Philips Cordless phone answer machine

CD2352S

Cool comfort
Making a call with the CD2 series is as cool and relaxing as chatting with your best friend. The easy-to-handle handset with keypad & display backlight makes calling comfortable, wherever you are.
Never miss a call Digital answering machine 15-minute recording time Illuminated message-counter on base Clear calls around the house Virtually interference free Speakerphone - Talk handsfree Polyphonic ringtones for a more natural and realistic sound Pure convenience Display indicates the caller's name and number An ergonomic shape gives you the most comfortable control Backlit display & keypad Multi-pack for ultimate convenience Free intercom - Free internal calls between handsets Complete phone package including a base and 2 handsets
Cordless phone answer machine

CD2352S/19

Specifications

Picture/Display

Backlight Backlight color: Amber Display colors: Black & White Lines of text: 2 Main Display Resolution: n.a. pixel Main Display Technology: CSTN Main Display Type: Alphanumerical

Highlights

Network Features
Antenna: Integrated on base, Integrated on handset Compatible: GAP Dialing: Tone, Pulse
Digital answering machine
With a digital answering machine, you'll never miss a call or message.

Operator Requirements

Name and Caller ID Caller ID on call waiting

15-minute recording time

Virtually interference free
Handset ringers: Instrumental, Polyphonic Volume Control: Volume Control up/down

Answering Machine

Convenience
Plug & Play Answering machine Recording time capacity: Up to 15 min Message counter on base
Thanks to an advanced digital technology, calls are undisturbed by interferences from other devices.
Speakerphone - Talk handsfree
Alarms: Alarm Clock Base Station Features: Message counter on base Base Station keys: Answering machine keys, Paging key Battery charging indication Call Management: Call Waiting, Caller ID, Conference Call, Explicit Call Transfer, Microphone mute Ease of Use: Hands free mode, Keypad Lock, Menu Control Function: Baby Call Multi base capability: Up to 4 bases Multi handset capability: Up to 4 handsets Signal strength indication Backlit keypad: Yes (Amber)

Memory Capacity

Call log entries: 20 Phonebook: 50 names and numbers
Handsfree mode uses a built-in loud speaker to amplify the voice of the caller, allowing you to speak and listen during a call without holding the phone to your ear. This is especially useful if you want to share the call with others or take notes during a call.

Dimensions

Base dimensions: 125 x 118 x 56 mm Handset dimensions: 154 x 50 x 29 mm

Polyphonic ringtones

Battery capacity: 650 mAh Battery type: AAA Battery Type: NiMH Kind of Battery: Rechargeable Mains power: AC 220-240V - 50Hz Number of batteries: 2 Standby time: 120 hours Talk time: up to 10 hours
A polyphonic ringtone means that the phone can play many tones simultaneously, so that an accurate musical performance is possible, and when played through the phone's speaker these polyphonic ringtones sound like real music.
Caller name & number display
The name of the person or company calling is shown on the telephone display. The names of first-time callers can be displayed as well as those that are programmed into the telephone beforehand.

Free intercom

No need to stop what you are doing and go to give the handset to your son when his friend calls: you can freely and easily call the handset in his bedroom and transfer the call.
Plug & Play pack - 2 handsets
A convenient Plug & Play phone package including a base and 2 handsets.

Ergonomic Design

The smooth rounded shaped handset fits perfectly in your hand for natural, balanced operation. It looks great too!
Backlit display & keypad
Backlit display & keypad ensures easy viewing and operation in low light conditions
Illuminated message-counter
Just look at the base station to instantly know whether you have received any messages.
Issue date 2009-03-08 Version: 1.2.4
2009 Koninklijke Philips Electronics N.V. All Rights reserved. Specifications are subject to change without notice. Trademarks are the property of Koninklijke Philips Electronics N.V. or their respective owners. www.philips.com
12 NC: 06091 EAN: 97483 7

From bloodjournal.hematologylibrary.org by guest on June 6, 2011. For personal use only.

1991 77: 961-970

B-lineage colonies from normal, human bone marrow are initiated by B cells and their progenitors
K McGinnes, M Letarte and CJ Paige
Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
B-Lineage Colonies From Normal, Human Bone Marrow Are Initiated by B Cells and Their Progenitors
By Kimberley McGinnes, Michelle Letarte, and Christopher J. Paige
We have recently described a reproducible method whereby colonies containing cells that secrete immunoglobulin (lg) can be grown from normal, human, adult bone marrow samples. The present report characterizes the cells that initiate these colonies. It is shown that all clonogenic cells express the CD19 surface antigen, as removal of these cells before plating in the B-cell colony assay abolished the subsequent growth of plaque-forming, B-lineage colonies. Cells from both the CD10' and CD20' B-lineage subpopulations initiated the growth of B-cell colonies, as removal of either subset resulted in a 50% reduction in the number of resulting B-cell colonies. The removal of activated B cells (CD23+),plasma cells (PCA-I+), or myeloid cells (CD13') did not lead to a significant depletion in B-cell colony formation. Pie-B cells that were not yet committed to Ig light chain expression were also able to differentiate and proliferateinto 19-secreting colonies under the culture conditions used. Colonies initiated by these light chain uncommitted cells were distinguished using a replicate protein immunoblotting technique, which detects the simultaneous secretion of lgK and Igb from single colonies. These experiments provide evidence that the CDlO antigen is expressed on B-lineage cells before Ig light chain commitment, whereas CD20 is not. In conclusion, this B-cell colony assay provides a system for studying the differentiation of bone marrow-derived B cells and their precursors into 19-secretingcells. by The American Society of Hematology.
LYMPHOCYTES differentiate from a common, pluripotent, hematopoietic stem cell'.4 in the bone marrow of adult humans? Stages of B-cell maturation can be characterized by the rearrangement and expression of the Ig genes: and by the expression of surface antigens7 It is generally accepted that during B-cell differentiation the rearrangement of the Ig heavy chain gene precedes that of the light chain The first genetic event in B lymphopoiesis is the recombination of a heavy chain diversity (D) gene segment with a joining (JH) segment, and the subsequent joining of this gene product to a variable (V,)-region segment.6 If this process results in the producheavy chain protein is tion of a functional gene, a expressed in the cytoplasm." In humans, pre-B cells at this stage of differentiation are found in fetal liver, fetal bone marrow, and adult bone m a r r o ~. ' ~The next major molec-'~ ular event in B-cell lymphopoiesis is the recombination of an Ig light chain variable (V,)-region gene segment with a joining (JL) segment. Once this has occurred, a complete Ig molecule can be assembled and expressed on the cell surface. Due to the mechanism of allotype exclusion, a single B cell usually expresses only one Ig heavy chain.I5 Isotype exclusion results in a single B cell expressing either IgK or IgX light chain, but not b ~ t h. ' ~ , ' ~ ~ ' ~ It is apparent that during B-cell differentiation a series of surface antigens are sequentially expressed and lost as this pathway proceeds from an immature progenitor to a mature B cell, and finally to a terminally differentiated plasma cell. Studies of human B-lymphocyte surface antigens have identified CD19 (B4, Leu-12) as a lineagespecific marker for all B lymphocytes.I8 CD34 (My-10, HPCA-1) is a progenitor cell antigen associated with cells of the myeloid lineage and is present on the earliest recognizable B-lineage CDlO (CALLA, J5) is expressed on a subset of immature B cells and is found on most cases of common acute lymphoblastic leukemia (CALL)." As B cells mature they acquire CD20 (Bl, Leu-16): lose CD10, express IgM on the surface, and then acquire CD21 (B2, C3d receptor, CR,).'' When mature human B lymphocytes are activated, several antigens, including CD23 (Fce receptor, L ~ U - ~ O are ~ ~ , ~ ~ ) , expressed. Finally, terminal differentiation into Ig-secreting plasma

Blood, Vol77, No 5 (March l), pp 961-970 1991:
cells is association with the loss of most B-cell antigens, the expression of antigens such as PCA-1," and the secretion of Ig. In the adult bone marrow, B lymphopoiesis is under the control of soluble growth factors and accessory cells in the stroma. Most of the information regarding the regulation of this process comes from investigations of the murine system? In the human system, the lack of suitable assays has hampered the elucidation of the specific growth factor and stromal cell requirements for in vivo stimulation of B-cell mat~ration.2~ phenotype and Ig gene rearrangeThe ment status of B-cell precursors in fetal liver and fetal bone marrow samples have been examined by first immortalizing the cells using Epstein-Barr virus tran~formation,~'~' by and cloning CD10+ CD19' and CD10+ CD19- fetal liver lymphoid ~ e l l s.Inferences regarding normal B-cell progeni~~,~~ tors and the control of differentiation have also been made from cloning fresh ALL sample^^^.^^ and from ALL-derived cell lines.37 In contrast to the differentiation of B-cell precursors, the control of mature human B-cell activation and terminal plasma cell differentiation have been well characteri~ed.~~-~' recently reported an experimenWe have tal system that permits the clonal growth of normal, bone marrow B-lineage cells into Ig-secreting plasma cells.42In
From the Division of Cell and Molecular Biology, Ontario Cancer Institute; the Division of Immunology and Cancer, The Research Institute, Hospital for Sick Children; and the Departments of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada. Submitted August 15, 1990; accepted October 23, 1990. Supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. K.M. is a Commonwealth Scholar. M.L. is a Teny Fox Scientist of the Cancer Society. Address reprint requests to Emberley McGinnes, PhD, Division of Cell and Molecular Biology, Ontario Cancer Institute, 500 Sherboume St, Toronto, Ontario, Canada M4X lK9. 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. by The American Society of Hematology. 0006-497119117705-0015$3.00/0
MCGINNES, LETARTE, AND PAIGE
this system, colonies containing cells that secrete Ig can be detected using novel in situ methods, such as colony plaque and protein immunoblotting assays. In the present report, we show that these assay conditions support the differentiation of both CD10 and CD20 B-lineage cells into colonies containing Ig-secreting cells. We also demonstrate that Ig commitment can be associated with surface antigen expression, and report that Ig light chain uncommitted cells express the CD19 and CD10, but not the CD20, surface antigens. Therefore, the B-cell colony assay described here and p r e v i o ~ s l y ~ ~ provides a suitable system with which to study the differentiation of human, bone marrow-derived B-lineage cells.

MATERIALS AND METHODS

Bone marrow sample collection and preparation. Normal human bone marrow samples were obtained by iliac crest aspiration from bone marrow transplantation donors or from the femur of osteoarthritis patients undergoing hip replacement surgery. These protocols were approved by the Ontario Cancer Institute and the Wellesley Hospital, Toronto, Ontario, Canada, respectively. After collection, bone marrow samples were washed in phosphate-buffered saline (PBS), and the cells were fractionated on a discontinuous, percoll (Pharmacia, Baie dUrfe, Canada) gradient? Bone marrow cells with a density of greater than 1.050 g/mL and less than 1.065 g/mL were collected and washed in PBS before further manipulation. This fraction was chosen because it represented a significant enrichment of clonogenic B-lineage cells, as reported previo~sly.~ Monoclonal antibodies (MoAbs). Purified mouse MoAbs recognizing CD19 (Leu-12), CD20 (Leu-16), CD21 (CR,), and CD34 (HPCA-1) were purchased from Becton Dickinson Monoclonal Center (Mountain View, CA). Purified anti-CD23 was purchased from AMAC (Bio/Can Scientific, Mississauga, Canada), and PCA-1 and CD13 (My7) from Coulter (Hialeah, FL). The antLCD10 (CALLA) MoAb used (44C10) has been described previously by Quackenbush and Letarte. Isotype controls, namely mouse IgG, and IgG,, were purchased from Becton Dickinson, and IgG,, from Coulter. F(ab), goat anti-mouse IgG-phycoerythrin (PE) was purchased from Tago Immunologicals (Burlingame, CA). MoAbs used for replicate protein blotting included biotinylated antihuman IgK and anti-Igh, and horseradish peroxidase (HRP0)conjugated anti-human IgK and anti-Igh (Southern Biotechnology, Birmingham, AL). Immunofluorescence andflow cytometry. Cells, 3 x los, in 50 pL PBS containing 2% fetal bovine serum (FBS) were incubated on ice for 30 minutes with MoAbs at a concentration that gave maximal fluorescence. These concentrations were 25 ng of antiCD19 and -CD21; 50 ng of anti-CD10, -CD20, -CD34, and isotype IgG, and IgG,,; 100 ng of anti-CD23, anti-CD13, and isotype IgG,,; and 125 ng of anti-PCA-1. After this incubation, the cells were pelleted, washed with 2 mL PBS, 2% FBS, and incubated with F(ab)z goat anti-mouse IgG-PE (30 p b 1:300) for 30 minutes on ice. The cells were washed before analysis. Quantitative fluorescence analysis was performed using a FACSCAN flow cytometer (Becton Dickinson). For the analysis of surface antigen expression, a minimum of 10,000 events for each sample was collected on a FACSCAN Research Management System (Becton Dickinson). Cells with a low side and forward light-scatter profile were gated and analyzed in the manner described by Loken et a]. These gates included all CD19 cells. To examine depleted populations, only 1,500 to 2,000 events were collected because of the small number of cells available.

B-cell colony assay. Percoll-fractionated cells were cultured in the top layer of a double-agar B-cell colony assay? Briefly, each layer consisted of 1mL of 0.3% melted agar (Difco, Detroit, MI) in culture media supplemented with 10% to 20% phytohemagglutinin (HA15: Wellcome, Dartford, UK) stimulated human T-cellconditioned media (TCM); and 10 &mL Staphylococcus aureus (Cowan strain) protein A (SPA) (Pharmacia) and/or 20 pg/mL dextran sulphate (DXS) (Sigma, St Louis, MO). TCM was included as a source of growth factors, and SPA/DXS as a source of mitogen necessary for the induction of Ig secretion from mature B cells. The culture media used was Opti-MEM (GIBCODRL, Burlington, Canada) supplemented with 5% FBS (GIBCO), 50 U/mL penicillin, 50 pg/mL streptomycin (GIBCO), and 5 x m o l n 2-mercaptoethanol (GIBCO). After a 10-day incubation at 37C in 5% CO,, each 35-mm culture dish was examined for the growth of B-lineage colonies. Colonyplaque assay. B-lineage colonies were enumerated using a previously described colony plaque assay that allows the detection of colonies containing cells that secrete Ig. Briefly, the top layer of a double-agar culture was transferred onto a glass slide, dehydrated, and overlaid with plaquing mixture containing 50 p L SPA-coupled sheep red blood cells (SRBC) (Woodlyn Labs, Guelph, Canada) (diluted 1:7 in Hanks buffered salt solution [HBSS]), 25 pL guinea pig complement (GIBCO) (diluted 1:3 in HBSS), 25 pL rabbit antiserum raised against human Igs (Cappell, Teknika Corporation, West Chester, PA) (diluted 1:25 in HBSS), and 500 pL 0.5% agar (BiTek; Difco). After a 5-hour incubation at 3TC, plaques were scored in the following manner. The SRBC were lysed with a 5% acetic acid:95% ethanol solution, and the plaque-forming colonies were stained with Giemsa (Fluka Chemicals, Beuchs, Switzerland) and enumerated microscopically. Those colonies that were within plaques and which contained more than 20 cells were designated as B-cell colonies. The colony-plaquing technique ensures that the colonies detected were Ig-secreting B-cell colonies! This is important because myeloid cells also clone under these assay conditions; and secondly, single, Ig-secreting cells remain viable during the incubation period. Depletion of B-lineage subpopulations. Percoll-fractionated bone marrow cells were depleted of cells expressing CD19, CD34, CD10, CD20, CD21, CD23, PCA-1, or CD13 with immunomagnetic beadsM using a modification of a previously described method!s Cells, 2 x lo6to 20 x lo6, at a concentration of 2.5 x lo7cells/mL PBS, 2% FBS, were incubated for 30 minutes on ice with MoAb (60 ng/l x lo6 cells of anti-CD19 and -CD21; 125 ng/l X lo6 cells of anti-CD10, -CD20, -CD23, -CD34, and IgG, isotype; 250 ng/ 1 x lo6 cells anti-PCA-1 and isotypes IgG,, and IgG,,). The cells were then pelleted, washed with PBS, 2% FBS, and incubated for 45 minutes on ice with magnetic beads (M-450; 4 x 108/mL)coated with sheep anti-mouse IgG (Dynal; P&S Biochemicals, Gaithersburg, MD) on ice for 45 minutes. These beads were washed with at least 300 vol of PBS, 2% FBS before use to remove all traces of preservative. Magnetic beads were added to the cells at a ratio of 2 beads to 1 cell (or approximately 15 beads:l target cell). Following this incubation, 8 mL PBS, 2%FBS was added to each tube and the tubes were placed in a magnetic particle concentrator (MPC-1; Dynal; P&S Biochemicals) for 1 to 2 minutes. Positive cells, which were rosetted with magnetic beads, were immobilized on the side of the tube next to the magnet, while the negative cells were in suspension and could be removed by pipetting. These negative or depleted cells were pelleted and incubated a second time with magnetic beads. After another 45-minute incubation on ice, the negative cells were harvested and washed twice. In all experiments the efficiency of this depletion procedure was assessed by immunofluorescence and flow cytometry. Depleted cells were incubated with F(ab), goat anti-mouse IgG-PE alone, or

FACSCAN analysis of percoll-enriched bone marrow. Percoll-fractionation of normal human bone marrow resulted in an enrichment in the percent of B-lineage cells,42 namely those expressing CD19, CD10, and CD20 surface antigens (data not shown). Plasma cells that expressed the PCA-1 antigen were more frequent in higher density percoll fractions. Cells in percoll-enriched bone marrow samples (5.5% 2 2.8% of nucleated bone marrow cells4*) were heterogeneous in their forward-angle and side lightscattering characteristics. Greater than 95% of CD19' cells have been previously shown to be confined to a well-defined cluster,' which allowed the gating of cells for further immunofluorescence analysis. The cells within these gates constituted approximately 50% of the cells with a density of between 1.050 g/mL and 1.065 g/mL. The distribution of cells in percoll-enriched bone marrow samples expressing specific B-lineage antigens is shown in Table 1. CD19' and CD20' cells fell almost exclusively within the population gated to include cells with low side and forward light-scatter properties. However, only a proportion of cells expressing CDlO or CD34 exhibited a lymphoid profile. Most of the PCA-1' plasma cells were not within the gated region. Effect of depletion of B-lineage subpopulations on B-cell colony growth. The B-cell colony assay allows the clonal growth of B-lineage cells.4*To characterize the clonogenic cells, the input population was depleted of specific B-lineage subpopulations, as determined by surface antigen expression, before plating in the B-cell colony assay. In these experiments, the contribution of a specific subset of B-lineage cells was determined by comparing the plaqueforming colony growth of depleted populations with that of undepleted, isotype control populations. Depletion was achieved by the use of MoAbs and magnetic immunobeads. FACSCAN analysis showed that this procedure led to a depletion of greater than 95% of the relevant cells in all experiments presented.
Table 1. Distribution of Surface Antigen Expression in Bone Marrow B-Lineage Cells

Percent Positive

Analysis
Gated cells-mean Gated cells-range Total-mean Total-range
12.5 2.9 2.5-27.6 0.2-9.3 6.6 3.0 0.4-12.6 0.7-10.8
6.9 7.4 1.7 0.5-11.5 1.7-17.3 0.0-5.1 6.5 3.5 1.1-23.8 1.0-7.4 5.2 2.0-12.6
The percent of percoll-enriched cells expressing the surface antigens shown were determined by FACSCAN analysis of cells with a density of 1.050 g/mL to 1.065 g/mL. The percent of positive cells is given for the total cell population and for those cells gated to have low forward and side light-scatter properties. The percent positive values shown have been adjusted for the specific isotype control. Approximately 50% of the cells collected fell within the established gates. These results represent the mean and range from 13 individual bone marrow samples.

INPUT POPULATION

CD19 NEG CD34 NEG

CD10 NEG

CD20 NEG

CD23 NEG PCA-1 NEG

No. B CELL COLONIES/ lb INPUT CELLS
Fig 1. Effect of B-lineage subpopulation depletionon 6-cell colony formation. Percoll-enrichedbone marrow cells were depleted of cells expressing either CD19, CD34, CD10, CD20, CD23, or PCA-1 using MoAbs and magnetic immunobeads. These cell populations (designated " N E G in this figure) were plated in the double-agar 6-cell colony assay and examined for plaque-forming colonies after a 10-day incubation. The number of resultant plaque-forming colonies (mean f SD for three replicatecultures) in depleted cultures is shown by solid bars; and in the isotype control cultures by hatched bars. MoAbs recognizing CDl9, CD20, CD23, and CD34 were mouse IgG,; anti-PCA-1 was an IgG, antibody; and anti-CD10 was an IgG,, antibody. The percent depletion of plaque-forming colonies relative to isotype controlcultures in this representativeexperimentwas 91% for CD19-; 37% for CD34-; 51%for CD10-; and 42% for CD20-depleted populations. No significant depletion (P >.05) of plaque-forming colonies was observed when PCA-1' or CD23' cells were depleted from the input populationbefore plating.
Figure 1 shows a typical depletion experiment. The results of a series of such experiments are presented in Table 2. It is demonstrated that the depletion of CD19' cells from percoll-enriched bone marrow samples resulted in a reduction of 96.1% 5.7% (n = 10) in the number of resultant plaque-forming colonies. This result indicates that the clonogenic cell in this assay is a B-lineage cell which expresses the B-cell-specific surface antigen, CD19. The removal of CDlO+ cells from the input population led to a reduction in the growth of plaque-forming colonies by 51.5% ? 13.3% (n = 8) relative to isotype control cultures (Fig 1 and Table 2). CDlO is expressed on a subset of immature B-lineage cells, and is acquired after the expression of CD19 and before the expression of surface IgM. Therefore, the results of this depletion experiment suggest that these assay conditions allow CD10' B-cell
precursors to differentiate into colonies containing cells that secrete Ig. The removal of CD20+ cells from the input population led to a depletion of 51.3% 5 6.0% (n = 8) of the number of resultant plaque-forming colonies when compared with isotype control cultures (Fig 1 and Table 2). During differentiation, CD20 is acquired after CD19 and CD10; therefore, this result suggests that more mature B-lineage cells also differentiate into Ig-secreting colonies under these culture conditions. In one experiment, the depletion of cells expressing the mature B-cell antigen, CD21, resulted in a similar reduction of plaque-forming colonies to that observed with CD20 depletion (Table 2). No significant reduction in the number of B-cell colonies was observed after the depletion of either plasma cells expressing PCA-1, or activated B cells expressing CD23, from percoll-enriched bone marrow samples in three experiments (Fig 1 and Table 2). Some depletion of plaque-forming colonies was observed when CD34- cells were plated in the B-cell colony assay (Fig 1 and Table 2). This result was variable, with a range of 27% to 51% depletion of plaque-forming colonies in four experiments. Monocytes and granulocytes express CD13 on their cell surface.% Depletion of CD13' cells from percoll-enriched bone marrow samples before plating in the B-cell colony assay did not significantly affect the number of resultant plaque-forming colonies in three experiments (Table 2). Identification of colonies secreting both K and A Ig light chains. Replicate colony protein blotting experiments were performed to assess whether the culture conditions described allowed the clonal growth of Ig light chain uncommitted B-cell precursors. In these experiments, cultures were analyzed for colonies that simultaneously secreted both IgK and IgA. This experimental approach is based on the premise that a single colony containing some cells secreting IgK and others secreting IgA originated from a progenitor cell that was not yet committed to the expression of a particular Ig light chain. Figure 2 shows the positive signals obtained after replicate protein blotting with anti-IgK and anti-IgA MoAbs. It can be seen that the autoradiographs of the first and the second antibodies can be aligned, allowing the enumeration of double K + A-colonies by counting the number of times positive signals precisely overlap on both autoradiographs. Figure 2 and Table 3 demonstrate that no active, residual

Table 2. Mean Depletion of Plaque-FormingColoniesAfter Removal of B-Lineage Subpopulations From a Series of Bone Marrow Samples
% Depletion of Plaque-Forming Colonies per 1 x lo5 Input Cells

Input Population

PCA-1-
Mean SEM Range No. experiments

96.1 5.7 85-100 10

41.2 11.4 27-51 4

51.3 13.3 37-76 8

51.3 6.0 42-62 8

9.6' 3.1 4-8 3

7.0* 5.7 0-14 3

5.3* 3.9 0-9 3

Percoll-enriched bone marrow samples were depleted of B-lineage subpopulations using MoAbs and magnetic immunobeads. These cells were then plated in the B-cell colony assay and resulting plaque-forming colonies enumerated. The percent depletion of plaque-forming colonies was calculatedfor each experiment and the mean, SEM (standard error of the mean), and the range of a series of experiments are presented in this table. 'Number of plaque-forming colonies was not significantly different (Student's t-test: 95% confidence interval) from the appropriate isotype control in each experiment.
GROWTH OF BONE MARROW B CELLS AND PROGENITORS

(ii) A

blotting technique is that single, Ig-secreting cells remain viable during the culture period and result in a positive signal. These cells could not be totally removed by prior depletion of cells expressing CD19, CD20, CD23, or PCA-1. Thus, while it is not possible to determine the precise number of positive Ig-secreting B-cell colonies from single protein blots at present, it is feasible to enumerate double IgK + IgA-producing colonies from replicate blots. The estimation of the frequency of Ig light chain uncommitted cells by counting the number of double K + A-positive signals assumes that isotype exclusion occurs and that K + A-producing colonies arise from a single cell. To determine whether the assay conditions allow for this clonal growth of K + A-colony progenitors, titration experiments were performed in which 5 x lo4 to 2 x los percollenriched bone marrow cells were plated and the resultant K + A-producing colonies enumerated. The least squares regression line fitted to these data had a slope of approximately 1(range 0.89 to 1.14, in four experiments), as shown in Fig 3. This result supports the assumption that the growth of K + A-producing colonies under these assay conditions is dependent on a single progenitor cell. In addition, time course experiments showed that the majority of double K + A-positive signals did not appear until at least the seventh day of culture. It was observed that single
Table 3 Representative Replicate Protein-Blotting Experiment.

No. of Positives

FirstAElgK Second ABAgK Igh None Double positive -K h

172 162

107 95
Fig 2. Replicate protein blotting. Percoll-enriched bone marrow cells were cultured in the 6-cell colony assay for 10 days. The Ig secreted from 6-cell colonies was transferred onto nitrocellulose filters and these filters were analyzed for IgK and Igh light chain isotype secretion using the replicate protein blotting technique. Autoradiographs produced with the first antibody (i) could be aligned to that of the second antibody (ii) by orienting the positive plasma marker signals in the corners of each filter. The first, biotinylated anti-human Ig antibodies used were anti-lgK (row A) and anti-lgh (rows B and C). Filterswere then incubated with earavidin-HPRO and developed using the luminographic method (2-minute exposure) (column i). The same filters in each row were then treated with sodium azide and incubated with HPRO-conjugatedanti-lgh (rows A and C ) or no antibody (row E), followed by luminographic development (2-minute exposure) (column ii). In row A, the white circles denote some of the signals that were positive for both IgK and Igh. Row B demonstrates that sodium azide treatment completely removed the signal caused by HRPO bound to the first antibody on the filter. In row C, it can be seen that this sodium azide inactivation process does not interfere with subsequent binding of a second antibody (anti-lgh)to the filter. In this example (C),greater than 90% of the Igh-positivesignals detected with the first antibody (i) could be detected with a second anti-lgh antibody (ii).

expected because of coincidence now becomes 4,and this is approximately 10-fold less than the actual number of K + A positives observed. It is also apparent that the larger the number of positive signals per filter, the greater the number of expected double positives due to coincidence (Table 4). In these replicate protein blotting experiments, the ratio of IgK to IgA expression was maintained at approximately 60%:40%,4' as can be seen in Table 4. This K:A ratio remained largely unchanged whether determined by singleor double-protein blotting (Table 3). The frequency of Ig light chain uncommitted B-lineage cells that were able to generate plaque-forming colonies ranged from 1 in 1.42 x lo4 to 1 in 1.33 x lo3 percollenriched bone marrow cells. The number of plaque-forming colonies was consistently higher than the number of observed K + A-producing colonies (Table 4) when the two were assessed in parallel. This result indicates that the clonogenic cell that gave rise to K + A-producing colonies is not the only cell type that can differentiate and proliferate into Ig secreting B-cell colonies in this assay. This observation is supported by the depletion experiments outlined above. Light chain uncommitted B-lineage cells express CD19 and CD10. The preceding results suggest that the clonogenic cell that initiates K + A-producing colonies has an immature phenotype. This was verified by replicate protein blotting analysis of cultures in which the input population was
Table 4. Relationship Between the Number of Input Cells Plated and the Number of Resultant K

+ A-Producing Colonies

of Ign

No. IgA + ve

Ign:A Ratio

Observed + A + ve

Coincidence + A + ve

5 x 1 x 2 x 15 0

9.1.0 21.1 t 1.9 42.6 t 5.5
33.42 5.5 62.8t 4.7 110.0 c 9.7
24.0 t 4.8 46.7 c 5.2 82.3 c 8.6

57:43 57x3 57:43

5.7 ? 1.2 9.7 f 0.9 18.9c 1 9.

0.4 16. 4.9

Three concentrations of percoll-enriched bone marrow cells (5 x l' 1 x lo5,and 2 x lo5) o, were cultured in the double-agar 8-cell colony assay. After a 10-day incubation the lg secreted from colonies growing in the agar was transferred onto nitrocellulose filters. The filters were incubated with either anti-human Igr-biotin or anti-human IgA-biotin antibody, then extravidin-HPRO, and developed using the luminographic technique. The HPRO on the filters was inactivated and each filter was subsequently incubated with either anti-human IgA-HRPO or anti-human IgK-HRPO antibody, respectively,and developed. The mean number of observed single lgK, single IgA, and double IgK IgA-positive(+ve) signals is given. The maximum number of double positives expected due to coincidence is also shown. The number of plaque-forming colonies (PFC) was estimated in parallel.

depleted of cells expressing specific B-lineage surface antigens (Table 5). The depletion of CD19' cells resulted in the removal of K + A-producing colonies, indicating that the clonogenic cell expressed CD19. Depletion of CD10' positive cells also led to a significant reduction in the number of resultant K + A-producing colonies. On the other hand, depletion of CD20' cells from percoll-enriched bone marrow samples did not result in a significant decrease in the number of resultant K + A-producing colonies. Taken together, these results suggest that the cell which gave rise to double IgK + IgA-producing colonies is an immature B-lineage cell that expresses CD19 and CDlO surface antigens but has not acquired the CD20 antigen. In addition, this implies that CD19 and CDlO antigens are acquired before Ig light chain commitment, whereas CD20 is not.

DISCUSSION

In this report we have determined that the culture conditions used in the previously described B-cell colony assay4' support the proliferation and differentiation of B-cell precursors, as well as more mature B lymphocytes. Specifically, we have characterized the clonogenic B-lineage cells from normal, adult, human bone marrow. The results presented here show that the culture conditions described allow the clonal growth and differentiation of both CD10' and CD20' B-lineage cells into Ig-secreting cells. Further, it is demonstrated that clonogenic cells which are uncommitted to Ig light chain expression have already acquired the CD19 and CD10, but not the CD20, surface antigens. The expression of surface antigens by bone marrow B-lineage cells has enabled to divide these cells into maturation stages. The earliest recognizable B-lineage cells express HLA-DR, CD19, and possibly CD34. During the next stage these cells gain CDlO and lose CD34. Next CD20 is acquired, CDlO is lost, and surface IgM is expressed. More mature B cells express CD21, on activation acquire CD23, and after terminal differentiation
Table 5. Effect of B-Lineage Subpopulation Depletion on K A-Producing Colony Formation
No. of K + A Colonies11 x lo5 Cells
Population Mean SEM t-Test: P value

IgG17.1 1.0

CD190.75 1.0.0002

CD207.7 1.8.42

IgGZb7.1 0.6

CD101.5 0.3

Percoll-enriched bone marrow samples were depleted of cells expressing CD19, CD20, or CDlO surface antigens. These depleted populations were plated in the double-agar B-cell colony assay and incubated for 10 days. The cultures (n = 8 replicates) were then assayed using the replicate protein blotting method to detect colonies containing both cells that secrete IgK and cells that secrete IgA. lsotype control cultures were set up and analyzed in parallel. The mean and the standard error of the mean (SEM) are given for four separate experiments. The Student's t-test was used to compare CD19- and CD20cultures with the lgGl isotype control cultures; and CDlO- cultures to A-secreting the IgG2b control cultures. The number of double kJK colonies was significantly reduced when the input population was depleted of CD19' cells or of CD10' cells.

to plasma cells express antigens such as PCA-1. To further characterize the clonogenic cells in the B-cell colony assay we chose to deplete bone marrow samples of B-lineage subpopulations before plating. Depletion, rather than positive enrichment, was selected because the binding of some B-lineage antibodies to their respective surface antigen has been reported to deliver either activation (CD20;' CD21,S1 CD2352)or inhibition (CD19,53CD207) signals to the cell. Also, we observed that considerable cell death resulted from an overnight incubation of cells rosetted with immunobeads; and lastly, cell sorting by flow cytometry was not considered feasible due to the low frequency of relevant cells in the bone marrow, and the lengthy periods of time required for this procedure. It was demonstrated that depletion of CD19' cells before plating in the B-cell colony assay led to an almost complete abolition of plaque-forming colony growth. This result indicates that the clonogenic cells express the CD19 surface antigen. CD19 is present on all B-lineage cells, including immature precursors and mature B cells. The results of these experiments led us to investigate whether immature B-lineage cells could initiate the formation of colonies containing cells that secrete Ig. The removal of CD10' cells led to a reduction of 50% in the number of resulting plaque-forming colonies, showing that bone marrow cells with an immature, CD10' surface phenotype could form colonies. Depletion of other B-lineage subpopulations showed that cells expressing the CD20 antigen could also initiate B-cell colony formation. On the contrary, activated B cells and plasma cells could not generate B-cell colonies under these culture conditions. Because the results of depletion experiments indicated that B-lineage cells with an immature surface phenotype could differentiate into Ig-secreting cells, we examined the status of Ig commitment of the clonogenic cells. By studying the pattern of Ig light chain isotypes secreted from daughter cells in the resultant colony, inferences could be made concerning the Ig commitment status of the parent cell which initiated that clone. We used the replicate protein blotting technique to examine single colonies for the simultaneous expression of IgK and IgA. The results showed that some colonies contained cells secreting IgK as well as some secreting Igh. These double K + A-positive colonies could not be accounted for solely by the coincidental overlap of single K-positive signals and single A-positive signals. This observation, together with the results of titration and time course experiments, is consistent with the clonal growth of single, Ig light chain uncommitted B-cell precursors, which can differentiate into colonies containing some cells that secrete IgK and others that secrete Igh. The frequency at which B-lineage cells, uncommitted to Ig light chain expression, give rise to colonies simultaneously containing cells that secrete IgK and those secreting IgA has been estimated to be a maximum of 1 in 1.33 x lo4 percoll-enriched bone marrow cells. It is difficult to determine the true "cloning efficiency" of Ig light chain uncommitted precursors as the input population is a heterogeneous collection of hematopoietic cells of similar buoyant

density. It is possible that the true cloning efficiency of determined. For example, the frequency of total B-cell these B-cell precursors is actually quite high. colony progenitors that grow under the described culture Further experiments, in which the depletion of B-lineage conditions was previously estimated to be a maximum of 1 in 1 X lo3 (0.1%),42while the maximum frequency of subpopulations was combined with replicate protein blotting analysis of the resultant colonies, allowed us to correclonogenic cells initiating IgK + A-producing colonies was late surface antigen expression with the status of Ig light M.33 x lo3 (0.03%) percoll-enriched bone marrow cells in chain commitment in clonogenic B-lineage cells. Depletion the experiments presented here. The normal, human B-cell of CD10' or CD19' populations resulted in the elimination colony assay may also prove useful for detecting B-lineage of double IgK A-producing colonies, whereas depletion of cells in bone marrow colonies of mixed lineages (CFU-mix), thereby affording a means for studying human pluripotent CD20' cells did not affect the growth of these colonies. stem cells that are capable of giving rise to myeloid and Thus, these experiments provide evidence that B-lineage cells that are not yet committed to the usage of a particular T-cell as well as B lymphocytes. The expression Ig light chain, express the CDlO surface antigen but do not of the CD34 antigen may be important for the study of such stem cells because (1) this antigen is expressed on early express CD20. This suggests that B-lineage cells in the normal, adult bone marrow acquire the CD19 and CDlO myeloid progenitor cells,'9363 it is also expressed on B-cell (2) surface antigens before Ig light chain commitment, whereas precursors, and (3) the depletion experiments reported here indicate that CD34 is expressed on some of the the CD20 antigen is expressed after this commitment event. Replicate protein blotting analysis may provide a useful clonogenic cells that give rise to plaque-forming colonies. tool in future studies aimed at determining the effects of Replicate RNA colony blotting4' will be an effective technique for studying multiple RNA transcripts in such single, growth factors and stromal cells on the differentiation of Ig light chain uncommitted progenitor cells in vitro. Unfortumulti-lineage colonies. nately, two drawbacks with this detection system should be In summary, we have demonstrated that under these kept in mind. Firstly, functional cells cannot be recovered culture conditions, cells expressing CD19, CD10, and CD20 after the detection procedure. Secondly, Ig-secreting cells surface antigens are able to differentiate in vitro to form colonies containing cells that secrete Ig. In addition, we are sustained during in vitro culture, as reported by others3' have shown that B-lineage cells uncommitted to Ig light These single Ig-secreting cells also cause a positive signal on autoradiographs after protein blotting. Therefore, input chain expression have already acquired the CD19 and CD10, but not the CD20 surface antigens. Studies are cell numbers must be kept low to minimize coincidental ongoing to assess the regulatory influences of known growth overlap of signals and alignment of autoradiographs should factors and bone marrow stromal cells on normal human B be carefully performed. lymphopoiesis using the B-cell colony assay. The results presented here, together with our previous describe culture conditions that allow the clonal growth and differentiation of B-lineage cells, including ACKNOWLEDGMENT immature CD10' cells, into Ig-secreting cells. Using this We thank Dr Hans Messner and Nazir Jamal, Ontario Cancer system it will be possible to investigate the direct effect of Institute, and Drs Earl Bogoch and David Hastings, Wellesley growth factors and stromal cells on human B-lineage colony Hospital, for providing bone marrow specimens. We also acknowlprecursors, and hence further our current understanding of edge Maryke Koekebakker for expert technical advice on immunonormal, human B l y m p h o p ~ i e ~ i ~ In~ ~ ~ ~ ~ ~ " ~ ~ ~fluorescence~ methods and FACSCAN analysis of human bone. addition, this '~~~~ ~ marrow samples; and Dr Mark Minden for critical comments. assay allows the frequency of the responding cell to be

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