Veterinary Immunology and Immunopathology, 14 (1987) 297-307 297 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

CYTOTOXIC CAPABILITIES OF BOVINE LYT4PHOCYTES AFTER LECTIN STIMULATION

MANUEL CAMPOS and CHARLES R. ROSSI

Animal Health Research, Auburn University, AL 36849, U.S.A.

(Accepted 26 September 1986)

ABSTRACT

Campos, M., and Rossi, C.R., 1987. Cytotoxic capabilities of bovine lymphocytes after lectin stimulation. Vet. Immunol. Immunopathol., 14, 297-307.

Bovine peripheral blood mononuclear leukocytes (PBML) were stimulated in vitro with the mitogenic lectins concanavalin A (Con A) and phytohemagglutinin. Their cytotoxlc capabilities were evaluated in a 51Cr release assay. Lectin-actlvated bovine effector cells did not mediate antibody dependent cellular cytotoxlcity (ADCC) nor direct killing against cultured tumor target cells. Nevertheless, activation of PBML with lectins consistently generated effector ceils able to mediate lectin-dependent cellular cytotoxicity. Cultivation of Con A stimulated-PBML for 3 to 4 weeks in the presence of' lymphokines-containing IL-2 generated cells with the ability to mediate lysis without using Con A-coated target cells. However, cytotoxic c u l t u r e s c a p a b l e of m e d i a t i n g d i r e c t l y s i s of t a r g e t c e l l s were no t a b l e to med ia t e ADCC.

INTRODUCTION

A m p l i f i c a t i o n of t he immune r e s p o n s e i n i t i a t e d by t h e t n t e r e c t i o n of immune

c e l l s and f o r e i g n a n t l g e n i s an i m p o r t a n t arm in the r e g u l a t i o n o f d i s e a s e

p P o c e s s e s . G e n e r a t i o n of n o n . s p e c i f i c k i l l e r c e i l s t h a t accompany t h e

e x p a n s i o n of t h e immune r e s p o n s e i s an a r e a of t h e a m p l i f i c a t i o n p r o c e s s t h a t

has g e n e r a t e d i n t e n s e r e s e a r c h in r e c e n t y e a r s (Welsh, 1978; Welsh and Doe,

1980) . N o n - s p e c i f i c c y t o t o x l c c e l l s a r e b e l i e v e d to p l a y an i m p o r t a n t r o l e in

c o n t r o l l i n g v i r a l s p r e a d ( S t e i n - S t r e l l e i n and 6 u f f e e , 1966) and tumor g rowth in

a h o s t (Mule e t a l . , 1966). However, p r e v i o u s work In ou r l a b o r a t o r y has

d e m o n s t r a t e d t h a t l y m p h o c y t e s t h a t can m e d i a t e c y t o t o x l c i t y a r e n o t d e t e c t a b l e

in b o v i n e p e r i p h e r a l b lood (Canpos e t a l . , 1982; Campos and R o s s t , 1985) and

t h a t c u l t u r e o f p r e c u r s o r c e l l s i s n e c e s s a r y f o r g e n e r a t i o n of some

non-specific cytotoxlc cells (Brigham and Rossl, 1986) Chung and Rossl, 1986)

whereas activation by lymphoklnes (LK) is necessary to generate other

non-speciflc cytotoxlc populations able to mediate direct lysis and

antibody-dependent cellular cytotoxicity (ADCC) of cultured tumor cells and

virus-lnfected cells (Campos and Rossi, 1986). The effects of other

stlmulatory factors in the generation of the cytotoxic function of bovine

lymphocytes have not been determined. In humans, lectin activation of

0165-2427/87/$03.50 © 1987 Elsevier Science Publishers B.V.

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l y m p h o c y t e s h a s been u s e d t o g e n e r a t e c y t o t o x i c c e l l s a g a i n s t a w ide v a r i e t y of

t a r g e t c e l l s ( H u n n i n g h a k e and F a u c i , 1977; Mazumder e t a l . , 1983) and u s e d t o

augmen t t h e a b i l i t y of ] y m p h o c y t e s t o m e d i a t e ADCC ( C o n n o l y e t a l . , 1975) .

F u r t h e r m o r e , l e c t i n a c t i v a t i o n of human PBML h a s been u s e d a s an a l t e r n a t i v e

a p p r o a c h f o r a d o p t i v e i m m u n o t h e r a p y i n c a n c e r p a t i e n t s (Mazumder e t a l . ,

1984 ) . I t was t h e r e f o r e of i n t e r e s t t o i n v e s t i g a t e t h e c y t o t o x i c

c h a r a c t e r i s t i c s o f b o v i n e PBML a f t e r l e c t i n a c t i v a t i o n .

METHODS

T a r g e t c e l l s

Human c e l l l i n e s HSB-2 and K562 were c u l t u r e d a t 38oc i n a h u m i d i f i e d

a t m o s p h e r e of 95% a i r and 5~ C02 and m a i n t a i n e d i n s u s p e n s i o n i n RPMI-1640

c o n t a i n i n g 10% f e t a l b o v i n e se rum (FBS), 200 U/ml of p e n i c i l l i n , 10 u g / m l of

s t r e p t o m y c i n , 2 mM of L - g l u t a m i n e , and 0 .1 mM of 2 - m e r c a p t o e t h a n o l (2-ME).

A n t i s e r u m

A n t i s e r u m a g a i n s t HSB-2 o r K562 c e l l s was p r o d u c e d i n c a l v e s by r e p e a t e d

i n t r a m u s c u l a r i n j e c t i o n of 108 c e l l s a t m o n t h l y i n t e r v a l s . P o s i t i v e se rum,

a s d e t e r m i n e d by a g g l u t i n a t i o n , was h e a t - i n a c t i v a t e d (56oc , 30 min) and

s t o r e d a t - 2 5 o c u n t i l u s e d .

P r e p a r a t i o n of c r u d e I n t e r l e u k i n 2 ( I L - 2 )

PBML were c u l t u r e d i n 25 -30 ml i n 75 cm2 t i s s u e c u l t u r e f l a s k s a t 3 . 5 X

106 c e l l s / m l i n 0pti-MEM I medium (GIBCO, Grand I s l a n d , N.Y. ) s u p p l e m e n t e d

w i t h 3% FBS, a n t i b i o t i c s , and 0 . I mM 2-ME. C u l t u r e f l a s k s were i n c u b a t e d f o r

24 h r s a t 38oC i n an a t m o s p h e r e o f 95~ a i r and 5% CO2. A f t e r i n c u b a t i o n ,

c u l t u r e d c e l l s were s u p p l e m e n t e d w i t h 5 u g / m l o f c o n c a n a v a l i n A (Con A) and I0

n g / m l of p h o r b o l m y r l s t a t e a c e t a t e (S igma Chem. Co . , S t . L o u i s , Mo. ) . The

s u p e r n a t a n t s were h a r v e s t e d a f t e r 18 h r s of f u r t h e r i n c u b a t i o n . IL-2 was

a s s a y e d on I L - 2 - d e p e n d e n t l y m p h o b l a s t s wh ich were o b t a i n e d e i t h e r by Con A

s t i m u l a t i o n o r by one -w a y mixed l y m p h o c y t e c u l t u r e . The a s s a y c e l l s were

washed f r e e of g r o w t h medium and r e s u s p e n d e d a t 1 X 105 c e l l s / m l i n RPMI-1640

s u p p l e m e n t e d w i t h 15% FBS, a n t i b i o t i c s , and 2 mM L - g l u t a m i n e . IL-2 d e p e n d e n t

l y m p h o b l a s t s (100 u l / w e l l ) were c u l t u r e d f o r 18 h r s i n 9 6 - w e l l m i c r o t i t e r

p l a t e s w i t h s e r i a l 2 - f o l d d i l u t i o n s of c r u d e I L - 2 . S i x h o u r s b e f o r e t h e

c u l t u r e s were t e r m i n a t e d , 1 uCi of [ 3 H ] - t h y m l d i n e was a d d e d t o e a c h w e l l t o

a s s e s s c e l l u l a r p r o l i f e r a t i o n . C u l t u r e s were h a r v e s t e d o n t o g l a s s f i b e r f i l t e r

s t r i p s and [ 3 H ] - t h y m i d i n e u p t a k e was d e t e r m i n e d u s i n g a l i q u i d s c i n t i l l a t i o n

c o u n t e r . O p t i m a l p r o l i f e r a t i o n was u s u a l l y p r e s e n t In w e l l s t h a t r e c e i v e d 10%

(V/V) c r u d e I L - 2 .

299

P r e p a r a t i o n o f e f f e c t o r c e l l s

P e r i p h e r a l b l o o d m o n o n u c l e a r c e l l s (PBffL) were o b t a i n e d f rom b l o o d o f normal

s t e e r s w i t h d r a w n by v e n l p u n c t u r e a s p r e v i o u s l y d e s c r i b e d (Campos and R o s s i ,

1985).

For l e c t t n a c t i v a t i o n of l y m p h o c y t e s , f r e s h PBNL were seeded a t 3 .5 X 106

c e l l s / m l In 12 -we l l p l a t e s in RPMI-1640 c o n t a i n i n g 15~ FBS, a n t i b i o t i c s , 2 mN

L - g l u t a m l n e , 0.1mM 2-ME, and e i t h e r 5 ug/m] of Con A o r 1 ug /ml of

p h y t o h e m a g g l u t t n i n (PHA). Two days a f t e r l e c t i n s t i m u l a t i o n , c e l l s were

l a y e r e d o n t o H l s t o p a q u e (Sigma Chem. Co.) ( s p gr= 1 .077g/cm3) and c e n t r i f u g e d

to s e p a r a t e dead and l i v e c e l l s . L y m p h o b l a s t s were r e s e e d e d a t 1 X 105

c e l l s / m l in 12 -we l l p l a t e s (1 m l / w e l l ) In c u l t u r e medium. Crude IL-2 was added

a t c o n c e n t r a t i o n s o p t i m a l f o r p r o l i f e r a t i o n a s d e t e r m i n e d by a s s a y of IL-2

b i o l o g i c a l a c t i v i t y . In t h o s e i n s t a n c e s where l e c t i n - a c t t v a t e d c e l l s were

t e s t e d f o r c y t o t o x i c i t y a f t e r l o n g i n c u b a t i o n , c u l t u r e medium was removed e v e r y

2 days and f r e s h medium and c r u d e IL-2 were added. In t he a b s e n c e of IL-2 ,

c u l t u r e s d i ed w i t h i n 5 d a y s .

For lymphokine (LK) a c t i v a t i o n o f l y m p h o c y t e s , f r e s h PBNL were seeded a t 3 .5

X 106 c e l l s / m l in 12 -w e l l p l a t e s in RPMI-1640 c o n t a i n i n g 15~ FBS,

a n t i b i o t i c s , 2 mM L - g l u t a m i n e , 0 .1 mM 2-ME, 25 mM a l p h a m e t h y l - 0 - m a n n o s t d e

(NDN) (Sigma Chem. Co.) to b ind f r e e Con A ( S i t k o v s k y , e t a l . , 1982) , and

10-20~ c r u d e I L - 2 - c o n t a t n i n g s u p e r n a t a n t s . C u l t u r e medium was removed e v e r y 2

days and f r e s h medium added. A f t e r c u l t u r e t h e c e l l s were l a y e r e d on to

H l s t o p a q u e and c e n t r i f u g e d to s e p a r a t e dead from l i v e c e l l s which were used as

e f f e c t o r c e l l s .

C e l l u l a r morpho logy was e v a l u a t e d on c e l l p r e p a r a t i o n s a f t e r c e n t r i f u g a t i o n

on a c y t o c e n t r i f u g e ( C y t o s p l n c e n t r i f u g e , S o u t h e r n I n s t r u m e n t s , S e w l c k l e y , Pa . )

and s t a i n e d w i t h D i f f Quick ( H a r l e c o , G ibbs town , N . J . ) . V i a b i l i t y was

d e t e r m i n e d by t r y p a n b l u e e x c l u s i o n .

C y t o t o x i c l t y a s s a y

C y t o t o x l c t t y a s s a y s were p e r f o r m e d in q u a d r u p l i c a t e in r o u n d - b o t t o m

m l c r o t i t e r p l a t e s a s d e s c r i b e d p r e v i o u s l y u s i n g 5 1 C r - l a b e l e d t a r g e t s (104

c e l l s / w e l l ) In a 4 - h o u r a s s a y (Campos and R o s s i , 1985). For ADCC a s s a y s ,

a n t t - H S B - 2 o r a n t i - K 5 6 2 a n t i b o d y (25 u l ) was added t o t h e w e l l s b e f o r e a d d i t i o n

o f e f f e c t o r c e l l s . For l e c t l n - d e p e n d e n t c e l l u l a r c y t o t o x l c i t y (LDCC) a s s a y s ,

t a r g e t c e l l s were p r e l n c u b a t e d w i t h 25 ug of Con A f o r 0 .1 t o 0 .5 h r s b e f o r e

t h e l a s t wash. In a l l e x p e r i m e n t s , 100 u l of e f f e c t o r c e l l s (106 c e l l s / w e l l )

were added to each w e l l t o o b t a i n an e f f e c t o r : t a r g e t c e l l r a t i o of 10:1 . E i g h t

w e l l s were t r e a t e d w i t h 100 u l o f T r i t o n X-IO0 (Sigma Chem Co.) t o d e t e r m i n e

maximum r e l e a s e a b l e 51Cr. At t h e t e r m i n a t i o n o f t h e a s s a y , t h e s u p e r n a t a n t

300

was collected with the Tltertek harvesting device (Skatron Inc., Sterling, Va.)

and the amount of 51Cr released determined in a gamma counter. Wells without

effector ceils were used to determine spontaneous 51Cr release. Standard

deviation of quadruplicate wells was usually less than 6~. Specific

cytotoxlcity was calculated as follows: ~ specific SlCr release= i00 X [(mean

cpm experiment - mean cpm spontaneous release)/(mean cpm detergent lysis - mean

cpm spontaneous release)]. To facilitate understanding of results, the terms

direct, LDCC and ADCC are used as follows: The direct cytotoxicity assays were

done wlth uncoated target cells, i.e., targets were neither coated with

antibody nor lectin. The LDCC assays were done with target cells precoated

with Con A by incubating 1 ml of target cells (3 x 108) with 25 ug of Con A

before mixing target and effector cells. The ADCC assays were done by coating

the target cell with specific anti-HSB-2 antibody; (25 ul) was included in the

assays.

RESULTS

The m i t o g e n i c l e c t i n s Con A and PHA c a u s e T c e l l b l a s t o g e n e s i s i n c a t t l e

s i m i l a r t o t h a t d e s c r i b e d f o r o t h e r s p e c i e s . However , t h e r e i s no a v a i l a b l e

i n f o r m a t i o n a b o u t t h e c y t o t o x i c c h a r a c t e r i s t i c s o f t h e p r o l i f e r a t i n g c u l t u r e s .

In humans, ADCC as well as direct killing of target cells can be significantly

augmented if effector cells are prestlmulated with lectins (Hunninghake and

Faucl, 1977; Mazumder et al., 198S; Connoly et al., 1975). To investigate

whether bovine lymphocytes were able to respond to lectin stimulation in a

similar way, PBML were stimulated with Con A or PHA for 5 days and their ADCC

capabilities against HSB-2 cells compared with that of PBML stimulated with LK

(Table 1). In all instances, as demonstrated previously (Campos and Rossi,

1986), Lg stimu]atlon generated cells able to mediate ADCC, whereas neither Con

A nor PHA stimulation generated ceils with similar cytotoxic characteristics.

To further evaluate the cytotoxic capabilities of bovine lymphocytes after

Con A stimulation, normal PBML were stlmulated with Con A for 24 hours and

maintained in culture in the presence of crude IL-2 for 5 additional days.

Proliferating cultures were tested in 3 different cytotoxic assay systems

against HSB-2 and R562 target cells (Table 2). Stimulated cultures were not

able to mediate direct killing nor ADCC against either target cell.

Nevertheless, significant cytotoxlcity was obtained if the target cells were

precoated with lectin in an LDCC assay.

In order to investigate the effect of time on cytotoxicity mediated by Con

A-stimulated cultures, PBML were stimulated with Con A for 24 hrs and

maintained in culture medium supplemented with crude IL-2 for different time

intervals. Cytotoxiclty was evaluated by measuring percentage specific 51Cr

301

r e l eased f rom K562 and HSB-2 t a r g e t c e l l s by d l r e c t assay and by LDCC (Table

3 ) . Fresh u n s t i m u l a t e d PBNL f rom 6 d i f f e r e n t ca l ves were no t ab le to mediate

LDCC or d i r e c t k i l l i n g o f t a r g e t c e l l s . LDCC mediated by Con A - s t i m u l a t e d

c u l t u r e s was f i r s t d e m o n s t r a t e d 2 days a f t e r s t i m u l a t i o n and was p r e s e n t a t a l l

s u b s e q u e n t t i m e s . D i r e c t c y t o t o x i c i t y was n o t e v i d e n t a t 0, 2, 5, and 13 days ,

b u t was p r e s e n t a t 21 and 35 days o f c u l t u r e . HSB-2 c e l l s were n o t l y s e d in

t h e a b s e n c e of l e c t t n . To f u r t h e r a n a l y z e t h e d e v e l o p m e n t o f e f f e c t o r c e l l s

capable of mediating direct lysls, Con A-stlmulated PBML were maintained in

culture medium containing crude IL-2. Seventeen-day-old cultures mediated

LDCC, dld not mediate direct lysis of K562 or HSB-2 cells and were not able to

mediate ADCC (Table 4). In contrast 50-day-old cultures mediated LDCC against

K562 target cells, and were able to lyse K562 in the presence or absence of

lectln. However, cytotoxic effector cells were never capable of mediating ADCC

against HSB-2 cells.

TABLE 1 C F t o t o x i c i t y o f PBHL a f t e r 5 days o f l e c t i n o r LK a c t i v a t i o n *

HSB-2

Expt Ca l f S t i m u l a n t D i r e c t ADCC

120 LK 0 . 0 " * 18.0 Con A 3.1 4.2 PHA 0.6 7 .5

125 LK 2.5 23.3 Con A 1.7 1 .0 PHA 3.8 1.2

128 LK 7 . 9 2 4 . 9 Con A - 0 . 4 4 .5

187 LK 2.5 3 9 . 8 Con A -1 .2 5.8 PHA 3.8 - 1 . 3

188 LK ] . 7 16.0 Con A - 1 . 0 2.0 PHA -0 .1 - 1 . 2

189 LK 5 . 4 2 6 . 9 Con A 3.1 5.1 PHA 4 . 4 4 . 5

, PBML were t r e a t e d w i t h LK, Con A o r PHA. F ive days l a t e r t h e e f f e c t o r c e l l c a p a b i l i t y o f t h e c u l t u r e s was m e a s u r e d .

** P e r c e n t a g e s p e c i f i c l y s i s .

302

TABLE 2 Cytotoxiclty of PBML after stimulation with Con A*

NSB-2 K562

Calf Direct ADCC LDCC Direct ADCC LDCC

120 - 2 . 0 * * 2 . 5 2 2 . 7 0 . 8 2 . 8 13 .8 125 3 . 8 1 .3 2 8 . 5 1 . 5 3 . 9 2 2 . 8 128 2 . 5 - 1 . 9 2 2 . 2 - 0 . 4 3 . 1 3 7 . 9 187 - 0 . 2 0 . 3 3 6 . 4 0 . 0 1 . 5 26 .7 1 8 8 - 0 . 6 - 5 . 0 24 .1 - 0 . 8 1 .1 2 7 . 5 189 0 . 9 - 0 . 9 27 .7 2 . 6 0 . 8 2 6 . 6

* PBML were t r e a t e d w i t h Con A f o r 24 h o u r s , washed and k e p t i n c u l t u r e i n t h e p r e s e n c e of c r u d e I L - 2 . F i v e d a y s a f t e r i n i t i a l t r e a t m e n t , t h e e f f e c t o r c e l l c a p a b i l i t y of t h e c u l t u r e s was m e a s u r e d .

** Percentage specific lysis.

TABLE 3 E f f e c t o f c u l t u r e t i m e on c y t o t o x l c l t ¥ m e d i a t e d by Con A - a c t i v a t e d PBML*

LDCC Direct Days in Number culture of calves K562 HSB-2 K562 HSB-2

0 5 2 . 5 + 1 . 1 " * 4 . 1 + 1 . 6 0 . 7 + 1 . 3 - 0 . 8 + 3 . 4 2 8 2 0 . 9 + 5 . 9 2 0 . 5 + 7 . 5 - 0 . 5 + 1 . 8 0 . 1 + 1 .4 5 4 42.0+14.2 34.9+_13.5 - 1 . 2 + 1.6 O. 1+ 0.5

13 12 54.6+._10,9 5 9 . 7 + 7 . 3 3 . 0 + 3 .1 - 0 . 4 + 1 .5 21 6 60.7+.13.2 5 9 . 3 + 1 3 . 8 8 . 4 + 5 .4 - 0 . 7 + 0 . 8 35 8 6 2 . 8 + 8.5 6 1 . 6 + 9.0 18 ,6+ 9.3 1 . 7 + 2.1

* PBML w e r e t r e a t e d w i t h Con A f o r 24 h o u r s , washed and k e p t i n c u l t u r e i n t h e p r e s e n c e o f c r u d e I L - 2 up t o 35 d a y s . At s p e c i f i e d i n t e r v a l s , t h e e f f e c t o r c e l l c a p a b i l i t y o f t h e c u l t u r e s was m e a s u r e d .

** P e r c e n t a g e s p e c i f i c l y s l s e x p r e s e d a s mean + SD from t h e number o f c a l v e s t e s t e d a t t h e s p e c i f i e d t i m e .

TABLE 4 Development o f NK-1ike c y t o t o x i c i t y in l ong t e rm c u l t u r e s of Con A-activated PBML*

HSB-2 K562 Days in

C a l f C u l t u r e D i r e c t ADCC D i r e c t LDCC

125 17 - 1 . 1 " * - 1 . 6 - 0 . 3 1 0 . 4 50 7 .5 B.0 37 .5 51 .3

128 17 - 0 . 8 - 0 . 6 - 1 . 6 14.9 50 3 .5 5 .5 7.1 11.2

187 17 - 0 . 5 3 .4 5 .3 20.7 50 3 .7 5.7 27.9 39.7

188 17 2 .5 1.5 3 .1 26 .5 50 7 .7 8 .1 37 .2 58 .5

303

* PBML were t r e a t e d w i t h Con A f o r 24 h o u r s , washed and k e p t in c u l t u r e in t h e p r e s e n c e of c r u d e IL-2 up to 50 d a y s . At 17 and 50 d a y s , t h e e f f e c t o r c e l l c a p a b l I t t y o f t h e c u l t u r e s was measu red .

. . P e r c e n t a g e s p e c i f i c l y s i s .

DISCUSSION

P r e v i o u s work in ou r l a b o r a t o r y has d e m o n s t r a t e d t h a t f r e s h n o n - s t i m u l a t e d

b o v i n e PBML l ack a d e t e c t a b l e k i l l e r (K) c e l l t h a t m e d i a t e s ADCC (Campos and

R o s s i , 1985) , and t h a t L K - a c t i v a t t o n of t h e e f f e c t o r c e l l p o p u l a t i o n i s

n e c e s s a r y to d e t e c t ADCC as we l l a s d i r e c t c y t o t o x i c i t y of t a r g e t c e l l s (Campos

and R o s s t , 1986) . In t h e p r e s e n t r e p o r t we i n v e s t i g a t e d t h e e f f e c t of l e c t i n

s t i m u l a t i o n of b o v i n e PBML and d e m o n s t r a t e t h a t b o v i n e PBHL do n o t r e s p o n d to

l e c t i n s t i m u l a t i o n by m e d i a t i n g d i r e c t c y t o t o x i c i t y o r ADCC as do human PBML.

In o r d e r to d e m o n s t r a t e LDCC w i t h b o v i n e PBML, p o l y c l o n a l a c t i v a t i o n of

b o v i n e PBML w i t h l e c t i n p r i o r t o t h e c y t o t o x l c i t y a s s a y was r e q u i r e d

(Tab le 2 ) . Somewhat s i m i l a r r e s u l t s have been o b t a i n e d in mice in which LDCC

can be med ia t ed by a n t i g e n s p e c i f i c CTL, bu t i s n o t o b s e r v e d in n o n s t i m u l a t e d

c e l l s ( B r a d l e y and Bonavlda , 1985) . However, in humans, LDCC can be med la t ed

by n o n s t i m u l a t e d normal PBHL. I t ha s been s u g g e s t e d t h a t LDCC can p r o v i d e a

v a l u a b l e method of a s s e s s i n g a c t i v a t e d c e l l p o p u l a t i o n s to unkown a n t i g e n s

(Bonav tda e t a l . , 1977) . A n a l y s i s a t t h e s i n g l e c e l l l e v e l (Bonavtda and Katz ,

1985) and d o u b l e t a r g e t c o n j u g a t e e x p e r i m e n t s ( B r a d l e y and Bonavida , 1984) have

d e m o n s t r a t e d t h a t n a t u r a l k i l l e r (NK)/K c e l l s a r e n o t i n v o l v e d in LDCC and t h a t

t h e m a j o r i t y o f c e l l s m e d i a t i n g LDCC a r e T l y m p h o c y t e s . N e v e r t h e l e s s ,

c o n t i n u o u s c u l t u r e of c e l l s a b l e t o m e d i a t e e i t h e r LDCC or NK a c t i v i t y can

modu la t e e f f e c t o r c e l l s to p e r f o r m b o t h c y t o t o x l c f u n c t i o n s ( B r a d l e y and

Bonav ida , 1984; R o b e r t s and Moore, 1985) . Bovine PBML d id n o t r e s p o n d

304

to lectln activation with enhanced direct killing o~ R562 target cells unless

they were maintained in culture wlth crude IL-2 for prolonged periods of time

(Tables 3 and 4). It is possible that continuous exposure of stimulated

cultures to crude IL-2 induced the LDCC effector cells to undergo functional

changes. Recent studies with cell lines developed from murine CTL clones have

revealed that exposure of CTL to medium rich in IL-2 leads these specific

killer cells to acquire new lyric speclficitles similar to those of splenic NK

cells (NK-like) (Brooks et al., 1983; Brooks et al., 1985).

In humans, incubation of human PHML wlth PHA for 2 days generates

non-specific cytotoxic cells that lyse autologous and allogeneic fresh solid

tumor cells (Mazumder et al., 1983). Similarly, lectin stimulation of hamster

lymphocytes results In activation of non-specific cytotoxic effector cells

(Stewart et al., 1985). IL-2 activation of human PBML also generates cells

able to mediate lysis of autologous fresh tumor cells, allogenelc fresh tumor

cells, and all cultured tumor cells tested, including NK-reslstant target

cells. The effector cells involved in this phenomenon are referred to as LK

activated killer (LAK) cells (Grimm eta]., 1982). Some investigators consider

LAK cells to be a cytotoxic cell distinct from cytotoxic T lymphocytes (CTL) or

NK cells (Grimm et al., 1983). However, cells with comparable characteristics

have been generated by stimulation of NK cells with IL-2 (Itho et al., 1986;

Trinchlerl et at., 1984). It has also been demonstrated that ADCC can be

significantly augmented by activation of human lymphocytes with PHA, Con A, and

(Hunninghake and Faucl, 1977; Connoly et al., 1975). It has been suggested

that IL-2-dependent activation is a common mechanism involved in LAK cell and

lectin-activated killer cell cytotoxicitles (Mazumder et al., 1983). However,

in the present study lectln activation of bovine PBML did not produce a similar

effect, suggesting several possible differences between bovine and human PBML:

(1) different cells in these species are responsible for these activities, (2)

similar cells in different stages of activation mediate these activities, (3)

and/or lectins have different effects on bovine and human cell populations.

In summary, It'appears that activation of bovine cytotoxic cells from

inactive precursors in peripheral blood can be accomplished by a number of

ways: by contact with virus-~infected cells (Brigham and Rossi, 1986; Chang and

Rossi, 1986} by lymphokines stimulation (Campos and Rossl, 1986), by lectin

stimulation as described herein, and by long-term cultures from

lectln-activated and crude IL-2 stimulated PBML (Table 5). It will be of

considerable interest to further define the effector populations with regard to

origin, activation signals, and role in antl-viral and antl-tumor immunity.

305

TABLE 5 Summary of a n t i g e n n o n s p e c l f l c c e l l u l a r c y t o t o x l c l t e s of bov lne PBNL b e f o r e and a f t e r s t i m u l a t i o n .

Cyto tox lc c a p a b i l i t i e s

S t i m u l a t i o n D i r e c t LDCC ADCC

None ( F r e s h PBNL) Neg L e c t i n a l o n e (2 days ) Neg L e c t l n ÷ c rude IL-2 (1 week) Neg L e c t i n + c rude IL-2 (abou t 3 weeks) Pos

Neg Neg Pos Neg Pos Neg Pos Neg

ACKNOWLEDGMENTS

Suppor ted in p a r t by t h e Alabama A g r i c u l t u r a l Exper iment S t a t i o n and U. S.

Depar tment of A g r i c u l t u r e Sc i ence and Educa t i on Grant 83-CRSR-22146.

P u b l i s h e d as p u b l ] c a t t o n No. 1810 C o l l e g e of V e t e r i n a r y Medic ine and Alabama

A g r i c u l t u r a l Exper iment S t a t i o n J o u r n a l S e r i e s No. 5-86982, Auburn U n i v e r s i t y ,

AL.

The a u t h o r s thank Roger Br]dgman f o r t e c h n i c a l a s s i s t a n c e .

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