[CANCER RESEARCH 47, 1105-1110, February 15, 1987]

Induction of "Innocent Bystander" Cytotoxicity in Nonimmune Mice by AdoptiveTransfer of L3T4+ Lyt-l+2~ Mammary Tumor Immune T-Cells1

Ronald D. Paul and Diana M. Lopez2

Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33101

ABSTRACT

Spleen cells from BALB/c mice, bearing a syngeneic mammary adenocarcinoma, nonspecific-ally lyse xenogeneic target cells following invitro reexposure to mammary tumor-associated antigens. The effectorsof this reaction have been shown to be Thy-1* I yt-I*2+ nylon-adherent

cells. Tumor-immune spleen cells are also able to induce nonimmunespleen cells to express nonspecific cytotoxicity. Studies were undertakento determine whether this inducer activity is mediated by the effectorpopulation or by a functionally distinct subset.

Cell separation studies demonstrated that the inducers and effectorsof innocent bystander cytotoxicity can be separated based upon theproperty of adherence to nylon wool. Further examination of the nylon-nonadherent inducer population indicated that the phenotype is 1314 +I,yi-l+2 . By flow cytometry the inducer subset was shown to express a

higher density of Thy 1 antigen than that expressed by the cytotoxiceffectors. When adult thymectomized mice were implanted with mammary tumors, nonspecific effectors were not generated. In contrast, spleencells from the same experimental animals were able to induce nonspecificcytotoxicity in normal mice following adoptive transfer of their spleencells. Thus, these data support the conclusion that during the course ofmammary tumor growth, at least two functionally and phenotypicallydistinct cell populations interact for the expression of "innocent bystander" cytotoxicity.

INTRODUCTION

Cytotoxic lymphoid cells have traditionally been characterized based on functional and phenotypic criteria (1, 2). Recently, a greater understanding of target recognition structureshas provided additional knowledge regarding the function ofcytolytic effectors (3). Based on the existing evidence, cytotoxicT-lymphocytes and natural killer cells have emerged as the twowell-described major populations of cytolytic effectors. Nevertheless, there is an increasing body of data regarding cytolyticcells which do not fit neatly into either category but appear toshare some of the attributes of both populations. These effectorsbear the description of anomalous killer cells (4), activatedkiller cells (5), or "innocent bystander" effectors (6) and share

the need to be further studied in order to determine theircontribution to the immune response and their relationship toother cytotoxic mechanisms.

In this context, previous reports from this laboratory haveshown that mice bearing a syngeneic mammary adenocarci-noma develop a novel cell-mediated reaction characterized as"innocent bystander" cytotoxicity. In this reaction, spleen cellstaken from tumor-bearing mice are rendered capable of lysingxenogeneic target cells following a secondary in vitro exposureto a cell-free preparation of homologous TAA3 (7). Parallel

Received 9/16/85; revised 8/26/86, 10/24/86; accepted 11/7/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This research was supported by Grant R37-CA-25583 from the National

Cancer Institute.2To whom requests for reprints should be addressed, at Department of

Microbiology and Immunology 1)4 4. University of Miami School of Medicine,P. O. Box 016960, Miami, FL 33101.

'The abbreviations used are: TAA, tumor-associated antigens; FBS, fetalbovine serum; CRBC, chicken red blood cells; FITC, fluorescein isothiocyanate;ATx, adult thymectomized, CTL, cytotoxic T lymphocytes.

studies demonstrated that spleen cells from normal mice failedto function as killers as did spleen cells from tumor bearerswhen exposed to preparations of normal tissues or to a syngeneic nonimmunogenic tumor. Therfore this reaction was shownto consist of a specific afferent or inductive limb followed by anonspecific efferent response. Since specific recognition ofmammary TAA was restricted to the inductive phase, the targetswere considered "innocent bystanders."

In a preceding paper, the cell population mediating thisnonspecific cytotoxicity was thoroughly characterized (8). Inthis context, the effectors or their immediate precursors wereshown to be Thy 1* Lyt-l+2+, could be easily enriched based

on their property of adherence to nylon wool, and were shownto lack classical natural killer activity. Because previous studieshad shown that naive mice or their lymphoid cells could beinduced to generate nonspecific effectors following exposure totumor-immune spleen cells or their products (9, 10), it was ofinterest to further investigate this inducer activity. Therefore,the present study demonstrates that the inductive phase of thisresponse is mediated by a subset of T-lymphocytes that is bothfunctionally and phenotypically distinct from the effector population. Following in vivo priming to mammary tumor-associated antigens, this cell population is able to confer "innocentbystander" cytotoxicity upon nonimmune mice via the induc

tion or recruitment of nonspecific killers. The potential cellularinteractions invoked during the generation and elicitation ofthis response are discussed.

MATERIALS AND METHODS

Mice and Tumors. Young adult (8-12 weeks old) male and/or femaleBALB/cCrgl mice were used throughout these studies. The colony wasoriginally established from The Cancer Research Genetics Laboratoryof the University of California and has been maintained in our animalfacilities by sister-brother matings. The tumor used in these studies wasa chemically induced mammary adenocarcinoma (Dl-DMBA-3), originally induced in BALB/c mice with 7,12-dimethylbenzanthracene (11)and sustained in vivo by serial s.c. transplantation in syngeneic mice.Dl-DMBA-3 grows progressively until the death of the host, whichusually occurs between 5 and 6 weeks following implantation. Thetumor has previously been shown to be immunogenic to the host oforigin (12).

Preparation of TAA. The method used for preparing purified membrane fractions of Dl-DMBA-3 mammary tumor tissue, which elicitimmunologically specific activity in Dl-DMBA-3 mammary tumorbearing mice, has been the subject of a previous publication (13).

Preparation of Spleen Cells. Experimental animals were sacrificed bycervical dislocation. Tumor-bearing mice had been transplanted withDl-DMBA-3 mammary adenocarcinoma between 21 and 28 days priorto use. At this time the tumors weighed between 7 and 10 g. Spleenswere removed aseptically, and single cell suspensions were prepared byhomogenization in a Teflon-on-glass tissue grinder containing 2 ml ofRPMI 1640 supplemented with 5% FBS. The cell suspensions werethen centrifugea and washed once. RBC were eliminated by hypotonieshock, as described previously (7). Spleen cell pools prepared fromeither normal or tumor-bearing mice were washed three additionaltimes in RPMI 1640. The cells were counted and resuspended in theappropriate medium and at the desired concentration, depending uponthe procedure to be used.

Nylon Wool Column Purification. Spleen cell suspensions were run

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INDUCERS OF CYTOTOXICITY

over nylon wool columns for fractionation into T- and B-lymphocytepopulations (14). The no nail herent cells were eluted with 30 ml ofRPMI 1640 containing 10% FBS. The adherent cells were then recovered by mechanical agitation of the nylon wool, followed by washingwith an additional 30 ml of medium. The recovered fractions werecharacterized by fluorescent antibody techniques and fluorescence-activated cell sorter analysis. Thus, the resulting nylon-nonadherentand nylon-adherent spleen cell populations were consistently determined to be 80-90% Thy 1.2 positive or surface immunoglobulinpositive, respectively.

Cell Depletion Studies. Antibody-dependent, complement-mediatedlysis was used to further purify nylon-adherent and nylon-nonadherentspleen cell populations. Monoclonal anti-mouse Lyt-1 arsanilate conjugate, Lyt-2 arsanilate conjugate, and affinity-purified rabbit antiar-sanilate were purchased from the Becton Dickinson Monoclonal Antibody Center (Sunnyvale, CA). The final concentration of each antibodyused was predetermined by titration for maximal killing. Viability wasdetermined and quantitated by a fluorescein diacetate staining procedure (IS) and flow cytometric analysis. Briefly, spleen cells were resus-pended in serumless RPMI 1640 at a concentration of 1 x IO7cells/

ml. The appropriate immunoglobulin was added at a predeterminedconcentration, and the mixture was incubated at 4°Cfor 1 h. For

indirect cytotoxicity, the cells were washed twice and resuspended at 1x io" cells/ml and a predetermined amount of rabbit antiarsanilate wasadded. Following an additional l h at 4"( '. the cells were washed twiceand resuspended at 1 x IO7cells/ml, and guinea pig complement (Pel-

Freeze Biochemicals, Rogers, AR) was added to a final 1:10 dilution.The cells were washed three times prior to use in succeeding assays.

Cytotoxicity Assays. The "innocent bystander11cytotoxicity assay was

performed as previously described (7). Briefly, CRBC were labeled withradioactive sodium enrómate ("Cr) (16) and used as target cells. Cy

totoxicity assays were performed in replicate cultures containing 0.4ml volumes of spleen cell suspensions at 3 x IO6 cells/ml in RPMI1640 with 5% heat-inactivated FBS, 0.25 ml volumes of 51Cr-labeledCRBC, and 1.0 ^g of purified Dl-DMBA-3 mammary TAA. Thecultures were incubated for 18 h at 37'C in a 5% CO2 atmosphere.

Following this incubation, the cultures were centrifuged (350 x g forIO min), and the supernatants were separated from the pellets. Radioactive counts were measured in a Packard model 500 gamma counter."Innocent bystander11cytotoxicity data are expressed as the percentageof "Cr released by the CRBC target cells in cultures stimulated withmammary TAA, minus the percentage of "Cr released by the CRBC

targets in control (spontaneous release) cultures containing lymphoidcells in the absence of TAA. The percentage of 5lCr release wascomputed by measuring radioactivity in supernatants (free 9lCr) andthe cell pellets (bound :'Cr) according to the following formula:

free "Cr (exp)% of "Cr release = -—rr- .,„ , x 100

free "Cr + "Cr (contro )

free "Cr (control)free "Cr + "Cr (control) x 100

Fluorescence Staining and Analysis. Analysis of i -cell subsets wasaccomplished by flow cytometry using a fluorescence-activated cellsorter (FACS III; Becton Dickinson FACS Systems, Sunnyvale, CA)interfaced to a Hewlett Packard model 86 computer system. Dataanalysis was aided by Flow System Software (Ritter Systems, Can.NC). Monoclonal anti-Thy 1-FITC, anti-Lyt-1-FITC, anti-rat-n-FITC,hint in conjugated monoclonal anti-Lyt-2, unconjugated anti-L3T4, andphycoerythrin avidin were purchased from the Becton Dickinson Monoclonal Antibody Center.

All incubations for fluorescent staining were performed for 45 minat 4°Cusing phosphate buffered saline containing 0.03 M NaN3. Forsingle phenotype analysis, I x 10'' spleen cells were incubated with

various dilutions of anti-Thy 1-FITC in a total reaction volume of 100¡i\.The samples were washed in the same buffer three times andresuspended in 1 ml volumes prior to analysis. For dual phenotypeanalyses, l x 10'1spleen cells were incubated with 2 ng of anti-Lyt-2-biotin-conjugated antibody and 5 pg of ami I,\t I ITI ('-conjugated

antibody. Following three washes the cells were incubated an additional45 min with 2 Mgof phycoerythrin avidin. Alternatively, dual stainingfor L3T4 and Lyt-2 was performed by sequential incubations with 2 jig

of anti-L3T4, 2 ng of anti-rat-it-FITC, 2 ¿tgof rat immunoglobulins, 2Mgof anti-Lyt-2-biotin, and 2 ^g of phycoerythrin avidin. Rat immunoglobulins were used in this assay to block any potential open bindingsites of the anti-rat-ic-FITC since both anti-L3T4 and Lyt-2 are ratmonoclonals. Single fluorochrome controls (blocked and unblocked)served as positive controls in this system and monoclonals of irrelevantspecificity served as negative controls. The cells were washed threetimes following each incubation.

Fluorochrome excitation was achieved with 500 mW of output froman argon ion laser (Spectra Physics) tuned to the 488-nm line. Separation of the emission spectra of fluorescein and phycoerythrin wasaccomplished with a 500-nm Dichroic mirror mounted in a beamsplitter support. The signals were separated further by the use of a 525-nm band pass filter in front of the photomultiplier tube interceptingthe FITC emission and a 575-nm band bass filter in front of thephotomultiplier tube receiving the phycoerythrin signal. A 515-nm longpass filter was used to block the laser line.

Thymectomy. Adult thymectomy was performed according to theprocedure described by Roubinian (17). Briefly, 8-12-week old, maleand/or female mice were anesthetized with a dilute solution of sodiumnembutal. The anesthesia was prepared by diluting the stock solution(60 mg/ml) 1/20 in physiological saline. Individual mice were weighedand administered 60 mg/kg of the dilute preparation by i.p. injection.Once anesthetized, mice were immobilized ventral side up on a dissecting board and disinfected locally with 70% ethanol. An incision approximately 1 cm in length, running anterior-posterior over the anteriortip of the sternum was made. The anterior tip of the sternum was thenretracted and a small section of the underlying tissue cut away to exposethe thymus. Both lobes of the thymus were removed by suction with asterile Pasteur pipet attached to a suction pump. The incision wasclosed with surgical staples and sprayed with an antiseptic surgicaladhesive. The mice were kept warm beneath a 60-W lamp until theyrecovered from anesthesia. Sham thymectomized aimais were treatedin the same fashion, however the thymus was left intact. Prior to usein cytotoxicity studies, thymectomized mice were sacrificed by cervicaldislocation and examined for the presence of a thymic remnant. Anysuspect animals were excluded from the studies.

RESULTS

Functional Separation of Inducer and Killer Cell Activities byNylon Wool Column Fractionation. Since the effectors of "innocent bystander" cytotoxicity had previously been shown toconsist of a nylon-adherent subset of Thy 1+ Lyt-1+2* lympho

cytes, initial studies were concerned with the functional isolation of inducer activity in mammary tumor-bearing hosts. Forthis purpose, nylon wool column fractionation was utilized toprepare enriched T- and B-cell populations from mammarytumor-bearer spleen cell pools. These cell populations alongwith unseparated spleen cells from normal and tumor-bearinghosts were tested for effector function in cytotoxicity assaysprior to being used as donor cells in adoptive transfer experiments into normal recipient mice. As shown in Fig. 1, only the

P!RC£NI5l-Cr

RELEASE

NOBUNSP til Ni NOW« l U NT AW

SOURCEOFEFFECT«CELLS

Fig. 1. "Innocent bystander" cytotoxic effector function of tumor bearers'

donor cells used in the adoptive transfer experiments. Nor. Unsep., normal miceunseparated cells; Tu. Unsep., tumor bearers' unseparated cells; Tu. NY. NonAdh.,tumor bearers' nylon-nonadherent cells; Tu. NY. Adh., tumor bearers' nylon-adherent cells. Results are expressed in mean % 5'Cr release of four separate

experiments ±SD.

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unseparated and nylon-adherent, B-cell-enriched fractions fromtumor-bearing mice, could act as killers in this cytotoxic reaction. The nylon-nonadherent, T-cell-enriched fraction was notcapable of effecting bystander cytotoxicity and responded at thesame level as the negative control cells from normal mice. Incontrast, spleen cells taken from normal recipient mice whichhad received tumor donor nylon-nonadherent cells 5 days earlier, were capable of acting as effectors in this reaction as werethe splenocytes taken from recipients of unseparated tumordonor spleen cell pools (Fig. 2). However, recipients of nylon-adherent tumor-immune spleen cells did not act as effectorsand the percentage of 51Cr release was comparable to that of

recipients of normal spleen cells. Therefore, these data indicatethat inducer and effector activities may be attributed to distinctspleen cell populations based on the property of adherence tonylon wool columns.

Phenotype Comparison of Inducer and Effector Populationsby Treatment with Anti-Lyt-1 or Anti-Lyt-2 Antibodies andComplement. The studies presented above demonstrated that"innocent bystander" inducer and effector activities were restricted to the nylon-nonadherent (80-90% Thy 1+) and nylon-adherent (80-90% SIg+) spleen cell populations respectively,when isolated from tumor-bearing mice. In order to prove thatthese functions are indeed performed by different subsets oflymphocytes, studies were conducted to determine the sensitivity of these cells to treatment with anti-Lyt-1 or anti-Lyt-2antibodies and complement. The results of these experimentsare presented in Fig. 3. It can be seen that treatment of nylon-nonadherent inducers or nylon-adherent effectors with arsani-

50

CfLL POPULATION TRANSFER«»

Fig. 2. Adoptive transfer of "innocent bystander" cytotoxicity to normalrecipient mice by unseparated or nylon-nonadherent cells from tumor-bearingmice. Nor. Unsep., recipients of normal mice unseparated cells; Tu. Unsep.,recipients of tumor bearers' unseparated cells; Tu. N. NA., recipients of tumorbearers' nylon-nonadherent cells; Tu. NA., recipients of tumor bearers' nylon-

adherent cells. Results are expressed in mean % cytotoxicity ±SD of eightindividual determinations per group.

TPERCENTS I-Cr

RELEASE

C C

INDUCERFUNCTION

Lyt-1 Lyt-2 C' C Lyt-1

EFFECTORFUNCTION

Lyt-2

Fig. 3. Phenotypic differences between inducer and effector cells of "innocentbystander" cytotoxicity as determined by antibody-mediated complement-dependent lysis. Nylon-adherent (•)and nylon-nonadherent (G) cells from mammarytumor-bearing mice were tested for inducer and effector activity in the "innocentbystander" cytotoxicity assay in the presence of complement (C"), or anti-Lyt-1and complement (Lyt-I), or anti-Lyt-2 and complement (Lyt-2). Effector functionwas assayed by testing the ability of various populations to lyse the xenogeneictargets. Inducer activity was tested by the ability of the various populations toadoptively transfer cytotoxic activity to naive recipients. The values representmean ±SD.

late-conjugated anti-Lyt-1 monoclonal antibody, followed by

antiarsanilate and complement, abrogated both of these activities when tumor bearers or their secondary recipients wereassayed. On the other hand, treatment of these populationswith arsanilate-conjugated anti-Lyt-2 followed by antiarsanilateand complement revealed that only the effector activity wasabrogated by this treatment while inducer activity remainedunaffected. The responses of donor nylon-nonadherent andnylon-adherent cells, or secondary recipients of these populations which were treated with complement only, served ascontrols. In no instance did treatment with antibody alone alterthe expression of cytotoxicity (data not shown). Thus, thesedata indicate that the inducers and effectors of "innocent bystander" cytotoxicity differ with respect to the expression of

the Lyt-2 antigen. The inducers are nylon-nonadherent Lyt-l+2~ cells, whereas the effectors are nylon-adherent Lyt-l+2+

cells.Flow Cytometric Analyses of Nylon Wool Separated Inducer

and Effector Populations. During the course of previous studiesaimed at characterizing the effector population, it was observedthat in spite of their sensitivity to treatment with anti-Thy 1and complement, the effectors appeared to fluoresce weaklywhen stained with FITC-conjugated monoclonal anti-Thy 1. Inorder to confirm this casual observation, experiments wereperformed in which the surface expression of the Thy 1 antigenwas compared in effector and inducer populations. In thesestudies nylon-adherent and nylon-nonadherent populationswere stained with various amounts of the anti-Thy 1 reagentranging from 2 to 10 ng of monoclonal antibody per sample.The results of these studies are summarized by the log-fluorescence histograms shown in Fig. 4 where it is evident that thenylon-adherent effectors (histogram B) express significantly lessThy 1 (mean channel, 37) compared to the nylon-nonadherentinducers (histogram C; mean channel, 61). Furthermore, thefluorescent distributions obtained by treatment with 2 fig ofmonoclonal antibody (Fig. 4) were typical of the fluorescencedistribution of these populations when they were treated withincreasing amounts of antibody up to 10 /¿gof anti-Thy 1 (datanot shown). Therefore, these results suggest that the inducerand effectors of "innocent bystander" cytotoxicity can be further

distinguished by the level of expression of the Thy 1 antigen.Since the antibody-mediated depletion studies had already

shown that effector and inducer populations could not be distinguished based on their sensitivity to anti-Lyt-1 and complement, dual fluorescence, multiparameter flow cytometric analyses were employed in order to more thoroughly characterizethe nylon-nonadherent and nylon-adherent populations withrespect to the expression of L3T4 and Lyt-2 antigens. Theresults of two individual experiments are given in Table 1

LOG FLUORESCENCE

Fig. 4. Differential density of Thy 1 antigen on the surface of nylon-nonadherent inducers and nylon-adherent effectors of the "innocent bystander" cytotoxicity from mammary tumor-bearing mice as determined by flow cytometricanalyses..-/, negative control consisting of cells treated with an FITC-conjugatedmonoclonal antibody of irrelevant specificity; H. histogram of nylon-adherenteffector cells treated with 2 ¿igof FITC-conjugated anti-Thy I reagent; C,histogram of nylon-nonadherent inducer cells treated with 2 pg of the anti-Thy 1antibody.

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Table 1 Dual fluorescence analyses of "innocent bystander" cytotoxicity inducerand effector cells from mammary tumor-bearing mice using monoclonal unii

L3T4 and anti-Lyt-2 reagents

Results are expressed as percentage of fluorescent positive cells based on10,000 cells analyzed per sample. Positive control samples included cells treatedwith either anti-L3T4 and FITC-conjugated anti-rat-«or biotin-conjugated anti-Lyt-2 and phycoerythrin avidin. Negative controls consisted of samples treatedwith FITC-conjugated anti-rat-»or phycoerythrin avidin.

Fluorescent positive cells (%)

Cell populationtestedExperiment

1Nylon-nonadherent

cellsNylon-adherentcellsExperiment

2Nylon-nonadherent

cellsNylon-adherent cellsL3T4-

Lyt-2*18.8

(30)"

3.8(40)9.0(11)

1.6(19)L3T«*

Lyt-2"42.6

(67)

3.7(39)66.6

(84)

5.1 (61)L3T4*

Lyt-2*2.0

(3.0)

1.9(20)3.7

(5)

1.6(19)" Numbers in parentheses represent the positive cells expressed as the percent

age of total T-cells.

Table 2 Effect of adult thymectomy on the ability to express"innocent bystander" cytotoxicity

Animals were thymectomized 2 months prior to tumor implantation. Experiments were performed 3 weeks after tumor passage.

AnimalsNormal

mice, sham operatedNormal mice, thymectomizedTumor bearers, sham operatedTumor bearers, thymectomizedDonor

response(% "Crrelease)"1.70

±0.722.96 ±0.61

69.37 + 3.312.90 ±0.49

' Mean ±SD.

expressed as either percentage of total cells or percent of T-cells. For this purpose T-cells were determined to be all cellsexpressing L3T4 and/or Lyt-2 antigens. Analyses of theseexperiments indicate that nylon column separation resulted inan enrichment of the helper-inducer (L3T4+) and cytotoxiceffector phenotypes (Lyt-2+) in the nylon-nonadherent andnylon-adherent fractions, respectively. In addition, it appearsthat L3T4+ Lyt-2* cells were greatly enriched in the nylon-

adherent fraction when the populations are expressed as percentage of total T-cells.

Effect of Adult Thymectomy on the Induction and Expressionof "Innocent Bystander" Cytotoxicity. Adult thymectomy wasused in order to study the differentiation of T-cells and T-cellprecursors involved in the generation of cytotoxicity in mammary tumor-immune mice. Therefore, ATx and sham ATxBALB/cCrgl mice were implanted with Dl-DMBA-3 mammary adenocarcinoma from 3 to 6 months following surgeryand their spleen cells were subsequently tested for the ability toact as effectors in cytotoxicity assays. Normal sham ATx andATx mice served as additional controls. The results of a representative experiment are shown in Table 2. It is evident fromthe data that spleen cells taken from tumor-bearer ATx miceare incapable of acting as effectors in "innocent bystander"

cytotoxicity assays. The responses of these mice were comparable to those of the normal sham or normal ATx mice whichserved as negative controls. In contrast, spleen cells taken fromtumor-bearing sham ATx mice demonstrated significant levelsof cytotoxicity. Spleen cell pools from the preceding experimental groups were also tested for inducer function in adoptivetransfer studies using normal recipient mice. Five days following adoptive transfer, recipient mice were sacrificed and theirspleens tested in cytotoxicity assays. As shown in Fig. 5, normalrecipients of ATx tumor-bearer or sham ATx tumor-bearerspleen cells responded equally well as effectors of cytotoxicity.The percentage of "Cr release of these groups was significantly

P£«C£HT51-Cr

RELEASf

NOR.stwi

we TX Tine*SW1

CEU PffUUTKM IBAKSf[ RUED

Tuna)IX

Fig. 5. Effect of adult thymectomy on the induction of the "innocent bystander" cytotoxicity reaction. Vor. Snam., recipients of cells from sham-operated

normal mice; Nor. Tx., recipients of cells from thymectomized normal mice;Tumor sham, recipients of cells from sham-operated tumor bearers; Tumor Tx.,recipients of cells from thymectomized tumor-bearing mice. Results are expressedas mean % cytotoxicity ±SD.

CELL WPUWTWN IRANSf FRBED

Fig. 6. Ability of unseparated and nylon-nonadherent cells from thymectomized tumor-bearing mice to act as inducers of the "innocent bystander" cytotoxicity reaction. Sham Unsep., recipients of unseparated cells from sham-operatedtumor bearers; Tx. Unsep., recipients of unseparated cells from adult thymectomized tumor-bearing mice; Tx. N. NA., recipients of nylon-nonadherent cellsfrom adult thymecotmized tumor bearers; Tx. NA., recipients of nylon-adherentcells from adult thymectomized tumor-bearing mice. Results are expressed asmean % cytotoxicity ±SD of eight determinations.

different from that demonstrated by normal recipients of eithersham ATx normal or ATx normal donors spleen cell pools.Furthermore, nylon wool separation of ATx tumor donorspleen cells prior to adoptive transfer, demonstrated that in-ducer function remained a property of nylon-nonadherent cells.As shown in Fig. 6, the ability to induce cytotoxicity in normalmice was enriched in the nylon-nonadherent population. Recipients of nylon-adherent spleen cells failed to generate a cytotoxic response as was previously shown. Therefore, the preceding studies indicate that a differential sensitivity to the effectsof adult thymectomy exists for the precursors of inducer andeffector T-cells operative in the "innocent bystander" cytotox

icity reaction.FlowCytometrie Analyses of T-Cell Subsets in Sham ATx and

ATx Tumor Donors. In order to gain further insight about thecells responsible for inducer function, phenotype analysis of T-cell subsets was performed on ATx and sham ATx donor spleencell pools at the time of adoptive transfer. Lyt-l+2~ and Lyt-l+2+ populations were defined by dual color immunofluores

cence. The results of a representative experiment are shown inTable 3. The data indicate that the Lyt-l'f2~ subset is greatlyreduced in tumor-bearer ATx mice as compared to their shamcounterparts. The reduction in this T-cell subset is minor inthymectomized normal mice. In contrast, a small concomitantincrease in the Lyt-l+2+ pool was observed in normal ATxmice. The increase in this T-cell subset was not observed in thespleens of tumor-bearer ATx mice. Since these data are expressed as a percentage of splenic lymphocytes, the failure toobserve a substantial increase in the percentage of Lyt-l+2+cells is indicative of a loss of this subset following adult thymectomy. In conjunction with the functional studies, these dataindicate that despite a reduction of both Lyt-l+2+ and Lyt-l+2~

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Table 3 T-cell subsets in the spleens of normal or tumor-bearing mice followingadult thymectomy

Thymectomized mice were assayed 2 months following surgery. Tumor-bearingmice transplanted with Dl-DMBA-3 mammary adenocarcinoma 21 days priorto use.

Percentpositives"Donor

groupsNormalsham TX*

Normal TXTumor sham TXTumor TXLyt-l*2-45.0

39.048.032.0Lyt-l+2*11.0

16.015.013.0

"Analysis of 2 x 10* cells per sample. Experimental groups consist of the

pooled spleens of at least five mice.* TX, thymectomized.

cells in tumor-bearing mice, the inducers of "innocent bystander" cytotoxicity appear to be more resistant to the effects

of adult thymectomy than the effector cell population.

DISCUSSIONThe mediators of "innocent bystander" cytotoxicity are de

tectable in the spleens of BALB/c mice approximately 14 daysfollowing transplantation of the Dl-DMBA-3 mammary adenocarcinoma (18). Although this cytotoxic activity is not expressed by splenocytes from normal mice, these mice willrespond following implantation of plasma clot diffusion chambers (9) containing tumor-immune spleen cells or after adoptivetransfer of tumor-bearer spleen cells. Alternatively, spleen cellsfrom naive mice will express bystander cytotoxicity after treatment with culture supernatants from tumor-immune spleencells (10). Since the effector cells had already been characterized, it was of interest to determine whether this inducer-likeactivity was expressed by a distinct cell population as well asits role in the generation of this cytotoxic response.

Our previous studies had shown that effector activity wasrestricted to the nylon-adherent fraction of nylon-separatedcells (8). Therefore, nylon-column separated spleen cells frommammary tumor-immune mice were tested for both inducerand cytotoxic activity. The results of these studies clearly demonstrate that inducer activity is confined to the nylon-nonad-herent fraction and suggest that inducer and cytotoxic activitiesare the properties of functionally distinct cell populations.Depletion studies using anti-Lyt antibodies and complementfurther demonstrate that both inducer and effector activitieswere susceptible to treatment with anti-Lyt-1 and complementwhile only effector function was sensitive to treatment withanti-Lyt-2 and complement. Inducers of suppressor cells andclassical cytotoxic T-lymphocytes have been shown to share theLyt-l+2" phenotype (19, 20). Treatment with anti-Lyt-1 and

complement has also been shown to abrogate CTL activitypresumably by the elimination of the inducer subset (21).

The phenotypes of inducer and effector cells were also studiedusing single and dual-fluorescence flow cytometric analyses.The results of these studies revealed that the inducer andeffector populations could be distinguished by the difference insurface expression of the Thy 1 antigen. "Innocent bystander"effectors were shown to be Thy 1+ dull in comparison to theinducer population. This result was true even when the nylon-adherent cells were stained with five times the amount ofantibody used to stain the nylon-nonadherent cells. Dual fluorescence analyses of these populations with ami L3T4 and anti-Lyt-2 antibodies indicates that cells bearing the inducer phenotype (L3T4+ Lyt-2~)were enriched in the nylon-nonadherentfraction while cells bearing the Lyt 2+phenotype were enrichedin the nylon-adherent fraction. This study also revealed that asmall population of L3T4+ Lyt 2+ cells were detectable in the

spleens of tumor-bearing mice. These phenotypes, which arenormally mutually exclusive in the spleens of normal mice (22),appeared to be enriched in the nylon-adherent population whenthe positives were expressed as the percentage of total T-cells.Therefore, these results support our previously published conclusions regarding the phenotype of the "innocent bystander"

effector cell. However, these studies can not yet clarify whetherthe effectors are Lyt-P2+, L3T4+, or Lyt-l+2+ L3T4~. The

former population bears the phenotype of immature thymocytes(23) and would be consistent with the low density of Thy 1expression.

The depletion studies with antibody and complement indicated that the inducer population was sensitive to treatmentwith anti-Lyt-1 and complement, but not affected by treatmentwith anti-Lyt-2 and complement. These data suggest that theinducer is a Lyt-2-negative cell. Since the dual fluorescenceanalyses show that all nylon-nonadherent Lyt-2-negative cellswere L3T4 positive, it follows from these studies that theinducers of the "innocent bystander" reaction are L3T4+ Lyt-1+ Lyt-2~ lymphocytes. In the preceding report (8) we could

not entirely rule out the possibility that elimination of cytotoxicactivity from the nylon-adherent population with anti-Lyt-1and complement was due to the elimination of inducers. However, the present data may explain the observation that nylonseparation sometimes results in a slight loss of cytotoxic activityeven though the effector and inducer populations are individually enriched.

The technique of adult thymectomy has allowed further differentiation between inducer and effector subsets as well as abetter understanding of their interactions. In our experiments,BALB/c mice that had been thymectomized from 2 to 6 monthsprior to tumor transplantation, failed to generate cytotoxiceffector cells even though cells from these same mice werecapable of adoptively transfer ing this cytotoxic reaction to naiverecipients. These results support the conclusion that a differential sensitivity to adult thymecotmy exists for the precursorsof inducer and effector T-cells operative in "innocent bystander"cytotoxicity. Early investigations had shown that the Lyt-1+2+subset was rapidly depleted following adult thymectomy (2,24).These conclusions were based on sequential killing mediated byantisera and complement. More recent studies utilizing thesensitive technique of dual fluorescence flow cytometry in combination with monoclonal antibody and complement-mediatedlysis have shown that most, if not all Lyt-2+ cells, express lowlevels of the Lyt-1 antigen. However, these low density Lyt-l +cells were not all subject to lysis with anti-Lyt-1 and complement (25). These findings have resulted in a reclassification ofT-cell phenotypes as wellas a réévaluationof previous depletionstudies using antibody and complement.

In accordance with this literature and our depletion studies,the thymectomy data support the conclusion that the precursorsof effectors are derived from a relatively short-lived Lyt-l*2+T-cell pool. It follows that thymectomized mice can inducecytotoxicity in normal recipients because these recipients contain the full component of Lyt-1+2+cells with whom the inducers must interact. Flow cytometric analyses of thymectomizedtumor donor spleen cells demonstrated a 32% reduction in thelevel of Lyt-l+2~ cells when expressed as the percentage of

splenic lymphocytes (Table 3). Since a compensatory increasein the percentage of Lyt-1+2+cells was not observed, as was thecase in normal mice, the data indicates a severe reductionoccurred in both of these subsets. Scollay (26) has reported thatthe proportion of splenic Lyt-l*2+ cells in normal mice doesnot change following adult thymectomy. In those studies theassessment of T-cell phenotypes was performed no later than 4

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INDUCERS OF CYTOTOXICITY

weeks following adult thymectomy. Our data in normal mice,where minor differences were observed in Lyt-1+2~ and Lyt-1*2"'"subsets, are in agreement with this observation. In the

tumor-bearing mice, however, the functional studies in conjunction with the phenotypic analyses indicate a relative resistance of the inducer subset to the effects of adult thymectomyas compared to the effector subset. Therefore, our data withtumor bearers support the conclusion that the effectors andinducer functions of "innocent bystander" cytotoxicity reaction

corespond to distinct T-cell subsets. Our studies both corroborate previous investigations regarding the effects of adult thymectomy in normal mice and provide new information aboutthe effects of adult thymectomy in tumor systems. These resultsshould not be interpreted to mean that all cytotoxic T-cells arederived from this same precursor pool even though similaritiesexist between "innocent bystander" effectors and other cyto

toxic lymphocyte populations. In our studies, the generation oflectin-induced cytotoxic effectors was not affected by adultthymectomy, thus indicating a distinct precursor subset for thiseffector population (data not shown). While classical CTLprecursors may bear a similar phenotype as well as similargeneration kinetics, it is clear that the "innocent bystander"

effector cell is phenotypically and functionally distinct. In contrast, lymphokine-activated killer cells are derived from a Thy1* asialo GMi+ precursor pool although the effectors may beThy T Lyt-2+ or Thy 1+ Lyt-2~ (27). Natural cytotoxic cells,

on the other hand, appear to represent a function which maybe attributed to phenotypically heterogeneous populations ofcells (28).

In conclusion, these studies have shown that the generationof tumor-induced "innocent bystander" cytotoxicity involvesthe interaction of at least two distinct subsets of T-cells. Inaddition to the phenotypic differences, the L3T4+ Lyt-1+2~inducer and the Lyt-1+2* effector can be further distinguished

based on properties of nylon adherence, density of expressionof the Thy 1 antigen, functional capabilities, and sensitivity toadult thymectomy. Based on the previously discussed findingsit is evident that a population of tumor-immune L3T4+ Lyt-l+2~ cells, in the presence of specific antigen, can induce asubset of Lyt-1+2+ precursors to express nonspecific killer

activity. In light of recent findings in which it has been shownthat murine anomolous killers and CTL are derived from thesame precursor pool (29), similarities in the generation of"innocent bystander" effectors and classical CTL should not be

surprising. However, these studies and our previous reportsindicate that neither anti-Thy 1 nor anti-Lyt antibodies aloneinhibit this cytotoxic reaction, indicating that these receptorsare not required for target recognition and destruction as is thecase with CTLs.

ACKNOWLEDGMENTS

We gratefully appreciate the excellent technical assistance of LynnHerbert and Mantley Dorsey, Jr., and we thank Frances Susi for typingthe manuscript.

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1987;47:1105-1110. Cancer Res   Ronald D. Paul and Diana M. Lopez  Immune T-Cells

Mammary Tumor-2+ Lyt-1+Mice by Adoptive Transfer of L3T4Induction of ''Innocent Bystander'' Cytotoxicity in Nonimmune

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