cellular distribution of a b-cell specific surface antigen ... · of immunoglobulin m. lymphoblasts...

[CANCER RESEARCH 46, 5431-5437, October 1986]

Cellular Distribution of a B-Cell Specific Surface Antigen (gp54) Detected by aMonoclonal Antibody (Anti-BL4)1

Labib I lushinii, Chang Yi Wang, Ayad Al-Katib, and Benjamin Koziner2

Lymphocyte Surface Markers Laboratory fL. H., A.A-K., B. K.J and Laboratory of Molecular Immunology Â¡C.Y. W.Â¡,Memorial Sloan-Kettering Cancer Center,New York, New York 10021

ABSTRACT

A monoclonal antibody (anti-BL4) recognizing a previously characterized M, 54,000 glycoprotein (gp54) was developed by immunizing BALB/c mice with cells from a precursor B-cell line (Josh-7). In normalindividuals, this antigenic molecule was present on tonsillar B-cells (60-80%) and on a fraction of peripheral blood B-cells (5-25%). BL4 (gp54)expression was investigated in 186 patients with a variety of hematolog-ical malignancies using indirect immunofluorescence and flow cytometricanalysis. Twenty-six of 37 cases of B-cell chronic lymphocytic leukemia(CLL) and 18 of 33 cases of B-cell non-Hodgkin's lymphoma were HI -4

positive. Surface expression of BL4 on reactive cases of CLL and non-Hodgkin's lymphoma was brighter than those of Bl, B2, and B4. HI 4

positive CLL cases expressed a higher proportion of mouse rosetteforming cells and I on-1 positive cells than the BL4 negative subgroupand were not associated with elevated serum immunoglobulin levels. Fourof 7 HI 4 negative CLL cases were associated with increased serum levelsof immunoglobulin M. Lymphoblasts from 14 of 14 cases of non-T acutelymphoblastic leukemia and 3 of 3 pre-B lymphoid blast crisis of chronicmyeloid leukemia were BL4 negative. Neoplastic cells from 2 of 3 casesof Waldenstrom's macroglobulinemia and 4 of 7 cases of hairy cell

leukemia were HI.4 reactive. None of 7 cases of multiple myeloma andplasma cell leukemia were HI 4 positive. All 11 T acute lymphoblasticleukemia cases, 6 other T-cell malignancies, 5 cases of Hodgkin's disease,

51 cases of acute nonlymphocytic leukemia, and 9 cases of chronicmyeloid leukemia in chronic phase thus far studied were Bl .4 negative.An in vitro induction experiment using phorbol ester on a case of B-CLLdemonstrated disappearance of HI.4 accompanied with further B-celldifferentiation. Our study further substantiates the previous finding thatgp54 is a differentiation antigen restricted to the B-cell lineage andexpressed during the intermediate stage of B-cell ontogeny.

INTRODUCTION

Since the introduction of the hybridoma technology by KÃ¶hler and Milstein (1), Mo Ab3 have been utilized to supplement

conventional cell surface markers in the diagnosis and classification of lymphomas and leukemias. However, the phenotypiccharacterization of B-cell neoplasias has been hampered by thelack of lineage specificity of most conventional surface markers(2-10), and Mo Ab developed against B-cells.

Among the previously reported B-cell associated MoAb areBAI and BA2, raised against a pre-B ALL cell line (Nalm-6-

Received 10/8/85; revised 5/14/86; accepted 7/7/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by Grants CA-08748. CA-20194, and CA-05826 from the National Cancer Institute: Grant Al-18321 from the NIH; Grants CH-322 and PDT-246 from the American Cancer Society, and by the Norman and Rosita WinstonFoundation, Inc., Dr. Burton J. Lee, and the Julie Gould Foundation for MedicalResearch.

2To whom requests for reprints should be addressed, at Memorial Sloan-

Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.3The abbreviations used are: MoAb. monoclonal antibodies; PB. peripheral

blood; BM, bone marrow; LN, lymph node: slg, surface immunoglobulin; ALL,acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin's lymphoma; CML, chronic myelogenous leukemia; HCL; hairy cellleukemia. ANLL, acute nonlymphoblastic leukemia; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; WM, Waldenstrom's macroglobuli

nemia: MM, multiple myeloma; CALLA, common acute lymphoblastic leukemiaantigen; MRFC, mouse rosette forming cells; CMFI, channel number of meanfluorescence intensity: CPF1, channel number of peak fluorescence intensity:TPA, 12-O-tetradecanoylphorbol-13-acetate.

Ml). BAI identifies a determinant expressed primarily on PBB-lymphocytes, B-CLL, B-NHL, and the majority of non-TALL cases (11). However, it has been also found to be expressedon granulocytes. BA2 detects an antigen expressed on BMprogenitor cells, 50% of slg positive CLL, 77% of non-T ALL,and 17% of T-ALL cases (12).

Other MoAb reported thus far include those reacting withBl, B2, B4, PCI, BL1, BL2, BL3, and BL7 antigens (13-18).Their diagnostic value lies predominantly in relating B-celltumors to specific differentiative stages. Although all B-cellantigens are preferentially expressed on PB slg positive B-cellsand B-CLL cells, Bl and B4 are also present on pre-B ALLblasts exhibiting immunoglobulin gene rearrangements but reported to be absent from blasts of non-B cell hematologicalmalignancies (19). With regard to the various BL antigens (Fig.1), previous studies have indicated that BL1 and BL2 areexpressed during the early stages of B-cell development (17,20). It was subsequently reported that 46 of 49 cases of non-TALL were anti-BL2 and/or anti-BLl reactive (21). In addition,both antigens were expressed on B-cell neoplasias, includingCLL and NHL; however, their expression has been shown tobe heterogeneous. Unlike BL1 and BL2, BL3 has been shownto be expressed primarily at the later stages (17) and BL7 atthe intermediate stages of B-cell differentiation (18). Althoughthe expression of these BL antigens is mostly restricted to theB-cell lineage, BL2 and BL7 have been reported to be presenton blasts of a proportion of ANLL cases studied (60 and 20%,respectively). Among the other described BL antigens are BL5(Pro-IM2) and BL6 (Pro-IMl), both detecting nonlymphoidassociated determinants (22).

In this study, we describe a new murine MoAb (anti-BL4)directed against a previously described B-cell antigen termedgp54, a glycoprotein with preferential expression on a subsetof tonsillar and PB B-cells, yet distinct from slg, la-like antigens, C3 receptors, and Fc receptors as determined by rabbitantisera (23). Reactivity of this MoAb was found to be B-celllineage specific and limited to distinctive stages of B-cell differentiation. Cellular distribution of BL4 in various hematologicalmalignancies and its correlation with the surface expression ofother B-cell associated antigens are subjects of this report.

MATERIALS AND METHODS

Cell Surface lodination and Immunoprecipitation. MoAb anti-BL4,an IgGjb antibody, was developed by immunization of BALB/c micewith the cell line Josh-7, a cell line established from non-T BM cells ofa young patient with X-linked agammaglobulinemia, and is thought tobe composed of precursors of B-lymphocytes (24). To characterize thetarget antigen recognized by anti-BL4, Josh-7 cells were radiolabeledexternally with 125Iby the lactoperoxidase catalyzed reaction and lysedwith Nonidet P-40 detergent, and the resulting solubilized surfacemembrane proteins were reacted with anti-BL4, anti-BL7, and controlmouse IgGi, respectively. The immunoprecipitates were analyzed bySDS-PAGE under both nonreduced and reduced conditions.

Peripheral Blood and Tissue Specimens. PB from 10 different healthydonors was studied. Tonsillar tissue was obtained from tonsillectomyspecimens in 10 patients ages 3 to 35 years. Samples from PB, BM,LN, spleen, and malignant pleural fluid were obtained, either at the

5431

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BL4 (gp54) EXPRESSION ON LYMPHOID MALIGNANCIES

abed

BL2

Fig. 1. Hypothetical scheme for sequence of BL antigen expression on B-lymphoid cells and their precursors. BLI expression starts at the earliest stageand disappears at the plasma cell level. Ill _ is non-B-cell lineage specific. BL3 isexpressed at the later and HI." at the intermediate stages of B-cell differentiation.HI4 is restricted to a distinctive phase along the intermediate stages of B-cellontogeny.

time of diagnosis or at relapse, from 186 patients with different typesof hematological neoplasias. The breakdown of cases was: ANLL, 51;B-CLL, 37; NHL, 33; T-ALL, 11; chronic phase CML. 9; non-T ALL,14; HCL, 7; Hodgkin's disease, S; T-cell hematological malignancies,

6; lymphoid blastic crisis of CML, 3; WM, 3; MM, 5; and plasma cellleukemia, 2. LN from 5 patients with reactive lymphadenopathy werealso studied. Two of these 5 patients were diagnosed as having acquiredimmunodeficiency syndrome and a third one was a healthy homosexual.The remaining 2 patients were known cases of NHL with hyperplasticlymph nodes.

All patients were seen at the Memorial Sloan-Kettering CancerCenter during the period of February 1983 through April 1986. Thediagnosis of leukemia or lymphoma was based on standard clinical,morphological, histochemical, and cytogenetic criteria. This was furthersubstantiated by conventional surface marker studies and reactivitieswith other MoAb identifying B- or T-cell lymphoid or myeloid relatedantigens. The French-American-British system (25) was used for theclassification of acute leukemia and the Rappaport scheme (26) for theclassification of NHL. In acute leukemia cases the proportion of blastsin the involved specimen ranged from 30 to 97%. In chronic leukemiasthe WBC exceeded 15,000/mmJ and was up to 350,000/mm\ with a

lymphocytic proportion of 55 to 98% in CLL, while in CML cases thepercentage of myeloid precursors was 60% or more. In patients withother types of neoplastic lymphoid diseases, PB or BM showed 30-100% malignant cells. In cases where pleura! fluid was studied, involvement was documented by cytomorphological examination. Representative portions from histopathologically involved LNs or spleens werestudied in selective cases of NHL, HCL, CLL, and Hodgkin's disease.

Cultured Human Cell Lines. Nineteen established human B/pre-Bhematopoietic cell lines (Josh-7, RAMOS, HPB-NULL, RPMI 8226,U937, SK-LY-16, B46M, NK-9, BALL-1, DG-75, Nairn-12, Nairn-17,LAZ-221, KOPN-8, REH, AUR, NAK, RPMI 1788, and RPMI 8866),and 5 non-B-cell lineage leukemic cell lines (HL-60, KE37, HSB-2,HPB-ALL, and CEM) were studied for their reactivity with anti-BL4by indirect immunofluorescence. These cell lines were kindly providedby Dr. Jun Minowada (VA Hospital, HiÃ±es,IL) and Dr. L. J. Old(Memorial Sloan-Kettering Cancer Center).

Other Monoclonal Antibodies. MoAb identifying B-cell antigens BLI,BL2, BL3, and BL7 were developed by one of us (C. Y. W.) andreported earlier (17, 18). MoAb to Bl, B2, and B4 were obtained fromCoulter Diagnostics (Hialeah, FL); anti-Leu-1, anti-Leu-4, and anti-Leu-5 were from Becton Dickinson & Co. (Mountain View, CA): andBA3 (anti-CALLA) was from Hybritech, Inc. (San Diego, CA). MoAbrecognizing myelocytic and monocytic lineages include aim HI 5 (anti-Pro-IM2), reported earlier (22); 5F1 and IG10, previously described byBernstein et al. (27); and OKM1, reported by Breard et al. (28). Allthese MoAb were used in saturating concentrations.

Mononuclear Cell Isolation. Cell suspensions were prepared fromLN, BM. and spleens by mincing the tissue in Hanks' balanced salt

solution until the cells were separated from the connective tissuestroma. Tonsillar tissues were dispersed as described before (29). Mono-nuclear cells were isolated from all cell suspensions, PB, BM, or pleuralfluid specimens by centrifugation on Ficoll-Hypaque gradient, as de-

94-

Ixo 43-69

30-

20-

Fig. 2. SDS-PAGE analysis of immunoprecipitates from 125I-labeledJosh-7cell lysates under nonreduced conditions, a, .nui HI 4; A.anti-BL7; r. anti-gp54;</.sequential precipitation ot .mii HI 4 after precipitation with anti-gpS4.

scribed previously (30). Monocytes were removed by a carbonyl ironingestion method. Preparations of enriched T- and B-lymphocyte populations were made by Ficoll-Hypaque gradient separation of lymphocytes rosetted with neuraminidase-treated sheep erythrocytes. Viabilityalways exceeded 80%, as determined by trypan blue dye exclusion test.

Surface Marker Analysis and Immunofluorescence. Spontaneous rosette formation with mouse erythrocytes (MRFC) was performed asdescribed previously (31) and with sheep erythrocytes at 4Â°Caccording

to the method of Bentwich et al. (32).Direct immunofluorescent staining for slg was carried out using

fluorescein conjugated rabbit I ul>) >fragments of antibodies directedagainst various human immunoglobulin fractions as reported previously(31).

Reactivity with anti-BL4 and the other described MoAb was assayedby indirect immunofluorescence with fluorescein conjugated, affinitypurified goat anti-mouse IgG and analyzed on a cytofluorograph system30L (Ortho Diagnostic Systems, Westwood, MA) utilizing the 488 mmband of a 50-mW argon laser as described previously (18). Backgroundfluorescence was obtained using normal mouse IgG. A minimum of10,000 cells/test were analyzed. MoAb reactivity was considered positive when 25% of the leukemic cells or more displayed immunofluorescence. CMFI or CPFI were chosen as indicators for density of antigenexpression. CMFI was calculated using a 2140 Data Handling System(Ortho).

Phorbol Ester Induced Differentiation. Neoplastic B-cells derivedfrom a case of CLL were used for induction experiments with TPA(Sigma Chemical Co., St. Louis, MO). Cells were cultured in a regularmedium at 37Â°Cin humidified atmosphere of 5% CO2, as describedpreviously.4 TPA was stored at -20Â°C at 1 mg/ml and was used in afinal concentration of 1.6 x 10"' M. Control cultures received an equal

amount of acetone without TPA. Cultures were maintained and replenished with new media daily for 6 days with viability always exceeding80%. Aliquots of cells were harvested on days 1, 3, and 6 and analyzedfor phenotypic changes.

RESULTS

Biochemical Characterization of BL4 Antigen and Its Distribution on Normal Cells. Immunoprecipitates obtained by reacting the radiolabeled and Nonidet P-40 solubili/ai surface membrane proteins of Josh-7 cells with both MoAb anti-BL4 andanti-BL7 were analyzed by SDS-PAGE under both nonreducedand reduced conditions. A single polypeptide with molecularweights of 54,000 under nonreduced conditions and 45,000under reduced conditions was precipitated by anti-BL4, whereasrepeated attempts to precipitate the BL7 antigen, known to beheat stable, from the cell lysate failed (Fig. 2). A glycoproteinwith an identical molecular weight was precipitated by thepreviously described rabbit anti-gp54 serum (23). Sequentialprecipitation with MoAb anti-BL4 after prior precipitation with

4 A. AI-Katib, C. Y. Wang, and B. Koziner. Interferon--/ (IFN--y)-inducedphenotypic changes in chronic lymphocytic leukemia and non-HodgkinÂ°s lymphoma cells in vitro: evidence of B-cell differentiating activity, submitted forpublication.

5432




rabbit anti-gp54 serum failed to bring down any discerniblecomponent suggesting that MoAb anti-BL4 recognized thesame B-cell specific gp54 molecule described previously.

Normal distribution of BL4 antigen was determined by testing 10 different PB samples obtained from healthy donors and10 tonsillectomy specimens. As illustrated in Fig. 3, BL4 wasfound to be strongly expressed on a major subset of tonsillarB-cells (60-80%) and on a variable fraction of PB B-cells, (5-25%). On the contrary, anti-BL4 reactivity was absent from allPB T-cells, monocytes, macrophages, and granulocytes in allindividuals tested. This distribution was identical to that ofgp54 antigen reported earlier (23).

BL4 Expression on Leukemias and Malignant Lymphomas.Anti-BL4 reactivity on all malignant cases studied is summarized in Table 1. Thirty-seven patients with B-CLL were tested.Cells in all cases expressed la antigens and faint surface im-munoglobulin (slg positive) and formed rosettes with mouseerythrocytes. Furthermore, cells in almost all CLL patientstested expressed Leu-1 (33 of 35) and BL7 (23 of 25). BL4 wasfound to be expressed in 26 of 37 cases with a mean percentageof positive cells of 60.7%.

Table 2 illustrates the phenotypic features of the two differentsubgroups of B-CLL, as determined by their surface expressionof BL4 antigen. Despite bright expression of la antigen in bothsubgroups, a larger number of cells expressed Leu-1 and MRFCin the BL4 positive subgroup.

Serum immunoelectrophoresis studies were available on 15

T-cellsp (PB, Tonsil,Thymus)lÃ¬

AMÃŒ-BL4If

Control

V,.

Monocytes/Mlti-BI_4Control

PB B-cells

Control

Tonsillar B-cells

Anti-BL4Control

Immunofluorescence Intensity (Linear)

Fig. 3. Representative immunofluorescen.ee profile from a normal individualshowing anti-BL4 reactivity of the different cellular components of the hemato-poietic system. A. T-cells. 100% negative; B, monocytes. 100% negative; C. PBB-cells. positive (faint); /'. tonsillar B-cells. positive (bright).

patients in the BL4 positive CLL subgroup and none hadevidence of paraproteinemia. Serum IgM, IgG, and IgA levelswere either normal or decreased. On the contrary, of the 7 BL4negative CLL patients studied, 4 had increased serum IgMlevels of 314-2420 mg/dl (normal, 50-280 mg/dl).

Leukemic cells from all CLL cases were surveyed for theirreactivity with anti-BL7. It was shown that both BL4 positiveand BL4 negative subsets of CLL express BL7 antigen.

Neoplastic cells from 33 cases of B-cell NHL (including onecase of pre-B lymphoblastic lymphoma) were evaluated for theirreactivity with anti-BL4. The B-cell lineage of these neoplasmswas determined by the presence of la antigens, bright expressionof monoclonal slg, and reactivity with MoAb identifying B-celllineage, i.e., Bl, B2, B4. Eighteen of 33 (54%) of these caseswere reactive with ami HI.4 with a mean positive reactivity of47.4% (Table 1).

Correlation between BL4 expression and the histologicalsubtypes according to the Rappaport classification is shown inTable 3. Five of 6 cases of nodular poorly differentiated lym-phocytic lymphoma and 7 of 9 cases (77%) of all nodularlymphomas reacted with anti-BL4, whereas only one-half of alldiffuse lymphoma cases were BL4 positive, including 4 of 8cases of diffuse poorly differentiated lymphocytic lymphomaand 6 of 12 cases of diffuse histiocytic lymphoma. Only 1 of 3cases of Burkitt's lymphoma tested was anti-BL4 reactive (pro

portion of positive cells, 32%). Concomitant testing of malignant cells from NHL cases with anti-BL7 showed that almostall BL4 positive cases (15 of 16) and the majority of the BL4nonreactive subgroup (9 of 12) were BL7 positive regardless ofhistological subtype.

Other B-cell hematopoietic malignancies examined included3 cases of WM, 2 of which reacted with anti-BL4. One casehad 55% abnormal lymphoid cells in the BM; these were ofintermediate size and a few displayed clefted nuclei. These cellsexpressed the following surface phenotype: Ia+, Bl+, B2+ andB4+, CALLA", BL4+, BL7+, BL3~. slg expression was of mod

erate intensity and of IgM* isotype. Paraproteinemia in thispatient was of two different isotypes; the main component wasIgMx (520 mg/dl) and in addition there was an IgG spike (2640mg/dl). In another WM patient there was a serum IgMÂ«paraproteinemia. slg fluorescence was intense and of IgM* isotype.Surface membrane phenotype in this case was as follows: Ia+,BT, B2-, B4+, CALLA", BL1+, BL2+, BL4+, BL7+, BL3~. Inthe remaining BL4' WM patient, 90% of BM cells were lymph

oid. There were also atypical plasma cells. Malignant cells didnot express slg and showed the following phenotype: Ia~, BL1",

Table 1 Reactivity of various hematopoietic malignancies with anti-BL4

DiagnosisCLLNHLWMHCLMM/PCLALLCM

L-lymphoblasticALLT-cell

malignanciesHDANLLCML-CPCell

lineageBBBBBNull

(pre-B)Null(pre-B)TT7Myeloid/monocyticMyeloidPhenotypeIa*,slg*,

E-*,MR*,Leu-r,BL7*ia*,sig*",E-.MR-ia+/-,sig*'-,E-.MR-Ia*,slg**,E-,MR-,Latex*lausig*'',clg*'-,E-Ia*.slg-,

CALLA*'",c!g*'-,E-Ia*.slg-,cIg*'-.E-,CALLA+/-Leu-r,Leu-5*

orLeu-4*.E*'-,sIg-Leu-l*.Leu-5*orLeu-4*.E*E-.sIg-BL5*

or OKM1*. ICIO*.5F1*BL5+or OKM1*. ICIO*. 5F1*No.

of patientsstudied3733377143II65519Ã•86Reactivities

withanli-BL4'+26IX2400000000-1115137143II65519BL4*

(% ofcases)70.054.02/357.00.00.00.00.00.00.00.00.0

â€¢+ (positive), 25% positive cells or more with a medium to high mean fluorescence intensity; - (negative), less than 25% positive cells with low mean fluorescence

intensity.* E. sheep erythrocyte receptor; clg, cytoplasmic immunoglobulin: MR, mouse erythrocyte rosette; Latex, attachment or ingestion of latex particles; HD, Hodgkin's

disease; CML-CP chronic myelocytic leukemia, chronic phase; PCL. plasma cell leukemia.

5433




Table 2 Phenolypic comparison between BL4 positive and BL4 negativesubgroups ofCLL

BL4"

reactivityBL4*

BL4-No.

ofpatients26

11Mean

proportion (%) of cells expressing themarkerBL4*60.7

10.6la*

Leu-1*78.S

71.2P = 0.026

78.8 52.3MRFC*44.0 26.8P= 0.03

* + (positive), 25% positive cells or more; - (negative), less than 25% positive

cells.

Table .1 Correlation of BL4 reactivity with the morphological subtype ofnon-Hodgkin's lymphoma

Subtype oflymphomaNodularNPDL"NHILNMLTotalDiffuseDPDLDHLTotalBurkitt's

lymphomaLBL(pre-B)TotalNo.

of casesstudied612~9812203133No.

ofBL4*

cases511~746Toi0ÃŽ8

" NPDL, nodular poorly differentiated lymphocy tic lymphoma; NHIL, nodu

lar histiocytic lymphoma: NML, nodular mixed lymphoma; DPDL, diffuse poorlydifferentiated lymphocytic lymphoma: DHL, diffuse histiocytic lymphoma: LBL,lymphoblaslic lymphoma.

BL2~, BL7~, BL3+. There was marked elevation of IgMx in the

serum (55,600 mg/dl).The neoplastic plasma cells of 7 cases with MM and plasma

cell leukemia failed to express the BL4 antigen.The "hairy cells" in 4 of 7 patients with HCL were BL4+,

and all expressed la and high phagocytic activity (30-66%) andwere MRFC negative. Acid phosphatase stain was done in 4patients and was resistant to tartaric acid in all.

None of the 17 cases of non-T ALL, lymphoblastic crisis ofCML, or Ph1 positive ALL studied reacted with anti-BL4.

Lymphoblasts from these cases expressed la but lacked slg andsheep erythrocyte receptors. Ten of these 17 cases were CALLApositive.

Lymphoblasts in all 11 patients with T-ALL were BL4 negative; 9 of these 11 patients were sheep erythrocyte receptorpositive and in all there was positive reactivity with at least oneMoAb identifying T-cell lineage, including anti-Leu-1, anti-Leu-5, and anti-Leu-4. In addition, 6 other cases of T-cellmalignancies, including 4 NHL, 1 CLL, and 1 SÃ©zarysyndrome, were studied and cells in none of these cases were foundto express BL4. Five LN from patients with Hodgkin's disease

were uniformly nonreactive with anti-BL4.Fifty-one cases of ANLL were studied. These included 2

myeloid blastic phase of CML and 5 with myelodysplasticsyndrome evolving into ANLL. The morphological breakdownof the remaining 44 cases according to French-American-British morphology was as follows: 2 MO, 5 Ml, 11 M2, 7 M3, 7M4, 11 M5, and l M6. Malignant cells isolated from thesecases were found to react with MoAb identifying myelomono-cytic lineage and none were BL4 positive.

Nine patients with Ph1 positive CML in chronic phase were

also evaluated, and again none of these reacted with am i lÃ¬I 4.Reactivity of mononuclear cells isolated from 5 patients with

reactive lymphadenopathy revealed la antigen expression andlight chain polyclonality. The majority of these LN (4 of 5)reacted with either anti-BLl, anti-BL2, or anti-BL7. Contrariwise, none of these cases expressed BL4 antigen.

Comparison of BL4 Antigen Expression with Bl, B2, and B4Antigens. Anti-BL4 reactivity on a selected number of caseswith hematological malignancies was correlated for lineagespecificity with MoAb detecting other B-cell specific antigensincluding Bl, B2, and B4. Results of such comparison areshown in Table 4. The percentage of CLL cases expressing BL4antigen was equivalent to those expressing Bl (22 versus 23 of26 cases studied), while B2 and B4 were less frequently expressed than BL4. Similar findings were noticed in NHL. Onlya minor subset of NHL cases demonstrated reactivity with anti-B2 (5 of 15 studied) and anti-B4 (7 of 18 studied). None of the17 ALL cases studied showed BL4 reactivity, while only 3 of14 non-T ALL cases reacted with Bl and another 3 expressedB4. Among the nonlymphoid leukemias, none of 31 cases testedreacted with ami Bl 4 and anti-B2, whereas 4 of 7 cases withacute monoblastic leukemia (M5) expressed Bl and B4.

For objective estimation of the BL4 antigen density of expression on leukemic cells, CMFI was measured on representativeCLL cases that expressed BL4 and correlated with that of BL7,Bl, B2, and B4. In 16 BL4 positive CLL cases, the mean valueof CMFI was 109.0, compared to 225.4 for BL7. Mean CMFIfor Bl, B2, and B4 in reactive CLL cases were 84.7, 73.3, and70.4, respectively. Representative analysis in a case of CLL anda case of NHL illustrating such correlation is shown in Fig. 4.BL7 was the most brightly expressed antigen on both CLL andNHL (CMFI = 240.0 and 711.0, respectively), followed by BL4(115.5 and 247.0, respectively). All B antigens (Bl, B2, and B4)found to be expressed on CLL and NHL were much weaklyexpressed than BL7 and BL4, with Bl being the brightest(CMFI = 90.2 and 94.9, respectively).

Reactivity with Hematopoietic Cell Lines. Table 5 shows anti-BL4 reactivity with 24 well characterized cell lines includingthe Josh-7 from which anti-BL4 was prepared. None of the 6pre-B ALL cell lines expressed bright reactivity with anti-BL4;however, 3 of 6 (REH, Nairn-12 KOPN-8) showed faint reactivity and the other 3 were negative. Among the B-cell hema-topoietic cell lines, those from Burkitt's lymphoma either were

BL4 negative (RAMOS, DG-75) or expressed faint reactivity(B46M, NK-9). Three of these 4 cell lines reacted with anti-BL3 and anti-BL7. On the other hand 2 Epstein-Barr virustransformed B-cell lines (RPMI 1788, RPMI 8866) expressedbright reactivity with anti-BL4 (CMFI = 532.0 and 447.0,respectively). Other B-cell lines tested were SK-LY-16 andU937, which are diffuse histiocytic lymphoma cell lines, andboth lacked BL4 expression. The myeloma RPMI 8226 cellline was BL4 negative; also all non-B leukemic cell lines tested

Table 4 Comparison ofBL4 expression to other B-cell antigens

DiagnosisCLLNHLHCLWMMM/PCL*ALL

(pre-B)Leukemia/lymphoma(T)ANLLM1-M4MSM6CML-CPCML-lymphoblasticNo.

of casestested26185241412237163No.

reactive with monoclonalantibodyBL422133200000000Bl2313420e3004000B2125'3100000000B41574203004000

Â°Fifteen cases studied.* PCL, plasma cell leukemia; CML-CP, chronic myelocytic leukemia-chronic

phase.' Three cases studied.

5434




A

11â€¢â€¢â€¢â€¢Background

BL4BL7B1B2B4

1 200 400 600 800Immunofluorescence Intensity

1000

Fig. 4. Immunofluorescence profiles from (A) a patient with CLL and (B) apatient with NHL. showing the differences in intensity of expression of variousBL and B antigens. BL7 expression is the brightest of all on CLL and NHL(CMFI = 240.0 and 711.0, respectively). BL4 and Bl are moderately bright(CMFI for BL4, 115.5 and 247.0, respectively; CMFI for Bl, 90.2 and 94.9,respectively); B2 and B4 are weak. Thickly dotted line, background fluorescence.

Table 5 Expression ofBL4 on cultured human cell lines

Reactivitywith

Cell line Origin Type of cell anti-BL4Â°

Josh-7KOPN-8REHNairn-

12HPB-NULLLAZ-221Nairn-

17BALL-1B46MNK-9RAMOSDG-75SK-LY-16U937RPMI

1788RPMI8866AURNAKRPMI

8226HL-60KE37CEMHSB-2HPB-ALLX-linked

agammaglobulinemiaALLALLALLALLALLALLALLBL'BLBLBLDHLDHLEBV

transformedEBVtransformedEBVtransformedEBV

transformedMMAPLALLALLALLALLPre-BPre-BPre-BPre-BPre-BPre-BPre-BBBBBBBBBBBBBMyelomonocyticTTTT+Â±Â±Â±â€”â€”â€”Â±Â±Â±â€”â€”â€”â€”++â€”Â±â€”-â€”â€”â€”-

" +. more than 25% positive cells; -, less than 10% positive cells; Â±,between

10 and 25% positive cells.* BL. Hin kin's lymphoma; DHL. diffuse histiocytic lymphoma; EBV, Epstein-

Barr virus; APL, acute promyelocytic leukemia.

thus far (HL-60, KE-37, CEM, HSB-2, and HPB-ALL) lackedBL4 expression. However, the latter cell line, HPB-ALL, whichis a T-ALL cell line, was B2 positive.

Disappearance of BL4 upon TPA Induction of B-Cell Differentiation. Fresh cells from one patient with B-CLL were subjected to TPA induction experiments and monitored for phe-notypic changes as detected by anti-BL4 and other MoAb.

Prior to TPA culture, only 40% of cells expressed low densityBL4 antigen (CPFI = 100.0), whereas 75% of the cells brightlyexpressed BL7 (CPFI = 220.0). As shown in Fig. 5, TPAinduced disappearance of BL4 expression after 3 days of culture. Concomitantly, the proportion of BL7 positive cells decreased sharply in a fashion parallel to that of BL4. However,the overall BL7 reactivity remained positive. On the contrary,BL3 expression was low on day 0 and increased progressively

BL7 (cont.;

BL3 (TPA)

BL7 (TPA)BL4 (cont.)

BL3 (cont.)

BL4(TPA)

Days in culture

Fig. 5. Decrease in BL4 and BL7 expression after TPA induction and increasein BL3 reactivity in a case of CLL. cont.. control.

from 10% to a peak of 91.0% on day 3 accompanied by anincrease in the CPFI from 90.0 to 125.0. Other phenotypicchanges noticed during this culture period included a 20%increase in Leu-1 expression on day 1, reaching a plateau onday 3 to decline thereafter but remaining strongly positive. Bland B2 were expressed on day 0. Their expression increased onday 1 to decline thereafter. On day 6 of TPA culture, Blexpression disappeared completely, while B2 showed only marginal expression (33.0%).

DISCUSSION

This paper describes the reactivity of a monoclonal antibody(anti-BL4, IgG2b) directed at a previously defined glycoproteinwith a molecular weight of 54,000 on normal and malignanthuman B-lymphoid cells. This murine MoAb was derived byimmunization of BALB/c mice with cells from cell line (Josh-7) from which another monoclonal antibody (anti-BL7) wasdeveloped. Like BL7, BL4 antigen is expressed on a subset oftonsillar and PB B-cells, but not on earlier lymphoid precursors,pre-B cells, or more terminally differentiated elements, i.e.,plasma cells.

Previous immunoprecipitation studies using surface iodina-tion and SDS-PAGE analysis have shown that the BL4 (gp54)antigen comigrates with the HLA heavy chain under reducedconditions, however, which resolved into a distinct moleculewith a molecular weight of 54,000 under nonreduced conditions(23). In addition, previous studies utilizing direct surface im-munofluorescence on fresh leukemic cells and leukemic celllines showed that gp54 positive cells have a characteristicspeckled and patchy appearance and simultaneously revealedmarked cell to cell variation in the staining intensity of thesepositive cells suggesting extreme heterogeneity of the gp54antigen expression.

In the present study we further confirm that BL4 is a B-lineage specific antigen. Expression of BL4 antigen on leuke-mias and lymphomas as detected by indirect immunofluores-cence with anti-BL4 was shown to be absent in all T-cell andmyeloid leukemias and leukemic cell lines. In comparison withthe other BL antigens, BL4 expression is mostly restricted tothe slg-bearing intermediate stages of B-cell differentiation asshown in Fig. 1. These include CLL, HCL, and the NHLspectrum of lymphoproliferative neoplasias. All non-T ALLcases studied, including pre-B-cell leukemias defined by expression of cytoplasmic immunoglobulin n heavy chain, were BL4negative. However, 3 of 6 non-T ALL cell lines studied (REH,

5435



BL4 (gp54) EXPRESSION ON LYMPHO1D MALIGNANCIES

Nairn-12, and K.OPN-8) showed faint reactivity with anti-BL4.The other pre-B-ALL cell lines studied (HPB-NULL, LAZ-221, and Nairn-17) were BL4 negative. This may suggest a lowlevel of antigen expression in a small proportion of theseleukemias. Unlike BL3, which is expressed at later stages of B-cell differentiation (Fig. 1) (17), BL4 expression was absent inthe terminally differentiated B-cells (plasma cells). All 7 MMand plasma cell leukemia cases studied were BL4 negative. Thelatter subgroup of lymphoproliferative disorders represents themost "differentiated" type of B-cell neoplasms according to

several proposed hypothetical schemes for B-cell ontogeny (19,33, 34).

BL4 expression was also correlated for lineage specificitywith BL7. Although both of these surface antigens are limitedin their expression to the intermediate stages of B-cell differentiation, BL7 is present on blasts of 20% of ANLL cases,while none reacted with anti-BL4. In contrast to BL7, BL4expression in CLL and NHL was variable. Only 70% of CLLand 54% of NHL cases were BL4 positive with mean reactivitiesof 60.7 and 47.4%, respectively. Al-Katib et al. (18) previouslyreported that all CLL (63 of 63) and the overwhelming majorityof NHL (22 of 28) expressed BL7. In addition, although bothBL4 and BL7 were among the most densely coexpressed antigens on the surface of B-CLL and NHL cells, the CMFI foranti-BL7 was significantly higher than that of anti-BL4 in themajority of the cases studied (240 and 711.0 versus 115.5 and247.0, respectively), as exemplified in Fig. 4. These data indicate that BL4 expression is more restricted than BL7 at theintermediate stages of B-cell differentiation. Furthermore, anti-BL4 apparently identifies a distinctive phenotypic subgroup inCLL, since the proportion of MRFC and Leu-1 positive cellswere higher in the BL4 reactive than the BL4 nonreactive cases.Additionally, it was noticed that the cases studied in thissubgroup were not secretory of immunoglobulins, while a proportion (4 of 7) of the BL4 negative subgroup were associatedwith elevated serum levels of IgM.

In comparison with Bl, B2, and B4 antigen expression, BL4resembled Bl regarding their frequency of expression in CLLand NHL. Both antigens were present on an equivalent numberof cases tested. Furthermore, among the B-antigens, Bl, although weaker (mean CMFI for reactive CLL cases, 84.7), wasthe closest in surface density of expression to BL4. However,these two molecules are structurally different, since Bl wasdescribed by immunoprecipitation to be a nonglycosylatedphosphoprotein with a molecular weight of 35,000 (35). Additionally, Bl was present on a proportion of the non-T ALL (3of 14 cases studied), yet none expressed BL4. As far as lineagespecificity, both Bl and BL4 seem to be B-cell restricted.However, in several instances in our patient population (4 of7), leukemic cells from acute monoblastic leukemia (M5) expressed Bl and B4, in addition to strong expression of nonlym-phoid associated antigens as detected by monoclonal antibodiesanti-BL5 and OKM1 (22, 28). In these Bl and B4 positiveANLL cases, BL4 was negative. Histochemical and cytofluo-rometric analysis of leukemic cells in the these patients did notreveal any evidence of biphenotypic leukemia. Bl and B4 werenot detected on leukemic blasts from all 148 cases of ANLLreported by Anderson et al. (19); however, none of these ANLLcases was consistent with M5 by morphology.

B2 and B4 were less frequently expressed in CLL than BL4and Bl. These antigens were present in approximately one-halfof the cases tested. In NHL, only a minor subset of casesexpressed B2 and B4 (5 of 15 and 7 of 18, respectively), whilethe majority reacted with anti-BL4 and expressed Bl (13 of 18studied). In CLL, NHL, and other B-lymphoid malignancies

when B2 and B4 molecules were present on the surface ofmalignant cells, their expression was usually weaker as compared to BL4 (average CMFIs for B2 and B4 on reactive CLLcases, 73.3 and 70.4, respectively). These differences in densityof expression are exemplified in Fig. 4.

An attempt was made to correlate anti-BL4 reactivity withspecific histolÃ³gica! patterns of malignant lymphoma. A trendwas noted in which BL4 was detected in LN suspensions fromthe majority of nodular lymphomas (77.0%) but in only 50%of diffuse lymphomas regardless of cellular subtype. Since mostpathologists attribute the cellular origin of nodular lymphomato follicular center B-cells (36, 37), it is reasonable to assumethat BL4 bearing cells are more prevalent at the germinalcenters than in the other anatomical and functional compartments of LN. Unlike nodular lymphomas, diffuse lymphomasare a heterogeneous group of tumors that encompass wide rangeof morphological and immunological diversity (38) and therefore tend to variably express BL4 antigen. Another noteworthyfinding was the relative absence of BL4 expression in freshBurkitt's lymphoma cells and in cultured Burkitt's lymphoma

cell lines.It should be emphasized here that, although the malignant

B-cells of WM are lymphoplasmacytoid in morphology andsecretory in function (features of advanced B-cell differentiation), they most often express a cell membrane immunoglobulinisotype similar to that of CLL, i.e., IgM (39, 40). The exactderivation of these cells from the proposed stages of B-celldifferentiation is a subject of controversy. For example, Anderson et al. (19) considered the lymphoid cells of WM as transformed B-cells at a pre-plasma cell level, whereas Jansen et al.(34) related lymphoplasmacytoid leukemias phenotypically toa post-CLL stage. Our analysis has shown that this group oflymphoproliferative disorders is morphologically and phenotypically heterogeneous. Neoplastic cells in this condition mayexpress intermediate stage phenotype (sIgM+, BL4+, BL3~) and

secrete variable amounts of paraprotein or express a plasmacell phenotype (slgM", BL4~, BL3+) and secrete large amounts

of immunoglobulin.In this study, it was also noticed that BL4 was least frequently

expressed by reactive LN. Only 1 of 5 LN showed faint (Â±)reactivity and the remaining were negative, while almost alltested LNs expressed either positive or faint (Â±)reactivity withany one of the BL antibodies (BL1, BL2, BL7) and with Bland B2. Therefore, this study suggests that anti-BL4 is leastlikely to cross-react with reactive LNs as compared with theother BL antibodies, when the examined LNs did not showevidence of slg monoclonality.

The relationship of BL4 antigen expression to B-cell differentiation was further investigated by in vitro TPA inductionstudies. It has been reported that TPA has the capacity toinduce B-CLL cells to differentiate into more mature cyto-plasmic immunoglobulin-containing B-cells (41). In this study,results of TPA induction in a case of B-CLL have shown thatthe expression of both BL4 and BL7 decreased with further B-cell differentiation while there was a concomitant increase inreactivity with anti-BL3. The disappearance of BL4 was accompanied by decrease in BL7 expression, both substantiating theconclusions which were drawn from the studies on fresh cellsabout the sequence of BL antigen expression in B-cell neoplasms and on their normal counterparts. This indicates thatBL4 and BL7 are both expressed in B-cell malignancies andduring normal B-cell differentiation in a sequential fashion.BL7 disappearance usually follows that of BL4 but in an orderlymanner.

In addition to TPA induction experiments, preliminary stud-

5436




ies using purified F(ab')2 fragments of gp54 rabbit antisera orthe whole antisera and thymidine labeling of B-lymphocytesrevealed that these fragments can induce T-cell independent liceli proliferative response but cannot induce immunoglobulinsynthesis (23). These studies suggested that B-cell activationinduced by the anti-gp54 serum was probably related to thebinding of these antibodies to the BL4 surface antigen. Parallelstudies using monovalent Fab fragments of the anti-gp54 reagent demonstrated marked inhibition of T-cell dependent B-cell differentiation. In vitro experiments with anti-BL4 on various mitogen stimulated cultured lymphocytes are under wayto substantiate the regulatory role of this antigenic molecule inB-cell proliferation and differentiation.

Collective analysis of the presented data suggests that anti-BL4 is B-cell lineage specific MoAb, limited in reactivity to theslg positive intermediate stages of B-cell differentiation and ofclinical usefulness in the diagnosis and classification of B-

lymphoid malignancies.

ACKNOWLEDGMENTS

The authors are most grateful to Dr. Jun Minowada of the VeteransAdministration Hospital, HiÃ±es,IL, for providing the leukemic celllines; to Patricia Black for her kind assistance in data gathering; andto Mary Lilly for typing the manuscript.

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1986;46:5431-5437. Cancer Res Labib Hashimi, Chang Yi Wang, Ayad Al-Katib, et al. Detected by a Monoclonal Antibody (Anti-BL4)Cellular Distribution of a B-Cell Specific Surface Antigen (gp54)

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