Normal cellular counterparts of B cell chronic lymphocytic ......tion were enriched for B cells by E...

1987 70: 418-427

AS Freedman, AW Boyd, FR Bieber, J Daley, K Rosen, JC Horowitz, DN Levy and LM Nadler Normal cellular counterparts of B cell chronic lymphocytic leukemia

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418 Blood, Vol 70, No 2 (August). 1987: pp 4 18-427

Normal Cellular Counterparts of B Cell Chronic Lymphocytic Leukemia

By Arnold S. Freedman, Andrew W. Boyd, Frederick R. Bieber, John Daley, Karen Rosen,

Jack C. Horowitz, David N. Levy, and Lee M. Nadler

In an attempt to compare B cell chronic lymphocytic

leukemia (B-CLL) with its normal cellular counterpart, thecell surface phenotype of 1 00 cases of B-CLL was deter-

mined by using a panel of monoclonal antibodies (MoAbs)

directed against B cell-restricted and -associated anti-

gens. The majority of B-CLL cells expressed Ia. B4 (CDi9).

Bi (CD2O). B2 (CD21 ). surface immunoglobulin (slg). and

Ti (CD5) but lacked C3b (CD35) receptors. In contrast. the

overwhelming majority of small unstimulated B cells

expressed Ia, B4, Bi . B2. 51g. and C3b receptors but lacked

detectable Ti . Small numbers of weakly slg + cells could

be identified in peripheral blood and tonsil that coex-

pressed the Bi and Ti antigens. Approximately i 6% of

fetal splenocytes coexpressed Bi . Ti , weak 51g. B2, and Ia

but lacked C3b receptors and therefore closely resembled

most B-Cu cells. With the phenotypic differences

between the majority of small unstimulated B cells andB-Cu cells, we examined normal in vitro activated B cells

G REATER THAN 95% of chronic lymphocytic leuke-

mias (CLL) are of B cell lineage by virtue of their

expression of monoclonal cell surface immunoglobulin

(slg),’3 clonally rearranged immunoglobulin genes,4’5 and

the expression of B cell-restricted cell surface antigens

(Ags).�” Although the B cell origin of CLL is generally

accepted, the subset of normal B cells from which B cell CLL

(B-CLL) is derived remains unresolved. The difficulty in

identifying the normal cellular counterpart of B-CLL is

based upon its unique cell surface phenotype and functional

properties. B-CLL cells differ from morphologically identi-

cal unstimulated small B lymphocytes in that they lack C3b

receptors (CD35),’2”3 express the 67-kilodalton (kD) T

cell-associated TI (CD5) Ag,’�2#{176}and form rosettes with

mouse erythrocytes (MRBC-R).21’23 Moreover, B-CLL cells

demonstrate variable responsiveness to mitogens that acti-

vate most normal small B lymphocytes.2�27 These phenotypic

and functional differences suggest that B-CLL appears to be

derived from a unique subset of B lymphocytes.

From the Division of Tumor Immunology, Dana-Farber Cancer

Institute; Department ofPathology, Brigham and Women’s Hospi-

tal,’ and Departments ofMedicine and Pathology, Harvard Medical

School, Boston.

Submitted January 2. 1 987,’ accepted March 28. 1987.

Supported by National Institutes of Health Grants No.

CA25369, CA34183. and CA40216. A.S.F. is supported by Public

Health Service Grant No. lKO8 CAOI 105-01 awarded by the

National Cancer Institute, DHHS.

Address reprint requests to Arnold S. Freedman. MD, Division of

Tumor Immunology. Dana-Farber Cancer Institute, 44 Binney St.

Boston, MA 02115.

The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked

“advertisement” in accordance with /8 U.S.C. §1734 solely to

indicate this fact.

(C) 1987 by Grune & Stratton, Inc.

0006-4971/87/7002-0013$3.00/0

and B-CLL cells for the expression of B cell-restricted and

-associated activation antigens. Of 20 cases examined.

virtually all expressed B5, and approximately 50% of the

cases expressed interleukin-2 receptors (lL-2R) and Blast-

i . Normal B cells were activated with either anti-Ig or

i 2-O-tetradecanoylphorbol-$-acetate (TPA) and then were

examined for coexpression of Bi . Ti , and the B cell

activation antigens B5 and IL-2R. Only cells activated with

TPA coexpressed Bi and Ti as well as B5 and lL-2R. B cells

activated with either anti-Ig or TPA proliferated in the

presence of lL-2, whereas B-CLL cells did not, although

they all expressed the identical 60-kilodalton proteins by

immunoprecipitation. These studies are consistent with

the notion that B-CLL resembles several minor subpopula-

tions of normal B cells including a population of B cells that

are activated in vitro directly through the protein kinase C

pathway.

S 1987 by Grune & Stratton, Inc.

Previous attempts to identify the subset of B cells from

which B-CLL cells arise have been largely based upon

morphology and analysis of cell surface phenotype. Until

recently, it was generally believed that B-CLL cells were

derived from small unstimulated peripheral blood B lympho-

cytes since they were morphologically identical and weakly

coexpressed 1gM and IgD. Moreover, both populations also

expressed HLA class II molecules as well as other B cell-

restricted Ags including B4 (CD19),” Bi (CD2O),6 and B2

(CD21),9 the C3d/Epstein-Barr virus (EBV) receptor. How-

ever, the coexpression of Ti and MRBC-R and B-CLL cells

led investigators to abandon the peripheral blood B cell in

search of another B cell subset that might express these

structures. Several studies have now identified subpopula-

tions of normal B cells that resemble B-CLL cells.28’29 For

example, a very small subpopulation of slg-positive B cells

(2% to 3%) was identified at the edge of the germinal center

of the secondary follicle in tonsil and lymph node material

that coexpressed Tl and formed MRBC-R. Similarly, Tl +

B cells have been identified in human fetal lymph node and

spleen tissue, and these cells appear to be a major subpopula-

tion of fetal B cells. These studies provided evidence for one

or more subpopulations of normal B cells that phenotypically

resembled B-CLL cells.

In the present report, we have attempted to extend these

studies by relating B-CLL cells to subpopulations of normal

B cells. In the studies to be reported later, we will demon-

strate that small populations of B cells can be identified in

adult peripheral blood, adult lymphoid tissue, and fetal

lymphoid tissue that coexpress the Bi and Ti Ags. More-

over, B-CLL cells express several cell surface Ags that are

expressed on activated B cells but not resting B cells, thereby

suggesting the B-CLL cells may be the neoplastic counter-

part of a subpopulation of activated B cells. Further evidence

for this notion is derived from the observation that 12-

0-tetradecanoylphorbol-�3-acetate (TPA) but not anti-Ig can

induce small unstimulated B lymphocytes to express Bi and

TI as well as B cell activation Ags.




NORMAL CELLULAR COUNTERPARTS OF B-CLL 419

MATERIALS AND METHODS

Adult Human Tissues Cells

Normal spleen, tonsil, and bone marrow were obtained after

appropriate Human Protections Committee validation and informed

consent. Normal spleen was obtained from operative specimens of

patients not known to have any systemic or malignant disease. Tonsilcells were obtained at the time of routine tonsillectomy. Nucleated

bone marrow cells were recovered by Ficoll-Hypaque centrifugation.Single-cell suspensions of spleen and tonsil tissue were prepared bydissolution in Hanks’ balanced salt solution (HBSS) with forcepsand scissors and extrusion through a stainless steel mesh. Mononu-

clear cells isolated by Ficoll-Hypaque density gradient centrifuga-tion were enriched for B cells by E rosetting to deplete T cells. Cellswere either used fresh or cryopreserved in 10% dimethyl sulfoxideand 20% fetal calf serum (FCS) at - 196#{176}C in the vapor phase of

liquid nitrogen until the time of study. Cells were recovered at

viabilities of 70% to 90%.

Isolation ofPeripheral Blood Cells

Human peripheral blood mononuclear cells (PBMC) were iso-lated from healthy volunteer donors by Ficoll-Hypaque densitysedimentation (Pharmacia Fine Chemicals, Piscataway, NJ).

Unfractionated mononuclear cells were separated into slg+ andslg- populations by Sephadex G-200 (Pharmacia) anti-F(ab’)2chromatography with modifications designed to minimize monocyteretention by columns as previously described.6 T cells were recovered

by E rosetting the slg- population with 5% sheep erythrocytes.Normal human monocytes were removed by adherence to plastic

culture dishes.

Fetal Tissues

Fetal tissues were obtained within one hour of prostaglandin-

induced abortion. All patients undergoing therapeutic abortion hadlast menstrual periods and diagnostic ultrasound imaging that

suggested that the fetal age was less than 24 weeks. To standardize

gestational age, age determination postmortem was determined bycrown-rump length and fetal foot length. Procurement of tissue wasapproved by the Brigham and Women’s Hospital Committee onHuman Subjects in Research, and informed consent was obtained

from all patients undergoing therapeutic abortion.Fetal bone marrow and single-cell suspensions of spleen and liver

were prepared as previously described.30

Patients Samples

Tumor cells were obtained from peripheral blood of previously

untreated patients with CLL from Brigham and Women’s Hospital

and Dana-Farber Cancer Institute after appropriate Human Protec-tion Committee validation and informed consent. The diagnosis was

based on a total peripheral blood lymphocyte count of 15 x 109/Land an infiltration of well-differentiated lymphocytes in the bone

marrow. B cell lineage was established by the presence of mono-clonal slg or the pan-B cell antigen BI as determined by indirectimmunofluorescence with the use ofanti-k, A, IgG, 1gM, IgD, and BImonoclonal antibodies (MoAbs).

MoAbs

The preparation and characterization of MoAbs used in this studyhave been described. The Ags to which these antibodies are directedare summarized and referenced in Table 1 . All MoAbs used in thisstudy were ascites fluid at saturated binding concentrations.

Table 1 . Expressi on of Lineage-Restri cted and -A ssociated Ags

Molecular

CD Norm& Weight

Ag Designation Cellular Reactivity (kD) Reference

Bi 20 pan-B 35 6

82 21 LimitedB 140 9

84 19 pan-B 95 11

Ia pan-B 29, 34 31

slg Limited B lgM-900

lgG- 150

gD- 150

31

B5 Activated B 75 32

Blast-i Activated B 45 33

Blast-2 23 Activated B 45 34

BB1 Activated B 37 35

lL-2R 25 Activated T. B 55 36

T9 Proliferating cells!

nonlineage re-

stricted

90 37

C3bR 35 B, RBC. monocyte.

�‘anulocyte

220 38

Mo2 14 Monocyte 39

Ti 5 T, thymocytes. sub-

set of B

67 14-20

Ti 1 2 T, thymocyte 50 40

Fluorescent Staining

Indirect. Cells were prepared in 10% pooled AB serum inHBSS; when the cells had been incubated with anti-Ig coupled to

beads, they were incubated in human serum for one hour at 37#{176}Cto

remove the beads from the cell surface. Aliquots of I 06 cells wereincubated with each antibody (generally a 1/100 to 1/400 dilution

of ascites) for 30 minutes at 4#{176}C.After washing, the cells wereincubated with a 1/50 dilution of fluorescein-conjugated goat anti-

mouse Ig antibody (Tago Inc. Burlingame, CA) for 20 minutes at4#{176}C.The cells were washed and were either analyzed fresh or were

fixed in 1% formaldehyde for subsequent analysis.6 A reaction wasconsidered positive when greater than I 0% of the test cells were more

fluorescent than the number of cells positive with isotype-identicalcontrol ascites. For each sample, a quantitative assessment of thenumber of positive cells was made (number of cells reactive with the

test antibody minus the number of cells reactive with unreactiveisotype-identical monoclonal antibody/lO,000 total cells tested).

Direct and dual-fluorescence staining. Directly fluorescein con-jugated MoAbs were prepared as previously described.’ For directlyphycoerythrin (PE) conjugated MoAbs, I .0 mg of protein A-

purified anti-Bl, anti-interleukin-2 receptor (IL-2R) antibody, or aeuglobulin precipitate purification of anti-B5 were reacted with 0.5mg of R-phycoerythrin (pyridyldisulfide derivative) as described

elsewhere.4’MoAbs were biotinylated by standard techniques.” The specific-

ity of each of these conjugated antibodies was tested on appropri-ately reactive normal tissues and cell lines and found to be identicalwith that of the unconjugated antibodies. To define the percentage of

cells that expressed the BI and Tl Ags within each cellularpopulation, the number of cells with positive fluorescence was

compared with the number of cells stained with negative controlantibody out of a total of 500 to I ,000 cells counted on a fluorescentmicroscope (Carl Zeiss, West Germany). Cells with two or morediscrete positive clumps per cell or cells with clear-cut peripheral

rims were scored as positive. To enumerate the number of Bl + cellsthat coexpressed the TI Ag, 100 Bl + cells were counted, and thepercentage of these cells expressing TI was determined. To charac-terize the BI + F(ab’)2 column nonadherent population, the number




420 FREEDMAN ET AL

ofcells expressing the BI and Tl Ags was first defined. The number

of directly fluoresceinated B I + cells coexpressing other cell surface

determinants was next enumerated by counting the number of Bi +

cells that stained with a second directly biotinylated MoAb and weredeveloped with avidin Texas red dye.” By using these techniques, itwas possible to accurately identify the number of BI + cells that

coexpressed several other cell surface determinants. All flow cyto-metric analysis was performed on either an EPICS V or C cell sorter(Coulter Electronics, Hialeah, FL). Dual-fluorescent-stained cellswere analyzed after initial calibration of the machine with cells

stained with each individual fluorochrome-labeled antibody andwith other controls as outlined previously.” When beads were

present in the cell suspensions, they could be excluded from theanalysis by setting the forward-angle light scatter gates to exclude

particles less than 5.tm in diameter. MRBC-R were enumerated by

the method previously described.22

B Cell Cultures

Large-scale B cell cultures. The E - fraction of splenic mononu-

clear cells was further enriched for B cells by two treatments withthe MoAbs anti-Mo!, anti-Mo2, anti-T4, and anti-T8 followed by

rabbit complement to deplete all but B cells from the spleen cellsuspensions. These highly enriched B cells were cultured for twodays at 1.5 x l06/mL in RPMI 1640 supplemented with 10% FCS, 2mmol/L glutamine, and I mmol/L sodium pyruvate in tissue culture

flasks (Corning Glass Works, Corning, NY), with either affinity-purified F(ab’)2 rabbit antihuman Ig coupled to Affigel 702 beads

(anti-Ig) (Bio-Rad, -Richmond CA) as previously described42 orTPA (Sigma Chemical Co. St Louis) used at a final concentration of10 ng/mL, and l0� mol/L 2-mercaptoethanol.

Microcultures. Highly enriched splenic B cells were preparedfrom the E - fraction of splenic mononuclear cells by anti-T cell and

antimonocyte MoAb and complement lysis. CLL cells were highlypurified by a similar depletion of T cells and monocytes. Cells were

cultured in 96-well, round-bottom microtiter trays (Costar, Cam-bridge, MA) at 50,000 per well. Anti-Ig beads and recombinant IL-2

(rlL-2) (a gift of the Biogen Corp. Boston) were added to yield a

final volume (per well) of 200 zL. Previous studies have demon-strated that maximal stimulation of normal B cell 3H-thymidine

incorporation with RI-L2 is at 200 U/mL.43 T cell conditionedmedium (TCM) was prepared as previously described.42 TPA wasused at a final concentration of 10 ng/mL.

Thymidine Uptake Assay

Thymidine uptake was used as an index of mitogenic activity.

Microcultures were pulsed with 0.2 �.tCi of 3H-thymidine (Amer-sham Corp. Eastbourne, England) per well and were harvested IS

hours later. Dried filters were counted on a Packard Tri-carb

scintillation counter (Downers Grove, IL).

Labeling ofCells With Radioisotopes

A modification of the lactoperoxidase iodination technique was

used. The labeled cells included IL-2R+ CLLs that were further

enriched for B cells by lysis with anti-T cell and antimonocyteantibodies and complement. T cells were activated with anti-TI 12

and -TI 13 antibodies for three days.” Highly enriched splenic B cellswere activated with TPA (10 ng/mL) for two days. The iodinated

cells were washed twice with cold phosphate-buffered saline andlysed on ice with cell lysis buffer (50 mmol/L Tris-HCI, 0.4 mol/L

NaCI, 1% Triton X-I00, 2 mmol/L phenylmethylsulfonylfluoride, 5mmol/L EDTA, 50 mmol/L iodoacetamide, pH 8). After 30minutes the lysate was centrifuged at 800 g for ten minutes toremove unlysed cells, nuclei, and other insoluble material. The

supernatant was frozen at -80#{176}Cuntil analyzed.

Immunoprecipitation

Cell supernatants and lysates were centrifuged at 100,000 g for IS

minutes and then precleared four times: twice for one hour at 4#{176}Cwith either Sansorbin (for Ig precipitations) or Pansorbin (Calbio-chem-Behring Corp, La Jolla, CA), once with Sepharose 4B beads,

and once with an irrelevant antibody conjugated to Sepharose 4B.The precleared lysates were mixed with anti-IL-2R antibody conju-gated to Sepharose 4B beads. The mixtures were held on ice for 2hours and then washed four times with 1% Triton X-iOO and 1%

sodium deoxycholate in 12 mmol/L sodium phosphate, 5 mmol/LEDTA, 5 mmol/L ethylene glycol tetraacetic acid (EGTA), and Immol/L NaF, pH 7.4 (RIPA buffer). Precipitates were analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%). Ig precipitates were performed in an identical

manner except that in the precipitation of the precleared superna-

tant we used antihuman Ig antibody bound to protein A-Sepharose

4B (Pharmacia, Uppsala, Sweden).

RESULTS

Expression ofB Cell-Associated Ags on B-CLL Cells

Tumor cells from 100 patients with the clinical and

morphological features of B-CLL were tested for reactivity

with a panel of MoAbs. Tumor cells in all cases were of B cell

derivation by the expression of Bl and B4 (Table 2). All but

five cases expressed Ti . Ninety of 100 cases expressed the

EBV/C3d receptor B2.4547 The two major phenotypic sub-

groups were defined by the expression of slg and the receptor

for C3b (C3bR). Monoclonal slg was not detectable on the

tumor cells of 21 patients. Only 19 of the 100 patients with

B-CLL expressed the C3b receptor. The overwhelming

majority of B-CLL cells coexpressed Ia, Bi, B4, Ti, and B2.

Three major subgroups could be identified by the examina-

tion of expression of slg and C3bR: (a) slg+, C3bR-

(n = 51); (b) slg+, C3bR+ (n = 17); and (c) slg-, C3bR-

(n = 15).

Examination of the intensity of Ag expression was also

undertaken (Table 2). Ia and Bl were strongly expressed on

all tumor cells (Fig 1). The B2 and B4 Ags were less

intensely expressed but were clearly positive on most tumor

cells (60% to 80%) in the neoplastic population. Cell slg, Ti,

and C3bR were much less intensely expressed on the cell

Table 2. B Cell Ag E xpression of CLL

.No. of PatientsExpressing Ag

Ag Intensity

Ia B 1 B2 B4 sig Ti C3bR

51 +++ +++/++ ++ ++ + +/++

17 +++ +++/++ ++ ++ + +/++ +

15 +++ +++/++ ++ ++ +/++

2 +++ +++/++ ++ ++ +/++ +

6 +++ +++/++ ++ + +/++

4 +++ +++/++ ++ +/++

5 +++ +++/++ ++ ++ +

The degree of positivity was qualitatively assessed by flow cytometry.

Blank space, no detectable reactivity over background; + , weak (slgM on

peripheral blood B cells. Fig 1); + + , moderate (B2 on peripheral blood B

cells, Fig i); + + + , strongest (B 1 on peripheral blood B cells, Fig 1).

The range of percent positive cells for each antigen was as follows: Ia,

65% to 85%; Bi, 70% to 90%; B2, 50% to 70%; B4, 60%-80%; Ti,

60% to 80%; C3bR, 20% to 40%. The percentage of cells expressing

slg was difficult to quantitate due to weak expression.




Log Green Fluorescence Intensity


a)-‘:3

E

z

a)

0

Fig 1 . Fluorescence-activated cell sorter histogram of the reactivity of anti-lgM. -lgD. -Ia. -B2. -Bi . -Ti . and C3bR with normal

peripheral blood B cells (top) and B-Cu cells (bottom). Background fluorescence was obtained by incubating the cells with isotype-identical unreactive MoAb and developing with goat antimouse Ig FITC.

surface. When compared with small resting B cells isolated

from peripheral blood or spleen tissue that express Ia, B4,

Bl, B2, slg, and C3bR, the intensity of sig and C3bR was

significantly less intense on B-CLL cells.

Identification of Ti Ag-Positive B Cells in Normal

Lymphoid Populations

With the observation that most B-CLL cells express Ia,

B4, Bl , sig, and TI , we attempted to identify normal B

lymphocytes that expressed the B-CLL phenotype. Mononu-

clear cells isolated from peripheral blood, tonsil, and bone

marrow were examined for the presence of cells that coex-

pressed Bi and TI Ags. In these experiments, the number of

Bl- and TI-positive cells was first enumerated, and then the

number of cells coexpressing T! was determined (Table 3).

In the PBMC fraction, few if any BI +Ti + cells were

identified. When T cells were removed by E rosetting, the

resulting E- fraction demonstrated approximately 3% of

cells that coexpressed Bi and TI, whereas no Bi + cells

coexpressed T3. B cells enriched by anti-F(ab’)2 column

chromatography isolated from peripheral blood and tonsil

tissue were 70% Bl + , and approximately 10% to 20% of

these BI + cells coexpressed TI . Of great interest was the

observation that approximately 5% of the column-nonadher-

ent sig- cells isolated from either peripheral blood or tonsil

tissue expressed Bi . These “slg-” cells were passed over the

column a second time in an attempt to remove all cells that

expressed slg; nonetheless, 5% of the slg- or very weakly

slg+ population remained BI +. Approximately 25% of

these column-nonadherent BI + cells were TI +, which

supports the notion that a population of normal B cells exists

that coexpresses Bi, TI, and very faint slg. Of note was the

observation that in normal adult bone marrow, none of the

BI+ cells wereTl+.

Since very small numbers of B cells isolated from adult

lymphoid organs coexpressed B 1 and T 1 , fetal tissues were

also examined for the presence of cells that coexpressed B I

and Ti . As seen in Table 3, approximately 40% of fetal

splenocytes expressed BI . In contrast to adult BI + cells,

Table 3. IdentifIcation of Bi + Ti + Cells Within Normal Adult

Lymphoid Populations and Fetal Lymphoid Organs

Percentage

Cell Populetion

No. ofTests B 1 (%) Ti (%) T3 (%)

of theB 1 -Positive CatsCoexpressing Ti

Adult

PBMC 3 6±3 60±10 67±8 i±i

E- 3 25±9 17±7 12±6 3±2

E+ 3 i±i 95±ii 96±8 0

PBslg+ 3 80±10 8±5 4±3 10±4

PBslg- 3 5±2 65±10 60±5 28r7

Tonsilslg+ 3 70±10 20±5 8±6 12±5

Tonsilslg- 3 6±3 84±9 81±8 27±9

Bone mar-

row 3 3±2 8±3 7±5 0

Fetal

Spleen 10 40 ± 8 40 ± 10 ND 40 ± 5

Bone mar-

row 4 15±6 5±3 ND 1±1

Liver 3 iO±5 3±2 ND i±i

Abbreviation : ND, n ot determined.




422 FREEDMAN ET AL

approximately 40% of these Bl + fetal spleen cells coex-

pressed TI. Similar to adult bone marrow, very few fetal

bone marrow B 1 + cells expressed T I . Moreover, fetal liver

cells that expressed Bl did not appear to express Ti.

Isolation and Phenotypic Characterization of Anti-F(ab’)2

Column-Nonadherent Bi -Positive Fetal Splenocytes

To define a population or populations of normal B cells

that correspond to B-CLL cells, fetal splenocytes were

isolated that expressed BI, Tl, and faint slg. Fetal spleno-

cytes were fractionated into the slg+ and slg- fractions.

The column-adherent cells were 80% slg+ with strong slg

expression. In contrast, cells that were nonadherent to the

column contained approximately 10% to 20% Bi + cells.

These cells, like most B-CLL cells, expressed such faint slg

that they readily passed through the anti-F(ab’)2 column.

Due to the phenotypic similarity of these anti-F(ab’)2

column-nonadherent BI + fetal splenocytes to B-CLL cells,

further characterization of these cells was undertaken by

using dual fluorescence. Table 4 depicts three experiments

examining the cell surface phenotype of these cells. The

overwhelming majority of these Bl + cells coexpressed 1gM,

IgD, Ia, B2, and TI, but very few, if any, expressed T3.

Although column-nonadherent BI-positive fetal splenocytes

were Tl +, the majority of these cells lacked C3bR. The

relative intensity of Ag expression on these cells was compa-

rable to that seen with B-CLL cells.

Expression ofB Cell Activation Ags on Activated B Cells

and B-CLL Cells

We and others have previously observed that B-CLL cells

express B cell activation Ags. In an attempt to compare

B-CLL cells with the stages of normal B cell activation,

unstimulated normal splenic B cells were first cultured with

the B cell-specific mitogen anti-Ig, and a temporal sequence

of antigenic changes was evaluated. As shown in previous

studies, anti-Ig-activated B cells lose sIgD, B2, and B3

(CD22).48 Moreover, they acquire a number of Ags not

expressed on resting B cells. Very few, if any, unstimulated B

cells expressed the B cell-restricted activation Ags B5,

Blast-I, Blast-2 (CD23), and BBI or the B cell-associated

activation Ags T9, the transferrin receptor, and IL-2R

(CD25). After activation, however, all were expressed and

increased for approximately three days. On day 1 , although

small numbers of cells (10% to 15%) expressed activation

Ags, only B5 and IL-2R were consistently expressed. By day

2, between 30% and 55% of the cells expressed B5, IL-2R,

T9, and BBI , whereas the number of cells expressing Blast-i

and Blast-2 were consistently less (iO% to 25%). By day 3,

the number of cells expressing each Ag was maximal, with

40% to 75% of cells expressing B5, IL-2R, and BB I , and 75%

to 90% of cells were T9 + . Blast-l and Blast-2 were

unchanged from the level of expression seen at two days. By

six days of culture, significantly fewer cells expressed any of

the activation Ags (10% to 40%), although none had

returned to the background level. The maximal expression of

activation Ags closely paralleled our previous studies of

3H-thymidine incorporation of in vitro-activated B cells.42

Tumor cells from 20 patients with B-CLL were examined

for the expression of these activation Ags. These cases all had

total peripheral lymphocyte counts of greater than I 5,000/

�.tL. Eleven of 20 of these cases expressed Ia, BI , B4, TI , B2,

and slg and lacked C3bR. Four additional cases coexpressed

the aforementioned Ags but in addition coexpressed C3bR.

Of the B cell activation Ags examined, the B5 Ag was most

frequently expressed (19 of 20) and was present on 60% to

90% ofcells within a given population (Table 5). Blast-I and

IL-2R were expressed on approximately 50% of the cases,

with 40% to 60% and 20% to 50% of cells within a given

population expressing the Ags, respectively. Blast-I and

IL-2R were generally present on the same cases. Blast-2 was

expressed on six of 20 cases, and BBi was infrequently

expressed (with 20% to 50% and 40% to 70% of cells

Ag-positive, respectively). T9 was not detected on any of the

tumor samples examined.

The intensity of Ag expression of three of these activation

Ags was different from their expression on normal anti-

Ig-activated B cells. Both the IL-2R, Blast-l, and BB1 were

less strongly expressed, and fewer Ag-positive cells were

present on B-CLL cells (20% to 40%) than on normal

anti-Ig-activated B cells (40% to 70%). In contrast, although

the Ag intensity of B5 on CLL cells was similar to normal

activated B cells, B5 was expressed on essentially all the cells

in each case of B-CLL, whereas normal anti-Ig-activated B

cells are more heterogeneous in their expression of B5, with

40%-60% of cells within a population expressing this Ag.

Induction of Ti Ag on Normal B Cells After Activation

With TPA

With the demonstration that B-CLL cells express Ti and

B cell activation Ags, we then attempted to induce unstimu-

lated normal B cells to express the B-CLL cell phenotype. It

has been recently observed that the TI Ag could be induced

on normal peripheral blood B cells with TPA.49 We therefore

activated highly purified resting splenic B cells with either

anti-Ig or TPA in an attempt to induce the coexpression of

TI and the B cell activation Ags B5 and IL-2R. For the 48

hours ofculture with anti-Ig, recovery of B cells was between

70% and 80%. Recovery of B cells after culture with TPA

Table 4. Phenotypic Characterizati on of the Bi -Pos itive F(ab’)2 Co lumn-No nadher ent Cells Isolat ed From Feta I Spleen

Ag

slgM sIgD a B2 Ti T3 C3b Receptor

Percentage of B i + cells

coexpressingAgs

Relative fluorescence in-

tensity

80 ± i2

+

70 ±

+

17 96 ± 4

+ + +

67 ±

+

i5 72 ±

+ +

12 2 ±

ND

1 15 ± 5

+ +




C

z

- B5

Ti


Results are one of three representative experiments.

TabI a 5. Ac tivation Ag Expression of CLI

.No. of Patients

Expressing Ag

Ag Intensity

B5 IL-2R Blast-i Blast-2 BB-i T9

7 ++ + +

5 ++

3 ++ +

2 ++ + + +

i ++ + +

1 ++ + +

1 + +

The range of percent positive cells for each Ag was as follows: B5.

60% to 90%; IL-2R, 20% to 50%; Blast-i , 40% to 60%; Blast-2, 20%

to 50%; BB-i. 40% to 70%.

was approximately 50%, probably due to residual adherence

of cells to the culture flasks.

Before stimulation, a population of highly enriched splenic

B cells that were greater than 90% BI + contained less than

1 % T cells and monocytes as assessed by the expression of

TI I and Mo2, respectively (Table 6). This population con-

tamed about 5% B5+ and IL-2R+ cells and less than 1%

TI + cells. As seen in Table 6, 34% of the TPA-activated

cells expressed T 1 , with 78% and 66% of the cells expressing

B5 and IL-2R, respectively. In this experiment, Blast-i was

expressed on 27%, Blast-2 on 38%, T9 on 31%, and BB-I on

54%, respectively. These TPA-activated cells failed to form

MRBC-R. In contrast, splenic B cells cultured for one to five

days with anti-Ig also expressed B5 and IL-2R but failed to

express Ti . The absence of contaminating T cells and

monocytes together with the high percentage of cells express-

ing the B cell-restricted Ag Bi support the notion that the B

cells were the population undergoing the observed antigenic

changes and were not indirectly due to effects of TPA on T

cells and monocytes. The recovery of adequate cell numbers

after stimulation also supports the view that we were observ-

ing antigenic changes and not selecting minor subpopulations

of B cells.

To further clarify the phenotype of TPA-activated B cells,

cells were examined before and after culture with TPA by

using dual-laser flow cytometric analysis. Splenic B cells

were dual fluorochrome labeled with fluorescein isothiocya-

nate (FITC)-conjugated anti-Ti and PE conjugated to

either anti-BI, anti-B5, or anti-IL-2R. As seen in Fig 2A, no

cells expressing either TI alone or TI and Bl were detectable

before culture with TPA. However, after two days of culture

with TPA, Ti +Bi + cells were detected as well as cells

expressing only BI (Fig 2B). Similarly, within the population

ofTPA-activated cells, subpopulations expressing B5 and Tl

(Fig 2C) were observed as well as IL-2R and Ti (Fig 2D).

Moreover, virtually all Tl + cells were B5+ and IL-2R+.

These studies suggest that the B cells that are induced to

express Ti also coexpress B5 and IL-2R and that

Table 6. Percentage of Cells Ex pressing Ag (Ag Intensity)

Bi Ti B5 IL-2R Tli Mo2

Control.day0 90(+++) 1 6(+/-) 6(+/-) 1

TPA.day2 9i(+++) 34(+/++) 78(++) 66(+) 1

Anti-Igday2 85(+++) 1 46(++) 31(+) 1

Fig 2. Highly enriched splenic B cells examined before (A) andafter stimulation with TPA. 10 ng/mL. for two days (B. C. D). Cellswere stained with directly fluoresceinated anti-Ti (anti-Ti F) anddirectly PE conjugated anti-Bi (panels A and B). anti-Ti F anddirectly PE conjugated anti-B5 (C). and anti-Ti F and directly PEconjugated anti-IL-2R (D). Cells were then examined by dual-laserflow cytometric analysis.

BI +Tl +B5+IL-2R+ B cells are subset of normal acti-

vated lymphocytes.

In an attempt to definitely demonstrate that B-CLL cells

express IL-2R, peripheral blood lymphocytes from two

patients with B-CLL that were greater than 90% BI + were

further enriched for B cells by anti-T cell and antimonocyte

MoAb and complement lysis. B-CLL cells were precipitated

with either anti-IL-2R, anti-B4, or anti-B2. TPA-activated

normal B cells were precipitated with anti-IL-2R. Activated

T cells were immunoprecipitated with anti-IL-2R and 4B4,

which identifies a T cell-associated Ag. The immunoprecipi-

tates were analyzed by 10% SDS-PAGE. Autoradiography

of the gel (Fig 3) showed that a single band corresponding to

a protein with a mol wt of 60,000 was precipitated from T

Fig 3. SDS-PAGE (iO%) of ‘2�l-labeled normal T cell blasts

(lanes A and B immunoprecipitated with 484 [T cell-associatedAg] and anti-IL-2R. respectively); CLI cells from two patients(lanes C. D. E. and F immunoprecipitated with anti-lL-2R [lanes Cand E]. anti-B4 [lane D]. and anti-B2 [lane F]; and normal TPA-activated B cells immunoprecipitated with anti-lL-2R (lane G).




424 FREEDMAN ET AL

cell blasts (lane B), cells from two patients with B-CLL

(lanes C and E), and normal activated B cells (lane G).

These studies suggested that the 60,000-dalton structures

immunoprecipitated from normal activated T cells, TPA-

activated B cells, and B-CLLs were identical.

Differential Effect ofRecombinant IL-2 on IL-2R-Positive

B-CLL Cells and In Vitro-Activated B Cells

In an attempt to functionally compare B-CLL cells with in

vitro-activated normal B cells, we examined IL-2R+ B-

CLL cells and normal activated B cells for proliferation in

response to recombinant IL-2 (rIL-2). Previous studies have

demonstrated that normal anti-Ig-activated B cells that

express IL-2R proliferate in response to rIL-2.43 The peak of

proliferation occurs at about three days of in vitro culture

and decreases to baseline by approximately six days. Normal

resting splenic B cells were cultured with media alone, rIL-2,

anti-Ig, TPA, anti-Ig and rIL-2, or TPA and rIL-2. As seen

in Table 7, resting B cells could be induced to proliferate with

anti-Ig or TPA. In contrast, rIL-2 alone induced minimal

proliferation, probably secondary to small numbers of in

vivo-activated B cells. The addition of rIL-2 to anti-Ig or

TPA led to a consistent augmentation of between two- and

fivefold. We next examined tumor cells from five patients

with B-CLL that were all IL-2R+ (3 cases were slg+,

C3bR-, 1 was slg-, C3b-, and I was slg+, C3b+). In

contrast to resting B cells, tumor cells from these five

patients with B-CLL did not proliferate to either rIL-2,

anti-ig, or a combination of them (Table 7) when examined

at three days. One patient (CLL 2) had a minimal prolifera-

tive response to TPA with some augmentation by rIL-2. By

six days of culture, 3H-thymidine incorporation of both

normal B cells and CLL cells was at the background level.

Previous investigators have noted proliferation of CLL cells

in response to anti-Ig and TCM. The substitution of TCM

for rIL-2 yielded results similar to those seen with rIL-2

(data not shown). These studies suggested that although

some B cell CLLs express IL-2R they fail to proliferate when

cultured in the presence of this growth factor.

DISCUSSION

In the present report, we have examined the cell surface

phenotype of 100 cases of B-CLL and attempted to identifynormal B cells that might represent its normal cellular

counterpart. The overwhelming majority of B-CLL cells

expressed Ia, B4 (CD19), Bl (CD2O), B2 (CD21), slg, and

TI (CD5) but lacked C3bR (CD35). Although, unstimu-

lated small B cells expressed Ia, B4, B!, B2, and slg, in

addition they expressed C3bR and lacked detectable Ti

expression. B cells isolated from normal lymphoid popula-

tions were then examined for the expression of Ags that were

present on B-CLL cells. Small numbers of weakly slg+ cells

expressing B 1 and T 1 were observed in adult peripheral

blood and tonsil tissue but not bone marrow. Greater num-

bers of these cells could be identified in fetal spleen material.

Weakly slg+, Bi + fetal splenocytes were further examinedand were shown to coexpress TI, sIgM, sIgD, Ia, and B2 but

lacked C3bR and thus closely resembled B-CLL cells. Con-

sidering the phenotypic differences between small unstimu-

lated B cells and B-CLL cells, we examined B-CLL cells and

normal in vitro-activated B cells for the expression of Ags

expressed on activated but not resting B cells. Of 20 cases of

B-CLL examined, virtually all expressed the B cell-restricted

activation Ag B5 and approximately 50% expressed Blast-i

and IL-2R (CD25). Unstimulated small splenic B cells were

then stimulated with anti-Ig or TPA. Although both anti-Ig

and TPA induced B5 and IL-2R expression on normal

stimulated B cells, only TPA-activated cells coexpressed Ti.

With the observation that TPA-activated B cells and some

B-CLL cells expressed receptors for IL-2, we attempted to

determine whether these cells could respond to IL-2 and

whether the receptor on B-CLL cells was identical to that

found on activated T and B cells. Although B-CLL cells and

activated B cells express IL-2R, only in vitro-activated B

cells proliferated to rlL-2. Immunoprecipitation demon-

strated that the identical 60-kD cell surface proteins were

expressed on B-CLL cells and activated T and B cells,

thereby demonstrating that both cells express IL-2R. These

results are consistent with the notion that small numbers of

normal B cells either isolated from fetal or adult lymphoid

tissues or induced by TPA activation appear to be candidates

for the normal cellular counterpart of the B-CLL cell.

Classically the activation of B lymphocytes occurs by the

cross-linking of cell slg by either Ag or anti-Ig.5#{176}This initial

event induces resting B cells to increase pools of intracellular

calcium and inositol triphosphate. Subsequently these cells

then synthesize RNA, increase in size, and finally become

competent to respond to a variety of B cell growth factors

(BCGF) including low- and high-molecular weight BCGF,

IL-2, and -y-interferon.5’ Alternatively, EBV, TPA, and C3d

as well as MoAbs directed against Bp35 (CD2O) also induce

several of these events as they trigger resting B cells,

probably through alternative pathways of activation, to leave

the G0 phase of the cell cycle.52’53 For example, TPA stimu-

lates B cells via direct activation of protein kinase C without

increases in intracellular calcium. After activation with

Table 7. Res ponse of Normal B Cells and CLI Cells to rlL-2

3H-Thymidine Incorporation (cpm)

Media rlL-2 WA Anti-Ig rIL-2 + Anti-Ig WA + rIL-2

NormalBcells

CLL1

CLL2

CLL3

CLL4

CLL5

476 ± 67

123±27

i14 ± 43

lii ± i2

153±23

102±23

2524 ± 266

246±53

146 ± 29

i76 ± 23

i86±24

211±53

i5,i92 ± 2,028 5,204 ± 702

218±97 i38±51

8i2 ± 176 378 ± 18i

131 ± 89 i79 ± 48

98±42 254±26

326±98 136±13

22,430 ± 5,i78

176±105

205 ± 23

176 ± i5

i89±57

166±29

37,466 ± 2,597

89±17

i,2i2 ± 245

264 ± 29

175±37

344±36





anti-Ig, a distinct sequence of antigenic changes has been

consistently observed.42 The earliest activation Ags can be

detected by 24 hours, with peak expression observed by 72

hours. By six days most activation Ags are either lost or

significantly decreased. The phenotype of the B-CLL cell

with its expression of Ia, slg, B4, BI, and B2 as well as the

activation Ags B5, Blast-I, Blast-2, and IL-2R suggests that

this cell might be the neoplastic counterpart of a major

subpopulation of activated B cells. However, B-CLL cells

express Tl, thereby demonstrating that this cell does not

correspond to a population of anti-Ig-activated normal B

cells. The observation that TPA-stimulated B cells that

coexpress B! and TI as well as the B cell activation Ags B5

and IL-2R suggests that direct activation via the protein

kinase C pathway induces a minor subpopulation of acti-

vated B cells that are phenotypically similar to most B-CLL

cells.

Although TPA-activated B cells resemble B-CLL cells,

they do not appear to be an identical population. Although

virtually all B-CLL cells express receptors for MRBCs,

resting B cells activated with either anti-Ig or TPA do not

express detectable numbers of MRBC-R. Moreover,

although B-CLL cells express IL-2R, they do not proliferate

to IL-2. Preliminary studies from our laboratory suggest that

TI +, TPA-activated normal B cells that express IL-2R

proliferate in response to IL-2. Our data are in contrast to

several previous reports where responsiveness to IL-2 has

been seen in B-CLL cells.5�57 Whether this is due to patient

selection or to the presence of very small numbers of contam-

mating normal activated T cells is unclear. This lack of

responsiveness may be due to a predominance of low-affinity

IL-2R on B-CLL cells.58 A recent report suggests that the

high-affinity IL-2R may consist of two subunits of 55- and

70-kD.59 B-CLL cells as well as hairy cell leukemia cells and

neoplastic pre-B cells that also express lL-2R but fail to

proliferate to IL-2 may lack the 70-kD subunit that appears

to be necessary for proliferation.�#{176}�2 The differences in

MRBC-R and responsiveness to lL-2 suggests that the

B-CLL normal cellular counterpart may be a subpopulation

of the Bl +Tl + population.

The cell surface phenotype of B-CLL cells appears to be

remarkably homogeneous, with approximately 90% of

tumors expressing Ia, B4, BI, B2, and Ti . Heterogeneity was

seen in the expression of sIg and C3bR and to a lesser extent

B2. The demonstration of this heterogeneity of B-CLL cells

may be reflective of the various normal Tl + B cell popula-

tions from which B-CLL cells are derived or states of

activation of the BI +Tl + population. Several investigators

have identified normal B cells that resemble B-CLL cells. In

situ studies have suggested that these cells are present in very

small numbers at the periphery of the germinal center of

normal adult lymph node28 whereas larger numbers of these

cells could be identified in fetal spleen and lymph node,21 in

the peripheral blood of patients after allogeneic bone marrow

transplantation,63 and in peripheral blood of patients withrheumatoid arthritis.” In murine systems, Lyl + B cells,

which may be the counterpart of human T I + B cells, secrete

autoantibodies and therefore may represent activated auto-

reactive B cells.65 In the present study, we identified

Bi +Tl + cells in adult peripheral blood and tonsil tissue but

not bone marrow. Because Tl + B cells are a major popula-

tion of fetal splenocytes, we used this tissue as a source of this

subset of B cells. We observed that these BI + cells weakly

expressed both sIgM and sIgD as well as Ia, B2, and Tl but

lacked T3 and that only 1 5% of these cells expressed C3bR.

In preliminary experiments, small numbers of these cells

coexpress B5 but do not appear to express Il-2R. This

immunophenotype as well as the intensity of Ag expression

on these cells was remarkably similar to that observed for the

majority of B-CLL cells.

In summary, the present study provides evidence that

B-CLL is derived from minor populations of Bl +TI +

lymphocytes. Moreover, the observation that this phenotype

could be induced via the direct activation of protein kinase C

is consistent with the hypothesis that subpopulations of

normal B cells may be activated via distinct pathways of

activation. Future studies will be directed toward identifying

the subset of unstimulated B cells that are induced to become

Tl +. Studies comparing TI + normal B cells with B-CLL

cells and anti-Ig-activated normal B cells (TI -) for their

responses to various growth and differentiation factors may

also lend insight into the defect in humoral immunity of

B-CLL.

ACKNOWLEDGMENT

The authors thank Dr K. lida for providing anti-C3bR MoAb andDr Ed Clark for BB-l MoAb. The authors also wish to thank MarieSweeney for excellent preparation of the manuscript. We appreciate

the excellent technical assistance of Herbert Levine.

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