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U M E A U N I V E R S I T Y M E D I C A L D I S S E R T A T I O N

N e w S e r i e s N o 1 5 8 - I S S N 0 3 4 6 - 6 61 2

F U N C T I O N A L A N D M O L E C U L A R A S P E C T S O F I N T E R F E R O N A C T I O N

I N H U M A N N A T U R A L K I L L E R C E L L S A N D O T H E R L E U C O C Y T E S

B Y

A K E G U S T A F S S O N D E P A R T M E N T O F V I R O L O G Y

U M E A U N I V E R S I T Y U M E A 1 9 8 5

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ERRATA:

The following corrections should be made in the thesis 'Functional and molecular aspects of in te rfero n action in human natural k i l l e r c e lls and other leucocytes':

Page present expression:

7, lin e 14 Isaccs 12, lin e 27 is diminished 13, lin e 16 response + /-

in table 13, l i ne 4 rlFN-y

20, lin e 9 Pross & Jondal, 1975 22, lin e 1 correlated to th e ir

a n tiv ir a l e ffe c t.

28, lin e 10 120 kD

29, lin e 20 and therefore were 31, lin e 2 MC

31, lin e 12 about 32, lin e 5 U llb e rg , 1984 32, lin e 35 43 kD

33, lin e 32 lin e (equivalent to th e ir p80-81 and p58) are 35, lin e 14 inconstantly present p58 36, lin e 7 (c)

41, lin e 24 These two proteins 44, lin e 10 mechanism which is

should read:

Isaacs

i s ge ne ral ly di mi n i s hed response ( + ) / - recombinant IFN-y delete reference v ira l e ffe c t 150 kD

and the proteins therefore we re

monocytes over

U llb e rg , 1983 44 kD

lin e are equivalent to th e ir p8Q-81 and p58 and are p58

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These proteins

mechanism ( i e . by IFN-y) which is

Paper I :

368, lin e 27 ccrp3-cem CCRF-CEM

376, lin e 5 20. Creasey, A ., . . . Hinuma, Y. and Grace J r . , J.

T. Proc. Soc. Exp. B io l. Med 1 2 4 ,1 0 7 7 19677 ... ...

376, lin e 6 21. Minowada, J . , . . . 227 Minowada, J . , . . . 376, lin e 7. 22. Foley, G. E... 21. Foley, G. E ., . . . Paper I I I :

6 , 7 , 10 MC monocytes

7 , lin e 2 percent delete word

11, lin e 2 p38 p36

11, lin e 4 NK c e lls LGL/NK c e lls

Paper IV:

4 , lin e 1 Top lin e missing and fu rth e r p u rifie d by a f ­ f i n i t y chromatography as de­

scribed by A llen e t 8 , lin e 12 ppLGL p a rtly p u rifie d LGL

10, lin e 14 can induce two proteins can induce several proteins * (Mr 80-81 and 58 kD) human fib ro b la s ts . Two of in human fib ro b la s ts . these (Mr 80-81 and 58 kD) These proteins are, are,

10, lin e 18 references (2 4 ); (25) (2 3 ); (24)

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FUNCTIONAL AND MOLECULAR ASPECTS OF INTERFERON ACTION IN HUMAN NATURAL

KILLER CELLS AND OTHER LEUCOCYTES

Akademisk avhandling som med vederbörligt tillstånd av Rektorsämbetet vid Umeå universitet för avläggande av medicine doktorsexamen kommer att offentligen

försvaras i hörsal B, Samhällsvetarhuset, Fredagen den 13 december 1985, klockan 10.30

Av

AKE GUSTAFSSON

UMEA 1985

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k i l l e r c e lls and other leucocytes. Åke Gustafsson, Department of V iro ­ logy, U niversity of Umeå, S-901 87 Umeå, Sweden. Umeå U niversity Medical D iss ertatio n s, New Series No 158 - ISSN 0346-6612.

Interferons comprise a class of s tru c tu ra lly related proteins which e x ert several regulatory e ffe c ts in responsive c e lls . These e ffe c ts include the establishment of an a n tiv ira l s ta te , the in h ib itio n of c e llu la r p ro life ra tio n and the a lte r a tio n of d iffe re n t immune reac­

tio n s . In p a rtic u la r , the IFN:s rapid ly augment the ly t ic a c tiv ity of the natural k i l l e r (NK) c e lls . In the present th esis, some of the func­

tio n a l and molecular mechanisms by which IFN: s act on NK c e lls and other leucocytes are studied. A good co rre la tio n is found between the a b i li t y of d iffe re n t tumor c e ll lin es to induce IFN production among peripheral blood lymphocytes and t h e ir s e n s itiv ity to NK c e ll c y to to xi­

c i t y , indicating th a t IFN might regulate the a c tiv ity of NK c e lls thro­

ugh a p ositive feed-back mechanism. When studying the in te ra c tio n bet­

ween the NK c e lls and two ta rg e t c e ll lin es i t is demonstrated th a t the two c e ll lines are not recognized by the same receptors. The augmenta­

tio n of NK c e ll c y to to x ic ity by IFN is shown to involve both a lte ra tio n of receptor structures on the NK c e ll and enhancement of steps in th e ir l y t i c machinery. The e ffe c ts of IFN on the synthesis of individual proteins is then studied by two-dimensional electrophoresis. I t is demonstrated th a t IFN-a and IFN-ß w ith in 1.5 hours induce the synthesis of nine proteins (Mf80, 75, 62 , 58, 53, 38, 36, 33 and 30 kD) in human lymphocytes. Tne induction is dependent on a rapid de novo RNA synthesis, which is in it ia t e d less than 30 minutes a f t e r the addition o f IFN. The expression of the nine proteins is well correlated to the development of augmented NK c e ll c y to to x ic ity . Four of the proteins (Mr 80, 62, 38 and 33 kD) are found to be expressed in a panel of ten hematopoetic and two anchorage-dependent c e ll lin e s , whereas the remai­

ning proteins seem to be expressed in leucocytes only. IFN induce the synthesis of the same proteins in both p u rifie d large granular lympho­

cytes (responsible fo r the main NK c e ll a c tiv ity in man), T c e lls and monocytes, demonstrating th a t the augmentation of NK c e ll a c tiv ity does not involve the formation of unique 1NK-cel11 sp ec ific proteins. Rath­

e r , the augmentation of the ly t ic a c tiv ity of both NK c e lls , cytotoxic T c e lls and monocytes seem to involve common stages in th e ir ly t ic mechanisms. In contrast to IFN-a and IFN-ß, IFN-y, does not induce any detectable proteins in e ith e r NK c e lls or T c e lls . This lack of e ffe c t o f IFN-y on the protein synthesis is not a general phenomenon, since the e ffe c ts of IFN-a and IFN-y are s im ila r 1n a glioma c e ll lin e . These re s u lts demonstrate th a t there ex ists a t le a s t one pathway to augment the NK c e ll c y to to x ic ity which does not involve the increased synthesis of the nine IFN -a/IFN -ß inducible proteins and indicates th a t e ith e r these proteins are mainly involved in other e ffe c ts of IFN, or th a t the augmentation by IFN -a/IFN-ß and IFN-y involve d iffe re n t pathways. When the e ffe c ts of IFN-a on the synthesis of membrane-associated proteins is studied, i t is demonstrated th a t only the 80 kD IFN-a inducible p ro tein is associated with the c e ll membrane. In ad d itio n , IFN-a seems to induce three a d d itio n a l, me mb rane-as so ci a ted proteins (Mr 94, 76 and 66 kD) which are not detected in whole c e ll lysates.

Key words: natural k i l l e r c e ll/la r g e granular lym phocyte/interferon/

in te rfe ro n production/i nterferon-induced/protein synthes is/two-dimen­

sional electro p ho resis/

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UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New S eries No 153 - ISSN 0346-6612

From The Department of V iro lo g y U n iv e rs ity o f Umeå, Umeå, Sweden E d ito r: The Dean o f the F aculty of Medicine

FUNCTIONAL AND MOLECULAR ASPECTS OF INTERFERON ACTION IN HUMAN NATURAL KILLER CELLS AND OTHER LEUCOCYTES.

by

ÅKE GUSTAFSSON Department of V iro lo g y

U n iv e rs ity o f Umeå

UMEÅ 1985

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To the memory of ny mother, In g rid Marta Gustafsson, 1910-1956

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3

CONTENTS:

1 . INTRODUCTION

1 .1 . THE INTERFERON SYSTEM.

1 .1 .1 . The discovery o f in te r fe r o n ... 7

1 .1 .2 . D e fin itio n of in te r fe r o n ... 7

1 .1 .3 . H eterogeneity of in te r fe r o n s ... 8

1 .1 .4 . Production of in te r fe r o n s ... 10

1 .1 .5 . .E ffe c ts o f i in te rfe ron s^ 1 .1 .5 .1 . A n t iv ir a l- e fT e c ts ...11

1 .1 .5 .2 . A n ti p r o lif e r a t i ve e f f e c t s ... 11

1 .1 .5 .3 . Immun oregul a tiv e e f f e c t s ...12

1 .2 . NATURAL KILLER CELLS. 1 .2 .1 . General background...14

1 .2 .2 . The discovery of the n a tu ra l k i l l e r (NK) c e l l ...14

1 .2 .3 . C h a ra c te ris tic s of the NK c e l l ... 15

1 .2 .4 . The l y t i c mechanism of the NK c e l l ... 18

1 .2 .5 . S p e c ific ity of NK c e l l s ...20

1 .2 .6 . Augmentation o f NK c e ll c y t o t o x ic it y by in te r fe r o n s ... 21

2. RESULTS 2 .1 . E ffe c ts o f IFN-g on d if f e r e n t phases o f the in te r a c tio n between NK c e lls and ta rg e t c e lls (paper I ) ... 23

2 .2 . E ffe c ts o f IFN-g and IFN-p on the p ro te in synthesis in human leucocytes (papers I I and I I I ) ...25

2 .3 . D is tr ib u tio n o f the IF N -in d u c ib le p ro te in s among human le uco­ cyte subpopulations and c e ll lin e s (papers I I and I I I ) . . . . T T . . .29

2 .4 . E ffe c ts o f IFN-y (paper I V ) ... 31

2 .5 . S u b c e llu la r lo c a lis a tio n o f IF N -in d u c ib le p ro te in s ...34

3. GENERAL DISCUSSION 3 .1 . Functional e ffe c ts o f IFN on NK c e ll re c o g n itio n and l y s i s 36 3 .2 . The ro le o f the IF N -in d u c ib le p ro te in s in leucocyte fu n c tio n s . 3 .2 .1 . Role in a n t iv ir a l e f f e c t s . . . ...40

3 .2 .2 . Role in a n t i p r o l i f e r a t i ve e f f e c t s ...41

3 .2 .3 . Role in immunoregulative e f f e c t s ... 41

3 .3 . Summary and concluding rem arks...43

4. ACKNOWLEDGEMENTS...45

5. REFERENCES ,46

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ABBREVIATIONS:

CTI Cold ta rg e t in h ib it io n IFN In te rfe ro n

IL -2 In te r le u k in 2

MAL Nylon wool non-adherent lymphocytes kD k ilo d a lto n

LGL Large g ra n u la r lymphocytes Mr R e la tive m olecular mass PBL P eripheral blood lymphocytes PHA Phytohem agglutinin

pi Is o e le c tr ic p o in t

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5

ABSTRACT

Functional and m olecular aspects of in te rfe ro n a c tio n in human natural k i l l e r c e lls and o th e r leucocytes. Åke G ustafsson, Department of V ir o ­ lo g y , U n iv e rs ity of Umeå, S-901 87 Umeå, Sweden.

In te rfe ro n s comprise a cla ss of s t r u c t u r a lly re la te d p ro te in s which e x e rt several re g u la to ry e ffe c ts in responsive c e lls . These e ffe c ts in clu d e the establishm ent of an a n t iv ir a l s ta te , the in h ib it io n of c e l lu l a r p r o l i f e r a tio n and the a lte r a t io n of d if f e r e n t immune reac­

tio n s . In p a r tic u la r , the IFU:s r a p id ly augment the l y t i c a c t iv it y of the na tural k i l l e r (UK) c e lls . In the present th e s is , some of the func­

tio n a l and m elecular mechanisms by which IFN:s act on UK c e lls and o th e r leucocytes are stu d ie d . A good c o r r e la tio n is found between the a b i l i t y of d iff e r e n t tumor c e ll lin e s to induce IFU pro du ction among p e rip h e ra l blood lymphocytes and t h e ir s e n s it iv it y to UK c e ll c y to to x i­

c i t y , in d ic a tin g th a t IFU m ight re g u la te the a c t iv i t y o f UK c e lls th r o ­ ugh a p o s itiv e feed-back mechanism. When studying the in te r a c tio n b e t­

ween the UK c e lls and two ta r g e t c e ll lin e s i t is demonstrated th a t the two c e ll lin e s are not recognized by the same re ce p to rs . The augmenta­

t io n o f UK c e ll c y to to x ic ity by IFU is shown to in v o lve both a lte r a tio n o f re ce p to r s tru c tu re s on the UK c e ll and enhancement o f steps in t h e ir l y t i c machinery. The e ffe c ts of IFU on the synth esis of in d iv id u a l p ro te in s is then studied by two-dimensional e le c tro p h o re s is . I t is demonstrated th a t IFU-a and IFU-ß w ith in 1.5 hours induce the synthesis o f nine pro te in s (Mr 80, 75, 62, 58, 53, 38, 36, 33 and 30 kD) in human lymphocytes. The in d u c tio n is dependent on a ra p id de novo RUA s y n th e s is , which is in it ia t e d le ss than 30 minutes a f t e r the a d d itio n o f IFU. The expression of the nine p ro te in s is w e ll c o rre la te d to the development of augmented UK c e ll c y t o t o x ic it y . Four of the p ro te in s (Mr 80, 62, 38 and 33 kD) are found to be expressed in a panel of ten hematopoetic and two anchorage-dependent c e ll lin e s , whereas the remai­

ning p ro te in s seem to be expressed in leucocytes o n ly . IFU induce the syn th e s is of the same p ro te in s in both p u r ifie d large g ra n u la r lympho­

cytes (re sp o n sib le f o r the main UK c e ll a c t iv i t y in man), T c e lls and monocytes, demonstrating th a t the augmentation o f UK c e ll a c t iv it y does not in vo lve the form ation o f unique 'U K - c e ll1 s p e c ific p ro te in s . Rath­

e r , the augmentation of the l y t i c a c t iv it y of both UK c e l ls , c y to to x ic T c e lls and manocytes seem to in volve common stages in t h e ir l y t i c mechanisms. In c o n tra s t to IFU-a and IFU-ß, IFU -y, does not induce any de te cta b le p ro te in s in e ith e r UK c e lls o r T c e lls . T his lack of e ffe c t o f IFU-y on the p ro te in synth esis is not a general phenomenon, since the e ffe c ts o f IFU-a and IFU-y are s im ila r in a glioma c e ll lin e . These r e s u lts demonstrate th a t the re e x is ts a t le a s t one pathway to augnent the UK c e ll c y t o t o x ic it y which does not in vo lv e the increased synthesis o f the nine IFU-a/IFU-ß in d u c ib le p ro te in s and in d ic a te s th a t e ith e r these p ro te in s are mainly in vo lve d in o th e r e ffe c ts of IFU, o r th a t the augmentation by IFU-a/IFU-ß and IFU-y in v o lv e d if f e r e n t pathways. When the e ffe c ts of IFU-a on the syn th e sis of membrane-associated p ro te in s is s tu d ie d , i t is demonstrated th a t only the 80 kD IFU-a in d u c ib le p ro te in is associated w ith the c e ll membrane. In a d d itio n , IFU-a seems t o induce three a d d itio n a l, membrane-associated p ro te in s (Mr 94, 76 and 66 kD) which are not detected in whole c e ll ly s a te s .

Key words: na tural k i l l e r c e ll/ la r g e g ra n u la r ly m p h o c y te /in te rfe ro n / i n te rfe ro n p r o d u c tio n /in te r fe r o n - i nd uce d/pro te in synthesis/tw o-dim en- sio na l e le c tro p h o re s is /

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T his th e s is is based on the fo llo w in g p u b lic a tio n s , which are re fe rre d to by t h e ir roman numerals in the te x t:

I . G ustafsson, Ä. and E. Lundgren. 1981. Augmentation of na tural k i l ­ l e r c e ll a c t iv it y in volve s both enhancement of l y t i c machinery and expression of new re ce p to rs. C e llu la r Immunology, 62:367-376.

I . G ustafsson, Å . , S. Sundström, T. Ny and E. Lundgren. 1982. Rapid in d u c tio n of seven p ro te in s in human lymphocytes by in te r fe ro n ; c o r r e la tio n to natural k i l l e r c e ll a c t iv i t y . Journal of Immuno­

logy, 129:1952-1959.

I I I .G ustaf sson, Å . , T. Hy and E. Lundgren. 1985. S im ila r e ffe c ts of treatm e nt w ith a - in te rfe r o n on the p ro te in synth esis of human la rg e g ra n u la r lymphocytes, T - c e lls , and manocytes. Scandinavian Journal of Immunology, 22:000-000.

IV . G ustafsson, Å . , S. Sundström and E. Lundgren. The augmentation of the na tural k i l l e r c e ll a c t iv it y by y in te r fe r o n is not associated w ith the in d u c tio n of the a - in te rfe r o n in d u c ib le p ro te in s . Sub- mi tte d .

V. Gustafsson, Å. and E. Lundgren. The 80 kD a - in te r fe r o n in d u c ib le p ro te in in human leucocytes is associated w ith the c e ll membrane.

Submitted.

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7

1. INTRODUCTION.

1 .1 . THE INTERFERON SYSTEM:

1 .1 .1 . The discovery of in te rfe ro n .

The class of p ro te in s which have la t e r been c a lle d 'in te rfe ro n s ' was independently discovered by Nagano e t al and Isaacs e t a l . The phenomenon of v ir a l in te rfe re n c e , ie . th a t animals in fe c te d w ith one v ir u s were in s e n s itiv e to the simultaneous o r immediate challenge w ith s im ila r or unrelated viruses was known since the 1930:s (Hoskins, 1935;

F ind la y & MacCallum, 1937). Nagano (Nagano & Kojim a, 1954; Nagano e t a l , 1954) demonstrated the existence of a v i r u s - in h ib i tin g substance, present in homogenates from tis s u e s , in o c u la te d w ith e ith e r liv e or U V -irra d ia te d va ccin ia v iru s . The 'c la s s ic a l' experiments concerning the nature of v ir a l in te rfe re n c e were performed by Isaacs and Linden- mann in 1957 (Isaccs & Lindenmann, 1957; Isaacs e t a l , 1957). The au­

th o rs tre a te d chick c h o rio a lla n to ic nembranes w ith h e a t-in a c tiv a te d in flu e n z a v iru s , and showed th a t t h is treatm ent lead to the production o f a solub le fa c to r which, when tra n s fe rre d to fre sh c e lls protected these a g a in st subsequent v ir a l in fe c tio n s . The name 'in te rfe ro n ' was f i r s t used by these authors.

I t was soon found th a t the in te r fe r o n (IFN) did not d ir e c t ly in a c tiv a te the viru s e s , but induced the form atio n of an 'a n t i v i r a l ' s ta te in the tre a te d c e lls . I t was also shown th a t IFN was a p ro te in . The p ro te c tin g e f f e c t of IFN was u n s p e c ific as regards the in fe c tin g v iru s ( ie . the e f f e c t was not lim ite d to c e rta in v ir a l f a m ilie s ) , but lim ite d to c e lls from the same species as the one in which the IFN was produced. This s p e c ie s - s p e c ific ity was la t e r found to be less s t r i c t ( see S tew art, 1979).

1 .1 .2 D e fin itio n o f in te r fe r o n .

To d is tin g u is h IFN from o th e r a n t iv ir a l substances a d e f in it io n of IFN was s e t up by Lockhart (1973):

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2. I t s a n t iv ir a l e f fe c t must not r e s u lt from n o n -s p e c ific to x ic e ffe c ts on the ta rg e t cel 1.

3. I t must be a c tiv e a g a in st a wide range of un related viru se s .

4 . I t must i n h ib it v iru s re p lic a tio n through an in t r a c e llu la r e f f e c t , which must in v o lve synthesis by the c e lls of both RNA and p ro te in s .

1 .1 .3 . Heterogeneity o f in te r fe r o n s .

I t was soon found th a t the re e x iste d two main species of IFN, d iff e r in g in a n tig e n ic as w e ll as in o th e r p ro p e rtie s . The two species were la t e r named IFM-a and IFN-ß. IFN-a is mainly produced by c e lls of haematopoe- t i c o r ig in , whereas IFN-ß is produced by f ib r o b la s ts and o th e r ancho­

rage-dependent c e lls . At pre sen t, several d iff e r e n t subspecies of IFN-a have been demonstrated ( see below).

Wheelock (1965) f i r s t described the existe nce of a t h ir d species of IFN, la t e r named IFN -y. This species, which d iffe re d from the o th e r two by being la b ile a t pH 2, was f i r s t detected in PHA-treated lymphocytes c u ltu re s . L a te r stud ies showed th a t t h is IFN was produced mainly by immunocompetent T c e lls in response to the a p p ro p ria te antigen stim u­

lu s . Recent in v e s tig a tio n s in d ic a te th a t IFN-y have immunoregulative e f f e c t s , d if f e r e n t from those exerted by IFN-a and IFN-ß ( see s e c tio n 1 .1 .5 .3 ) . Some of the main biochemical p ro p e rtie s of the three species o f IFN-.s is lis te d in ta b le I .

In 1980 a gene coding fo r IFN-a was is o la te d by Nagata e t al (1980).

T his work was la t e r repeated by o th e r groups, and i t soon became e v i­

dent th a t several genes, coding f o r d if f e r e n t IFN-a subtypes were pre­

sent in the genome. At pre sen t, more than 20 d if f e r e n t genes or gene lik e sequences are known fo r IFN-a. Several of these seem to c o n s titu te pseudogenes. (G oedell et a l , 1981; Nagata e t a l , 1981; Lundh e t a l , 1984; C o llin s , 1983 (ove rvie w ) ). IFN-ß (Taniguchi e t al_, 1979;

1980; Derynck e t a l , 1980;) and IFN-y (Gray e t a l , 1982) have also been

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9

TABLE I .

C haracteristics of d iffe re n t human IFN species.

IFN species Main source Mr (kD) Acid 1ab i1e

Gene sequences known

IFN-a lymphocytes,

monocytes

18.000- 21.400

no >20

IFN-ß anchorage-

dependent cel Is

21.000 no 1

IFN-y immuno­

competent T c e lls , NK cel Is

20.000 yes 1

is o la te d , but in these cases only one gene sequence have been associa- ted w ith each of the two IFN speci es.

Although d iff e r in g in t h e ir a n tig e n ic and chemical p ro p e rtie s , large s im i la r i t i e s between the d iff e r e n t IFN -a:s as w e ll as between IFN-a :s and IFN-ß on both nu cle o tid e and amino-acid le v e l. Thus, on the peptide le v e l IFN-ß show about 35 % homology to the consensus IFN-a sequence ( C o llin s , 1983, p. 3 9 ).

On the peptide le v e l, IFN-y is much le ss re la te d to the o th e r two spe­

c ie s of IFN, showing le ss than 10 % homology to IFN-a and IFN-ß (C o l­

l i n s , 1983, p. 3 9 ). IFN-y also seems to u t i l i z e a re c e p to r, d is t in c t from th a t used by IFN-a and IFN-ß (Branca & B a g lio n i, 1981; Joshi e t a l , 1982; Sarkar e t a l , 1984), and the genes f o r the IFN-y and IF N -a /- IFN-ß rece ptors are not s itu a te d on the same chromosome (Raziuddin e t a l , 1984). The genes fo r IFN-a and IFN-ß are located on chromosome 9 Meager e t a l , 1979; Owerbach e t a l , 1981), whereas the gene f o r IFN-y have been mapped to chromosome 12 ( C o llin s , 1983, p. 4 7 ).

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1 .1 .4 . Production of in te rfe ro n s .

The c la s s ic way to induce form ation of IFN-a o r IFM-ß is to challenge the producing c e lls w ith e ith e r s u ita b le viruses or fo re ig n double­

stranded n u cle ic acids such as poly inos in e -c y to s in e (p o ly lC ). In ad­

d it io n to these defined inducing agents, several o th e r, le ss w e ll-d e ­ fin e d substances in c lu d in g b a c te ria and b a c te ria l products of d iff e r e n t o r ig in have been reported to induce IFN in c e rta in s itu a tio n s . For exte nsive reviews of t h is area, see Burke (1983) and W ilkinson &

M o rris , (1983). Production of IFN as a concequence of c o c u ltiv a tio n of leucocytes and tumor c e lls was f i r s t described by T ri neh ie r i e t a l , ( 1977; 1978a). In such c o c u ltu re s , both IFN-a (Timonen e t a l , 1980) and IFN-y (Grönberg e t a l , 1984) can be demonstrated. Although d ire c t e v i­

dence fo r IFN pro du ction by conjugating NK c e lls have been presented (Timonen e t a l , 1980), lymphoid c e lls , devoid of NK a c t iv i t y , also produce IFN when c o c u ltiv a te d w ith tumor c e lls (Weigent e t a l , 1981;

Rönnblom e t a l , 1983; Abb e t a l , 1984). F u rth e r, IFN -a a n d/or IFN-y is produced in the mixed lymphocyte c u ltu re (MLC) (K irc h n e r e t a l , 1979;

P erussia e t a l , 1980; Peterman e t a l , 1984), and IFN-y p ro du ction can be induced by treatm ent of lymphocytes w ith the T c e ll growth fa c to r , in te r le u k in 2 ( IL -2 ) (F a rra r e t a l , 1981; Kasahara e t a l , 1983; Kawase e t a l , 1983; T ri neh ie r i e t a l , 1984). The production of IFN is depen­

dent on de novo syn th e s is of RNA and p ro te in s , and the peak s e cre tio n o f IFN is g e n e ra lly reached about 12 hours a f t e r challenge (S tew art, 1979, p. 65 -6 7 ).

During the f i r s t twenty years of research in t h is f i e l d , h ig h ly p u r i­

f ie d pre pa ration s of in te rfe ro n s were d i f f i c u l t to o b ta in . T his can be a t tr ib u te d to the high in t r i n s ic a c t iv it y of IFN, being a c tiv e in con­

c e n tra tio n s as low as 10-13 m. ß0th IFN-a and IFN-ß have now been p u r i­

f ie d to homogeneity (K n ig h t J r . , 1976; Berg & Heron, 1980). F u rth e r, a l l three species of IFN have no/ been expressed in heterologous sys­

tems, such as E sche rich ia C o li, p e rm ittin g the pro du ction and p u r i­

f ic a t io n of sin g le subspecies of d iff e r e n t in te rfe ro n s . In g e n e ra l, the e ffe c ts of recombinant and na tive in te rfe ro n s are s im ila r . However, d iffe re n c e s in the r e la tiv e e ffic a c y of in d iv id u a l subspecies of IFN-a e x is t ( see Kingsman & Kingsman, (1983), p. 238).

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11

1 .1 .5 . Effects of in te rfe ro n s .

1 .1 .5 .1 . A n tiv ira l e ffe c ts .

Most stud ies concerning the a n t iv ir a l e ffe c t of IFN have, so f a r , been performed using IFN o f the a o r ß ty p e . In ge ne ral, IFN:s of these species in h i b i t the v ir a l m u ltip lic a tio n by inducing a lte r a tio n s in the p h y s io lo g ic a l s ta te of the r e c ip ie n t c e l l, which leads to in h ib it io n of the tr a n s c r ip tio n and/or the tr a n s la tio n of v ir a l p ro te in s (th e 'a n t i ­ v i r a l ' s ta te ) . For a general review of t h is area, see McMahon and M o rris , (1983). The m olecular basis of the IFN a c tio n is only p a r tly understood. IFN binds to a s p e c ific c e llu la r re c e p to r, but whether the i nte rn al iz a tio n of the IFN i t s e l f is necessary, or whether a second messenger is in volved is not known. The form ation of the a n t iv ir a l s ta te is dependent on i n i t i a l de novo RNA and p ro te in synth esis (H e l­

l e r , 1963; Lockhart, 1964; T a y lo r, 1964; Dianzani e t a l , 1976).

Several authors have shown th a t IFN s tim u la te s the a c t iv it y of a s e t of th re e enzymes, a p ro te in kinase, an ( 2 '- 5 ‘ ) o lig o (A) synthetase and a phosphodiesterase. Some of these enzyrres are a c tiv e in the presence of double-stranded RNA, and are capable of degrading RNA and/or in h ib it in g t r a n s la t io n . For extensive reviews of t h is area, see Revel e t a l , ( 1979) and Lengyel e t a l , (1981). Although the a n t iv ir a l a c t iv it y of IFN seems to be mediated mainly by the in h ib it io n o f the tr a n s la tio n of v ir a l p ro te in s , d ir e c t e ffe c ts on the tr a n s c r ip tio n of v ir a l RNA have been described (Metz e t a l , 1976). In a d d itio n , e ffe c ts on o th e r p a rts o f the v ir a l r e p lic a tio n such as e a rly steps in the Simian V iru s 40 r e p lic a t io n cycle (Yamamoto e t a l , 1975) and the release of r e t r o v iral p a r tic le s ( B i 1 l i eau e t a l , 1976; Naso e t a l , 1982) have been demonstra­

te d .

1 .1 .5 .2 A n tip ro life ra tiv e e ffe c ts .

The a b i l i t y of IFN to i n h ib it c e llu la r m u ltip lic a tio n was f i r s t desc­

rib e d by Paucker e t al (1962). For a general review , see T aylo r-P a- p a d im itrio u , (1983). In general , IFN in h ib it s c e ll p r o li fe ra tio n by delaying the e n try in to the S phase (Sokawa e t a l , 1977; Creasey e t a l ,

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1980). In some cases, IFN a ffe c ts o th e r p a rts of the c e ll c y c le . Thus, c e lls of lymphoid o r ig in such as the P3-HR-1 c e ll lin e seem to accumu­

la te in a p o s tm ito tic G Q -like re s tin g s ta te (Leanderson & Lundgren, 1982), whereas the e ffe c ts of IFN on the glioma c e ll lin e 251 MG in ­ volves a p ro lo n g a tio n of the passage through the S phase (Lundblad &

Lundgren, 1981).

Several re p o rts in d ic a te th a t the a n t i p r o l i fe r a tiv e and a n t iv ir a l e f­

fe c ts of IFN a t le a s t p a r tly in vo lve d if f e r e n t pathways. Studies by Leanderson e t al (1982) show th a t a su b lin e of the P3-HR-1 c e ll lin e , w h ile r e s is ta n t to the a n t ip r o lif e r a tiv e e ffe c ts of IFN, s t i l l is able to reach an a n t iv ir a l s ta te . In a d d itio n , IFN induce the synthesis of some IF N -in d u c ib le p ro te in s also in these re s is ta n t c e lls . The s p e c ific mechanism f o r the a n t ip r o life r a tiv e e ff e c t of IFN is not known. Reduced steady s ta te le v e ls of the c - nyc gene t r a n s c r ip t have been demonstrated (Jonak e t a l , 1984; E in a t e t a l , 1985). Also the presence of elevated in tr a c e l lu la r le v e ls of 2 1- 5 1 o lig o (A) n u cle o tid e have been c o rre la te d to the decrease in c e ll p r o li fe r a tio n (Kimchi e t a l , 1979).

1 .1 .5 .3 . Immunoregulative e ffe c ts .

IFN:s a ffe c t many immunological fu n c tio n s , and in many cases both en­

hancement and in h ib it io n of the same fu n c tio n have been re p o rte d , appa­

r e n tly depending on the experimental c o n d itio n s . Some of these re s u lts are summarized in tab le I I . For a general review , see Moore, (1983). IFN-a and IFN-ß a lte r s the s e c re tio n o f an tib o d ie s both in vivo and in v i t r o . IFN also a ffe c ts the g ra ft-v e rs u s -h o s t re a c tio n as w e ll as the a llo g r a f t s u r v iv a l. IFN-a and IFN-ß a lso increases the ra te of s yn th e sis and expression on MHC cla ss I a n tige ns. The p r o l i fe r a tiv e response of T c e lls is dim inished by IFN-a and IFN-ß in both the MR and m itog en -stim ula te d p r o li fe r a tiv e responses. In c o n tra s t, the c y to ­ t o x ic i t y displayed by T c e lls , monocytes and NK c e lls is ra p id ly aug­

mented by IFN-a and IFN-ß.

IFN-y seem to d i f f e r from the o th er two species of IFN in th a t the p r o li fe r a tio n in the MR o r in mi to g e n -stim u la te d p r o lif e r a t iv e respon­

ses are g e n e ra lly less a ffe c te d a t comparable doses (Shalaby e t a l ,

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TABLE I I .

Examples of e ffe c ts of d iffe re n t interferons on immune responses.

E ffe c t IFN species

oc/ß Y

References

Antibody s e c re tio n in vivo

+/-1 ? Braun & Levy, ( 19 72) Strannegård e t a l, (1978) Antibody se cre tio n

in v it r o + /- + H ä rfa s t e t a l , ( 1981 )

Shai a by e t a l, (1984) Delayed type hyper­

s e n s it iv it y

+ /- ? DeMeyer e t a l , ( 1975) DeMeyer & DeMeyer, (1980) MHC cla ss I

expression

+ + Lindahl e t a l, (1973; 1974) Heron & Berg , ( 1978) Rosa e t a l , ( 1983) MHC cla ss I I

expression

0 + Basham & Merigan, (1983) Rosa et a l, 1983

P r o lif e r a tiv e response i n MLR

- 0 Heron & B e rg , (1976; 1979) Z a r li ng e t a l , ( 1978) Shai aby e t a l , ( 1984) P r o l ife r a tiv e response

in response to le c tin s + /- 0 Mi ö rn e r e t a l , (1978) Shai aby e t a l , ( 1984) C y to to x ic ity of

T c e lls

+ ? Heron & Berg, (1976; 1979) Z a r li ng e t a l , ( 1978) C y to to x ic ity of mono­

cytes

+ + Herberman e t a l, (1982)

Lee & V il çeck, ( 1984) C y to to x ic ity of NK-

cel Is

+ (+ )/+ T r in c h ie r i & S a n t o li, (1978) T r i neh ie r i e t a l, (1984) T h is th e s is , paper IV Platsoucas, (1984) r ) + = increased by IFN; - =

? = no in fo rm a tio n a v a ilib le .

decreased by IFN; 0 = not a lte re d by IFN;

1984). IFN-y increases the expression of both cla s s I and I I NHC a n t i­

gens. Native or recombinant, Escherishia Coli produced, IFN-y also augments the c y to to x ic ity of NK c e lls and monocytes. However, the aug­

m entation of NK c e ll a c t iv it y caused by rlF N -y seems to be lower and fo llo w in g a slower k in e tic s as compared to e ith e r n a tive IFN-y o r IFN-a o r IFN-ß (P latsoucas, 1984; K ie s s lin g , personal communication).

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1 .2 . NATURAL KILLER CELLS.

1 .2 .1 . General background.

A ll liv in g organisms are c o n s ta n tly exposed to the in flu e n ce s from the exte rn a l w o rld . To m aintain t h e ir in t e g r i t y , d if f e r e n t means of defence towards such in flu e n c e s have been developed. In mammals, these func­

tio n s are maintained by a system of s p e c ia liz e d c e lls : the immune system. This system seems to operate by a combination of non- s p e c i­

f i c and s p e c ific mechansisms.

The sp ecific p a rt of the immune system is only a c tiv a te d a f t e r exposure to the a p p ro p ria te a n tig e n . This p a rt of the immune system is dependent on two in h e re n t p ro p e rtie s : A la rge ava i l ib le re p e rto ire of re c e p to r s tru c tu re s , capable of s p e c ific in te ra c tio n w ith the s tim u la ­ tin g antigen and the a b i l i t y of rap id expansion of the a p p ro p ria te clones of responding c e lls . This kind of immunity is exerted by the T and B c e l ls , . In both cases, the c e lls have a large re p e rto ire of c lo ­ nal ly d is tr ib u te d receptors on t h e ir c e ll surface . Upon contact w ith an a n tig e n , these c e lls undergo a rap id clonal expansion and subsequent d i f e r e n tia tio n in to e ffe c to r c e lls , s p e c if ic a lly d ire c te d towards the s tim u la tin g a n tig e n . The B c e lls mature in to im m unoglobulin-secreting plasma c e lls , whereas the T c e lls mature in to e ith e r lym phokine-produ­

cing T he lp er c e lls or c y to to x ic T lymphocytes.

In c o n tra s t to the T and B c e lls , the non-specific p a rt of the immune system needs no p r io r exposure to antigens to become a c tiv a te d . T his p a rt of the immune system, which includes the gra nu locytes, phago­

c y t ic macrophages and monocytes, uses a d iff e r e n t s tra te g y : i t is de­

pendent on the presence of a large number of n a tu ra lly re a c tiv e c e lls , which can mount a rap id but u n s p e c ific in te r a c tio n w ith fo re ig n mate­

r i a l s .

1 .2 .2 . The discovery of the natural k i l l e r (NK) c e l l .

Although c le a r ly a c tiv e and fu n c tio n a l in the defense a g a in st invading microorganisms, the ro le of the immune system in the defence a g ainst

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15

tumor development is more obscure. The immunosurveil lance th e o ry , ie . the idea th a t a ris in g neoplasms are co n tin o u sly e lim in a te d from the body through the immune system was proposed al ready in 1909 by E rlic h ( E r lic h , 1957). The theory was la t e r reform ulated by Burnet (1970), who proposed th a t the immunosurveil lance was mediated by the c e lls which were responsible fo r the delayed type h y p e r s e n s itiv ity , ie . the c y to ­ to x ic T c e lls . Although several authors demonstrated th a t the immuni­

z a tio n of inbred mice w ith tumor c e lls from chem ically or v ir a l induced tumors in h ib ite d the la t e r tra n s p la n ta tio n of these (F o le y, 1953; K le in e t a l , 1960; Sjogren e t a l , 1961), attem pts to fin d s im ila r mechanisms a c tiv e towards spontaneously a ris in g tumors have, in g e ne ral, been unsuccessful (H e w itt e t a l , 1976), and t h is kind of immune response m ight be lim ite d to c e rta in exogenously induced tumors ( f o r a review , see K le in , 1980).

Beginning in 1975, several authors demonstrated th a t also lymphocytes from unimmunized animals showed c y to to x ic ity towards tumor c e lls (K ie s s lin g e t a l , 1975a; 1975b; Sendo e t a l , 1975; Herberman e t a l , 1975a; 1975b; Jondal & Pross, 1975). I t was shown th a t the c e lls media­

tin g t h is e ffe c t were phenotypical ly d iff e r e n t from the c y to to x ic T c e lls mediating the s p e c ific re s is te n c e . Since these c e lls were a c tiv e in the absence of previous exposure to the recognized a n tig e n (s ), they were termed ' natural k i l l e r 1 c e lls . L a te r studies in d ic a te d th a t these c e lls might be im p ortan t not only in the immunological response to a ris in g tumors (K ärre e t a l , 1980; Kasai e t a l , 1979), but th a t they m ight also be a p a rt of the e a rly response of the body to v ir a l in fe c ­ tio n s (Lopez e t a l , 1982 ; She!1am e t a l , 1982; Bishop e t a l , 1983), as w e ll as having an in flu e n c e on the growth of hematopoetic stem c e lls (Hansson e t a l , 1981; 1982; Kolmberg e t a l , 1984). For reviews on t h is area, see Herberman, (1980; 1982).

1 .2 .3 . C haracteristics of the NK c e l l .

The NK c e ll was o r ig in a lly defined as a c e ll of lymphoid o r ig in , which, upon d ir e c t c e l l- t o - c e l l contact ra p id ly k il le d c e rta in c e ll lin e s of hematopoetic o r ig in . The ta rg e t c e lls most commonly used in these s tu ­ dies are the K 562 erythro le ukem ia c e ll lin e (Lozzio & L o zzio , 1979)

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in the human system and the YAC-1 mastocytoma c e ll in the mouse system (Cikes e t a l , 1973). Early stud ies in d ic a te d th a t the NK c e ll was a bone-marrow d e rive d , p re -th y m ic , non-adherent c e ll of the s o -ca lle d n u ll c e ll lin e a g e , ie . not being a p a rt of e ith e r the B o r T c e ll l i ­ neage. I t was however c le a r th a t the e f f e c t was mediated by a heteroge­

nous p o pu latio n o f c e lls in respect to most parameters s tu d ie d .

The presence of both fu n c tio n a l membrane s tru c tu re s and o th e r c e ll surface markers on the NK c e ll have been stud ied e x te n s iv e ly . Some of the r e s u lts concerning human NK c e lls are shown in ta b i e I I I . As can be seen, the NK c e ll( s ) are h ig h ly heterogenous, d is p la y in g s tru c ­ tu re s associated w ith both T c e ll and monocyte linea ge s. The surface s tru c tu re s which are, a t pre sen t, most w e ll c o rre la te d w ith the NK c e ll a c t iv i t y in man are the e n t it ie s defined by the Leu 7 (HNK-1) (Abo &

Balch, 1981) and Leu 11 (NK-15) (P h il lip s e t a l , 1983) monoclonal a n t i- bodi es.

By use of the p u r if ic a tio n technique described by Timonen e t al (1981) i t became possible to o b ta in , in the human system, c e ll preparations h ig h ly enriched fo r NK c e ll a c t iv i t y . The NK c e ll a c t iv it y in man was shown to be h ig h ly c o rre la te d to the presence of a c e ll w ith p a r tic u la r morphology, the large g ra n u la r lymphocyte (LGL). F u rth e r, the presence o f the Leu 7 and Leu 11 markers is w e ll c o rre la te d to the existence of c e lls w ith t h is morphology (Abo & Balch, 1981; P h illip s e t a l , 1983).

S im ila r granulated c e lls have la t e r been shown displa y NK c e ll a c t iv it y both in the r a t (Reynolds e t a l , 1981) and the mouse (Kumagai e t a l , 1983). Although most NK c e lls are associated w ith c e lls d isp la y in g the LGL morphology, the two e n t it ie s are not id e n tic a l. Less than 80% o f a l l LGL d is p la y NK c e ll a c t iv it y (Timonen e t a l , 1982a). Abo e t al (1982) show th a t only about 70 % o f the Leu 7+ c e lls in p e rip hera l blood displa y the 'm ature1, h ig h ly c y to to x ic Leu 7+/0KT3” /0KMl+ pheno­

ty p e , w h ile the remaining 30 %, d is p la y in g the Leu 7+0KT3+/0KMl“ pheno­

ty p e , show minimal NK c e ll a c t iv i t y . F u rth e r, only 70-90 % o f LGL bear t h is marker, and a Leu l l +/Leu 7" subpopulation of PBL is even more c y to to x ic than t h e ir Leu l l +/Leu 7+ c o u n te rp a rt (L a in e r e t a l , 1983).

The lineage of the NK c e ll is s t i l l a m atter of debate. The NK c e lls d is p la y c e ll surface markers, associated w ith both monocytes and lym-

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17

TABLE I I I .

Surface markers on human NK c e lls , T c e lls , B c e lls and monocytes.

NK c e lls

T c e lls

B mono­

c e lls cytes

References (NK c e lls )

SIg _1 - , + Timonen e t a l , 1979

Cr - - + + West e t a l , 1977

FcyR - /+ -/+ + + West e t a l, 1977

Masucci e t a l, 1983

SRBC-R - / ( + ) + - West e t a l , 1977

Masucci e t a l, 1983

LGL-morph. ( - ) / + - - Timonen e t a l, 1979

Ia - / ( + ) -/+ + + Breard e t a l, 1981

Platsoucas, 1982

OKM-1 - /+ - + Breard e t a l, 1981

Platsoucas, 1982

OKT-3 - + - Breard e t a l , 1981

Platsoucas, Ì982

0KT-10 - /+ - - O rtal do e t a l, 1982

Platsoucas e t a l , 1982

Leu-7 + - / ( + ) - Abo & Bai eh, 1981

Leu-11 + - - P hil l i ps e t a l , 1983

1) + = presence of marker, - = marker absent, (+) = marker weakly ex- pressed or detected only under c e rta in c o n d itio n s .

phoid c e lls . I t has been suggested th a t NK c e lls are re la te d to the T c e ll lineage (P o lla ck e t a l , 1979; Kaplan & C a lla w a e rt, 1978; Timonen e t a l , 1979) o r to monocytes (Lohmann-Mattes e t a l, 1982). The a l t e r ­ n a tiv e pro po sal, ie . th a t the NK c e ll represents a c e ll lineage of i t s own has been argued by O rtal do (1982) as w e ll as by Abo e t al (1983).

The l a t t e r au thors, studying the expression of NK c y t o t o x ic it y in foe­

t a l lymphocytes, re p o rt th a t fo e ta l NK c e lls appear e a r lie r than fun c­

tio n a l T c e lls , and th a t these c e lls do not displa y e ith e r T o r MC linea ge markers.

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Fresh NK c e lls are able to p r o lif e r a t e in response e ith e r to p o ly c lo ­ nal ly a c tiv a tin g le c tin s and IL -2 (Abo & Balch, 1983; Timonen e t a l , 1982b) o r to IL -2 alone (Suzuki e t a l , 1983). However, bulk c u ltu re s of such c e lls seem to d e te rio ra te and lose both t h e ir s p e c ific markers and c y to to x ic capacity upon prolonged c u ltu re in v it r o (Timonen e t a l , 1982b; Abo & B alch, 1983). Long term c u ltu re s of 'N K -lik e ' c e ll c lo n e s, obtained by the expansion of e ith e r CTL o r enriched LGL in the presence o f IL-2 co n d itio n e d madia have been described (Dennert, 1980; 1981;

Hercend e t a l, 1983; Acha-Orbea e t a l, 1983; A llavena & O rta ld o , 1984;

Roberts & Moore, 1985). These 'N K -lik e ' clones displa y varying amounts o f T c e ll associated markers, supporting the hypothesis th a t the NK c e lls are re la te d to the T c e ll lin e a g e . However, t h e ir exact r e la t io n ­ ship to fre s h NK c e lls remains to be e s ta b lis h e d .

A d d itio n a l sets of n a tu ra lly c y to to x ic c e lls , d is tin g u is h a b le from the NK c e lls , e x is t in both the human and the murine system. Stutman e t al ( 1978) describe the na tural c y to s ta tic (NC) c e ll in the mouse system, whose primary ta rg e t seems to be fibrosarcoma c e lls and which d i f f e r from the NK c e ll as concerns c e ll surface markers and o th e r characte­

r i s t i c s (Paige e t a l , 1981). Burton e t al (1981) describe two subsets o f murine NK c e lls (NK-a and NK-b), which d i f f e r as concerns surface markers and ta r g e t s p e c if ic it y . Among o th e r, Seeley e t al (1979) and Grimm e t al (1982) have demonstrated the existence of 'le c t i n a c tiv a ­ t e d 1 or 'anomalous' k i l l e r c e lls , which are induced in MR and PHA s tim u la te d PBL c u ltu re s , and which, although demonstrating N K -like s p e c if ic it y , can be d is tin g u is h e d from the 'n a t u r a lly ' a c tiv e NK c e ll by bearing markers fo r CTL, and by being detected only a f t e r several days of c u ltu r e .

1 .2 .4 . The l y t i c mechanism of the NK c e l l .

The d iff e r e n t phases in the in te r a c tio n between the NK and ta rg e t c e lls have been studied in d e ta il by several authors. I t appears th a t the l y t i c in te r a c tio n c o n s is ts of three defined phases:

1) An i n i t i a l re c o g n itio n /c o n ju g a tio n phase. During t h is phase, the e f f e c t o r and ta r g e t c e lls bind to each o th e r, forming conjugates. This

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19

step is dependent on the presence of Mg^+ but not Ca^+ ions (H ise ro d t e t a l , 1982). By a d ire c t de term in ation of the number of in d iv id u a l NK c e l l/ t a r g e t c e ll conjugates and the number of conjugates which proceed to ta r g e t c e ll ly s is , several authors (S ilv a e t a l , 1980; Targan &

Dorey, 1979; U llberg e t al 1981a; Timonen e t a l , 1982a) have shown th a t not a l l ta r g e t binding (NK) c e lls are c y to to x ic . The two l a t t e r authors also demonstrate th a t the NK c e ll have a 're c y c lin g ' c a p a c ity , ie . th a t each NK c e ll can k i l l more than one ta r g e t c e l l.

2) A secondary in te r a c tiv e phase ( ' programming f o r l y s i s ' ) . During t h is phase the NK c e ll seems to induce ir r e v e r s ib le a lte r a tio n s in the phy­

s io lo g ic s ta te of the ta rg e t c e l l , w h e re a fte r the conjugate d isso lve s.

T h is step have is a b so lu te ly dependent on the presence o f Ca^+ and Mg^+

ions in the medium (H iserod t e t a l , 1981).

3) A t e r t ia r y l y t i c phase ( ' k i l l e r c e ll independent l y s i s ') in which, a f t e r a time la g , the detached ta rg e t c e lls ly s e . T his phase is inde­

pendent on e ith e r the presence o f fu n c tio n a l NK c e lls o r d iv a le n t ca­

tio n s (H ise ro d t e t a l , 1981).

Newman et al (1982) and Burns e t al (1984) describe two monoclonal a n tib o d ie s (Mab) which i n h ib it the ly s is of several s e n s itiv e ta r g e t c e lls , and which are d ire cte d towards epitopes on the T 200 s tru c tu re o r adjacacent s tru c tu re (s ) on the NK c e l l . None of these Mab:s in h ib it s the i n i t i a l NK c e l l/ t a r g e t c e ll c o n ju g a tio n . The Mab described by New­

man, (1 3 .1 ), in h ib it s the subsequent c y to to x ic re a c tio n also when added to already formed conjugates, but only i f allowed to in te r a c t w ith these in the absence of Ca2+ (Targan & Newman, 1983). In c o n tra s t, the o th e r Mab (9.1C3) seems to a ffe c t a la t e r stage during the Ca2+-depen- dent secondary in te ra c tio n s (Burns e t a l , 1984). This group also show t h a t the pre in cu b a tio n of K 562 ta r g e t c e lls w ith an a n ti- id io ty p e antibody to the 9.1C3 Mab render these c e lls r e s is ta n t to ly s is w ith o u t a ffe c tin g the i n i t i a l c on ju gatio n between the e ffe c to r and ta rg e t c e lls (W erkm eister e t a l , 1984). Taken to g e th e r, these data in d ic a te th a t the i n i t i a l in te ra c tio n between the NK and ta rg e t c e lls c o n s is ts of a two- step in te r a c tio n , where the s p e c if ic it y of the re a c tio n m ight reside in the l a t t e r step.

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1 .2 .5 . S p e c ific ity of NK c e lls .

The ta rg e t c e ll s p e c if ic it y of the NK c e ll is broad. Most e sta b lish e d c e ll lin e s of hematopoetic o r ig in , in clu d in g T and B lymphomas ( I , K ie s s lin g e t a l , 1975a; Herberman e t a l , 19 75a; Sendo e t a l , 1975;

C a lla w a ert e t a l , 1977; 1979), non-lymphoid tumor c e lls (Nunn, e t a l , 1977; Welsh, e t a l , 1979) as w e ll as a llo g e n e ic fo e ta l fib r o b la s ts (Timonen & Saksela, 1977) and vi r u s - i nfected c e lls (S a n to li e t a l , 1978; Welsh e t a l , 1980) are s e n s itiv e . Even xenogenous c e lls are s e n s itiv e (Pross & J o n d a l, 1975; H a lle r e t a l , 1977; Hansson e t a l , 1973). However, the k i l l i n g of xenogenous ta rg e ts is g e n e ra lly less e f fe c tiv e (Hansson, e t a l , 1978). F u rth e r, NK c e lls have, in some in s ­ tances, been found to be a c tiv e not only a g a in s t c u ltu re d c e ll lin e s , bu t also a g a in st fre sh tumour and leukemia c e lls (Vanky e t a l , 1980, Za r i ing e t a l , 1979). Although NK c e lls are a c tiv e toward a v a rie ty of d if f e r e n t c e lls , the c y t o t o x ic it y displayed by NK c e lls is not unspeci­

f i c , since c e rta in c e ll lin e s are com pletely r e s is ta n t to NK c e ll c yto ­ t o x ic it y ( I ; K ie sslin g e t a l , 1975a; Herbe rman e t a l , 1975a; U llberg e t al_, 1982)

Dennert e t al (1981), studying cloned c e ll lin e s w ith 'N K -lik e ' beha­

v io u r, of CTL o r ig in , as w e ll as Roberts & Moore (1985), using clones o rig in a tin g from h ig h ly p u r ifie d LGL, s ta te th a t the ta r g e t spectrum of a l l e sta b lish e d clones are id e n tic a l and s im ila r to the spectrum of fre s h NK c e lls . In c o n tra d ic tio n to t h is , Brooks e t al (1982) and Her- cend e t al (1983) re p o rt th a t a l l such clones displa y d iff e r e n t ta rg e t c e lls s p e c if ic it ie s . N e ith e r of these re s u lts exclude th a t each NK c e ll cou ld carry more than one re ce p to r s p e c if ic it y .

The nature of the s tru c tu re s recognized on the ta rg e t c e ll by the NK c e lls are not known. The NK c e ll is not M HC -restricted (Welsh e t a l , 1979; Becker e t a l , 1976), and also c e lls which lack MHC s tru c tu re s are re a d ily k il le d (Stern e t a l , 1980). Attem pts to study the c e ll reco gn i­

t io n of ta rg e t c e ll s tru c tu re s by c o ld ta r g e t in h ib it io n experiments, (O rta ld o e t a l , 1977; Koide e t a l , 1979; C allaw aert e t a l , 1979) o r by s e le c tiv e d e p le tio n of NK c e lls on ta r g e t c e ll rronolayers (Jensen &

Koren, 1978; P h illip s e t a l , 1980) demonstrate a p a tte rn c o n s is te n t w ith several d iff e r e n t s p e c if ic it ie s of the NK c e l l . However, the popu­

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21

la tio n s were shown to overlap co n s id e ra b ly. In experiments d ir e c tly determ ining the number of conjugate-form ing c e lls , Ul 1 berg e t al (1982) found th a t id e n tic a l numbers of PBL conjugate to several d iff e r e n t ta r g e t c e ll lin e s , regardless of t h e ir s e n s it iv it y to NIK c e ll ly s is . T his suggests th a t i f d iff e r e n t s p e c if ic it ie s do e x is t w ith in the NK c e ll p o p u la tio n , these show a high degree of c r o s s r e a c t iv it y .

Several c e ll surface s tru c tu re s have been c o rre la te d to the NK c e ll r e c o g n itio n , but so f a r no d e f in ite evidence fo r t h e ir involvem ent in t h is in te ra c tio n have been shown. Examples of such s tru c tu re s are shown in ta b le IV . A few attem pts to bioch em ica lly de fin e the s tru c tu re (s ) which are recognized by NK c e lls have been performed, using membrane p re p a ra tio n s (Roder e t a l , 1979a; 1979b; Obexer e t a l , 1981) or super­

n a tants from ta rg e t c e lls (Zaunders e t a l , 1981). These experiments show th a t several d is t in c t p ro te in s w ith Mr between 80 and 240 kD can in te r fe r e w ith the ly s is of s e n s itiv e ta rg e ts . Some of these p ro te in s s e le c tiv e ly in h ib ite d the binding of some, but not a l l ta rg e t c e lls , in d ic a tin g th a t the NK c e lls do not recognize the same s tru c tu re s on a l l ta rg e t cel Is .

In co n clu sio n , i t appears th a t no d e f in ite 'NK ta rg e t' s tru c tu re seems t o e x is t. In ste a d , the NK c e ll seems to recognize a heterogenous, comp­

le x p a tte rn of c e ll surface s tru c tu re s . Probably, some of these are shared between c e lls , whereas some are more r e s tr ic te d to c e rta in c e ll types or c e ll lin e s .

1 .2 .6 . Augmentation of NK c e ll c y to to x ic ity by IFN:s.

T r in c h ie r i and coworkers ( T r in c h ie r i & S a n to li, 1978; T r in c h ie r i e t a l , 1978b) f i r s t described th a t sh o rt time treatm ent of lymphocytes w ith IFN o r IFN inducers lik e poly IC could augment t h e ir NK a c t iv i t y . These re s u lts have la t e r been confirm ed in v i vo (Senik e t a l , 1979; Ein­

horn e t a l , 1978). F u rth e r, the augmenting e ffe c t have been shown to be id e n tic a l to both IFN-a and IF N -ß ( see se c tio n 1 .1 .5 .3 f o r re fe re n ­ c e s ). Using p u r ifie d preparations of d iff e r e n t recombinant IF N -a :s, Or- t a l do e t al (1984) have re c e n tly shown th a t the NK augmenting e ffe c t of d if f e r e n t subspecies of IFN-a is not always c o rre la te d to t h e ir a n t i-

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TABLE IV .

Examples of structures, claimed to be involved in the NK ce11:target c e ll In te ra c tio n .

Structure Evidence fo r in v o l- volvement (references)

Evidence against in ­ volvement (references)

C type v iru s an tig e n

H a tz fe ld e t a l , 1981 Kende e t a l, 1979

Becker e t a l , 1976

D iff e r e n t ia tio n antigens

Werkmeister e t a l, 1932 Garson e t a l, 1983 Stern e t a l , 1980 Trans f e r r i n

re c e p to r

V od in elich e t a l, 1983 Newman e t a l, 1984

Dokhelar e t a l, 1984 Werkmeister e t a l, 1983b Carbohydrate

antigens

Brunda e t a l , 1983 Forbes e t a l, 1981 W erkmeister e t a l, 1983a Roder e t a l , l979a

Haubeck e t a l, 1985

S ia lic acid Yogeeswaran e t a l, 1981 W erkmeister e t a l, 1983b

V ira l antigens Bishop e t a l, 1983 (herpes simplex v iru s ) B la zar e t a l, 1980 (E p s te in -B a rr v iru s )

c o rre la te d to t h e ir a n t iv ir a l e f f e c t . Also IFN-y can augment the NK c e ll a c t iv it y ( T r i neh ie r i e t a l , 1984; Platsoucas, 1984). The aug (ren­

tin g e ffe c t of IFN-a and IFN-ß is dependent on i n i t i a l de_novo synthe­

s is of RNA and p ro te in , as judged from experiments where these func­

tio n s have been blocked by a d d itio n of in h ib ito r s of RNA and p ro te in syn th e sis (Djeu e t a l , 1981; O rtaldo e t a l , 1980). The NK c e ll reaches a maximally augmented s ta te w ith in a few hours of in c u b a tio n w ith IFN, and continue to displa y augmented l y t i c a c t iv it y about s ix hours a f t e r the removal of IFN (S ilv a e t a l , 1980). As shown by T ri neh ie r i and coworkers (T ri neh ie r i & S a n to li, 1978; T ri neh ie r i e t a l , 1981) and Welsh e t al (1981), pretreatm ent of ta rg e t c e lls w ith IFN render these le s s s e n s itiv e to NK c e ll ly s is . IFN-y have an even mare pronounced p ro te c tiv e e f f e c t , being demonstrable a t con cen tra tion s w e ll below those a c tiv e in i t s a n t iv ir a l e ffe c t (W allach, 1983). The mechanism f o r the development of t h is re sista n ce is not known, but de novo RNA and p ro te in synth esis is needed ( T r in c h ie r i & S a n to li, 1973; Welsh e t a l , 1981).

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23

2. RESULTS.

2 .1 . Effects of IFN-g on d iffe re n t phases of the in te rac tio n between NK c e lls and ta rg e t c e lls (Paper I ) .

As mentioned in sectio n 1 . 1 . 4 . , c o c u ltiv a tio n of tumor c e lls and PBL leads to the form atio n of IFN, mainly IFN-a. This a b i l i t y to induce IFN is not noted when PBL are c o c u ltiv a te d w ith fe ta l f ib r o b la s ts , which d is p la y low s e n s it iv it y to NK c e ll c y to to x ic ity (Saksela e t a l , 1979).

To study whether the a b ili t y of a given c e ll lin e to induce IFN produc­

t io n was associated w ith i t s s e n s it iv it y to NK c e ll c y t o t o x ic it y , we compared these p ro p e rtie s , using a panel of s ix c e ll lin e s of hemato- p o e tic o r ig in . In these experiments, a good c o r re la tio n between the s e n s it iv it y of the c e ll lin e s to both unstim ulated and IFN-augmented NK c y t o t o x ic it y and t h e ir a b ili t y to induce IFN pro du ction in 24 hours c o c u ltu re s w ith PBL was noted. The strong c o rr e la tio n between these two p ro p e rtie s of the c e ll lin e s in d ic a te s th a t the IFN -inducing s tru c tu re s and the s tru c tu re s which determine NK c e ll s e n s it iv it y are re la te d . T h is suggests th a t the in d u c tio n caused by the in te r a c tio n between the NK c e lls and s e n s itiv e ta rg e ts represents a p o s itiv e feed-back system, r e c r u itin g new NK c e lls and/or increasing the a c t iv it y of the 'm ature' NK c e lls . Conjugating NK c e lls have, as judged from the determi n a tion o f in tr a c e l lu la r IFN by immunofluorescence, been d ir e c t ly shown to respond to tumor c e ll contact by producing IFN (Timonen e t a l , 1980).

However, also o th e r, n o n -c y to to x ic , lymphoid c e lls produce IFN in the presence of tumor c e lls (Weigent e t a l , 1981; Rönnblom e t al 1983, Abb e t al 1983), in d ic a tin g th a t the re g u la tio n of NK c e ll a c t iv it y by IFN c o u ld in v o lve a d d itio n a l, re g u la to ry , c e lls .

Since a considerable number of NK c e lls binds to ta rg e t c e lls which do not induce IFN, (Saksela e t a l , 1979; I ) , the trig g e r in g step f o r the IFN produ ction among the NK c e lls cannot reside w ith in the i n i t i a l c o n ju g a tio n step, but is trig g e re d by the secondary in te ra c tio n s be­

tween the conjugated c e lls . This in d ic a te s th a t the i n i t i a l in te r a c tio n in v o lv e s a two-step re c o g n itio n mechanism. This view is supported by the data obtained by Targan & Newman (1983), ie . th a t the a d d itio n of an antibody s p e c ific fo r re a c tiv e s tru c tu re s on the NK c e ll can in h i b i t

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the trig g e r in g of the d e liv e ry of the ' l y t i c h i t ' even when added a f t e r the i n i t i a l co n ju g a tio n ( see s e c tio n 1 .2 .4 ) .

We th e re a fte r stud ied the in te r a c tio n between NK c e lls and two of these c e ll lin e s (one h ig h ly s e n s itiv e (K 562), and one m a rg in a lly s e n s itiv e (HL 60)) in d e t a il. C o c u ltiv a tio n of HL 60 and PBL lead to minimal IFN p ro d u c tio n . The NK c e ll a c t iv it y of the PBL was not augmented, and the c e lls were s t i l l s e n s itiv e to the subsequent s tim u la tio n by exogenous IFN. In c o n tra s t, co cu ltu re s of PBL and K 562 lead to both IFN produc­

t io n and maximal s tim u la tio n of the PBL, as measured from t h e ir r e fr a c ­ to rin e s s to fu r th e r s tim u la tio n by exogenous IFN. When studying the number of PBL which bound to the d if f e r e n t ta rg e t c e lls , IFN treatm e nt was found to increase the number of ta rg e t binding c e lls (TBC) which bound to the HL 60 c e lls . In c o n tra s t, no increase in the number of TBC which bound to the K 562 c e lls was noted. A s im ila r increase in the nurrber of TBC towards m arg in a lly s e n s itiv e adherent ta r g e t c e lls have been reported by Timonen e t a l , ( 1982a). This demonstrates th a t, when m a rg in a lly s e n s itiv e c e lls are s tu d ie d , IFN can a ffe c t the i n i t i a l c o n ju g a tio n between the e ffe c to r and ta r g e t c e lls .

Cold ta rg e t in h ib it io n (CTI) experiments demonstrated th a t, w h ile the a d d itio n of co ld HL 60 c e lls a t a 1:1 r a t io a ffe c te d the k i l l i n g of the K 562 c e lls only m a rg in a lly , the a d d itio n of co ld K 562 c e lls s i g n i f i ­ c a n tly increased the k i l l i n g of the HL 60 c e lls . This was probably due to augmentation of the NK c e lls by the IFN-a which was produced w ith in the c y to to x ic assay i t s e l f . The CTI experiments were la t e r repeated as k in e t ic experim ents, in the manner o u tlin e d by P ollack & Emmons (1979), using K 562 c e lls as labeled ta rg e ts . Using t h is approach, we were able t o determine the maximal ra te of k i l l i n g ( V ^ x ) as w e ll as the apparent a f f i n i t y constant (K^) f o r the l y t i c re a c tio n . As discussed in d e ta il by P ollack & Emmons (1979), the V ^ x can be regarded as a measure of the o v e ra ll 'v e lo c it y ' of the c y to to x ic re a c tio n , whereas the K^ probably r e f le c ts the r e la tiv e a f f i n i t y of the re a c tio n be­

tween the NK and ta rg e t c e lls . Thus, K^ probably measures events associated w ith the i n i t i a l con ju g a tio n phase in the l y t i c in te r a c tio n , whereas V ^ x measures the combined e ffe c ts of in te r a c tio n s , taking place during both the con ju gatio n and the secondary in te ra c tiv e phases.

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25

As shown in fig u re 1 (page 2 6 -2 7 ), treatm e nt of the e ffe c to r c e lls w ith IFN leads to an increase in the Vm x , a re s u lt co n s is te n t w ith a change in the ra te of the re a c tio n ra th e r than a change in the binding p ro p e rtie s of the e ffe c to r c e l l. The a d d itio n of cold K 562 c e lls to the re a c tio n , le ad , (as expected) to a cooperati ve in h ib i­

t io n , ie . a ffe c tin g the b u t not the Yrnax» in d ic a tin g th a t the MK c e ll recognized the same s tru c tu re on both labeled and unlabeled c e lls . In c o n tra s t, when co ld HL 60 c e lls were added to the re a c tio n , reg ard less of whether untreated or IF N -tre a te d e ffe c to r c e lls were used, an in h ib it o r y p a tte rn c o n s is te n t w ith noncooperati ve i n h ib i­

t io n ( ie . lowering V p ^ ) was noted. This is c o n s is te n t w ith the hypothesis th a t the NK c e ll does not recognize s im ila r s tru c tu re s on the two c e ll lin e s (Forbes & Oldham, 1980).

Taken to g e th e r, these re s u lts in d ic a te th a t the in te ra c tio n between the two c e ll lin e s and PBL is not mediated through id e n tic a l nechanisms, s i nee :

1) The two c e ll lin e s show d iff e r e n t a b i l i t y to induce IFN among the PBL.

2) IFN tre a te d PBL bind more fre q u e n tly to HL 60 c e lls , but not to K 562 c e lls .

3) A d d itio n of co ld HL 60 c e lls in h ib it s the k i l l i n g of K 562 c e lls only m a rg in a lly , and w ith an noncooperative k in e t ic s .

2 .2 . Effects of IFN-g and IFN-ß on the protein synthesis in human leucocytes (papers I I and I I I ) .

As concluded in the preceding s e c tio n , the augmentation of NK c e ll a c t iv i t y by IFN seemed to in vo lve the a lte r a t io n of several c e ll fun c­

tio n s . Experiments by Djeu e t al (1981) and O rta ld o e t al (1980) in d i­

cated th a t the augmentation of NK c e lls by IFN-a i s dependent on de novo RNA and p ro te in synthesis during the f i r s t three to fo u r hours o f in cu b a tio n . Thus, the increased NK c e ll c y to to x ic ity involve s an increased ra te of synthesis of re le v a n t p ro te in s . To understand the

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