Common tumor-Specific surface antigens on cells of different species transformed by avian RNA tumor viruses

VIROLOGY 49, 145-149

(1972)

Common Tumor-gpecific Surface Antigens on Cells of Different Species Transformed by Avian RNA Tumor Viruses REINHARD

K U R T H AND H E I N Z B A U E R

Robert Koch-Institut, Abteilung fiir Virologie, 1 Berlin-West 65, Nordufer 20, Germany Accepted March 16, 1972 The presence of avian tumor virus (ATV)-induced tumor-specific surface antigen (TSSA) has been demonstrated on the surface of ATV-transformed chicken, mouse, and hamster cells by humoral and cellular microcytotoxicity tests, by the chromiumrelease test, and by indirect immunofluorescent staining. The TSSA is at least partially cross-antigenic on cells from all three species. With a standard mouse antiTSSA serum the antigen could be demonstrated in different quantities on the surface of cells from different species, indicating the existence of only a partial cross-antigenicity or the expression of the antigen in different quantities. Even within a given species (mouse) the antigen is expressed in varying amounts on the surface of cells from different cell lines, possibly reflecting antigenic modulation or antigenic conversion. No correlation could be found between group-specific (gs) antigen content and virogenicity of the cells and the quantitative expression of TSSA. INTRODUCTION I n two previous papers (Kurth and Bauer, 1972; Gelderblom et al., 1972) the appearance of new antigens on the surface of avian t u m o r virus-infected chicken cells was studied. I t was concluded t h a t a subgroup specific, presumably viral envelope (Ve) antigen exists on the surface of all infected cells. I n addition, the presence of a tumor~ specific surface antigen was demonstrated on transformed chicken cells only. This TSSA is group-specific, i.e., common to all avian t u m o r virus-transformed chicken cells regardless of the subgroup or the pathogenicity of the virus used for transformation

are not virus constituents and which are cross-antigenic in different species. M a m m a l i a n tumors caused b y the same R N A t u m o r virus also exhibit cross-antigenic cell surface antigens, as demonstrated b y Geering et al. (1966) for the Gross cellsurface antigen appearing on infected mouse and rat t u m o r cells. Similarly, Ferrer and Kaplan (1968) and Maruyama and D m o -

chowski (1971) demonstrated cross-antigenic tumor antigens induced by radiation leukemia virus or the murine leukemia viruses, respectively, in different species. However, the nature of the cross-reacting antigen, e.g., whether it is a virion or a nonvirion antigen or both, remains to be elucidated. A cross and is not found on the surface of Rous antigenicity for TSSA has been demonsarcoma virions (RSV). Very little is known about the nature and strated in vivo between mouse and rat Rous origin of t u m o r virus-induced cell surface sarcomas (Jonsson, 1966). However, in the antigens. There is evidence t h a t in virus- same study no specific isograft resistance induced tumors the TSSA is cross-antigenic could be demonstrated after p r e t r e a t m e n t of on t u m o r cells of different animal species. mice with xenografts of Rous hamster or For example, it has been shown in the SV 40 rabbit tumors. In addition, no significant (Girardi, 1965) and the polyoma virus cross-antigenicity could be demonstrated (Habel, 1963) systems t h a t these viruses between Rous sarcomas from mice and induce tumor-specific surface antigens which chickens (Jonsson and SiSgren, 1965). The 145 Copyright © 1972 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

146

KURTH AND BAUER

failure to detect neutralizing antibodies in Rous sarcoma-bearing mammals was interpreted as an indication that Ve antigen does not act as transplantation antigen in mammalian tumors (Koldovsky et al., 1966; Bauer et al., 1969). The question as to whether ATV induce cross-reacting cell surface antigens in birds and mammals seems to be of great importance for an understanding of the origin and nature of these antigens. For this reason the previously described TSSA induced by ATV on chicken cells was compared by several in vitro techniques with the tumor-specific surface antigens induced on mouse and hamster cells. Furthermore, we reinvestigated the question of whether there exists any correlation between the expression of TSSA, virogenicity, and the expression of gs-antigen. Cellular and humorM cytotoxic microtests, a 51chromium-release test, and indirect immunofluorescence were used to demonstrate specific TSSA on ATV-transformed cells of different species. MATERIALS AND METHODS Viruses. The Schmidt-Ruppin strain 1 (SRV-1) of RSV (subgroup A) and the B 77 sarcoma virus (subgroup C) were kindly supplied by Dr. P. K. Vogt, USC, Los Angeles, CA. RAV-1 was isolated by endpoint dilution technique from RSV (RAV-1) which was obtained from Dr. P. M. Biggs, Houghton, Huntingdon, UK. SRV-H, a member of subgroup D, and MAV-B (myeloblastosis-associated virus of subgroup B) were isolated as previously described (Bauer and Graf, 1969). Two nontransforming mutants, NC-SRV-1 and NC-SRV-H, were isolated after hydroxylamine treatment of SRV-1 and SR¥-H, respectively (Graf et al., 1971), and show envelope properties identical to the parental sarcoma virus strains. They have lost the capability to transform chicken embryo cells (CEC) in vitro. Stocks of all viruses were prepared in C/O or C/A type CEC (Vogt, 1965) derived from embryos of chicken line 15. Animals. L-15 chickens have been maintained at the Max Planck Institut fiir Virus Forschung, Tiibingen, since 1964 and are now also kept at the Robert Koeh-Institut,

Berlin. Random inbreeding within the relatively small flock should have eliminated major histoincompatibility differences. The chickens have repeatedly been found to be free of spontaneous leukosis virus particles of subgroup A through D. STU mice represent an inbred strain of mice (started by Dr. W. Sch~fer, Tiibingen) presently in the 75th inbred generation. They were tested during their first year of life and were found to be free of demonstrable C-type particles (Gelderblom et al., 1970) and spontaneous tumors. Golden Syrian hamsters are kept as a small flock in our institute and are not totally inbred. Tumor cells. All cells were grown in a modified Eagle's medium supplemented with 5% heat-inactivated calf serum. Chicken embryo cells were transformed as growing secondary cultures by various strains of ATV. The D4 mouse cell line was previously derived from a STU sarcoma induced by SRV-H. This permanent cell line has been maintained in our laboratory for 3 years with two passages a week. The cells are highly oncogenic in mice and kill the animals within 2 weeks after intraperitoneal injection of 103 cells. During the first year in passage the SRV genome could be rescued upon injection of the cells into chickens, thus causing a virus-producing flbrosarcoma. Gs-antigen as well as tumor-specific transplantation antigen (TSTA) were also demonstrable (Bauer et al., 1969). In recent tests D4 cells did not reveal gs antigen and had lost virogenicity but had retained their capability to induce antitumor resistance in syngeneic mice. RVP3 cells were derived from a C57/BL sarcoma induced by RSV-Prague strain and originally obtained from Dr. J. Bubenik, Prague. ATV group-specific antigen could not be detected in these cells and the RSV genome could not be rescued after injection of the cells into chickens or cocultivation with chicken cells in vitro. However, RVP3 exhibited RSV-specific transplantation antigen (Bubenik and Bauer, 1967). This cell line was later shown by electron microscopy to carry murine C-type particles (Gelderblom et al., 1970) and to contain the gs antigen of murine leukemia viruses (Schiller and Seifert, 1968).

TUMOR CELL SURFACE ANTIGENS

147

TABLE 1 PROPERTIES OF ASV-TRANSFORMEDMAMMALIANCELL LINESa Properties in 1971

Properties in 1967-1968 Species

Transformed by

gs Viroantigen genicity

TSTAb

gs Viroantigen genicity TSSAc

D4

Mouse (STU) d

SRV-H

q-

-4-

q-

-

-

q-

P~¥Pa

Mouse (C57/BL) d

RSV Prague strain

--

-

q-

-

--

q-

RSH

Hamster

SRV

-k

q-

N.T. e

-

q-

q-

a Complement-fixation using anti-gs rabbit sera (Bauer et al., 1965) was used in testing gs antigen. Virogenicity was demonstrated by injection of the cells into chickens and recovery of transforming virus from the tumor. TSTA was demonstrated in vivo in syngeneic animals by the induction of antitumor immunity after injection of the cells. b TSTA = tumor-specific transplantation antigen (demonstrated in vivo). c TSSA = tumor-specific surface antigen (demonstrated in vitro). e Phenotype. e Not tested. R S H cells are hamster cells transformed in vitro b y SRV (Vesel:~ et al., 1966). S R V - H was later isolated from these cells after cocultivation with chicken cells. I n contrast to experiments made earlier (Bauer and Janda, 1967), R S H cells were now found to be gs antigen negative but had retained their virogenicity. T h e properties of the transformed m a m m a l i a n cells used are summarized in Table 1. Sensitization of animals. Sensitization of L-15 chickens for the production of antisera and immune spleen cells was previously described in detail (Kurth and Bauer, 1972). S T U mice were sensitized against S R V - H b y two subcutaneous injections of 5 X 10 a living D4 cells at 4-week intervals into the tail. T u m o r s developing in the tails of some animals were a m p u t a t e d . After 7 weeks all animals received one or two additional intraperitoneal injections of 107 lyophilized D4 cells. T h e animals were sacrificed 5 days after the last booster to obtain antisera and spleen cells for cytotoxic tests.

derived from normal or sensitized chickens to test their activity on target cells f r o m different species. The procedure to prepare white spleen cells has been described ( K u r t h and Bauer, 1972). Chicken target cells were prepared b y transforming C E C as secondary cultures b y A T V and were used when cell cultures were judged b y microscopic observation to be fully transformed. Uninfected C E C served as controls. Mouse embryonic cells (MEC), D4 (STU phenotype) and RVP3 (C57/BL phenotype) cells were used as mouse target cells. R S H and h a m s t e r embryonic cells (ttaEC) served as h a m s t e r target cells. T h e assay for testing cell-mediated immunity on a microseale has been published in detail (Kurth and Bauer, 1972). W e used microtest I tissue culture plates (No. 3034, Falcon Plastics) with the wells of the plates having a volume of 10 ul. Target cells were delivered in 10-ul vol b y a H a m i l t o n syringe (Hamilton Company, Whittier, CA) and allowed to adhere to the b o t t o m of the wells for 24 hr. The target cells were then washed, Assays for Cell Surface Antigens counted microscopically, overlayed b y effec(a) Cellular cytotoxic microtest. Because of tor cells at the desired cell ratio, and a g a i n the inability of avian antibodies to activate incubated for 2 days. T h e test was t e r m i the C ' I component of m a m m a l i a n comple- nated b y washing the cells with medium t(~ ment (Benson et al., 1961) we used lympho- preserve only viable cells which were stained cytic spleen cell suspensions as effector cells after fixation with methanol b y Giemsa and

148

KUI~TFI AND BAUER

then counted. A survival index was calculated as follows:

and stained with trypan blue before or Giemsa after fixation with cold methanol for 15 rain. Survival index The number of surviving target cells in target cells incubated with each well was counted under an inverted immune lymphoid cells microscope using 80 times magnification. X 100 Cytotoxic effects were manifested in general target cells incubated with by detachment and subsequent loss of target normal lymphoid cells cells during the final washings. Highly (b) Humoral cytotoxic microtest. To test for damaged target cells still adhering to the humoral immunity we usually used a micro- bottom of the wells had a much smaller apcytotoxicity assay described by E. Bloom pearance and were stained by trypan blue, (1970). Target cells were cultured in Eagle's whereas living cells were always elongated medium supplemented with 15 % inactivated having a clear cytoplasmatie ring around calf serum and seeded into the 10-gl wells of the nucleus. Falcon microtest I tissue culture plates by a (e) 51Chromium-release test. More recently Hamilton syringe and incubated overnight we have also made use of the ~lehromium in a humidified atmosphere containing 5 % release test to check for surface antigens COs to allow the cells to adhere to the bot- (Sanderson, 1964). This method is more tom. The cells were then washed by over- rapid and avoids microscopic counting of laying the wells with medium, inverting the surviving target cells. On the other hand, one plate, and applying a sudden, horizontal needs at least five times as much antiserum shake to remove loose cells and debris. when compared with the microtest described ATV-transformed CEC were seeded out at above. Target cells were collected with a 1000 cells/10 gl, avian leukosis virus (ALV)- rubber policeman, sedimented (4 rain at infected CEC, normal CEC, D4, and RSH, 600g and 4 °) and resuspended in Eagle's respectively, at 500 cells/10 gl. The plating Medium supplemented with 5 % inactivated efficiency of transformed CEC ranged be- calf serum. The concentration of the cells was tween 15% and 30%, whereas all other adjusted to about 107/ml, and 100 uCi radiotarget cells had a plating efficiency between active sodium chromate (sp. act. 100-300 30 % and 80 % in individual experiments de- gCi/t~g chromium; Radiochemical Center, pending on the cell line. Especially with the Amersham, England) was added. The susmammalian target cells a very constant pension was incubated in plastic tubes for 1 plating efficiency was observed. hr at 37 ° in a 5% CO2 atmosphere with Inactivated antisera were diluted to the gentle rocking. Incubation was stopped by desired concentration and added to the the addition of 9 ml cold medium and sediwashed target cells in 5-ul vol by a Hamilton mentation. The cells were washed three syringe. Usually each dilution was tested in times, recounted, and adjusted to a concentriplicate. The plates were incubated for 30 tration of usually 4.106/ml. The viability of min at 37 ° before the addition of guinea pig these target cell suspensions as determined complement. The complement has always by trypan blue exclusion was always above been absorbed with chicken, mouse, and 90 %. 25 gl portions of the target cell suspenhamster fibroblasts (2 hr/4 °) after the re- sions (25 gl, 105 cells) were incubated with moval of Ca 2+ from the complement by 25 gl serum of different dilutions for 30 rain EDTA to avoid loss of complement activity at 37 ° in 5% CO2 atmosphere before the b y antibody activation (Boyse et al., 1970). addition of 25 gl of guinea pig complement in Complement was also delivered in 5-t~l vol the final dilution of usually 1:12. The cells usually at a dilution of 1 : 16 in medium. The were then incubated with gentle agitation for plates were again incubated for 4 hr. In another 45 rain at 27 °. At the lower temperapreliminary experiments it had been found ture a higher activity of the complement can that longer incubation periods did not increase cell damage by cytotoxic a.ntisera. The be observed (Leon, 1956). Incubation was cells were again washed as described above terminated by the addition of 1 ml ice-cold

TUMOR CELL SURFACE ANTIGENS medium and sedimentation of the cells at 600g for 4 rain at 4 °. One-tenth milliliter of the supernatant fluid was taken off for assessment of radioactivity in a Packard Scintillation Counter (Packard Instruments, Downers Grove, I1). Maximal (= 100%) release of ~lCr was determined by freezethawing several times an equal portion of labeled target cells in an equivalent volume of distilled water and determining the radioactivity in the supernatant fluid after sedimentation of the cell debris at 1000g for 10 min. The experimental tubes were usually set up in triplicate and the average counts were divided by the average counts for the freezethawed preparations and expressed as percentage of cytotoxicity. (d) Immunofluorescence. For the indirect immunofluorescence we modified the procedure given by Vogt and Rubin (1961). It appeared to be very important to absorb at least twice the neutralizing chicken sera with normal chicken, mouse, and hamster cells to remove proteins which otherwise combined nonspecifically with target cells and were stained. Target cells were seeded out on coverslips in petri dishes and allowed to adhere to the glass overnight. They were then washed by immersion in balanced salt solution (BBS, pH 7.2) and placed for staining on a steel net in a moist chamber. A drop of usually undiluted chicken antiserum was applied and left on the cells for 5 rain. This was followed by two washings of 10 rain each in BSS, the addition of 1 drop of undiluted fluoresceinisothioeyanate-conjugated rabbit anti-chicken serum for 5 min and washing of the cells again in BSS for 15 rain with one change of the buffer. The unfixed cells were mounted in buffered glycerol and studied with a Leitz Ortholux fluorescence microscope with an automatic camera attached to it. This microscope has a vertical illuminator with interchangeable dichroie mirrors using incident light that has to pass the eoverslip only to reach the obiect (Ploem, 1967). The exciter filter combination used was 1.5 mm BG 12 and $470, an interference filter. As barrier filter we used the fluorescenceselection filters K 510 or AL 525, thus vir-

149

tually omitting M1 autofluorescence and having a practically black background. Photomicrographs were taken using an Ilford HP 4 film at usually 1000 X magnification, thus focusing all exciting light energy on very few cells at a time. Exposure times remained relatively constant between 35 and 45 sec. RESULTS

Demonstration of ATV-Specific TSSA on Trans]ormed Mouse Cells After having developed a technique that allowed the detection of surface alterations on virally transformed chicken cells (Kurth and Bauer, 1972), we intended to look for surface changes on transformed mammalian cells in order to compare cells from different species for possible common tumor-specific surface properties. For this purpose we introduced additional methods allowing the in vitro detection of cell surface alterations by use of antibodies. A standard mouse antiserum and sensitized mouse spleen cells prepared against a non-virus-producing ATV tumor were desirable. Therefore, mice were immunized with D4 cells and were tested for their humoral and cellular activity in microcytotoxicity tests. Normal sera or spleen cell suspensions were used as controls. Figure 1 shows the effects of a normal mouse serum and of a mouse D4 antiserum on D4 target cells. Normal mouse serum had no significant effect on the survival of D4 target cells when compared to the growth of target cells in medium and complement only (set as 100 % survial). In contrast, the anti-D4 serum exerted a strong cytotoxic effect on the D4 cells. In later experiments it became evident that the strength of the individual antisera varied usually between 2v and 29 antiserum dilution for a 50 % survival of target cells. Both sera exerted no effect on normal MEC. Mouse hyperimmune sera prepared for other purposes against different proteins, e.g., ovalbumin or bovine serum albumin, served as negative control and showed no effect on either MEC or D4. Likewise, rabbit anti-mouse hyperimmune serum was used as positive control in the experiments. To exclude the possibility that Forssmann antigen, which is known to be contained in

150

KURTH AND BAUER °/°surviving ceI{s

120. 110. ~o~

100

~- - ~ 7 ° -

°~°~--~.

~o

/

°

normal m o u s e serum

90. 807060-

o

50403020-

o-

~ouse

10-

log 2 antiserum dilution

Fio. 1. Survival of D4 target cells after incubation with doubling dilutions of normal or ~nti-D4 mouse serum plus complement (dilution 1 : 16). 100% = growth of target cells in medium and complement only. avian myeloblastosis virus (Eckert et al., 1955), is acting as the relevant tumor antigen, we absorbed mouse anti-D4 serum twice with an equal volume of sheep red blood cells for 1 hr at 4 ° and observed no reduction in the cytotoxic titer afterward. In the cellular cytotoxic assay using spleen cells as effector cells and M E C and D4 cells as target, a constant background of toxicity was observed when spleen cells of normal mice were used as effectors with D4 as target cells. We therefore introduced a survival index as a parameter for tumor-specific cytotoxic effects. Normal M E C are not affected b y either normal or specifically sensitized spleen cell suspensions. The specific cytotoxic effect of anti-D4-sensitized spleen cells on D4 cells is shown in a titration curve of the spleen cells (Fig. 2). The slight increase of the survival index at higher cell ratios is probably caused b y the greater degree of destruction of target cells due to the increased number of normal spleen cells (denominator in the survival index formula), whereas the destruction of target cells by sensitized spleen cells remained constantly high. The percentage of survival in a typical experiment is shown in the left part of Table 2.

Comparison of ATV-Specific TSSA on Transformed Cells of Different Mammalian Species After the in vitro demonstration of an ATV-induced TSSA on D4 cells a comparison was made between ATV-induced antigens on isogeneic (mouse) and xenogeneic (hamster) transformed mammalian cells. RVP~ (mouse) and RSH (hamster) were used as corresponding target cells. A standard mouse anti-D4 serum was used for humoral cytotoxic microtests. The results are shown in Fig. 3. Normal mouse serum had no effect on any of the target cells, indicating that natural antibodies against mouse or hamster histocompatibility antigens were not present to a demonstrable amount. A very strong cytotoxic effect was exerted by anti-D4 serum on D4 and RSH target cells, whereas RVP3 cells were killed only to a moderate extent. We also studied the existence of a common TSSA on D4 and RSH cells b y the 51chromium-release technique. The results are summarized in Table 3 and show a clear, complement-dependent cytotoxic effect of the mouse anti-D4 serum on both kinds of cells, whereas normal mouse serum had no effect.

TUMOI~ CELL SUI%FACE ANTIGENS

151

survival index |

1.1

~o/O

~

t0

o MEC target ce[ls

.90.80.70-

.60,50-

o

.~0-

"30 ,20.1t0

/o o

j

Dz, forget ceils

o~o~o~.~"

ratio target ce(ls:effector celts

Fro. 2. Survival of M E C and D4 target ceils after incubation w i t h different numbers of mouse spleen effeetor cells. 1.0 = growth of t a r g e t cells in medium only. TABLE 2

SURVIVALOF D4

Mouse

TARGETCELLSAFTERINCUBATION~VITHEFFECTORCELLSFRON~[7~V~OUSEAND CHICKENSPLEENSu Effector cells from

Ratio of effector to target cell

Mice Not immunized

Immunized Not with immunized D4 cells

Chickens immunized with

RAV-1

NC-SRV-1

SRV-1

B 77

SRV-H

25:1 85.2 4- 6.9 b i28.0 4- 5.2 71.0 ± 7.5 50.3 4- 7.~ 33.1 4- 6.4 28.4 ± 6.2 19.9 ± 7.7 16.6 ± 3.1 50:1 65.4 ± 4.3

21.4 ± 2.9 ~7.1 ± 8.8 24.6 4- 5.4 17.9 4- 4.3 16.9 ± 3.8 11.8 ± 1.9 9.6 4- 2.7

Figures r e p r e s e n t p e r c e n t a g e of surviving t a r g e t cells after i n c u b a t i o n w i t h spleen cells compared to the growth of t a r g e t cells in m e d i u m only. b S t a n d a r d deviation.

Comparison of ATV-I~duced TSSA Mammalian and Chicken Cells

on

The next question that came up was whether one might detect cross-antigenic tumor-specific surface antigens on mammalian and chicken cells transformed by various ATV strains. Cellular and humorM eytotoxic tests as well as indirect immunofluorescent staining were used for this purpose. Chicken spleen cells were first tested as effector cells on D4 target cells. The destruc-

rive effect of anti-SRV-1 spleen ceils on D4 ceils is illustrated in Fig. 4. When compared with effector cells from unsensitized chickens, effector cells derived from avian sarcoma virus (ASV) as well as avian leukosis virus (ALV)-sensitized chickens exerted a much more pronounced eytotoxic effect on D4 target cells as expressed by the survivat index (Fig. 5). The absolute percentage of surviving target cells compared to t he growth of these cells in medium only is shown in the right part of Table 2. The back-

152

K U R T H AND BAUER

surviving cells

120t

- -

I\\ 30- l\ 706o-

_

_,.

-,

i

~Ix\\..

/

/

/

/

~ \

/

50-

X~

40-

\\

3020-

.

/P" f

fl ~\

T--V

RVP3 o--o o_o~RSH ,--,~

/

10-

log2 a0tiserum dilution FIG. 3. Survival of D4, RVP,, and RSH target cells after incubation with increasing dilutions of normal and anti-D4 mouse serum plus complement. The three upper lines represent incubation of target cells with normal mouse serum, the three lower broken lines with anti-D4 mouse serum. 100% = growth of target cells in medium only.

E F F E C T OF _-ViouSE A N T I - D 4 SERUM ON

TABLE 3 D4 A~D RSH T A R G E T

C E L L S IN TttE 51CHROMIU1VI-RELEASE T E S T

Incubation with Target cells

Medium only

Medium + complement

Medium + complement + NMS ~

Medium + complement + anti D4 MSb

Distilied water

D 4

990 c ~ 83d

815 ~ 121

1011 =t= 103

1715 ~ 96

8216~ ~ 556

RSH

640 ± 68

583 ~ 81

795 =t= 75

1340 ~ 146

5827 i

472

a Normal mouse serum. b Anti-D4 mouse serum. Counts per minute released. Standard deviation. e Freeze-thawed preparations. g r o u n d r e d u c t i o n of t a r g e t cells b y n o r m a l spleen cells is c o n s i s t e n t l y lower t h a n t h e r e d u c t i o n b y A T V - s e n s i t i z e d spleen cells. T h e r e s p e c t i v e c r o s s - a n t i g e n i c i t y of T S S A on c h i c k e n a n d m o u s e cells could be t e s t e d also b y t h e effect of m o u s e a n t i - D 4 sera on c h i c k e n cells, w h ic h were m o c k - i n f e c t e d or i n f e c t e d b y different A L V or A S V strains. U n i n f e c t e d , as well as R A V - I - , M A V - B - ,

an d N C - S R V - H - i n f e c t e d C E C were n o t affected b y m o u se a n t i - D 4 serum. H o w e v e r , a significant c y t o t o x i e effect could be d e m o n s t r a t e d on S R V - l - t r a n s f o r m e d cells b y t h e a n t i - D 4 serum, as sh o w n in Fig. 6. W h i l e D 4 m o u se cells were killed in a t y p i c a l m a n n e r w i t h a 50 % t a r g e t cell s u r v i v a l at an an t i s e r u m d i l u t i o n of m o r e t h a n 27, t h e S R V - 1 t r a n s f o r m e d C E C were killed a t an an t i -

TUMOR CELL SURFACE ANTIGENS

153

Fio. 4. Photomicrograph of a cellular microcytotoxicity test. Above : D4 cells grown in medium only. Below: D4 cells after incubation with chicken spleen cells sensitized against SRV-1. Growth in Falcon plastic microtest I plates. Magnification 80X. serum dilution of 2s only. This weaker cyto~ toxic effect could be demonstrated repeatedly and seems to be significant. Normal mouse serum had no effect on the growth of any of the target cells. Since Gelderblom et al. (1972) were able to

detect ASV-induced TSSA by ASV antisera on transformed chicken cells, we tested chicken antisera prepared against ASV in indirect immunofluorescent staining to demonstrate TSSA on cells from mice and hamsters. Using a series of antisera prepared

154~

KURTK AND BAUER

survival index t 1.1 10 .80 .go~.70 I I

.3o-

• RAV-1

\\

.20-

~7 "~-:a ~

~

• ~

. ~ "

sRv-.

o

.10-

;o

do

,~

,60

llo

t;.o

,;o

)

ratio effector cells : target cells

Fro. 5. Survival of D4 mouse target cells after incubation w i t h spleen cells from normal or A T V -

sensitized chickens. Spleen cells were sensitized against: RAV-1, NC-SRV-1, SRV-1, B 77, SRV-H.

%survivir~ ceils "[ t2oJ

---'-'-~--"

110 °

-~ ~V.

~.o~mockinfected

I00-

•

~v.;~v_

s

90.

70" 60. 50' z,O

20 I0-

log 2 antiserum dilution

Fro. 6. Survival of ATV-infected chicken embryo and mouse Rous sarcoma cells after incubation with mouse anti-D4 serum. Chicken target cells were mock infected or infected by RAV-1, SRV-1, MAV-B, o r NC-SRV-H. D4 = SI~V-H-transformed mouse cells. 100% = growth of target cells in medium and complement'only. against SRV-1 and SRV-tt in experiments with normal or SRV-H-transformed mouse and hamster cells, a comparatively constant degree of staining of the transformed cells was found. The unfixed transformed cells were stained to more t h a n 95 % (Fig. 7), whereas the normal control cells were hardly stained at all. Under the technical conditions used, all autofluorescence was omitted and a virtually black background in the controls was obtained. Distribution of cells on the

coverslips was checked b y phase-contrast microscopy using transmitting light. Sera prepared against RAV-1 and N C - S R V - H did not stain D4 or R S H cells. Since antiN C - S R V - H chicken sera recognize Ve antigen on the surface of chicken cells infected b y subgroup D virus (Gelderblom et al., 1972), it can be concluded t h a t Ve antigen is not expressed on the surface of m a m m a l i a n cells transformed b y ASV of subgroup D, e.g., D4 and R S t t .

155

TUMOR CELL SURFACE ANTIGENS

cells. First of all, the existence of TSSA on transformed mouse cells had to be demonstrated in vitro. For this purpose, antisera and sensitized spleen lymphocytes prepared against D4 cells in mice were tested for antitumor activity on M E C and D4 cells in microcytotoxieity assays. Since immunization and tests were performed within a syngeneie system (inbred S T U mice), we concluded t h a t any cytotoxie effects are exerted only via tumor-specific surface alterations. This notion is supported b y the lack of any effect of normal mouse serum on M E C or D4 cells and of normal or sensitized spleen cell suspensions on M E C . I n addition, mouse hyperimmune sera prepared against a series of proteins had no effect on growth of M E C and D4. Mouse antisera prepared against D4 cells had no effect on uninfected M E C , b u t consistently killed 50 % of D4 target cells even at dilutions of up to 29. We considered this to be a rather high antiserum titer, especially in light of the fact that living t u m o r cells can be used for immunization only with caution since they are highly oncogenie for our mice and readily kill the animals. FIG. 7. Positive fluorescent staining of D4 (above) and I%SI-I(below) cells. Growth on glass coverslips. Magnification 1000X: R S H cells, in contrast to D4 cells, tended to detach from the surface and to grow in suspension; it, therefore, proved to be difficult to focus these cells for photography since they usually floated in the glycerol buffer used for mounting the coverslips. The rounded appearance of D4 cells in Fig. 7 is typically for this test (compare with Fig. 4), this probably being due to reduced attachment of the unfixed cells to the glass coverslips. The results of the fluorescent staining are summalized in Table 4. DISCUSSION For the comparison of ATV-induced TSSA on cells of different species, several techniques were adapted in addition to the previously described cell-mediated cytotoxic assay (Kurth and Bauer, 1972) which revealed ATV-induced TSSA on chicken

TABLE 4 IMMUNOFLUROESCENT STAINING OF ATV-Tm~NSFORMED MOUSE AND HAMSTER CELLS BY CHICKEN ANTISERA PREPARED AGAINST A T V a Sera

Cells stainedb

Normal chicken serum

Antisera prepared against RAV-1

.

.

SRV-H

NCSRV-H

+

÷

--

SRV-1

M E C

.

D4

-

H a E C

--

N T

-

--

-

RSH

-

NT

q-

-t-

--

--

.

.

Positive staining means that more than 95% of the cells showed a clear surface fluorescence. Cells considered negative were stained very weakly to less than 5%. b MEC = mouse embryo cells; D4 = SRV-Ittransformed mouse cells; I~aEC = hamster embryo cells; RSH = SRV-transformed hamster cells.

156

KURTH AND BAUER

Using mouse spleen lymphocytes as effector cells on D4 target cells a certain degree of cytotoxicity was exerted by normal spleen ceils on D4 after the 2 days of incubation. A survival index was therefore introduced to demonstrate the much greater cytotoxieity of D4-sensitized spleen cells compared to normal spleen cells on D4 cells. It can be assumed that the cytotoxic effect of normal spleen cells is due to in vitro sensitization by tumor antigens since neither normal nor anti-D4 spleen cells have any effect on the survival of uninfected MEC (Fig. 2). The anti D4 mouse serum was used in the search for TSSA on isogeneic (RVP3) and xenogeneic (RSH) tumor cells. TSSA was detected on RVP3 cells only at very low antiserum dilutions. This might either reflect a low amount of the antigen expressed on the cell surface or indicate a partial eross-antigenicity of the respective tumor antigens. It cannot be decided whether the phenomenon is due to the different genotype (C57/BL) of the RVP3 cell compared to the STU cell or due to the fact that this line has recently been found to produce murine leukemia virus (Gelderblom et al., 1970). In the latter ease one could imagine that the presence of another TSSA induced by MLV could lead to antigenic conversion or antigenie modulation with a decrease of ATVinduced TSSA. Cells of the hamster cell line RSH could also be shown to possess an ATV-induced surface antigen which cross-reacts with the RSV-induced TSSA on mouse cells. This cross-antigenieity was demonstrated by all three kinds of eytotoxie tests, i.e., humoral and cellular mierocytotoxicity tests and the 51chromium release test. Uninfected hamster embryonic cells were only sometimes affected by mouse anti-D4 sera (Table 2) and in these eases to a moderate extent only as compared with the RSH cells. This slight destruction was exclusively demonstrable in the 5'chromium release test and we believe this to be due to the oecassional occurrence of natural mouse anti-hamster antibodies. A eross-antigenieity of TSSA on mammalian and chicken cells could also be

demonstrated by cellular and humeral cytotoxic tests as well as by indirect immunefluorescence. In the cell-mediated tests, again a certain background reduction of target cells by normal spleen cells was noted (Table 2), but the cytotoxic effects of specifically ATV-sensitized spleen cells were always at least twice as high as those exerted by normal spleen cells. It had been shown before (Kurth and Bauer, 1972) that NC-SRV-1 and NC-SRV-H, in vitro nontransforming mutants of SRV-1 and SRV-H (Graf et al., 1971), induced a surprisingly strong antitumor response in chickens. In concordance, NC-SRV-1- and NC-SRV-Hsensitized spleen cells as well as spleen ceils sensitized against other ALV-strains exerted a specific cytotoxic effect on D4 mouse cells. The capability of the two mutant strains to induce a strong antitumor response in chickens even after having lost the ability to transform CEC in vitro is very probably due to the existence of at least two different target cells that can be transformed by SRV-1 and SRV-H. This assumption has gained support by the recent observation that in vivo both NC-SRV-1 and NC-SRV-H have the capability to induce erythroid and lymphoid leukosis. In addition, NC-SRV-H causes osteopetrosis and osteochondrosarcoma after a latency period of several months (Biggs et al., submitted for publication). These findings indicate that part of the viral genome can be inactivated in a way that the transformation of a certain cell type is selectively inhibited, whereas transformation of another histologically different cell type is still possible. Antisera prepared in chickens against ALV or nontransforming mutants did not recognize ATV-induced TSSA on the surface of D4 and RSH cells in immunofluorescence tests, which is quite in agreement with earlier results obtained in our laboratory (Gelderblom et al., 1972). But this might well be a quantitative rather than a qualitative difference, especially since our sera are not hyperimmune sera and since TSSA is a surface antigen preferentially triggering a cell-mediated immune response. In humeral cytotoxic tests using mouse anti-D4 serum and chicken target cells, only trasnformed chicken cells were killed. The

TUMOR CELL SURFACE ANTIGENS comparatively good survival of transformed chicken cells at antiserum dilutions higher than 23 might reflect a partial cross-reaction only or is the expression of a relatively low amount of TSSA on the surface of chicken cells. On the other hand, it could also be due to the large amount of mucopolysaccharides synthesized by ATV-transformed chicken cells which may in part block the respective antigenic receptors. Similarly, transformed chicken cells are stained to a lower degree than D4 and RSH cells in indirect immunofluorescent tests. The finding that TSSA is only demonstrable on transformed cells is well in agreement with our earlier findings (Gelderblom et al., 1972; Kurth and Bauer, 1972). It had been shown there that neither uninfected nor ALV-infected chicken ceils could be stained by the immunoferritin technique by antisera specific for ATV-induced TSSA, indicating the absence of these tumor antigens from these cells. In concordance, the cells could not be killed by ATV- or ALV-sensitized spleen cells. The indirect immunofluorescent staining of ATV-transformed mouse and hamster cells by chicken antisera prepared against ATV and the lack of staining of the corresponding uninfected or ALV-infected cells seems also to be strong evidence that the TSSA demonstrated is indeed tumor specific, i.e., absent from nontransformed cell surfaces. Therefore, the assumption can be made that the lack of cytotoxicity of the anti-TSSA sera and lymphoid cells for nontransformed chicken, mouse, and hamster cells is not due to, e.g., a lowered amount of TSSA or a different arrangement of the respective antigenic determinants on normal cells, but instead to the absence of this TSSA from the normal cell surface. Probably no correlation exists between gs antigen content and virogenicity of the cells and the quantitative expression of TSSA, since the mammalian tumor cell lines (D4, RVPs, RSH) express different amounts of TSSA on their cell surface while all three lines are gs antigen negative and only RSH is virogenic. This is in good agreement with earlier in vivo experiments reported from our group (Bubenik and Bauer, 1967).

157

These studies demonstrated by in vitro techniques a common TSSA on the surface of ATV-transformed chicken, mouse, and hamster cells. The results streng%hen the general opinion that tumor viruses induce a cross-antigenic surface antigen on transformed cells even in different species. In contrast, chemically induced tumors express individual tumor antigens, and cross-reactions between chemically induced tumors have been reported only occasionally (Prehn and Main, 1957; Basombrio, 1970). From these studies we cannot decide at present whether the TSSA is coded for by the viral genome. Besides that, it is very well conceivable that what we tentatively describe as TSSA is not an antigenic entity. We should be aware that the reexpression of embryonic antigens and the rearrangement of receptor sites for plant agglutinins (Nicholson, 1971) as demonstrated for other tumor systems may account for at least part of the cross-antigenicity of the TSSA described above. Working in the defined ATV-system, one has now the opportunity to study the questions about the synthesis of TSSA, its antigenic determinants, its relation to embryonic antigens and plant agglutinin receptor sites and its biological function. It becomes also extremely important to compare cell surface alterations induced by different oncogenic viruses in the same species. For example, comparing tumor antigens induced by murine tumor viruses, avian tumor viruses, and also by oncogenic DNA viruses in mouse, rat, or hamster will be of interest. In addition, solubilization and biochemical characterization of the tumor antigens should now be attempted, in order to establish a correlation between the biochemical (Hakomori et al., 1971) and immunological surface alterations demonstrated on ATV-transformed cells. ACKNOWLEDGMENT One of the authors (R.K.) was recipient of a Volkswagen-Foundation fellowship. Part of the work was done at the Max Planck-Institut fiir Virusforschung, Ttibingen. We thank Mrs. Ch. Hormozdiaryfor helpful technical assistance.

158

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