Interleukin 2 (IL-2) production by mitogen stimulated bovine peripheral blood lymphocytes and its assay

Veterinary Immunology and Immunopathology, 7 (1984) 201--212 Elsevier Science Publishers B.V., A m s t e r d a m -- Printed in The Netherlands

I N T E R L E U K I N 2 (IL-2) P R O D U C T I O N BY M I T O G E N P E R I P H E R A L B L O O D L Y M P H O C Y T E S AND ITS A S S A Y

201

STIMULATED

BOVINE

G. O L D H A M 1 and L I N D A W I L L I A M S 2 iDepartment Diseases,

of Immunology, Compton,

2Statistics Diseases, (Accepted

Newbury,

Section, Compton,

26 M a r c h

AFRC

AFRC

Institute

Berks.,

RGI60NN

Institute

Newbury,

for R e s e a r c h

(Great Britain)

for R e s e a r c h

Berks.,

on A n i m a l

RGI60NN

on A n i m a l

(Great Britain)

1984)

ABSTRACT Oldham, G. and Williams, Linda., 1984. I n t e r l e u k i n 2 (IL-2) p r o d u c t i o n by m i t o g e n s t i m u l a t e d bovine p e r i p h e r a l blood l y m p h o c y t e s and its assay. Vet. Immunol. Immunopathol., 7: 201-212. C u l t u r e m e d i u m from b o v i n e p e r i p h e r a l blood m o n o n u c l e a r cells s t i m u l a t e d w i t h the m i t o g e n s p h y t o h a e m a g g l u t i n i n (PHA) or Conc a n a v a l i n A (Con A) was found to m a i n t a i n the p r o l i f e r a t i o n of Con A b l a s t s in vitro. The factor r e s p o n s i b l e for this a c t i v i t y was not a b s o r b a b l e w i t h b o v i n e e r y t h r o c y t e s or fresh p e r i p h e r a l b l o o d l y m p h o c y t e s but was r e m o v e d by Con A blasts. P r o d u c t i o n of this factor was d e p e n d e n t on the dose of m i t o g e n used and was g r e a t e s t after 24 h c u l t u r e c o m p a r e d to 48 h. Quantitative d e t e r m i n a t i o n s of factor a c t i v i t y in s u p e r n a t a n t s were c a r r i e d out by r e g r e s s i o n a n a l y s i s of logit t r a n s f o r m e d data from assays m e a s u r i n g the m a i n t e n a n c e of Con A b l a s t p r o l i f e r a t i o n by supernatants.

INTRODUCTION In v i t r o

assessment

of the

inductive

has until

recently

been restricted

responses

of cells

cultured

T-cells unable

activated

to u n d e r g o

to p r o d u c e antigen), 1979; cells,

2

(IL-2)

is also p r e s e n t et al.,

helper

(Baker et al., 1980).

proliferation

interleukin

Smith

1979;

0165-2427/84/$03.00

unless

Clonal

analysis

of b o v i n e of b o v i n e

1979;

proliferation

et al.,

1980;

IL-2 may T cell

or antigens.

cell

type,

to m i t o g e n

T cells

are able

(or

Coutinho

et al.,

of c y t o t o x i c

T

is IL-2 d e p e n d e n t MacDonald

et al.,

a l l o w the d e v e l o p m e n t

clones

© 1984 Elsevier Science Publishers B.V.

function

blastogenic

(or antigen)

a second

in r e s p o n s e

and s u p p r e s s o r Schreier

of T-cell

either m i t o g e n s with m i t o g e n

(Bonnard et al.,

1979).

T cells

The a v a i l a b i l i t y

and f u n c t i o n a l

with

by contact

phase

to m e a s u r i n g

as well

as

202

p r o v i d i n g an a l t e r n a t i v e assay for T-cell activation. of IL-2 by h u m a n

(Morgan et al.,

mouse lymphocytes

1976;

(Gillis and Smith,

m i t o g e n s has been described.

Production

Ruscetti et al.,

1977)

1977)

and

in r e s p o n s e to T-cell

More r e c e n t l y Baker and K n o b l o c k

(1982) have d e s c r i b e d the p r o d u c t i o n of a factor w i t h costimulator

a c t i v i t y by bovine l y m p h o c y t e s

This paper c o n f i r m s natants

in r e s p o n s e to m i t o g e n s

this finding but uses the ability of super-

to m a i n t a i n Con A blast p r o l i f e r a t i o n to detect factor

a c t i v i t y and gives further s t a t i s t i c a l c o n s i d e r a t i o n s quantitative

for its

assay.

M A T E R I A L S AND M E T H O D S P r e p a r a t i o n of cattle 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 cells H e p a r i n i s e d venous blood

(20 i.u. h e p a r i n / m l blood)

c e n t r i f u g e d at 900 x G for 20 min. l a y e r e d onto a F i c o l l - M e t r i z o a t e 1.086)

p r e p a r e d by m i x i n g

w i t h 36 parts Norway). lO°C.

64 parts

32.8% m e t r i z o a t e

at lO°C.

gradient

was

The buffy coat was

(specific g r a v i t y

8% Ficoll

(Ficoll 400, Sigma)

(Nyegaard and Co A/S, Oslo,

The g r a d i e n t was c e n t r i f u g e d at 900 x G for 40 min.

The cells at the i n t e r f a c e were collected,

times w i t h p h o s p h a t e b u f f e r e d saline tissue c u l t u r e medium.

(PBS) and r e s u s p e n d e d

The m e d i u m used was RPMI 1640

glutamine

(30 mM),

(2 mM),

penicillin

sodium b i c a r b o n a t e

(200 u/ml)

(27 mM),

and s t r e p t o m y c i n

in

(Gibco)

s u p p l e m e n t e d w i t h 10% heat i n a c t i v a t e d foetal calf s e r u m Labs.),

at

w a s h e d three

(Flow

Hepes

(i00 ~g/ml).

This cell p o p u l a t i o n was found to be greater than 95% v i a b l e by trypan b l u e dye e x c l u s i o n and c o n t a i n e d 90-95% m o n o n u c l e a r cells as d e t e r m i n e d by Giemsa stained cell smears.

P r e p a r a t i o n of m i t o g e n - a c t i v a t e d

cell s u p e r n a t a n t 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 cells volumes Labs.,

in L i n b r o m u l t i - w e l l plates Scotland)

the c u l t u r e s

(PHA, reagent grade, Wellcome)

Mitogen, or

(Con A, type IV, Sigma) was added to the cells and

i n c u b a t e d at 37°C for 1 h.

w e r e then r e c o v e r e d from the wells, iO min.

76-033-05 F l o w

at a cell d e n s i t y of 4 x 106 cells/ml.

either phytohaemagglutinin Concanavalin A

(PBM) w e r e c u l t u r e d in 2 ml

(cat. no.

The cell suspensions

centrifuged

and the s u p e r n a t a n t m e d i u m discarded.

at 350 x G for The cells w e r e

w a s h e d once w i t h PBS and then r e s u s p e n d e d in 2 ml fresh medium.

203

The w e l l s

were

added

back

24 or

48 h at

The

cell

stored

by

latex

ml

and Con

until

uptake)

a density

of

cells

latex

particles.

at the

Interleukin

and

for

of

the

The

cells

were

a further

5% C 0 2 : 9 5 %

air.

supernatants

lymphocytes

10-20%

phagocytic

suspended

ml/flask,

washed

2 x 105

these

atmosphere

of

4 or

times

cells/ml

end

of

culture for

three

2.5

5 ~g/ml.

x 106 The

(3024,

5 days

at

with

culture

of

(as d e t e r m i n e d

flasks

in t i s s u e

the

cells

at a d e n s i t y

to a c o n c e n t r a t i o n

15-20

then

of PBS.

and c u l t u r e d

centrifuged

in 75 cm 2 t i s s u e

Ca.),

2 ml

assayed.

were

A added

were

The

were

containing

cultured

Oxnard,

wells

of Con A - a c t i v a t e d PBM

with

in a h u m i d i f i e d

suspensions

Bovine

once

original

37°C

at - 2 0 ° C

blasts

washed

to t h e i r

Preparation

were

also

culture

were

able

Falcon,

37°C.

PBS and

cells/

cells

The

Con

resuspended medium.

A

to

None

of

to p h a g o c y t o s e

2 assay

assay

by L a f f e r t y

used et

to m e a s u r e

al.

(1980)

IL-2 w a s

basically

to m e a s u r e

that

'maintenance

described

factor'

activity. Serial natants

2-fold

were

micro-test were

1 ~Ci

The

with of

in t i s s u e (Nunclon

cells

was

contents

were

added

to e a c h

scintillation PBM

The

incorporation

and w i t h

with

well

high

was

The

plate

added 37°C

D N A was

A supernatant

seen

with

this

2 x 104

Radiochemical

onto

was

supernatants

at w h i c h

discs

Incorporation

produced

supernatant

with

medium either

and

by

by Con A

in e a c h was

assay.

considered

cells control

an e q u a l

medium

volume

containing

of w a s h e d

A

18 h c u l t u r e

determined

included

Con time

Centre,

glass-fibre

harvester.

cellular

activity

All

a further

harvested

super-

in f l a t - b o t t o m e d

24 h at

After

test

activity.

A__bsorption of m e d i a

absorbed

into

counting.

stimulated

Conditioned

well.

micro-test

(3H-TdR)

to be the m a x i m u m

for

the

Denmark).

(5 C i / m M o l ,

were

of

medium

Nunc,

To each

thymidine

a semi-automated

3H-TdR

culture

Delta,

cultured

of the w e l l s

3H-thymidine

liquid

(iOO ~i volumes)

in t r i p l i c a t e .

]methyl-3HI

Amersham) the

made

wells

assayed

blasts.

dilutions

PHA were

packed

bovine

204

erythrocytes

(Ox rbc)

or fresh PBM

(1 x 107 c e l l s / O . 5

supernatant)

or Con A blasts

(i x 107 cells/0.5

supernatant)

for 2 h at 370C

in s t e r i l e

(2054 tube, were

then

Falcon)

with

centrifuged

periodic

ml culture

ml culture

round b o t t o m e d

mixing.

and the s u p e r n a t a n t

The

cell

stored

tubes

suspensions

at -20°C

until

assayed.

Experimental Three calves

design

experiments

(nos.

75,

Experiment

i.

concentrations collected

collected

40 and

of 45 and

value

package curves. relevant) analysis table

with

PHA at

and culture

supernatants

were

stimulated

with

and c u l t u r e

Con A at supernatants

were

90 ~g/ml

stimulated and culture

with

PHA at

supernatants

analysis

(cpm)

obtained

sigmoidal

from 4

24 and 48 h culture.

for each

per m i n u t e

stimulated

900 ~g/ml

160 ~g/ml

Lymphocytes

For each e x p e r i m e n t measured

cells

from 3 to 6 months.

24 h incubation.

3.

after

Statistical

aged

were

450 and

Lymphocytes

of 20,

concentrations

out each u s i n g

24 h incubation.

after

Experiment

collected

Lymphocytes

2.

concentrations

carried

104 and 610)

of 225,

after

Experiment

were

90,

shape

the t h y m i d i n e

supernatant were

expressed

for that

effects

on t h e s e

Since

1978)

of m i t o g e n

transformed

of variance.

for E x p e r i m e n t

the data

transformation

(Baker and Nelder, The

for each

as a p e r c e n t a g e

assay.

the iogit

incorporation

dilution

was used dose

3 was

Source

the

average among

slope calves

24 v 48 h c u l t u r e among

doses

slope x dose

of the m a x i m u m a roughly

in the G L I M s t a t i s t i c a l to l i n e a r i s e

the

time

if

investigated

by

analysis

of the f o l l o w i n g

of v a r i a t i o n

in counts

showed

(and c u l t u r e

lines w e r e

As an e x a m p l e

values

calf

form:-

df 1 3 1 2 2

residual

84

total

93

of v a r i a n c e

205

RESULTS Quantitative Medium mitogens

raeasurement

f r o m bovine

not

lymphocytes

Con A or PHA was

proliferation medium

of I n t e r l e u k i n

found

of Con A blasts

however

failed

2 in culture

stimulated

to be able

in v i t r o

to induce

with

supernatant

the T cell

to m a i n t a i n

(Fig i).

proliferation

the

This

conditioned

of fresh

PBM

(data

shown).

16

®

26

14

,,,

22

'0

×

12

,,

10

5:

8

c~

6

:9

IS 14

I0

r,

4

'-

o

(D 2

n

O-

l 2

I 4

J

J

5

163264

A

J

2

-

I

I

I

I

4

8

16

32

I

2

64

Reciprocal of Dilution

Fig i. I n c o r p o r a t i o n of 3 H - t h y m i d i n e by 4 day Con A blasts after 24 h culture with i n c r e a s i n g d i l u t i o n s of culture s u p e r n a t a n t s in 4 calves. S u p e r n a t a n t s w e r e o b t a i n e d from lymphocyte c u l t u r e s s t i m u l a t e d w i t h e i t h e r (a) Con A (40 ~g/ml) or (b) PHA (225 pg/ml).

When

thymidine

culture) dilution) for some (Figs data The

of these

allowed

The

curves

were

(represented

dilutions plotted

to a p p r o x i m a t e

application

straight

of each

values

of d o u b l i n g

of any s u p e r n a t a n t

1-3).

titre

incorporation

from a series

lines

to be fitted was

there were

defined

log 2

shape

transformation through

as

tendencies

to a sigmoidal

of the logit

supernatant

as cpm/

(represented

to these

the points.

as the d i l u t i o n

giving

206

50% of m a x i m u m activity. different s u p e r n a t a n t s

Interestingly

lines g e n e r a t e d from

assayed s i m u l t a n e o u s l y w e r e non-parallel.

Thus all titre d e t e r m i n a t i o n s were carried out u s i n g separate lines fitted to each supernatant.

Table I c o m p a r e s titres

c a l c u l a t e d from the best fitting parallel

lines with those c a l c u l a t e d when

lines were fitted.

TABLE I C o m p a r i s o n of titres of s u p e r n a t a n t IL-2 a c t i v i t y o b t a i n e d using either i n d i v i d u a l lines or p a r a l l e l lines fitted to logit t r a n s f o r m e d data

Calf no.

Con A ~ (~g/ml)

IL-2 titre* d e t e r m i n e d by:Individual lines P a r a l l e l lines

90

20 40 160

5.6 9.4 21.4

6.1 9.6 22.8

104

20 40 160

<1% 4.6 12.7

1.9 6.6 13.5

~Dose of m i t o g e n used to s t i m u l a t e lymphocytes. Supernatants c o l l e c t e d after 24 h culture (Experiment 2). *IL-2 titre is taken as the r e c i p r o c a l of the d i l u t i o n giving 50% of m a x i m u m 3H-TdR incorporation. A c t i v i t y p r e s e n t but s u p e r n a t a n t w o u l d n e e d to be c o n c e n t r a t e d before 50% of m a x i m u m 3 H - T d R i n c o r p o r a t i o n would be reached.

Conditions

for p r o d u c t i o n of I n t e r l e u k i n

2 by m i t o ~ e n s t i m u l a t e d

bovine l y m p h o c y t e s M i t o g e n dose s i g n i f i c a n t l y a f f e c t e d factor p r o d u c t i o n for both Con A and PHA).

(p
The higher the c o n c e n t r a t i o n of

m i t o g e n used for s t i m u l a t i o n the greater was the activity in the supernatant

(Fig 2).

No o p t i m u m c o n c e n t r a t i o n for either m i t o g e n

was found over the ranges tested. Table II d e m o n s t r a t e s b e i n g the exception) supernatants

that in almost all cases

c o m p a r e d to the 48 h supernatants.

was s i g n i f i c a n t for animals no. but not for no.

(calf no.

75

there was higher a c t i v i t y in the 24 h

610 and no.

75.

90

(p
This d i f f e r e n c e

and no.

104

(p
207

36 9_____0 no.

24

no__104 2B

~0

IG

20

X

g

12

e-

"

'i

6

¢.-

o U

O

_

I

I

I

2

4

B

I

t

16 32

J

I

I

I

64

2

4

5

Reciprocol

of

I

16 32

64

Dilution

Fig 2. I n c o r p o r a t i o n of 3 H - t h y m i d i n e by 4 day Con A blasts after 24 h culture w i t h i n c r e a s i n g d i l u t i o n s of s u p e r n a t a n t s from lymphocyte c u l t u r e s of (a) calf no. 90 stimulated w i t h 20 ~g/ml (H), 40 ~g/ml (~----W) and 160 ~g/ml (e-----e) Con A and (b) calf no. 104 s t i m u l a t e d with 225 ~g/ml ( H ) , 450 Hg/ml (~ ~) and 900 Hg/ml (e-----e) PHA.

TABLE II IL-2 titres in s u p e r n a t a n t s from cattle lymphocytes s t i m u l a t e d with PHA at v a r i o u s c o n c e n t r a t i o n s and c u l t u r e d for 24 and 48 h.

Calf

PHA (~g/ml)

IL-2 titre* after culture for:24 h

48 h

no.

610

45 90

2.4 20.8

<1% 18.7

no.

75

45 90


i.i 16.7

no.

90

45 90

3.2 25.6

1.4 16.9

no.

104

45 9O

3.5 18.3


*as for Table I %as for Table I

2O8

A b s o r p t i o n of c o n d i t i o n e d media w i t h Ox rbc, resting

lymphocytes

and Con A b l a s t s and its effect on IL-2 activity In order to e x a m i n e the c e l l u l a r affinity of the active m o i e t y in c o n d i t i o n e d medium,

the m e d i u m was absorbed with Ox rbc,

u n s t i m u l a t e d PBM or Con A blasts and r e - t e s t e d for its ability to m a i n t a i n Con A blast proliferation. As can be seen in Fig 3 a b s o r p t i o n of c o n d i t i o n e d m e d i u m w i t h Ox rbc had no effect on m a i n t e n a n c e activity. control m e d i u m c o n t a i n i n g PHA culture)

A b s o r p t i o n of

(but not h a v i n g been used for cell

w i t h Ox rbc reduced its ability to induce p r o l i f e r a t i o n

in fresh PBL

(data not shown).

This would suggest that the

a c t i v i t y in c o n d i t i o n e d m e d i u m was not due to r e s i d u a l mitogen. F r e s h PBL were also unable to absorb out the a c t i v i t y in c o n d i t i o n e d m e d i u m but a b s o r p t i o n with Con A blasts reduced the activity markedly

(Fig 3) .

DISCUSSION The factor d e s c r i b e d in this paper w o u l d appear to be e q u i v a l e n t to the T cell growth factors p r o d u c e d by m i t o g e n stimulated murine R u s c e t t i et al.,

and human lymphocytes 1977)

in v i t r o p r o l i f e r a t i o n of Con A blasts. out this factor w i t h Ox rbc do not have IL-2 receptors) IL-2 r e c e p t o r s

(Gillis and Smith,

1977;

as shown by its ability to m a i n t a i n the The i n a b i l i t y to absorb

(which b i n d PHA)

or fresh PBL

w h i l e Con A blasts,

(Robb et al., 1981),

(which

w h i c h possess

are able to effect its removal

w o u l d s e e m to c o n f i r m this. W a s h i n g the cells free of u n b o u n d m i t o g e n one hour after its a d d i t i o n p o s s i b l y results in s u b - o p t i m a l amounts of m i t o g e n r e m a i n i n g for the rest of the culture p e r i o d as shown by the f a i l u r e to find o p t i m a l m i t o g e n doses.

This p r o c e d u r e h o w e v e r

allays the n e e d to r o u t i n e l y remove m i t o g e n from s u p e r n a t a n t s e x a m p l e by s a l t i n g - o u t

(Baker and Knoblock,

a b s o r p t i o n w i t h Ox rbc

(Paganelli et al., 1983) b e f o r e being

assayed.

A recent publication

1982)

for

or by

(Baker and Knoblock,

1982) has

d e s c r i b e d the p r o d u c t i o n of a c o - s t i m u l a t o r factor by bovine lymphocytes

in r e s p o n s e to mitogens.

smaller c o n c e n t r a t i o n s

These w o r k e r s used m u c h

of Con A than e m p l o y e d h e r e and found that

1 ~g/ml g a v e o p t i m a l c o - s t i m u l a t o r production.

This may indicate

how l i t t l e m i t o g e n is left in the cultures d e s c r i b e d p r e s e n t paper.

in the

209

90

80

70 A

'O -- 6 0 X

50 .c_

4O 30

==

\ 20

IO 0

I

I

2

4

I

I

I

I

B

16

32

64

Reciprocal

I

I

128 2 5 6

of Dilution

Fig 3. I n c o r p o r a t i o n of 3 H - t h y m i d i n e by 4 day Con A blasts after 24 h culture with u n t r e a t e d (0----0), Ox rbc absorbed (I----8), fresh PBL a b s o r b e d (A---~) and Con A blast absorbed ( H ) 24 h culture s u p e r n a t a n t from calf lymphocytes s t i m u l a t e d with 900 ~g/ml PHA

It is p r o b a b l e

that the Con A blast assay d e s c r i b e d in this

paper,

like that d e s c r i b e d by Smith and Ruscetti

mouse,

detects only IL-2 and not IL-I.

failure of the b o v i n e Con A blasts to p r o l i f e r a t e of a s u p e r n a t a n t

(1982)

for the

This is suggested by the in the p r e s e n c e

from a c t i v a t e d bovine macrophages.

The p r e s e n c e

of IL-I in this s u p e r n a t a n t is i n d i c a t e d by its ability to induce p r o l i f e r a t i o n by p e r i p h e r a l blood m o n o n u c l e a r cells in the p r e s e n c e of a s u b - o p t i m a l c o n c e n t r a t i o n of Con A observations).

(unpublished

210

Also other

the titres

assays

for IL-2,

the s u p e r n a t a n t produced

of IL-2 m e a s u r e d

that

reflect

by this

only

the amount

is the d i f f e r e n c e

and the amount

consumed

assay,

as in all

of free

between

IL-2

in

the amount

(or absorbed)

by a c t i v a t e d

lymphocytes. Occasionally stimulation dilution This

we o b s e r v e d

of Con A blasts

(Fig 2) e s p e c i a l l y

phenomenon

finding 1982)

supernatants

either

was

supernatants

(Lafferty

Conditioned including

then

supernatants by others

of Con A

as well

factors

amounts

of m i t o g e n

that we b e l i e v e

seen

supernatants becomes

have

of m e t h o d s

of t r e a t i n g

Dauphin~e

et al.,

1981).

of IL-2

lines w e r e

fitted

lines w e r e

in fact p a r a l l e l

were

clearly

chose

to analyse

et al. shape

to t r a n s f o r m e d

so m a k i n g

on the a r b i t r a r y

in cell assay

comparisons

choice

similar

of our data

showed

curves were

linearised

did not cross

lines

considered

and u s e f u l

previously

stated,

titres w e r e difference and those

using

found b e t w e e n

calculated

using

still be made.

using

the

fitted

We

sigmoidal

fitted

of d i l u t i o n s calculated

In a d d i t i o n

as

because

the

no p r a c t i c a l

calculated

non-parallel

evidence

not parallel.

titres

50% point,

those

1980;

either

by translines

lines were

in the range

comparisons,

could

calculated was

that

lines

a roughly

of the s t r a i g h t

However,

the

showed

a titre

et al.,

to that of Gillis

sigmoidal

analysis

activity

of s u p e r n a t a n t

The plots

data

IL-2

of the end point.

(1978).

Regression

culture

data w i t h o u t

or else the

our data by a m e t h o d

to the t r a n s f o r m e d

to

of supernatants.

Lafferty

and these

formation.

not

for the

in these p u b l i c a t i o n s

the

non-parallel

be

than toxic

and o b t a i n i n g

1978;

that

dependent

tested here

comparisons

the data

parallel

titres

will

It w o u l d

dilutions.

et al.,

However

factors

them.

are r e s p o n s i b l e

to p e r m i t

(Gillis

in the c u l t u r e

of factors

rather

to q u a n t i t a t i v e l y

in o r d e r

been d e s c r i b e d

media

it is this

the p r e s e n c e

the a b i l i t y

necessary

A number

to m e a s u r e

at low s u p e r n a t a n t

demonstrated

factor

a variety

for the c o n d i t i o n e d

inhibitory

Having

the

and Knoblock,

as IL-2 and more

are d e v e l o p e d

contain

inhibition

activity.

1980).

contain

and

at a 1/4

who ascribed

(Baker

of an i n h i b i t o r y

media

and IL-3

as assays

be s u r p r i s i n g

than

w i t h high

et al.,

culture

IL-I

identified

at a 1/2 d i l u t i o n

amounts

or to the p r e s e n c e

lower

with

also o b s e r v e d

to toxic

that gave

lines

using (Table

parallel I).

The

lines

211

reason

for this

natants with

with

lower

stimulated

These

cells

(data not

would

in the

The a b i l i t y production

findings

shown)

seem to be that

supernatants

are

to d e m o n s t r a t e

test

true

for culture

cells

The most

factors,

and antigen

other with

adjunct

T cell

of b o v i n e

IL-2 will

in v i t r o

of T cell

lines

permit

than

to

IL-2,

the assay. assay

to the

function

of cattle

not due

likely

and q u a n t i t a t i v e l y a useful

Super-

than those

and are t h e r e f o r e

the a v a i l a b i l i t y culture

hold

interfering

in a s s e s s i n g

clear.

out faster

stimulated

supernatants.

of IL-2 may prove

transformation

is still not

diluted

from both m i t o g e n

in the culture

explanation present

activity

activity.

supernatants

mitogen

lack of p a r a l l e l i s m

greatest

the

lymphocyte

in cattle

the cloning

and

and

origin.

ACKNOWLEDGEMENTS We w o u l d technical

like to thank Mrs.

assistance

and Miss

April Jenny

Warrick Howard

for e x c e l l e n t

for typing

the

manuscript.

REFERENCES Baker, P.E., Gillis, S. and Smith, K.A., 1979. Monoclonal c y t o l y t i e T-cell lines. J. exp. Med., 149: 273-278. Baker, P.E. and Knoblock, K.F., 1982. Bovine co-stimulator. I. P r o d u c t i o n kinetics, p a r t i a l p u r i f i c a t i o n and q u a n t i f i c a t i o n in s e r u m - f r e e Iscove's medium. Vet. Immunol. Immunopathol. 3: 365-379. Baker, R.J. and Nelder, J.A., 1978. The GLIM system, release 3, g e n e r a l i s e d linear i n t e r a c t i v e modelling. Numerical A l g o r i t h m s Group, Oxford. Bonnard, G.D., Yasaka, K. and Jacobson, D., 1979. Ligand a c t i v a t e d T cell g r o w t h f a c t o r - i n d u c e d p r o l i f e r a t i o n : a b s o r p t i o n of T cell g r o w t h factor by a c t i v a t e d cells. J. Inununol., 123: 2704-2708. Coutinho, A., Larsson, E-L., Gronvik, K-O. and Andersson, J., 1979. Studies on T l y m p h o c y t e activation. II. The target cells for Con A - i n d u c e d g r o w t h factors. Eur. J. Immunol., 9: 587-592. Dauphin6e, M.J., Kipper, S.B., Wofsy, D. and Talal, N., 1981. I n t e r l e u k i n 2 d e f i c i e n c y is a common feature of a u t o i m m u n e mice. J. Immunol., 127: 2483-2487. Gillis, S., Ferm, M.M., Ou, W. and Smith, K.A., 1978. T cell g r o w t h factor: p a r a m e t e r s of p r o d u c t i o n and a q u a n t i t a t i v e m i c r o a s s a y for activity. J. Immunol., 120: 2027-2032.

212

Gillis, S. and Smith, K.A., 1977. Long term culture of tumorspecific cytotoxic T cells. Nature 268: 154-156. Lafferty, K.J., Prowse, S.J., Ai-Adra, A., Warren, H.S., Vasalli, J. and Reich, E., 1980. An improved assay for interleukin 2 (lymphocyte growth factor) produced by mitogenactivated lymphocytes. Aust. J. Exp. Biol. Med. Sci., 58: 533-544. MacDonald, H.R., Cerottini, J-C., Ryser, J-E., Maryanski, J.L., Taswell, C., Widmer, M.B. and Brunner, K.T., 1980. Quantitation and cloning of cytolytic T lymphocytes and their precursors. Immunol. Rev., 51: 93-123. Morgan, D.A., Ruscetti, F.W. and Gallo, R.C., 1976. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 193: 1007-1OO8. Paganelli, R., Aiuti, F., Beverley, P.C.L. and Levinsky, R.J., 1983. Impaired production of interleukins in patients with cell-mediated immunodeficiencies. Clin. exp. Immunol., 51: 338-344. Robb, R.J., Munck, A. and Smith, K.A., 1981. T cell growth factor receptors. Quantitation, specificity and biological relevance. J. exp. Med., 154: 1455-1474. Ruscetti, F.W., Morgan, D.A. and Gallo, R.C., 1977. Functional and morphologic characterisation of human T cells continuously grown in vitro. J. Immunol., 119: 131-138. Schreier, M.H., Iscove, N.N., Tees, R., Aarden, L. and von Boehmer, H., 1980. Clones of killer and helper T cells: growth requirements, specificity and retention of function in long term culture. Immunol. Rev., 51: 315-336. Smith, K.A., Gillis, S., Baker, P.E. and McKenzie, D., 1979. T cell growth factor-mediated T cell proliferation. Ann. N.Y. Acad. Sci., 332: 423-432. Smith, K.A. and Ruscetti, F.W., 1982. T cell growth factor and the culture of cloned functional T cells. Adv. Immunol., 31: 137-175.