malignant B lymphocytes. expressed on normal ...

B4, a human B lymphocyte-associated antigen expressed on normal, mitogen-activated, and malignant B lymphocytes. L M Nadler, K C Anderson, G Marti, M Bates, E Park, J F Daley and S F Schlossman J Immunol 1983; 131:244-250; ; http://www.jimmunol.org/content/131/1/244

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 1983 by American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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0022-1767/83/1311-0244$02.00/0 JOURNAL OF IMMUNOLOGY Copyright 0 1983 by The American A s s o c i a t i of Immunologists

THE

Vol. 131,No. 1, July 1983 Printed in U S .A.

84, A HUMAN B LYMPHOCYTE-ASSOCIATED ANTIGEN EXPRESSED ON NORMAL, MITOGEN-ACTIVATED, AND MALIGNANT B LYMPHOCYTES LEE M. NADLER, KENNETH C. ANDERSON,GERALD MARTI, MICHAEL BATES, EDWARD PARK, JOHN F. DALEY, AND STUART F. SCHLOSSMAN From the Division of Tumor Immunology, Sidney Farber Cancer Institute and the Department of Medicine, Harvard MedicalSchool, Boston, MA02 7 15


Thecharacterizationof a new B cell-specific antigen appearsbeforetheconventionalcytoplasmicp-positive pre-8 (84) is described in this report. With the use of a mono- cell and is lost from the cell surface when presecretory cytoclonal antibody to 84, it was shown that8 4 is present on plasmic IgG appears (22,23,25). 82, in contrast, appears shortly B cells isolated from peripheral blood and lymphoid orafter the pre-B celland is lost when cells transform into lymphgans, on cell lines derived from normal and malignant B oblasts, lose cell surface IgD, and develop presecretory cytocells,andontumor cells isolated frompatientswith B plasmic IgM (23). Neither B1 nor 82 is present on normal or cell-derived neoplasms.B4, in contrast, was not detected malignant plasma cells. Moreover, the localization of these antion normal, activated, or malignant cells of T or myeloid gens in primary and secondary lymphoid follicles supports the origin. The B4 antigen is distinct from known B cell anti- hypothesis that these antigens are expressed in a limited number gens, including slg, la, 61, 82, Fc, and C3. Examination of B cell differentiation steps(26). of mitogen-stimulated B lymphocytes suggests that the In the present study, we report the preparation and character8 4 antigen initially increases with B cell activation and ization of a monoclonal antibody that defines aBnew cell antigen. then is lost at the terminal stage of B cell differentiation. This antigen, termed 84, is B cell-specific within the hemato8 4 is expressedon almost Moreover, the observation that all early B cell tumors suggests that it may precede B I , poietic system, increases with B cell activation, and is distinct from surface lg (slg), la, B1, 82, C3,and Fc. Like B1, 84 can be CALLA, cytoplasmic p, and 8 2 in early B cell ontogeny. detectedon allnormal,mitogen-stimulated,andmalignant B cells, excluding the plasma cell. Moreover, in contrast to B1 and Human B lymphocyteontogenyanddifferentiationhas,in 82, 84 is expressedonthemajority of early B celltumors, general,been investigated by thesequentialexpressionofa suggestingthat 84 precedes B1 and 82 inanearly B cell series of B cell markers, including the heavy chain isotypes of ontogeny. immunoglobulin (lg)(1-3), HLA-D-related la-like antigens (la)' (4MATERIALS AND METHODS 6), receptors for complement (C) components(7,8) and the Fc portion of lg (9-1 l), and receptorsformurineandmonkey lmmunization and somatic cell hybridization. A 6-wk-old female BALB/c 5 erythrocytes (E) (1 2, 13).Although these markers have provided mouse (The Jackson Laboratory, Bar Harbor, ME) was immunized i.p. with x IO' cryopreserved B cell chronic lymphocytic leukemia (CLL) cells (WBC > important insight into the stages of normalB cell differentiation, 300,000) in phosphate-buffered saline (PBS). Twentyeight days later, the their utility has been limited because they may be expressed on mouse was boosted with 5 x 10' tumor cells i.v., and somatic cell hybridicells of other lineages. The recent development of monoclonal zation was carried out 4 days later by the method of Kohler and Milstein (27) modifications (28). Mouse splenocytes (1.5 x IO') were fused in 30% antibodies defining uniqueB cell-associated antigens has begun with x IO7 P3/NSl/l-Ag4-1 polyethyleneglycolandDulbecco'sMEMwith2 to provide a series of reagents capable of elucidating the semyeloma cells. Selection and growth of hybridomas. After fusion, cells were cultured in quence of normal B lymphocyte differentiation(14-21). In previous studies, we reported the preparation and charac- aminopterincontaining media at37OC in a 7% C02 humid atmosphere (29). Ten to 28 days later, 100 @Iof supernatants from cultures exhibiting cell terization of several monoclonal antibodies that identify B cell- growth were tested for the presence of hybridoma antibodies reactive with associatedantigens (15,18,19,21). Two oftheseantigens, immunizingtumorcellbyindirectimmunofluorescenceasdescribed(30). termed B1 and 82, have been shown to be phenotypically and Hybridoma cultures containing antibodies reactive with the immunizing tumor selected and cloned three times by the limiting dilution method in the molecularly distinct from known B cell determinants; within the were presence of irradiated BALB/c splenic feeder cells (31). hematopoietic system they are restricted to cells of B lineage Normal human tissues. After securing appropriate Human Protection Com(15, 18). Considerable evidence on normal, mitogen-stimulated, mittee Validation and informed consent, human samples were obtained. /solation of peripheral blood cells. Human peripheral blood mononuclear andmalignant B cellshasbeenaccumulatedsupportingthe cells (PBMC) were isolated from healthy volunteer donors by Ficdl-Hypaque notion that the expression ofB1 and 82 antigens are limited to (Pharmacia Fine Chemicals, Piscataway, NJ) density gradient centrifugation into slg-positive distinct stages of B cell differentiation (22-24). The B1 antigen (32).Unfractionatedmononuclearcellswereseparated

(slg'XB) and slg-negative (slg-XT plus null) populations by Sephadex G-200 anti-F(ab'kchromatography (33) with modificationsdesigned to minimize Received for publication January 3, 1983, (34).T cells were recovered monocyte retention by the column as described Accepted for publication March14, 1983. by E rosetting the slg- population with 5% sheepE (Microbiological AssociThe costs of publication of this article were defrayed in part by the payment of ates, Bethesda, MD). Normal human monocytes were obtained by adherence pagecharges.Thisarticlemusttherefore be herebymarkedadvertisementin to plastic culture dishes as described (35) or by isolation from PBMC by accordance with 18 U.S.C. Section 1734 solely to indicate this fact. centrifugation in 50% Percoll in 0.5 M NaCl (density 1.064 g/mlX36). GranuAbbreviations used in this paper:slg, surface immunoglobulin; Fc, Fc receptoc locytes were isolated from the sediment formed during Ficoll-Hypaque density forIgG;C3.C3receptor;la,HLA-DR-relatedla-likeantigen;PBMC,peripheral blood mononuclear cells; CLL. chronic lymphocytic leukemia; E-, E rosette-nega- gradient centrifugation of peripheral blood. Contaminating E were excluded TR, Texas red dye; ALL, acute lymphoblastic by gravity (1 x G) sedimentation in the presence of 0.4% dextran according tive; FITC, fluorescein isothiocyanate; to the techniqueof Levy et a/. (37). For determination of the reactivity of antileukemia; AML, acute myeloblastic leukemia; CML, chronic myelocytic leukemia; B4, 1 x IO' of each cell population were subjected to indirect immunoflue CALLA, common acute lymphoblastic leukemia antigen: PWM. pokeweed mitogen; G/M-FITC. fluoresceinated goat anti-mouse IgM and IgG; NP-40, Nonidet P-40. rescence analysis.

244

(B4)

245

ANTIGEN B-ASSOCIATED HUMAN

'9 '


Normal tissues. Nucleated bone marrow cells were recovered by Ficdl- fluorochrome conjugate. The conjugate was then ultracentrifuged at 100,000 x G for 20 minto remove aggregated protein. Hypaque centrifugation. Tonsil cells were obtained at the time of routine Biotinylation of purified antibodies was undertakenas follows: 1.7 mg of tonsillectomy. Lymph node tissue was taken for diagnostic biopsy and was (+)biotin N-hydroxysuccinimide ester (Calbiochem-Behring, La Jdla, CA) was considered normal on the basis of histology and cell surface markers. Splendissolved in 1.O ml of N.Ndimethyl formamide (Fisher Scienti, Fair Lawn, ocytes were obtained at the time of traumatic rupture. Normal human thyNJ). Then, 200 pI of this biotin solution were added to an aliquot of affinitymocytes were obtained from patients who had portions of their thymuses purified monoclonal antibody (4.3 mg/ml 0.05 M carbonate buffer, pH 9.1). removed during corrective cardiac surgery. All tissue specimens were immeThis mixture was incubated at room temperature for 4 hr without mixing. The diately placed in media containing 5% fetal calf serum (FCS), finely minced with forceps and scissors, and made into single c e l l suspensions by extension incubation mixture was then extensively dialyzed against 1x PBS (pH 7.5). to through stainless steel mesh. C e l l samples were used fresh or Cryopreserved Afterfourdialyses,thebiotinconjugatesolutionwasultracentrifuged remove aggregates. The conjugate was tested for specificity, and a serial and thawed as needed. Lymphoid tissues were fractionated into lg' and lgdilution was performed to find the optimal dilution by using indirect imrnunopopulations as described above. fluorescence with an Epics V c e l l sorter or fluorescence microscope (Zeiss, Human leukemia, lymphoma, and myeloma samples. All tumor cells were cryopreserved in -196°C vapor-phase liquid nitrogen in 10% dimethyl sulf- West Germany). Dual fluorescencewas undertaken with 1to 2 x 10' cells used per sample. oxide and 20% human serum until the time of surface characterization. Tumor with the directly fluoresceinated antibody, washed twice, Cells were incubated cells were obtained from patients with acute and chronic leukemias, nonincubatedwiththebiotinylatedantibody,andthenincubatedwithavidin Hodgkin's lymphomas, and plasma c e l l dyscrasias. Diagnoses were deterand histochemistry. In all instances, the tumorconjugated to Texas red dye(TR). After two subsequent washes, the number mined by standard morphology of cells expressing and coexpressing each antigen was enumerated under a populations utilized contained more than 75% neoplastic cells by WrightGiemsamorphology.Bcelllineagewasestablishedbythepresence of fluorescence microscope. At least 200 to 500 cells were counted for each sample. monoclonal slg, determined by indirect immunofluorescence utilizing anti& 84 antigen.ADEAEcelluloseIgG p , 7 , and X hybridoma antibodies (15). Additional determinants expressed on Biochemical characterization of the B cells included Fc and/or C3 receptors-la-like antigens tested by reactivity fraction was obtained from 3.5 ml of ascites fluid; the IgG fraction was eluted It was coupled directly to with 20 mM potassium phosphate buffer, pH 7.4. with a monoclonal antibody directed against a nonpdymorphic la determinant, CNBr-Sepharose 46 (2 mg/ml wet gel). Approximately 16 mg of protein were designated 1-2 (38), and by reactivity with monoclonal anti-61 (15) and anticoupled to 7.5 ml of Sepharose (2.5 g dry weight). The immunoabsorbent 62 (18).Tcelltumorswere identied byreactivitywith speCmc Tcell monoclonal antibodies (39). Myeloid cells were enumerated by using the was MY7precycled with Nonidet P-40 (NP-40) buffer (0.5% NP-40, 10 mM Tris, pH 8.0in normal saline) and treated once with DEAE (0.5%) (43). and MY8 (40) and Mol (41) monoclonal antibodies. The monocyte-specific Three cell lines were radiolabeled intrinsically with [3H]arginine (10' cells, monoclonal antibody Mo2 (41) was used to enumerate monocytes. Human cell lines. Epstein Barr virus- (EBV) transformed B lymphoblastoid 5 mC, -60% incorporation of label). The cell lines were TRAL and Hom-2 An NP-40(0.5%) lysate lines (SB, Laz 007, 388,467,468,471,475,803), T cell lymphoblastoid lines (EBV-transformed homozygous typing cell) and Daudi. (CCRF-CEM and HSB-2), null cell leukemia lines( L a z 221 and 207),chronic (approximately 5 x l @ cells) was prepared and a 100,000 x G x 60 min myelocytic leukemia (CML) in blast crisis line Nalm-1 , Burkitt's lines (Daudi, supernatant was isolated(44). Approximately5mlofthe100,000 x G x 60minNP-40lysatewere Ramos, Raji), the promyelocytic line HL-60, the erythrocytic line K562, and histiocytic lymphoma line U937 were kindly provided by Dr. Herbert Lazarus applied directlyto the immunoabsorbentand were incubated in thecokl on a with NP-40bufferand2% (Sidney Farber Cancer Institute, Boston, MA). The myeloid leukemiac e l l line rotatorovernight.Afterwashingexhaustively deoxycholatebuffer,theirnmunoabsorbentwaselutedwith0.5%NP-401 KG-1 was supplied by Dr. David Golde (University of California School of to the method of Medicine, Los Angeles, CA). Antigen-activated T cell clone lines were kindly 0.5% DEAE. Autoradiographs were prepared according Bonner and Laskey (45). provided by Dr. Stefan Meuer (Sidney Farber Cancer Institute). Antibdy sbsorption and blockingexperiments. One hundred microliters of The specifically bound and eluted material (reduced and nonreduced) was of 1O0/o sodium anti-64 antibody at a1/10,000 or 1/40,000 dilution were absorbed with 10' analyzed on lO-crn cylindrical and slap gels with the use viable cells from variable cell populations, including CLL cells, B cells, T cells, dodecyl sulfate-polyacrylamide el electrophoresis (SDS-PAGE) according to granulocytes, monocytes, and reactive and unreactivec e l l lines. After a l-hr the method of Laemmli (46). C-hlgh m.w. standards were obtained from Bethesda Research Laboratories (Bethesda, MD). incubation on ice with agitation, absorbing cells were removed by centrifugation. Residual antibody activity in the supernatant was assessed by indirect In vitro stimulation: pokeweed mitogen (PWM). One hundred microliters of B cells (lO'/ml in tissue culture medium) were plated in round-bottomed 96immunofluorescence on known reactive cells. The number of positive cells wellplates(Linbro Scientiic, Hamden.CT),and 100 pl oftissueculture withabsorbedanti-64antibodywascompared with thenumberofcells staining with the unabsorbed antibodies. medium (TCM)or TCM containing 25to 50 cg/ml PWM (Difco Laboratories, The possibility that anti-64 binds to C3 or 7SFc receptors was examined Detroit, MI) were added to each well. Twenty percent irradiated E rosetteby attempts to inhibit competitively EAC and EA rosette formation (42) by positive (€+KT) cells and 5% adherent macrophages were addedto facilitate pretreatment of reactive cells with anti-64 antibody. Known positive CLL cells B cell activation by PWM (47). Plates were cultured in 95% air and 5% Con cells wereharvestedaftervaryingintervalsfor humidatmosphere,and (l@/ml) were incubated with anti-I34 (1/250) or with an unreactive control antibody for 30 rnin at 4'C. After a wash step, 100 pl containing 1 x 108 immunofluorescence analysis. pretreatedcellswerethenincubatedwith100 pI ofeitherEAC(0.5% EACl95Clgp4,2,3gp) or 0.5%EA 7 s (Cordis LaboratoriesCorp., Miami, FL) RESULTS for 30 min at 37%. and the suspensions were centrifuged at 200x G for 5 min. The c e l l sediments were gently resuspended, and a drop was placed on Development of the 84 monoclonal antibody. BALB/c mice a slide for microscopic examination. The number of cells forming rosettes wereimmunized with circulatingtumorcells(WBC300,000/ with the sheep E were counted. mm3) from a patient with B cell CLL. These tumor cells were Indirect immunofluorescence. Cryopresewed cells or cultured cells from activation studies were analyzed for surface phenotype by indirect immunoidentifiedas B cells by their expression of monoclonal cell surface fluorescence as described (30). In brief, 0.5 to 1 x 10' viable washed cells IgM, IgD, K , as well as the expression of the B cell-associated were treated with 100 pI of a 1 /250 dilution of specific or control antibodies antigensla, B1, and 82. Incontrast,thesetumorcellswere (saturating concentrations), incubated at 4'C for 30 min, and washed three unreactive with monoclonal antibodies directed against the comtimes. The cells were then treated with 100 pl of a 1/40 dilution of goat antimouse IgG and goat anti-mouse IgM conjugated with fluorescein isothiocya- mon acute lymphoblastic leukemia antigen (CALLA) (48); T cell 30 nate (G/M-FITC; Coulter Electronics, Hialeah, FL), incubated at 4°C for antigensT3, T4, T6, T8, orT11(39);andmyeloid/monocyte min, washed three times, and analyzed on an Epics V cell sorter (Coulter and MY8 (40). antigens Mol (41), Mo2 (41), MY7 (40), Eiectronics). The percent positivecells were determined by using the EASY system (Coulter Electronics) Immuno-program. Aftersomaticcellhybridization,approximately250macroDual fluorescence microscopy.To carry out direct fluorescent staining of scopic clones were identified, one-half of which were reactive allpopulations,directlyfluoresceinatedandbiotinylated anti-61, -62, and with the immunizingCLL ceils, measuredby indirect immunoflu-64 wereprepared.Conjugationofpurifiedmonoclonalantibody(DEAEpurified) to FlTC was undertaken by standard methods. Monoclonal antibody orescence. Producer clones were then screened on a panel of was dialyzed against 0.05 M carbonatebicarbonate (pH 9.0) in the cold forfractionated 4 peripheral blood and tumor cells. One hybrid clone, hr. The protein was then removed from dialysis and adjusted with carbonatedesignated anti-B4, was foundto react with the immunizing CLL bicarbonatebuffer(pH 9.0) to giveafinalconcentration of 12.5 mg/ml. BcellCLL.Intheinitialscreen, 84 Fluorescein-5-isothiocyanate (Molecular Probes, Junction City,OR) dissolved Cellsandseveralother in carbonatebicarbonate buffer (1.0 mg/ml) was then added in 100-pl incre-appeared to beuniquebecause it wasweaklyreactive with ments over 30 min until a 1O:l ratio (w/w) of protein to dye was achieved. peripheral blood lg' cells, unreactive with T cells, and strongly The mixture was then incubated on a rotary mixer in the dark for 1.5 hr at reactive with tumorcellsfrompatientswithnon-Tcellacute roomtemperature.Theincubationwasstoppedbypassingthereaction mixture over a Sephadex G-25 columnto remove free dye from the protein- lymphoblasticleukemia(ALL).Hybridcloneanti-B4wasthen

246

ANTIGEN B-ASSOCIATEDHUMAN

(84)


subclonedthreetimesbylimitingdilutionandpassaged into cells may be due to our inability to detect cells with low 84 BALB/c mice to produce a malignant ascites. Supernatant and antigendensitybyindirectimmunofluorescenceandflowcyascites anti-B4 were shownto have similar reactivity by indirect tometry. immunofluorescence.The 84 ascitesdemonstratesreactivity To address these two alternatives, the coexpression of B1, with theimmunizing CLLcells to adilutionof1/50,000that 62, and 84 on B cells was investigated by dual fluorescence. diminishes to background at 1/200,000. The 84 antibody is of Anti-81,-B2,and -84 weredirectlyconjugated to FITC. In the murine lgGl subclass. In all subsequent experiments, ascites addition, each antibody was also conjugatedto the N-hydroxy84 was utilized. succinimide ester of biotin. Surface lg+ cells were then directly Anti-64 reacts with B lymphocytes. The reactivity of anti-B4 stained with FITC antibody, washed, incubated with a second with cells isolated from normal peripheral blood and lymphoid biotinylated antibody, and then developed with avidin conjugated tissues was evaluated by indirect immunofluorescence and flow to TR.Byutilizingthismethod,80 to 90% ofslg'cellsin cytometry. Approximately 6% k 3 of PBMC isolated by Ficollperipheral blood, lymph node, andtonsil coexpress B1,82, and Hypaque density sedimentation expressed the 84 antigen (Table 64 as assessed by dual fluorescence microscopy. Nevertheless, I).The number of cells that expressed 84 was almost identical it should benoted that these dual fluorescence studies detected to the number of cells that expressed B1 and 82. All the B4- a small number of cells (10 to 20%) that did not coexpress all reactive cells were confinedto the E- fraction (Table I).This E- three antigens. Further enumeration and functional characterifraction is the conventional B cell-enriched population used to zationofthesesmallsubsetsof B cells ispresentlyunder studytheexpressionofBcellantigensaswellasBcell lg investigation. Overall, these findings support the view that the production.Asseen in Table I, thenumberofE-cellsthat majority of slg+ cells coexpress B1, 82, and 84 and that the express B cell-associated antigens (B1 ,B2, and 84) ranged from lower percentage of B2+ and B4+ cells is apparently due to the 5 to 28%. The larger number of cell reactive with anti-r,A reflects lower density of these antigens compared with B1 or la. cytophilic IgG bound to monocytes. The incubation of cells with To characterize further the lymphoid cellular expression 84, of serum-free media for 2 to 12 hr at 37OC to remove cytophilic lg the number of cells reactive with anti-B4 was enumerated by decreases the number of cells reactive with slg(.,X) (PBMC, 8%; indirect immunofluorescence and flow cytometry. Normal human E-, 25%; monocyte,4%;andgranulocyte,3%) but does not tonsil, lymph node, and spleen contain 38% f 6, 19% k 5, and change the number of cells reactive with anti81, -82, or -84. 18% f 7 B4' cells, respectively. Small numbers of B4'cells (5% The majority of cellsin the E- fraction are in fact monocytes, null k 3) (n = 5) could be detected in normal adult bone marrow. In cells, and a small number of T lymphocytes. Anti-B4 was uncontrast, no BCreactive cells(fewerthan 1%) were found in reactive with T cells, monocytes, granulocytes, erythrocytes, andnormal thymus (n= 6). platelets. In addition PHA or Con A-activated T cells were unTo confirm the specificity of anti-B4 further, absorption studies reactive with anti-B4. were undertaken in which 100 MIof anti-B4 were diluted to 1/ 10,000 or 1/25,000 and incubated with lo7 or 10' immunizing To define the reactivityof anti-B4 further, B cells were isolated SB, lymphoma from peripheral blood by utilizing a G-200 anti-F(ab')n lg affinity CLL cells, slg+ tonsillar B cells, B cell lineBurkitt's line Daudi, E' T cells, adherent macrophages, granulocytes, T column. Slg' cells isolated from peripheral blood, lymph node, cell lines HSB and CEM, or murine splenocytes. After 1 hr of tonsil,andspleenexpressed 84 (Table 11). Incontrast,B4reactive cells could not be identified in the slg- population (less incubation, the cell suspension was centrifuged, and the superthan 3% in all preparations). Again, cytophilic binding of lg is natants were tested for reactivityon the immunizing CLL cells. Anti-B4 reactivity was completely absorbed by the immunizing responsible for the larger number of cells bearing slg ( K J ) compared with the number of cells expressing B1, 82, and 84. As CLL cells, slg' B cells, and B cell lines(SB and Daudi),but could seen in Figure 1, the84 antigen is weakly expressed on periph- not be absorbed by T cells or T cell lines, macrophages, granueral blood slg' cells and is more strongly expressedon slg' cells locytes, or murine splenocytes. These studies further confirm that 84 expression is limitedto B cells and B cell lines. isolated from tonsil, lymph node, and spleen. The antigen density Biochemical characterization of the B4 antigen. With the eviof 84 on peripheralblood slg' cells was similarto that of 82 (Fig 1). but was consistently less than the expression of B1 (Fig. 1) dence that 84 expression was restricted to cells of B lineage, it or la (intensity of la was consistently equal to or greater than was important to demonstrate that it was distinct from other Bl). Similarly, the intensity of B4 expression on slg' cells from previously defined B cell surface determinants. To demonstrate that 84 was distinct from slg, la, B1, and 82, the biochemical tonsil, lymph node, and spleen was slightly less than the expresof the 84 sion of 82 and considerably less than the expression of slg,B1, characterizationof 84 wasundertaken.Isolation and la. One possible explanation for the above findings is that antigen was attempted from the B lymphoblastoid cell lines HomBurkitt's lymphoma cell line Daudi. Previous there are fewer B2+ andB4' cells in peripheral blood, tonsil, and 2 and TRAL and the studies had indicated that by indirect immunofluorescence and spleen than B1+ cells. Alternatively, the smaller number of B4+ TABLE I Expression of 8 cell-associated antigens on fractionated peripheral blood cells Cell Type

Number of Tests

% of Cells Reactive with MonodonalAntibody

84

81

82

T3

fl,A

la

Mol

Mo2

16 f 5 PBMC 8 6 + 31 2 25 r t 26 + 2 5 17 f 6 70% 6 26 t 8 E10 20 f 8 13k8 18+9 35 f 8 53 8 7f5 49k10 37k12 221 95 f 5 12t5 I fl T (E') 10 1f 1 1+I 021 021 Activated T (Con PHA) A or 4 1+1 0+1 1 +1 021 39 f 5 92 f 7 ND 1f O Monocyte 8 2 + 12 + 12 + 1 20 2 7 82 2 5 222 79 f 10 78+6 5 +3 Granulocyte IO 1+7 1+ 2 0+1 15f8 3r2 3Cl 87 f 5 2+1 Erythrocyte 4 1 f1 1 +1 1+0 2-cl I f 1 1 fl 1 +1 2+1 Platelets 2 12 fl +1 2 5 21 fl2 2 1 2+1 1t 2 * Mean percentage positivecells f percent standard error of the mean assayed by Epics flow cytoflletfY and calculated by the Coulter EASY Immun*PrWram. 'ND. not done.

*

ANTIGEN B-ASSOCIATED HUMAN

247

(84)

TABLE II Expression of B cell-associated antigenson slg' column-adherent cells isolatedby anti-F(ab')* affinity column chromatography % of Cells Reactive with "mhdAntibody

Cellular ongin of B Cells

Number of Tests

84

lg"

PB. node,Lumph Tonsil, spleen. lg+

lg' lg'

4

6

5 3

40 + 7b 90 + 5 62 + 7 41 + 5

€31

82