minutes, then washed in 2× 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, 2× SSC pH 7 at 42°C, followed by 1× SSC at 60°C. Blocking solution (3% [wt/vol] BSA, 4× 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, 4× SSC, 0.1% [vol/vol] Tween 20) was applied and slides incubated at 37°C for 40 minutes. Slides were washed in 4× SSC, 0.1% (vol/vol) Tween 20 at 42°C and counterstained with 200 ng/mL DAPI, followed by 2 minutes in 2× 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).
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 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.
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 are2,3-, 2,6- or
2,8-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 CD34+ 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 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 28.9 ± 6.8 for N6B6, 41.5 ± 15.6 for 67D2, 22.3 ± 27.1 for 103B2/9E10, and 7.5 ± 4.8 for 105A5, after subtraction of the MFI values for isotype-matched negative controls and where the values are means ± SD of three independent experiments. This compares with MFI values for lymphocyte staining of 7.9 ± 3.4 for N6B6, 8.3 ± 2.7 for 67D2, 2.7 ± 2.0 for 103B2/9E10, and 3.6 ± 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 CD34+CD164+
Subset Contains Phenotypically Primitive Cells
CD164 Expression Is Maintained on Nucleated Erythroid Cell Subsets,
but Is Lost on Terminal Erythroid Differentiation
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Abstract
Introduction
or that coexpressed AC133, CD90(Thy-1),
CD117(c-kit), or CD135(FLT-3) were
CD164(103B2/9E10)+. This CD164 epitope was generally
detected on a significant proportion of
CD34+CD71lo/
Abstract
