Feline oncornavirus-associated cell membrane antigen: A viral and not a cellularly coded transformation-specific antigen of cat lymphomas

VIROLOGY

124, 445-461

(1933)

Feline Oncornavirus-Associated Cell Membrane Antigen: A Viral and Not a Cellularly Coded Transformation-Specific Antigen of Cat Lymphomas SHARANJIT S. VEDBRAT, **l SURAIYA RASHEED,? MATTHEW A. GONDA,§ SANDRA RUSCETTI,” MURRAY AND WOLF PRENSKY*

HANS LUTZ,* B. GARDNER,?’

*Molecular Cytogenetics Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021; tDepartmeni of Pathology, University of Southern California, School of Medicine, Los Angeles, Cal&rnia 90038; $Departm,ent of Medicine, School of Veterinary Medicine, University of Zurich, 8057 Zurich, Swit.zerlan& OElectrolz Microscopy Laboratory, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701; and “Laboratory of Tumor Virus Genetics, National Cancer Institute, Bethesda, Maryland 20205 Received June 9, 1982; accepted September 10, 1982 The feline oncornavirus-associated cell membrane antigen (FOCMA) was defined as a tumor antigen common to cat lymphomas and fibrosarcomas induced by feline leukemia virus (FeLV) and feline sarcoma virus (FeSV), respectively. The antigen was recognized by sera from cats thought to be resistant to leukemogenesis. We report here that a common denominator in the activity of naturally occurring viremic cat antisera to FOCMA is, in fact, their reactivity to FeLV C antigenic determinants. The cat antisera, monoclonal antibodies to FOCMA, and monoclonal antibodies to FeLV C, all reacted in immunofluorescence assays with FeLV C-infected cells and immunoprecipitated a molecule electrophoretically indistinguishable from envelope glycoprotein of FeLV. Viremic cat antisera to FOCMA bound to budding virus particles of FeLV C-infected cells, even though some of them could not be absorbed by mature virion proteins. Thus, the unusual feature of cat antibodies to FOCMA is their binding to nascent but not to mature virus particles. FOCMA-positive cat lymphomas expressed antigenic determinants of FeLV-C gp70, with or without productive infection. FeLV-negative tumors not expressing FeLV C gp70 were also FOCMA negative. Furthermore, most of the viremic cat sera and the monoclonal antibodies to FOCMA did not react with FeSV-transformed nonproducer cells. The absence of FOCMA from these cells and from FeLV-negative lymphoid tumors and its nresence in FeLV-C infected fibroblasts indicated that this antigen is virus encoded and not a cellular tumor-specific antigen.

cats suggested that it is the product of the FeSV-cmc gene, and might be the common transforming factor in both types of tumors (Stephenson et al., 1977b; Snyder et al., 1978,198O; Fleissner and Snyder, 1982). However, this thesis is inconsistent with the data of other investigators (Frankel et al., 1979; Barbacid et al., 1980; Gardner et al., 1980; Sherr et al., 1980). FOCMA was also reported to be distinct from FeLV structural proteins on the basis of (a) absence of FOCMA expression in nontransformed, FeLV-infected fibroblasts or lymphocytes (Hardy et al., 1977,1978), (b) failure of FeLV proteins to absorb FOCMA activity from naturally occurring viremic

INTRODUCTION

The feline oncornavirus-associated cell membrane antigen called FOCMA was thought to be a transformation-specific antigen present in cells transformed by feline leukemia virus (FeLV) (Essex et al., 1971; Hardy et al., 1977) and by feline sarcoma virus (FeSV) (Sliski et al., 19’77,Sliski and Essex, 1979; Hardy 1980). The presence of FOCMA in FeLV-induced leukemias and FeSV-induced fibrosarcomas of 1 To whom reprint 2 Current address: versity of California 95616.

requests should be sent. Department of Pathology, School of Medicine, Davis,

UniCalif.

445

0042-6822/83/020445-17$63.00/O Copyright All rights

0 1933 by Academic Press, Inc. of reproduction in any form reserved.

446

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cat sera (Hardy et al., 1977, 1978; Stephenson et al., 1977a; Synder et al., 1980), (c) lack of correlation between virus neutralizing activity and anti-FOCMA activity in cat sera (Essex et al., 1975; Schaller et al., 1975; Hardy et al., 1976, 1978), and (d) FOCMA expression on FeLV-negative cat leukemia cells (Hardy et al., 1977,1978, 1980). In these studies, the data were obtained with the most prevalent subgroups of FeLV, A or AB, and the possible role of FeLV C, found only in l-2% of all infected eats (Jarrett et al., 1978), was not considered. However, eat lymphoma FL74 cells, used as the standard FOCMA-positive target cells for selection of cat sera with antiFOCMA activity, release FeLV C (Sarma and Log, 1973). These cells also express FeLV C as the most predominant FeLV envelope antigen on their cell surface (Russell, 1977). Strong support for the expression of FOCMA in FeLV-negative cat lymphomas was based on the properties of a cell line 3191. These FeLV-negative 3191 cells yielded a 70,000-dalton FOCMA protein (Snyder et al,, 1978) which had a tryptic digest map identical to the analogous protein isolated from FL74 cells (Snyder et al., 1980). This p70 was also used as a reference FOCMA protein in radioimmunoassays to determine FOCMA expression in unknown cell types (Snyder et al., 1979a, b). FeLV infection in the genesis of FeLV-negative but FOCMA-positive cat lymphomas (Hardy et al., 1980) was implied from the epidemiological evidence and was considered to be an attractive model for the possible viral etiology of otherwise virus-negative human leukemias (Essex et d., 1978). In our efforts to develop monoclonal antibodies to FOCMA, we screened for hybridomas producing antibodies reacting with cell surface antigens of both FL74 cells and FeSV transformed nonproducer mink lung epithelial 64F3C17 cells (VedBrat et al., 1980), since FOCMA expression was reported in both the cell types (Sliski et al., 1977; Sliski and Essex, 1979; Snyder et at., 1978). However, on cloning of such hybridoma cultures, two classes of antibodies were segregated. One class, frequently found, bound to trans-

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formation-related antigens expressed in normal fibroblasts and lymphocytes but not in normal epithelial cells. These antigens, however, were ectopically or inappropriately expressed in transformed epithelial cells (VedBrat et al., 1979; Prensky and VedBrat, 1983). None of these antigens were specific to FeSV-transformed cells and thus had no relation to FOCMA. The second class, represented here by antibodies 2-124 and Z-88, reaeted not only with lymphoma cells infected with FeLV C but also with FeLV C-infected normal fibroblasts and with FOCMA positive cat lymphoma cells, like F422, not known to produce infectious FeLV C (Sarma and Log 1973). FeLV shed by F422 cells was infectious but did not lead to FeLV C expression in fibroblasts infected with it. F422, therefore, expressed noninfectious FeLV C antigenic determinants (epitopes) cross-reactive with cat antisera to FOCMA, while FL74 eells expressed determinants of a replication-competent form of FeLV C on their cell surface (VedBrat et al., 1980). Moreover, the FeLV-negative 3191 cat lymphoma line was found to be negative for FeLV C antigen as well as for FOCMA expression (VedBrat et al., 1980). Worley and Essex (1980) also failed to find FOCMA in 3191 cells, thus also contradicting the reports of Snyder et al. (1978,1979a, b, 1980) on the presence of FOCMA in virus-negative cells. Since observations in several laboratories could not confirm most of the properties ascribed to what was defined as FOCMA, they led to a partial reconstruction of some of the earlier studies on the nature of FOCMA expression. We report here that the common denominator of FOCMA antisera obtained from viremie cats, including the antisera used in previous studies, is their reaction with FeLV C envelope gp70 epitopes. These antisera do not react with cell surface antigens of FeSV-transformed mink cells. MATERIALS

AND

METHODS

Cells, viruses, and tumors. The origin of most cell lines used in this study was pre-

FOCMA:

AN

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C-RELATED

viously described (VedBrat et al., 1980; Rasheed and Gardner, 1980). The feline lymphoid cell lines include: FL74 cells (Theilen et al., 1969), producing FeLV as a mixture of subgroups A, B, and C (Sarma and Log, 19’73), and has been designated as FeLV ABC/KT, F422 cells producing Rickard strain of FeLV A (Sarma and Log 1973) but expressing FeLV C-related antigenic determinants on their cell surface (VedBrat et al., 1980); and the FeLV-negative 3191 cells (Snyder et al., 1978, 1980; VedBrat et al., 1980; Worley and Essex, 1980). Nonlymphoid cells were FeLV-infected and uninfected feline embryo fibroblasts, designated FLFS, FEA and CF927; human rhabdomyosarcoma RD cells, mink lung epithelial cell line (CCL64) and its FeSV-GA transformed nonproducer derivative 64F3C17. The putative cat lymphoma line, 3403 (VedBrat et al., 1980; Grant et al., 1980), infected by a mixture of at least two subgroups of FeLV, A and C, was later identified as human lymphoma line by karyotype (Dr. S. Jhanwar, our institute) and DNA restriction fragment analyses (Dr. J. Mullins). The human B cell, GM1500/6TG (Croce et al., 1980), free of FeLV, was used as a control. Lymphocytes were grown in RPM1 1640 (Gibco), and all other cells were maintained in a medium containing a 50% mixture of Ham F12 (Gibco) and DME high glucose (Gibco). All media were supplemented with 10% fetal calf serum, 1% glutamine, 2.5 pg/ml Gentamycin, and 2.5 pg/ml Fungizone. Two viral preparations, FeLV ABC/KT, lot No. 19-76, produced from FL74 cells, and RD114 virus, lot No. 13-94, were obtained from Pfizer Inc. FeLV A (Rickard), lot No. 579-90-6, produced from F422 cells, was obtained from Virgo Reagents. FeLV A, lot No. 31-62-4P, FeLV B, lot No. 31-124P, and FeLV C, lot No. 31-66-4P, were prepared from A7573 dog fibroblasts each infected with the respective cloned virus preparation by Hem Research, Inc. FeLV C (Sarma) is a cloned derivative of FeLV ABC/KT (Sarma and Log, 1973). Three feline FeLV-negative primary lymphomas from New York area (3508, 3576, and 3618) were generously provided by Dr. W. D. Hardy, Jr. The other four

ANTIGEN

447

FeLV-negative lymphomas (CT1220, CT636, CT649, and CT1311) were obtained from Southern California. The New York tumors were identified as virus negative by IF assays alone, while California tumors were characterized by IF assays and immunoelectron microscopy. Controls consisted of naturally occurring FeLVpositive lymphomas, FeLV-positive and negative fibrosarcomas, and carcinomas of cats. Fibrosarcoma XC4A was induced experimentally by infecting a young kitten with the Gardner-Arnstein (GA) strain of FeSV. Antibodies. Two previously used FOCMA antisera, obtained from naturally viremic cats 3192A and 24640 (Snyder et al., 1978, 1979a, 1980; Hardy et al., 1978; VedBrat et al., 1980) and rabbit anti-FeLV serum, were provided by Dr. W. D. Hardy, Jr. It may be pointed out that same lot or bleeding of 3192A serum was used for both immunoprecipitation and the virus absorption assays. Criteria for the selection of eight naturally occurring viremic cat sera from California, CT1145, CT1201, CT1203, CT1112, CT1113, CT1328, CT921, CT369, were as described by Snyder et al. (1980), and some were used in earlier studies (Gardner et al., 1980). Goat antisera to FeLV total protein, to p30 and to gp70 envelope proteins, and fluorescein-labeled second antibodies for cellular IF assays were obtained from the Office of Program Resources, National Cancer Institute. AntiIgG antibodies used in immunoprecipitation studies were from Cappel Laboratories (Cochranville, Pa.) and from Antibodies, Inc. (Davis, Calif.). Derivation of the murine monoclonal antibodies was described before (VedBrat et al., 1980). Antibodies 2-124 (IgGz,) and 2-88 (IgGi) from cloned (twice) hybridoma cells were made against FL74 cell surface antigens and were selected for their ability to react with FOCMA-positive cat lymphoma cells but not with FOCMA-negative normal cat lymphocytes, fibroblasts, or cells infected by FeLV A and B. Monoclonal antibodies B12 and C3 were raised against FeLV ABC/KT. The latter antibodies neutralized FeLV subgroup C (FeLV C), and to some extent also FeLV B, but

448

VEDBRAT

not FeLV subgroup A (0. Jarrett and H. Lutz, unpublished observations). Immunc$?uorescence (IF) assays. Viable and acetone-fixed cat lymphoma cells and FeSV-transformed cells were analyzed for FeLV and FOCMA expression by IF assays as described elsewhere (VedBrat et al., 1979, 1980). Normal lymphocytes and fibroblasts, both FeLV infected and uninfected, were assayed as controls. IF assays were also performed with antibodies absorbed with different antigen preparations. Absorption assays. Three antibody absorption assays were done, with viable cells (Table 2), disrupted FeLV (Table 3), and with extracts prepared from homogenized tumor or normal tissues (Table 4). FOCMA anti-sera were absorbed by mixing 100 ~1 of serum with an equal volume (about 9 X 107) of viable packed cells. The mixture was incubated, with gentle shaking, at 37” for 1 hr and then overnight at 4’. The absorbed serum was then titered for FOCMA activity on FL74 cells by IF assay. Tissue extracts for the absorption of sera were prepared in phosphate-buffered saline (PBS, pH 7.4) as 10% homogenate suspensions. Tissues were homogenized and sonicated at 4” and centrifuged at 2000 g for 10 min, and the solute was used for serum absorption. The FOCMA antisera were serially diluted with tissue extracts and incubated for 1 hr at 37” and then overnight at 4’, and controls were diluted with equal volumes of PBS. The absorbed sera were tested by IF assays on FL74 cells. Endpoint titer of the absorbed sera was defined as the reciprocal of highest dilution at which about 50% of the test cells showed bright and dense sectorial or ring fluorescence. An absorption was considered to be significant when there was a twofold or greater reduction in the endpoint titer of the absorbed sera. For absorption with purified virions, 0.1 ml of serum, at a dilution fourfold lower than its endpoint IF titer, was mixed with an equal volume of freeze-thawed virus preparation. Protein concentration of different viral preparations varied from 200 to 500 lg/ml. The mixture was incubated for 1 hr at 37”, 2 hr at room temperature,

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and then centrifuged at 2000 g for 5 min. Activity of the absorbed serum was evaluated by IF assays on FL74 cells. Immunoelectron microscope studies. The indirect immunoferritin technique used in all electron microscopic assays to demonstrate the antibody binding sites was the same as described earlier (Gonda et al., 1979). Polyvalent hyperimmune goat antiFeLV gp70, viremic cat antisera to FOCMA, and control cat sera from pathogen free cats were reacted with FL74, FEA, FEA-C, CCL64, and 64F3C17 cells. The primary sera were used at a 1:4 to 1:lO dilution of their original concentration. Specifically bound goat or cat antibodies were detected using ferritin-conjugated, affinity-purified rabbit anti-goat or rabbit anticat IgG at 0.35 mg/ml. Primary and secondary antibody incubations were each carried out for 10 min at 4’. After labeling, the cells were fixed in 1.25% glutaraldehyde and 1% OsOl followed by dehydration in a graded series of alcohols. After three final changes in absolute alcohol, the cells were embedded in Epon 812 and polymerized at 60’ for 48 hr. Ultrathin sections were cut and double stained with uranyl acetate and lead citrate. Radioimmunoprecipitations. FL74,3191, and a series of mink CCL64 cells transformed by each of three strains of FeSV (Gardner-Arnstein, Sarma-McDonough, and Snyder-Theilen) were pulse-labeled for 30 min with r5S]methionine, and all extracts were immunoprecipitated with different antibodies as previously described (Ruscetti et al., 1979). ‘251-radioiodinated FeLV ABWKT was immunoprecipitated in the presence of 90 ~1 of 0.2 M NaCl, 0.1 M Tris-HCl, pH 7.4, 0.5% Triton X-100, and 0.1% bovine serum albumin. Antigen-antibody complexes were precipitated with a 10% suspension of Staphylococcus aurew A (Kessler, 1975), with or without prior coating of the complex with rabbit anti-mouse IgG, rabbit anti-goat IgG, or goat anti-cat IgG antibodies. Immunoprecipitates were electrophoresed on 7% polyacrylamide-sodium dodecyl sulfate (SDS) gels, the gels were dried and exposed to X-ray film (Kodak X-O-Mat) for suitable lengths of time.

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RESULTS

Speci$city of Murine Monoclmal and Feline FOCMA Antibodies in Cellular IF Assays The activity of cat sera to FOCMA and of monoclonal antibodies 2-124 and 2-88, raised against FOCMA-like cell surface antigens of FL74 cells (VedBrat et al., 1980), was compared to that of FeLV B and FeLV C neutralizing monoclonal antibodies B12 and C3, raised against gp70 of FeLV ABC/ KT. Our observations on three standard cat lymphoma cell lines and on a series of

TABLE

other cat, human, and mink cells are summarized in Table 1. As expected from our preliminary report (VedBrat et al., 1980), cells infected with FeLV C (FL74 cat and 3403 human lymphoma cells and CF927C and FEA-C cat fibroblasts) were also positive for FOCMA, either in assays with cat antisera to FOCMA, or with monoclonal antibodies 2-124 and 2-88. Uninfected feline 3191 and human GM150016TG lymphoma cells and fibroblasts which were either uninfected or which were infected with FeLV A or FeLV AB were FOCMAnegative with all the FOCMA antibodies

1

VIABLECELLIMMLJNOFLUORESCENCEASSAYSFOR FOCMA

Cat antisera

Cells

FeLV subgroups

449

ANTIGEN

AND FeLV

31924

DETERMINANTS Monoclonal antibodies

to FOCMA”

24640

C

CT1145

2-124 2-88

c3 B12

Cat lymphocytes FL74 F422 3191 NCL” NCL

ABC A t-1 C-1 A. AB

80 80
80 80
92 NT NT <4 <4

2000 2000 <50 t50 40

2000 <50 <50 <50 t50

Human lymphocytes GM1500 3403

t-1 A, C



NT NT

<50 4000

t50 4000

t-1

t10

<4 NT <4 <4 20 20

40 40 40 <50 NT 1000

t50 4% 40 40 NT 1000


t10
<4 <4 <4

<50 t50 NT

<50 t50 NT

Cat fibroblasts FLF3 FLF3AB CF927, FEA CF927A, FEA-A CF927B CF92’7C, FEA-C Other cells CGL64 (mink) 64F3CL7 (mink) XC4A (cat)

AB t-1 A B C

c---j (-) FeSV B, FeSV

Note. Numbers represent reciprocal of the highest dilution factor (titer) of sera at which more than 90% of the cells fluoresce. New York sera not tested at dilutions less than 1:lO. High background fluorescence did not permit the analysis of monoclonal antibodies from ascites fluids at dilutions less than 1:50. NT, Not tested. a Five other FOCMA typing sera from naturally occurring viremic cats from California (CT1112, CT1328, CT1201, CT1113, and CT1203) gave similar results. *NCL, Normal cat lymphocytes, obtained from spleen, bone marrow, thymus, peripheral blood and lymphnodes of one nonviremic and two viremic cats.

VEDBRAT

450 LlSW!d.

In these assays, all the monoclonal ant ibodies mimicked the activity of cat antisc ?ra to FOCMA. However, the two groups

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of monoclonal antibodies reactive with FeLV C-infected fibroblasts differed with respect to their reaction with the FOCMA-

FIG. 1. Cell surface immunofluorescence of FeLV-negative 3191 cat lymphoma and of FeLVpositive 3403 human lymphoma cells. The two cells were mixed in 41 (3191:3403) ratio and treated with appropriate dilutions of each antibody. (A, C, and E) Phase contrast; (B, D, and F) xenonarc illumination for fluorescence. Human 3403 have larger diameters than 3191 cells, and arrows point to some of the antigen-positive cells within each panel. (A and B): Cells treated with FOCMA typing serum 24640; (C and D) with monoclonal antibody 2-124; (E and F) with rabbit antiserum to FeLV group specific antigens. Similar results were obtained on mixtures of 3191 with FeLVpositive FL74 and F422 cells and on human FeLV-negative GM1500/6TG cells mixed with 3403 cells. All observations were confirmed on unmixed-cell preparations.

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positive F422 cells. Antibodies 2-124 and 2-88 bound to F422 cells while B12 and C3 did not. Consequently, the FOCMA determinants recognized by cat antisera to FOCMA were more closely related to those recognized by monoclonal antibodies 2-124 and 2-88 than to those reactive with B12 and C3. The surprising observation was that cat antisera to FOCMA as well as the monoclonal antibodies failed to bind specifically to 64F3C17 FeSV-transformed mink lung epithelium cells. The negative reaction of FOCMA antibodies with 3191 as well as with 64F3C17 cells contradicted earlier work based on the same cat antisera (Snyder et al., 19’78, 1979a, 1980). Moreover, except in our preliminary report (VedBrat et al., 1980), the cross-reaction of FOCMA antibodies with antigens of FeLV C-infected cells had not been documented before. The controversial nature of our IF data led to the further investigations reported below.

Correlation of FOCMA FeL V C Infection

Expression

with

The finding that FOCMA expression is not specific to feline leukemias and is correlated with FeLV C infection of cells is illustrated in Fig. 1, where FeLV-negative feline lymphoma 3191 cells are FOCMA negative while FeLV C-infected human lymphoma cells are FOCMA positive. The IF data on the correlation of FeLV C infection with FOCMA expression were also substantiated by antibody absorption assays. Cat embryo CF-927 fibroblasts and human RD cells, whether free of or infected with FeLV A or FeLV AB, failed to absorb the antibody activity of viremic cat antisera to FOCMA. However, infection of these cells with FeLV C(Sarma), or with FeLV ABC/KT, led to absorption of antibody activity (Table 2). The same correlation was observed when FeLV-negative 3191 cells were infected with different isolates of FeLV and tested by IF assay (VedBrat et al., 1980), or when mink lung epithelial CCL64 cells were infected with FeLV C and assayed by radioimmunoprecipitation (data not shown). Thus, FOCMA expression was not specific to feline lymphoid tumor cells and could be observed

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TABLE

2

ABSORPTIONOFFOCMA POSITIVECATSERA WITHVIABLECELLPREPARATIONS

Cells and their FeLV status

Titer of absorbed cat antisera to FOCMA on FL74 cells CT369

CT1145

CT921

Cat cells No absorption FL74/(ABC/KT) CF927 CF927/(A) CF927/(B) CF927/(C) CF927/(ABC/KT) CF927/MNNG” CF927/SPT” XC4A/(B, FeSV-GA)

32 3 24 20 20 8 3 NT NT NT

92 <3 92 46 20 <2 NT 92 92 92

>64

Cells of other species RD RD/(AB) RD/(ABC/KT) 64F3CL7/(FeSV-ST)

20 20 3 32

92 NT NT 92

6 164 NT NT NT 8 NT NT NT

NT NT NT 64

Note. Data represents the reciprocal of serum dilution (titer) still active by fluorescence assay on 50% or more of FL74 target cells. NT, Not tested. a CF927/MNNG and CF9271SPT are carcinogen and spontaneously transformed CF927 cells, respectively (Rasbeed and Gardner, 1980).

in FeLV C-infected tissue cultured normal or transformed cells of any type (lymphoid, fibroblastic, or epithelial) and of any species (feline, mink, or human).

Absorption of FOCMA Activity Structural Proteins

by FeLV-C

Appropriate dilutions of two cat antisera, 24640 and 31928, and of two monoclonal antibodies 2-124 and 2-88, were mixed with suspensions of purified and disrupted FeLV virions. Twelve micrograms of viral protein of ABWKT, 12 pg of FeLV A (Rickard), and 15 pg of FeLV C abolished the activity of 0.05 ml of all the antibody preparations tested, except that of serum 3192A. By contrast, an equal volume of 12 pg RD114 virus suspension, used as a control, did not absorb any of the antibodies tested (Table 3). Absorption

VEDBRAT

452 TABLE ABSORPTION WITH

3

OF A~BODIES TO FOCMA VIRUS PREPARATIONS FOCMA

typing Virus ABWKT FeLV FeLV FeLV FeLV RD114

(cells)

(FL74, eat) A (F422, cat) A (A7573, canine) B (A7573, canine) C (A7573, canine) (RD. human)

24640 +a + + +

-

MOIlOClOn~l

serum 3192A -

-

antibodies 2-124 and 2-88 + + f +

-

a +, Serum was fully absorbed with respect to its activity on FL74 cells; -, not absorbed; +, partially absorbed.

of the antibodies was considered to be complete when 10% or fewer cells were weakly fluorescent. When serum 3192A was tested after absorption with different viral preparations, more than 90% of the cells gave positive fluorescence with no reduction in its intensity. Similar observations were also made on the activity of the viremit cat sera from California, namely that some sera could be absorbed by FeLV ABC/ KT and others could not (data not shown). FeLV B proteins (19 pg) absorbed the activity of appropriate dilution of 0.05 ml of cat antiserum 24640 but not that of 3192A (Table 3). Monoclonal antibody 2-124 was fully absorbed by FeLV C but only partially with FeLV B, since 50% of the cells still showed positive fluorescence with relatively reduced intensity after absorption. Absorption of the antisera with 13 pg of FeLV A resulted in an insignificant reduction in the intensity of target cell fluorescence (Table 3). Thus, activity of some of the cat antisera to FOCMA was absorbed by those isolates of FeLV which contain FeLV C epitopes while that of sera like 3192A was not. Absorption by Rickard virus (FeLV A) indicated that F422 cells produced FeLV A virions phenotypically mixed with proteins containing the epitopes recognized by antibodies 2-124 and 2-88. Binding of FOCMA Antibodies to Budding Virm of FL74 C&s FL74 cell binding of 15 cat antisera to FOCMA from several sources was visu-

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alized by immunoelectron microscopy using ferritin-labeled second antibodies. Whereas sera from pathogen-free eats gave no binding (Fig. 2A), a series of antiFOCMA cat sera including serum 3192A and goat antiserum to gp’70 of FeLV ABC/ KT resulted in ferritin deposits, mainly locaiized to budding virus particles (Figs. 2B, C, and D) though some were also on areas of the cell membrane free of budding virus. There was a quantitative but not a qualitative difference between the reactions obtained with cat antisera to FOCMA and with goat antiserum to FeLV gp70. The titer of one of the CT sera was low and gave only marginal ferritin label on FL74 cells. In agreement with observations made by cellular IF and by antibody absorption assays, ferritin binding due to cat antisera to FOCMA was also seen on budding virus of feline fibroblas~ FEA cells infected with FeLV C (Sarma) but not on the cell membrane of FEA fibroblasts free of virus or infected with FeLV A (Rickard). Also, 14 out of 15 antisera to FOCMA failed to bind to FeSV-GA transformed 64F3C17 mink cells (data not shown). Thus, even sera like 3192A which were not absorbed by mature viral protein preparations bound to budding virus of cells infected with FeLV C and did not react with cell surface antigens of 64F3C17 cells in this assay system. Pr~~~at~ FOCMA

of FeLV Envelope Protein Antibodies

bg

These studies were undertaken to determine which protein of FeLV C bear the antigenie determinants or epitopes. reacting with FOCMA antibodies. All the four monoclonal antibodies immunoprecipitated an ‘?-labeled molecule similar to envelope glycoprotein gp’70 from the disrupted virus preparation of FeLV ABC/ KT (Fig. 3). No other viral proteins were immunoprecipitated by these sera. Ascites fluid from mice injected with the parent mouse myeloma SP2/0 cells, used as a control, failed to precipitate this molecule. None of the four monoclonal antibodies bound to FeLV A in ELISA assay or by immunoprecipitation experiments (data not shown). When FL74 cells were pulse-labeled with

FOCMA:

FIG 2. antibody FOCMA, (Snyder

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~-R~ATED

ANTIGEN

453

Binding of ferritin-labeled second antibodies to FL74 cell membranes treated with different preparations. (A) Control serum from pathogen-free cats; (3) CT1201 eat antiserum to from California; (C) goat antiserum to FeLV gp7& (D) 3192A cat antiserum to FOCMA et al., 1978,1979, 1980) from New York.

[~S~methionine, monoelonal antibodies C3 and 2-124 and FOCMA typing sera 24640 and 3192A immunoprecipitated a protein

similar to that precipitated by goat antiserum to FeLV gp’70 (Fig. 4). Cell lysates of cat lymphoma 3191, or its subline 32OlB,

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VEDBRAT 1

2

3

4

5

6

FIG. 3. Reactivity of various antisera with FeLV virion proteins. ‘%I-labeled disrupted FeLV KT/ABC virus was immunoprecipitated and electrophoresed on polyacrylamide gels. Lane 1: control, ascites fluid from mice inoculated with SP-2 cells; lane 2: murine antiserum to whole FeLV; lanes 3-6: monoclonal antibodies C3, B12, 2-88, and 2-124, respectively.

did not yield a readily detectable immunoprecipitate, even when tested with serum 3192A used by Snyder et al. (1978, 1979a, 1980) to isolate “~70 FOCMA protein” from these cells. A similar protein was readily precipitated with antibody 2-124 from FeLV C (Sarma)-infected mink CCL64 cells but not from normal mink cells or from

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mink cells transformed by three independent isolates of FeSV (data not shown). However, rat sera recognizing each of the three “gag-one” polyproteins (Ruscetti et al., 1980b) did precipitate out the respective polyproteins in the same experiments (data not shown). The data suggested that the reaction of FOCMA antibodies with FeLV C infected cells or with FeLV virions was due to their binding to the gp70 molecule of FeLV C and that the lack of FOCMA expression in 3191 cells was due to the absence of this or a similar molecule in lysates of 3191 cells.

Lack of FOCMA Expression in Virus-Negative Lymphomas and in FeSV-Transformed Mink Cells Our observation that cat lymphoma 3191 cells were FOCMA-negative by IF assays (Fig. 1 and Table 1) was followed by an attempt to detect the antigen by a more sensitive assay. We compared the level of FOCMA expression on 3191 cells with that on FL74 cells by absorbing the activity of serum 24640 with varying numbers of cells of each type. Ability of 0.1 ml of the serum to bind to FL74 cells at a dilution fourfold

234567

FIG. 4. Reactivity of various antisera with FeLV-encoded proteins. FL74 (A) and 3191 cells (B) were pulse-labeled with [%]methionine and cell lysates immunoprecipitated and analyzed by gel electrophoresis. Lane 1: goat anti-FeLV gp ‘70 serum; lane 2: FOCMA antiserum 31924; lane 3: FOCMA antiserum 26460; lane 4: monoclonal antibody C3; lane 5: antibody 2-124, lane 6: murine control @P-2) serum; lane 7: normal cat serum.

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less than its endpoint titer was partially eliminated by 5 X lo5 FL74 cells and completely eliminated by 3 X lo6 FL74 cells. On the other hand, as many as 3 X lo7 3191 cells did not affect the binding capacity of this serum. Therefore, if 3191 cells express FOCMA, the level of expression was at least 60-fold lower than that in FL74 cells. FeLV-negative primary cat lymphomas obtained from New York and California were tested for FOCMA expression, the former by viable cell IF assay, and since the latter tumors were B-cell lymphomas, they were tested by absorption assays. Monoclonal antibodies 2-124 and 2-88, and serum 24640 bound to two out of the three New York tumors (Table 4). Similarly, three out of four California tumors failed to absorb the activity of antisera to FOCMA and one absorbed FOCMA activity only partially (Table 4). This tumor was no longer on hand by the time the monoclonal antibodies became available. Thus, TABLE EXPRESSION

4

OF FOCMA IN FeLV-NEGATIVE CAT LYMPHOMAS Viable cell IF assaya

Cat lymphomas 3508 3576 3616 CT649 CT636 CT1220 CT1311

24640

2-124

-

-

+ + NT NT NT NT

+ + NT NT NT NT

Absorption assay” CT1201 and CT1145 NT NT NT + -

Note. First three tumors were determined to be FeLV negative by Dr. W. D. Hardy using IF assays (Hardy et al., 1980). California tumors of CT series were FeLV-negative by EM analysis as well. “New York tumors were identified as T-cell lymphomas and could be assayed by viable cell assays. FL74 and 3403 cells were used as positive controls. NT, Not tested. +, Positive, -, negative in IF assay. * Partial list of CT sera given, others gave similar results. Some of the CT tumors were B-cell lymphoma and could not be examined by direct IF assays. Absorption assays were done according to Hardy et al. (1978). Positive control consisted of absorption with extracts of FL74 cells. -, No absorption; +, partial absorption.

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four out of seven FeLV-negative lymphomas were FOCMA negative, two were FOCMA positive, and one could not be definitively categorized. Specificity of monoclonal antibodies implied that expression of FOCMA in the New York tumors could be correlated with the expression of gp70 determinants of FeLV C missed by IF assays with rabbit anti-FeLV AB serum (Hardy et al., 1980) used to determine the FeLV status of these tumors. FeSV-transformed mink lung epithelial cells (64F3C17) were also negative in viable cell IF assays using cat antiserum 3192A, monoclonal antibodies, and other cat antisera (Table 1). FOCMA antibody activity of viremic cat sera CT1145, CT1201, CT1203, CT369, and CT921 could not be absorbed with equal volumes of 64F3C17 cell pellets while all the sera were completely absorbed by relatively smaller volume of cell pellets of FL74 cells (Table 2). These results are supported by the immunoelectron microscopy data where 14 out of 15 cat sera showed no ferritin binding to 64F3C17 cell membranes. The aberrant serum showed marginal binding, presumably due to some anti-gag antibodies in that serum. We conclude, therefore, that FeSV-transformed mink cells do not express FOCMA, i.e., do not express any of the antigens carrying the epitopes which are specifically recognized by cat antisera to FOCMA in FL74 cell membranes. DISCUSSION

Our studies show that a common denominator of naturally occurring FOCMA positive viremic cat sera is their reaction with FeLV C gp70 epitopes expressed on the cell surface of cat lymphomas and FeLV C-infected cells. A similar conclusion, correlating FOCMA antibody activity to envelope protein of FeLV, was reached from the data of Ruscetti and Parks (1976,1977). The notion that FOCMA was a tumor-specific antigen was developed on the basis of the following three lines of evidence: (1) It was reported to be expressed in all cat lymphomas, including FeLV-negative cat lymphomas, and FeSV transformed nonproducer mink cells, (2) nontransformed cells infected by FeLV

456

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were reported to lack FOCMA expression, and (3) disrupted FeLV proteins were shown to be unable to absorb FOCMA activity of the viremic cat serum tested. Except for the last item, our data failed to substantiate a number of crucial attributes previously associated with FOCMA as a tumor-specific antigen. Instead, it appears that FOCMA activity is due to gp70 epitopes expressed both on replication competent and defective FeLV C and that some of the FOCMA activity can also be attributed to FeLV C epitopes which are altered after mature virions are released from the cell membrane. Lymphomas That Lack Viral Antigen pression also Lack FOCMA

Ex-

About one-third of cat leukemias positive for FOCMA were reported to be FeLV negative (Hardy et aZ., 1977, 1980). Some cats diagnosed as FeLV negative, on the basis of IF assays on peripheral blood leukocytes could, however, have an occult infection and thus have FeLV antigens circulating in their plasma (Lutz et al., 1980). In fact, Saxinger et al. (1980) found that 60% of the FeLV-negative cats with tumors had detectable antigens related to FeLV ABC/KT in their sera. Since two out of three FOCMA-positive tumors from FeLV-negative cats identified by IF assay with rabbit anti-FeLV gsa serum (Hardy et al., 1980) were found to be positive for FeLV C epitopes with the monoclonal antibodies 2-124 and 2-88 (Table 4), and since our studies indicate that viremic cat sera reactive with FL74 cells also react with FeLV C, the FeLV status of the rest of the 40% of the FeLV-negative, FOCMA-positive lymphomas needs to be reexamined. It is not clear why five different sublines derived from the FeLV-negative 3191 tumor were reported to be FOCMA-positive by Snyder et al. (1979a, 1980). We found two of them to be both FeLV C negative and FOCMA negative by IF, antibody absorption, and radioimmunoprecipitation assays. Similar observations were reported earlier by VedBrat et al. (1980) and Worley and Essex (1980). Unfortunately, none of the original 3191 cells are available for reanalysis. Since the available FOCMA-

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negative 3191 cells are capable of forming tumors in nude mice (data not shown), and since FOCMA-negative cat lymphomas were also identified in this study (Table 4), tumorigenicity of cat lymphomas cannot necessarily be associated with FOCMA expression. Thus, while infection of cats by FeLV is correlated with higher risk of leukemogenesis (Hardy et al., 1977, 1978), the absence of FeLV and FOCMA in some cat lymphomas indicates that in cats, as in other mammalian species, agents other than FeLV infection also play a role in leukemogenesis. Lack of Correlation of FOCMA Expression with FeSV Transfomu;ction Lack of reaction of FOCMA antisera, including 31928, with 64F3C17 cells in IF assays confirmed the results of the monoclonal antibody screening experiments. Inability of these mink cells to absorb activity of “FOCMA positive” antisera was also demonstrated by Ruscetti and Parks (1977). The quantitative studies on the reaction of cat antisera with FOCMA of both mink and cat cells was given by Sliski and Essex (1979), who found that 13 cat sera had equivalent titers of activity on both FL74 and 64F3C17 cells. We failed to identify the common occurrence of such cat sera when 50 viremic cat sera reactive with FL74 cells were screened for their reaction with 64F3C17 cells in IF assays. Similarly, Dr. R. G. Olsen (Columbus, Ohio) found 1 out of over 40 viremic cat sera which was reactive with both FL74 and 64F3C17 cells (personal communication). Moreover, 90% of viremic cat sera specifically selected for their negative reaction with FL74 cells were reported to react with 64F3C17 cells (Chen et al., 1980). These observations make it difficult to rely on the assumption that a small percentage of viremic cat sera which react with both the cell types do, indeed, detect the same antigen called FOCMA. Serum 3192A was reported to precipitate a ~85 protein from 64F3C17 cells (Snyder et al., 1978, 1979a). However, its titer in viable cell IF assays was not given, and in our hands it did not react with cell surface antigens of the transformed mink

FOCMA:

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ANTIGEN

457

cells. Our observations are also consistent with more recent data based on 24 FOCMA antisera from both viremic and nonviremit cats, none of which could immunoprecipitate an FeSV-encoded polyprotein from 64F3C1’7 cells (Gardner et al., 1980; Barbacid et al., 1980). Conversely, rat sera specific to one gene products of different isolates of FeSV (Ruscetti et al., 1980b) did not immunoprecipitate “FOCMA protein ~70” from FL74 cells (Sherr it al., 1980). Frankel et al. (1979) reported the absence of FeSV-specific RNA from FL74 cells. Rosenberg et al. (1980) found that transection of FeSV (Snyder-Theilen strain) DNA into NIH3T3 mouse fibroblasts failed to induce FOCMA expression by IF assays, though the presence of gag-one polyprotein was readily demonstrable. We are, therefore, forced to conclude that FOCMA, as a tumor-specific antigen common to both FeLV and FeSV transformed cells, does not exist. Furthermore, lack of FOCMA expression in unambiguously classified FeLVnegative lymphomas, and its expression in FeLV C-infected nontransformed cells relieves FOCMA of its role as a cellularly coded tumor-specific antigen.

broblasts infected by this virus are negative in assays with monoclonal antibodies 2-124 and 2-88 (VedBrat et al., 1980),yet the parent F422 cells express the FeLV C antigens recognized by these antibodies. Moreover, they produce virus which absorbs the activity of these monoclonal antibodies. They must, therefore, contain envelope proteins of both FeLV A and FeLV C. Since infectious FeLV C is found in a very small percentage of FeLV-positive cats (Sarma et al., 1973; Jarrett et al., 1978), F422 cells could be considered a prototype for those FOCMA-positive tumor cells which either shed infectious FeLV A, FeLV AB, or which shed no infectious FeLV at all. As in the case of Grx expression of MuLV gp70 on normal murine thymocytes (Fleissner and Snyder, 1982), FOCMA expression in F422 and in FeLV-negative primary cat lymphomas appears to be correlated with the expression of the viral envelope gene which is not part of a replication-competent genome of FeLV.

Correlation fection

It seems that we have an experimental system that can differentiate between the envelope proteins of infectious and noninfectious FeLV C, since antibodies 2-124 and 2-88 bind to F422 cells while antibodies B12 and C3 do not (Table l), and both groups of antibodies precipitate a molecule similar to gp70 of FeLV C. Similarly cat antisera like 24640 can bind to mature virus particles while sera like 3192A react with antigenic components found in cell membrane or in budding virus particles only. On this basis, three different antigenie determinants of the gp70 molecule of FeLV C can be identified. (1) The 3192Aspecific epitopes, recognized by a large proportion of cat antisera to FOCMA and found only on cell membrane and on budding virus, and which are somehow altered, or masked, in mature virion particles, (2) B12/C3 type epitopes, found on envelope of infectious FeLV C virions, and (3) 2-124/2-88 type epitopes, also identified by cat sera like 24640, and found on protein of both replication-defective and replica-

oj’ FOCMA

with FeLV C In-

Our data on the binding of standard FOCMA typing sera to budding virus particles are supported by earlier observations made with sera from both viremic and nonviremic cats (Calafat et al., 1979). We have demonstrated that FOCMA expression in cat fibroblasts, human rhabdomyosarcoma RD cells, or human 3403 lymphoma cells in culture reflects FeLV C infection, an observation consistent with data of Rice et al. (1981), who found FOCMA expression in FeLV-infected feline embryo fibroblasts. Strong FOCMA expression on human lymphoma 3403 cells is also consistent with the similar observation of Essex et al. (1972) on FeLV ABC/ KT-infected human lymphocytes. FOCMA and Noninfectious FeLV C Antigen Expression in Cat Lymphomas F422 cells produce infectious FeLV A but not FeLV C (Sarma and Log, 1973). Fi-

IdentiJication of Three Antigenic Camp nents of FeLV C gp70 Molecule by FOCMA Antibodies

458

VEDBRAT

tion-competent forms of FeLV C. We cannot, however, rule out the possibility that BlZ/C3 epitopes represent a variable portion of gp70 (thus observed on FL74 cells and not on F422 cells), while Z-124/2-88 epitopes represent a conserved portion of FeLV C gp’70.

Antigenic Variability, Recmnbinant ruses, and Letdcemogenesis

Vi-

Immunofluorescence and absorption data showing cross-reactive antigenic determinants on FeLV B and C are consistent with the observation of Sarma and Log (1973) and of Russell and Jarrett (1978a) who found that FeLV A is monotypic and probably represents the infectious exogenous agent, while FeLV B and C were always found in association with FeLV A and showed a spectrum of overlapping antigenic variability. The antigenie variability of FeLV B and C, and production of FeLV C neutralizing antibodies by cats infected by cloned isolates of FeLV A, led to the hypothesis that infectious (Russell and Jarrett, 1978a, b) as well as noninfectious FeLV C expression (VedBrat et al., 1980) may arise as a result of a recombination event between exogenous FeLV-A and endogenous FeLV-related sequences in the cat genome. A similar model was provided by Ruscetti et al. (1980a) as an intermediate step for the generation of FeSV. Alternatively, infection of cats with FeLV A may, in some instances, induce the expression of one of the endogenous FeLV sequences, leading to expression of FeLV C envelope protein. Either mechanism could lead to the expression of a number of replication-competent and replicationdefective viral products, depending on which of the multiple copies of endogenous FeLV sequences took part in a given recombination or gene activation event. In the chicken system, recombination between endogenous viral sequences and exogenously infecting viruses has been shown to result in the formation of recombinants, like RAV-60, expressing envelope glycoproteins of endogenous virus (Hanafusa et al., 1970; Hayward and Hanafusa, 1975). Recombination between endogenous and exogenous virus was recently documented

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to be the critical event in the generation of highly oncogenic murine MCF viruses both in inbred and in outbred feral strains of mice (Chattopadhyay et al., 1982; Rasheed et al., 1982). Whether defective FeLV C type recombinants can be isolated as highly tumorigenic agents from F422 type of cells is yet to be determined. It has been seen, however, that infection with FeLV C increases the frequency of spontaneous transformation in feline embryo fibroblasts in culture (Rasheed and Gardner, 1980; Rice et al., 1981; VedBrat and Prensky, unpublished results). Thus, the expression of FOCMA in cat lymphomas may be due to the frequent expression of recombinant FeLVs which are most of the times replication defective. ACKNOWLEDGMENTS The work by S.S.V. and W.P. was supported by National Cancer Institute Contract NOl-CP-71059 and Grants CA 08748 and CA-16599. H.L. was partly supported by Grant 3.748-0.39 from the Swiss National Science Foundation, and S. Rasheed and M.B.G. by Public Health Service Contract NOl-CPB-1963, with the National Cancer Institute, U. S. A. Immunological reagents and virus were obtained from the Office of Program Resources, National Cancer Institute. We thank Dr. W. D. Hardy, Jr., for generously providing us with FeLV negative cat lymphoma cells and FOCMA typing sera and Drs. M. Essex and R. G. Olson for some of the FOCMA typing sera used in electron microscope studies. We are grateful to Dr. J. Mullins for the restriction endonuclease cleavage analyses of 3403 cell DNA and to Drs. S. Jhanwar and R. Chaganti for the karyotype analyses, which made the identification of 3403 cells possible. H.L. is indebted to G. Theilen and N. Pedersen in whose laboratories the monoclonal antibodies to gp70 of FeLV C were established. The excellent assistance of Alexander Barkas, Lynda Wijcik, Marcia McCalla, and Yaroslav Eliseev with FOCMA immunofluorescence and absorption assays is gratefully acknowledged. We are especially grateful to Dr. H. Hanafusa for a critical review of the original manuscript. REFERENCES BARBACID, M., LAWER, A. V., and DEVAFZ, S. G. (1930). Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen strains of feline sarcoma virus. J. V+ol 33, 196-397. CALAFAT, J., WEIJER, R., HAGEMAN, P. C., and DAAMS, H. (1979). Studies on antibodies against feline leukemia virus (FeLV) in cat sera and rabbit anti-

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FeLV sera: Cross reaction and differences. .I. Gen. Viral 45, 217-222. CHATTOPADHYAY,S. K., CLOYD,M. W., LINEMEYER, D. L., LANLIER,M. R. RANDS,E., and LOWY, D. R. (1982). Cellular origin and role of mink cell focus-forming viruses in murine thymic lymphomas. Nature (Lendon) 295,25-31. CHEN, A. P., ESSEX, M., MIKAMI, T., ALBERT, D., NIEDERKORN, J. Y., and SHADDUCK, J. P. (1980). The expression of transformation related proteins in rat cells. In “Feline Leukemia Virus” (W. D. Hardy, Jr., M. Essex, and A. J. McClelland, eds.). Develop. Cancer

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major virion glycoprotein. Pwx. Nat. Acad Sci USA 74,1219-1223. STEPHENSON,J. R., KHAN, A. S., SLISKI, A. H., and ESSEX, M. (197713). Feline oncornavirus-associated cell membrane antigen (FOCMA): Evidence for an immunologically crossreactive feline sarcoma virus-coded protein. Proc. Nat. Ad Sci USA 74. 5668-5612. THE&EN, G.H., KAWAKAMI, T. G., RUSH, J. D., and MUNN R. J. (1969). Replication of cat leukemia virus in cell suspension cultures. Nature (London) 222, 589-590. VEDBRAT, S. S., HAMMERLING, II., HARDY, W. D., JR., BORENFREUND,E., and PRENSKY, W. (1979). Monoclonal antibodies: Detection of transformation re-

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VEDBRAT, S. S., MCCALLA, M. E., LUTZ, H., and PRENSKY, W. (1980). Monoclonal antibodies to FOCMA: binding to FeLV-C infected cells. In “Feline Leukemia Virus” (W. D. Hardy, Jr., M. Essex, and A. J. McClelland, eds.). Develop Cancer Res. 4, 457-470. WORLEY, M., and ESSEX, M. (1989). Identification of membrane proteins associated with transformation related antigens shared by lymphoma cells and feline sarcoma virus-transformed fibroblasts. In “Feline Leukemia Virus” (W. D. Hardy, Jr., M. Essex and A. J. McClelland, eds.). Develop. Cancer Res. 4.431440.