(CANCER RESEARCH 48. 1312-1318, March 1, 19881

ABSTRACF

A T-lymphomacell line wasestablishedfroma lymphnodebiopsyofa boy currentlyalive in completeremission.Neoplasticcells fromthisbiopsydid not growin @i1ro,whereasthey formeda progressivelygrowings.c. tumor in splenectomized and subletbally irradiated nude mice andbecame serially transplantable in splenectomized and sublethally irradiated nude mice with a stable latency time. After the fourth transplant,cells were stored in liquid nitrogen and referred to as ST-4 cells. ST-4cells display a membrane phenotype and a karyotype similar to that ofthe biopsy cells. After thawing, ST-4 cells grow both in splenectomizedand sublethally irradiated nude mice and in vitro. They do not secreteinterferon or interleukin 2, do not have natural killer activity, and do notrespond to mitogen or alloantigen stimulation. The stable features ofthese T-lymphomacells and the availability of normal autologous lymphocytes from the patient make this in vivosystem quite uniqueand ofimportance for studies in tumor immunotherapy.

INTRODUCI1ON

Athymic nude mice (nu/nu) have been used for the transplantation of various human tumors, including a few T- or B-cellleukemias (1—3).This model has provided a tool for evaluationof the effects of therapeutic agents (4, 5) and immune reactivityagainst human tumors (6) in vivo. However, hemopoietic malignancies have consistently proved difficult to transplant in vivoand there are still few reports on their serial maintenance innude mice (1).

The difficulty in serially heterotransplanting lymphatic tumors into nude mice may depend on the expression of a highdensity of membrane antigens that make them particularlysensitive to thymus-independent host reaction mechanisms (6,7). To facilitate their growth, intraocular and intracerebral (2,8) inoculations have been used to take advantage of the immunological privilege of these sites. However, these maneuvers arecumbersome and place a limit on the range of experimentalobservation. Alternatively, sublethal whole body irradiation (3,

9) and splenectomy (10) have been used to decrease host immune reactivity.

We report here the serial transplantability into S-Is nudemice of T-lymphoma cells obtained from a lymph node biopsyof a boy currently alive and in complete remission. These cellsgrow with a stable pattern in S-I nude mice. Those recoveredfrom the fourth serial transplant were extensively characterizedand preserved in liquid nitrogen. They maintain most of themarkers of the biopsy lymphoma cells. Moreover, autologous

Received8/14/87; revised I 1/23/87; accepted12/1/87.The costs of publication of this article were defrayed in part by the payment

of pagecharges.Thisarticlemustthereforebe herebymarkedadvertisementinaccordancewith 18 U.S.C. Section 1734solely to indicate this fact.

I This work was supported by grants from Ric. Finaliz. Reg. Piemonte,

National Research Council, Italy (PF No. 86.00414.44), Comit. Piem. “G.Ghiroui,―andItalianCancerResearchAssociation.

2 To whom requests for reprints should be addressed, at Istituto di Microbio

logia, Via Santena9, 10126Tonno, Italy.3 The abbreviations used are: S-I, splenectomized and sublethally irradiated;

IL-2, interleukin2; PBS, phosphate-bufferedsaline;HBSS, Hanks' balancedsaltsolution; PHA, phytohemagglutinin; PBL, peripheral blood lymphocytes NK,naturalkiller, IFN, interferon;MoAb, monoclonalantibody;NHL, non-Hodgkin's lymphoma FACS, fluorescence-activatedcell sorter.

lymphocytes from the patient in full remission are available.These features make this system quite unique for studies ofTlymphocyte differentiation induced by lymphokines and immune reactivity against human T-cell neoplasms.

MATERIALS AND METhODS

Case Report. A 12-year-old boy presented in June 1984 with a 2-yearhistory of asthenia and dyspnea. He had been treated for a few monthsfor pulmonarytuberculosiswithout any improvement.Then,corticosteroids were given for a presumptive diagnosis of sarcoidosis. Someshort-lived improvement was obtained. When the child was referred tothe ReginaMargherita Children's Hospital, his condition was fair.Physical examination discloseda slenderboy with no acute distress.Ahypophonetic area was present at the right pulmonary base and X-rayexamination revealed a right pleural effusion. At thoracentesis, torbidfluid was obtained. Cytological examination revealed a composite cellular population with a predominance of both small and activatedlymphocytes,histiocytes, mesothelial cells, and a fewgranulocytes. Nomicroorganisms grew from the pleural effusion. Since no definitediagnosiscouldbeobtained,a biopsywasmadeof anenlargedaxillarylymph node (Fig. 1A).The histological diagnosis was malignant lymphoma ofT-cell lineage (convoluted type) and the boy was treated witha high risk protocol. At the time of writing, he had been in completeremission for 28 months.

Histopathology. Fragments of the biopsy specimen and the tumorgrowing in mice were transferred to media for microbiological andtissue cultures. Part of the tissues was fixed in Formol-Zenker fixative(B5) and then routinely processed for histological evaluation (11).Tissue fragments from tumor grown in mice were also processed forelectron microscopy by fixing in 2.5% glutaraldehyde in PBS at 4°C.They were then washed for 2 h in PBS and postfixed in 1.33%osmiumtetroxide in PBS at 0°C,dehydrated in alcohol, transferred into toluene,and embedded in Fisher Epon 812 resin contained in Beem capsules.The resin was allowed to polymerize in a dry oven at 60°Cfor 24—36h. Thin and ultrathin sections were cut on a Porter-Blum microtomeand stained with lead hydroxide. A cell suspension was obtained byteasing the lymph node with blunt scissors and gently pressing the cellsthrough a steel mesh in HBSS. Touch preparations (imprints) werestained with May-Grunwald-Giemsa stain and for cytochemical reactions, including periodic acid-Schiff, peroxidase, acid phosphatase, anddiaminopeptidase IV (12). Slices of fresh tissue were embedded inoptimal cutting temperature compound (Miles Italiana, CavenagoBrianza, Italy), snap-frozen in —70°C-cooledisopentane and kept at—70Cuntil just prior to sectioning. Cryostat sections were air-driedfor 1 h, fixed for 5 mm in 4°Cacetone, and then immunostained usingappropriate dilutions of mouse MoAb. A peroxidase-conjugated rabbitsecondary antibody (Dakopatts, Labometrics, Milan, Italy) directedagainst mouse immunoglobulins was then applied. Single-cell suspensions oflymphoma cells obtained from the biopsy, from tumors grownin nude mice, and from in vitro cultures were also labeled by indirectimmunofluorescenceas described in detail previously(13). The following MoAb were used for both sections and cell suspensions: pan-T,pan-B, leukocyte common, and dendritic reticular cells (Dakopatts);OKT3, OKT4, OKT6, OKT8, OKT9, OKT1O,0KT26 (OrthoDingnostics, Milan, Italy); Leu-7, Leu-9 (Becton Dickinson Italia, Milan,Italy);TEC-Calla, TEC-anti-la and TEC-HLA A,B,C (Technogenetics,S. Mauro Torinese, Italy). Cell suspensions were analyzed according toimmunofluorescencereactivitywith a FACS IV flowcytometer (BectonDickinson).

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A New Childhood T-Cell Lymphoma Established in Nude Mice and in Vitro'

Roberto Arione, Cristina Jemma, Marco Forni, Cristiana Marchese, Gabriella Benetton, Cnstina Giubellino,Andrea Modesti, Maria Giovanna Martinotti, Luca Cordero di Montezemolo, Tiziana Musso, and Guido Forni2

Institute ofMicmbiolosj@ [R. A., C. J.. G. B.. M. G. M., T. M., G. F.J and DepartmentsofGenetics IC. M., C. G.Jand Pediatrics IL C. M.J. University ofTurin andDepartment ofPathology, Regina Margherita Children's Hospital, 10126 Turin (M. F.J; and Department ofExperimensal Medicine, University ofRome, 00161 Rome(A. M.J, italy

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HUMAN T-CELL LYMPHOMA GROWING IN NUDE MICE

Fig. 1. (A) Original biopsy. Lymphomacells are intermingled with lymphocytes, a fewplasma cells, and prominent epithelioid-likehistiocytes. (B) Histological and (C) ultrastructural appearanceof the neoplastic massgrownin the s.c. adipose tissue of S-I nude micefollowingthe injection ofl x l0@cells obtainedfrom lymphnodebiopsy.Lymphomacellsgiveriseto a tumorwith the typicalmorphologyofthe childhood convoluted T-cell lymphoma(33). No areasof necrosisor histiocytesarepresent.The tumor cells from the first passagein vivo (C) and ST-4 cells cultured for tenpassagesin vitro(D) maintain the sameultrastructural morphology. A and B, H & E. Original magnification:A, B, x 40; C, x 7500 D,x 3000.

In Vitro Cultures. All the in vitro cultures were maintained at 37°Cin a 5% CO2 humidified atmosphere in basic culture medium RPMI1640 supplemented with 5 x 10' M, gentamicin, L-glutamine, 10 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, and 10%heat-inactivated fetal bovine serum (all reagents were from Flow Laboratories, Opera, Italy). For electron microscopy observations, the cellsuspension was centrifuged and the pellet was processed as describedabove.

Chromosome Analysis. Leukemic cells (106) were incubated for 72 hin basic medium supplemented with 20% fetal bovine serum with orwithout 0.2 @@g/mlofPHA (purified; Wellcome Research Laboratories,Beckenham, Kent, United Kingdom),and synchronized with high dosesof thymidine. At harvest, colchicine was added for 30 mm. Hypotonictreatment was done with a 0.075 Msolution ofKCl for 10 mm at 37°C.Cells were then fixed with methanol:acetic acid 3:1 and the cell suspension was dropped on slides and air dried. Chromosomes were bandedwith trypsin (14) or Q banded (15).

Southern Blot Analysis.High molecular weight DNA extracted fromST-4 cells or from a human fibroblast line was digested overnight withBamHI restriction endonuclease. The DNA fragments were fractionated by electrophoresis on 0.8% agarose gel and transferred to anitrocellulose filter, according to the method of Southern (16). Thefilter was hybridized to a nick-translated “P-DNAprobe, which hybridizes with both genes of the constant regions of the@ chain of T-cellreceptor (400-base pair BgIII complementary DNA fragment fromJUR-@ clone) (17), and washed at high stringency. Hybridizationsignals were visualized after 3—7days of autoradiography with intensifying screens. Non-T-cell control DNA was run simultaneously toidentify the germ line position of the genes.

Mice. Six-week-oldmale normal BALB/c and Swiss nude mice werepurchased from Charles River Laboratories, Calco, Italy, and maintamed under specific-pathogen-freeconditions. They receivedsterilized

pellets and water ad libitum. Splenectomy was performed on anesthetized mice by performing a 5-mm-long incision in the left upper flank.This usually healed within 1 week. Three days before challenge, untreated mice and mice splenectomized 1 week earlier received a totalbody irradiation of 450 rads from a ‘37Cssource providing a dose rateof 50 rads/min. The splenectomized and irradiated mice are referredto hereafter as S-I mice.

Transplant of LeukemicCells in Nude Mice. A fragment ofthe lymphnode biopsy or of the tumor growing in nude mice was minced asdescribed above. The cells were filtered over sterile gauze and stratifiedover a Lymphoprep (Nyegaard, Oslo, Norway) gradient in order toisolate the living cells. Cellular suspensions obtained in HBSS (0.3 ml)were then injected s.c. in the anterior lateral thoracic wall of recipientmice. After challenge, mice were palpated twice weekly to note tumorappearance, after which neoplastic masses were measured with calipersin the two perpendicular diameters and the average value was recorded.Latency and survival time were defined respectively as the period (indays) between challenge and the growth of a neoplastic mass of 4 and14 mm mean diameter, when mice were killed for humane reasons. Thecell preparation obtained from the fourth serial transplant into S-I nudemice was divided into aliquots and preserved in liquid nitrogen. Thesecells are called ST-4 cells.

PBL. PBL were isolated from a healthy allogeneic donor (GF) orfrom the patient in complete remission by Lymphoprep density gradientcentrifugation (13). After 3 washings with HBSS, PBL were resuspended in basic medium at the specified concentrations. The patient'sPBL werestoredin liquidnitrogen.

HLA Typing.This was performed on leukemic cells and on GF PBLin accordance with the standard NIH lymphocytotoxicity techniquewith a set of sera of proved specificity. All defined specificities weretested for (18).

Cytotoxicity Assay. All in vitro experiments were performed mdc1313

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Table 1 FACSanalysisofthe surfacemarkerexpressionby lymphomacellsobtainedfromlymphnodebiopsy.by thefourths.c.transplantinS-inude

mice, and by in vitro-grown ST-4cells%

of positive cellsfromCluster

of differentiation The fourths.c. ST-4 grownandMoAb Biopsy transplant° invitrobCDI

(OKT6) 75 9833CD2(OKT1 1) 83 <5<5CD3(OKT3) <5 <5<5CD4(OKT4) <5 28<5CD8(OKT8) 80 9613CD7(Leu-9) 75 2528OKT9

83 4768CD38(OKT19) 91 9438Leu-7

19 1011CD25(0KT26) <5 <5<5CDIO(Calla) <5 <5<5TEC-HLA-A,B,C

79 8426TEC-anti-la<5 <5 <5

HUMAN T-CELL LYMPHOMA GROWING IN NUDE MICE

pendently at least three times; a representative experiment is shown.The cytotoxicityassay was carried out in U-bottomed 96-wellmicrotestplates in a 0.2-mI volume of basic medium. Target cells (5 x 10@)werelabeledwith 200 @iCiof sodium l―Crlchromate(New England Nuclear,Dreieich,WestGermany)at 37°Cfor 1 h, washedthree times,andadded at 1 x l0@cells/well to various numbers of effector cells. After a4-h culture, the plates were spun down at 150 X g for 10 mm. Supernatant(0.l ml)wascollectedand its radioactivity(in cpm)was evaluatedin a gamma counter. The specific lysiswas then calculated as describedpreviously in detail (19).

Proliferation Assays. These were performed in flat-bottomed 96-wellmicrotest plates. Responder PBL or ST-4 cells were seeded at a concentration of 2 x 10' cells/well in basic medium and stimulated with0.2 @tgof PHA, 2 @gof pokeweed mitogen (Grand Island BiologicalCo.), and 2@ of concanavalin A (Pharmacia, Uppsala, Sweden).Alternatively they were stimulated with stimulator cells preincubatedwith 50 @g/l07cells/mI of mitomycin C (Sigma Chemical Co., St.Louis, MO) at 37°C for 30 mm and added at different responder.stimulator cell ratios. The cultures were incubated respectivelyfor 72 and 96 h, then pulsed with [3Hjthymidine (New England Nuclear), and harvested 8 h later. [3HjThymidineuptake was evaluated byliquid scintillation spectrometry. The results were expressed as thearithmetic mean ±SD of total cpm of quadruplicate cultures or as astimulation index (20).

Lymphokine Secretion. ST-4 cells (2 x l0@)were cultured in flatbottomed microplate wells in 0.2 ml basic medium and stimulated with1 @tg/mlPHA or I @zg/mlPHA plus l0@ M l2-O-tetradecanoylphorbol13-acetate (Sigma) for 72 h. Supernatants were harvested and testedfor IFN-'y and IL-2 production. IFN-'y was titrated by evaluating theantiviral activity in a cytopathic assay on human amnion cells (WISH),as described previously in detail (21). Titers are expressed as thegeometricmeanof the reciprocalof thesupernatantdilution causinga50% reduction in the number of lytic plaques as compared with thecontrols.An internal laboratorystandardcalibratedagainstNIH G002-90451 1 standard was included in each assay. The IL-2 titer wascalculated by using a probit analysis computer program to calculate thereciprocal titer of the test samples that gave 50% of maximal cpm ofour laboratory standard (21, 22), which was calibrated against theBiologicalResponse Modifiers Program reference reagent human IL-2(Jurkat) (Lot ISDP-84l, containing 500 reference units/mi).

RESULTS

Histopathology. The histological evaluation of the primarylesion disclosed a lymph node extensively infiltrated by a malignant neoplasm of lymphoid origin. Neoplastic cells wereintermingled with mature lymphocytes and a few plasma cells.They had large irregular nuclei with deep indentations. Chromatin was delicate and no nucleoli were seen. A few smallfollicles were still present (Fig. 1A). On frozen sections, neoplastic cells were decorated with several MoAb detecting T-cellsurface antigens (OKT8, OKT4, OKT3, Leu-9, OKT6, andleukocyte common). The percentage of positivity ranged from50% to over 75% for all markers except OKT3 (10%). Dendriticreticular cells and pan-B were positive only in spared follicles(data not shown). The FACS analysis of the single-cell suspension obtained from lymphoma-involved areas showed the presence of a pattern of surface antigens mostly similar to thatobserved in the histopathological sections (Table 1). On imprints, the lymphomatous cells were positive for acid phosphatase (90%, occasionally with “dot-like―pattern) and negativefor diaminopeptidase IV, periodic acid-Schiff, and peroxidase.The combined histological and immunological data pointed toa convoluted T-cell lymphoma (23) with surface markers according to the second stage ofdifferentiation (24, 25). Morphological diagnosis, however, was rendered difficult by prominentclusters of epithelioid histiocytes and necrotic areas.

In Vitro and in Vivo Growth of Cells from Biopsy. Part of the

a Several aliquots of this cell preparation were stored in liquid nitrogen andreferred to as ST-4 cells.

aST-4 cellsthawedandanalyzedafter the tenth in vitropassage.

Table 2 in vivogrowthoflymphomacells

Leukemic cellsLatencyRecipient

miceSourceNo. (xlO')Takes/totalinjected mice°time(days)NudeuntreatedBiopsy300/160NudeS-I

Nude S-IBiopsy I transplantb30 301/1 2/225 23 ±3CNudeS-III transplant303/320 ±2NudeS-IIII transplant305/520±4NudeS-IIII transplant152/222 ±3NudeS-IIII transplant102/228 ±4NudeS-IIII transplant52/253 ±SNudeS-IIIItransplant10/2>60NudeuntreatedIIItransplant300/4>60NudesplenectomizedIIItransplant300/4>60NudeirradiatedIIItransplant300/4>60BALB/c

untreatedIIItransplant300/4>60BALB/csplenectomizedIIItransplant300/4>60BALB/cirradiatedIIItransplant300/4>60BALB/cS-I

Nude S-IIIItransplant

ST-4 cells'30 100/4 12/12>60 30 ±SNudeS-IinvitroST-4'102/228±3

a Takes at day 60 after challenge.b Lymphoma cells recovered from subsequent transplants into S-I nude mice.CMean ±SD.d Thawedjust before challenge.e ST-4 cells cultured in vitro for ten subsequent passages.

cell suspension from the biopsy was cultured in basic mediumat different cell concentrations ranging from 1 x 1O@to 1 x106/ml in flasks maintained in the horizontal or vertical position. No cell growth was observed and more than 99% of thecells were dead after 10 days ofculture. In parallel experiments,3 x lO@of the same cell preparation were injected s.c. into anuntreated and a S-I nude mouse. As shown in Table 2, no tumorgrowth was found in the untreated nude mouse, whereas the 5-I nude mouse displayed a progressively growing s.c. mass at thechallenge site. The autopsy, performed when the tumor was 14mm in mean diameter, showed a semisolid capsulated off-whitemass, shiny at the cutting surface and very fragile. Histologicalsections disclosed lymphomatous proliferation infiltrating adipose s.c. tissue. No clusters of histiocytes nor areas of necrosiswere seen (Fig. 1B). At electron microscopy, the cells showedan irregular nuclear envelope with several undulations of thenuclear membrane and a scanty cytoplasm poor in membraneorganelles and rich in polyribosomes. A few scattered, smallrough endoplasmic reticulum cisternae could be seen. Small,round mitochondria were usually aggregated toward one sideofthe cytoplasm. Lipid vacuoles were also seen (Fig. 1C). Thus,while the tumor morphology was atypical on the original biopsy, following transplant into nude mice the biopsy cells

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HUMAN T-CELL LYMPHOMA GROWING IN NUDE MICE

originated a neoplastic mass that fulfilled the diagnostic criteriafor “convolutedT-cell lymphoma,―as originally described byBarcos and Lukes (23). Chromosome analysis ofthe lymphomacells derived from the mouse, performed without mitogen stimulation, showed that 47 human chromosomes were present inthe majority of the metaphases. All the metaphases had anapparently balanced translocation t(2;6)(q2l;q23). The Y-chromosome was occasionally missing, several metaphases showedan extra X-chromosome, and an extra chromosome 2 withdeletion of the long arm as a result of the translocation wasalways present. Random gains and losses were also observed(Fig. 2). By contrast, the karyotype of cells obtained fromresidual pleural effusion during remission was normal (46,XY).

Serial Transplants of Lymphoma Cells in Nude Mice. Tumorcells recovered from the first s.c. transplant were serially transplanted s.c. three more times. All S-I mice eventually displayedprogressively growing solid leukemic masses with a latencytime of about 20 days (Table 2). By using the leukemic cellsrecovered from the fourth in vivo passage, the minimal tumorinducing dose and the growth pattern of the lymphoma cells invarious recipient mice were evaluated 60 days after the challenge. Tumor growth was still observed in S-I nude mice only.The latency time was progressively increased by decreasing thenumber of injected tumor cells, and no tumor growth wasobserved after inoculation of 1 x 106cells or less. The leukemiccells recovered from the fourth transplant in S-I nude micewere then divided into aliquots and stored in liquid nitrogen.

This cell preparation was called ST-4 and further extensivelycharacterized.

Characteristics ofThawed ST-4 Cells. ST-4 cells are still ableto form a s.c. tumor mass in S-I nude mice with the samelatency time as the in vivo-transplanted lymphoma cells (Table2). FACS analysis showed that they display almost the samemembrane markers as the biopsy cells. The only striking difference is the almost complete loss ofOKTI 1 positivity (Table 1).ST-4 cells display a human karyotype similar to that of lymphoma cells recovered from the first transplant in S-I nudemice. However, when they are synchronized with high doses ofthymidine most metaphases are tetraploid. Both No. 6 chromosomes are apparently normal, while the der(2) chromosomeand the supernumerary X are still present. In several casestrisomy of chromosome 16 and monosomy (or loss) of chromosome 15 are also evident. Random gains and losses werealso observed (data not shown). Southern blot analysis of ST-4DNA showed two rearranged segments (18 and 15 kilobases)of the fi chain ofT-cell receptor, with no germ line counterpart,indicating that a new DNA configuration had replaced that ofthe germ line on both chromosomes. By contrast, the controlDNA from non-T-cells showed a germ line configuration, witha single 24-kilobase BamHI fragment (Fig. 3).

ST-4 cells cultured at 1O@cells/ml in basic medium withoutexogenous T-cell growth factor grew with a doubling time ofabout 36 h. This remained constant during subsequent passages.The ultrastructural morphology of ST-4 cells after ten subsequent in vitro passages was almost identical to that observed in

C

As UFig. 2. Typical G-banded human karyotype of lymphoma cells recoveredafter the first transplant into S-I nude mice. 47,X,—Y,+X,t(2;6@q2l;q23),

+der(2),t(2;6@q2l;23). Arrow (A), 2q—;arrow (B), 6q+; arrow (C), supernumerary X.

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A

t(III H 1111@1

I,I ‘ II h II U 11

$1

•1

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Table 3 Proliferativeresponseand lymphokinereleasebyST-4 celisfollowingmitogenstimulationMedium

only PHA (0.2 gsg/ml) Pokeweedmitogen(2 @tg/ml) ConcanavalinA(2ig/ml)cpm°IL-2

IFN-@y cpm IL-2 IFN-@y cpm IL-2 IFN-@ycpmIL-2IFN—y(l0@)(units/mi)(lU/mI) (10@) (units/mi) (lU/mI) (l0@) (units/mI) (lU/mi)(l0@)(units/mI)(lU/mI)NormaIGFPBL0.30

3 72 30 256 13 19 321616128ST-4cells1.203 0.9 0 3 1.2 0 31.703a

[3HlThymidine uptake is cxpressedas mean total cpm for quadruplicate cultures. SD wasconstantly lessthan 10%and is not shown.

HUMAN T-CELL LYMPHOMA GROWING IN NUDE MICE

lymphoma recently stabilized in our laboratory from a biopsyof a young boy. In S-I nude mice, biopsy T-lymphoma cellsgrew as a solid mass and became serially transplantable with aconsistent growth pattern. The cells recovered from the firstand the fourth transplants have a human karyotype and displaylittle differences in cell membrane markers from the biopsycells. Those from the fourth transplant were divided into numerous aliquots, stored in liquid nitrogen, and referred to asST-4 cells.

Acute T-cell neoplasias of childhood, usually associated witha mediastinal mass, include acute lymphoblastic leukemia ofTorigin and childhood NHL of T-cell origin (T-convoiuted orlymphoblastic acid phosphatase positive). These diseases arerelated and are difficult to separate on a clinical and morphological basis. Moreover, the distinction between leukemia andlymphoma may appear arbitrary, since in the natural history ofNHL a leukemic phase is present as the disease progresses. Thecharacterization of leukemic cell surface markers permits thedistinction of 3 maturation stages (24, 25) reflecting the stepsof normal T-cell maturation. However, a significant fraction ofleukemias have a more immature membrane phenotype ascompared to NHL, and they seem to form a distinct entity withan initial or very early leukemic phase (24, 25).

Even if one takes into account the dishomogeneity of childhood T-cell leukemias/lymphomas, the case presented here wasunusual. The prolonged chronic picture was in contrast withthe acute and sometimes life-threatening onset ofthese diseases.The cytological evaluation ofthe cells from the pleural effusion,augmented by immunology and cytochemistry, was inconclusive. A specific diagnosis was finally obtained from axillarylymph node biopsy, although the histology was still somewhatunusual. Interestingly, histological comparison of the biopsyand the tumor growing in S-I nude mice disclosed a passagefrom an unusual pattern for a childhood lymphoma, as in thebiopsy, to a typical morphology of T-eonvoluted NHL. Thus,when interfering factors such as previous therapy, especiallywith steroids, and massive necrosis alter tumor morphology onthe surgical specimen, heterotransplantation into S-I nude micemay become of diagnostic help by offering a more easily interpretable morphological pattern.

Biopsy cells appear to be of a convoluted T-cell lymphomaat the second stage of differentiation, as suggested by thehistochemical, immunological, and chromosomal markers (23—25). They retain the CD! antigen characteristic of the corticalthymocytes (24), do not express the CD3 antigen related to theexpression of the T-cell receptor (26), and display a clonal generearrangement for the fi chain of Ti receptor (27). The only

striking difference between biopsy cells and those recoveredfrom the fourth transplant into S-I mice is the loss of OKT1 1positivity. This loss has also been observed with another Tlymphoma cell line growing in vitro (28) and can be ascribed toantigenic modulation or by clonal evolution (29).

The karyotype of the cells recovered from the first transplantin S-I mice was characterized by an apparently balanced translocation between chromosomes 2 and 6, loss ofthe chromosomeY in some metaphases, and the presence of an extra chromo

A B

Fig. 3. Southern blot analysis of DNA from ST-4 cells. (A) germ line configuration of the control DNA from human fibroblasts with a single 24-kilobaseBamHI fragment.(B) two rearrangedsegmentsof 18 and I 5 kilobases,with nogerm line counterpart from DNA of ST-4 cells.

the cells growing in nude mice (Fig. 1D). Their membranephenotype, too, was almost unchanged apart from a decreasedexpression of CD1, CD4, and CD8 surface antigens as compared to ST-4 cells recovered from S-I nude mice. Moreover,when injected in S-I nude mice, they formed a lymphomatousmass with the same latency time of that of the cells seriallytransplanted in vivo.

ST-4 cells do not release IFN-'y and IL-2 spontaneously, afterPHA or PHA plus 12-O-tetradecanoylphorbol-13-acetate stimulation (Table 3). They do not display a proliferative responseto PHA, pokeweed mitogen, and concanavalin A mitogens(Table 3) or against allogeneic PBL differing at multiple HLAantigens (Table 4). They are able to stimulate a vigorous proliferative response by allogeneic GF PBL. In effect, they displayHLA-A l,29:HLA-B 5,12 alloantigens, whereas DR and DQantigens are not expressed. By contrast, they do not stimulatea proliferative response by autologous PBL obtained from thepatient in complete remission. These PBL, however, are ableto proliferate in response to allogeneic GF PBL. Lastly, ST-4cells do not show NK activity against K562, although whenused as target cells they are sensitive to NK activity by normalPBL, although less than K562 (Table 5).

DISCUSSION

In the present study, we describe the serial transplantabilityin nude mice and the in vitro growth of a new human T-cell

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TabIc 4 ProliferativeresponseofST-4 cells,GFcilogeneicPBL, andpatient PBL inMLCResponderStimulatorResponder:stimulator

cell ratio(3H@Thymidineup

take°ST-4

cellsbST-4 cellsST-4 cellsST-4 cellsGF allogeneicPBLGFallogeneicPBLGF allogeneicPBLGF allogeneic PBLGF allogeneicPBLPatient PBLdPatient PBLPatient PBLPatientPBLPatient PBLPatientPBLPatientPBLGF

allogeneicPBLCOF allogeneicPBLGF allogeneicPBL

ST-4cellsST-4 cellsST-4 cellsST-4 cells

GF allogeneicPBLGF allogeneicPBLST-4cellsST-4 cellsST-4cellsST-4cells1:1

5:110:1

1:1 -10:120:1

100:1

1:120:1

1:110:120:1

100:11,097

±701,037 ±221 (0.9)1,437 ±191 (1)1,085 ±83 (0.9)1,290 ±85

10,164±950(8)20,773 ±2,@80(16)29,960 ±2,245 (23)24,387 ±1,951(19)

170 ±21882 ±67 (5)

14,456 ±I 10 (17)887± 130(1)195 ±25 (1)180±50(1)111 ±15(0.6)

EffectorTarget100:150.l25:112:1ST-4

cellsK5620.90.70.40.1GFallogeneicPBLK56275.443.318.513.3GFallogeneicPBLST-435.112.36.23.5

HUMAN T-CELL LYMPHOMA GROWING IN NUDE MICE

a Expressedasmeancpm ±SD for quadruplicatecultures.Numbersin parentheses,stimulationindex.b HLA performed on PBL ofthe patient in full remission: A3, 9, B35, DR5, DRw52l (DR and DQ alloantigens, however, are not expressed by ST-4 cells).C HLA: Al, Aw19, Aw33, B8, B35, Bw6, Cw4, Cw7, DR3, DRw1O, DRwS2, DQw1, DQw2. The [‘HJthymidine uptake by stimulator cells was: GF allogeneic

PBL, 85 ±10 cpm;ST-4 cells, I 10 ±15 cpm.d Patient PBL were stored in liquid nitrogen and thawed just before the experiment.

Table 5 NK activityofST-4 cellsand normalPBL

% of specific lysis at following effector:target cell ratio

an efficient immune selection of lymphoma cells during thelatency period probably also accounts for the stability of lymphoma cell features we observed during serial transplants.Moreover, morphological observations show that human leukemic cells growing in S-I mice are no more than very slightlycontaminated by mouse lymphocytes.

This stabilization of a well-characterized human T-cell lymphoma, growing with a constant pattern in S-I nude mice, aswell as in vitro, provides the scientific community with a newfoundation for studies on leukemic T-cell differentiation induced by various agents in vivo. Moreover, the availability ofnormal lymphocytes from a patient in complete remissionmakes this in vivo system quite unique for studies of humantumor immunology.

ACKNOWLEDGMENTS

We wish to thank A. M. Del Monte and M. T. Vigna-Docchio forexcellent histological assistance, Dr. G. Ghisolfi for skilled immunostainings of frozen sections, Dr. M. Meregalli for FACS analysis, Dr.A Amoroso for HLA typing, and Dr. J. Iliffe for careful revision ofthe manuscript.

REFERENCES

1. Fogh, J., and Giovanella, B. C. The Nude Mouse in Experimental andClinical Research,Vol. 2. New York: Academic Press, 1982.

2. Lozzio, B. L., and Machado, E. A. Transplantation and dissemination ofhuman haematopoietic malignant cells in nude and lasat mice. 1n.@Nomuraet a!. (eds.), Proceedings of the Second International Workshop on NudeMice, pp. 587—594.Tokyo: University ofTokyo Press, 1982.

3. Nara, N., andMiyamoto,T. Direct andserialtransplantationof humanacutemyeloidleukemiainto nudemice.Br. J. Cancer,45: 778—782,1982.

4. Zaino, R. J., Satyaswaroop,P. G., and Mortel, R. Hormonal therapy ofhumanendometrialadenocarcinomain a nude mousemodel.Cancer Res.,45:539—541,1985.

5. Balkwill, F. R., Goldstein, L., and Stebbing, N. Differential action of sixhumaninterferonsagainsttwo humancarcinomasgrowingin nudemice.Int.J. Cancer,35:613—617,1985.

6. Campanile, F., Crinó,L, Bonassard,E., Houchens,D., and Goldin, A.Radioresistentinhibitionoflymphomagrowthin congenitallyathymic(nude)mice. Cancer Res., 37: 394—398,1977.

7. Cudkowicz, G. Rejection of bone marrow allografts by irradiated athymicnudemice.Proc.Am. Assoc.CancerRes.,16: 170, 1975.

8. Schaadt,M., Kirchner,H., Fonatsch,C., and Diehl, V. Intracranialheterotransplantation of human haemopoieticcells in nude mice. Int. J. Cancer,23:751—761,1979.

9. Ohsugi, Y., Gershwin, M. E., Owens, R. B., and Nelson-Rees,W. A.Tumorigenicity of human malignant lymphoblasts: comparative study withunmanipulated nude mice, antilymphocyte serum-treated nude mice, and Xirradiated nude mice.J. NatI. Cancer Inst., 65: 715-718, 1980.

10. Watanabe, S., Shimosato, Y., Kuroki, M., Sato, Y., and Nakajima, T.

some X. The role of this translocation in the mechanisms ofneoplastic transformation remains uncertain, although a c-mybprotooncogene location on human chromosome 6 at q22—q24(30) may be involved in the rearrangement. Even though ST-4lymphoma cells no longer display the rearranged chromosome6, and both No. 6 chromosomes appear normal, the rearrangedchromosome 2, possibly carrying the myb protooncogene, as aresult of the translocation, is maintained. Loss of the chromosome Y is frequently seen in leukemias and lymphomas, but itssignificance is uncertain, since it is observed in bone marrow ofnormal males over the ages of 60 years. In our case, this lossmay reflect the “aging―of the cell line, as suggested by Pierreand Hoaglund (31), since it was always observed in later passages.

ST-4 cells stimulated by mitogens or alloantigens do notshow augmented proliferation or release IFN or IL-2. Moreover, they do not have spontaneous NK activity. This lack ofimmune responsiveness fits in well with their rather immaturemembrane phenotype.

Neither biopsy cells nor cells recovered from the third serialtransplant into S-I nude mice were able to grow in S-I euthymicmice, untreated nude mice, and nude mice irradiated only orsplenectomized only. This inability may be due to their susceptibility to NK activity, which is particularly high in untreatednude mice (13, 20). Moreover, residual T-dependent reactivitymay be boosted by the heterotransplanted lymphoid cells (32).By contrast, the use of S-I nude mice markedly increases theprobability of human leukemia outgrowth, as shown by thepresent data, by our previous data with a T-leukemia line (28),and by the work ofother laboratories (9, 10). In effect, S-I nudemice have almost no residual T-lymphocyte reactivity and marginal NK activity during the first 20 days after irradiation (datanot shown), a lapse that corresponds to the lymphoma latencytime. This is the most critical phase of tumor-host immunerelationship, since during this period the host reactivity can stillimpair the growth of the incipient tumors (33). The absence of

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HUMAN T-CELL LYMPHOMA GROWING IN NUDE MICE

Transplantability of human lymphoid cell line, lymphoma and leukemia insplenectomized and/or irradiated nude mice. Cancer Res., 40: 2588—2592,1980.

11. Lennert, K. Histopathologyofnon-Hodgkin's Lymphomas.Berlin:SpringerVerlag, 1981.

12. Chilosi, M., Pizzolo, G., Menestrina, F., lannucci, A. M., Bonetti, F., andFiore-Donati, L. Dipeptidyl(amino)peptidase IV (DAP IV) histochemistryon normal and pathologiclymphoidtissues.Am. J. Clin. Pathol., 77: 714—719,1982.

13. Bernengo,M. G., Meregalli, M., Jemma,C., et at. FunctionalpropertiesinSézarycells with an unusual phenotype. Clin. Immunol. Immunopathol., 37:298—311,1985.

14. Seabright,M. A. A rapid banding technique forhumanchromosomes. Lancet,2:971—972,1971.

15. Evans, H. J., Buckton, K. G., and Sumner, A. T. Cytological mapping ofhumanchromosomes:resultsobtainedwith quinacrinefluorescenceand theacetic-saline-Giemsatechniques.Chromosoma (Ben.), 35: 310—325,1971.

16. Southern, E. M. Detection of specific sequencesamong DNA fragmentsseparatedby gel electrophoresis. i. MoL BioL, 98: 503—517,1975.

17. Yoshikai, Y., Anatoniou, D., Clark, S. P., Yanagi, Y., Sangster,R., Van denElsen, P., Terhorst, C., and Mak, T. W. Sequenceand expression of transcripts of the human T.cell receptor $.chain genes. Nature (Lond.), 312:521—524,1984.

18. Brandt, D. L, Ray, I. G., and Hare, D. B. Preliminarytrials towardstandardization oflymphocyte typing. In: P. I. Terasaki (ed.), HistocompatibilityTesting, p. 357. Copenhagen:Ejnur Copenhagen: 1970.

19. Forni, G., Bindoni, M., Santoni, A., Belluardo,N., Marchese,A. E., andGiovarelli,M. Radiofrequencydestruction of the tuberoinfundibularregionof the hypothalamus permanently abrogates NK activity in mice. Nature(Lond.), 306: 181—184,1983.

20. Meo, T. The MLR test in the mouse: in.@1. Lefkovits and B. Pernis (eds.):Immunological Methods, pp. 227—239.New York: Academic Press, 1979.

21. Foa, R., Giovarelli, M., Jemma, C., Fierro, M. T., Lusso, P., Ferrando, M.L., Lauria, F., andForni, G. Interleukin2 (IL-2)and interferon-ayproductionby T lymphocytes from patients with B chronic lymphocytic leukemiaevidence that normally released IL-2 is absorbed by the neoplastic B cellpopulation.Blood,66: 614—619,1985.

22. Sette,A., Adorini, L, Marubini, E., and Doria, G. A microcomputer program

analysis of interleukin (IL-2) titration data. J. Immunol. Methods, 86: 265—271,1986.

23. Barcos,M. P., and Lukes, R. J. Malignant lymphoma ofconvoluted lymphocytes:a new entity of possibleT cell type. In: L F. Sinks and J. 0. Godden(eds.): Conflicts in Childhood Cancer and Evaluation of Current Management,Vol 4, p. 147. New York: Alan R. Lisa, 1975.

24. Reinherz, E. L., Kung, P. C., Goldstein, G., Levey, R. H., and Schlossman,S. F. Discretestagesofhumanintrathymicdifferentiation:analysisof normalthymocytes and leukemic lymphoblasts of T cell lineage. Proc. NatI. Acad.Sci. USA, 77: 1588—1592,1980.

25. Roper, M., Crist, W. M., Metzgar, R., Ragab,A. H., Smith, S., Starling, K.,Pullen, J., Leventhal, B., and Bartolucci, A. A. Monoclonal antibody characterization of surfaceantigens in childhood T-cell lymphoid malignancies.Blood, 61: 830—837,1983.

26. Meuer, S. C., Fitzgerald, A. K., Hussey,R. C., Hodgdon, J. C., Schlossman,S. F., and Reinherz,E. L. Clonotypicstructuresinvolvedin antigen-specifichumanT cell functions..1.Exp. Med., 57: 705—719,1983.

27. Sangster,R. M., Minowada, J., Suciu-Foca, N., Minden, M., and Mak, T.w. Rearrangementandexpressionof the a, @,and @ychainT cellreceptorgenesin human thymic leukemia cells and functional T cells. J. Exp. Met,163:1491—1508,1986.

28. Pegoraro,L, Fierro, M. T., Lusso,P., Giovanazzo, B., Lanino, E., Giovarejli,M., Matera, L., and Foa, R. A novel leukemicT-cell line (PF-382) withphenotypic and functional featuresofsuppressor lymphocytes. J. Nati Cancer Inst., 75:285—290,1985.

29. White, S., Meyer, P. R., and Benedict,W. F. Establishmentand characterizationof a humanT-ceHleukemialine (LALW-2) in nudemice..1.NatI.CancerInst., 72: 1029—1034,1984.

30. Harper, M. E., Franchini, G., Love, J., Simon, M. 1., Gallo, R. C., andWong-Staal, F. Chromosomal sublocalization of human c-myb and c-leacellular oncogenes.Nature (Lond.), 304: 169—171,1983.

31. Pierre, R. V., and Hoaglund, H. C. Age-associatedaneuploidy: loss of Ychromosome from human bone marrow cells with aging. Cancer (Phila.), 30:889—894,1972.

32. Dosch, H. M., White, D., and Grant, C. Reconstitution ofnude mouseT cellfunction in vivo: IL 2-dependenteffect of human T cells. J. ImmunoL, 134:336—342,1985.

33. Forni, G., Varesio, L, Giovarelli, M., and Cavallo, G. Dynamic state ofspontaneousimmunereactivitytowardsa mammaryadenocarcinoma.In: F.Spreafico and R. Arnon (eds.): pp. 167—192.New York: Academic Press,1979.

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1988;48:1312-1318. Cancer Res   Roberto Arione, Cristina Jemma, Marco Forni, et al.  

in Vitroand A New Childhood T-Cell Lymphoma Established in Nude Mice

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