CELLULAR IMMUNOLOGY 141,342-35 I (1992)

Different Sensitivity to lnterleukin 4 of lnterleukin 2- and Interferon a-Induced CD69 Antigen Expression in Human Resting

NK Cells and CD3+, CD4-, CD8- Lymphocytes

FRANCA GEROSA, *J MARINA TOMMA%,* GIUSEPPE CARRA,~ GIORCIO GANDINI,~ GIUSEPPE TRIDENTE,* AND CLAUDIA BENATI*

*Is&to di Scienze Immunologiche and tlstitulo di Chirnica Biologica, Universitd di Verona.. $Centro Tra&sionale, Policlinico di Borgo Roma, 37100 Verona, Italy

Received October 24, 1991: accepted December 9. 1991

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-cu on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-a-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rlNF-a-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the 0 chain of IL-2 receptor, evaluated in NK cells by indirect im- munofluorescence. Expression of the LY chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of 1y and 0 chains of IL-2 IXCeptOr. % 1992 Academic Press, Inc.

INTRODUCTION

Natural killer (NK) cells constitute a population of lymphocytes which lyse a broad spectrum of tumor cells and virus-infected cells. The lytic activity of NK cells is po- tentiated both in vitro and in vivo by IL-2 (1) and by other soluble cytokines such as interferons (2, 3). Activation of the lytic function of NK cells induced by IL-2 is accompanied by a succession of events that lead to production of cytokines, cell pro- liferation, and the capacity to lyse NK-resistant tumor cells (LAK function) (4, 5). Recently, an antigen, defined as CD69, has been described that is acquired early after activation of T lymphocytes with PMA or with mitogenic stimuli (6,7) and is induced on NK cells after stimulation with IL-2 (8). In a previous report we demonstrated that CD69 antigen is rapidly induced on NK cells also after stimulation with rINF-cu (9).

IL-4, originally described as B cell stimulatory factor, is a cytokine with pleiotropic effects. Among the multiplicity of effects, IL-4 induces proliferation of activated B and T cells (10) and is responsible for the secretion of specific Ig isotypes (11). In

’ To whom correspondence should be addressed at Istituto di Scienze Immunologiche, Policlinico di Borgo Roma, 37 100 Verona, Italy.

342

0008-8749/92 $5.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproductnx~ in any form reserved.

EFFECTS OF IL-2/IL-4 AND INF-a/IL-4 ON NK CELLS 343

addition to activatory functions, IL-4 exerts a strong inhibitory activity on different cellular functions in various cell types: for example, it antagonizes the growth of human B cell chronic lymphocytic leukemia cells mediated by IL-2 ( 12) and inhibits superoxide production in human phagocytes ( 13). In human NK cells, IL-4 inhibits IL-2-induced LAK generation ( 14- 16).

In the present report we investigated the role of rIL-4 on CD69 antigen expression and on the lytic activity induced in human resting NK cells by rIL-2 and rINF-ol. We found that rIL-4 selectively inhibits IL-2-mediated expression of CD69 activation antigen on NK cells, and their lytic activity, while it did not alter INF-a-mediated activation of NK cell function. Moreover, the antagonism of IL-2-mediated activation was not related to downregulation of o( and 0 chains of IL-2 receptors. Finally, we analyzed the stimulatory activity of rIL-2 and rINF-a and their sensitivity to rIL-4 on CD69 antigen expression in CD3+, CD4-, CD8- cells, a fraction of T PBL that express the y/S form of the T cell receptor and share many functional activities with NK cells (17). We found that rINF-a induced CD69 antigen expression in this subset of T lymphocytes and that rIL-4 has the same pattern of modulatory activity as in NK cells.

MATERIALS AND METHODS

Isolation and stimulation of NK cells and CD3+, CD4-, CD8- PBL. NK cells were purified from PBL of normal donors by a double treatment with anti-CD3 (CBT3- M, kindly provided by F. Malavasi, Torino, Italy), anti-CD4 and anti-CD8 (B73.41 and 1 D8, respectively, kindly provided by S. Carrel, Lausanne, Switzerland). and anti- HLA-DR (Dl- 12, kindly provided by R. S. Accolla, Verona, Italy) and complement. Dead cells were removed by centrifugation on Ficoll-Hypaque density gradient. Pu- rified cells were more than 90% CD16+, less than 3% CD3+, less than 8% Leu-M I+, and less than 2% OKpanB+. Where indicated under Results, NK cells were purified from PBL, after depletion of adherent cells by plastic adhesion, by a double treatment with anti-CD3 and anti-monocyte (Leu-M I and Leu-M3, Becton-Dickinson, Moun- tain View, CA), mAb, and complement. Cells were more than 90% CD16+, less than 1% Leu-M l/Leu-M3+, and less than 3% CD3+. NK cells were incubated at 2 X 1 06/ ml in RPM1 1640 containing 10% FCS in microwells with medium alone or with medium containing the following cytokines: rINF-cu (kindly provided from Takeda Pharmaceuticals Co., Osaka, Japan by I. Suzuki, Verona, Italy), rINF--y (kindly pro- vided from Glaxo, Geneva, Switzerland by R. S. Accolla, Verona, Italy), rTNF-cu (Genetech Corp., South San Francisco, CA), and rIL-6 (British Bio-Technology, Ox- ford, UK). To evaluate the effect of rIL-4 (Genzyme, Cambridge, MA), NK cells were incubated with medium or with rIL-4 at the final concentration of 2000 U/ml. After 15 min, human rIL2 (kindly provided by Glaxo, Geneva, Switzerland) or human rINF-a at the concentrations indicated under Results were added and cells cultured for 18 hr. Fractions of PBL enriched in CD3’, CD4-. CD8- cells were obtained by rosetting with neuraminidase-treated SRBC cells and centrifugation on Ficoll-Hypaque (Et cells). After lysis of SRBC, E+ cells were treated twice with anti-CD4, anti-CDS, and anti-HLA-DR mAb and complement. Dead cells were removed by centrifugation on Ficoll-Hypaque.

Immunofiruorescence andflow cytometry. Samples of cultured NK cells were stained by indirect immunofluorescence with the following mAb: Cl. 18 (anti-CD69) (18) Marl08 (anti-CD25) (6) and FITC goat anti-mouse Ig (Becton-Dickinson). To detect

344 GEROSA ET AL.

the ~75 subunit of IL-2 receptor cells were stained by a three-step immunofluorescence using the Mikp3 mAb (IgGi , kindly provided by M. Tsudo, Tokyo, Japan) (19), bio- tinilated goat anti-mouse IgGl (Amersham), and phycoerythrin-conjugated strepa- tavidin (SAPE) (Becton-Dickinson). To detect CD69 antigen expression on CD3’-, CD4--, CD8--enriched PBL, after incubation with the cytokines, cells were stained by double immunofluorescence with biotinilated C 1.18 mAb and SAPE- and FITC- conjugated anti-Leu4 mAb. Samples were analyzed on a flow cytometer (Profile, Coul- ter, Hialeah, FL).

Assayfor cytolytic activity. Cytolytic activity of cultured NK cells was assayed against K562 or Raji cell lines labeled with 5’Cr at the effector:target (E:T) ratios indicated under Results. Plates were incubated for 4 hr at 37°C. After incubation, the plates were centrifuged and cytolysis was tested by counting the radioactivity of 0.1 ml su- pernatant. Lysis (%) was calculated as described (16).

RESULTS

We analyzed the capacity of rINF-a, rINF-y, rTNF-cq and rIL-6 to induce the expression of the CD69 early activation antigen, associated with the described increase of lytic activity against the NK-sensitive K562 target cell line (20,2 1). The incubation of NK cells for 18 hr with rINF-cu induced CD69 antigen expression in the large majority of NK cells (Table 1). In contrast, the expression of CD69 antigen after incubation with rINF-y, rTNF-or, and rIL-6 was comparable to CD69 antigen expres- sion of NK cells incubated with medium alone. Although CD69 antigen expression of NK cells did not appreciably increase after incubation with rINF-y or rTNF-a, the cytolytic activity of NK cells against the K562 target cell line increased after incubation with these cytokines; however, the cytolytic activity induced by rTNF-a and by rINF- y was much lower than the cytolytic activity induced by rINF-a. Cytolytic activity of cells incubated with rIL-6 did not differ, in our study, from control values. To avoid the loss of CD8+ NK cells and to completely deplete monocytes, in some experiments NK cells were purified from PBL by plastic adherence and treatment with anti-CD3 and anti-monocyte mAb and complement (Table 1, Experiments 2 and 3). The results did not differ employing NK cells obtained by the two different methods of enrichment.

The effect of rIL-4 on the expression of CD69 activation antigen induced in NK cells by incubation with rIL-2 or rINF-Lu is shown in Fig. 1. The expression of CD69 antigen induced by rIL-2, at concentrations as high as 500 U/ml, was almost completely inhibited by rIL-4. In contrast, the expression of CD69 antigen induced by rINF-a (1000 U/ml) was not affected by the presence of rIL-4. Additionally, rIL-4 did not affect CD69 antigen expression induced by suboptimal ( 100 U/ml) rINF-oc concen- trations (49 and 5 1% CD69+ cells in the absence and in the presence of rIL-4, respec- tively).

The same results were obtained with NK cells purified from PBL by plastic adherence and treatment with anti-CD3 and anti-monocyte mAb and complement (data not shown).

The cytolytic activity against Raji target cell line of NK cells incubated with rINF- a alone was comparable to the cytolytic activity of cells incubated with rINF-a plus rIL-4, whereas the cytolytic activity induced by rIL-2 was strongly reduced by the presence of rIL-4 (Table 2).

TABL

E 1

Effe

ct o

f Diff

eren

t C

ytok

ines

on

CD

69 A

ntig

en E

xpre

ssio

n an

d C

ytol

ytic

Act

ivity

ag

ains

t K56

2 Ta

rget

Cei

ls o

f Pur

ified

NK

Cel

ls

i .>

Expe

rimen

t 1’

Ex

perim

ent

2’

Expe

rimen

t 3’

3 cn

%

Lys

is

% L

ysis

%

Lys

is

g Tr

eatm

ent“

(U/m

l) %

CD

69+

30: 1

IO

:1

3:1

9% CD

69+

20: 1

IO

:5

5:l

% C

D69

+ 30

: 1

IO:1

3:

l F L

Med

ium

35

36

16

I

20

6 5

2 29

53

36

27

-s

I * rIN

F-cx

(200

0)

74

100

8-l

41

71

60

41

21

IO

93

71

57

rINF-

mu (5

00)

67

100

82

42

51

nd

67

83

72

5o

52

0 rT

NF-

ol(5

000)

29

53

24

5

22

21

21

14

42

71

49

31

rTN

F-ol

(200

0)

42

40

21

II 19

21

20

13

35

65

44

28

2

rINF-

y (2

000)

28

53

28

15

11

24

16

13

36

64

55

37

4

rINF-

7 (5

00)

35

61

29

14

18

21

13

8 nd

nd

7

rIL-6

(20

00)

30

32

14

I 16

5

I 3

36

48

33

28

; rIL

-6 (

500)

32

34

16

9

nd

nd

33

44

33

19

z

a NK

cells

wer

e in

cuba

ted

with

med

ium

con

tain

ing

the

diffe

rent

cyt

okin

es. A

fter

18 hr

of c

ultu

re, e

xpre

ssio

n of C

D69

ant

igen

was

det

ecte

d by

indi

rect

imm

unof

luor

esce

nce;

3

the

cyto

lytic

act

ivity

was

det

ecte

d ag

ains

t 5’C

r-lab

eled

K56

2 ta

rget

cel

ls.

I; * N

K ce

lls (m

ore

than

90%

CD

l6+)

w

ere

obta

ined

by

treat

men

t w

ith a

nti-C

D3,

ant

i-CD

4, a

nti-C

D8,

and

ant

i-HLA

-DR

m

Ab a

nd c

ompl

emen

t. Si

mila

r re

sults

wer

e F ~

obta

ined

in th

ree

diffe

rent

exp

erim

ents

. ’ N

K ce

lls (m

ore

than

90%

CD

16+)

wer

e ob

tain

ed f

rom

adh

eren

t cel

l-dep

lete

d PB

L by

trea

tmen

t w

ith a

nti-C

D3,

ant

i-Leu

-Ml,

and

anti-

Leu-

M3

mAb

and

com

plem

ent.

346 GEROSA ET AL.

medlum

rll-4

rlL-2

rlL-2 + rlL-4

rlNF-Cl

rlNF-CJ + rlL-4

Log. fluorescence intensity

FIG. I. Effect of rIL-4 on surface expression of CD69 activation antigen induced in NK cells by rIL-2 or by rlNF-a. NK cells (more than 90% CD16+) were incubated for 15 min with medium (thick line) or with rIL-4 (2000 U/ml) (thin line) before addition of medium or of medium containing rIL-2 (500 U/ml) or rINF-a (1000 U/ml), respectively. After I8 hr of culture, expression of CD69 antigen was detected by indirect immunofluorescence. The background fluorescence (dotted line) is given by cells stained with FITC anti- mouse reagent alone. Values (%) of CD69+ cells were medium, 20; rIL-4, 18; rIL-2, 66; rIL-2 + rIL-4, 25; rINF-cu, 78; rINF-a + rIL-4, 77. Similar results were obtained in six different experiments.

The expression of CD69 activation markers and cytolytic activity, therefore, clearly showed that stimulation of NK cells by rIL-2 is sensitive to the inhibitory effect of rIL-4, while stimulation of NK cells by rINF-a was not inhibited by rIL-4.

To evaluate whether rIL-4 might inhibit IL-Zinduced activation of NK cells through modulation of LY (CD25) and 0 (~75) subunits of IL-2 receptors, NK cells, incubated for 18 hr with medium alone or with rIL-2 in the presence or in the absence or rIL- 4, were stained by indirect immunofluorescence with Mar108 (anti-CD25) or with Mik@3 (anti-p75) mAb (Fig. 2). It can be seen that NK cells did not express detectable CD25 antigen, under any culture condition. The expression of ~75 p chain of NK cells cultured with medium or with rIL-2 was superimposable on the expression of ~75 /3 chain of NK cells cultured with rIL-4 or rIL-2 plus rIL-4, respectively. Therefore, the selective inhibitory effect of rIL-4 on the activation induced by rIL-2 was not due to downmodulation of the ~75 p chain of IL-2 receptor. The different expression of ~75 /3 chain detectable between cells incubated with or without rIL-2 was not due to competition with rIL-2 since the Mik/33 mAb did not compete with rIL-2 for binding

EFFECTS OF IL-2/IL-4 AND INF-a/IL-4 ON NK CELLS 347

TABLE 2

Effect of rIL-4 on Cytolytic Activity Against Raji Target Cells Induced in NK Cells by rIL-2 and by rINF-n

o/o “0 release

Treatment W/ml) E:T

Experiment I Experiment 2

Medium rIL-4 Medium rIL-4

Medium

rIL-2 (500)

rlNF-n (2000)

IO:1 3

5:l 0 1O:l 71 5:1 40

IO:1 32 5:l 25

rINF-a (1000) IO:1 5:l

nd nd

rINF-cu (100) IO:1 4 5:l 3

I 0

30 I7 35 20

9

7 0

70 50 29 18 I7 I2

0

29 16 20 14 16 I4

nd nd

No(P. NK cells (more than 90% CD16+) were treated as described in the legend to Fig. I. After 18 hr of culture. the cytolytic activity of NK cells was tested against S’Cr-labeled Raji target cells. rlL-4 was used at the final concentration of 2000 U/ml.

to ~75 p chain (19). However, this difference could be explained by modulation of the receptor in cells cultured in the presence of rIL-2. Indeed, in the presence of NaN3, NK cells incubated with i-IL-2 or with medium alone did not show considerable dif- ferences in the expression of ~75 fl chain (data not shown).

The proliferative activity of NK cells cultured with rIL-2 or rINF-a in the presence or in the absence of rIL-4 was also evaluated (Table 3). In contrast to the strong inhibitory activity exerted by rIL-4 on IL-2-induced CD69 antigen expression and cytolysis, i-IL-4 only slightly reduced NK cell proliferation in response to rIL-2. No effect of rINF-cu, alone or in combination with rIL-4, was detectable.

We have recently reported that rINF-cu did not induce CD69 antigen expression in purified T lymphocytes (9). In the present report we evaluated whether rINF-a could induce CD69 antigen expression in CD3+, CD4-, CD8 lymphocytes, a fraction of T lymphocytes consisting of less than 2% of T cells in the majority of normal donors. To obtain populations of cells enriched in CD3+, CD4-, CD8 cells, E+ PBL were treated with anti-CD4, anti-CD8, anti-DR mAb, and complement. The population obtained was composed of 30-50% CD3+, CD4-, CD8 lymphocytes and 50-70% CD16+ cells. These cells were incubated for 18 hr with rINF-cu. Expression of CD69 antigen was evaluated on CD3’ cells by double immunofluorescence. Figure 3 shows that rINF-a (1000 U/ml) induced CD69 antigen expression in CD3+, CD4-, CD8.~ cells. Moreover, in analogy with data obtained with NK cells, expression of CD69 antigen induced by rINF-oc was not altered by the presence of rIL-4 (Fig. 3). rIL-4 inhibited CD69 antigen expression induced by a wide range of concentrations of rIL- 2: the effect of 500 U/ml rIL-2 was completely inhibited by rIL-4 (data not shown): inhibition was also observed employing very high amounts of rIL-2 (2000 U/ml), as in the case of the experiment represented in Fig. 3.

348 GEROSA ET AL.

p75 CD25

h,, _ kL medium

‘,,-. 1 r’L-2

LA. 1 rll-4

Le. ,,, rll-2 l rll-4 1,

Log. fluorescence Intensity

FIG. 2. Expression of ~75 p chain and CD25 @ chain of IL-2 receptor on NK cells incubated for 15 min with medium or rIL-4 (2000 U/ml) before addition of medium or rIL-2 (500 U/ml). Cells were stained after I8 hr of culture with Mikp3 or with Marl08 mAb. Background fluorescence (dotted line) is given by cells stained with biotinilated goat anti-mouse IgG, , SAPE, and FITC goat anti-mouse Ig, respectively. Values (%) of p75+ cells were medium, 95; rIL-2, 78; rIL-4, 92; rIL-2 + rIL-4, 79. Values (%) of CD25+ cells were medium, 3; rIL-2, 6; rIL-4, 3; rIL-2 + rIL-4, 3. Similar results were obtained in three different experiments.

DISCUSSION

In the present report we have investigated the effect of rIL-4 on CD69 antigen expression and lytic activity induced by rIL2 and by rINF-or on human resting NK cells. We found that activation of NK cells induced by rINF-cu was not inhibited by rIL-4, while both expression of CD69 activation antigen and cytolytic activity against the NK-resistant Raji target cell induced by rIL-2 were drastically inhibited. It has been previously reported that rIL-4 may act differently on different functions of NK cells: indeed, this cytokine drastically inhibits the generation of cytolytic activity against NK-resistant tumor target cells (LAK function); in contrast, the lytic activity against NK-sensitive target cells (NK function) is not influenced by rIL-4 (15, 16). Therefore, the drastic inhibition of rIL-4 on CD69 antigen expression suggests that the expression of this antigen is related to the generation of LAK function rather than to an increase of NK cell function. This view may be supported by the finding that no appreciable increase in CD69 antigen expression was associated with increased lytic activity of

EFFECTS OF IL-2/IL-4 AND INF-n/IL-4 ON NK CELLS 349

TABLE 3

Effect of rIL-4 on Proliferative Activity of NK Cells in Response to rIL-2 or rINF-cu

[‘H]Tdr incorporation

Medium rIL-4

Medium rIL-2 rlNF-tu

Experiment I

100 5800

200

100 4100

nd

Medium rIL-2 rINF-cu

Experiment 2

100 250 7700 5300

250 300

/Vote. NK cells, treated as described in legend to Fig. 1, were cultured for 72 hr. [3H]TdR was added 18 hr before cell harvesting. Results are expressed as means from triplicate cultures. Cytokines were used at the following final concentrations: rIL-4, 2000 U/ml; rIL-2. 500 U/ml; rINF-‘-N, 2000 U/ml. CD3+ cells at the end of the culture were less than 4% in both experiments.

rINF-y and rTNF-cr against the NK-sensitive K562 target cell. Regarding this last point, however, the involvement of residual contaminating cells, that could be activated by rINF-y or by rTNF-cu, cannot be ruled out. It has been recently reported that rIL- 6 increases NK lytic activity without stimulating other functions such as LAK activity or cytokine production from NK cells (21). In our study, however, rIL-6 did not increase the lytic activity of NK cells against the K562 target cells. The discrepancy might be explained by differences in the effector population of NK cells due to dif- ferences in the methods of enrichment employed.

In contrast to the drastic effect on IL-2-induced CD69 antigen expression and lytic activity, we found that IL-2-induced proliferative activity of NK cells was only slightly reduced. A complete abrogation of IL-2-induced proliferative activity by rIL-4 has been previously reported (15). This discrepancy remains to be explained; however, the doses of rIL-4 employed were largely different, which could explain the different results.

The finding that rIL-4 selectively inhibited IL-2-generated CD69 antigen expression and the lytic activity of NK cells, while not altering the functions generated by rINF- N, could be explained by several hypotheses: a downmodulation of CY and p chains of IL-2 receptor in the presence of rIL-4; a selective activation of different subpopulations of NK cells; and the triggering of resting NK cells through different activation pathways. Data obtained from the expression of (Y chain of IL-2 receptor demonstrated that this antigen was not expressed on the population of NK cells employed in this study, nor was it induced after culture with rIL-2. Therefore, modulation of CD25 antigen could not account for the inhibitory effects of rIL-4. It has been previously reported that a small subset of NK cells constitutively expresses CD25 antigen (22). Indeed, when NK cells were purified from adherent cell-depleted PBL by treatment with anti-CD3 and anti-monocyte mAb and complement, a small fraction of CD25+ cells (ranging from 8 to 12% in three different experiments) could be observed. Data obtained on

350 GEROSA ET AL.

rlNF-o

rlNF-a + We-4

FIG. 3. Effect of rIL-4 on surface expression of CD69 activation antigen induced in CD3+, CD4-, CD8- lymphocytes by rIL-2 or by rINF-cu. Cells that consisted of 30% CD3+, CD4-, CD8-, and 67% CD16+ lymphocytes were incubated as described in legend to Fig. 1. After 18 hr of culture, cells were stained by double immunofluorescence with FITC-conjugated anti-Leu-4 mAb and biotinilated C 1. I8 mAb and SAPE. Electronic gates were set on CD3+ cells and data were reprocessed to determine CD69 antigen expression in the CD3+ population. The background fluorescence is given by cells stained with FITC-conjugated anti- Leu-4 mAb and SAPE. Cytokines were used at the following final concentrations: rIL-4, 2000 U/ml; rIL-2, 2000 U/ml; rINF-a, 1000 U/ml. Values (%) of CD69+ cells in the CD3+ fraction were medium, 22; rIL-2, 84; rIL-2 + rlL-4, 65; rINF-a, 79; rINF-ru + rIL-4, 82.

the effects of rIL-4 on the expression of p75 /3 chain of IL-2 receptor indicate that this antigen was not downmodulated by rIL-4. Taken together, data obtained on 01 and 0 chains of IL-2 receptor strongly suggest that inhibition exerted by rIL-4 on NK cells stimulated by rIL-2 is not mediated by interaction with IL-2 receptors. This suggestion is further strengthened by the report that rIL-4 exerts its inhibitory activity also on IL-7-generated LAK activity (23). Previous results on the effect of rIL-4 on IL-2- mediated and rINF-a-mediated cytolytic activity alone (15) could not discriminate between the last two hypotheses. Our combined analyses of the expression of an ac- tivation marker and of a functional assay provide strong evidence that their induction in NK cells by rINF-a and by rIL-2 might be mediated by the triggering of different activation pathways. In this context, it has been recently suggested that rIL-4 exerts its antagonistic action on intracytoplasmic second messenger associated with stimu- lation by rIL-2 (24).

It is known that CD3+, CD4-, CD8- lymphocytes, a fraction of T cells expressing the r/s form of the T cell receptor, can lyse NK-resistant tumor target cells similarly to NK cells and can express CD69 activation antigen after incubation with rIL-2 (8,

EFFECTS OF IL-2/IL-4 AND INF-n/IL-4 ON NK CELLS 351

17). The finding that rINF-cw induced CD69 antigen expression in CD3+, CD4+, CD8- T lymphocytes and that rIL-4 has the same pattern of activity in CD3+, CD4-, CD8- PBL and in NK cells contributes to strengthening the similarity of the two populations of cells.

Finally, the finding that the activity of rINF-cu, a potent stimulator of NK cell function, is not downregulated by rIL-4 could be important in view of the employment of combinations of cytokines in the therapeutic approach to human cancer.

ACKNOWLEDGMENTS

This work was supported by CNR projects “Oncology” and “Biotechnology and Bioinstrumentations.” by Minister0 Pubblica Istruzione by A.I.R.C. and by Regione Veneto.

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