two mouse monoclonal antibodies detecting two different … · mouse monoclonal antibodies were...

[CANCER RESEARCH 45,1314-1319. March 1985]

Two Mouse Monoclonal Antibodies Detecting Two Different Epitopes of anActivated Lymphocyte Antigen on Adult T-Cell Leukemia Cells1

RyuzoUeda,2 Keiko Nishida, YukioKoide, IkuyaTsuge, MasaoSeto, Mitsuaki Yoshida, IsaoMiyoshi,

KazuoOta, and Toshitada Takahashi

Laboratoriesof Chemotherapy[R. U., K. N., K. O.], ExperimentalPathology[M. S.Â¡,and Cell Biology [T. T.], Aichi CancerCenterResearchInstitute, Chikusa-ku,Nagoya,464: Departmentof Microbiology, HamamatsuMedical School, Handa-cho, Hamamatsu431-31 [Y. K.]; Departmentof Pediatrics, Nagoya UniversitySchool of Medicine,Tsurumai-cho,Nagoya 466 [I. T.J;Department of Viral Oncology, Cancer Institute, Toshima-ku,Tokyo, 170 [M. Y.]; and Third Department of Internal Medicine, KochiMedical School, Okatoyo-cho,Nangoku, 781-51[I. M.], Japan

ABSTRACT

Mouse monoclonal antibodies were produced against MT-2cell line derived from adult T-cell leukemia or human T-cellleukemia virus-rich fraction therefrom. Two lgG1 antibodies,

Ta60a and Ta60b, were found to be reactive not only with celllines derived from adult T-cell leukemia or cutaneous T-cell

lymphomas, but also with activated peripheral blood lymphocytes, suggesting the similarity of Ta60 antigen group to Tacantigen which is present on interleukin 2 receptor. Thus, therelationship among these antigens was studied. Two Ta60 antibodies and Tac antibody immunoprecipitated the molecule withalmost identical electrophoretic mobility, approximately a M,60,000 antigen from [3H]glucosamine-labeled activated periph

eral blood lymphocytes or MT-2, MT-1, or ATN-1 cells from adultT-cell leukemia and a M, 53,000 antigen from HUT-102 cellsderived from cutaneous T-cell lymphomas. Further, Tac antibodywas found to immunoprecipitate Ta60b molecule on 125l-labeled

MT-2 cells by sequential immunoprecipitation, indicating that

these two epitopes are on the same molecule. Antibody bindinginhibition assays with either 3H-labeled Ta60a or Ta60b antibody

demonstrated that Ta60a and Tac are the same epitope, butdifferent from Ta60b. Thus, at least two epitopes were demonstrated to be present on interleukin 2 receptor molecule. However, Ta60b antibody showed almost no blocking effects onproliferation of an interleukin-2-dependent cell line, whereas

TaGOaantibody did.Various hematopoietic tumor cells were typed with these two

antibodies, but the results with Ta60b antibody were described,because they showed a similar specificity. Ta60b antibody reacted with all adult T-cell leukemia cases, but did not react withT-cell acute lymphoblastic leukemia, lymphoblastic lymphoma,or mature T-cell lymphoma. Interestingly, 3 of 12 acute myelo-

blastic leukemia and 2 of 5 chronic myelocytic leukemia in blasticcrisis showed positive reactions. One-third of B-cell chroniclymphocytic leukemia and B-cell lymphoma as well as a few B-

cell lines were also weakly reactive with this antibody. A part ofthe results with direct tests was confirmed by the absorptiontests. The results obtained demonstrated the presence of Ta60bon a certain fraction of malignant hematopoietic cells of otherthan T-cell origin.

1This study was supported in part by the Ministries of Education, Science,andCulture, and Health and Welfare (Comprehensive 10-year strategy for cancercontrol) in Japan, and by the Cancer Research Institute, Inc., New York, NY.

2To whom requests for reprints should be addressed.ReceivedJune 15, 1984; accepted November9, 1984.

INTRODUCTION

In Japan, Takatsuki er al. in 1977 proposed a new diseaseentity, ATL,3 for a particular type of T-cell lymphoma/leukemia,

which has a peculiar geographic distribution, in that ATL is foundpredominantly in the southwestern part of Japan (24). The celllines were established from ATL patients by Miyoshi ef a/. (11,12), and many particles resembling C-type retrovirus observed

by electron microscopy were named ATL viruses (6). Furthermore, an antigen associated with ATL virus was detected in anMT-1 cell line by most ATL patients' sera and were called ATL

cell-associated antigen (6). In the United States, Poiesz ef a/. (15)succeeded in the isolation of a C-type retrovirus, HTLV, from

CTCL cell lines in 1980. Subsequent studies revealed that ATLvirus and HTLV are quite similar to each other (16, 17, 28, 29,32). Thus, both viruses are now called HTLV (31).

In this study, mouse monoclonal antibodies are producedagainst MT-2 cell line (12) derived from ATL or HTLV-rich fractionfrom MT-2 cells, in order to detect antigens associated with ATL

cells. Two hybridoma clones producing lgG1 antibodies reactivepredominantly with ATL cell lines were selected and established.Both antibodies, however, were found to react also with activated lymphocytes, but not with resting lymphocytes. Accordingly, the relation of the antigens recognized to Tac antigen ofUchiyama ef a/. (22, 23) is studied. The results revealed thatthese 2 antibodies showed a specificity quite similar to that ofTac antibody; Ta60a epitope is identical to Tac but different fromTa60b, whereas both Ta60a/Tac and Ta60b are on the samemolecule. Further, typing of various hematopoietic tumors withthese antibodies was carried out. Ta60b was found not only onATL but also on a part of tumor cells derived from cell lineagesother than T-cells.

MATERIALS AND METHODS

Preparation of Normal and Malignant Hematopoietic Cells. Peripheral blood and bone marrow aspirate obtained from patients with hematopoietic cancer or normal volunteers were separated by Ficoll-Paque

(Pharmacia, Uppsala, Sweden) density gradient centrifugation, and interface layers were collected. Single-cell suspensions of normal and malig

nant lymphoid tissues were obtained by gentle manual squeezing of thespecimens. Only the samples from the patients consisting of more than80% tumor cells were studied.

Preparation of Activated Lymphocytes. Ten million PBLs or ATL

' The abbreviations used are: ATL, adult T-cell leukemia; HTLV, human T-cellleukemia virus; PBL, peripheral Wood lymphocyte; IL-2, interleukin 2; Con A,concanavalinA; CTCL,cutaneous T-cell lymphoma;M-MHA,mouse mixed hemad-sorption; AML, acute myelocytic leukemia;CML-BC, chronic myelocytic leukemiain blastic crisis; null-ALL.null cell acute lymphocytic leukemia.

CANCER RESEARCH VOL. 45 MARCH 1985

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TWO EPITOPES ON ACTIVATED LYMPHOCYTE ANTIGEN

cells were cultured in 10 ml of the RPMI Medium 1640 supplementedwith 20% pooled human serum and 10% (v/v) partially purified IL-2(Biotest, Frankfurt, West Germany) for 72 hr at 37Â° in 5% C02 in

humidified air. PBL were also cultured with phytohemagglutinin (50 ng/ml; Difco, Detroit, Ml) or Con A (50 mg/ml; E. Y. Laboratory, San Mateo,

CA).Cultured Cell Lines. MT-1 cell line was derived from ATL cells (11),

while MT-2 cell line (12) was established from cord lymphocytes cultivated with MT-1 cell lines. ATN-1 cell line was established from ATLcells by Naoe et al. (14). HUT-102 and HUT-78 cell lines derived from

CTCL were established by Gazdar ef al. (5). Other cell lines were derivedfrom hematopoietic tumors and other cancers (see Refs. 13 and 25-27).

Cultures were maintained in RPMI Medium 1640 supplemented withstreptomycin (100 ^g/ml), penicillin (100 units/ml), 2 mw glutamine, and10% heated (56Â°,30 min) fetal bovine serum.

Both NK3.3 (7) and 4E8 cultured lines were maintained in the mediumdescribed above containing IL-2.4E8 was derived from a purified proteinderivative of tuberculin-stimulated PBL cultures.4

Production of Monoclonal Antibodies. Eight-week-old female BALB/c mice were immunized with either MT-2 cells, or HTLV particle-rich

fraction from MT-2 culture supernatant fluid which was separated on a

sucrose density gradient as described previously (32). The mice wereimmunized 3 times with 107 MT-2 cells and sacrificed for hybridization 4

days after the last immunization. When HTLV fraction was used forimmunization, it was treated with 6 cycles of freezing and thawing, andapproximately 0.5 mg of HTLV protein was injected s.c. with 20 n\ ofcomplete Freund's adjuvant for the first immunization. Four weeks later,

the mice were given s.c. injections of 0.9 mg of the antigen mixed with20 /il of incomplete Freund's adjuvant. Three weeks later, 0.9 mg of the

antigen were injected Â¡.p.,and 4 days later, mice were sacrificed for cellfusion. Cell hybridization was performed according to the method de

scribed (25).For detection of antibodies against cell surface antigens, M-MHA (25)

was mainly used. Generally, 10-fold dilutions (starting from 1/103) of

ascites from hybridoma-bearing mice were used to test normal and

malignant hematopoietic cells. When more than 10% of the target cellsplated into the wells of Terasaki microplates (Falcon Plastics, Oxnard,CA) showed rosette formation at a dilution of 1/103, the reactions were

regarded as positive. In each experiment, control wells incubated withmouse nonimmune immunoglobulin (19) alone were included; as a rule,these had less than 5% positive cells. Antibody titer refers to the highestserum dilution that showed 10% positive cells. Indirect immunofluores-cence assays were also used to confirm the results with M-MHA.

Absorption assays were performed as described previously (25).Tac antibody was kindly provided by Dr. T. Uchiyama, Kyoto University

School of Medicine, Kyoto, Japan. NL-1 antibody (27) was produced in

our laboratories, and B1 antibody (20) was purchased from Coulter

Electronics (Hialeah, FL).Blocking Effects by Monoclonal Antibodies on Proliferation of IL-

2-dependent Cultured Cell Line. 4E8, an IL-2-dependent cell line, was

used for this assay. Aliquots (0.2 ml) of the 4E8 cell suspension (1 x105/ml) in RPMI Medium 1640 with 10% heat-inactivated fetal bovine

serum were distributed into wells of a 96-well flat-bottomed microtiter

plate (Costar, Cambridge, MA). After preincubation with various antibodies for 30 min at room temperature, partially purified IL-2 was added at

a final concentration of 10% (v/v). Triplicate cultures were incubated for72 hr at 37Â°in 5% C02 in air in a humidified incubator. Six hr beforeharvest of culture, 0.4 ÃŸC\of [mef/?y/-3H]thymidine (New England Nuclear,

Boston, MA) was added to each well. Radioactivity of the cultured cellswas measured by a liquid scintillation counter (Beckman, Fullerton, CA).Results are expressed as percentage of inhibition of [3H]thymidine incor

poration as follows:

4 Y. Koide and T. O. Yoshida, submitted for publication.

% of inhibition =

/ cpm of sample - cpm of medium control \

\ cpm of IL-2 alone - cpm of medium control/

Immunoprecipitation Procedures. Appropriate cultures cells weremetabolically labeled with [3H]glucosamine (New England Nuclear) asdescribed previously (25) or with '25I using lodogen (Pierce Chemical,

Rockford, IL). A glass vial was coated with 100 ^g of lodogen that hadbeen dissolved in 100 n\ of mÃ©thylÃ¨nechloride. The solvent was evaporated under a stream of nitrogen. Twenty to 100 million cells resuspendedwith 1 ml of Dulbecco's phosphate-buffered saline (pH 7.4) were added

to the vial, followed by 1 mCi of Na125l(New England Nuclear). The vial

was gently agitated for 20 min at room temperature, then the mixturewas transferred into 10 ml of cold phosphate-buffered saline to stop thereaction. The cells labeled with [3H]glucosamine, or 125Iwere solubilized

with 0.5% Nonidet P-40, and immunoprecipitation was carried out as

described (25).For sequential immunoprecipitation, lysates of MT-2 cells were treated

with a saturating amount of Ta60b or Tac antibody which had beenreacted with the rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup,Denmark) prereacted on formalin-fixed Staphylococcus aureus. After

centrifugation, each supernatant was treated again with a saturatingamount of Ta60b or Tac antibody and an excess of rabbit anti-mouse

immunoglobulin. Precipitates were recovered with S. aureus and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Binding Inhibition Assay. [3H]Leucine (New England Nuclear) labeling

of Ta60a and Ta60b antibodies and binding inhibition assays were carriedout as described elsewhere (19).

RESULTS

Production of Ta60a and TaGOb Monoclonal Antibodies.Two hybridoma clones were established from the fusion betweenNS-1 cells and the spleen cells obtained from the mice immunizedeither with HTLV-rich fraction from MT-2 cells (clone Ta60a) orwith MT-2 cell line (clone Ta60b). Both Ta60a and Ta60b antibodies were found to have 7"! -heavy chain, and the antibody

activity was detected either by M-MHA or by membrane immu-

nofluorescence. The results with Ta60b antibody will be described because of their similar specificity.

Reactivity of Ta60b Antibody Tested against Culture CellLines. The serological reactivity against cultured cell lines by M-

MHA was shown in Table 1. Ta60b antibody reacted stronglywith the ATL-CTCL cell lines up to the dilution of 1/10", except

Table 1Reactivity of Ta60b monoclonal antibody against cultured cell lines by M-MHA

assay

The other antibody, TaSOa, showed an almost identical reactivity.

Positive cell Negative cell

ATL/CTCL-related ATL/CTCL-relatedMT-2 (HTLV) HUT-78 (CTCL)"

MT-1 (ATL) T-cellATN-1 (ATL) RPMI-8402 (T-ALL), CCRF-CEM (T-ALL), MOLT-3 (T-HUT-102 (CTCL) ALL), Jurkat (T-ALL), HPB-ALL (T-ALL)

B-cellIL-2 dependent Daudi (Burkitt), Wa (EBV), LICR-LON-HMy2 (EBV),

NK-3.3 BALL-1 (B-ALL), SK-Ly-16 (B-lymphoma), RPMI-4E8 8226 (myeloma)

Non-T, non-BB-cell NALL-1 (null-ALL), NALM-6 (null-ALL)

Raji (Burkitt) Myelo-MonoRPMI-1788 (EBV) HL-60 (APL), K-562 (CML-BC)

Walk (EBV)* No HTLV production was observed, but one proviral DNAcopy was contained

(32).


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HUT-78 line derived from CTCL, which does not produce HTLV(29). It reacted also with 2 IL-2-dependent cell lines, NK3.3 and4E8, but not with the cell lines derived from T-cell acute lympho-cytic leukemia. Three of 9 B-cell lines, Raji, RPMI 1788, andWalk, were also reactive, but less so than against the ATL-CTCL

cell lines. None of the solid tumor cell lines tested was positive(data not shown).

Reactivity of Ta60b Antibody Tested against Normal He-matopoietic Cell Populations. The M-MHA reactivity against

various lymphoid populations is shown in Table 2. None of the12 PBL samples showed any positive reactions with Ta60bantibody. However, PBL cultured either with IL-2, phytohemag-

glutinin, or Con A for 3 days displayed positive reactions withTaSOb antibody. Antigen-positive cells detected by this antibody

were not present on other normal hematopoietic cell populations.Thus, Ta60b antibody was found to react predominantly withactivated PBL.

Blocking Effect by Ta60a and Ta60b Antibodies on Proliferation of 4E8 IL-2-dependent Cell Line. The experiments were

carried out to determine whether these antibodies could inhibitthe proliferation of an IL-2-dependent cell line, 4E8, and one of

the results was presented in Chart 1. Generally, Ta60a antibodyshowed more than 70% inhibition of [3H]thymidine incorporation

at the concentration of 10 to 100 Mg/ml, whereas Ta60b antibodydid not.

Reactivity of Ta60b Antibody Tested against MalignantHematopoietic Cells. Leukemia and lymphoma cells collectedfrom 104 patients were tested (Table 2). All ATL cases showedthe reactivity with Ta60b antibody; two-thirds of the casesshowed strong reactivity (i.e., titer of more than 105 by M-MHA),

whereas the rest showed weak reactivity. No difference in reactivity was observed between fresh and short-term cultured cells

(data not shown). This antibody exhibited no significant reactivityagainst other T-cell cancers except one T-cell chronic lympho-cytic leukemia case, which was OKT3+, 4~, 8* phenotype. How

ever, Ta60b antibody reacted with sporadic cases among cancers other than T-cell origin. Three of 12 AML and 2 of 5 CML-

TaWe2Reactivity of Ta60bantibody tested against normal and malignant hematopoietic

cells by M-MHA assay

Normal hematopoieticcellsCell

typePBLCultured

withMedium"aloneIL-2PHAConAMonocyteGranulocyteThymocyteBone

marrowcellSpleencellNo.tested161286644633No.positive0086600000Malignant

hematopoieticcellsCell

typeATLT-CLLCT-ALLLLTrlymphomaB-CLLB-lymphomaNull-ALLCML-BCAMLAMoLNo.tested181*975912185128No.positive11(7)"0(1)0000(3)0(4)1(2f2(2f3(0)'0

* RPMI medium 1640 containing 10% FBS and IL-2, PHA, or Con A for 3 days.6 Numbers in parentheses,showed weakly positive, i.e., M-MHA titer, 1/103to

1/104.c T-CLL, T-cell chronic lymphocytic leukemia;AMoL, acute monocytic leukemia;

T-ALL, T-cell acute lymphocytic leukemia; PHA, phytohemagglutinin; LL, lympho-blastic lymphoma;T2,mature T-cell; B-CLL, B-cell chronic lymphocytic leukemia.

"OKT3+, 4-, 8+T-CLL.8These Ta60b-positive cases were included in common ALL antigen (NL-1)

positive group.' These Ta60b-positivecases were not reactive with B1 antibody.

0.1 1 10 100

Purified Antibody Added ( pg/ml )

Chart 1. Blocking effects of Ta60a and Ta60b antibodieson proliferationof 4E8IL-2-dependent cell line. 4E8 cells were treated with various concentrations ofTa60a (O) or Ta60b (A) antibody and cultured for 3 days in the presence of IL-2.Incorporationof [3H]thymidineby each culture was measured.Data are expressedas percentageof inhibitionof the response obtained in culture receiving IL-2 alone(4666 Â±137 cpm). Bars, S.D.

80

3o>>

I40

Unabsorbed

80

40

Unabsorbed

AML(KX)

1/4 1/64i

â€¢AMLW08)AMLÃ•231) /MT"2

/ATU631)/

1/4 1/16 1/64Dilution of Ta60b Antibody after Absorption ( xlO' )

Chart 2. Absorption tests with Ta60b antibody. Fifty Mlof 1/10" diluted Ta60bantibody were absorbed with 2 x 107of various hematopoietic tumor cell lines (A)or cryopreserved cells (8). Then the residual reactivity was tested against MT-2target cells.

BC were positive. One-third of B-cell chronic lymphocytic leukemia and B-cell lymphoma reacted weakly with Ta60b antibody.

The reactivity of Ta60a and Tac antibodies was also testedagainst all these samples, and the results were concordant withthose with Ta60b antibody.

In order to confirm the presence of Ta60b antigen on cells ofother than T-cell lineage, absorption tests were performed. Asillustrated in Chart 2, 2 AML cases and 2 B-cell lines, Raji and

RPMI 1788, removed the reactivity of Ta60b antibody againstMT-2 cells.

Immunoprecipitation of Ta60a and TaGOb Molecules. MT-2, MT-1, ATN-1, and HUT-102 cell lines as well as Con A-stimulated PBL were labeled with [3H]glucosamine, and immu-

noprecipitation experiments were carried out. Ta60a and Ta60bantibodies precipitated a M, 60,000 glycoprotein from NonidetP-40 lysates of MT-2, MT-1, or ATN-1 cells, and a M, 53,000


1316




molecule from HUT-102 line as illustrated in Fig. 1 (data withMT-1 and ATN-1 not shown). In the case of HUT-102 cells, a

weaker band with a molecular weight of approximately 60,000was generally observed. Ta60a and Ta60b antibodies werefound also to precipitate a molecule of approximately M, 60,000from Con A-stimulated PBL (data not shown). Immunoprecipita-tion with these 4 cell lines labeled with 125Idisplayed results quitesimilar to those with [3H]glucosamine-labeled cells.

Five ATL, 2 AML, and 2 null-ALL samples were selected from

the leukemias which were positive with Ta60a and Ta60b antibodies and labeled with 125I,and then immunoprecipitation with

Ta60b was carried out. One of the representative experimentswas illustrated in Fig. 2. The immunoprecipitates were observedwith 3 ATL, one AML, and 2 null-ALL cases. The molecular

weight of all these samples was approximately 60,000, similarto that of Con A blasts. Similar results were also obtained withimmunoprecipitation with Ta60a antibody (data not shown).

Sequential Immunoprecipitation. The results so far obtainedsuggested that Ta60a and Ta60b antigens are quite similar to

ABCa b a b a b

abc

92.5-

69-

46-

30-

1Fig. 1. Autoradiogram of radiolabeled proteins from NP-40 lysates of MT-2 and

HUT-102 cell lines immunoprecipitated by Ta60a, Ta60b, or Tac antibody andanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (reducedsamples). ['HJGIucosamine-labeled MT-2 (a) and HUT-102 (b) cells were immuno

precipitated with either Ta60a (A), Ta60b (B), or Tac antibody (C). The moleculeimmunoprecipitated from MT-2 was M, 60,000 (upper arrow), whereas the onefrom HUT-102 was 53k (lower arrow). Molecular weight markers: phosphorylaseb. M, 92,500; bovine serum albumin, M, 69,000; ovalbumin, M, 46,000; and carbonicanhydrase, M, 30,000.

92.5-69-

46-

30-

Fig. 2. Autoradiogram of radiolabeled proteins from NP-40 lysates of 3 leukemiasamples immunoprecipitated by TaSOb antibody and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (reduced samples). 125l-Labeled ATL(Case 1045; a), AML (Case 231; b), and null-ALL (case 516; c) samples were

immunoprecipitated with Ta60b antibody. Arrow, M, 60,000. For molecular weightmarkers, see legend of Fig. 1.

each other, and that the Tac antigen reported by Uchiyama efal. (23, 24) has similarities to the Ta60 antigen group. Accordingly, the relationship among these antigens was investigated:(a) Tac antibody was found to Â¡mmunoprecipitate molecules withalmost identical mobility to Ta60a or Ta60b antigen from [3H]-glucosamine-labeled MT-2 and HUT-102 cells in sodium dodecylsulfate-polyacrylamide gel electrophoresis (Fig. 1); and (b) bysequential immunoprecipitation (Fig. 3), Tac antibody was ableto immunoprecipitate Ta60b molecule, resulting in a faint precipitating band, when the MT-2 lysate pretreated with Tac antibody

was immunoprecipitated with Ta60b antibody. Ta60b antibodyalso removed Tac antigen molecule. The results indicated thatTa60b and Tac epitopes are on the same molecule on MT-2cells.

Binding Inhibition of 3H-labeled TaSOa or Ta60b Antibody.

For the analysis of antigenic determinants defined by each antibody, antibody-binding inhibition assays were carried out (Chart3). Tac antibody showed significant inhibition of the binding of[3H]leucine-labeled Ta60a antibody to MT-2 target cells, whereas

TaÃ©obantibody did not. When labeled Ta60b antibody was used,neither Ta60a nor Tac antibody evidenced any inhibition. Theresults suggested that Ta60a epitope was different from Ta60bbut identical to Tac. Thus, Ta60a/Tac and Ta60b epitopes weredemonstrated to be present on the same molecule (1.9., IL-2

receptor).

DISCUSSION

From analysis of monoclonal antibodies against MT-2 cells orHTLV-rich fraction from MT-2 cells, 2 antigens, Ta60a and


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A Babc abc

92.5-69-

46-

30-

I

Fig. 3. Sequential immunoprecipitation of Ta60b and Tac antigens. NP-40 ly-sates of 125l-labeledMT-2 cells were treated twice with insolubilized NL-12 antibodydetecting la-like antigen (27) (a; negative absorption control), Ta60b (6), or Tac (c)antibody. Then, each supernatant was recovered and immunoprecipitated withTa60b (A) or Tac antibody (B). Arrow, immunoprecipitate from MT-2 cells with a

molecular weight of 60,000. For molecular weight markers, see legend to Fig. 1.

xlO3Â«K)3g1I

6i0~

4aI

2I8in*642n

._LiTn*NMSTo60oTa60b Toc NMS Ta60a 10606TacpH]Ta60a

pH]Ta60b

Chart 3. Binding inhibition of 3H-labeled Ta60a and Ta60b antibody by monoclonal antibodies. Fifty Â¡Â¿of MT-2 cell suspension (2 x 107/ml) were incubated with

50 n\ of monoclonal antibodies (1 mg/ml) or nonimmune IgG prepared from normalmouse serum (/VMS) (1 mg/ml) at 4Â°for 60 min. After the incubation, 50 nl of[3H]leucine-labeled TaÃ¨Oa(A) or Ta60b antibody (B) were added to each tube andincubated again for another 60 min at 4Â°.The cells were then washed 4 times, and

the radioactivity bound to the cells was counted. Bars, S.D.

Ta60b, with similar antigen distribution and almost the samemolecular weight, were defined, probably because Ta60a membrane antigen was contaminated in HTLV preparation used forthe present experiments. It was demonstrated by sequential

immunoprecipitation and antibody binding inhibition assays thatTa60a and Tac are the same epitope, but are different fromTa60b epitope on the same antigen molecule. Since severalevidences suggested that Tac antigen is present on IL-2 receptoron activated T-cells (3,4,9,10,18), there are at least 2 epitopes,

Ta60a/Tac and Ta60b, on this molecule. In this connection, it isnoted that only Ta60a antibody, but not Ta60b antibody, wasable to inhibit the proliferation of an IL-2-dependent cell line in a

similar manner to Tac antibody (10), suggesting that Ta60a/Tacepitope is involved in immunobiological function of IL-2 receptor,

whereas Ta60b epitope is not.One of the interesting observations with immunoprecipitation

was that both TaGOaand Ta60b antibodies precipitated antigenswith 2 different molecular weights, I.e., M, 60,000 from MT-2,MT-1, or ATN-1 cells, and Mr 53,000 from HUT-102 cells. The

results by Leonard et al. (9) and the present finding that Ta60a/Tac and Ta60b on Con A blasts have a molecule of approximatelyM, 60,000 suggested that HUT-102 cells might have an abnormalIL-2 receptor molecule due to malignant transformation by HTLV

infection. However, it is probable that the results reflect theheterogeneity in molecular weight of IL-2 receptor. In this re

spect, Yodoi ef al. (30) proposed an attractive hypothesis thatthe unregulated or "constitutive" expression of IL-2 receptor is

primarily responsible for the unrestricted proliferation of ATL andCTCL cells, by demonstrating that the physiological regulationof IL-2 receptor on ATL-CTCL cells is disturbed. This hypothesis,however, may not explain all cases of ATL-CTCL, because HLTT-

78 CTCL line, which was shown to contain one proviral DMAcopy of HTLV (29), did not react with Ta60a or Ta60b antibody.A detailed biochemical analysis of IL-2 receptor molecule de

tected either by Ta60a or by Ta60b antibody on ATL cells andnormal activated T-cells might provide clues to determinewhether dysregulation of IL-2 receptor caused by HTLV infectionmay result in ATL-CTCL (1, 30).

The analysis of reactivity against various leukemias and lym-phomas revealed that, among T-cell cancers, ATL cases werereactive with Ta60b antibody. Three AML cases, 2 CML-BCcases, and 3 B-cell lines showed also weak, but definite reac

tions, and a part of the results with direct tests was confirmedby absorption tests and immunoprecipitation experiments. Kors-

meyer et al. (8) reported recently the presence of Tac antigen onB-cell-type hairy cell leukemia cases, which were positive withB1 antibody detecting B-cell differentiation antigen, and suggested that IL-2 receptor may be expressed on normal B-cells

at certain stages of development. This might be supported byour present finding that 4 positive CML-BC cases (including 2weakly positive ones) and one positive null-ALL case werereactive with NL-1 antibody detecting common ALL antigen (27),which is regarded as a pre-B-cell marker (8). However, the 3AML cases studied belong to M1 group by French-American-British classification (2), and they did not react with B1 or NL-1

antibody. Further, Ta60b antibody immunoprecipitated a M,60,000 antigen molecule from one of 2 cryopreserved AMLsamples. Preliminary experiments, however, revealed that IL-2

did not stimulate to Ta60 positive AML samples so far examined.There are at least 3 possibilities to explain this unexpectedobservation: (a) Ta60b antibody might react with IL-2 receptoras well as other molecules which are not IL-2 receptor and areexpressed on cells of other than T-cell lineage; (b) IL-2 receptoritself is present on a certain population of cells other than T-cells


1318




and it functions as an IL-2 receptor; and (c) IL-2 receptor appeared abnormally on malignant cells of other than T-cell lineage

as a result of transformation. At this moment, it is very difficultto determine which possibility is most likely. However, recently,IL-2 molecule produced by gene engineering is available (1, 21),and the use of well-characterized IL-2 molecule as such might

serve to elucidate whether the molecule detected by Ta60b orby Ta60a antibody on cells of other than T-cell lineage mightfunction as IL-2 receptor. It is also very important to obtain genecoding IL-2 receptor molecule by use of these monoclonal anti

bodies in order to understand the immunobiological function ofIL-2-IL-2 receptor system as well as the pathogenesis of ATL-

CTCL.

ACKNOWLEDGMENTS

We wish to thank the following investigators for providing cultured cell lines: Dr.L. J. Old and Dr. B. Dupont of Memorial Sloan-Kettering Cancer Center, New York.

NY; Dr. R. C. Gallo of National Cancer Institute, Bethesda, MD; and Dr. T. Naoeand Dr. H. Shiku of Nagoya University School of Medicine, Nagoya, Japan. Theexcellent technical assistance of R. Moriwaki, Y. Matsudaira, M. Noritake, and K.Mizutani is also acknowledged.

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1985;45:1314-1319. Cancer Res Ryuzo Ueda, Keiko Nishida, Yukio Koide, et al. Leukemia CellsEpitopes of an Activated Lymphocyte Antigen on Adult T-Cell Two Mouse Monoclonal Antibodies Detecting Two Different

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