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From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. 1987 70: 418-427 Normal cellular counterparts of B cell chronic lymphocytic leukemia AS Freedman, AW Boyd, FR Bieber, J Daley, K Rosen, JC Horowitz, DN Levy and LM Nadler 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. From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. Normal Cellular By Arnold Counterparts S. Freedman, Andrew Jack In an attempt leukemia cell to (B-CLL) surface mined using directed Bi (CD2O). Ti (CD5) B2 but of B-CLL (CD21 C3b identified in and splenocytes lacked most B-Cu between cells. the B-Cu cells, G With majority we expression (slg),’3 of clonally weak , normal monoclonal rearranged accepted, the subset of normal (B-CLL) is derived remains unresolved. the normal cellular counterpart upon its unique properties. cell B-CLL cells surface differ from coex- The phenotype from lL-2, the notion tions and Ti that activated B-CLL resembles including S by Grune 1987 morphology B cell CLL in is functional (MRBC-R).21’23 Moreover, and functional derived differences from a unique suggest subset that C3b B-CLL B-CLL cells that actiphenotypic appears recently, Institute; Division of to be of B lymphocytes. Department tal,’ and analysis of cell surface believed that 1gM and HLA class January by CA25369, 2. 1 987,’ accepted National CA34183. Health Service National and Grant Cancer Address reprint Women’s Hospi- Harvard Medical Ags including led of normal example, B4 (CD19),” Institute, requests MA of CA40216. Health A.S.F. lKO8 CAOI Grants is supported 105-01 No. awarded by to Arnold S. Freedman. Cancer MD, Institute, the Division 44 Binney of St. 02115. publication charge payment. “advertisement” indicate this fact. (C) 1987 by Grune costs ofthis article This article must in accordance & Stratton, 0006-4971/87/7002-0013$3.00/0 with Inc. were defrayed therefore /8 U.S.C. be in part hereby §1734 by page marked solely subpopulaof B cells protein that kinase C from upon phenotype. Until B-CLL cells Tl have been tissue, and of fetal subpopulations resembled B-CLL B cells. In the to that were this appear cells These as well cells.28’29 provided lymph center material that Tl + node and subpopula- evidence B cells For B cells Similarly, fetal to be a major studies of normal for one phenotypically report, we have attempted to extend these B-CLL cells to subpopulations of normal to be reported populations cells express cell can cells may of activated from will demon- be identified surface in Ags B cells, that are thereby be the neoplastic B cells. Further counterevidence the that observation (TPA) B lymphocytes Vol we tissue, and fetal and Ti Ags. More- but not resting as B cell activation Blood, lymphoid the Bi several B cells is derived later, of B cells blood, adult that coexpress 0-tetradecanoylphorbol-3-acetate induce small unstimulated TI B-CLL MRBC-R. these on activated notion express these subpopula- of slg-positive formed B2 How- and B-CLL cells blood B cell in might identified resemble in human suggesting the B-CLL part of a subpopulation for that now and receptor. at the edge of the germinal in tonsil and lymph node and studies small B-CLL expressed subset have also B cell- cells. adult peripheral lymphoid tissue over, (CD2O),6 and MRBC-R the peripheral identified B cells. or more populations as other (EBV) subpopulation identified follicle coexpressed Bi virus that small (2% to 3%) was of the secondary strate by Public DHHS. Dana-Farber B cells a very In the present studies by relating 28. 1987. March Institutes No. Immunology. The 418 and Pathology, minor IgD. Moreover, both II molecules as well the C3d/Epstein-Barr tion Cancer Boston. Supported Boston, and by with the subset of B cells been largely based generally (CD21),9 that Dana-Farber Brigham ofMedicine Submitted Tumor Immunology, ofPathology, Departments School, Tumor the although consistent the and restricted spleen the through in not, proteins are it was coexpressed expressed B cells From did several to identify arise have tions erythrocytes B cells Inc. mouse demonstrate variable responsiveness to mitogens vate most normal small B lymphocytes.227 These lL-2R. proliferated cells cell with a population & Stratton, ever, the coexpression identilack B derived from small unstimulated peripheral blood B lymphocytes since they were morphologically identical and weakly difficulty of B-CLL they the pathway. B cell studies in that were activated 60-kilodalton directly search of another structures. Several B lymphocytes TPA or then and , the Blast- anti-Ig and cells studies B cells in vitro Ti of and either as B5 and or identical receptors (CD35),’2”3 express the 67-kilodalton (kD) T cell-associated TI (CD5) Ag,’2#{176}and form rosettes with small as well B-CLL of Ti to abandon unstimulated Only These Previous attempts which B-CLL cells and Bi . anti-Ig the of normal are and of whereas expressed Ia leukeof their morphologically all Bi 50% with IL-2R. and examined. (lL-2R) (TPA) and either immunoprecipitation. and B5 cases approximately activated investigators cal and 20 receptors were coexpressed they B cells and Of coexpression of resembled which TPA for of cell surface antigens of CLL is generally B cells of B cell-restricted antigens. antigens presence immunoglobulin genes,4’5 Rosen, expression B5, cells could B cells Karen interleukin-2 B cells activated cell surface immunoglobulin B cell-restricted the B cell origin identifying activation cells 51g. B2, the expressed with lymphocytic by virtue the expression of (Ags).” Although based examined the differences in vitro all expressed Normal . for activation activated closely 95% of chronic of B cell lineage i Daley, Nadler cells -associated Leukemia i 2-O-tetradecanoylphorbol-$-acetate i 6% unstimulated B-CLL M. lacked that phenotypic of small REATER THAN mias (CLL) are tonsil therefore the examined slg + and John Lee but Approximately Bi . Ti and B receptors and and In contrast. and antigens. receptors (slg). Levy, cases (CDi9). Lymphocytic R. Bieber, virtually anti- Ia. B4 of weakly coexpressed C3b deter(MoAbs) unstimulated C3b blood Ti N. -associated receptors. numbers David the was immunoglobulin peripheral Bi B-CLL antibodies small Frederick lymphocytic expressed (CD35) of Small . the of Chronic Boyd, counterpart, and cells ). surface lacked Ti pressed but cases Ia, B4, Bi . B2. 51g. and detectable chronic cellular of monoclonal majority expressed fetal 1 00 W. C. Horowitz, cell B cell-restricted majority overwhelming be of a panel against The B its normal phenotype by gens. compare with of B Cell but not anti-Ig to express Bi 12can and Ags. 70, No 2 (August). 1987: pp 4 18-427 From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. NORMAL CELLULAR COUNTERPARTS MATERIALS Adult Human Tissues OF AND 419 B-CLL Table METHODS 1 . Expressi on of Lineage-Restri cted and -A ssociated Ags Molecular Cells CD 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. Tonsil cells 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 by dissolution in Hanks’ balanced salt solution (HBSS) with forceps and scissors and extrusion through a stainless steel mesh. Mononuclear cells isolated by Ficoll-Hypaque density gradient centrifugation were enriched for B cells by E rosetting to deplete T cells. Cells were either used fresh or cryopreserved in 10% dimethyl sulfoxide and 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%. Ag Designation Norm& Cellular Reactivity Bi 20 pan-B 82 21 LimitedB 84 19 pan-B Ia pan-B slg Limited Weight (kD) 35 6 140 9 95 11 29, B Reference 34 lgM-900 31 31 lgG- 150 gD- 150 B5 Activated B 75 32 Blast-i Activated B 45 33 Activated B 45 34 Activated B 37 35 Activated T. B 55 36 90 37 Blast-2 23 BB1 lL-2R 25 T9 Proliferating cells! nonlineage re- stricted Isolation ofPeripheral Blood C3bR Cells Tissues Fetal tissues were obtained within one hour of induced abortion. All patients undergoing therapeutic last menstrual periods and diagnostic ultrasound suggested that the fetal age was less than 24 weeks. gestational age, age determination postmortem was crown-rump length and fetal foot length. Procurement approved by the Brigham and Women’s Hospital Human Subjects in Research, and informed consent from all patients undergoing therapeutic abortion. Fetal bone marrow and single-cell suspensions of were prepared as previously described.30 Patients B, RBC. monocyte. 220 38 ‘anulocyte Human peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteer donors by Ficoll-Hypaque density sedimentation (Pharmacia Fine Chemicals, Piscataway, NJ). Unfractionated mononuclear cells were separated into slg+ and slgpopulations by Sephadex G-200 (Pharmacia) anti-F(ab’)2 chromatography with modifications designed to minimize monocyte retention by columns as previously described.6 T cells were recovered by E rosetting the slgpopulation with 5% sheep erythrocytes. Normal human monocytes were removed by adherence to plastic culture dishes. Fetal 35 prostaglandinabortion had imaging that To standardize determined by of tissue was Committee on was obtained spleen and liver 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 Protection Committee validation and informed consent. The diagnosis was based on a total peripheral blood lymphocyte count of 15 x 109/L and an infiltration of well-differentiated lymphocytes in the bone marrow. B cell lineage was established by the presence of monoclonal slg or the pan-B cell antigen BI as determined by indirect immunofluorescence with the use ofanti-k, A, IgG, 1gM, IgD, and BI monoclonal antibodies (MoAbs). MoAbs The preparation and characterization of MoAbs used in this study have been described. The Ags to which these antibodies are directed are summarized and referenced in Table 1 . All MoAbs used in this study were ascites fluid at saturated binding concentrations. Mo2 14 Monocyte Ti 5 T, thymocytes. Ti 1 2 T, thymocyte 39 sub- 67 14-20 50 40 set of B Fluorescent Staining Indirect. Cells were prepared in 10% pooled AB serum in HBSS; when the cells had been incubated with anti-Ig coupled to beads, they were incubated in human serum for one hour at 37#{176}C to remove the beads from the cell surface. Aliquots of I 06 cells were incubated with each antibody (generally a 1/100 to 1/400 dilution of ascites) for 30 minutes at 4#{176}C. After washing, the cells were incubated with a 1/50 dilution of fluorescein-conjugated goat antimouse Ig antibody (Tago Inc. Burlingame, CA) for 20 minutes at 4#{176}C. The cells were washed and were either analyzed fresh or were fixed in 1% formaldehyde for subsequent analysis.6 A reaction was considered positive when greater than I 0% of the test cells were more fluorescent than the number of cells positive with isotype-identical control ascites. For each sample, a quantitative assessment of the number of positive cells was made (number of cells reactive with the test antibody minus the number of cells reactive with unreactive isotype-identical monoclonal antibody/lO,000 total cells tested). Direct and dual-fluorescence staining. Directly fluorescein conjugated MoAbs were prepared as previously described.’ For directly phycoerythrin (PE) conjugated MoAbs, I .0 mg of protein Apurified anti-Bl, anti-interleukin-2 receptor (IL-2R) antibody, or a euglobulin precipitate purification of anti-B5 were reacted with 0.5 mg of R-phycoerythrin (pyridyldisulfide derivative) as described elsewhere.4’ MoAbs were biotinylated by standard techniques.” The specificity of each of these conjugated antibodies was tested on appropriately reactive normal tissues and cell lines and found to be identical with that of the unconjugated antibodies. To define the percentage of cells that expressed the BI and Tl Ags within each cellular population, the number of cells with positive fluorescence was compared with the number of cells stained with negative control antibody out of a total of 500 to I ,000 cells counted on a fluorescent microscope (Carl Zeiss, West Germany). Cells with two or more discrete positive clumps per cell or cells with clear-cut peripheral rims were scored as positive. To enumerate the number of Bl + cells that coexpressed the TI Ag, 100 Bl + cells were counted, and the percentage of these cells expressing TI was determined. To characterize the BI + F(ab’)2 column nonadherent population, the number From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. FREEDMAN 420 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 were developed with avidin Texas red dye.” By using these techniques, it was possible to accurately identify the number of BI + cells that coexpressed several other cell surface determinants. All flow cytometric analysis was performed on either an EPICS V or C cell sorter (Coulter Electronics, Hialeah, FL). Dual-fluorescent-stained cells were analyzed after initial calibration of the machine with cells stained with each individual fluorochrome-labeled antibody and with other controls as outlined previously.” When beads were present in the cell suspensions, they could be excluded from the analysis 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 mononuclear cells was further enriched for B cells by two treatments with the MoAbs anti-Mo!, anti-Mo2, anti-T4, and anti-T8 followed by rabbit complement to deplete all but B cells from the spleen cell suspensions. These highly enriched B cells were cultured for two days at 1.5 x l06/mL in RPMI 1640 supplemented with 10% FCS, 2 mmol/L glutamine, and I mmol/L sodium pyruvate in tissue culture flasks (Corning Glass Works, Corning, NY), with either affinitypurified F(ab’)2 rabbit antihuman Ig coupled to Affigel 702 beads (anti-Ig) (Bio-Rad, -Richmond CA) as previously described42 or TPA (Sigma Chemical Co. St Louis) used at a final concentration of 10 ng/mL, and l0 mol/L 2-mercaptoethanol. Microcultures. Highly enriched splenic B cells were prepared from the E - fraction of splenic mononuclear cells by anti-T cell and antimonocyte MoAb and complement lysis. CLL cells were highly purified by a similar depletion of T cells and monocytes. Cells were cultured in 96-well, round-bottom microtiter trays (Costar, Cambridge, 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 demonstrated that maximal stimulation of normal B cell 3H-thymidine incorporation with RI-L2 is at 200 U/mL.43 T cell conditioned medium (TCM) was prepared as previously described.42 TPA was used 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 (Amersham 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 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}C with either Sansorbin (for Ig precipitations) or Pansorbin (Calbiochem-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 conjugated to Sepharose 4B beads. The mixtures were held on ice for 2 hours and then washed four times with 1% Triton X-iOO and 1% sodium deoxycholate in 12 mmol/L sodium phosphate, 5 mmol/L EDTA, 5 mmol/L ethylene glycol tetraacetic acid (EGTA), and I mmol/L NaF, pH 7.4 (RIPA buffer). Precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (10%). Ig precipitates were performed in an identical manner except that in the precipitation of the precleared supernatant we used antihuman Ig antibody bound to protein A-Sepharose 4B (Pharmacia, Uppsala, Sweden). RESULTS Expression ofB Tumor cells with Cell-Associated from morphological a panel patients with of B-CLL of MoAbs. on B-CLL Tumor were cells receptor were the B2.4547 defined The two B-CLL expressed the C3b of B-CLL cells coexpressed (n = (n = for in all cases major by the expression majority tion clinical tested and reactivity were of B cell phenotypic of slg and for C3b (C3bR). Monoclonal slg was not tumor cells of 21 patients. Only 19 of the Three Cells by the expression of Bl and B4 (Table 2). All expressed Ti . Ninety of 100 cases expressed EBV/C3d groups Ags 100 features derivation five cases major subgroups of expression 51); (b) slg+, receptor. could of slg C3bR+ The be identified and (n = sub- the receptor detectable 100 patients Ia, Bi, but the on the with overwhelming B4, Ti, and B2. by the examina- C3bR: (a) slg+, 17); and (c) slg-, C3bRC3bR- 15). Examination of the intensity of Ag expression was undertaken (Table 2). Ia and Bl were strongly expressed all tumor intensely cells (Fig 1). The B2 expressed but were clearly cells (60% to 80%) and C3bR were Table 2. less Ti, the cell of CLL Ag Intensity Ag Ia B1 B2 B4 sig Ti +++ +++/++ ++ ++ + +/++ 17 +++/++ +++/++ ++ ++ + +/++ 15 +++ +++ ++ ++ 2 +++ +++/++ ++ 6 +++ +++/++ 4 +++ +++/++ 5 +++ +++/++ of positivity was degree no detectable space, peripheral blood cells, i); Fig The range B cells. of percent Bi, 70% 60% to 80%; C3bR, assessed 20% to quantitate cells for to each antigen B2, 50% 40%. due +/++ by flow The to weak cytometry. + , weak (slgM (B2 on peripheral (B 1 on peripheral to 90%; + +/++ + over background; Fig 1); + + , moderate positive to 85%; difficult qualitatively reactivity ++ + +/++ + ++ ++ C3bR +/++ ++ ++ + + + , strongest 65% slg was slg, on 51 The Blank Cell expressed B Cell Ag E xpression also on Ags were less on most tumor population. intensely No. of Patients Expressing and B4 positive in the neoplastic much . 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 antimonocyte antibodies and complement. T cells were activated with anti-TI 12 and -TI 13 antibodies for three days.” Highly enriched splenic B cells were activated with TPA (10 ng/mL) for two days. The iodinated cells were washed twice with cold phosphate-buffered saline and lysed on ice with cell lysis buffer (50 mmol/L Tris-HCI, 0.4 mol/L NaCI, 1% Triton X-I00, 2 mmol/L phenylmethylsulfonylfluoride, 5 mmol/L EDTA, 50 mmol/L iodoacetamide, pH 8). After 30 minutes the lysate was centrifuged at 800 g for ten minutes to remove unlysed cells, nuclei, and other insoluble material. The supernatant was frozen at -80#{176}Cuntil analyzed. ET AL to 70%; percentage expression. blood was B cells, B Fig 1). as follows: B4, 60%-80%; of cells on blood expressing Ia, Ti, From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. NORMAL CELLULAR COUNTERPARTS OF B-CLL 421 a) -‘:3 E z a) 0 Log Green Fluorescence Fig 1 Fluorescence-activated cell sorter histogram of the reactivity of anti-lgM. -lgD. -Ia. blood B cells (top) and B-Cu cells (bottom). Background fluorescence was obtained unreactive MoAb and developing with goat antimouse Ig FITC. . peripheral identical surface. When compared peripheral from Bl, significantly C3bR, the less intense on B-CLL of Ti the , Bl marrow and of sig and supports isolated express Ia, C3bR B4, that was observation TI B Cells that , we in Normal examined were TI Ags. B-CLL cells express for the presence experiments, TI-positive cells was of cells coexpressing In PBMC identified. resulting fraction, of cells if any B Mononuand bone that BI +Ti and + coexpressed Bi and TI, T3. B cells enriched chromatography from isolated were 70% these BI + cells coexpressed that approximately observation cells expressed Bl , tissue + and whereas no by anti-F(ab’)2 peripheral blood TI . Of 5% isolated from either Bi . These “slg-” great cells splenocytes the 3). were tonsil to 20% was blood passed of the +, were that coexpressed . In contrast to adult of Bi + Ti + Cells Populations and Fetal 40% BI of BI + Within Normal Lymphoid Organs fetal cells, Adult which of No. of Tests B 1 (%) Ti (%) T3 (%) Percentage of the B 1 -Positive Cats Coexpressing Ti 3 PBslg+ 3 PBslg- 3 5±2 65±10 Tonsilslg+ 3 70±10 20±5 8±6 12±5 Tonsilslg- 3 6±3 84±9 81±8 27±9 6±3 60±10 67±8 i±i 17±7 12±6 3±2 i±i 95±ii 96±8 80±10 8±5 3 3±2 8±3 7±5 25±9 4±3 60±5 0 10±4 28r7 Bone mar0 Fetal 10 Spleen column-nonadherent 25% TI adult E+ these were from tissues 3 Liver cells isolated T 1 , fetal 3, approximately BI the of the 3 population BI + of cells none E- slg+ Approximately B 1 and exists was PBMC Bone +. B cells Of note marrow, of B cells in Table IdentifIcation slg. Adult a second time in an attempt to remove all cells that expressed slg; nonetheless, 5% of the slgor very weakly BI 3. row or tonsil over the column remained seen bone presence expressed Cell Populetion of the column-nonadherperipheral cells were As Lymphoid Bi + cells column interest . for the faint adult coexpressed C3bR with normal cells with isotype- of normal very numbers and the of then (Table and 10% approximately Ti Table When T cells were removed by E rosetting, the Efraction demonstrated approximately 3% of cells that coexpressed and and . coex- the number first enumerated, T! was determined few Ia, normal examined a population TI, in normal organs also -B2. -Bi . -Ti by incubating that Bi, that lymphoid to identify In these Bl- and number the most attempted the notion coexpresses BI+ cells wereTl+. Since very small cells. that expressed the B-CLL phenotype. isolated from peripheral blood, tonsil, Bi and pressed tissue B cells that intensity Ag-Positive observation sig, lymphocytes clear cells sig- resting tissue Populations With ent small or spleen and Identification Lymphoid with blood B2, slg, B4, Intensity 40 ± 8 40 ± 10 ND 40 ± mar- row Abbreviation 4 15±6 5±3 ND 1±1 3 iO±5 3±2 ND i±i : ND, n ot determined. 5 From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. FREEDMAN 422 approximately pressed 40% TI. bone of these Bl + fetal to adult bone marrow, Similar B 1 + cells expressed marrow cells that expressed Bl did spleen T I . Moreover, not appear coex- the few fetal 40% fetal liver to cells very to express Ti. number unchanged from six days and Phenotypic Characterization Column-Nonadherent Bi -Positive Fetal To define a population correspond that isolated that cytes were The to B-CLL expressed BI, fractionated These cells, that dual the B2, Although were Tl the to that of these Cell and Cells B-CLL cells. IL-2R 1gM, T3. C3bR. cells was Activation on Activated mitogen changes of normal B cells were anti-Ig, was and evaluated. anti-Ig-activated The The the Ags (CD25). After and T9, transferrin the activation, (10% Ags, were T9, and only B5 and between and 30% BBI Blast-2 Table , IL-2R and 55% whereas were 4. or the all Phenotypic and consistently of the cells expressed. of cells less (iO% Characterizati B5, expressing to 25%). B-CLL intensity are more present the been most on 60% anti-Ig TI 70% 3, F(ab’)2 or TPA and hours Blast-i on of the Bi -Pos itive IL-2R, fewer the ofculture and Co lumn-No activation anti-Ig, nadher normal although to normal of B5, with this After Activation B-CLL cells express Ag could TPA.49 B cells to induce the B5 and recovery Ti Percentage a B2 coexpression of B cells Ti For was culture of the 80 coexpressingAgs ± i2 70 ± 17 96 ± 4 67 ± i5 72 ± 12 2 ± TPA C3b Receptor 1 15 ± in+ + + + + + + + ND 48 between with Feta I Spleen T3 It We therefore with either of B i + cells Relative fluorescence tensity sIgD and be induced IL-2R. after Isolat ed From Ag. to induce unstimucell phenotype. the TI of B cells ent Cells on B Cells Ags Recovery were were expressing attempted the B-CLL that anti- expression blood B cells with purified resting splenic with 80%. activation normal on essentially all the cells normal anti-Ig-activated B in their in an attempt B cell on cells of the Blast-l, and BB1 Ag-positive cells a population that of on any of these expression and was Ag slgM to infrequently 70% cells (20% to 40%) than B cells (40% to 70%). In contrast, of B5 on CLL cells was similar observed peripheral Blast-I Blast-2 not detected of three their Ag on Normal highly activated By day to T9 was within recently on normal IL-2R, was 40% Ags, we then B cells to express normal has BBi and demonstration activation lated and heterogeneous of cells With and By day was B2, C3bR. B5 Ag was 50% B cells, B5 was expressed case of B-CLL, whereas B cell 1 , although activation expressed and , coexpressed coexpressed the cases, to from Induction of Ti With TPA IL-2R expressed three days. On day to 15%) expressed the number consistently were 20 B cells. Both the expressed, and 40%-60% B cell-associated receptor, however, increased for approximately small numbers of cells 2, BBI Ag cells sequence B2, different on activated in each not B B5, activation was anti-Ig-activated in previous sIgD, all had I 5,000/ B4, TI , cases in addition examined. of Ag expression Ig-activated less strongly (CD22).48 Moreover, they acquire a number of Ags expressed on resting B cells. Very few, if any, unstimulated cells expressed the B cell-restricted activation Ags (CD23), lose of 20% respectively). samples intensity tumor compa- B3 Blast-2 B cells six Ag-positive, and Blast-I, (with B cell activation, first cultured with a temporal than Ia, BI of examined cases of greater examined, of 20) were the Ags, respectively. present on the same cases. generally on B-CLL cells to compare As shown (19 expressing expressed B Cells observed that In an attempt but Ags of studies B cells.42 These additional of had expression B-CLL expressed Four Ags expressed were Ags Ags cases C3bR. By any none maximal Ags. counts activation expressed splenocytes lacked B cell population The expressed fetal cells aforementioned expressed although with activation with 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 by coexpressed if any, on these the BB I , and 75% Blast-2 were seen at two days. The 20 patients of these lacked maximal, and and cells 40%), level. was paralleled our previous of in vitro-activated of 20 of these and Of the experiments these to lymphocyte slg frequently undertaken from peripheral present B cell-specific studies, slg Ag fewer (10% closely incorporation cells and cells. with the stages normal splenic of antigenic cells. faint Ags .tL. Eleven column. three of few, and others have previously B cell activation Ags. B-CLL cells unstimulated Bi + such was Bl + cells very of these with 20% BI-positive majority seen ofB We express cells slg to the these anti-F(ab’)2 to B-CLL cells, 4 depicts of Ag expression Expression B-CLL to expressed Ags to the background Tumor total strong 10% phenotype but with nonadherent significantly for the expression fractions. the anti-F(ab’)2 Table TI, slg- slg+ were each level of expression the activation activation 3H-thymidine spleno- expressing of culture, returned B cells Fetal were cells, surface and +, and 80% that of these cell intensity rable were column-nonadherent relative slg+ through majority Ia, the phenotypic similarity of BI + fetal splenocytes overwhelming IgD, slg. and B-CLL fluorescence. examining the most characterization using faint cells passed Due to the column-nonadherent further splenocytes approximately like readily they fetal cells In contrast, contained column of normal cells, into column-adherent expression. or populations Tl, the of Anti-F(ab’)2 Splenocytes Isolation of cells to 75% of cells expressing B5, IL-2R, 90% of cells were T9 + . Blast-l ET AL + + 5 From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. NORMAL CELLULAR COUNTERPARTS TabI a 5. Ac tivation Ag Expression Expressing Ag B5 7 ++ 5 ++ 3 ++ 2 IL-2R Blast-i + + ++ + + i ++ + 1 ++ + + of percent to 90%; IL-2R, BB-i. positive 20% 40% T9 + + + The range BB-i Blast-2 + 1 60% of CLI Ag Intensity . No. of Patients to 50%; 423 OF B-CLL cells to 50%; + each for Blast-i 40% , C Ag was as follows: to 60%; Blast-2, B5. z 20% to 70%. B5 - was approximately cells B 1% T TI I that probably 50%, of cells to the culture Before stimulation, were greater than cells and monocytes and Mo2, respectively tamed about 5% B5+ and T 1 , with B5 and respectively. expressed 54%, seen on 27%, with express Ti monocytes . The 78% after stimulation ing antigenic also of B cells. To further clarify examined using dual-laser flow dual fluorochrome nate (FITC)-conjugated anti-BI, cells expressing after cytometric labeled alone express- In an attempt express IL-2R, patients with MoAb with observ- TPA B cells are subset normal demonstrate peripheral blood B-CLL enriched and either to definitely that for B cells complement anti-IL-2R, B cells were of normal acti- that B-CLL cells were analyzed by anti-T precipitated by 10% of the gel (Fig 3) showed that a protein a mol with than cell from 90% and two BI + were antimonocyte lysis. B-CLL cells were precipitated anti-B4, or anti-B2. TPA-activated with cells were immunoprecipitated which identifies a T cell-associated were lymphocytes greater anti-IL-2R. Activated with anti-IL-2R and 4B4, Ag. The immunoprecipi- T tates B cells, with +B5+IL-2R+ lymphocytes. further B subpopulations culture SDS-PAGE. a single wt of 60,000 Autoradiography band was corresponding precipitated to from T by analysis. Splenic B cells with fluorescein isothiocyaand or anti-IL-2R. TI and that the antigenic we were BI +Tl vated of TPA on T cell numbers minor anti-Ti either cells of TPA-activated and on to form for one to five but failed to indirectly due to effects The recovery of adequate anti-B5, before culture with with TPA, Ti +Bi failed Fig 2. Highly enriched splenic B cells examined before (A) and after stimulation with TPA. 10 ng/mL. for two days (B. C. D). Cells were stained with directly fluoresceinated anti-Ti (anti-Ti F) and directly PE conjugated anti-Bi (panels A and B). anti-Ti F and directly PE conjugated anti-B5 (C). and anti-Ti F and directly PE conjugated anti-IL-2R (D). Cells were then examined by dual-laser flow cytometric analysis. was BB-I of cells that 1% expressing T percentage the view than and cells not selecting of con- Blast-i contaminating the high before either of the cells experiment, T9 on 31%, the phenotype were less Ag Bi support the notion undergoing the observed and cells were of splenic TPA-activated B cells cultured B5 and IL-2R supports changes and the TPA-activated with not 66% on 38%, ing the B cell-restricted cells were the population changes and were cells and monocytes. and of Ti less than by the expression This population 34% In this splenic expressed enriched cells 6, adherence BI + contained IL-2R+ absence together of highly 90% Table These In contrast, anti-Ig also MRBC-R. days in Blast-2 respectively. to residual as assessed (Table 6). TI + cells. As cells expressed IL-2R, due flasks. a population or TI TPA. However, + cells were PE conjugated As seen and to in Fig 2A, Bl were no detectable after two days of culture detected as well as cells 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). expressing Moreover, virtually all Tl + cells were These studies suggest that the B cells express Table Ti 6. also coexpress Percentage of Cells Bi Control.day0 90(+++) TPA.day2 9i(+++) Anti-Igday2 85(+++) B5 Ti 1 34(+/++) 1 Results are one of three representative and Ex pressing B5 6(+/-) B5+ and IL-2R+. that are induced IL-2R and Ag (Ag Intensity) IL-2R 6(+/-) Tli 1 78(++) 66(+) 1 46(++) 31(+) 1 experiments. to that Mo2 Fig 3. SDS-PAGE (iO%) of ‘2l-labeled normal T cell blasts (lanes A and B immunoprecipitated with 484 [T cell-associated Ag] and anti-IL-2R. respectively); CLI cells from two patients (lanes C. D. E. and F immunoprecipitated with anti-lL-2R [lanes C and E]. anti-B4 [lane D]. and anti-B2 [lane F]; and normal TPAactivated B cells immunoprecipitated with anti-lL-2R (lane G). From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. 424 FREEDMAN cell blasts (lane (lanes C These studies B), cells and suggested and from immunoprecipitated activated B cells, Differential and Effect B-CLL Cells cells response normal B-CLLs normal and express proliferate IL-2R occurs proliferation decreases resting splenic anti-Ig, TPA, anti-Ig led next that C3bR-, 1 was contrast to The slg-, B-CLL did not at three One patient to TPA with days. response tive six days of culture, B cells normal Previous investigators in response for rIL-2 (data not some B cell cultured CLLs TCM. express presence from cases either of background seen suggested they with the phenotype normal present report, of cases B cells counterpart. The Ia, expressed (CD5) TI lated 100 small we have rIL-2 when cell B4 (CD19), Bl but lacked C3bR B cells expressed majority of B-CLL (CD2O), B2 (CD21), (CD35). Although, B!, B2, Ia, B4, Table that expressed thereby on B-CLL demonstrating 476 CLL1 123±27 ± 7. Res ponse 2524 ± 246±53 for on B-CLL cells B cells. Although on normal coexpressed B cells and IL-2, Ti. some we attempted respond was to to IL-2 identical and to that B-CLL cells and with either cell and both the notion isolated surface proteins were activated T and B cells, cells express IL-2R. These that from fetal small numbers or adult of lymphoid of cell slg by either Ag or anti-Ig.5#{176}This initial resting B cells to increase pools of intracellular calcium inositol the and triphosphate. including and ± low- -y-interferon.5’ as MoAbs of these Subsequently increase in size, and to a variety of B cell directed events and high-molecular Alternatively, against as they B Cells Cells Incorporation 218±97 5,204 BCGF, TPA, and C3d of activation, to leave example, TPA stimukinase C without activation with to rlL-2 (cpm) Anti-Ig 2,028 become factors Bp35 (CD2O) also induce trigger resting B cells, through alternative pathways G0 phase of the cell cycle.52’53 For and CLI cells these finally growth weight EBV, lates B cells via direct activation of protein increases in intracellular calcium. After of Normal i5,i92 60-kD cells that probably WA 266 cells Blast-i B cells were both anti-Ig expression could identical consistent B cells several cells slg, and rlL-2 67 are as well unstimuslg, in and expressed of cross-linking event induces 3H-Thymidine Media NormalBcells proliferated the and of Ags express IL-2R, only in vitro-activated B to rlL-2. Immunoprecipitation demon- strated IL-2, surface B-CLL and attempted to identify might represent its normal cellular overwhelming IL-2R cells synthesize RNA, competent to respond the 50% receptors T and B cells (BCGF) examined receptor cells expression not resting B cells. Of 20 cases all expressed the B cell-restricted these then factor. of that the Con- unstimu- B-CLL for the B2 but cells. 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 although fail to proliferate growth examined small we examined and expressed whether num- material. Ia, and B-CLL B cells, only TPA-activated observation that TPA-activated on activated normal level. that sIgD, TPA cells cells peripheral were further approximately B5 were Greater then induced that slg+ spleen between B cells B5 and marrow. and results both cells, adult in fetal resembled and B-CLL of Ags in sIgM, differences Ti popula- of weakly (CD25). Unstimulated small splenic stimulated with anti-Ig or TPA. Although cells rIL-2, closely vitro-activated activated examined TI, phenotypic Ag detectable lymphoid IL-2R found In five thus on activated but examined, virtually whether DISCUSSION In observed expressed B-CLL five patients were slg+, to to those IL-2R of this T 1 were in determine and C3b+). these B cells stimulated With the or two- 7) when at the studies numbers normal and proliferation of CLL cells The substitution of TCM similar These to anti-Ig the activation with incorporation noted and results shown). in the cells (3 was Small to coexpress and lacked normal for the expression cells. shown B-CLL 2) had a minimal proliferaaugmentation by rIL-2. By cells have to anti-Ig yielded culture to proliferate (Table some CLL of Normal of between 3H-thymidine and peak six days. proliferate (CLL and from examined B 1 and C3bR lated that of in vitro and I was slg+, tumor cells from of them or a combination in sidering have cells The of rIL-2 tumor IL-2R+ C3b-, B cells, lacked alone induced minimal to small numbers of in addition examined were all B- media alone, rIL-2, and rIL-2. As seen augmentation resting with patients anti-ig, rIL-2 secondary B cells. fivefold. We with B-CLL days be induced In contrast, to a consistent studies to rIL-2.43 three could then on B-CLL were and in proliferation B by approximately probably vivo-activated TPA in response B cells or TPA. proliferation, for with IL-2R+ Previous were cultured with and rIL-2, or TPA anti-Ig 7, resting cells B-CLL B cells at about B cells on IL-2R-Positive anti-Ig-activated to baseline were present C3bR isolated blood and tonsil tissue but not bone bers of these cells could be identified Weakly slg+, Bi + fetal splenocytes (rIL-2). normal tions TPA- expressed B cells IL-2 we examined activated that T cells, they expression. expressing compare IL-2 addition B-CLL (lane G). structures B Cells B cells, normal with identical. were to functionally to recombinant in Table patients activated ofRecombinant demonstrated and two activated B cells the 60,000-dalton and In Vitro-Activated In an attempt vitro-activated CLL from normal that E), ET AL ± rIL-2 702 i38±51 22,430 + Anti-Ig ± 5,i78 176±105 WA 37,466 + rIL-2 ± 2,597 89±17 CLL2 i14 ± 43 146 ± 29 8i2 ± 176 378 ± 18i 205 ± 23 i,2i2 ± 245 CLL3 lii ± i2 i76 ± 23 131 ± 89 i79 ± 48 176 ± i5 264 ± 29 CLL4 153±23 i86±24 98±42 254±26 i89±57 175±37 CLL5 102±23 211±53 326±98 136±13 166±29 344±36 From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. NORMAL CELLULAR COUNTERPARTS anti-Ig, a distinct sequence observed.42 consistently detected by hours. By 24 with peak most its expression Ags cell B5, Blast-I, might be subpopulation express thereby to that suggests C pathway kinase vated B cells are do not appear virtually all B-CLL B cells express of this cell a minor cells not normal B B cells that activation Ags B5 via the protein B cells to be an identical cells express with activated although B-CLL to IL-2. Preliminary cells previous they our normal suggest responsiveness IL-2R neoplastic pre-B proliferate to IL-2 normal +Tl The cell remarkably seen cells to IL-2 has is unclear. This A recent consist as well that may report also lack subunits cell express the cellular + 70-kD counterpart may reactive B + cells lL-2R subunit may In sIgM T3 and that on these cells the In is but Lyl present with + B cells, we and tonsil blood secrete activated study, in marrow T I + B cells, represent peripheral and autoidentified tissue only do that these BI sIgD + cells as Ia, B2, and as well 1 5% of these cells small not as well was be induced consistent normal activation. the subset but as the remarkably to lend the of express intensity to that study provides activation C3bR. these cells Il-2R. This of Ag similar hypothesis expression observed for the evidence of this that Bl +TI phenotype of protein that weakly Tl but expressed numbers appear via the direct with kinase subpopulations of unstimulated B cells comparing that TI + normal are induced B cells anti-Ig-activated to various growth normal B cells and differentiation insight defect into the in humoral (TI + C of B cells may be activated via distinct pathways Future studies will be directed toward identifying Studies +. cells and responses also node,21 bone is derived from minor populations lymphocytes. Moreover, the observation that Tl in the lymph B-CLL and appears of cells. blood of patients of human majority of B-CLL cells. In summary, the present and to and systems, may experiments, B5 cells that In in adult preliminary of 55fail B-CLL allogeneic murine therefore cells.65 both the but popula- marrow. Because Tl + B cells are a major populaof fetal splenocytes, we used this tissue as a source of this that leukemia In and spleen after be the counterpart autoantibodies could of of low-affinity suggests of two as hairy lack B cell or states investigators resemble in peripheral and immunophenotype that for proliferation.#{176}2 The differences responsiveness to lL-2 suggests that of to become with B-CLL for their factors may -) immunity of B-CLL. be a subpopulation population. surface phenotype homogeneous, expressing in the expression B2. The may cells to be necessary MRBC-R and T cells cells.58 B-CLL tumors which in fetal of patients arthritis.” coexpress do not proliferate be due to a predominance may high-affinity Bl rheumatoid lacked do not B cells that express IL-2R Our data are in contrast to where activated on B-CLL B-CLL transplantation,63 expressed Moreover, laboratory that subset of B cells. We observed cells, or TPA MRBC-R. IL-2R, from normal to IL-2. reports responsiveness of the blood Bi +Tl been seen in B-CLL cells.557 Whether this is due to patient selection or to the presence of very small numbers of contam- 70-kD.59 be identified not bone population. Although receptors for MRBCs, of express studies TPA-activated proliferate in response IL-2R could peripheral B-CLL anti-Ig B cells the of acti- B-CLL normal cells subpopulation resemble either numbers +, mating identified Tl + derived Several 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 similar to most normal various have that does the or a major B-CLL activation phenotypically detectable several direct TPA-activated they TI suggests of 72 tion cells. Although resting that TPA-stimulated as the B cell induces that IL-2R anti-Ig-activated of and lost However, demonstrating that as well either be reflective tions from which B-CLL cells are activation of the BI +Tl + population. be by are may been can observed counterpart B cells. a population has Ags of the B-CLL cell and B2 as well as the and neoplastic cells. The observation coexpress B! and TI IL-2R Ags phenotype B4, BI, of activated Tl, correspond changes expression Blast-2, the 425 activation activation The of Ia, slg, decreased. activation this earliest with days B-CLL of antigenic The hours, six significantly OF demonstration Ia, of with B-CLL cells approximately appears 90% B4, BI, B2, and Ti . Heterogeneity of sIg and C3bR and to a lesser of this heterogeneity of B-CLL to be of was extent cells ACKNOWLEDGMENT The authors thank Dr K. lida for providing anti-C3bR MoAb and Dr Ed Clark for BB-l MoAb. 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