Depletion of protein kinase C induced by an anti HLA class I monoclonal antibody in phytohemagglutinin activated human T cells

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 95]-956

Vol. 152, No. 3,1988 May 16,1988

D E P L E T I O N OF PROTEIN KINASE C INDUCED BY AN ANTI HLA CLASS I M O N O C L O N A L A N T I B O D Y IN PHYTOHEMAGGLUTININ ACTIVATED HUMAN T CELLS Licia Poltronieri*, Edon Melloni**, Michele Rubini*, Rita Selvatici*, C r i s t i n a Mazzilli***, Roberto Baricordi* and Enrico Gandini* *Istituto di Genetica Medica, Universit& degli Studi di Ferrara, Ferrara, Italy **Istituto P o l i c a t t e d r a di Chimica Biologica, Universit~ degli Studi di Genova, Genova, Italy ***Dipartimento di Biologia Sperimentale, Roma, Italy

Universit~ La Sapienza,

Received March 22, 1988

SUMMARY: Anti HLA Class I Monoclonal Antibody depletes Protein Kinase C (PKC) to 20% of control value in PHA activated human T cells. The effect is reversible: in 24 hours the enzymatic activity returns to 58% of control value. Removal of antibody from the culture medium increases the rate of recovery. Implications of this finding for the modulation by HLA Class I antigens of the p r o l i f e r a t i v e response of T cells to lectins are discussed. ® 1988 A c a d e m i c Press, Inc.

Gene Complex in

products

in

the

Major

Histocompatibility

(MHC) play a central role in immune response in

other

animal

cytolytic

T

antigens

species.

lymphocyte

in the mouse

(2,3,4,5), antigen

encoded

Class

(CTL)

I antigens

response

as

are shown

man,

as

involved

in

for

(i) and for HLA A and B antigens

H2K/H2D in

human

w h e r e a s MHC Class II antigens are largely involved in

presentation

cells and m a c r o p h a g e s

and antigen specific interactions between T (6).

Furthermore antibodies to MHC Class

I

Abbreviations: PKC, p r o t e i n kinase C; PHA, phytohemagglutinin; FCS, fetal calf serum; MoAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate; PMBC, peripheral mononuclear blood cells; SRBC, sheep red blood cells.

951

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc: All rights of reproduction in any form res#rved.

Vol. 152, No. 3, 1988

antigens affect human

have

been

natural

antigens vitro

T

of

such

(PMA)

T cell

of T cell

anti-T3

antibodies were

no i n h i b i t i o n

(PMBC)

the

an early T cells

Class

with

event

have I

stimulated

as

plays

cell

and

also

or o t h e r (9).

In

of

a

action

antibodies

phorbol

an

activation

complex

the

and

line of

of v i r u s e s

after

ionophores,

(6,10);

12-myristate

Phytohemagglutinin

and

the

the

down

adherent

regulate

(PHA),

can

cell

Calcium

was

subpopulation

response.

These that

anti

dependent

HLA

the

proliferative such

contrary,

observed plays

results

PKC,

(14).

In

an i m p o r t a n t blood

were

HLA Class

I

as PHA,

activates

mononuclear

pathway

Class

On the

directly

response

of p e r i p h e r a l

anti

agents,

(11,12,13).

by PMA w h i c h

hypothesis

that

of a g o n i s t i c

proliferative

of the PKC

reported

ionophores

stimulated

chosen

MoAb

been

(MoAbs)

inhibition

before

We

T

receptor

as a n t i g e n s

to a v a r i e t y

proliferative

consistent

response.

is o b s e r v e d

lectins

it has

of the

system

in

activation

antigen

function

of a c l o n e d

by the p r e s e n t a t i o n

agents

antibodies

T cells

killer

proliferation.

response

role

or

T cell

immune

as C a l c i u m

recently

monoclonal

the PHA

the

agonistic

13-acetate

More

(7,8).

proliferation

chemicals

induce

in

natural

proliferation

the T3 g l y c o p r o t e i n

cell

variety

cells

can be e l i c i t e d via

to m o d u l a t e

dependent

killer

role

"in vivo"

when

found

Interleukin-2

important

also

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cells

considered

I MoAbs

affect

of a c t i v a t i o n

of

is a c t i v a t e d . to

on PKC

study

the

effect

of a p u r i f i e d

by the m i t o g e n i c

lectin

MATERIALS

of a m o n o m o r p h i c

T lymphocytes

anti

HLA

sub-population

PHA.

AND METHODS

Materials DEAE-cellulose (DE-52) was obtained from Whatman. Phosphatidylserine (bovine brain), Dioleoylglycerol, Histone IIIS, Leupeptin, Phenyl~thylsulphonyl f l u o r i d e and ATP were p u r c h a s e d from Sigma. (~P ) A T P (3OOOCi/mmol) was from Amersham. PHA was from B u r r o u g h s W e l l c o m e . M o A b 01.65 (15) a n t i - H L A Class I

952

Vol. 152, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

antigens (HLA-A, -B and -C) and Mol anti m o n o c y t e s p r o v i d e d by Dr. F. M a l a v a s i . A l l o t h e r c h e m i c a l s w e r e r e a g e n t grade.

were

kindly

T cells p r e p a r a t i o n Peripheral blood l y m p h o c y t e s w e r e i s o l a t e d from b u f f y coats of normal adults by centrifugation over Ficoll/Hypaque density g r a d i e n t at 1000xg. for 20 min. at 20 C' Cells w e r e w a s h e d twice with RPMI 1640 m e d i u m and s u s p e n d e d in RPMI 1640 containing 5% FCS. Then, cells w e r e i n c u b a t e d in Petri d i s h e s at 37'C for 1 h o u r in 5% CO 2 for d e p l e t i o n of m o n o c y t e s / m a c r o p h a g e s . T h e n non a d h e r e n t 6 cells w e r e c o l l e c t e d in RPMI 1640 at the concentration of 6x10 cells/ml.. E r o s e t t e s w e r e f o r m e d w i t h sheep red cell~ (SRBC) treated with Neuraminidase (4 ml. SRBC treated/100xl0 v cells) and incubated at 37'C for 15 min.. The mixture was c e n t r i f u g e d at 1000xg. at r o o m t e m p e r a t u r e and i n c u b a t e d on ice for 1 hour. The p e l l e t s w e r e r e s u s p e n d e d g e n t l y and c e n t r i f u g e d o v e r F i c o l ! , at 1000xg • for 20 ~ m i n " " The B cells w e r e r e c o v e r e d from the interface, and the E cells w e r e obtained from the p e l l e t by lysis of the S R B C w i t h 5 mM. Ammonium Cloride followed by c e n t r i f u g a t i o n at 700xg. for 5 min.. The p u r i f i e d T cells w e r e r e s u s p e n d e d in RPMI 1640 a d d e d w i t h a n t i b i o t i c s . T p u r i f i e d cell p r e p a r a t i o n c o n t a i n e d less then 3% of Mol p o s i t i v e cells. I n c u b a t i o n of p u r i f i e d T c e l l s _ T cells w e r e s u s p e n d e d a t ixl0 b cells/ml, c o n c e n t r a t i o n in RPMI 1640 medium w i t h 5% fetal c a l f s e r u m (FCS) and a n t i b i o t i c s and incubated at 37'C w i t h P H A (2~g./ml.) a n d / o r MoAb 01.65 (0.I mg./ml.) at d i f f e r e n t times. F o l l o w i n g i n c u b a t i o n the T cells w e r e w a s h e d and lysed by s o n i c a t i o n as d e s c r i b e d below. I s o l a t i o n of s o l u b l e and p a r t i c u l a t e f r a c t i o n Lysate~ were obtained by s o n i c a t i n g the T cells suspension (15x10 v T c e l l s / m l , in 0.25 M. sucrose, I0 mM. H e p e s pH 7.5, 5 mM. EDTA, I0 mM. 2 - M e r c a p t o e t h a n o l , 2 mM. p h e n y l m e t h y l s u l p h o n y l f l u o r i d e and 0.01% leupeptin) w i t h six 10-sec. b u r s t s from a MSE model MK2 s o n i c a t o r , f o l l o w e d by a c e n t r i f u g a t i o n at 100.000xg. for 20 min.. T h e s u p e r n a t a n t was c o l l e c t e d as c y t o s o l i c f r a c t i o n and the p e l l e t w a s s u s p e n d e d in the same b u f f e r c o n t a i n i n g 0.1% T r i t o n X - 1 0 0 as p a r t i c u l a t e fraction. P a r t i a l p u r i f i c a t i o n of PKC from T cells PKC was partially p u r i f i e d from the c y t o s o l i c fraction by chromatography on a DEAE-cellulose column equilibrated with mM. H e p e s pH 7.5, 10 mM. 2 - M e r c a p t o e t h a n o l and 5 mM. EDTA. PKC was e l u t e d w i t h a 70 ml. linear 0-0.4 M. N a C I g r a d i e n t .

a

i0

Enzyme assay PKC w a s a s s a y i e d a c c o r d i n g to M e l l o n i et al. (16). O n e u n i t of PKC activity was def&ned a~^ the amount that causes the incorporation of 1 nmol. of Zp into H i s t o n e IIIS under the experimental conditions.

RESULTS Native of M o A b form

and

PKC

alone

activity

exists

is c o n s t a n t

in non

activated

T cells

as Ca +~ and P h o s p h o l i p i d during

24 hours

953

and

in p r e s e n c e

dependent

of e x p e r i m e n t .

cytosolic

Vol. 152, No. 3, 1988

In

PHA-added

observed. and

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Membrane

phospholipid

min..

culture

PKC tends

50% of the control PHA

detectable dependent of

the

form. control

eventually

When

In

value.

to decrease

within

PKC as

is Ca ++

after

24 hours

are added

together

at

is

as

and

found

human

Ca ++

PKC activity

in the medium is removed

to

during

i0

about

activity

is faster

faster

fraction

to about 6

20%

hours

if the

24 hours

the

and

MoAb

is

(Fig.l). the recovery

of

(Fig.2) .

is also

found to

be

(data not shown). cytosolic

in I0 min. T cells

value

O,

phospholipid

for

from the culture,

membrane

time

is down

constant

is significantly

experiments,

reversion

to cytosol

58% of original

insoluble

deprived

PHA activated

PKC returns

It remains

to

of e n z y m a t i c these

cytosolic

form and it is d e t e c t a b l e

i0 min.

activity

Triton

completely

and

After

present

the enzymatic

deprived

activity

antibody

The

MoAb

increases

continuously

of

value.

and

PKC

depletion

translocated

independent

Cytosolic

When

a

PKC

activity

after the addition

culture.

almost

of antibody

The d e p l e t i o n

after removal

is

to

is r e v e r s i b l e

of the a n t i b o d y

from

the

culture. DISCUSSION These PHA

data

stimulated

activity

is

well

cells

cells

anti HLA Class an

role

of PKC

induction

activity

activity

Exposure a rapid (17);

I MoAb

complete

is back

induces

depletion

PKC

is r e v e r s i b l e

cell p r o c e s s e s

erythroleukemia

(over 90% w i t h i n

the PKC depleted

by h e x a m e t h y l e n e - b i s - a c e t a m i d e

of

on

to 58% of normal.

of specific

of murine

decrease

954

01.65

The d e p l e t i o n

in the activation

established.

PKC

almost

of culture.

the enzyme

to PMA p r o d u c e s

of total to

that

in a few m i n u t e s

and in 24 hours The

show

cells and

(MEL)

2 hours)

are r e s i s t a n t

reacquire

their

Vol. 152, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20.

20 - - - - m -

•

2

6

•

•

12

24

6 >I-

F-

>-1o t-

pO cJ

2

0

6

1'2 TIME,

1'8

~

hr

TIME,hr

Fig.l

Effect of PHA and PHA.plus MoAb O1.65 on PKC activity in purified T cells iXlOb/ml.. T cells were incubated with PHA (2~g./ml.) or PHA plus MoAb O1.65 (O.Img./ml.). At the time indicated, the mixture of incubation was removed for determination of PKC activity. T cells only(O) and T cells plus MoAb(m) were used as control. The cytosolic PKC (PHA:O; PHA+MoAb:O) and the particulate PKC (PHA:~;PHA+MoAb:•) were determinated at different times.

Fig. 2

Effect of antibody removal on PKC depletion in PHA stimulated T cells in presence of MoAb O1.65. T cells were cultured with PHA (~ug./ml.) and MoAb O1.65 (O.Img./ml.). At 2 hrs. culture(O) was centrifuged and T cells were resuspended in fresh medium without PHA or MoAb. Culture was continued then for 24 hrs. as a second culture with PHA and MoAb(O). At different times, PKC activity was determined using T cells only(1) as control.

responsiveness when

following

the

level

PKC

activation

synthesis

in

of P K C

depletion

in P H A

with

observed

the

synthesis

in

however,

only

investigated synthesis requires cells

in

the

act

anti

continuous

these

cultures.

known

by

to

anti

without

of a s e r i e s of t h e

Class

presence After

955

induce

or

cultures

PMA,

I

(18).

culture;

on

which

DNA

must

be

fact,

DNA

dependent

and

In

is t i m e

antibody

substitution

Enzyme

it d e s c r i b e s ,

experiment.

I MoAbs

DNA

consistent

MoAbs

of e v e n t s

of the

augment

is t h e r e f o r e Class

lymphocyte

continuation by

incubation

T lymphocytes

stimulated first

of

lymphocytes

inhibition

inhibition the

furthermore

activated

the

hours

increases.

stimulated

lectin

in

activity

is

lectin

35-48

and of

the

adherent medium

Vol. 152, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

containing the antibody with antibody free medium,

DNA synthesis

recovers. Further

experiments

will clarify the role played

by

this

early

event in the inhibition of DNA synthesis by anti

Class

I

MoAbs

also in relationship with the reported effects of adherent

cells subpopulation not present in our system.

AKNOWLEDGEMENTS Dr. Licia Poltronieri is personally supported by the Associazione Italiana per la Ricerca sul Cancro, Milano - Italy. The autors thank Prof. F. Malavasi, Turin - Italy, for the generous supply of Antibody 01.65. This work was partially supported by C.N.R. Progetto Finalizzato "Oncologia", contratto n. 87.01297.44.

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