Cytotoxic immune response of puppies to feline sarcoma virus induced tumors

Veterinary Immunology and Immunopathology , 7 (1984) 131--138 Elsevier Science Publishers B.V., Amsterdam - - P r i n t e d in The Netherlands

131

CYTOTOXIC IMMUNERESPONSE OF PUPPIES TO FELINE SARCOMAVIRUS INDUCED TUMORS M.M. SUTER*, B.I. OSBURN and C.A. HOLMBERG Department of Pathology, School of Veterinary Medicine, U n i v e r s i t y of C a l i f o r nia, Davis, CA, U.S.A. * Supported by Swiss National Science Foundation (Accepted 7 February 1984) ABSTRACT Suter, M.M., Osburn, B.I. and Holmberg, C.Ao, 1984. Cytotoxic immune response of puppies to f e l i n e sarcoma virus induced tumors. Vet. Immunol. Immunop a t h o l . , 7:131-138.1 The c y t o t o x i c immune response of puppies to f e l i n e sarcoma virus induced tumors was studied. Neonatal puppies were compared with adolescent dogs. Three d i f f e r e n t types of c y t o t o x i c i t y were investigated: complement dependent cytot o x i c i t y , T-cell-mediated c y t o t o x i c i t y and antibody dependent c e l l u l a r cytotox i c i t y . The r e l a t i o n s h i p between the spontaneous regression of the sarcoma and the development of the immune system of the puppies is discussed. INTRODUCTION Various authors have described experimental

infection with f e l i n e sarcoma

virus (FeSV) and the development of sarcomas in puppies (Gardner, 1970, 1971; Slauson et a l . , 1976; Snyder, 1969). Slauson et al. (1976a,b) compared morphological changes in the tumor tissue in relation to several immunological parameters. They reported evidence of cell-mediated immunity, cytotoxic antibodies, serum blocking a c t i v i t y , and virus-neutralizing antibodies. However, the relationship of the "in v i t r o " tests to the "in vivo" tumor regression remained unclear. Spontaneous regression of the tumor appeared to be related to the development of the immunologic competence of the puppy, as adult dogs did not develop tumors (Gardner, 1970). Relatively few studies have been concerned with the development of the immune system in dogs. Humoral immunity to different antigens in fetal l i f e has been demonstrated (Jacoby, 1969; Shifrine et a l . ,

1971). Depending upon the

antigen, dogs are able to respond with antibodies after the 40th day of gestation. Cell-mediated immunity studies demonstrated acute rejection of skin allografts in 12-day-old neonatal puppies. Skin grafts, transplanted at days 40 and 48 of gestation, had a prolonged survival time of 42 days (Dennis and Jacoby, 1969). The development of cytotoxic immune responses in neonatal dogs has not been f u l l y characterized. 0165-2427/84/$03.00

© 1984 Elsevier Science Publishers B.V.

132

The primary goal of this study was to ascertain i f

and when cytotoxic immu-

n i t y was responsible for the defense of the host against a neoplasm. We report experiments on cytotoxic immune responses of puppies to FeSV-induced sarcomas. MATERIAL Four puppies (1 day old, nos. 1-4, 1 Beagle, 3 Australian sheepdogs) and 3 adolescent dogs (3-6 months old, nos. 5-7, 3 Beagles) were used. Six dogs (3 neonates and 3 adolescents) were infected with FeSV (Snyder-Theilen strain of f e l i n e sarcoma virus, k i n d l y supplied by Dr.G.Theilen, University of C a l i f o r nia, Davis). In 5 dogs the source Qf virus was concentrated, c e l l - f r e e supernatant of a FeSV-infected, transformed sheep f i b r o b l a s t cell neonate (no. cells). cells

3) was infected with

cells

of the same cell

line (tShfb). One line

(1.5 x 107

At the age of 4 weeks neonate no. I received a second i n j e c t i o n with (4 x 107 c e l l s ) .

A l i t t e r m a t e of the neonates was used as an uninocu-

lated control (no. 4). Preinoculation tests served as controls for the adolescent dogs. METHODS Tumors Tumor size was measured d a i l y f o l l o w i n g inoculations. Three measures were taken simultaneously: length, width, and thickness. Blood samples Weekly blood samples were taken from a l l the " i n v i t r o " tests.

infected and control

puppies for

The small blood q u a n t i t i e s obtained in the neonates did

not permit T-lymphocyte c y t o t o x i c i t y (TCC) evaluations. Tissue culture A cell

l i n e of tShfb was grown in RPMI 1640 medium (Grand Island Biological

Co., Santa Clara, CA) with 10% f e t a l c a l f serum (FCS) (Grand Island Biological Co., Santa Clara, CA). These c e l l s were used as source of virus inoculum and as target c e l l s in the c y t o t o x i c i t y tests against heterologous c e l l s . Biopsy material from tumors, and from c l i n i c a l l y

normal subcutaneous t i s -

sues, was minced and cultured on tissue culture flasks in Waymouth MB 752/1 medium (Grand Island B i o l o g i c a l Co., Santa Clara, CA) with 10% FCS. Cultures of f i b r o b l a s t s from normal subcutaneous tissue biopsies were grown in 20% c e l l free supernate of tShfb u n t i l

the growth and morphological features of trans-

formation to tumor c e l l s were evident. The " in v i t r o " used as target c e l l s for autologous lymphocytes.

cultures of c e lls were

133

Immunofluorescence The i n d i r e c t

immunofluorescent antibody test (IFA) for c e l l membrane a n t i -

gen, performed on a l l c e l l

lines before c y t o t o x i c i t y t e s t i n g ,

showed that a l l

infected c e l l s carried FeSV antigen on t h e i r surfaces. Cytotoxicity assay A 51Cr release

assay was used to measure cytotoxicity. The assay was a

modification of previously reported techniques (Cerottini and Brunner, 1971; Grant et a l . , 1977; Kaakinen et a l . , 1974; Kibler and Meulen, 1975). Complement-dependent c y t o t o x i c i t y (CDC), antibody-dependent cell-mediated cytotoxic i t y (ADCC), and T-cell-mediated cytotoxicity were measured. CDC and ADCC were tested against heterologous target cells ( i . e . tShfb), TCC against autologous target cells. Target cells were collected by trypsinisation from monolayers, washed and suspended to a concentration of 3 x 106 cells in 1 ml medium with 10% FCS, incubated in the presence of 300 pCi 51Cr (New England Nuclear, Boston, MA) for 90 minutes in a 37oc waterbath to allow labeling by diffusion. The target cells were separated from free 51Cr by a one-step centrifugation through 90% heat-inactivated FCS (Cerottini and Brunner, 1971). The cells were then resuspended

in

a concentration of 4 x 105 cells/ml

for

CDC or 2 x 105

cells/ml for ADCC and TCC. Antibody dilutions: Serum samples were diluted optimally 1:5 in CDC and ADCC. For TCC and ADCC, lymphocytes were separated according to described methods (English and Andersen, 1974). The lymphocytes were resuspended in RPMI 1640 medium with 20% heat-inactivated FCS at various concentrations and are henceforth called "effector cells". The target:effector cell ratio was 1:30 and 1:100 in TCC, and 1:100 as an optimal concentration in ADCC. All tests were run in t r i p l i c a t e . 0.05 ml of target cell suspension was pipetted into each well of round-bottom microtiter plates with 96 wells (Costar, Cambridge, MA 02139). 0.1 m] effector cells in ADCC and TCC, and 0.05 ml serum in CDC and ADCC, were added. For CDC 0.1 ml guinea pig serum (diluted 1:10) was the complement source. After an incubation of 16 hours (ADCC and TCC) and 6 hours (CDC), respectively, the supernatant was harvested (Flow Laboratories, Inglewood, CA) and r a d i o a c t i v i t y was counted in a gamma counter. Cytotoxicity was expressed according to the following formula: experimenta] release - highest control release % specific release =

x 100 maximal reTease - highest control release

The following controls were included: t o t a l , maximal and spontaneous release, as well as serum, lymphocyte, and C' control.

°°f

I

I

4

I

I

5

I

I

6

I

I

7

I

I

8

!

._0._..0.. ,0

A

1

2

3

4

5

6

7

8

I

A

0

1801 -

0

o.~>,.

10

0

I

3

I

_

10

I

2

1

0

0

0

I

I

/

10

5(

180 170

0 0

10 ~

1

I

1

I

2

I

2

I

.

.

3

I

3

I

. I

4

I

4

.

5

I

5

I

6

I

6

I

7

I

7

I

8

I

8

•

"o

0

A

0

I

1

1

I

I

2

I

2

--c~ "~°

100ill}

5O

180 170

0

10

50

!

I

3

I

3

I

4

I

4

I

I

I

I

I

I

5 w e e k s of age

I

5 w e e k s of age

I

3

Fig. I. CDC (O) and ADCC (0) t i t e r , and tumor size (A) in the neonatal puppies over a period of 8 weeks post infection (~). All 3 doqs developed CDC and low ADCC antibody t i t e r s sumultaneously with the sarcoma growth.

E

N

¢,

E

g

0

(O

03

_m 5

DOG

135

Karyotypin 9 The tumors induced by tShfb were karyotyped to d e f i n e the c e l l cells

origin.

The

were c o l l e c t e d from a monolayer and resuspended in 4 ml serum-free Way-

mouth medium. 0.25 ml Calcemid (Grand I s l a n d B i o l o g i c a l was added and the c e l l s centrifugation,

incubated f o r

2 hours

in

Co., Santa Clara,

CA)

a waterbath at 37oc. A f t e r

the supernatant was removed, and 4 ml 0.8% KCI was added s l o w l y

to the sediment. A f t e r

15 minutes at room temperature the supernatant was r e -

moved by a second c e n t r i f u g a t i o n

and 4 ml of a m i x t u r e 25% g l a c i a l

in 75% methanol was added. This f i x a t i o n

a c e t i c acid

procedure was repeated 3 times.

Fi-

n a l l y the c e l l s were resuspended in 0.5 ml supernatant which was dripped onto a glass

slide,

dried

and stained

with

Giemsa. The karyotype was determined by

chromosome count. RESULTS Tumor i n d u c t i o n in puppies In 2 neonatal puppies (no. 2 and no. 3) a sarcoma grew at the s i t e of inocuR e i n f e c t i o n of a t h i r d

puppy (no. 1) at 4 weeks w i t h tShfb r e s u l t e d in

tumor growth. The 2 sheep c e l l

lation.

induced tumors were shown by k a r y o t y p i n g to have

i n f e c t e d canine c e l l s tion

Figure 1 shows the r e s u l t s

of the 3 puppies and tumor size versus time. A l l

titer

of ADCC and CDC t i t r a 3 dogs developed a CDC

s i m u l t a n e o u s l y w i t h the onset of sarcoma growth. In dog no. 1 and no. 2

the t i t e r sion. ring

only.

persisted

throughout the growth of the tumor and a f t e r

Only a minimal ADCC t i t e r

(<10% s p e c i f i c

the tumor growth. Puppy no. i

titer

a f t e r the i n i t i a l

tumor and a high, size,

rising

but a f t e r

CDC t i t e r

the second i n o c u l a t i o n

occurred.

rose s l o w l y and r e -

low. Puppy no. 3 died of anemia 3 weeks a f t e r

had a l a r g e tumor and r a p i d l y r i s i n g

a small

Dog no. 2 had a medium

s l o w l y growing and regressing tumor; the CDC t i t e r

mained r e l a t i v e l y

regres-

showed no tumor growth and no c y t o t o x i c i t y

inoculation,

rapidly

its

r e l e a s e ) could be measured du-

infection;

CDC response. Dog no. 4 (the c o n t r o l

t e r m a t e ) d i d not develop a tumor. CDC and ADCC t i t e r s

it lit-

were negative throughout

the o b s e r v a t i o n p e r i o d . Adolescent dogs Figure 2 shows the c y t o t o x i c i t y

titers

of the 3 adolescent dogs. No sarcomas

were observed during a 2-month p e r i o d . A l l 3 developed a high CDC t i t e r

(70-90%

specific

The TCC

titer target

release)

and a medium ADCC t i t e r

(40-60% s p e c i f i c

release).

increased in the order no. 5, no. 6, no. 7. I n t e r e s t i n g l y cells

showed an i n c r e a s i n g frequency of c e l l

immunofluorescence in the same o r d e r .

the autologous

surface v i r a l

antigen by

136

80

50

Q. ffl ~

10 0

i

0 weeks

i

I

1 2 3 post infection

Fig. 2. CDC ( e ) , ADCC ( 0 ) , and TCC ([]) response to i n f e c t i o n w i t h FeSV. Bars r e p r e s e n t maxima and minima.

DISCUSSION The c y t o t o x i c i t y

experiments showed t h a t

age are unable to respond w i t h study ADCC f i r s t

cells

B-cell,

natural

killer

an ADCC to FeSV induced sarcoma c e l l s .

chester,

or monocyte (Kaakinen et

1976). A p p a r e n t l y ADCC r e q u i r e s a f u n c t i o n a l

mature e f f e c t o r with

In t h i s

cells

be

non-thymus-dependent, F c - r e c e p t o r - b e a r i n g c e l l ,

cell,

al.,

1974).

have also been shown to possess F c - r e c e p t o r s ( C o r d i e r et a l . ,

cocyte (Kaakinen et a l . , teract

less than 2 months of

appears in dogs o l d e r than 3 months. The ADCC appears to

mediated by a n o n - p h a g o c y t i c , i.e.

puppies of

Null

1976; Win-

Fc-receptor-bearing

leu-

1974). Therefore the neonatal puppies e i t h e r i ) lacked

f o r ADCC, i i )

failed

to produce antibody which could i n -

leucocytes to cause ADCC, or i i i )

the c e l l s

lacked f u n c t i o n a l

Fc-

receptors. CDC antibody appears e a r l i e r

than the ADCC. One-week-old puppies are able to

produce a high amount of c y t o t o x i c a n t i b o d y . In o l d e r dogs CDC antibody response showed g r e a t e r t i t e r s

than c e l l - m e d i a t e d c y t o t o x i c i t y

same phenomenon was demonstrated in c y t o t o x i c i t y line

leukemia v i r u s

(Grant,

(ADCC and TCC). This

assays in cats exposed to f e -

personal communication). In our study TCC was pre-

sent at an age of 3 months. The c y t o t o x i c

antibody r e a c t i o n to f e l i n e

leukemia v i r u s - i n f e c t e d

been demonstrated in i n f e c t e d cats (Grant et a l . , demonstrated

cytotoxic

antibodies

FeSV. Bech-Nielsen et a l . blocking antibodies

and lymphocytes

(1978) showed t h a t

cells

1977). Slauson et a l . in

puppies

the p o s t s u r g i c a l

in dogs is r e l a t e d to the c l i n i c a l

infected titer

has

(1976) with

of serum-

course of osteosarcoma,

137

i.e.

the development of metastases.

They did not find

a correlation

cell-mediated c y t o t o x i c i t y against the tumor c e l l s and survival

between

time. Antibo-

dies to f e l i n e oncorna virus cell membrane antigen (FOCMA) were recognized as protective

against

lymphomas in cats

(Grant et

al.,

1978;

Pedersen et

al.,

1977). In our experiments, CDC occurred simultaneously with tumor growth. CDC alone did not protect the dog against the neoplasia, but spontaneous regression of the tumor occurred. Elevated ADCC antibodies were not necessary for regression. The adolescent dogs did not develop tumors, but they had good CDC, ADCC, and TCC immune response. I t is conceivable that a combination of several components of the immune system is necessary to protect the host against the tumor.

REFERENCES 8ech-Nielsen, S., Reif, J.S. and Brodey, R.S., 1978. Pre- and postoperative studies of in v i t r o cell-mediated r e a c t i v i t y in canine osteosarcoma. Am. J. Vet. Res., 39: 87-93. C e r o t t i n i , J.C. and Brunner, K.T., 1971. In v i t r o assay of target cell l y s i s by sensitized lymphocytes. In: B.R. Bloom and P.R. Glade ( E d i t o r s ) , In V i t r o Methods in Cell-Mediated Immunity. Academic Press, New York, pp 369. Cordier, G., Samarut, C., Bronchier, J. and R e v i l l a r d , J.P., 1976. Antibody dependent c e l l c y t o t o x i c i t y (ADCC). Scand. J. Immunology, 5: 233-242. Dennis, R.A. and Jacoby, R.O., 1969. Development of immunity in the f e t a l dogs: Skin a l l o g r a f t r e j e c t i o n . Am. J. Vet. Res., 30: 1511-1516. English, D. and Andersen, B.R., 1974. Single-step separation of red blood c e l l s , granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque. J. Immunolog. Methods, 5: 249-252. Essex, M., K l e i n , G., Snyder, S.P. and Harrold, J.B., 1971. Correlation between humoral antibody and regression of tumors induced by f e l i n e sarcoma v i r u s . Nature, 233: 195-196. Gardner, M., 1970. Experimental transmission of f e l i n e fibrosarcoma to cats and dogs. Nature, 266: 807-809. Gardner, M., 1971. Feline sarcoma virus tumor induction in cats and dogs. J. Am. Vet. Med. Assoc., 158: 1046-1053. Grant, C.K., Worley, M.B. and DeBoer, D.J., 1977. Detection of complement-dependent l y t i c antibodies in sera from f e l i n e leukemia v i r u s - i n f e c t e d cats by the Chromium-51 release assay. J. Natl. Cancer I n s t . , 58: 157-161. Grant, C.K., Essex, M., Pedersen, N.C., Hardy, W.D., Stephensen, J.R., Cotter, S.M. and Theilen, G.H., 1978. Lysis of f e l i n e lymphoma c e l l s by complement dependent antibodies in f e l i n e leukemia virus contact cats: Correlation of l y s i s and antibodies to FOCMA. J. Natl. Cancer I n s t . , 60: 161-166. Jacoby, R.O., 1969. Development of immunity in f e t a l dogs: Humoral responses. Am. J. Vet. Res., 30: 1503-1510. Kaakinen, A., Bondevik, H., Kiss, E. and Thorsby, E., 1974. Antibody induced cell-mediated c y t o t o x i c i t y studies of human alloantibodies in a micro-platet e s t . Tissue Antigens, 4: 346-360. K i b l e r , R. and Meulen, V.T., 1975. Antibody-mediated c y t o t o x i c i t y a f t e r measles virus i n f e c t i o n . J. Immunology, 14: 93-98. Pedersen, N.C., Theilen, G.H., Keane, N.A., Fairbanks, L., Mason, T., Orser, L., Chert, C. and A l l i s o n , C., 1977. Studies of n a t u r a l l y transmitted f e l i n e leukemia virus i n f e c t i o n . Am. J. Vet. Res., 38: 1523-1531.

138

S h i f r i n e , M., Smith, J.B., Bulgin, M.S., Bryant, B.J., Zee, Y. and Osburn, B . I . , 1971. Response of canine fetuses and neonates to antigenic stimulat i o n . J. Immunology, 107: 965-970. Slauson, D.O., Osburn, B . I . , S h i f r i n e , M. and Dungworth, D.L., 1976a. Regression of f e l i n e sarcoma virus-induced sarcomas in dogs: I. Morphologic invest i g a t i o n s . J. Natl. Cancer I n s t . , 54: 361-370. Slauson, D.O., Osburn, B . I . , S h i f r i n e , M. and Dungworth, D.L., 1976b. Regression of f e l i n e sarcoma virus-induced sarcomas in dogs: I f . Immunologic inv e s t i g a t i o n . J. Natl. Cancer I n s t . 54: 371-377. Snyder, S.P. and Theilen, G.H., 1969. Transmissible f e l i n e fibrosarcoma. Nature, 221: 1074-1075. Winchester, R.J., Fu, S.M., Wang, C.Y., Hoffmann, T. and Kunkel, H.G., 1976. Lymphocyte surface markers: Evidence for three subpopulations of Fc-receptor lymphocytes. In: S.D. Litwin et al. ( E d i t o r s ) , C l i n i c a l Evaluation of Immune Function in Men. Grune and Stration I n c . , New York, pp 1-11.