Proliferation of CD30 T-Helper 2 Lymphoma Cells Can Be ...clincancerres.aacrjournals.org/content/clincanres/9/7/2744.full.pdf · Proliferation of CD30 T-Helper 2 Lymphoma Cells Can

Proliferation of CD30� T-Helper 2 Lymphoma Cells Can BeInhibited by CD30 Receptor Cross-Linking withRecombinant CD30 Ligand1

Jorg Willers, Reinhard Dummer, Werner Kempf,Thomas Kundig, Gunter Burg, andMarshall E. Kadin2

Department of Dermatology, University Hospital Zurich, Zurich,Switzerland [J. W., R. D., W. K., T. K., G. B.], and Department ofPathology, Beth Israel Deaconess Medical Center, Harvard MedicalSchool, Boston, Massachusetts 02215 [M. E. K.]

ABSTRACTPurpose: Cutaneous anaplastic large cell lymphomas

(ALCLs) are characterized by the expression of CD30, spon-taneous regression of skin lesions, and increased concentra-tion of CD30 ligand (CD30L). We hypothesize that CD30-CD30L interactions explain the unusual clinical behavior ofcutaneous ALCLs.

Experimental Design: Eight lymphoma cell lines estab-lished from four different patients were analyzed for T-cellclonality using PCR and subsequently denaturating gradientgel electrophoresis. The expression levels of CD30 wereassessed by flow cytometry. Secreted cytokines [IFN-�, in-terleukin (IL)-2, IL-4, IL-6, IL-8, and IL-10] were deter-mined in the supernatant after 3-day culture. Proliferationand apoptosis of cultured cells were measured by 5-bromo-2-desoxyuridine and propidium iodine incorporation.

Results: The results showed different levels of CD30expression and a predominant T-helper 2 profile. In a cellkinetic analysis we found that ALCL cell growth is effec-tively inhibited by CD30L but only in those cell lines ex-pressing CD30 molecules in sufficient amounts on the cellsurface. Cell cycle analysis revealed that growth regulationwas because of apoptosis and growth arrest, and was de-pendent on cell culture conditions. Comparison of effects ofligation with CD30L and anti-CD30 agonistic antibodyHeFi-1 revealed higher efficacy for CD30L in these ALCLlines.

Conclusions: CD30� ALCL cells can be growth inhib-ited by receptor ligation. Observed pleiotropic effects ofCD30 signaling are most likely dependent on cell cycle statusand signal strength. The binding of CD30 by its ligandprovides new opportunities for controlling cell growth andtreatment of CD30� ALCL.

INTRODUCTIONMalignant lymphomas can originate from lymphocytes at

any level of development and differentiation between pre-B/Tcells and peripheral differentiated B or T lymphocytes. There isgrowing evidence that a clonal disease may have differentclinical and histological features depending not only on the timepoint in the disease process but also probably on the correspond-ing state of lymphocyte activation (1). The precise mechanismsof lymphomagenesis are still obscure. However, it appears thatvarious factors are involved including environmental, especiallyinfectious agents, chromosomal translocations, insertions, orpoint mutations resulting from genetic instability of tumor cells(2, 3). The microenvironment in the skin contributes to thepeculiar behavior of these neoplasms by providing various sig-nals from adhesion molecules and cytokines. The CD30 antigen,a member of the tumor necrosis factor receptor superfamily,serves among others (4) as a prognostic marker in cutaneousT-cell lymphomas (5–9).

The biological function of tumor necrosis factor receptorfamily members is regulation of growth and elimination ofperipheral T cells during their expansion in response to anti-genic stimuli. CD30 is normally expressed on immunoblastslocated in the perifollicular region around germinal centers. Inaddition, CD30 is expressed by Reed-Sternberg cells in virtuallyall cases of classical Hodgkin’s lymphoma and ALCL3 (10).The cognate CD30L is expressed on neutrophils, histiocytes,eosinophils, mast cells (11, 12), and a small subset of activatedT cells (13). Furthermore, CD30L has been detected in Reed-Sternberg-like cells and smaller cells in lesions of patients withlymphomatoid papulosis (14). The human CD30L is a Mr

40,000, 234 aa residue transmembrane glycoprotein with 72%aa sequence identity to its ms counterpart.

Cutaneous CD30� ALCL often are accompanied by spon-taneous tumor regression (14–16). Although the underlyingmechanisms are still unknown, we hypothesize that a selectiveincrease in CD30L expression measured in clinical specimens of

Received 12/6/02; revised 2/6/03; accepted 2/20/03.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by the Swiss “Fonds fur medizinische Forschung,” and theSwiss Cancer League No. 983-02-2000 and NIH/NCI Grant P50-CA93683.2 To whom requests for reprints should be addressed, at Department ofPathology, Division of Hematopathology, Beth Israel Deaconess Med-ical Center, Harvard Medical School, Laboratory Medicine, YA-309,330 Brookline Avenue, Boston, MA 02215. Phone: (401) 624-2715;Fax: (617) 667-4533; E-mail: [email protected].

3 The abbreviations used are: ALCL, anaplastic large cell lymphoma;CD30L, CD30 ligand; aa, amino acid; Ab, antibody; TCR, T-cellreceptor; DGGE, denaturating gradient gel electrophoresis; mAb, mono-clonal antibody; ms, mouse; BrdUrd, 5-bromo-2-desoxyuridine; Th2,T-helper cell type 2; IL, interleukin; NF�B, nuclear factor �B.

2744 Vol. 9, 2744–2754, July 2003 Clinical Cancer Research

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cutaneous ALCL may play an important role (14). CD30 cross-linking with Abs may result in proteolytic degradation of theprotein and a release of soluble CD30 products into the extra-cellular compartment (17). However, not all of the anti-CD30Abs can cause the described pleiotropic effects, and many ofthem are unable to produce any detectable biological changes(18). To additionally elucidate the function of CD30 and itspotential impact on lymphoproliferative disorders, several mu-rine models have been established. CD30 transgenic mice haveshown enhanced cell death by CD30 receptor cross-linking (19),and CD30�/� mice showed an impaired negative selection inthe thymus (20). When CD30� ALCL cells were transplantedinto mice, tumor growth could be inhibited by administration ofthe anti-CD30 mAb HeFi-1 (21, 22).

In the present study, we compare eight lymphoma cell lineswith respect to surface marker expression, cytokine profile, andclonal TCR gene rearrangement. We correlate these findingswith functional assays in which we inhibit cell growth byreceptor cross-linking. In kinetic analyses we can clearly showthe efficacy of recombinant CD30L to inhibit lymphoma cellgrowth when the CD30 receptor is expressed on the cell surfacein sufficient amounts. Additional analysis revealed that celldeath observed in our cultures is only partly because of apo-ptosis. Moreover, it is evident that cell culture conditions verymuch influence the lymphoma cell susceptibility to CD30 cross-linking. We believe our data help to explain controversial resultspresented in the literature, showing differential effects on lym-phoma cells caused by CD30 activation (23, 24).

MATERIALS AND METHODSCell Lines. The cutaneous lymphoma cell lines Mac1,

Mac2A, and Mac2B were derived from different clinical spec-imens of one patient showing progression from lymphomatoidpapulosis to systemic ALCL (1, 15, 25, 26). In particular, theMac1 cell line was derived from circulating tumor cells in theblood of the patient during an indolent course of his disease,whereas the Mac2A and Mac2B cell lines were derived fromseparate rapidly growing skin tumors 3 years later in the pro-gression of the disease. The cell lines JK-A, JK-B, and JK-Xrepresent different subclones, with a splicing variant of CD30that lacks the extracellular and transmembrane domains ofCD30, derived in vitro from a biopsy of a patient with a CD30�cutaneous ALCL (26). The cell lines Karpas 299 and JB6 werederived from lymphoma cells of different patients with nodal/systemic CD30� ALCL (27). Table 1 summarizes known char-

acteristics of the cell lines from the literature (1, 25, 28–30). Thehuman T-lymphoblastic lymphoma cell line Jurkat was obtainedfrom the American Type Culture Collection (Manassas, VA).All of the cell lines were grown in RPMI 1640, supplementedwith 10% heat-inactivated fetal bovine serum (Seromed, Berlin,Germany), 1% of an antibiotic mixture containing 10,000 unitspenicillin/ml and 10,000 �g streptomycin/ml, 5 mM glutamine,and 5 mM sodium pyruvate (all Life Technologies, Inc., Paisley,United Kingdom).

PCR of TCR-� Genes. The method was performed ac-cording to Meyer et al. (31). Briefly, for PCR amplification 1 �gof total DNA was suspended in 50 �l PCR solution containing40 pM of each primer, 200 �M of deoxynucleoside triphosphate,50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 0.01%gelatin, and 2.5 units Taq DNA polymerase in a total volume of50 �l. In a first step a PCR was performed with a primer setdescribed elsewhere (32, 33). Initially, 25 cycles using theTCR-� variable region (V�1–8) and the TCR-� joining regionJ�1/2) primers were performed, then 10 �l of the first-roundPCR product was added to 100 �l of fresh PCR buffer contain-ing the nested set of V�1–8 and J�1/2 primers, and another 20cycles were performed. Cycle parameters in both rounds were:initial denaturation step at 94°C for 2 min, denaturation at 94°Cfor 30 s, annealing at 55°C for 30 s, and extension at 70°C for40 s, followed by a terminal extension step at 72°C for 10 min.To amplify rearrangements involving V�9, the single V�9primer replaced the two V�1–8 primers (31–33). This protocolhas been performed on all of the cases as a standard. Cases thatdemonstrated no clonality, when amplified with primers V�1–8,V�9, and J�1/2, were reamplified with consensus primers forvariable regions V�10/11 (34), joining region J1/2 (35) andJP1/2 (36). With these primers a single-round PCR was per-formed with the following thermocycling conditions: initialdenaturation step at 94°C for 4 min, 35 cycles of denaturation at94°C for 1 min, annealing at 58°C for 1 min, and extension at72°C for 1 min, followed by a terminal extension step at 72°Cfor 5 min. The reaction was performed in a Thermal Cycler 9600(Perkin-Elmer, Kuesnacht, Switzerland). Primers as shown inTable 2 were obtained from Microsynth (Balgach, Switzerland).

DGGE. The DGGE gel was performed with the D GeneSystem (Bio-Rad, Hercules, CA) according to the manufactur-er’s instructions and as described elsewhere (37). The PCRproducts were precipitated and resuspended in 10 �l of deion-ized water, heat-denatured at 95°C, and then reannealed for 30min at 30°C. The samples were loaded on a 6.5% polyacryl-amide gel containing gradients of 30–60% urea/formamide andelectrophoresed at 60°C for 6 h at 150V. The ethidium bromide-stained gel was photographed under UV light.

Cell Kinetic Analysis. Two 96-well flat-bottomed mi-crotiter plates were coated with 0.5 �g of a murine recombinantCD30L (R&D Systems, Minneapolis, MN), mAb HeFi-1 (a kindgift of Dr. Longo, National Cancer Institute, Frederick, MD),and ms IgG2a (Ancell, Bayport, MN) in 100 �l PBS/well. Themurine CD30L cross-reacts with human CD30 (13). The recom-binant CD30L was derived from a DNA sequence encoding aaresidues Gln 68-Asp 239 of ms CD30L, and fused to the signalpeptide of human CD30 and a polyhistidine-containing linker.After overnight incubation of the plates at 4°C, the coatingsolution was removed, and 200 �l RPMI 1640 containing

Table 1 Phenotypic cell line description

Cell line CD15 CD2 CD3 CD25 ALKa CLA

Mac1 � � � � � �Mac2A � � � � � �Mac2B � � � � � �Karpas n.d.a n.d. � n.d. � n.d.JB6 � � � � � n.d.JK-X � n.d. � n.d. � n.d.JK-A � n.d. � n.d. � n.d.JK-B � n.d. � n.d. � n.d.

a ALK, anaplastic lymphoma kinase; n.d., not done; CLA, cutane-ous lymphocyte antigen.

2745Clinical Cancer Research



10,000 cells plus 0.5 �g of CD30L, HeFi-1, or ms IgG2a wasadded to each well. Cell cultures have been in logarithmicgrowth conditions at the time of transfer. The plates wereincubated at 37°C in a 5% CO2 atmosphere. After 1, 4, 6, and 8days, aliquots of 20 �l were removed and stained with 100 �lPBS/trypan blue (Biochrom KG, Berlin, Germany). Living(nonstained) and dead (stained) cells were counted separatelywith a microscope (one visual field at �200 magnification). Atday 4, one volume of fresh RPMI 1640 was added to all of thewells, and all of the subsequent counts were multiplied by two.

DNA Staining. Microtiter plates were coated with 1 �gCD30L, HeFi-1, or ms IgG2a in 100 �l PBS/well overnight at4°C. Then, the supernatant was removed and �100,000 cells in100 �l (a) serum-free medium or (b) complete medium wereadded. Cells in serum-free medium were kept under starvedculture conditions for 12 h before use. After adding 100 �l offresh serum-free medium, the cells were incubated for 24 h at37°C in a 5% CO2 atmosphere. The next day, BrdUrd (Sigma,St. Louis, MO) was added to the cells. The thymidine analogueBrdUrd was added to the medium at a concentration of 2 mg/ml.The plates were kept light protected and incubated for an addi-tional 10 h. Then, the cells were harvested and stained with aFITC-conjugated anti-BrdUrd mAb (B44; BD Bioscience, Hei-delberg, Germany). Briefly, the single cell suspensions werecentrifuged for 10 min at 200 � g. The pellets were resuspendedin 200 �l PBS and injected into 5 ml of 70% methanol. The cellswere fixed for 20 min at room temperature and then centrifugedagain for 5 min at 500 � g. After removing the supernatant, thecells were resuspended in 2 ml 1 N HCl containing 0.5% Tween20 (Sigma) and incubated for 15 min at 37°C. After anothercentrifugation step, the acid was neutralized with 200 �l 0.1 M

sodiumtetraborate. Cells were washed in 1% BSA/PBS and thenstained with an anti-BrdUrd-FITC Ab. For detection of incor-porated BrdUrd, we used a FACScalibur (Becton Dickinson)and the CellQuestPro software. After measuring proliferation,the remaining cells in the tube were stained with 40 �g/mlpropidium iodide (Sigma) for cell cycle analysis and measuredagain in the FACScalibur. Acquisition and analysis were per-formed in the “doublet discrimination mode.”

Cell Surface Staining. For surface receptor detection, 106

cells were stained with anti-CD30 mAb (HeFi-1) at a concentrationof 5 �g/ml for 2 h on ice in a total volume of 20 �l followed by asecond staining with anti-ms IgG2a-FITC (Ancell) Aliquots of 106

cells were incubated twice in a total volume of 20 �l with theappropriate labeled mAb. Then, cells were washed, resuspended,and subjected to FACScalibur analysis.

Cytokine Profiling. One million cells per ml RPMI 1640were cultured for 3 days, then the supernatants were harvestedand analyzed in ELISA for IFN-�, IL-2, IL-4, IL-6, IL-8, andIL-10 in the Department of Clinical Immunology (UniversityHospital Zurich, Zurich, Switzerland). The amount of solublecytokines was measured by Hbt Human IFN-� ELISA test kit(HyCult Biotechnology, Uden, Netherlands), and IL-2, -4, -6,-8, and -10 ELISA test kit (R&D Systems Inc.), according to themanufacturer’s instructions. Appropriate reference material forall of the ELISA was included (Quantikine Controls, QC Con-trols Group 1; R&D Systems Inc.).

Fig. 1 Clonal TCR-V� chain rearrangement in lymphoma cell lines.PCR-DGGE analysis was performed for eight lymphoma cell lines withvarious primer combinations. Only positive results (Lanes 1, 3, 5, 7, and9) and a corresponding negative control (Lanes 2, 4, 6, 8, and 10) areshown. Lane 1 represent a V�10/11:J1/2 rearrangement; Lanes 3, 5, and7 show V�1–8:JP1/2 rearrangements; and finally Lane 9 revealedclonality with V�1–8:J�1/2 primers. Lymphoma cell lines printed inbold (JK-X, Mac1, Mac2B, Karpas, and JB6) are shown. Lymphomacell lines printed in italics below (JK-A, JK-B, and Mac2A) revealedsimilar/identical rearrangement pattern to the lymphoma cell linesabove (data not shown). M is a molecular size marker (Marker VI;Roche Applied Science, Basel, Switzerland). Arrows indicate bands ofclonality.

Table 2 Primer sequences

Name Sequence (5�33�) Length (bp)

V�1–8 GAA GCT TCT AGC TTT CCT GTC TC 23V�1–8 (nested) CTC GAG TGC GCT GCC TAC AGA GAG G 25V�9 GGA ATT CCA AAT TCT TGG TTT A 22V�10/11 CAC TGG TAC YGG CAG AAA C 19J�1/2 CGT CGA CAA CAA GTG TTG TTC CAC 24J�1/2 (nested) GGA TCC ACT GCC AAA GAG TTT CTT 24J1/2 CCC TCT ATT ACC TTG GAA ATG TTG 24JP1/2 GAA GTT ACT ATG AGC YTA GTC CCT T 25

2746 CD30L Inhibits Lymphoma Growth



RESULTSClonal TCR V� Rearrangements in Lymphoma Cell

Lines. All of the established lymphoma cell lines were sub-jected to clonal analysis with various combinations of TCR-�chain-specific primers. As shown in Fig. 1 the JK-family carries

a V�10/11:J1/2 rearrangement (very faint band). TheMac1,2A,2B-family showed a V�1–8:JP1/2 rearrangement. It isnoteworthy that Mac2B, derived from the same patient but froma different lesion as Mac1 and Mac2A showed a different bandpattern, thus emphasizing intraindividual heterogeneity of thetumor cells. Karpas 299 showed a V�1–8:JP1/2 rearrangementas well, and additionally V�1–8:J�1/2 rearrangements (data notshown). The lymphoma cell line JB6 gave positive results withthe same primer pair V�1–8:J�1/2.

Differential CD30 Expression of Lymphoma Cell Lines.Nine lymphoma cell lines were tested for CD30 expression (Fig.2). fluorescence-activated cell sorter analysis revealed threeexpression patterns. The cell lines JK-X, JK-A, and JK-Bshowed no detectable CD30 expression, whereas the cell linesJurkat and Mac2B showed weak CD30 surface expression. Incontrast to that, we found high CD30 expression on Karpas 299,Mac1, Mac2A, and JB6 cells. This is especially interestingbecause Mac1, Mac2A, and Mac2B were derived from differentbiopsies of the same patient, whereas JK-X, JK-A, and JK-Bwere derived from the same biopsy of another patient (Fig. 3).

Th2 Cytokines Are Dominantly Expressed in the Lym-phoma Cell Lines. The cytokine expression profile for IL-2,IL-4, IL-6, IL-8, IL-10, and IFN-� was tested for seven lym-

Fig. 2 Fluorescence-activated cell sorter staining of CD30 expressionof lymphoma cell lines. The thin line represents the isotype control; thethick line represents the HeFi-1 (anti-CD30) stainings. JK-X, JK-A, andJK-B are negative; Jurkat T-cell lymphoblastic lymphoma and Mac2Bare weakly positive, Mac1, Mac2A, and JB6 are strongly positive forCD30 expression and comparable with the known CD30 expression ofKarpas 299 and JB6, although the isotype control of JB6 exhibits a smallside peak. Peripheral blood leukocytes (PBL) of healthy donor served asa negative control.

Table 3 Cytokine expression profiles

Cell line

Th1-like cytokines Th2-like cytokines

IFN-�(units/ml)

IL-2(pg/ml)

IL-4(pg/ml)

IL-6(pg/ml)

IL-8(pg/ml)

IL-10(pg/ml)

JK-X 0.4 0 0 4034.0 5.3 2620.0JK-A n.d.a 0 0 n.d. n.d. 359.9JK-B 0.3 2.63 0.3 2297.0 5.3 499.2Mac1 1.2 0 3.8 0.2 10.7 0.3Mac2A 3.7 1.85 0.1 3947.0 6.4 478.0Mac2B 0 0 0 287.8 3.3 0Karpas 0 0 0 51.8 0 2568.0JB6 3.0 0 0.1 0.4 24.4 180.0Referenceb 0.1 10.00 0.1 3.1 0.1 8.0

a n.d., not done.b Normal serum levels.

Fig. 3 Origin and characteristics of the cell lines including their phe-notypes. � strong expression; �/� weak expression; � noexpression; n.d. not done.




Fig. 4 Kinetics of tumor cells. At indicated days (1, 4, 6, and 8) 20-�l aliquots of cells were removed from the culture, stained with 100 �l ofPBS/trypan blue and counted under a microscope. One visual field at �200 magnification has been counted. Living cells (A) and dead cells (B) werecounted separately. The experiments were done in triplicates. The symbols represent the mean. Statistically significant differences between the pairsCD30L versus % (nothing) and HeFi-1 versus iso (isotype) are indicated; bars, �SD.




Fig. 4 Continued.




phoma cell lines under standardized conditions, whereas JK-Awas tested only for IL-2, IL-4, and IL-10. Besides individualdifferences, the cell lines expressed more Th2 cytokines thanTh1 cytokines (Table 3). In detail, we see high levels (�100pg/ml) of IL-6 and IL-10, and low levels (10 units or pg/ml)of IFN-�, IL-2, IL-4, and IL-8 in Mac2A, JK-X, JK-B, andJK-A (note: JK-A was not tested for IFN-�, IL-6, and IL-8).Mac2B was high only for IL-6, whereas Mac1 showed noremarkable cytokine expression. Karpas 299 and JB6 showedhigh levels (�100 pg/ml) only for IL-10.

Specific Cell Growth Inhibition of CD30� LymphomaCell Lines by CD30L. After characterization of the cell lines,our main interest was to investigate the influence of CD30L andanti-CD30 mAbs on the proliferation of lymphoma cell cultures.As demonstrated in Fig. 4A, several effects could be observed.First, significant differences in tumor cell growth kinetics areseen in five cell lines (Karpas 299, Mac1, Mac2A, Mac2B, andJB6), all of them expressing CD30 on the surface. JB6 was theonly cell line showing significant growth inhibition with bothCD30L and HeFi-1. JK-X, JK-A, and JK-B, all CD30 negative,remained unaffected by CD30L or HeFi-1. Despite expressingsmall amounts of CD30, Jurkat cells remained unresponsive toboth reagents when compared with untreated or isotype controlcultures. All of the experiments were performed in parallel toassure comparable conditions. However, some lymphoma celllines, such as Mac2B, showed only marginal overall cell pro-liferation in this setting, whereas others (JK-B) reached an earlyplateau and subsequently decreased in numbers. Secondly, Fig.4A reflects different growth characteristics of the tested lym-phoma cell lines. In all of the cases the amount of starting cellswas the same. However, after transfer into microtiter plates theuntreated populations developed differently. JK-X, JK-A, Kar-pas 299, Mac1, and Jurkat showed increasing cell numberduring the first days in culture until a plateau was reached. Incontrast, JK-B, Mac2A, Mac2B, and JB6 became static or evendecreased in cell number probably because of adaptation prob-lems to the new culture conditions, because this phenomenonwas seen in both the treated and the untreated/isotype controlgroups of the respective cell cultures.

In correspondence to the kinetics of living cells we lookedat the development of the dead cell population. Significantdifferences were seen again for Karpas 299, Mac1, Mac2A, andto a certain extent for JB6 when treated with CD30L. However,treatment with HeFi-1 revealed no significant effect on the deadcell counts. In each case reduced amounts of dead cells wereobserved in the CD30L-treated cultures compared with thecontrol and HeFi-1 groups. Fig. 4B shows increasing numbers ofdead cells in all of the untreated or isotype-treated cultures overtime except for Mac2B. In Mac2B cultures we see high numbersof dead cells already after 1 day reflecting most likely adapta-tion of the population to new culture conditions, which isanalogous to what we have observed in the kinetics of livingMac2B cells. The overall increase of dead cell counts in thecultures (with the exception of Mac2B) took place after �4days, compared with the increase of living cells observed in Fig.4A. Therefore, we believe that exponential cell growth in ourcultures is limited by the experimental setting to �4 daysfollowed by an increased cell death that keeps the number ofliving cells at a relatively constant level. This hypothesis may

apply to Jurkat, JK-X, JK-A, Karpas 299, and Mac1. However,JK-B, Mac2A, Mac2B, and JB6 behave differently as describedabove.

To visualize the effect of CD30L on cell-cell interactions,we observed the lymphoma cells in vitro. CD30L inhibits theformation of colonies in CD30-positive Mac1 cells but not inCD30-negative JK-X cells as shown in Fig. 5. Additionally, theCD30L-treated Mac1 culture shows a large amount of celldebris.

CD30L-mediated Cell-Cycle Alterations. To addition-ally investigate the observed growth inhibition of lymphomacell lines by CD30L, we analyzed the DNA content of treatedcells and observed BrdUrd incorporation for a 10-h time inter-val. Karpas 299 and JB6 represent cell lines with high CD30expression levels, whereas Mac2B represents intermediateCD30 expression levels and JK-A represents cells with nodetectable CD30 expression, and serves as a negative control.Fig. 6A demonstrates a reduced proliferation of Karpas 299,Mac2B, and JB6 cells when incubated with CD30L, whereasJK-A remained unaffected by CD30L. At the same time, Fig. 6Brevealed differential results for apoptosis measurements. Here,under starving culture conditions, Karpas 299 showed signifi-cantly reduced apoptosis, whereas Mac2B and JB6 showedincreased apoptosis. Under starving culture conditions, CD30ligation reduces cell proliferation and simultaneously reducesapoptosis, but only in Karpas 299 (CD30 high) cells, whereasapoptosis in Mac2B (CD30 low) and JB6 (CD30 high) is in-creased. Under nonstarving culture conditions the proliferationof Karpas 299, Mac2B, and JB6 was inhibited by CD30L (Fig.6C), similar to what was observed in Fig. 6A for starving cells.A difference was observed for apoptotic values (Fig. 6D). Kar-pas 299 and, as a negative control, JK-A appeared unaffected byCD30L, whereas Mac2B and JB6 exhibited a slightly increasedapoptosis after CD30 ligation. Under nonstarving culture con-ditions, CD30 ligation inhibits proliferation of Karpas 299,

Fig. 5 Inhibition of colony formation on the culture plate bottommediated by CD30L. The pictures were taken at day 6 of culturing thecell lines with or without CD30L as described in “Materials and Meth-ods” under cell kinetic analysis. JK-X represents a CD30-negative andMac1 a CD30-positive lymphoma cell line.




Mac2B, and JB6 cells, and simultaneously increases apoptosisslightly in Mac2B and JB6 cultures, compared with the negativecontrol culture, JK-A. Taken together, we conclude that cellproliferation is reduced under both culture conditions byCD30L. Moreover, we found starving cells to be more suscep-tible to CD30L-mediated apoptosis than nonstarving cells withthe exception of Karpas 299 (Fig. 6E). Fig. 6E shows clearlythat Karpas 299 behaves differently than other cell lines. Here,under starving culture conditions Karpas 299 appears moreresistant to apoptosis upon CD30 ligation. Whereas BrdUrdincorporation, shown in Fig. 6, A and C, depict develop-ment (proliferation) of a population over a certain time period,propidium iodide staining with cell cycle analysis, as shown inFig. 6, B and D, represents a snapshot of the same population.

DISCUSSIONIn this study we analyzed eight anaplastic T-cell lymphoma

and one lymphoblastic T-cell lymphoma lines in terms of ge-notypic, phenotypic, and functional characteristics. Analysis ofTCR gene rearrangement is widely used for the detection of

T-cell clonality in lymphoproliferative diseases (31). We foundTCR-V� chain gene clonality in all eight of the anaplastic T-celllymphoma lines tested, although the band for JK-A was weak.The detected clones often underwent uncommon rearrange-ments using pseudogene JP1/2 as reported for Sezary patients(37), thus confirming the T-cell lymphoma origin of the celllines.

The marked increase in IL-6 secretion into the tissue cul-ture medium by Mac2A cells corresponds to our publishedresults on the comparative gene expression profile of the celllines. We could show a 20-fold amplification of IL-6 geneexpression by Mac2A cells over Mac1 cells (38). This findingcorresponded to the onset of systemic B symptoms, which arebest correlated with serum levels of IL-6 in large cell lymphomaand Hodgkin’s disease (39). In progression of disease, we alsoreported loss of response of Mac2A cells to growth inhibition bytransforming growth factor (25) because of an inactivatingmutation of the receptor type II (40).

Furthermore, cytokine profiling of the cell lines providednew results relevant to the ongoing debate about the T-helper1/2 dichotomy in the context of neoplastic T-cell disorders in theskin (41–45). In the murine model, two major subdivisions ofthe T-helper system have been defined. T-helper 1 clones se-crete mainly IL-2 and IFN-�, whereas Th2 clones produce IL-4,IL-5, IL-6, and IL-10 (46). This system of dichotomy is alsoapplicable to humans if Th 1/2 functions are understood aspredominant but not exclusive poles (41, 42, 47). Our cell linespreferentially express Th2-like cytokines (i.e., IL-4, IL-6, IL-8,and IL-10). This is in accordance with observations made byYagi et al. (48). They detected IL-10 mRNA in biopsies fromprimary cutaneous large cell lymphoma and lymphomatoidpapulosis. As a consequence, they could demonstrate beneficialeffects of a treatment with IFN-� that selectively inhibits thegrowth of Th2 cells (48). Th2 cytokine mRNA was detected inskin lesions at all stages of the disease (47). Indeed, IFN therapyhad become an important treatment modality in lymphoprolif-erative disorders (49–52). On the basis of our genotypic andphenotypic analysis, we feel encouraged that the lymphoma celllines we studied closely represent the original malignancy and,therefore, serve as a suitable in vitro model for the biology andtreatment of human cutaneous lymphoproliferative disorders.

Because CD30L was detected in regressing cutaneouslymphoid lesions and often exhibits coexpression with CD30(14), we explored the possible influence of recombinant CD30Lon established lymphoma cell lines. We hypothesized thatCD30-CD30L interactions in vivo may have an impact on themechanism of self-regression of cutaneous lymphoma lesions.In our cell kinetic studies the two endpoints we measured, livingcells and dead cells, complemented each other to provide adynamic picture of the tumor cell population. In general, we sawinhibition of proliferation when CD30-positive cell lines wereincubated with CD30L. These findings are supported by datapublished recently, showing that soluble trimeric CD153(CD30L) was effective in triggering cell death of target cellswith membrane anchored CD30 (53). This is in contrast to someof the effects reported with HeFi-1. Franke et al. (18) haveconcluded that HeFi-1 does not directly induce CD30 signaling,which additionally stimulated our interest to directly test theeffects of CD30L. The proliferative effect of cross-linking

Fig. 6 Proliferation and cell cycle analysis 36 h after CD30L treat-ment. In A and C, the amount of cells that have incorporated BrdUrdwithin the last 10 h of the experiment is expressed as % proliferation. InB and D, the hypodiploid peak of DNA content was measured andexpressed as % apoptosis. The bars represent the mean of three indi-vidual experiments; bars, �SD. E summarizes the results of A–D. Thedifference between nontreated and CD30L-treated cells is given for eachexperimental setting.




CD30 with HeFi-1 on cutaneous lymphoma cells, as describedin the literature, can be explained by nuclear translocation andactivation of NF�B (15). The intracellular domain of CD30 canbind tumor necrosis factor receptor-associated factor, resultingin activation of NF�B. According to Mir et al. (24), NF�B playsthe determining role in the sensitivity or resistance of lymphomacells to CD30-induced apoptosis. The quality of the signal(binding site, affinity) provided by CD30L or HeFi-1 binding toCD30 may be different and, thus, may influence the resultingbiological effect.

Pleiotropic effects of CD30 activation have already beendescribed by Gruss et al. (54). They found induction of celldeath of systemic CD30� ALCL after CD30L exposure. Incontrast, it has been shown that CD30L enhances proliferationof peripheral T cells and the Hodgkin’s cell line HDLM-2associated with tyrosine kinase phosphorylation of a cytosolicprotein but exhibit an opposite effect on ALCL cell lines with-out tyrosine kinase phosphorylation (55). Schneider and Hub-inger (56) stated recently that CD30-mediated signaling canpromote cell proliferation as well as cell death depending on celltype and costimulatory signals. In that light, we believe thatdifferent parameters influence the fate of lymphoma cells onCD30 activation. Firstly, the microenvironment or cell cyclestate at time of CD30 activation is of importance, whetherlymphoma cells are starving or in a logarithmic growth phase.This would be similar to the action of certain drugs, e.g.,methotrexate, and is important for planning treatments involvingCD30 activation. Secondly, the mode of receptor cross-linkageinfluences the transduced signal, and thirdly, the receptor den-sity on the lymphoma cell contributes to signaling effects. Itmight be possible that under starving conditions the level ofmembrane-bound CD30 changes, increasing susceptibility toCD30L-mediated cell death, as we have observed for Mac2Band JB6, or vice versa. These questions will be addressed inadditional experiments. Interestingly, Hubinger et al. (57) ob-served different effects of CD30 activation in Karpas 299 andJB6 lymphoma cell lines. Whereas Karpas 299 responded withgrowth inhibition, JB6 appeared unaffected by treatments withanti-CD30Abs. Consistent with our results (23), growth arrest ofKarpas 299 cells correlated with up-regulation of the cell cycleinhibitor p21 rather the induction of apoptosis (57).

Other experimental approaches indicate that CD30 canserve as a suitable target for tumor lysis (29, 58, 59). Hombachet al. (58) demonstrated that lymphoma cell cross-priming withrecombinant anti-CD30-� receptor-transfected T cells can effectspecific lysis of lymphoma cells. We had already shown that animmunotoxin consisting of Saporin-6 conjugated to an anti-CD30 Ab, Ber-H2, has potent antitumor activity in vitro and insevere combined immunodeficiency disease mice engraftedwith human CD30� systemic ALCL (29). Huhn et al. (59)found that a newer CD30L-based fusion toxin (Ang-CD30L)using the human RNAase angiogenin, exhibits specific cytotox-icity against CD30-positive Hodgkin’s lymphoma cell lines.Our current study suggests that naked CD30L, without toxin,also has the potential for treatment of human CD30� ALCL.We provide evidence that CD30 ligation can inhibit cell prolif-eration; additionally, we show a slightly increased rate of ap-optosis that can be enhanced when cells were kept under starv-ing culture conditions with the exception of Karpas 299 cells.

Karpas 299 cells, in contrast, exhibit reduced apoptosis levelsand proliferation under starving culture conditions indicating acell cycle arrest.

ACKNOWLEDGMENTSWe thank Geri Barmettler for excellent technical assistance and for

performing the DGGE-PCR analysis. Additionally, we thank the De-partment of Clinical Immunology at the University Hospital Zurich forcytokine analysis.

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2003;9:2744-2754. Clin Cancer Res Jörg Willers, Reinhard Dummer, Werner Kempf, et al. CD30 Ligand

RecombinantInhibited by CD30 Receptor Cross-Linking with Proliferation of CD30+ T-Helper 2 Lymphoma Cells Can Be

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Proliferation of CD30 T-Helper 2 Lymphoma Cells Can Be ...clincancerres.aacrjournals.org/content/clincanres/9/7/2744.full.pdf · Proliferation of CD30 T-Helper 2 Lymphoma Cells Can