Proc. Natl. Acad. Sci. USAVol. 78, No. 4, pp. 2502-2506, April 1981Immunology

Cytotoxic T lymphocyte hybridomas that mediate specifictumor-cell lysis in vitro

(allogeneic murine cytotoxic T lymphocytes/fusion/clones/immune killing)

YAEL KAUFMANN*, GIDEON BERKE *, AND ZELIG ESHHARtDepartments of *Cell Biology and tChemical Immunology, The Weizmann Institute of Science, Rehovot, Israel

Communicated by Michael Sela, December 29, 1980

ABSTRACT Cytotoxic hybridomas were generated by poly-ethylene glycol-induced fusion of cytotoxic T lymphocytes (CTL)and BW5147 lymphoma cells. The CTL populations used for fusionwere obtained from BALB/c (H-2d) mice primed with leukemiaEL4 ofC57BL/6 (H-2b) and restimulated either in vivo or in vitro.To circumvent possible CTL-mediated nonspecific lysis of BW5147cells during fusion, the CTL were transiently inactivated by tryp-sin prior to fusion. Four cytolytically active hybridomas were ob-tained, cloned, and subeloned. Hybrid clones lysed all H-2b leu-kemic target cells tested but not lipopolysaccharide- or concanavalinA-stimulated C57BL/6 lymphoblasts or non-H-2b target tumorcells. The mechanism ofhybridoma-mediated killing oftarget cellsin vitro appears to be similar to that of parental CTL, althoughsome differences have been observed. The hybridomas appear topossess neither natural killing nor antibody-dependent cytolyticactivity. Clones ofhybrids propagated in culture for over 6 monthswithout the addition ofknown external stimulus (i.e., independentof cell growth factor and antigen) exhibit specific lytic activityagainst H-2b tumor cells. Such autonomous hybridomas will pro-vide a tool for studying the mechanism of CTL-mediated lysis andthe nature of the CTL receptors.

Cytotoxic T lymphocytes (CTL) are well-known effector cellsin transplantation immunity, viral infection, and certain anti-tumor responses. Despite considerable investigation, themechanism by which CTL specifically interact with and lysetarget cells (TC) is still largely unknown. The development oftechniques for obtaining massive quantities of monoclonal CTLwould greatly enhance the analysis ofthe structure and functionof the CTL receptors. Recently, functional CTL have been suc-cessfully maintained in long-term culture by continuous anti-genic stimulation, addition ofT cell growth factors (TCGFs), orboth (1-3). However, the quantity of CTL obtained by suchmethods is limited, and repetitive stimulation with antigens orfactors is mandatory for CTL proliferation and lytic activity.Another approach that has proven very useful for obtaining con-tinuous cultures of functional lymphocytes is somatic cell hy-bridization. Fusion of antibody-forming lymphocytes with my-eloma cells has been successful for generation of hybridomassecreting monoclonal antibodies (4). Functional T cell hybri-domas mediating suppression (5, 6) or help (7) in antibody re-sponses have recently been obtained, but attempts to immor-talize CTL by somatic cell hybridization have been unsuccessful,possibly due to CTL-mediated nonspecific lysis of the fusionpartner during hybridization (8, 9). Recently a TCGF-depen-dent CTL hybridoma has been described (10). The present pa-per describes the generation offunctionally specific and TCGF-independent CTL hybridomas.t CTL obtained by secondaryin vivo or in vitro stimulation have been transiently inactivatedby trypsin and then hybridized with BW5147 (BW) thymoma

cells. The resulting hybridomas, capable of specific TC lysis invitro, have been cloned and propagated in culture for over 6months without the addition of any known antigenic stimulusor TCGF.

MATERIALS AND METHODSMice, Tumors, and Lymphoblasts. C57BL/6 gH-2b),

BALB.B10 (H-2b), BALB/c (H-2d), and DBA/2 (H-2 ) micewere provided by the Animal Breeding Center of this Institute.C57BL/6 leukemia ELM and thymoma ALC5, DBA/2 masto-cytoma P815, BALB. B10 thymoma ALB. B2, and BALB/c thy-moma ALB. 1 were maintained in ascitic form by passage in syn-geneic mice. The A. Sn (H-2a) leukemia YAC (obtained from R.Herberman, National Institutes of Health) and AKR/J (H-2k)thymoma BW5147 (a hypoxanthine phosphoribosyltransferase-negative line, obtained from G. Kohler, Basel Institute), werepropagated in culture. YAC cells were transferred in RPMI-1640 medium supplemented with 5% fetal calf serum (GIBCO)and BW cells in Dulbecco's modified Eagle's medium (DMEmedium) supplemented with 15% fetal calf serum. Lympho-blastoid cells were generated in culture by 3-day incubation of10 x 106 C57BL/6 spleen cells in 10 ml of mixed lymphocyteculture (MLC) medium (RPMI-1640 medium containing 5%fetal calf serum, 50 ,AM 2-mercaptoethanol, antibiotics, and 5mM sodium Hepes, pH 7.4) supplemented with either conca-navalin A (Con A; twice crystallized, Miles-Yeda) at 2 jig/mlor lipopolysaccharide (LPS; Difco) at 25 ,ug/ml.

Preparation of CTL, Generation of Hybridomas, and Clon-ing. Peritoneal exudate CTL (PEL) were prepared as described(13) from BALB/c mice 5 days after a secondary intraperitonealinjection of25 x 106 EL4 cells. MLC CTL were prepared fromBALB/c splenocytes, primed 6-9 weeks earlier with EL4 cellsand restimulated with irradiated [ 1300 roentgens (0. 33 coulomb/kg)] C57BL/6 splenocytes in vitro. After 6 days in culture, deadand adherent cells were removed by sedimentation through aFicoll/metrizoate gradient followed by passage through nylonwool columns. Prior to fusion, CTL were transiently inactivatedby controlled trypsin digestion. PEL (8 x 106 per ml) or MLCcells (5 x 106 per ml) were incubated in phosphate-bufferedsaline supplemented with 10% fetal calf serum and trypsin(trypsin-TPCK, Worthington, 246 units/mg) at 1.8 mg/ml for

Abbreviations: CTL, cytotoxic T lymphocyte(s); TC, target cell(s); PEL,peritoneal exudate lymphocyte(s); MLC, mixed lymphocyte culture;DME medium, Dulbecco's modified Eagle's medium; DME/20 orDME/15, DME medium with 20% or 15% fetal calfserum, antibiotics,and 2 mM L-glutamine; PHA, phytohemagglutinin M; Con A, conca-navalin A; LPS, lipopolysaccharide; TCGF, T cell growth factor; HATmedium, hypoxanthine/aminopterin/thymidine medium; Tos-LysCH2Cl, N-tosyl-L-lysyl chloromethyl ketone.t Preliminary reports of this work have been presented: (i) AnnualMeeting of the Israel Immunology Society, Haifa, May 1980 (ref. 11);(ii) Seventh International Congress of the Transplantation Society,Boston, June 1980 (ref. 12).

The publication costs ofthis 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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Proc. Natl. Acad. Sci. USA 78 (1981) 2503

30 min at 37°C. The cells were washed three times in standardmedium (RPMI-1640/10% fetal calf serum/10 mM sodiumHepes, pH 7.4) and kept on ice until used. Trypsin treated PELor MLC (40 X 106 cells) were allowed to fuse with 20 x 106 BWcells in the presence of 41% (wt/vol) polyethylene glycol 1500(Serva) (7, 14). After fusion, the cell mixtures were suspendedin DME medium supplemented with 20% fetal calf serum, an-

tibiotics, and 2 mM L-glutamine (DME/20) and plated in 24-well Costar plates, 5 x 105 cells per well. The plates were in-cubated at 37°C in a humidified atmosphere containing 10%CO2. After 24 hr, selection against BW cells was begun by theaddition of DME/20 medium containing hypoxanthine, ami-nopterin, and thymidine (14) (HAT selection medium). After3 weeks aminopterin was omitted from the selection medium,and a week later cultures were transferred to DME/15 (DMEmedium plus 15% fetal calf serum), a routine medium for long-term culture of hybrid cells. Clones and subelones of hybridcells were isolated on soft agar as described (15).

Cytotoxicity Assays. To determine cytolytic activity, 1 X 104to 4 X 105 hybrid cells were mixed with 1 x 104 51Cr-labeledTC in 0.2 ml ofstandard medium, in V-shaped microtiter plates,centrifuged (200 X g, 5 min, 20°C), and incubated at 37°C forthe indicated times (see legends). Cytolytic assays were stoppedby cooling. Aliquots (0. 1 ml) ofsupernatants were removed aftercentrifugation (450 X g, 10 min, 4°C) and their radioactivity wasmonitored. All assay points represent means of triplicate cul-tures. When indicated, 2 ,g of phytohemagglutinin M (PHA,Difco) was included in the assay medium. In some experiments(2 X 106) hybrid cells and 51Cr-labeled TC were treated sepa-rately with 15 units of neuraminidase (Behringwerke) in 0.2 mlof phosphate-buffered saline for 15 min at 37°C and washedprior to the lytic assay. Percent lysis was calculated as follows:

100 x

cpm released with effectors - cpm released spontaneously0.75 X total radioactivity - cpm released spontaneously

RESULTSGeneration and Characterization of the CTL Hybridomas.

Two measures were taken to increase the probability of CTLhybridoma formation. First, the CTL preparations used for fu-sion contained a high proportion (30-50%) of effector cells ca-

pable ofbinding to and lysingTC (ref. 16 and unpublished data).Second, the CTL were temporarily inactivated by trypsin diges-tion to minimize potential nonspecific lysis of the BW cells at-tached to the CTL during fusion (8, 9). Regeneration of lyticactivity was expected to occur after hybridization, because tryp-sin-treated CTL regain full lytic capacity after 3- to 4-hr incu-bation at 37°C (12, 16, 17).

Fusion of20 X 106 BW cells with 40 X 106 BALB/c anti-ELACTL generated in vitro (MLC) or in vivo (PEL) resulted in cellgrowth of 86/96 or 83/96 of the cultures originally seeded inHAT medium, respectively (12). The cultures were screenedfor lytic activity against 51Cr-labeled EM4 TC (Table 1). PHA,a lectin known to support non-specific CTL-mediated lysis (18),was added to one set of the test cultures to promote detectionof hybrid cells capable of lysis but not expressing functional re-

ceptors for specific TC binding. Several hybridoma culturesderived from both MLC and PEL CTL lysed TC in the presenceof PHA (Table 1), and two of such cultures derived from eachCTL preparation also lysed EL4 in the absence of the lectin.When lectin-independent hybrids were cloned and subclonedon agar, over 80% of the subclones were cytolytic (12). Twohighly reactive subclones, P. 13.47 (PEL hybrids) and M. 18.40(MLC hybrids), whose properties are described-in this report,

Table 1. Frequency of cytolytic hybridomasCytolytic cultures/

PHA in total cultures*Hybrid cells assay PEL hybrids MLC hybrids

Original cultures - 2/83 2/86+ 6/83 7/86

Third cloning cyclet - 6/9 46/47+ 9/9 20/20

* Cultures mediating over 10% lysis of EL4 TC in 3 hr were scoredpositive.

t Cytolytically active hybridomas derived from original (PHA-inde-pendent) cultures were cloned and subcloned on soft agar (12, 15). Thesubclones P.13.47 (PEL hybrid) and M.18.40 (MLC hybrid) were fur-ther subcloned by limiting dilution (0.2 cell per well in 0.1 ml ofDME/15 medium). Hybrid cells and EL4 were treated with neuraminidaseprior to the lytic assay (3 hr, effector-to-TC ratio 10:1).

were tested for homogeneity by cloning in limited dilution(Table 1). Most of the progeny of M. 18.40 and two-thirds ofP.13.47 lysed EIA TC. Moreover, all the progeny exhibitedlectin-dependent TC lysis, reflecting the homogeneity of theseCTL hybrid subclones.The hybrid nature of subclones P.13.47 and M. 18.40 was

verified by karyotype and surface antigen analyses (Table 2).Both lines contained aneuploid numbers of chromosomes, av-eraging 67 and 63 for P.13.47 and M. 18.40, respectively. Thehybrid cells co-expressed on their surfaces the H-2 and Thy 1alloantigens derived from both parental lines. Thus, all hybridcells were stained by anti-H-2k and anti-Thy 1.1 antibodies (BWphenotype) as well as by anti-H-2d and anti-Thy 1.2 antibodies(CTL phenotype). Another characteristic of the functional sub-clones is their rapid proliferation (Fig. 1): they reached densitiesof 3-4 x 106 cells per ml with a doubling time of about 10 hr.Thus, according to the above criteria, the newly derived cellsare true cell hybrids that acquired, along with the cytolytic ac-tivity ofthe parental CTL, the continuous rapid growth capacityof the BW tumor cells.

Table 2. Surface markers and karyotypes of functionalhybridoma subclones

% fluorescent cellsHybridoma

Surface Parental cells subclonesmarkers* PEL MLC BW P.13.47 M.18.40

H-2d 99 100 3 99 95Thy 1.2 96 92 1 100 99H-2k 1 2 98 99 96Thy 1.1 0 4 97 100 98

Chromosome numbert 40 40 41 67 63(41-45) (64-69) (60-65)

* Surface markers were examined by indirect immunofluorescence.Monoclonal anti-Thy 1.1 and anti-Thy 1.2 antibodies were a gift fromA. Marshak-Rothstein (Massachusetts Institute of Technology).Anti-H-2d serum was produced in C57BL/6 mice immunized againstP815 mastocytoma of DBA/2. Anti-H-2k serum was produced inC3H.SW (H-2b) mice immunized against C3H.DiSn (H-2k) spleno-cytes. Cells were first allowed to react with either anti-Thy 1 (1:250)or anti-H-2 (1:25) sera. Fluorescence was then developed with fluo-rescein-conjugated goat antibody against Fab fragments of mouseIgG (obtained from J. Haimovich). At least 200 cells were examinedfor cell surface fluorescence in each sample. Less than 7% ofthe cellswere stained by normal mouse serum (1:25). Hybridomas were ex-amined 15 weeks after fusion.

t Karyotypes were analyzed 17 weeks after fusion (19). Ranges aregiven in parentheses.

Immunology: Kaufmann et al.

2504 Immunology: Kaufmann et al.

c)

04

xCo

X5,

FIG. 1. Growth curves of theD 100 - hybrid cells. Hybridoma sub-

clones P.13.47 (E) and M.18.400z) t-/ (o) grown in culture for 20 weeks. were seeded at the indicated con-. centration in DME/15 medium.

Viable cells were scored in 0.1%10 trypan blue in phosphate-buff-0 20 40 ered saline; cell viability was

Time in culture, hr over 90%.

Cytolytic Activity of the Hybridomas. The kinetics of hy-bridoma-mediated lysis of 51Cr-labeled EL4TC is shown in Fig.2. After a 2-hr lag period, gradual increase in TC lysis was ob-served; .70-80% specific EMU. lysis was reached after 6 hr. Treat-ment ofthe hybridomas and EL4 cells with neuraminidase priorto the assay increased the rate of lysis but did not shorten thelag period. As shown in Tables 3 and 4,.the enhanced lysis ob-served following neuraminidase treatment was not due to stim-ulation of non-specific lysis. The capacity of the hybridomas tolyse two H-2b tumors, leukemia EL4 ofC57BL/6 and thymomaALB. B2 of BALB. BlO mice, was examined at various effector-to-TC ratios (Fig. 3) in a 3-hr assay. Lysis of both tumors wasmaximal at a hybrid-to-TC ratio of 5:1-10:1. Interestingly, athigher hybrid-to-TC ratios, lysis was markedly reduced. Inhi-bition at hybrid cell excess was less pronounced after neur-aminidase treatment.To test the specificity of lysis mediated by the CTL hybri-

domas, several tumors and normal target cells were used (Table3). Both MLC and PEL hybridomas lysed only tumor cells ofthe same phenotype as that of EL4 (H-2b), against which theparental CTLwere originally induced. Parental CTL and hybridcells lysed EL4 TC more effectively than two additional leu-kemias bearing the H-2b haplotype, ALC5 and ALB.B2. Nor-

6 8 0Time, hr

FIG. 2. Kinetics of hybridoma-mediated EL4 lysis. PEL hybridsP.13.47 andMLC hybrids M.18.40 (5 x 105 cells) were mixed with 51Cr-labeled EL4 (0.5 x 105 cells) in 1 ml of standard medium in 12 x 75mm Falcon tissue culture tubes, centrifuged (200 x g, 5 min, 20TC) andincubated at 37TC for the indicated time. Assays were stopped by ad-dition of 1 ml of ice-cold phosphate-buffered saline and the 5"Cr re-

leased to the supernatants was determined after centrifugation. Val-ues represent means of triplicate counts, which varied by less than 5%.* and *, Hybrid and EL4Mcells were pretreated with neuraminidase;r and a, no enzymatic pretreatment. Spontaneous release from treatedor nontreated EL4 cells was. 23% after 7-hr incubation.

60 '''I ' I ',I "I_EL4 ALB.B2

~40 -

~20-

Cl1 4 10 40 1

Hybrid-to-TC ratio4 10 40

FIG. 3.. Cytotoxicity ofhybridomas at various effector-to-TC ratios.Lysis ofEL4 and ALB.B2 TC by P.13.47 (circles) and M.18.40 (squares)hybridomas was tested in a 3-hr standard assay. a and *, Neuramin-idase pretreatment; o and o, no enzymatic treatment. Spontaneousrelease values from EL4 or ALB.B2 were 21% or 17% from treated TCand 20% or 15% from nontreated TC, respectively.

mal C57BL/6 spleen cells stimulated with Con A or LPS, andtumor cells derived from H-2d, H-2a, and H-2k mice, were notlysed by the CTL hybridomas. Pretreatment ofhybrids and TCwith neuraminidase increased the lysis of all the H-2b tumorcells examined but did not lead to lysis of normal H-2b lym-phoblasts or non-H-2b tumors. That neuraminidase pretreat-ment does not stimulate normal splenocytes and BW cells tolyse ELM TC is shown in Table 4. The-killing specificity of thehybridoma subelones was restricted to H-2b leukemic TC,whereas the parental CTL lysed normal H-2b blast cells as well(Table 3), suggesting that these hybrid clones originated froma subpopulation of the parental CTL. It should be noted thatthe tumor cells that were not susceptible to lysis all carry theH-2 antigen of the cytotoxic hybridomas; therefore additionalTC carrying other H-2 antigens should be examined. The ob-servation that YAC cells, known to be highly sensitive to naturalkillers (21), were not lysed by the hybridomas suggests that thehybrid cells are not natural killers. In addition, hybridoma sub-clones that specifically killed EL4 did not exhibit antibody-de-pendent cell-mediated cytotoxicity (Table 4), in contrast tospleen cells of nonimmunized mice, which did not kill EL4 TCbut efficiently lysed chicken erythrocytes coated with the ap-propriate antibodies.

In preliminary experiments the requirements of hybridoma-mediated TC lysis (Table 5) appeared similar to those for thelysis induced by the parental CTL (13, 23, 24); both lyses oc-curred at 370C but not at 21'C and both were inhibited byEDTA and cytochalasin B. In addition, the lytic activity of thehybridomas could be abolished by trypsin or by the trypsin in-hibitor N-tosyl-L-lysyl chloromethyl ketone (TosLysCH2Cl)(Table 5), as is the case with CTL (12, 17, 22). These similaritiessuggest that the hybridomas employ the CTL recognition andkilling mechanism in TC lysis.

'DISCUSSIONThe monoclonal CTL hybridomas described in this study arestable, functionally specific cell lines that provide a tool forstudying the mechanism ofCTL-induced lysis and the structureof the CTL receptor(s) for TC recognition. The hybrid clonesderived from PEL and MLC CTL multiply rapidly in culture,having a doubling time of 10 hr (Fig. 1) and reaching a densityof3-4 x 10' cells per ml, whereas antigen- or TCGF-dependentCTL lines have longer doubling times and reach considerablylower cell densities (1-3, 25). The cloned CTL hybridomas re-tain specific killing activity while growing continuously for overhalf a year in a standard medium independent of a known an-tigenic stimulus or detectable TCGF. Thus these hybrids are

Proc. Natl. Acad - Sci. VSA 78 (1981)

Proc. Natl. Acad. Sci. USA 78 (1981) 2505

Table 3. Specificity of hybrid-mediated TC lysisLysis, %*

Parental CTL Hybrid cells

H-2 PEL MLC P.13.47 M.18.40Mouse haplo- Stand- Neuramin- Stand- Neuramin- Stand- Neuramin- Stand- Neuramin-

TC strain type ard idase ard idase ard idase ard idaseEL4 C57BL/6 b 51 68 72 72 44 75 59 82ALC5 C57BL/6 b 38 48 64 63 37 47 52 63ALB.B2 BALB.B10 b 24 42 53 60 19 46 26 48

Con A blast C57BL/6 b 18 30 20 20 -5 -14 -19 -17LPS blast C57BL/6 b 25 38 37 34 -6 -7 0 0

BW5147 AKR/J k 3 9 8 9 1 1 1 1ALB1 BALB/c d 1 2 1 1 -18 -11 -20 -17P815 DBA/2 d 0 0 0 1 -1 0 -1 -1YAC A/Sn a 11 10 6 5 -2 0 -3 -2

* Cytotoxicity was tested in tubes (see legend to Fig. 2) at an effector-to-TC ratio of 9:1. Incubation was for 1 hr with CTL and for 6 hr with hybridcells. Spontaneous release values were 5-10% in 1 hr and 15-25% in 6 hr. Negative values represent experimental values lower than spontaneousrelase. Effector cells and TC were treated with neuraminidase in the nonstandard reactions.

in an activated stage, constitutively expressing the apparatus forTC recognition and killing.

The CTL hybridomas presented here are clearly differentfrom the TCGF-dependent hybridomas reported by Nabholzet al. (10). In the latter case, TCGF is essential for the expressionof hybrid cell cytotoxicity. The use of TCGF-dependent CTLlines for hybridization (10) may explain why the resultant hybridcells exhibit TCGF dependence, although this point remainsto be clarified. The successful production of cytotoxic hybri-domas in the present study may be attributed to the transientinactivation by trypsin of the CTL prior to fusion. However,because no trypsin treatment of the effector cells preceded fu-sion in the study by Nabholz et al. (10), and because Grutzmannand Hammerling (9) and Kohler et al. (8) reported that underfusion conditions BW cells are lysed nonspecifically by CTL,it is clear that the mechanism of CTL-BW cell fusion resultingin CTL hybrid formation needs to be studied in greater detail.The CTL lines reported here are genuine hybridomas re-

sulting from fusion of CTL and BW lymphoma cells. Four cri-teria support this conclusion: (i) The hybridomas co-express cellsurface antigens (H-2 and Thy 1) of both parental cells (Table2). (ii) Hybrid cells possess over 60 chromosomes, a substantialdeviation from the normal diploid chromosome number of 40.(iii) Hybrid cells display cytolytic activity that is independentof TCGF and yet grow in HAT selection medium. BW cells

Table 4. The hybridomas do not mediate antibody-dependentcell cytotoxicity

Lysis, %

EL4 lysis'Effector cells ADCC* Standard Neuraminidase-

P.13.47 hybridoma 2 23 63M.18.40 hybridoma 2 19 47Normal BALB/c spleen 65 -1 0BW5147 0 -3 -3

* Antibody-dependent cell cytotoxicity (ADCC) was tested on 1x 10551Cr-labeled chicken erythrocytes in the presence of rabbit anti-chicken serum (final dilution 1:100,000) with 1 x 10hybrid or spleencells in 0.2 ml of standard-medium. Incubation was for 18 hr at 37°C(20); spontaneous release was 3%.

t Lysis ofEL4 TC was tested for 3 hr at 10:1 effector-to-TC ratio; spon-taneous release was 14% for standard treatment and 17% for pre-

treatment of effector and ETA cells with neuraminidase.

neither grow in HAT medium nor express cytolytic activity.Parental CTL, on the other hand, require either TCGF or con-

tinuous antigenic stimulation for in vitro growth and expressionofcytotoxicity (1-3). (iv) The average diameter ofthe hybridomacells is about twice that of the BW thymomas and thrice thatof parental CTL. In addition, hybridoma cells appear pleo-morphic, displaying a distinct uropod structure.The killing ofTC by CTL hybridomas appears to be similar

to that mediated by parental CTL. This conclusion is based on

the following findings: (i) Hybridoma-mediated TC lysis, likeCTL-mediated killing, appears to require direct contact be-tween effector and TC and is not mediated by a soluble com-

ponent released into the medium (12). (ii) Trypsin and the tryp-sin inhibitor TosLysCH2Cl inhibit killing both by CTL (12, 17,22) and by the hybridomas (Table 5). (iii) Lysis of TC by CTLand by the hybridomas requires temperatures above 21°C (13,23) and is inhibited by EDTA and cytochalasin B at concentra-tions shown to affect CTL-mediated lysis (Table 5) (23, 24).These observations suggest that elevated temperatures, biva-lent cations (Ca2+, Mg2+, or both), and microfilaments are es-

sential for both killing processes. The similarities in the killingby CTL hybridomas and parental CTL are substantiated by lackof natural killing and antibody-dependent cell cytotoxicity ob-

Table 5. Requirements for hybrid-mediated TC lysis

Lysis, %Treatment Assay conditions* P.13.47 M.18.40

Standard 23 27Neuraminidase

(75 units/ml) Standard 53 47Trypsin

(2 mg/ml) Standard 0 NTTosLysCH2Clt Standard -1 -7Neuraminidase Low temp. (21°C) 0 0Neuraminidase EDTA (10 mM) 1 0Neuraminidase Cytochalasin B 0 8

(5 ,ug/ml)

NT, not tested.* Standard assay; 3 hr at 37°C, hybrid-to-EL4 ratio 10:1.t Hybrid cells were pretreated with 0.1 mM TosLysCH2Cl in standardmedium for 1 hr at 37°C, which inactivated parental CTL cytotoxicity(ref. 22 and unpublished data). After treatment the inhibitor wasremoved by washing and cytotoxicity was tested.

immunology: Kaufmann et al

2506 Immunology: Kaufmann et al.

served for both cells (Tables 3 and 4). Collectively these ob-servations suggest that the hybridoma cells utilize the CTL rec-ognition and killing mechanisms to bring about specific TC lysis.The kinetics of TC lysis (Fig. 2) showed that the majority

of ETA TC are lysed in 6 hr by both PEL- and MLC-derivedhybridomas. However, a 2- to 3-hr lag period was observedbefore considerable lysis could be detected. The reasons for thelag period and the relatively low efficiency of hybridoma-me-diated lysis are not known at present. Because the surface areaof hybrid cells is about 9 times larger than that of CTL, it ispossible that the number of receptors per unit surface area is9 times lower, thus accounting for a lower avidity for the TC.In addition, the reduced rate of lysis may be due to a rate-lim-iting step of receptor clustering necessary to achieve a criticalreceptor concentration prior to onset of killing. The rate of thehybridoma-mediated cytotoxicity increased after pretreatmentof both effectors and TC with neuraminidase (Fig. 2). This en-hancement was mainly due to pretreatment of targets rather'than treatment of hybrid cells (data not shown). It is, therefore,possible that removal of sialic acid residues from the TC surfaceenables hybrid cells to interact more effectively with TC, dueto exposure of masked antigens or reduction of repulsive forcesthat prevent effector-TC interaction. Maximal lysis ofEL4 andALB.B2 TC was obtained at hybrid-to-TC ratios of 5-10:1 (Fig.3). At large hybridoma excess (40:1) lower or no TC lysis wasdetected. Although all hybridoma cells in the assay may be con-sidered potential killers, as is evident from the activity of allhybrid clones (Table 1), it is conceivable that only a fraction ofthe population is cytolytic at a given stage of the cell cycle,whereas the rest may interfere with lysis by occupying TCthrough abortive binding or simple steric interference. Exam-ination of the killing activity of hybrid cells cultured at differentcell densities showed maximal killing with effector cells grownto densities of 0.5-2 X 106 cells per ml and less killing whenhybrid cells grew to 2-4 X 10. per ml, supporting the notionthat CTL hybrids may be more effective in TC lysis at certainstages of growth.

Hybridoma-mediated TC lysis appears to be immunologi-cally specific, because hybrid cells originated from BALB/canti-EL4 CTL (anti-H-2b) kill EL4, ALC5, and ALB. B2 (H-2b)tumor cells but not P815 and ALB1 (H-2d), BW (H-2k), or YAC(H-2a) cells (Table 3). Interestingly, hybrid-mediated killingappears to be restricted to H_2b leukemia cells, because normallymphoblastoid cells (Con A- or LPS-transformed spleen cells)were not susceptible to lysis (Table 3). Unlike the hybridomaclones tested, which exhibited restricted specificity, the het-erogeneous parent CTL populations kill normal and tumor TCof the appropriate H-2 type (Table 3). The existence of an ef-fector-cell subpopulation among BALB/c anti-EL4 CTL, whichkill EL4 but not normal H-2b TC, was previously suggested byKedar and Bonavida (26). The CTL hybrid clones describedhere might have originated from such a subpopulation whoseactivity is not solely directed against H-2b antigens but whichis specific for a viral or tumor antigen and restricted by H-2b.The nature of the TC antigenic determinants, whether majorhistocompatibility complex (K, D, or Ia) or viral products, whichare recognized by the hybrid cells, is not yet known. Detectionof hybrid cells that lyse tumor but not normal TC may be dueto preferential fusion oftumor-specific CTL, or it may be a resultof the selection procedure in which EL4 TC were exclusivelyutilized in the initial screening for cytotoxic hybridomas. Wewould predict that screening for lytic activity against normal TCwould reveal H-2b specific cytolytic hybridomas.

Some interesting subclones have been isolated by limitingdilution (Table 1); their lytic activity is expressed only in the

presence of lectin (PHA). These variants could have been de-rived from fully functional CTL hybridomas that lost the geneticinformation responsible for TC binding while retaining cyto-toxic potential. A similar lectin dependence ofcontinuous CTLlines (3, 25) and of several lymphomas (27) has been previouslyobserved. Dissociation of the binding from the killing mecha-nisms of CTL has previously been achieved by hyperthermiaor formaldehyde treatment (16). The detection of hybrid vari-ants that lyse TC only in the presence of lectin supports thenotion that this separation is genetically regulated. As soon asthe karyotype of these variants is available, specific chromo-somal assignments to CTL binding and killing functions will bepossible. Moreover, the lectin-dependent hybridomas and ad-ditional nonfunctional variants may serve as an ideal tool in re-lating CTL function to structure.

We thank Ms. T. Waks and Mrs. T. Oren for their skillful assistanceand Drs. C. Webb and B. Schick for critical reading of the manuscript.G.B. is an Isaac and Elsa Bourla Professor of Cancer Research. Z.E.is an incumbent of the Recanati Career Development Chair in CancerResearch. This research was supported in part by National Institutesof Health Contract CB-74183 to G.B.

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