[CANCER RESEARCH 50. 2582-2586. May I. 1990]

Inhibition of Tumor Cell Proliferation by Natural Suppressor Cells Present inMurine Bone Marrow1

Kikuya Sugiura, Muneo Inaba, Hajime Ogata, Ryoji Yasumuzu, Evelio E. Sardina, Kayo Inaba, Sho-ichi Ruma,Robert A. Good, and Susumu Ikehara2

Department of Pediatrics, All Children 's Hospital, University of South Florida, St. Petersburg, Florida 33701 [K. S., E. E. S., K. A. G.J; First Department of Pathology,Kansai Medical University, Moriguchi, Osaka 570, Japan [K. S., M. I., H. O., R. Y., S. A'., S. I.]; and Department of Zoology, Kyoto University, Kyoto 606, Japan

IK. 1.1

ABSTRACT

Natural suppressor (NS) cells, which are Thy-1", immunoglobuliiT,

and nonadherent cells with relatively low density (1.063 to 1.075 g/ml),inhibit not only the proliferation of spleen cells which have been stimulated by allogeneic cells or mitogens but also the proliferation of tumorcell lines. Cell-to-cell contact is not necessary for NS cells to exert NSactivity. Being radioresistant, DNA synthesis is not necessary for NScells to suppress proliferation. However, protein synthesis is necessary,since puromycin blocks NS cell activity. In addition, NS cells were foundto secrete a factor which inhibits DNA synthesis. Of the various cytokinestested, interleukin 3 and granulocyte-macrophage colony-stimulating factor enhance NS activity. These results suggest that NS cells play animportant role in the suppression of not only immune responses but alsotumor growth.

INTRODUCTION

NS' activity is defined as the ability of unprimed cells to

nonspecifically suppress various immunological responses (1).There has recently been an increase in data indicating that NScells play a crucial role in the induction of self tolerance andregulation of immune responses, including the prevention ofGVHD (2, 3). NS cells have been found not only in the spleensof neonatal mice (4, 5), adult bone marrow (6-8), and deciduaof pregnant mice (9-12) but also in the spleens of adult miceafter various treatments, such as TLI (13-15), the induction ofchronic GVHD across minor histocompatibility barriers (16),and the administration of cyclophosphamide (17), cyclosporine(18), 89Sr (19, 20), BCG (21, 22), and anti-allotype antibody

(23).NS cells were previously thought to be "null" cells that had

no markers of mature T-cells. B-cells, or macrophages, althoughthere are two reports indicating that la-positive macrophageprecursors (24) or that macrophages (25) in the neonatal spleenhave NS activity. We have previously found that there is acorrelation between NS cell activity and the enrichment ofhemopoietic stem cells; both hemopoietic stem cells and NS

Received 4/11/89; revised 10/27/89.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 work was supported in part by American Cancer Society InstitutionalGrant 179-08; a grant from the Japanese Ministry of Health and Welfare; a grantfrom the Naito Foundation; a grant from the Mitsubishi Foundation: a grant-in-aid from the Mochida Memorial Foundation for Medical and PharmaceuticalResearch: a grant from the Susuken Memorial Foundation: the Science ResearchPromotion Fund of the Japanese Private School Promotion Foundation (1987);(.um in \i.l for Cancer Research 62015088 (1987); Grant-in-Aid for GeneralScientific Research 63480147 (1988) from the Japanese Ministry of Education.Science, and Culture; and Grants AG05628 and AI22360 from the NIH.

2To whom requests for reprints should be addressed.J The abbreviations used are: NS. natural suppressor; GVHD, graft-v<rjuj-host

disease: TLI. total lymphoid irradiation: BCG. Bacillus Calmette-Guerin; WGA,wheat germ agglutinin; FCS. fetal calf serum: ADC. adherent cells; WGA-FITC,fluorescein isothiocyanate-conjugated wheat germ agglutinin; BM. bone marrow;BMC. bone marrow cell: DDC. double-diffusion chamber: MLR. mixed-lymphocyte reaction; IL, interleukin: rIL, recombinant interleukin: GM-CSF. granulocyte-macrophage colony-stimulating factor; IFN--y. -p-interferon; r!FN-->. recombinant -y-interferon; NC. natural cytotoxic: BM Fr.2, bone marrow cells inFraction 2; MHC, major histocompatibility complex; NK. natural killer.

cells belong to WGA-positive cell populations in the bonemarrow (26). In addition, we have found that NS cells aresensitive to 5-fluorouracil treatment. These results stronglysuggest that NS cells are hemopoietic stem cells in the cyclingphase. In this paper, we show that NS cells inhibit the proliferation of tumor cells, and we also characterize their mode ofaction and biological properties.

MATERIALS AND METHODS

Mice. C57BL/6J. DBA/2, and C3H/HeJ mice were purchased fromThe Jackson Laboratory (Bar Harbor, ME) or CLEA Japan, Inc.(Osaka. Japan) and maintained under specific pathogen-free conditionsuntil use. All mice were used when 6 to 12 wk old.

Tumor Cell Lines. Tumor cell lines used in this study were EL4(mouse lymphoma), P815 (mouse mastocytoma), X5563 (mouse myeloma), and WEHI 164 (mouse fibrosarcoma). All tumor cell lines weremaintained in vitro in RPMI 1640 medium containing 10% PCS(GIBCO. Grand Island, NY) until use.

Fractionation of Bone Marrow Cells. Isolation of hematopoietic stemcells or NS cells has been described elsewhere (26. 27). To remove T-cells, ADC, and B-cells, whole bone marrow cells were treated withanti-Thyl.2 antibody (clone 30-H12; Becton Dickinson Immunocy-tometry System, Mountain View, CA; or clone F7D5; Olac, Bicester,United Kingdom) plus baby rabbit complement (Pel-Freez, Roger, AR)and passed through a G10 column (Pharmacia Fine Chemicals, Uppsala, Sweden) and an anti-mouse immunogloblin-coupled Sepharose4B (Pharmacia) column. The cells were then fractionated by equilibriumdensity centrifugation on a discontinuous Percoli (Pharmacia) gradient.For density separation. Percoli solutions were prepared in densities of1.085. 1.075, and 1.063 g/ml. After centrifugation at 1500 x g for 30min, cells were collected in each fraction: < 1.063 g/ml (Fr. 1); 1.063 to1.075 g/ml (Fr.2); 1.075 to 1.085 g/ml (Fr.3); and >1.085 g/ml (Fr.4).Since Fr.l had almost no living cells and cells in Fr.4 were red bloodcells, cells in Fr.2 or Fr.3 were used in assays for NS activity.

Cell Sorting. Cells in Fraction 2 were labeled with WGA-FITC at0.4 Mg/ml and sorted on a flow cytometry system (Epics C; CoulterCorp., 11laicali. FL). Electronic windows were set to select highly WGA-FITC-labeled cells with moderate forward and low right-angle light

scatter.Assay of NS Activity. NS activity of BMCs was estimated by the

ability to inhibit the proliferation of tumor cells. Tumor cells (1 x 10"in 0.2 ml or 5 x IO4 in 1 ml) were cultured with various numbers of

BMCs or various concentrations of the supernatant from the culture ofBMCs in RPMI 1640 medium including 10% FCS and 5 x 10-5M 2-

mercaptoethanol. Forty-eight h later, the proliferation of tumor cellswas estimated by counting the DNA incorporation of [3H]thymidine.[3H]Thymidine was introduced during the last 4 h of the culture. The

percentage of suppression was calculated using the following formula.

% of suppression = 1 cpm (with BMCs)cpm (without BMCs)

x 100

In some experiments, the cell-to-cell contact between BMCs andtumor cells was prevented using a QA-pm DDC (M1LLICELL; Milli-pore, Bedford, MA). In brief, MILLICELL chambers were placed intowells of 24-well plates (Costar, Cambridge. MA). BMCs (1 x 10" in0.5 ml) were put into the chamber, and tumor cells (1 x IO6in 0.5 ml)

were put in the wells. The tumor cells (0.1 ml) were counted 48 h later

2582

INHIBITION OF TUMOR CELL PROLIFERATION

to estimate the degree of proliferation. NS activity was also determinedusing assays for MLR and mitogen responses, as previously described(26).

Culture Supernatant of NS Cells. BMCs (1 x 10"/ml) were cultured

with or without mouse cytokines in RPMI 1640 medium includingPCS (10%) and 5 x 10~5M 2-mercaptoethanol. The supernatant was

collected 48 h later and used for assaying NS activity.Cytokines. Murine rIL-2, rIL-3, GM-CSF, and IFN-r were purchased

from Genzyme Corporation (Boston, MA).All experiments were repeated 3 or more times, and reproducible

data were obtained. Representative data, therefore, are shown in "Results."

RESULTS

Nonspecific Suppressor Activity Is Enriched in a Low-DensityFraction of Mouse Bone Marrow Cells. Since NS cells have beendefined as "null cells" (1), BMCs from C57BL/6J mice were

purged of ADCs, T-cells, and B-cells. As shown in Table 1, thedegree of suppressor activity was enhanced at each stage ofpurging. The BMCs at each stage exerted suppressor activityeven when cell-to-cell contact with tumor cells was preventedby culturing in a DDC. Furthermore, BMCs suppressed notonly lymphoid lineage tumor cells but also solid tumor cells(WEHI 164, fibrosarcoma). WEHI 164 cells have been reportedby Djeu et al. (28) to be a target of NC cells. However, nocytolytic activity was observed in the experiment where cell-to-cell contact between BMCs and the tumor cells was preventedby the DDC system (data not shown).

As the next step in characterizing the suppressor cells, BMCspurged of ADCs, T-cells, and B-cells were fractionated byequilibrium density centrifugation on a Percoli gradient. Percolisolutions were designed to obtain the hematopoietic stem cell-or precursor cell-enriched fraction (Fr.2, 1.063 to 1.075 g/ml)as described in our previous paper (26) or reported by Visser etal. (29). As shown in Table 1, the suppressor activity wassignificantly enriched in cells from Fr.2 as compared with wholebone marrow cells or Fr.3 cells (P < 0.025).

We sought to determine whether the suppressor activity ofBM Fr.2 is effective in the context of the MHC. We analyzed3 murine tumor cell lines bearing different MHC (H-2) phe-notypes: EL4 (H-2b); P815 (H-2d); and X5563 (H-2k). Cells

from each of these cell lines were cultured with BM Fr.2 cellsfrom C57BL/6J mice or with syngeneic spleen cells (C57BL/

Table 1 .\'S activity is enriched in a low density fraction (Fr.2) of mouse BMCsTumor cells (5 x 10") were cultured with BMCs of C57BL/6J mice (1 x 10')

for 48 h at 37°C.The growth of tumor cells was estimated by counting theincorporation of (JH]thymidine.

25<>r

BMCsaddedNone

Whole BMCsBMCs purged of ADCsBMCs purged of ADCs

and T-cellsBMCs purged of ADCs,

T-, and B-cellsFr.2"Fr.a'Tumor

cell growth [(cpm x IO3)(cell-to-cell contact with (+) or without (-)BMCs)]EL4

(+) EL4 (-)" WEHI-164(-)"20.

1 ±0.3* 23.9 ±2.4 29.2 ±2.519.2 ±2.6 (5)' 20.2 ±2.5(15) 24.5+1.8(15)15.2 ±0.7 (25) 18.0 + 0.9(25) 19.2 + 2.2(34)13.7 ±2.8 (36) 15.0 + 2.9(37) 17.5 ±0.7(40)12.9

+ 0.5(36) 14.7 + 2.3(38) 17.0 ±1.1(42)9.1

±0.3' (55) 11.5 ±2.5' (52) 13.4 ±2.4' (54)21.5 ±1.7(0) 22.4 ±0.8 (6) 29.1 + 2.5(0)

°Tumor cells were separated from BMCs by MILLICELL as described in"Materials and Methods."

* Mean + SD.c Numbers in parentheses, percentage of suppression.* BMCs were purged of ADCs. T-cells, and B-cells and then fractionated with

discontinuous density gradients of Percoli solutions as described in "Materialsand Methods."

' Significantly different from whole BMCs or Fr.3 at P < 0.025 by Student's t

test.

2 6Number of Cells Added

2O

( X 10* )

Fig. 1. Three mouse tumor cell lines (1 x 10*) bearing different H-2 pheno-types (EL4 (•).H-2": P8I5 (•),H-2"; and X5563 (A), H-2k] were cultured

together with bone marrow Fr.2 cells ( ) of C57BL/6J mice or with syngeneicspleen cells ( ) for 48 h at 37°C.The proliferation of tumor cells wasestimated by counting the incorporation of |3H]thymidine.

6J spleen cells for EL4, DBA/2 spleen cells for P815, andC3H/HeJ spleen cells for X5563). As shown in Fig. 1, BM Fr.2cells from C57BL/6J mice demonstrated a suppressive effecton tumor cells. However, syngeneic spleen cell preparations didnot show a suppressive influence. It is noteworthy that the BMFr.2 cells suppressed the growth of allogeneic P815 cells andX5563 as well as syngeneic EL4 cells. The BM Fr.2 cells,however, did not exert any cytolytic activity against these tumorcell lines.

Wheat Germ Agglutinin-positive Cells in a Low-Density Fraction Have Potent Suppressor Activity against Tumor Cells. Wehave reported that low-density BM cells with high affinity towheat germ agglutinin (WGA-positive) exert strong suppressoractivity on the various immunological responses of mouse lymphocytes (26). BM Fr.2 cells from C57BL/6J mice were sortedinto Fr.2 WGA-positive cells and Fr.2 WGA-negative cells,and each cell population was assayed for NS activity. As thedata in Table 2 show, the Fr.2 WGA-positive cells exerted thestrongest suppressor activity against tumor cell growth whencompared with the Fr.2 WGA-negative cells or the unsortedFr.2 cells (P < 0.005). Also, the Fr.2 WGA-positive cellsshowed the strongest suppressor activity, even when the cellswere prevented from coming into direct contact with tumorcells. It therefore appears that murine BM Fr.2 WGA-positivecells are enriched for suppressor activity not only against immunological responses [as we previously reported (26)] but alsoagainst tumor cell growth.

Effect of Cytokines on Suppressor Activity of BM Fr.2 Cells.It has been reported that NS activity is enhanced by IFN-r orIL-2 (30). We examined whether the suppressor activity of BMFr.2 cells is increased by any cytokine. As shown in Fig. 2, theinhibitory effect of the BM Fr.2 cells was amplified in thepresence of murine recombinant IL-3 or GM-CSF, whereasthese cytokines themselves did not demonstrate any significant

2583

INHIBITION OF TUMOR CELL PROLIFERATION

Table 2 WGA-positive, low density cells (Fr.2 WGA*) have potent NS activityEL4 cells (1x4) were cultured with various concentrations of BMCs or spleen cells of C57BL/6J mice for 48 h at 37'C. The growth of EL4 cells was estimated

by counting the incorporation of [3H]thymidine.

Tumor cell growth (cpm x 10') when the following nos. of cells were added (x IO4)

Cells added0None2 1.3 ±3.5*

Fr.2 WGA*Fr.2 WGA-

Fr.2Unfractionated BMC/Spleen cells221.1

±0.6(20)c

26.3 ±4.1 (0)22.5 ±2.6(14)25.5 ±2.2 (3)25.9 ±3.5 (2)614.5

±1.0(45)34.2 ±6.3 (8)18.9 ±2.2(28)23.4 ±2.7 (11)25.5 ±2.9 (3)206.5

±0.5" (75)

22.8 ±2.2(13)13.2 ±0.4(50)17.8 ±I.I (32)25.6 ±2.7(3)0°

20°22.3

±2.76.0

19.911.415.421.00.7'

(73)

2.3(10)1.3(49)0.5(31)3.5 (6)

" Tumor cells were separated from BMCs or spleen cells by MILLICELL as described in "Materials and Methods."* Mean ±SD.c Numbers in parentheses, percentage of suppression.' Significantly different from Fr.2 WGA~ or Fr.2 (below) at P < 0.005 by Student's t test.' Significantly different from Fr.2 WGA~ or Fr.2 (below) at P < 0.01 by Student's t test.

s were purged of ADCs, T-cells. and B-cells, but not fractionated with discontinous density gradients of Percoli solutions.

Tumor Cell Growth(cpm x 1000)

Tumor Cell Growth(cpm x 1000) B

15 5O 15O 5OO

Concentration of IL-2 (U)

Tumor Cell Growth(cpm x 1000}

15 50 150 500Concentration of GM-CSF (U)

II15 60 150 500Concentrasen oíIL-3 (U)

Tumor Cell Growth p*(cpm«1000) *-"

150 5OO

Concentration of IFN-gamma (U)

Fig. 2. BM Fr.2 cells (2 x 10s) from C57BL/6J mice were added to culturesof EL4 cells (1 x IO4)along with various concentrations of murine recombinantcytokines (A, IL-2: B, IL-3: C. GM-CSF; and D, IFN-7) for 48 h at 37'C (•).

The proliferation of EL4 cells was estimated by counting the incorporation of[3H]lhymidine. As a control. EL4 cells were incubated along with cytokines but

without bone marrow Fr.2 cells (D). The counts of EL4 cells (cpm) in culturewithout any cytokines were 14,172 ±2,630 (EL4 cells plus BM Fr.2 cells) and27,674 ±2,426 (EL4 cells alone).

Table 3 Effect of cytokines on NS activityBone marrow cells in Fr.2 (1 x 106/ml) were incubated with various concen

trations of murine recombinant IL-2, IL-3, GM-CSF, or IFN-r in RPMI 1640medium with 10% PCS for 48 h at 37'C. The cells were then washed and cultured

BMCsaddedNone

Fr.2Preincubation

withMedium

IL-2 (500 units/IL-3 (500 units/ml)IL-3( 100 units/ml)GM-CSF (500 units/ml)GM-CSF( 100 units/ml)\FN-r (1000 units/ml)Tumor

cell growth (cpm X IO3)with thefollowing nos. of BMCs added (x10")027.8

±2.4°

20.9 ±26.6 ±14.6 ±16.2 ±15.4±17.5 ±21.8±62.2

(25)*

1.6(5)0.8C(48)

0.5(41)0.9' (44)

2.0(37)1.3(22)2014.7

r20.7

9.611.912.613.117.0t

2.6 (47)2.6 (24)1.2(65)1.4(57)1.2(54)2.7 (53)2.1 (39)

°Mean ±SD.* Numbers in parentheses, percentage of suppression.c Significantly different from medium at P < 0.05 by Student's t test.

when the cells were introduced to the assays 24 h after irradiation, the cells completely lost their NS activity. The treatmentof Fr.2 cells with puromycin blocked NS cell activity (Table 5).This shows that protein synthesis is necessary for the suppression by NS cells and also indicates that suppression by NS cellsis dependent on the synthesis of the suppressive factor(s).Experiments using the DDC system demonstrated that cell-to-cell contact was not necessary for NS activity (Table 1). Actually, the supernatant from the culture of BM cells inhibitedthe tumor cell growth. As shown in Table 6, the supernatantfrom the culture of Fr.2 WGA-positive cells exerted the mostsignificant suppressive activity. Also, the suppressive activity ofthe supernatant was enhanced when BM Fr.2 cells were culturedwith IL-3 or GM-CSF, although the supernatant from theculture of IL-3 or GM-CSF alone demonstrated almost nosuppressive activity. These results suggest that a soluble mediator is involved in the suppression.

inhibitory effect against the growth of tumor cells. In addition,the suppressor activity of BM Fr.2 cells was also enhanced bypreincubation with IL-3 or GM-CSF followed by extensivewashing (Table 3). Murine recombinant IL-2 or IFN-r did notenhance their suppressive activity; some reversal of suppressionwas observed in the cells pre-cultured with IL-2.

Mode of Action of NS Cells in Mouse Bone Marrow. C57BL/6J bone marrow cells in Fr.2 were irradiated to examine radi-osensitivity of NS cells and to determine whether DNA synthesis is necessary for the suppression by NS cells. As shown inTable 4, bone marrow cells in Fr.2 retained NS activity evenwhen the cells were added to the assays for mitogen responsesand MLR immediately after irradiation of 16 Gy. However,

DISCUSSION

In the present study, we have demonstrated that NS cells,which are Thy-1-negative, immunoglobulin-negative, and non-adherent cells with relatively low density (1.063 to 1.075 g/ml),inhibit not only the proliferation of spleen cells which havebeen stimulated by allogeneic cells or mitogens but also theproliferation of tumor cell lines. In addition, we have foundthat NS cells secrete a factor which inhibits DNA synthesis.Although NS cells have been studied extensively (1), this is, toour knowledge, the first report indicating that NS cells in thebone marrow can suppress the proliferation of tumor cells. NScells (WENS, Wehi-3-expanded neonatal splenocytes) in the

2584

INHIBITION OF TUMOR CELL PROLIFERATION

Table 4 \S cells are radioresistantBone marrow cells in Fr.2 (BM Fr.2) from C57BL/6J mice were irradiated

with 4 or 16 Gy from a To source (1.9 Gy/min). Irradiated/unirradiated BMFr.2 cells were used for NS assays in mitogen responses or MLR. In mitogenresponses, spleen cells (5 x 10*)of C57BL/6J mice were cocultured with variousnumbers of bone marrow cells in the presence of 2.5 /jg/ml of concanavalin Aand 25 Mg/ml of lipopolysaccharide made from Escherictiia coli for 3 days. InMLR. spleen cells (4.5 x 10*)of C3H/HeN mice were cocultured with irradiated(20 Gy) C57BL/6J spleen cells (7.5 x 10*) as stimulator cells and C57BL/6J

bone marrow cells as NS cells for 4 days.

Tumor cell growth (cpmx 10') with thefollowingnos.

of cellsaddedh

after ir-CellsAssayradiationaddedConcanavalin

A 0" NoneBM

Fr.224f

NoneBM

Fr.2Lipopolvsaccharide

0 NoneBMFr.224

NoneBMFr.2MLR

0 NoneBMFr.224

NoneBM Fr.2Radiationdose

(Gy)004160016004160016004160016(x

10'0

0.171.255.4(22)*60.2(15)62.7(12)64.952.1

(26)66.8(0)28.920.4

(29)18.9(35)21.1(27)28.216.8(40)27.0

(0)50.423.6(53)17.0(66)18.5(63)39.021.2(45)41.8(0))18.3

(88)12.9(82)10.0(86)14.1

(78)74.9(0)6.9

(76)7.2(74)8.0(72)2.1

(92)29.1(0)6.2

(88)3.6(93)3.3

(93)5.7(85)41.2(0)

°BM cells (Fr.2) were irradiated and immediately used for NS assays in

mitogen responses and MLR.* Numbers in parentheses, percentage of suppression.c BM cells (Fr.2) were irradiated and incubated at 37°C.Twenty-four h later,

the surviving cells were collected and used for NS assays in mitogen responsesand MLR.

neonatal spleen, which were described by Jadus and Parkman(5), suppress the growth of WEHI-164 cells only, whereas ourNS cells present in the bone marrow suppress not only WEHI-164 but also the other tumor cell lines (EL4, P815, and X5563).Since our NS cells suppress the proliferation of WEHI-164(fibrosarcoma), the antitumor effect is not restricted to tumorcells of hemopoietic lineage. In addition, the NS cells did nothave the killing activity of NK or NC cells, and they were also

Table 5 Puromycin blocks A'.Vactivity

Bone marrow cells in Fr.2 from C57BL/6J mice were incubated with puro-mycin (5 >ig/ml. in RPMI 1640 medium with 10% FCS) for 12 h. As a nontreatment control, the BM Fr.2 cells were incubated with medium alone. The cellswere washed and used for NS assays in mitogen responses or MLR as describedin Table 4. The cell viability following puromycin treatment was 80%.

Tumor cell growth (cpm x IO3)wilh

the following nos. of BM Fr.2 cellsadded (x10*)AssayLipopolysaccharideConcanavalin

AMLRPretreatmentNone

PuromycinNone

PuromycinNone

Puromycin0

0.128.2

16.8(40)°

36.1(0)64.9

52.1 (26)66.8(0)39.0

21.2(45)41.8(0)0.38.2(71)

30.1(0)27.9

(57)67.4(0)14.7

(62)44.6 (0)12.1

(92)23.1(18)14.1

(78)74.9(0)5.7(85)

41.2(0)" Numbers in parentheses, percentage of suppression.

found to be Thy-1 negative and asialo GM-1 negative (data notshown).

We examined NS activity using tumor cells instead of MLRor mitogen responses to exclude the possible direct suppressiveeffects of cytokines on the responding spleen cells used in MLRor mitogen response assays. As shown in Table 6 and in Fig. 2,only IL-3 and GM-CSF (although weaker than IL-3) enhancedNS activity against tumor cells when bone marrow cells in Fr.2were cultured or precultured with various cytokines. Since ithas been reported that IL-3 (rather than GM-CSF) acts on theproliferation of an early committed stem cell compartment (31,32), it is interesting that IL-3 has a stronger capacity to enhanceNS activity than GM-CSF.

It has been reported by Holda et al. (30) that NS activity isincreased by T-cell signals such as rlFN-r or rIL-2 (30). Whenwe measured natural suppression in MLR or mitogen responseassays with either rlFN-r or rIL-2 continuously present duringthe culture period (without washing out), NS activity was increased (data not shown). However, these cytokines did notenhance NS activity when assays were carried out using NScells (BM Fr.2) and a tumor cell line (EL4) (Table 6; Fig. 2).Also, no significant effect was observed on the proliferation oftumor cells when tumor cells were cultured with cytokinesalone. It therefore appears that rlFN-r or rIL-2 acts on different

Table 6 Bone marro»'cells produce NS factorEL4 cells (1 x IO4)were cultured with the supernatant from BMC culture described below for 48 h at il'C. The growth of EL4 cells was estimated by counting

the incorporation of ['Hjthymidine.

Supernatant from thecultureofNoneUnfractionated

BMCsFr.2Fr.2

WGA+Fr.2WGA-Fr.2

+IL-2*IL-2alone'Fr.2

+IL-3¿IL-3alone'Fr.2

+GM-CSF^GM-CSFalone'Fr.2

+IFN-/IFN-ralone'Tumor

cell growth (cpm x 10J) with the following concentrations ofsupernatant0

V,27.8±2.4"27.4

±2.2(1)*25.423.826.526.526.825.128.523.428.21.8(9)1.8(14)0.2

(5)1.6(5)2.3

(4)1.6(10)1.5(0)0.3(16)2.3

(0)26.5±1.6(5)28.4±0.6 (0)M26.0

±2.0(6)22.9±2.8(18)18.4±1.7(34)26.2±0.9(6)25.7±1.4(8)27.2±1.9(2)20.7±0.4(26)27.5±1.2(2)21.2±0.7(23)28.1±3.0(0)25.7±1.4(8)27.1±1.7(3)Vi23.3

±1.2(16)20.8±1.0(25)12.0±0.8C(57)25.6

±1.3(8)24.8±4.6(11)27.9±2.6(0)13.4±OV(52)26.8

±3.6(4)18.9±0.4(32)27.3±1.7(2)24.2±3.3(13)26.2+ 4.1 (6)

°Mean ±SD.6 Numbers in parentheses, percentage of suppression.' Significantly different from Fr.2 WGA". Fr.2. or unfractionated BM at P < 0.005 by Student's t test.d Fr.2 cells were cultured with murine recombinant IL-2 (500 units/ml), IL-3 (500 units/ml). GM-CSF (500 units/ml), or IFN-r (1000 units/ml) for 48 h at 37'C.' Murine recombinant IL-2 (500 units/ml), IL-3 (500 units/ml), GM-CSF (500 units/ml), or IFN-r (1000 units/ml) was incubated for 48 h at 37"C.1 Significantly different from Fr.2 at P < 0.005 by Student's t test.

2585

INHIBITION OF TUMOR CELI, PROLIFERATION

populations of cells leading to the enhanced suppression ofimmunological functions when NS activity was evaluated usingMLR and mitogen response assays. Indeed, Azuma and Kaplan(33) recently reported that a mouse NK cell line which wasestablished in culture containing rat IL-2 demonstrated potentNS activity. These findings support the theory that naturalsuppression may define an activity rather than a specific celllineage.

Though the exact suppressive mechanisms of NS cells havenot yet been clarified, the use of a double chamber culturesystem allowed us to observe that the suppressive activity wasmediated by soluble factor(s) released from the NS cells (Table1). This observation was confirmed by the finding that the NScell activity was abrogated by puromycin, a protein synthesisinhibitor (Table 5). Indeed, the culture supernatant of NS cellsin the Fr.2 WGA-positive fraction has NS activity againsttumor proliferation (Table 6). Other investigators have alsoreported that NS cell activity is governed by soluble mediator(s)released from NS cells (34-41). We are in the process of notonly purifying a suppressor factor released from the NS cells,but also clarifying the in vivo role of NS cells in the regulationof various immune responses, including tumor immunity.

ACKNOWLEDGMENTS

The authors wish to thank Susan Lay. Roberta Hill, and KayokoHiguchi for their expert technical assistance and Sachi Ohya and Jane

i for their help in preparing this manuscript.

REFERENCES

1. Maier, T.. Holda, J. H., and Claman, H. N. Natural suppressor (NS) cells:members of the IGL regulatory family. Immunol. Today, 7: .112-315, 1986.

2. Strober. S., Palathumpat. V., Schwadron. R., and Hertel-Wulff. B. Clonednatural suppressor cells prevent lethal graft-versus-host disease. J. Immunol..138: 699-703. 1987.

3. Van Bekkum. D. W., and Knaan-Shanzer. S. Characterization of a subpop-ulation in neonatal thymus which suppresses the graft-i'î.-hostreaction. Eur.J. Immunol.. 13: 403-409. 1983.

4. Rodriguez, G., Andersson. G., Wigzell. H.. and Peck. A. B. Non-T cell natureof the naturally occurring, spleen-associated suppressor cells present in thenewborn mouse. Eur. J. Immunol.. 9: 737-746. 1979.

5. Jadus, M. R., and Parkman, R. The selective growth of murine newborn-derived suppressor cells and their probable mode of action. J. Immunol., 136:783-792, 1986.

6. Duwe. A. K.. and Sharwan. K. S. The immunoregulatory role of bone marrow.II. Characterization of a suppressor cell inhibiting the in vitro antibodyresponse. Cell. Immunol.. 43: 372-381, 1979.

7. Córvese,J. S., Levy, E. M., Bennett, M., and Cooperband. S. R. Inhibitionof an in vitro antibody response by a suppressor cell in normal bone marrow.Cell. Immunol., 49: 293-306. 1980.

8. Dorshkind. K., and Rosse. C".Physical, biologic, and phenotypic properties

of natural regulatory cells in murine bone marrow. Am. J. Anat.. 164: 1-17,1982.

9. Clark, D. A., Slapsys, R., Croy, B. A., Kroek, J.. and Rossant, J. Local activesuppression by suppressor cells in the decidua. Am. J. Reprod. Immunol.. 5:78-83, 1984.

10. Clark, D. A., Chaput. A., and Tutton. D. Active suppression of host-vs-graftreaction in pregnant mice. VII. Spontaneous abortion of allogeneic CBA/Jx DBA/2 fetuses in the uterus in CBA/J mice correlates with deficient non-T suppressor cell activity. J. Immunol.. 136: 1668-1675, 1986.

11. Starkey, P. M., Sargent, 1. L., and Redman, C. W. G. Cell populations inhuman early pregnancy decidua: characterization and isolation of largegranular lymphocytes by flow cytometry. Immunology, 65: 129-134, 1988.

12. Lala. P. K.. Kennedy, T. G., and Parhar. R. S. Suppression of lymphocytealloreactivity by early gcstational human decidua. H. Characterization of thesuppressor mechanisms. Cell. Immunol., 116: 41 1-422. 1988.

13. Slrober, S. Natural suppressor (NS) cells, neonatal tolerance, and totallymphoid irradiation: exploring obscure relationships. Annu. Rev. Immunol..2: 2 19-237, 1984.

14. Weigensberg, M.. Morecki, S., Weiss, L.. Fuks, Z., and Slavin, S. Suppressionof cell-mediated immune responses after total lymphoid irradiation (TL1). I.Characterization of suppressor cells of the mixed lymphocyte reaction. J.Immunol., 132: 971-978. 1984.

15. May, R. D., Slavin, S.. and Vitella, E. S. A partial characterization of

suppressor cells in the spleens of mice conditioned with fractionated totallymphoid irradiation (TLI). J. Immunol.. 131: 1108-1114. 1983.

16. Holda. J. H., Maier, T., and Claman, H. N. Murine graft-versus-host diseaseacross minor barriers: immunosuppressive aspects of natural suppressor cells.Immunol. Rev., 88: 87-105, 1985.

17. Greeley, E. H., Segre. M., and Segre, D. Modulation of autoimmunity inNZB mice by cyclophosphamide-induced, nonspecific suppressor cells. J.Immunol., 134: 847-851. 1985.

18. Uhteg, L. C., Salomon, D. R.. Rocher. L. L., Kupiecweglinski. J. W.. Araujo,J. L., Rubin. M. F.. Tilney, N. L.. and Carpenter. C. B. Cyclosporine-inducedtransplantation unresponsiveness in rat cardiac allegrati recipients: in vitrodetermination of helper and suppressor activity. J. Immunol.. 135: 1800-

1805. 1985.19. Merlluzzi. V. J., Levy. E. M., Kumar. V.. Bennett. M.. and Cooperband. S.

R. In vitro activation of suppressor cells from spleens of mice treated withradioactive strontium. J. Immunol., 121: 505-512, 1978.

20. Levy, E. M., Bennett. M., Kumar. V.. Fitzgerald. P.. and Cooperband. S. R.Adoptive transfer of spleen cells from mice treated with radioactive strontium: suppressor cells, natural killer cells, and "hybrid resistance" in recipientmice. J. Immunol.. 124: 611-618, 1980.

21. Bennett, J. A., and Marsh. J. C. Relationship of Bacillus Calmette-Guerin-induced suppressor cells to hematopoietic precursor cells. Cancer Res.. 40:80-85, 1980.

22. Kalo. K.. Yamamoto. K., and Kimura. T. Migration of natural suppressorcells from bone marrow to peritoneal cavity by live BCG. J. Immunol.. 135:3661-3668. 1985.

23. Gilbert. K. M., and Dresser, D. W. Generation in vivo of non-T suppressorcells with the use of anti-allotype antibody. J. Immunol., 140: 15-20, 1988.

24. Snyder, D. S., Lu, C. Y., and Unanue, E. R. Control of macrophage laexpression in neonatal mice—role of splenic suppressor cells. J. Immunol..128: 1458-1465, 1982.

25. Piguet, P. F., Irle, C., and Vassalli. P. Immunosuppressor cells from newbornmouse spleen are macrophages differentiating in vitro from monoblasticprecursors. Eur. J. Immunol.. //: 56-61, 1981.

26. Silumin. K.. Inaba, M., Ogata, H.. Yasumizu. R.. Inaba. K., Good, R. A.,and Ikehara. S. Wheat germ agglutinin-positivc cells in a stem cell enrichedfraction of mouse bone marrow have potent natural suppressor activity. Proc.Nati. Acad. Sci. USA. 85: 4824-4826. 1988.

27. Miyama-lnaba. M.. Ogata. H., Toki, J.. Kuma. S.. Sugirua. K., Yasumizu.R.. and Ikehara. S. Isolation of murine pluripotent hemopoietic stem cells inthe Go phase. Biochem. Biophys. Res. Commun.. 147:687-694. 1987.

28. Djeu, J. Y., Lanza. E., Pastore, S.. and Hapel. A. J. Selective growth ofnatural cytotoxic but not natural killer effector cells in interleukin-3. Nature(Lond.), 306: 788-791, 1983.

29. Visser, J. W. M.. Bauman, J. G. J., Mulder, A. H., Eliason, J. F., and deLeeuw. A. M. Isolation of murine pluripotent hemopoietic stem cells. J. Exp.Med.. 59: 1576-1590, 1984.

30. Holda. J. H., Maier. T.. and Claman, H. N. Natural suppressor activity ingraft-i'i-host spleen and normal bone marrow is augmented by IL 2 andinterferon-v J. Immunol., 137: 3538-3543. 1986.

31. Suda. T.. Suda. J.. Ogawa. M., and Ihle. J. N. Permissive role of interleukin3 (IL-3) in proliferation and differentiation of multipotential hemopoieticprogenitors in culture. J. Cell. Physiol.. 124: 182-190. 1985.

32. Bender. J. G., Van Epps. D. E., and Stewart, C. C. A model for the regulationof myelopoiesis by specific factors. J. Leuk. Biol., 39: 101-111, 1986.

33. Azuma, E., and Kaplan, J. Role of lymphokine-activated killer cells asmediators of veto and natural suppression. J. Immunol., 141: 2601-2606.1988.

34. Hertes-Wulff. B.. and Strober. S. Immunosuppressive lymphokine derivedfrom natural suppressor cells. J. Immunol.. 740: 2633-2638. 1988.

35. Argyris. B. F. Suppressor factor produced by neonatal mouse spleen cells.Cell Immunol.. 62: 412-424. 1981.

36. Jadus, M. R., and Peck, A. B. Naturally occurring, spleen-associated suppressor activity of the newborn mouse: biochemical and functional identification of monokines secreted by newborn suppressor-inducer three mono-cytes. Scand. J. Immunol., 23: 35-44, 1986.

37. Almawi, W. Y., Dolphin, P. J., Thies, R. L.. McMaster, W. R., Levy, J. G.,and Pope, B. L. Suppressor cell-inducing factor: a new lymphokine secretedby a natural suppressor cell line with natural cytotoxic activity. I. Purificationto apparent homogeneity and initial characterization of suppressor cell-inducing factor. J. Immunol.. ¡41:2529-2535. 1988.

38. Knaan-Shanzer. S.. and Van Bekkum, D. W. Soluble factors secreted bynaturally occurring suppressor cells that interfere with in vivo graft-n-hostdisease and with T cell responsiveness in vitro. Eur. J. Immunol.. 17: 827-834. 1987.

39. Maes. L. Y., York, J. L., and Soderberg. L. S. F. A soluble factor producedby bone marrow natural suppressor cells blocks interleukin 2 production andactivity. Cell. Immunol., 116: 35-43, 1988.

40. Matsuo, T., Watanabe, S., Bouvet, J-P., Kolb. J-P., and Quan, C. P. Suppressor factor secreted by T hybridoma established from peripheral bloodlymphocytes of a bone marrow- transplantation patient. I. Establishment ofhuman T-cell hybridoma and partial characterization of suppressor factor.Cell. Immunol., 116: 450-466, 1988.

41. Cleveland. M. G.. Lane. R. G., and Kumpel. G. R. Spontaneous IFN-fíproduction. A common feature of natural suppressor systems. J. Immunol..141: 2043-2049. 1988.

2586