Lymphokine-induced Cytotoxicity: Characterization of Effectors, … · the Lyt T helper T-cells to produce interieukin 2. The Lyt 2+ T-cells play a negative role in the regulation

[CANCER RESEARCH 46, 513-518, February 1986]

Lymphokine-induced Cytotoxicity: Characterization of Effectors, Precursors,

and Regulatory Ancillary Cells

Chou-Chik Ting,1 Stringner S. Yang, and Myrthel E. Hargrove

Immunology Branch [C-C. T., M. E. H.J and Laboratory of Cellular Oncology [S. S. Y.], National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

ABSTRACT

In the present study, we have characterized the effectors,precursors, and regulatory ancillary cells involved in the in vitrogeneration of lymphokine-induced cytotoxicity. It was first shown

that at least two lymphokines are needed for the generation oflymphokine-induced cytotoxicity. They are interieukin 2 and a

novel lymphokine, the cytotoxic cell differentiation factor (CCDF).CCDF was produced primarily by the macrophages. The effectors of the lymphokine-induced cytotoxic cells thus generated

selectively killed tumor targets of different etiological origins. Theserological phenotype of lymphokine-induced cytotoxic cell effectors were found to be Thy 1+, Lyt 2~, and AGM1"; therefore,

they were neither classic natural killer (NK) cells nor cytotoxic T-

lymphocytes. Extensive characterization of the precursors bysequential column separation and antibody lysis and also bylimiting dilution analysis showed that they were AGM1 + and Lyt

2 ; thus they were NK-like cells. In addition to NK-like cells being

identified as the precursors, two other cell compartments wereidentified as ancillary cells which regulate the lymphokine-induced cytotoxicity. They were the macrophages and T-cells.

Macrophages were needed to produce CCDF and to activatethe Lyt T helper T-cells to produce interieukin 2. The Lyt 2+ T-

cells play a negative role in the regulation of the lymphokine-induced cytotoxic cell response. The process of lymphokine-

induced cytotoxicity thus involves a complex interaction betweenat least two lymphokines (interieukin 2 and CCDF) and three cellcompartments, namely, NK-like cells, macrophages, and T-cellsof Lyt 1+ and Lyt 2+ phenotypes.

INTRODUCTION

Nonspecific cytotoxic effector cells could be induced in theabsence of antigen by culturing nonprimed lymphocytes withlymphokines. These lymphokine-induced cytotoxic responses

have emerged as an important mechanism which may be responsible for a variety of so called "anomalous cytotoxicities"

(1-10). The lymphokine-induced cytotoxic effectors may also

play a role in the immune surveillance against spontaneousneoplasms (11).

In a previous report we demonstrated that cytotoxic lymphocytes were generated by culturing normal mouse spleen cellswith syngeneic peritoneal cells and indomethacin (12); this process was found to be mediated by the endogenous production of

Received 8/19/85; revised 10/28/85; accepted 11/1 /85.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.

1To whom requests for reprints should be addressed, at Immunology Branch,

Bldg. 10, Rm. 4B17, National Institutes of Health. Bethesda. MD 20205.

a putative new lymphokine, namely the "CCDF."2 It was later

found that at least two signals were needed to generate anoptimal level of the LICC. These two signals are IL2 and cytotoxiccell differentiation factor (13). The LICC selectively killed varioussyngeneic and allogeneic lymphoid or solid tumor targets, andthere was no cytotoxic activity against syngeneic or allogeneiclymphoblasts (14). In addition, these LICC were very effective inpreventing in vivo tumor growth in syngeneic hosts (14). Ourfindings on the requirement of at least two lymphokines to induceLICC appear to be quite different from a similar human system,by Grimm ef al. (15), in which IL2 was the only signal required toinduce a special class of cytotoxic effectors, namely the LAK.The difference in these results could possibility be explained onthe basis of two different classes of lymphokine-induced cytotoxic effectors. It appears that there is a need to determinewhether the lymphokine-induced cytotoxic effectors in various

systems represent a single family of effectors. In this paper, wehave characterized the effectors, the precursors, and the regulatory ancillary cells involved in our system (LICC). The differences between the cells in LICC system and the LAK systemwere also identified. Furthermore, we have also examined therole of immunoregulatory ancillary cells for the lymphokine-in

duced cytotoxic responses.

MATERIALS AND METHODS

Mice

Female C57BL/6, DBA/2, and CBA mice, 2 to 5 months of age, wereobtained from the Veterinary Resources Branch, Division of ResourcesServices, National Institutes of Health, Bethesda, MD.

Lymphokine Preparation

Interieukin 2. CM-IL2 refers to a supernatant prepared from W/Fu ratspleen cells cultured with concanavalin A-conjugated Sepharose beads.

It was essentially free of concanavalin A and contained IL2 (100 units/ml), when compared to a standard sample which contained IL2 (400units/ml). The IL2 activity was determined by a method developed byGillis et al. (16). This IL2 preparation contained no detectable level ofinterferon, determined by inhibition of the cytopathic effect of vesicularstomatitis virus on L929 cells (kindly performed by Dr. Julie Djeu, Bureauof Biologies, Bethesda, MD). It may contain one or more lymphokinesrelevant to the present study.

Interieukin 1. IL1 were prepared as described earlier (17) by culturingmacrophage cell line P388D,, at 5 x 10" cells'ml, in LPS (20 ng/m\)

(lipopolysaccharide; DIFCO, Detroit, Ml) for 48 to 72 h. The supernatantsthus harvested contained IL1 activity when determined by the thymocyte

2The abbreviations used are: CCDF, cytotoxic cell differentiation factor; CM-

CCDF, conditioned medium containing CCDF; IL2, interieukin 2; CM-IL2, conditioned medium containing IL2; CM-IL1, conditioned medium containing interieukin1; LAK, lymphokine activated killer; NK, natural killer; CTL, cytotoxic T-lympho-cytes; LICC. lymphokine-induced cytotoxic cells; |12Sl]dUrd. [12Sl]deoxyuridine; E/T,

effector to target cell ratio; AGM1, asiÃ³loGM1; NT, nylon wool column separatedenriched T-cells; CP, complement.

CANCER RESEARCH VOL. 46 FEBRUARY 1986

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CHARACTERIZATION OF LYMPHOKINE-INDUCED CYTOTOXICITY

proliferation assay (17) at 1:30 to 1:100 dilution of the CM-IL1 prepara

tion.Cytotoxic Cell Differentiation Factor. CM-CCDF refers to a super

natant prepared by culturing normal peritoneal cells with indomethacinor normal peritoneal cells plus normal spleen cells with indomethacin.The details are described in a previous report (13). There was nodetectable IL1 or IL2 in CM-CCDF using thymocyte proliferation (17) andT-cell proliferation (16) assays, respectively. It contained Interferon (1-

10 units/ml).

Induction of LICC

The complete lymphocyte culture medium was used throughout theexperiments. It was RPM11640 containing 5% fetal bovine serum (FlowLaboratories, Rockville, MD), 2 x 10~2M 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid buffer, and 5 x 10~2 M 2-mercaptoethanol. For

induction by CCDF, normal spleen cells were cultured with 30% (v/v) ormore CM-CCDF. For induction by CCDF and IL2, normal spleen cellswere cultured with 10% CM-CCDF and CM-IL2 (0.3 unit/ml). Inductionby culturing normal spleen cells at 1.5 x 106 cells/ml with normalperitoneal cells at 1-1.5 x 105 cells/ml and 1 x 10"6 M indomethacin (12)

was also done.

Allogeneic Mixed Lymphocyte Culture

Normal spleen cells prepared from C57BL/6 (H-2b) mice (responders)were cultured in 2000 rads X-irradiated spleen cells from DBA/2 (H-2d)

mice (stimulators) at a respondenstimulator cell ratio of 10:1.All cultures were established in 30-ml tissue culture flasks in a final

volume of 5 to 10 ml. They were incubated in a humidified atmospherecontaining 5% CO2. After 5 days, unless specified, the cells werecounted, washed once, and resuspended in RPM11640/5% fetal bovineserum at the appropriate concentrations to be tested in the [125l]dUrd

release assay.

Limiting Dilution Analysis

Graded numbers of precursors were dispersed into U-bottom micro-

plates; they were cultured in lymphocyte culture medium supplementedwith 20% CM-CCDF and IL2 (2 units/ml) obtained from an IL2 producingEL-4 cell line. After 5 days, cytotoxicity was determined by adding 2.5 x103 [126l]dUrd-labeled target cells to each well. A positive response was

defined as total percentage of lysis greater than 3 SD above spontaneousrelease (target cells in medium alone). The frequency of precursors wascalculated according to the method of Fazekas de St. Groth (18), it wasthe number of precursor cells which gave 37% nonresponsiveness.

Lysis with Antibodies

The following antibodies were used: (a) Â«Thy1.2 monoclonal antibody;(b) nLyt 2.2 monoclonal antibody (both purchased from New EnglandNuclear, Boston, MA); and (c) Â«AGM1 antibody (purchased from WakoChemicals, Dallas, TX). The Â«AGM1 antibody recognized a glycolipiddeterminant present on NK cells (19), which effectively eliminated the NKcytotoxicity against susceptible targets. The details for lysis with theseantibodies have been described elsewhere (12). The mitogenic responsesto T-cell mitogen concanavalin A or phytohemagglutinin were completely

abolished after the spleen cells were lysed by Â«Thy1 antibody plus CP.The ability to generate alloreactive CTL was reduced to 0-10% of the

untreated spleen cells after lysis with either Â«Thy1 or aLyt 2 antibodyplus CP. After lysis with the aAGM1 antibody, there were no appreciablechanges in the mitogenic responses to T-cell mitogens or the ability to

generate alloreactive CTL. The recovery of viable cells for the antibodyplus complement treatment of the fresh or cultured unseparated spleencells was 40-60%, 60-80%, 80-90%, and 85-95% for Â«Thy+CP, Â«Lyt

2+CP, Â«AGM1+CP, and CP alone, respectively.

Enriched T-cell Preparation

The spleen cells were passed through a nylon wool column as described by Julius et al. (20). The nonadherent cells were used as enriched

T-cells which contained 80-85% T-cells and less than 0.3% macro

phages as determined by phagocytosis of Latex beads.

Sephadex G10 Column Passage

Spleen cells were passed through a Sephadex G10 column (21). Afterpassage, there was less than 0.1-0.3% macrophages, as determined by

phagocytosis.

Target Cells for Cell-mediated Cytotoxicity Assay

Four murine leukemia cell lines were grown in suspension culture in1640/5% fetal bovine serum. These cell lines were Friend virus-inducedleukemias FBL-3 (22) and HFL/b (23) of the H-2" haplotype, radiationleukemia virus-induced leukemia RLÃŽ1(24) of the H-2" haplotype, andMoloney virus-induced leukemia YAC (25) of the H-2" haplotype.

Cell-mediated Cytotoxicity Assay

The [125l]dUrd release assay was used to measure the cell-mediated

cytotoxicity. The details of the technique have been described elsewhere(26). In brief, 0.05 ml of [125l]dUrd-labeled target cells at 1 x 105 cells/ml

and 0.15 ml of effector cells at an appropriate concentration were addedto the wells of 96-well, U-bottom microplates. All effector cells were

adjusted to contain the same number of viable cells for each E/T cellratio. Incubation was carried out at 37Â°Cin a 5% CO2 atmosphere. The

supematants were then harvested, and the radioactivity was determinedin a well-type 7-scintillation counter. The results are expressed as total

percentage of lysis and net percentage of lysis according to the followingformulas:

Total % lysis = (cpm in supernatant/total cpm in cells) x 100

Net % lysis = (total % lysis obtained with test effectors) - (total % lysis

obtained with normal spleen cells cultured alone for 5 days)

The spontaneous release obtained with different targets ranged from 5to 20% (total % lysis). The standard errors obtained with total percentageof lysis were usually between 0.5 and 3.

RESULTS

Generation of LICC

In a previous report (12) we demonstrated that LICC weregenerated by incubation of normal spleen cells with syngeneicperitoneal cells and indomethacin or were generated by incubating normal spleen cells directly with lymphokines. It was furthershown that at least two lymphokines were required for thegeneration of LICC, IL2, and CCDF (13). The generation of LICCcan be best depicted in Fig. 1, which showed that when normalspleen cells were cultured with a suboptimal dose of CM-CCDF(10%) alone, it failed to induce a high level of cytotoxic response.However, if a small amounts of CM-IL2 (0.1-0.3 units/ml) werealso added to these cultures, high levels of cytotoxic responseswere seen. There was no significant cytotoxic response whenspleen cells were cultured with CM-IL2 alone. These resultsindicated that CCDF synergized with IL2 to induce LICC. WhenCM-IL1 was used to substitute CM-IL2, there was no significantincrease of cytotoxic response. In other experiments, CM-IL1when used at a wide dose range (0.1-30%, v/v) also failed tosynergize with either CM-IL2 or CM-CCDF to induce LICC. Itshould be pointed out that the LICC effectors had been washedonce prior to the cytotoxicity assay, and thus there was verylittle lymphokine left in the effector preparations. Furthermore,the conditioned medium containing lymphokine (ILI, IL2, orCCDF) was not directly cytotoxic for the target cells. We found


514




Responding Cultures

ContainedNel Percent Lysis

CCDF IL 2 IL 1

(v/v) (u/ml) (v/v) 0

Table 1

Characterization of the effectors

Fig. 1. Induction of LICC by IL2 and CCDF. Normal C57BL/6 spleen cells at 1.5x 10" cells/ml were cultured with CM-CCDF, CM-IL2, CM-IL1 alone or in combi

nation at indicated amounts. Cytotoxicity was determined on day 5 by testingagainst three targets at two E/T ratios as indicated.

that the LICC selectively killed syngeneic or allogeneic lymphoidor solid tumor targets (14). There was no cytotoxicity againstsyngeneic or allogeneic lymphoblasts. In these experiments freshnormal spleen cells or cultured normal spleen cells did not givesignificant cytotoxicity against these targets, when comparedwith medium control (spontaneous release; data not shown).

Characterization of the LICC Effectors

After 5 days of culturing, the LICC induced by the lymphokines(IL2 and CCDF) in C57BL/6 responders were subjected to lysisby various antibody plus CP to determine their serological phe-

notype (Table 1). It was found that the LICC were susceptible toaThy 1 plus CP lysis but were completely resistant to the lysisby aLyt 2 antibody and were relatively resistant to the lysis ofaAGM1 antibody. On the other hand, alloreactive CTL weresusceptible to lysis by both aThy 1 and aLyt 2 antibodies butwere relatively resistant to lysis by aAGM1 antibody. In contrast,the NK activity was removed by lysis of the NK cells with aAGMtantibody but not by the aThy 1 or aLyt 2 antibody. These resultsshow that the LICC are neither classic NK cells nor classic CTL.

Characterization of the Precursors

Depletion Experiments. Different responder populations wereprepared from normal spleen cells by various column adsorptionand antibody treatments for the depletion analysis. These were:(a) untreated normal spleen cells; (b) normal T-cell-enriched

responders prepared by passing cells through a nylon woolcolumn; macrophages and B-cells were mostly depleted by thistreatment; (c) T-cell depleted responders prepared by treatmentwith aThy 1 antibody plus complement; and (d) NK cell depletedresponders prepared by treatment with aAGM1 antibody pluscomplement. These responders were cultured for 5 days with30% CM-CCDF or with 10% CM-CCDF and CM-IL2, after whichcytotoxicity was determined (Fig. 2). The results obtained bytesting against syngeneic target (HFL/b) and an allogeneic target(RLc51) were very similar. Untreated responders generated a highlevel of cytotoxic activity when cultured with a high dose of CM-CCDF (30%). The NT generated a significant but reduced level

Net % lysis at thefollowing E/T ratios"

Treatment ofeffectors'1LICCUntreatedÂ«Thy1

+CPÂ«Lyt2+CPÂ«AGM1

+CPCPaloneAlto-CTLUntreatedaThyl


+CPCPaloneNK

activityUntreatedCBAspiÂ«Thy1


+CPCPalone...

â€”/â€”75/128232223350/13V* ~/25/119522162015/1-175/120122171950/135312934150/13331341321" "" /25/110085815/124-10162250/1181816017

" The LICC were generated by culturing normal C57BL/6 spleen cells with CM -

IL2 (0.3 unit/ml) and 10% CM-CCDF. The alloreactive CTL were generated byculturing normal C57BL/6 (H-2") spleen cells with 2000 rads X-irradiated DBA/2(H-2") spleen cells at a responder:stimulator cell ratio of 10:1. After 5 days, the

effectors were treated with various antibodies, as indicated, plus CP or with CPalone. After treatment, they were washed, counted, and resuspended at appropriate concentrations to be tested in the [126l]dUrd release assay. The NK cells werethe spleen cells from 7-week-old CBA mice. These spleen cells were treated withantibody plus complement as above.

The untreated and treated LICC and allo-CTL were tested against HFL/b andYAC. The NK cells were tested against YAC cells.

Responders

NS â€¢CCDF 30%NT Â»CCDF 30%T-DpI . CCDF 30%NT â€¢T-DpI Â»CCDF 30%NK Dpi * CCDF 30%

NS * CCDF 10%NS - CCDF 10% Â»IL2HS * IL2

NT Â«CCDF 10%NT - CCDF 10% + 1L2NT Â»CCDF 10% Â»PMÂ»NT Â»IL2NT .PMÂ»

T-OpI * CCDF 10%T-DpI Â«CCDF 10% - ILJT-DpI * IL2

NK Dpi t CCDF 10's

NK Dpi Â«CCDF 10% Â»IL:NK-DpI, IL2

Net Percent Lysis

Target HFL/b (H-2&) Target RLrf 1 (H-2Â°)

10 20 30 10 20 30

Fig. 2. Characterization of precursors. The responders were prepared as follows: (a) Untreated normal spleen cells (NS) from C57BL/6 mice: (b) NT; (c) T-depleted responders (T-DpI), obtained by treating normal spleen cells with aThy 1antibody plus CP; and (d) NK-depleted responders (NK-Dpl), obtained by treatingthe normal spleen cells with ,.AGM1 antibody plus CP. These responders werethen cultured with 30% CM-CCDF or with 10% CM-CCDF and CM-IL2 (0.3 unit/ml). The controls included responders cultured alone or cultured with CM-CCDF orIL2 alone. The peritoneal macrophages (P-M)were the adherent peritoneal cellsafter treatment with aThy 1 antibody plus CP. The cell recovery after antibody+CPtreatment was similar to that described in 'Materials and Methods."

of cytotoxicity. Depletion of T-cells or NK cells completely abolished the cytotoxic response. Reconstitution of NT with T-celldepleted responders restored the response. In these experiments the generation of LICC by the addition of high dose (30%)


515




of CCDF alone in untreated spleen cells was presumably attributed to the presence of undetectable but sufficient amounts ofIL2 in the CCDF preparation; or alternatively, the endogenousproduction of 112by unseparated spleen cells might compensatefor the insufficient amounts of IL2. In contrast, depletion ofmacrophages by nylon wool column or by Sephadex G10 absorption or depletion of T-cells by antibody lysis reduced orabolished the ability of the remaining cells to generate LICCwithout exogenous IL2. These results suggested that endogenous production of IL2 was essential. These results also indicatethat at least three cell compartments were involved in the generation of LICC: Thy "T cells; nylon adherent cells; and AGM1 +

cells. When a suboptimal dose of CM-CCDF was used (10%),supplementation of CM-IL2 restored the cytotoxic responses inall responder populations except -the NK-depleted responders.

Addition of peritoneal macrophages instead of IL2 also restoredthe response of NT responders but not the T-cell depletedresponders (data not shown). These experiments showed thatthe cells depleted of AGM1+ cells were the only responders the

ability to induce LICC of which could not be restored by thesupplementation of exogenous lymphokines. AGM1 + cells, prob

ably NK cells, are therefore most likely to be the precursors,based on the results of these depletion analysis.

Sequential Column and Antibody Treatments. It has beenshown that Thy 1 antigen was also expressed on some hema-topoetic stem cells other than classic T-cells (27); therefore weelected to use aLyt 2 antibody to remove the classic allo-CTLprecursors (reacting against class I MHC antigens). Results fromthese studies are summarized in Table 2. Three different responders were used: the untreated spleen cells; the NT; and theSephadex G10 column separated macrophage-depleted responders. These responders were further subjected to variousantibody lysis. These different preparations were then culturedwith CM-IL2 and CM-CCDF to induce cytotoxic effectors. Depletion of Thy 1+ cells generally resulted in reduced levels ofcytotoxic responses (a 30-50% reduction at an E/T ratio of 100/1). Depletion of AGM1 + cells completely abolished the cytotoxicresponse. In contrast, depletion of Lyt 2+ cells increased the

cytotoxic responses in most cases; the levels of cytotoxicityremained at a plateau at an E/T ratio of 100/1 and 30/1 butdropped sharply at an E/T ratio of 10/1. Again, these responseswere completely abolished by further depletion of AGM1 + cells.

These results indicate that the precursors or essential ancillarycells are AGM1+ and Lyt 2' cells, which is consistent with their

being NK-like cells.Limiting Dilution Analysis. To further characterize the pre

cursors, limiting dilution analysis was performed (Table 3). Bulkcultures (in 30 ml tissue culture flask) were also set up forcomparison. In bulk culture, depletion of Lyt 2+ cells resulted in

a 3-fold increase of the cytotoxic response as determined by thelytic units generated in these cultures. Depletion of AGM1 + cells

eliminated most of the cytotoxic response. In limiting dilutionanalysis, depletion of Lyt 2* cells resulted in a 3-fold increase of

the frequency of cytotoxic precursors from 1/3300 to 1/980. Incontrast, depletion of AGM1 + cells resulted in a more than 13-

fold reduction of the frequency of precursors (1/43,000).Taken together, these experiments indicate that the precur

sors are AGM1+, Lyt 2~ cells; in short, they are probably NK-like

cells.

DISCUSSION

Lymphokine-induced cytotoxicity has been proposed to beresponsible for a variety of "anomalous cytotoxicities" induced

Table 2

Sequential column and antibody treatment of the precursors

Precursors8MS"NSNSNSNSNTNTNTNTNTG10SG10SG10SG10SG10SDepletion

by61st

2ndantibodyantibodyaAGM1

+CPÂ«Thy1+CPÂ«Lyt2+CPaLyt2+ CP aAGM1 +CPÂ«AGM1

+CPoThyl+CPÂ«Lyt2+CPoLyt2+ CP aAGM1 +CPÂ«AGM1

+CPaThyl+CPÂ«Lyt2+CPÂ«Lyt2+ CP aAGM1 + CPNet

%lysis0100/127-1153603102227-22101228130/1180532-21511525012-1818010/111021707-1614-141210-1

8 Spleen cells were obtained from normal C57BL/6 mice, and some were passed

through nylon wool columns to obtain an enriched T-cell preparation (NT), or theywere passed through a Sephadex G10 column to obtain macrophage-depletedresponders.

Â°The responders were treated with various antibodies and CP as described in"Materials and Methods." One group in each responder preparation received two

consecutive antibody treatments (Â«Lyt2+ CP and aAGM1 + CP). The recovery ofviable cells after each antibody plus CP treatment is as follows:(a). NS: the cell recovery was 85, 47, 76, 65, and 89% for Â«AGM1+ CP, aThy!

+ CP, aLyt2 -I- CP, aLyt2 + CP and then Â«AGM1 + CP, and CP alone,

respectively.(Â£>).NT: the cell recovery was 81, 13, 69, 57, and 85% for Â«AGM1+ CP, aThyl

+ CP, aLyt2 + CP, aLyt2 -I- CP and then 0AGM1 + CP, and CP alone,respectively,

(e). G10S: the cell recovery was 86,41, 72,59, and 87% for aAGM1 + CP, aThyl+ CP, aLyt2 + CP, aLyt2 + CP and then aAGM1 + CP, and CP alone,

respectively.The untreated and treated responders were counted and adjusted to the same

viable cell concentration (1.5 x 10*/ml) and then cultured with CM-IL2 (0.3 unit/ml)

and 10% CCDF. After 5 days, cytotoxicity was determined by testing against HFL/b (H-2b) at the indicated E/T ratios. Similar results were obtained by testing against

other syngeneic or allogeneic targets.d NS, normal spleen; G10S, Sephadex G10 column-separated spleen cells.

by lectin (1), fetal bovine serum (2-4), allosensitization (5-7), andmixed lymphocyte tumor cell interaction (8-10). To determine

whether these phenomena are truly attributable to a single familyof cytotoxic effectors, one needs to characterize the effectors,the precursors, and the ancillary cells as well as the varioushumoral mediators (lymphokines and cytokines) involved in thesecytotoxic reactions. In the present study, our major effort focusedon the characterization of the various cell compartments and onthe corresponding lymphokines involved in the induction andregulation of the cytotoxic responses.

The LICC generated in our experiments appeared to involveat least two lymphokines (CM-IL2 and CM-CCDF; Fig. 1). In a

previous report the direct involvement of IL2 was shown by thespecific blocking of the LICC response by the anti-IL2 receptor

antibody (13). It was further shown that IL2 alone tested at awide dose range failed to generate LICC; CM-CCDF was also

needed to induce significant LICC response. In fact, high dosesof IL2 induced lower levels of cytotoxicity when CM-CCDF was

present, indicating that lack of LICC response by adding IL2alone was not due to insufficient IL2 (13). These findings are insupport of the recent report of Lopez-Botet et al. (28) that IL2alone was insufficient to induce NK-like activity in human T-cell


516




Table3Characterizationof the precursors in bulk cultures and in limiting dilution analysisBulkculture"Treatment

ofprecursors"Â«Lyt2

+ CPÂ«AGM1+ CP100/151621230/125

45010/15

25-1Lytic

units05

15


role in the regulation of the lymphokine-induced cytotoxicity. We

reported that normal spleen cells cultured with increasingamounts of IL2 (from 1 to 30 units/ml) induced suppressor cellswith increasing suppressor activity (35). These suppressor cellswere characterized as Thy 1+ and Lyt 2+ cells, consistent with

classic suppressor T-cells. They suppressed the generation ofalloreactive CTL in mixed lymphocyte cultures and also suppressed the generation of LICC. These findings indicate that Lyt2+ cells are responsible for the negative regulation of lymphokine-

induced cytotoxicity.Our current findings further demonstrated that the complexity

of interaction between cells and soluble mediators is also seenin lymphokine-induced cytotoxicity. We established that in thelymphokine-induced cytotoxicity, two lyrnphokines (IL2 and

CCDF) and three cell compartments are involved (Fig. 3). In aprevious report (13) we showed that not only were both IL2 andCCDF involved in the generation of LICC, there was also a cleartemporal relationship between the activation of LICC and thesequential appearance of these two lymphokines. The LICCprecursors were first activated by IL2; the CCDF was neededlater to induce the differentiation of the activated precursors intocytotoxic effectors. The noncytotoxic NK-like cells are the pre

cursor cells. The macrophages are needed to produce CCDFand to activate the third cell compartment, the Lyt 1+ helper T-

cells which produce IL2 (34). The other cell type in the T-cellcompartment, the Lyt 2* T-cells, plays a negative role in the

regulation of LICC. The LICC not only killed tumor cells in vitro,they were also very effective in preventing the in vivo tumorgrowth in syngeneic hosts, and the ability to generate LICC waspreserved in the tumor bearing hosts until the terminal stage oftumor growth (14); therefore, an understanding of the regulatorymechanism for the generation of LICC may help us to designstrategies to strengthen the host's defense against autologous

tumor.

ACKNOWLEDGMENTS

We are very thankful to the critical review of this manuscript by Drs. H. Coding,D. Segal, and J. Wunderlich.

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1986;46:513-518. Cancer Res Chou-Chik Ting, Stringner S. Yang and Myrthel E. Hargrove Precursors, and Regulatory Ancillary CellsLymphokine-induced Cytotoxicity: Characterization of Effectors,

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Lymphokine-induced Cytotoxicity: Characterization of Effectors, … · the Lyt T helper T-cells to produce interieukin 2. The Lyt 2+ T-cells play a negative role in the regulation