Proc. Nati. Acad. Sci. USAVol. 80, pp. 7337-7341, December 1983Medical Sciences

Detection of lymphocytes producing a human retrovirus associatedwith adult T-cell leukemia by syncytia induction assay

(cat cell line/human T-cell leukemia virus/neutralizing antibody/Southern blot hybridization/virus transmission)

HIROo HOSHINO*, MASANORI SHIMOYAMAt, MASANAO MIWA*, AND TAKASHI SUGIMURA**Virology Division, National Cancer Center Research Institute, and tDepartment of Internal Medicine, National Cancer Center Hospital, Tsukiji, Chuo-ku,Tokyo 104, Japan

Contributed by Takashi Sugimura, August 25, 1983

ABSTRACT Recently 10 T-cell lines were established frompatients with adult T-cell leukemia (ATL). During establishmentof these cell lines, it was found that when T-cell lines expressingthe ATL-associated retroviral antigen were cocultivated with 8Ccat cells, multinucleated syncytia were formed. Retroviral anti-gen-negative T-cell lines did not induce syncytia. Peripheral bloodlymphocytes obtained from ATL patients did not express the ret-roviral antigen before cultivation in vitro but became positive forthe retroviral antigen after cultivation for a short period; theseretroviral antigen-positive lymphocytes, but not retroviral anti-gen-negative lymphocytes, induced syncytia upon cocultivation with8C cells. Peripheral blood lymphocytes isolated from patients withchronic lymphocytic leukemia of T-cell origin or Skzary syndromeor from normal adults and lymph node cells from a patient withimmunoblastic lymphadenopathy-like T-cell lymphoma did not ex-press the retroviral antigen even after cultivation in vitro and didnot induce syncytia upon cocultivation with SC cells. Thus, therewas complete agreement between the presence of the retroviralantigen in established T-cell lines or freshly isolated peripheralblood lymphocytes and their ability to induce syncytia. Syncytiaformation was enhanced 5- to 20-fold by the presence of Poly-brene and inhibited by addition of plasma of ATL patients to thecocultures. Syncytia were detected within 4 hr on cocultivation of8C cells with the retroviral antigen-positive T-cells, indicating thatmost syncytia were formed by early polykaryocytosis. After co-cultivation, a clone of 8C cells that harbored the AU virus ge-nome and had syncytia-inducing activity was isolated. These find-ings indicate that the retrovirus associated withAU has syncytia-inducing activity. Syncytia induction assay using 8C cells will beuseful for detection and characterization of human retrovirus as-sociated with T-cell malignancies.

Retroviruses have been found to be closely associated with hu-man T-cell malignancies (1). Human T-cell leukemia virus (HTLV)was isolated from patients with mycosis fungoides (2) and SezaryT-cell leukemia (3). A retrovirus is also associated with Japanesepatients with adult T-cell leukemia (ATL) and was named ATLvirus (4-6). There seems to be slight heterogeneity even amongATL viruses isolated in Japan-that is, analyses of nucleotidesequences of ATL viruses integrated in MT-1 cells (7) and inlymphocytes of an ATL patient (8) showed the presence of sev-eral base changes in the long terminal repeats of these two vi-ruses. Patients having ATL are clustered in southwestern Japanand the West Indies (1, 4, 9, 10). HTLV has also been detectedin patients from different parts of the world (11). Although thegeographical and racial distributions of HTLV-i.e., HTLV-I-and ATL virus were quite different, these two viruses are closelyrelated or identical (12-14). Another HTLV, HTLV-II, was iso-lated from a patient with hairy cell leukemia of T-cell origin

(15). Furthermore, a possible link was suggested between ac-quired immune deficiency syndrome (AIDS) and HTLVs (16-19). Differences have been found in HTLVs detected in T cellsof patients with AIDS: one isolate was identical to HTLV-I (18),whereas another isolate was different from HTLV-I or HTLV-11 (19). Thus, there are several closely related retroviruses thatinfect human T cells.

Several retroviruses isolated from mice, cats, monkeys, cat-tle, and a human induce formation of multinucleated syncytiaof certain indicator cells, and syncytial plaque assays have beenused for detection and characterization of these viruses (20-27).We tested whether ATL virus also induced syncytia of someindicator cells. We found that a cat cell line, 8C (28), formedsyncytia quite efficiently when these cells were cocultivatedwith T cells expressing the retroviral antigen associated withATL. Several lines of evidence indicated that syncytia were in-duced by ATL virus. Thus, it may be possible to detect andcharacterize retroviruses associated with human T-cell malig-nancies or AIDS by syncytia induction assay using 8C cells.

MATERIALS AND METHODSCells. Cells were obtained from the 10 patients with ATL

described elsewhere (6). Seven T-cell lines, ATL-1K, ATL-2M,ATL-3I, ATL4K, ATL-5S, ATL-6A, and ATL-7S, establishedfrom 7 ATL patients and peripheral blood lymphocytes (PBL)obtained from the other 3 ATL patients, KY, SY, and FK, weremaintained in vitro as described (6). PBL were also obtainedfrom patients with chronic lymphocytic leukemia of T-cell or-igin (T-CLL) and Sezary syndrome and from normal adults.Lymph node cells were obtained from a patient with immu-noblastic lymphadenopathy-like T-cell lymphoma (IBL-like T-L). Most of these cells had surface markers for suppressor/cy-totoxic T-cells. The lymphocytes were cultivated in RPMI 1640medium containing 20% fetal calf serum and an appropriateamount of T-cell growth factor. 8C cells were a clonal cell linederived from a feline kidney fibroblast line, CCC, and weretransformed by Moloney murine sarcoma virus (28). 8C cellswere obtained from H. Yoshikura (Tokyo University, Tokyo)and maintained in Eagle's minimal essential medium (EMEmedium) supplemented with 10% fetal calf serum. Epstein-Barrvirus-transformed lymphoblastoid cell line XPL- 15 (29) wasmaintained in RPMI 1640 medium containing 10% fetal calfserum.

Syncytia Induction Assay. 8C cells were seeded into 35-mmplastic plates (Falcon 3001) at 2-4 x 105 cells per plate in EME

Abbreviations: AIDS, acquired immune deficiency syndrome; ATL, adultT-cell leukemia; HTLV, human T-cell leukemia virus; IBL-like T-L, im-munoblastic lymphadenopathy-like T-cell lymphoma; PBL, peripheralblood lymphocytes; T-CLL, chronic lymphocytic leukemia of T-cell or-igin.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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medium containing 10% fetal calf serum and Polybrene (0-10jg/ml) (Sigma). The following day, T-cell lines established from

ATL patients were irradiated at 10,000 rads (6°Co) (1 rad = 0.01gray) in some experiments and were added at 2 x 104-2 X 106cells per plate to duplicate plates containing 8C cells. Freshlymphocytes or lymphocytes maintained in vitro for a short pe-riod were added without irradiation. One to three days later,the cells were fixed with methanol and stained with Giemsa.Numbers of syncytia containing >10 nuclei were counted un-der an inverted microscope. 8C cells rarely form syncytia spon-taneously, and almost all spontaneous syncytia in 8C cells con-tain <5 nuclei.

Immunofluorescence Assay. Lymphocytes expressing theretroviral antigen associated with ATL were detected by in-direct immunofluorescence assay and antibody titers of plasmaspecimens against the retroviral antigen were determined asdescribed (6).

Inhibition of Syncytia Induction by Plasma. Plasma speci-mens were obtained from a normal adult and patients with T-CLL, IBL-like T-L, and ATL and were heated for 30 min at560C before use. 8C cells were seeded into 24-well plates (Fal-con 3047) at 1 X 105 cells per well in EME medium containing10% fetal calf serum and Polybrene (2 .g/ml). The followingday, the medium was replaced by fresh medium, which con-tained 10% human plasma and had been filtered through a Mil-lipore filter (0.45 um). Then 'Co-irradiated ATL-2M or ATL-7S cells (1 x 1 cells) were added to each well. The cells werecocultivated for 3 days and then were fixed with methanol andnumbers of syncytia were counted as described above.

Southern Blot Hybridization. Cellular DNA was isolated, di-gested with EcoRI, and transferred to nitrocellulose filters. Thefilters were hybridized with nick-translated probes specific forATL virus or human Alu fimily DNA as described (6). 3P-La-beled, nick-translated probes that represented >80% of the ATLvirus genome (7, 8) were kindly supplied by M. Yoshida (Can-cer Institute, Tokyo). 32P-Labeled BLUR8 plasmid DNA thatcontained human Alu family DNA (30) was kindly supplied byT. Sekiya (this institute).

RESULTSInduction of Syncytia by Established T-Cell Lines. Seven T-

cell lines, ATL-1K, ATL-2M, ATL-3I, ATL-4K, ATL-5S, ATL-6A, and ATL-7S, harbored the ATL virus genome and had sur-face markers for an inducer/helper subset of T cells. All of thesecell lines except the ATL-1K cell line expressed the retroviralantigen and produced ATL virus as described (6). We noticedthat all of these lines except the ATL-1K cell line induced mul-tinucleated syncytia when they were cocultivated with 8C catcells (Fig. 1). Numerous syncytia were formed after coculti-vation for 1-5 days when viral antigen-positive ATL-2M, ATL-31, ATL-4K, ATL-5S, ATL-6A, and ATL-7S cells, but not viralantigen-negative ATL-1K cells, were assayed (Table 1). Duringcultivation, ATL-6A cells gave a negative reaction for the viralantigen for 4 months and then began to express the viral antigenas described (6). Viral antigen-negative ATL-6A cells did notinduce syncytia. Another four human cell lines, Molt-3 derivedfrom a case of acute lymphocytic leukemia (31), HL-60 from acase of promyelocytic leukemia (32), Raji from a case of Burkittlymphoma (33), and XPL-15 (29), did not induce syncytia. Thus,all of the retroviral antigen-positive cells, and only these cells,induced syncytia when they were cocultivated with 8C cells.ATL-2M or ATL-7S cells induced large syncytia containing

20-50 nuclei, whereas ATL-31 or ATL-5S cells induced smallsyncytia containing 10-20 nuclei. The nuclei in syncytia were3-4 times larger than those of ATL cell lines, indicating thatmost nuclei in syncytia were of 8C cell origin.

Polybrene is reported to enhance adsorption of retroviruses(34). Addition of Polybrene (0.4-10 Ag/ml) to the culture me-dium of 8C cells increased the numbers of syncytia induced byATL-5S or ATL-7S cells 5-20 times (Fig. 2). This finding sug-gests that ATL virus is involved in syncytia induction.

Next the time course of development of syncytia was studied(Fig. 3). Syncytia containing >10 nuclei were detected within4 hr on cocultivation of 8C cells with WCo-irradiated ATL-7Scells, and their number usually reached a maximum on cocul-tivation for 1-3 days. The number of syncytia decreased grad-ually with increase in cell passages. Thus, most of these syn-

FIG. 1. Multinucleated syncytia induced by cocultivation of 8C cat cells with irradiated (10,000 rads) ATL-2M cells. After cocultivation for 1day, cells were fixed with methanol and stained with Giemsa. (x365.)

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Table 1. Syncytia induction by T-cell lines established fromATL patients

Viral antigen-Cell line positive cells, % Syncytia, no.*ATL-1K <1 <5ATL-2M 80 740ATL-3I 7 8ATL-4K 70 200ATL-5S 50 44ATL6A 30 280ATL-7S 70 960

Each cell line was tested for expression of the retroviral antigen as-sociated withATL as described (6). Irradiated (10,000 rads)T cells wereoverlaid onto 8C cells seeded i day before. Numbers of syncytia con-taining >10 nuclei were counted after cocultivation for 3 days.* Number of syncytia induced by 1 x 10' T cells.

cytia seemed to be formed by early polykaryocytosis or cell fusion-from-without, not by replication of ATL virus in 8C cells.

Syncytia Induction by PBL of ATL Patients. We testedwhether freshly isolated PBL or lymphocytes maintained in vi-tro for a short period would induce syncytia upon cocultivationwith 8C cells (Table 2). PBL of three ATL patients, KY, FK,and SY, contained atypical cells at frequencies of >50%. ThesePBL did not express the retroviral antigen before cultivationbut became positive for the viral antigen after cultivation for 1-40 days as described (6). When fresh PBL isolated from ATLpatient KY were overlaid onto 8C cells, only small syncytia con-taining 4 or 5 nuclei were observed after 1 day, but numerouslarge syncytia containing >10 nuclei were detected after co-

cultivation for 3 days (Table 2). This indicated that fresh lym-phocytes became positive for the retroviral antigen during co-cultivation and accordingly induced syncytia. PBL of patientKY that had been maintained in vitro for 2 days before cocul-tivation expressed the retroviral antigen (Table 2) and inducedlarge syncytia after cocultivation for only 1 or 3 days. Lym-phocytes from ATL patients FK and SY induced syncytia onlyafter these lymphocytes became positive for the retroviral an-

tigen (Table 2). Lymphocytes obtained from patients with T-CLL(two cases), Sezary syndrome (one case), or IBL-like T-L (onecase) or from normal adults (four cases) did not express the ret-roviral antigen in vitro and did not induce syncytia of 8C cells(Table 2; data not shown). Most PBL of two T-CLL patients

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FIG. 2. Effect of Polybrene on syncytia formation. 8C cells wereseeded at 4 x 105 cells per plate in medium containing Polybrene. Oneday later ATL-5S orATL-7S cells were overlaid. After cocultivation for1 day, numbers of syncytia induced by 2 x 10' ATL-5S cells (e) or 1 x104 ATL-7S cells (o) were counted.

Time (hr)

FIG. 3. Time course of syncytia induction. 8C cells were seeded at2 x 10 cells per plate in medium containing Polybrene (2 pMg/ml). Oneday later irradiated ATL-7S cells were added. At the indicated times,cells were fixed with methanol and stained with Giemsa. Numbers ofsyncytia induced by 1 x 104 ATL-7S cells are shown.

were leukemia cells and showed surface markers for inducer/helper T cells like those of ATL patients (data not shown).However, these leukemia T cells of the T-CLL patients did notinduce syncytia. Thus, there was a complete correlation be-tween the syncytia-inducing activity of lymphocytes and theirexpression of retroviral antigen.

Inhibition of Syncytia Formation by Plasma of ATL Pa-tients. ATL patients have antibodies against the viral proteinsof HTLV or ATL virus-e.g., p19, p24, or p45 (5, 10, 13, 35,36). Therefore, we next examined whether syncytia formationwas neutralized by these antibodies. For this, the syncytia in-duction assay was carried out in the presence of plasma of ATLpatients. Table 3 shows that syncytia formation was specificallyinhibited by addition of heat-inactivated plasma of ATL pa-tients to cocultures of 8C cells and irradiated ATL-2M or ATL-7S cells. Plasma of a normal adult and of patients with T-CLLand IBL-like T-L, which did not contain antibodies against theATL-associated viral antigen, did not inhibit syncytia forma-

Table 2. Syncytia induction by lymphocytes maintained in vitrofor a short period

PBL ViralCulture antigen-

Source period, positive Syncytia,Number (patient) day cells, % no.*

1 ATL 0 <1 1,200(KY) 2 20 640

2 ATL 0 <1 <5(FK) 3 55 12

3 ATL 0-13 <1 <5(SY) 40 2 48

4 T-CLL 0-47 <1 <55 T-CLL 0-3 <1 <5

6-9 Normal 0-30 <1 <5adults

Lymphocytes from each individual were assayed for expression ofretroviral antigen and for syncytia-inducing activity. Assays were donetwo orthree times on the indicated days or during the indicated periodsof cultivation. PBL were overlaid onto 8C cells seeded in medium con-taining Polybrene (2 jig/ml) 1 day before. Numbers of syncytia werecounted after cocultivation for 3 days.* Number of syncytia induced by 1 x 10' lymphocytes.

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Table 3. Inhibition of syncytia induction by plasma ofATL patients

Plasma Syncytia, no.tSource (patient) Antibody titer* ATL-2M ATL-7SMedium alone 740 300Normal adult <10 560 300TCLL <10 520 280IBL-like T-L <10 720 200ATL.(FK) 40 300 240ATL (SK) 80 12 20ATL (KI) 160 8 4ATL (IS) 160 12 <2ATL (YS) 320 4 2

8C cells and irradiated (10,000 rads) ATL-2M cells or ATL-7S cellswere cocultivated in the presence of 10% human plasma. Three dayslater cells were fixed with methanol and numbers of syncytia werecounted.*Antibody titers against the retroviral antigen expressed in ATL-2Mcells were determined by indirect immunofluorescence assay (6). Re-ciprocals of end points of plasma dilution are shown.tNumber of syncytia induced by 1 x 10 ATL-2M or ATL-7S cells.

tion. These results suggest that ATL virus is involved in syn-.cytia formation.

Southern Blot Hybridization. 8C cells were seeded into Fal-con 3001 plates, and the following day irradiated ATL-2M cellswere added. After three cell passages, these 8C cells were clonedtwice in microtiter plates (Falcon 3042). One clone, c77, in-duced syncytia efficiently when overlaid onto 8C cells (Table4). c77 cells did not form syncytia with >10 nuclei sponta-neously. Southern blot hybridization analyses showed that c77cells contained the ATL virus genome (Fig. 4) but not the hu-man Alu family DNA, which can be detected with BLUR8 probe(data not shown). 8C cells and Epstein-Barr virus-transformedXPL-15 lymphoblastoid cells did not contain the ATL virus ge-nome. When two human cell lines, ATL-2M and XPL-15, wereexamined with the BLUR8 probe, hybridization was detectedall along the lanes. Thus, the syncytia-inducing activity of ATL-2M cells and ATL virus in ATL-2M cells were both transmittedto 8C cells, and human DNA containing Alu family DNA wasnot necessary for syncytia induction by c77 cells. These findingsindicate that the syncytia-inducing activity of the retroviral an-tigen-positive T cells was coded for by ATL virus and probablynot by human DNA.

DISCUSSIONWe found that syncytia were induced when 8C cat cells werecocultivated with six retroviral antigen-positive T-cell lines orfreshly isolated PBL from ATL patients that expressed the ret-roviral antigen (Tables 1 and 2). This formation of syncytia wasenhanced by treatment with Polybrene (Fig. 2) and was spe-cifically inhibited by plasma of ATL patients (Table 3). After

Table 4. Syncytia induction by clone c77 of 8C cells

Indicator Syncytia, no.*cells c77 None8C 3,300 <5c77 <5 <5

8Cand c77 cells wereeeeded at 2 x 105 cells perplate into 35-mmplates in medium.containing Polybrene (2 yg/ml). One day later c77cells were overlaidontothem in duplicate cultures. Afteran additional2-days, all plates were fixed with methanol and numbers of.syncytiacontaining >10 nuclei were counted.* Number of syncytia induced by 1 x 10' c77 cells or without overlayof c77 cells.

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FIG. 4. Detection oftheATLvirus-genome in the cat cell clone c77.Cellular DNA (5 ug) from each cell line was digested.with EcoRI andassayed as described (6). Lane 1, ATL2M; lane 2, XPL-15; lane 3, c77clone of 8C cells; and lane 4, 8C. kb, Kilobase pairs.

cocultivating 8C cells with irradiated ATL-2M cells, we isolateda clone of 8C cells, c77, that induced syncytia when it was over-laid onto 8C cells (Table 4). This clone contained the ATL virusgenome (Fig. 4) but not human Alu family DNA. These find-ings indicate that the retrovirus associated with ATL has syn-cytia-inducing activity. Thus far we have not succeeded in in-ducing syncytia by exposing 8C cells to cell-free concentratesof culture fluid of ATL-2M, ATL-4K, or ATL-7S cells or to cell-free homogenates of these cells. Transmission of ATL virus andHTLV-I to other cells has been achieved only by cocultivationwith irradiated virus-producing cells, not with cell-free mate-rials (11, 37, 38).

Viral envelope protein has been suggested to be responsiblefor the syncytia-forming activity of RD-114 cat retrovirus (39).Thus, the inhibition of syncytia formation by plasma of ATLpatients (Table 3) may possibly be due to the presence of an-tibodies against the envelope protein of ATL virus in this plasma.We found that ATL-2M or ATL-7S cells induced syncytiawhen

cocultivated with several cell lines of human, simian, murine,or feline origin-e.g., HeLa, FL, Vero, K-BALB, Fc2Lu, and8C cells. Of these lines, 8C cells were most susceptible to syn-cytia induction: the syncytia formed in 8C cells were the mostnumerous and the largest (unpublished data). Most retrovi-ruses-e.g., murine leukemia virus, baboon endogenous virus,and simian sarcoma-associated virus-induce syncytia in onlya few cell lines (20-25, 28), but, like ATL virus, bovine leu-kemia virus induces syncytia in many cell lines (26). Similaritiesbetween HTLV or ATL virus and bovine leukemia virus havealso been found in the amino acid sequences of p24 viral pro-teins (40) and the structures of the long terminal repeats of theseviruses (7). It is probable that the envelope proteins of theseviruses have similar properties, such as syncytia-forming activ-ity.

Several retroviruses have been detected in human T cells.HTLV and ATL virus are associated with cutaneous T-cell leu-kemia or Japanese and Caribbean ATL (1-6). HTLV-II was iso-lated from a patient with hairy cell leukemia (15). HTLV, whichwas similar-but not identical to HTLV-I or HTLV-II, was foundin a patient with AIDS (19). It remains to be determined whetherall of these retroviruses have syncytia-inducing activity on someindicator cells. Syncytia induction by bovine leukemia virus or

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ovine leukemia virus is crossneutralized by sera of leukemiccattle or sheep (41), indicating that these two viruses are closelyrelated. It will be interesting to test whether serum or plasmaof patients with cutaneous T-cell lymphoma-leukemia, hairycell leukemia, or AIDS inhibits syncytia formation induced bylymphocytes producing ATL virus. The syncytia induction as-say using 8C cells will be useful for clinical diagnosis and fordetection and characterization of retroviruses associated withhuman malignancies.Note Added in Proof. After submission of this paper, K. Nagy et al. (42)also reported induction of syncytia by. HTLV-I and inhibition by pa-tients' sera.

We thank Dr. M. Yoshida for supplying the nick-translated probesfor the ATL virus genome and Dr. T. Sekiya for supplying the nick-translated BLUR8 plasmid DNA. This work was supported in part byGrants-in-Aid for Cancer Research from the Ministry of Education,Culture and Science and from the Ministry of Health and Welfare ofJapan and by a Grant-in-Aid from the Society for Promotion of CancerResearch, Tokyo.

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