Characterization of mouse monoclonal antibodies specific for friend murine leukemia virus-induced erythroleukemia cells: Friend-specific and FMR-specific antigens

VIROLOGY

112.131-144

(1981)

Characterization of Mouse Monoclonal Antibodies Specific for Friend Murine Leukemia Virus-Induced Erythroleukemia Cells: Friend-Specific and FMR-Specific Antigens B. CHESEBRO,

Department National

of Health Institute

and

K. WEHRLY, M. CLOYD, W. BRITT, J. COLLINS, AND J. NISHIO Human and Mountain

of Allergy Rocky

J. PORTIS,

Services, Public Health Service, National Institutes Ir&ectious Diseases, Laboratory of Persistent Viral Laboratories, Hamilton, Montana 59840 Accepted

October

of Health, Diseases,

7, 1980

Monoclonal antibodies were derived from hybrid cell lines produced by fusing mouse myeloma cells with spleen cells from mice recovering from Friend virus-induced erythroleukemia. Of the 17 clones characterized, two appeared to have the Friend, Moloney, Rauscher pattern of specificity. One of these was specific for the envelope protein, gp70, and the other reacted with a core protein, ~15. Seven other anti-gp70 clones and one antip15 clone were restricted in reactivity to cells infected with Friend or Rauscher viruses only. One clone reacted with p15E and recognized this protein on many ecotropic and dual-tropic viruses. In addition, six IgM antibodies were obtained which appeared to recognize nonviral antigens present only on leukemia cells of the erythroid lineage. Six monoclonal antibodies of complement-fixing immunoglobulin classes with specificity for gp’i’0 or ~15 were compared for their ability to bind or lyse erythroleukemia cells in the presence of complement. Individual antibodies to the same viral protein appeared to differ markedly in their ability to mediate cytolysis.

mune response in this system by using the technique of Kohler and Milstein (1975, 1976) to produce monoclonal antibodies directed against FV-induced antigens. In previous reports anti-AKR-MuLV monoclonal antibodies were selected by screening against virion antigens, and a majority of positive clones was specific for the viral protein, p15E (Nowinski et al., 1979; Lostrom et al., 1979). In the present study, we were interested primarily in antibodies which recognized FV-induced leukemia cell surface antigens. Therefore, we selected most clones on the basis of reactivity with leukemia cell surface antigens in membrane immunofluorescence (MF) or cytotoxicity tests. We describe here the characteristics of 17 monoclonal antibodies specific for FV leukemia cells. Antibodies obtained had specificity for three different virus-induced proteins as well as for nonviral erythroleukemia-associated antigens. The results indicated that many clones were type-specific for Friend or

INTRODUCTION

The mouse humoral immune response to Friend leukemia virus (FV)-induced antigens has a marked effect on the course of virus infection and leukemia progression in some mouse strains (Chesebro and Wehrly, 1976). An autosomal non-H-2 linked gene, Rfv-3, influences the ability of mice to make an antiviral antibody response following inoculation with FV (Doig and Chesebro, 1979). Mice of the Rfv-3”” genotype make antiviral antibodies which result in decreased expression of virus-induced antigens, decreased infectious virus release from leukemic spleen cells, and elimination of FV viremia (Chesebro et al., 1979). Characterization of the biological activities of mouse antiviral antibodies of different Ig classes and with specificities for different FV-induced antigens has been difficult due to the heterogeneity and low titers of most antisera. Therefore, we have attempted to study the antiviral im131

0042-6822/81/090131-14$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Rauscher virus-induced proteins, but antibodies having the Friend-MoloneyRauscher (FMR) pattern of cross-reactivity (Old et al., 1964) were obtained which exhibited either anti-p15 or anti-gp70 specificities. MATERIALS

AND

METHODS

Sera. The following goat sera were kindly provided by Dr. J. Cole, Biological Carcinogenesis Branch, National Cancer Institute, Bethesda, Maryland: fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin (Ig) (‘733-000004), anti-Tween-ether-disrupted Rauscher leukemia virus (R-MuLV) (23-783), anti-RMuLV p10 (763-169), anti-R-MuLV p12 (53-37), anti-R-MuLV-p15 (788-450), antiR-MuLV p30 (23-658), anti-R-MuLV gp70 (53-167). Rabbit anti-mouse Ig serum was kindly provided by Dr. J. Coe, Rocky Mountain Laboratory. Mouse anti-FV sera were obtained from mice which had recovered from FV leukemia and were boosted with FV leukemic spleen cells as described (Doig and Chesebro, 1979). Viruses and cells. N-, NB-, and B-tropic strains of Friend virus complex were propagated and assayed as described previously (Chesebro et al., 1974). The Friend helper virus (F-MuLV) from the N-, NB-, and B-tropic complexes was passed once on SC-1 cells and then cloned by limiting dilution of SC-1 cells. Rauscher leukemia virus from JLS-V9 cells was obtained from Dr. J. Ihle, and was cloned by limiting dilution and propagated on SC-1 cells. SC-l cells infected with B-tropic FV complex were used for membrane immunofluorescence and [35S]methionine immunoprecipitation studies. The following cloned viruses were obtained from Dr. J. Hartley, National Institutes of Health, Bethesda, Maryland: Moloney leukemia virus (MMuLV) free of MCF viruses (J. Hartley, personal communication), AKR2a strain of ecotropic AKR-MuLV, Friend-MCF virus (Ishimoto strain), Moloney-MCF virus, AKR-13-MCF virus, BALB-IU-1 xenotropic virus, and 4070A amphotropic virus (Cloyd et al., 1979). Normal rat kidney cells (NRK) nonproductively infected with

ET AL.

spleen focus-forming virus (SFFV) were obtained from Dr. D. Troxler (1977). FV-induced erythroleukemia cell lines Y57 (clone 2C), YA97, and AA41 were obtained from (C57BL/lO X A.BY)F1, (BIO.A X A.BY)F1, and (BIO.A X A/WySn)F1 mice as described (Chesebro et al., 1976; Doig and Chesebro, 1979). Al3 leukemia cells were obtained from a BALB.B mouse after neonatal inoculation with BALB-Tennant leukemia (B/T-L) virus (Chesebro et al., 1976). YAC leukemia cells induced by MMuLV (Cikes et al., 1973) were obtained from Dr. Harvey Cantor, Harvard Medical School, Boston, Massachusetts. The G-line of EL4 carcinogen-induced leukemia cells was provided by Dr. R. Herberman, National Institutes of Health, Bethesda, Maryland. Although low in MuLV production, we have consistently isolated both ecotropic and xenotropic MuLV from these EL4 cells, and they are highly positive for cell surface gp70 (Portis and McAtee, 1981). The nonsecretor NSl clone (P3-NS-lAg4/1) of the HAT-sensitive mutant (P3X63 Ag8) of MOPC21 myeloma cells was obtained from the Salk Cell Distribution Center, LaJolla, California (Kohler and Milstein, 1976). Hybridoma production. At 10 to 30 days after intravenous (iv) inoculation of mice with B-tropic FV (1500 FFU) each spleen (1 to 2 x lo8 cells) was dissociated in phosphate-buffered balanced salt solution (PBBS) and washed three times in RPM1 1640 without serum and mixed with lo7 washed NSl cells in a 50 ml polypropylene tube (Corning). All manipulations were carried out at room temperature. After centrifuging at 250 g for 5 min, the supernatant was aspirated and replaced with 2 ml of 35% (w/v) polyethylene glycollOO0 (PEG) in RPM1 1640 without serum. Cells were resuspended by gentle swirling for 2 min. The suspension was centrifuged 3 min at 500 g and after 3 min more the supernatant was diluted to 50 ml with RPM1 1640 without serum. The cell pellet was again resuspended by swirling and the suspension was centrifuged 5 min at 250 g. The supernatant was aspirated and cells were resuspended in 24 ml RPM1 1640 with

MONOCLONAL

ANTIBODIES

15% fetal calf serum (FCS). One milliliter of cell suspension was placed in each well of a Linbro TC24 tray, and cells were placed in a CO2 incubator overnight. The following day 1 ml of HAT medium, RPM1 1640 with 15% FCS plus hypoxanthine 13.6 pg/ml, aminopterin 0.176 pg/ml, thymidine 3.9 kg/ml was added to each well. Thereafter, every 3 to 4 days one-half the supernatant was aspirated and replaced by fresln medium. After 14 days medium containing hypoxanthine and thymidine but no aminopterin was used. Colonies of fused cells were apparent in 10 to 20 days, and supernatant from wells with such colonies was assayed for antibody activity by indirect membrane fluorescence, cytotoxicity, virus binding, or immunoprecipitation. Cells from positive wells were cloned by limiting dilution in Linbro 96 well trays (‘76-003-05) using 2 to 4 X lo5 normal C57BL/lO spleen cells per well as a feeder layer. Wells containing colonies were tested for antibody activity, and desired cultures were expanded in larger flasks. In some cases, 1 to 5 X lo6 cells were inoculated intraperitoneally (ip) into (C57BL/lO X BALB/c)Fi mice, and ascites fluid was obtained by daily peritoneal drainage with a 19-gauge needle. Fourteen days prior to inoculation with cells, mice were given 0.5 ml 2,6,10,14-tetramethylpentadecane (Aldrich) ip. Membrane JEuorescence (MF). Indirect membrane immunofluorescence was done using washed leukemia cells or trypsinized washed. attached cell lines. Cells (1 to 2 X 106) were mixed with 50 ~1 of undiluted hybridoma tissue culture supernatant or hybridoma ascites fluid or antiserum dilutions in Linbro round bottomed 96 well microtiter trays. Diluent was PBS with 0.5% gelatin and 0.1% NaN3. After 30 min at 37”, cells were centrifuged and washed twice with diluent and resuspended in 50 ~1 of a l/50 dilution of FITC-conjugated goat anti-mouse Ig serum. After 30 min at 37”, cells were centrifuged and washed twice, ithen resuspended in PBS with 1% formalin and 50% glycerol and examined for me:mbrane fluorescence in a Leitz Orthoplan microscope. Cytotoxicity test. 51Cr-labeled target cells

TO FRIEND

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133

were lysed with antibody and absorbed rabbit complement as described previously (Chesebro and Wehrly, 1976). Immunoprecipitation. The details of the method used were described previously (Collins and Chesebro, 1980a). Cells were labeled in methionine-free minimal essential medium with 50 &i/ml [35S]methionine for 2 hr and the lysed in 0.5% NP40 in 0.01 M Tris-HCl, 0.14 M NaCl, pH 7.2, and stored at -70”. Lysates were precleared with normal goat serum and Staphylococcus aureue Cowan I strain (SA), ultracentrifuged, and immunoprecipitated with antibody overnight at 4” and with SA for 30 min. Immunoprecipitates with monoclonal antibodies were washed with lysing buffer, and those with antisera were washed with lysing buffer plus 0.1% SDS and 0.5% sodium deoxycholate. Precipitates were eluted and analyzed on slab SDS-polyacrylamide gel electrophoresis (SDS-PAGE). From 0.2 to 0.5 ml hybridoma supernatant and 50 ~1 10% SA was used. To detect antibodies of IgG1, IgM, or IgA classes, 50 ~1 10% SA was preincubated 10 min with 5 ~1 rabbit anti-mouse Ig serum and washed once before addition to the mixture of radiolabeled antigen hybridoma antibodies. Goat antisera to R-MuLV proteins ~10, ~12, ~15, ~30, gp70, and Tween-ether-disrupted RMuLV virions were used as controls. Gels were fixed, processed for fluorography, and exposed to Kodak X-Omat R-film at -70”. FV was obtained from infected SC-1 cells and was purified on sucrose gradients as described (Collins and Chesebro, 1980b). Ten micrograms of virus was dissociated in 100 ~1 0.5% NP40 in 0.01 M Tris-HCl, 0.14 MNaCl, pH 7.2 on ice for 30 min. Viral proteins were iodinated with 20 pg Iodogen (Pierce Chemical Co.) and 1 to 2 mCi lz51 for 10 min at 0” (Markwell and Fox, 1978). After addition of 1 mg KI and 1 mg gelatin, the solution was dialyzed 1 to 2 hr against Tris buffered saline, aliquoted, and stored at -70”. Aliquots were thawed, precleared, and used for immunoprecipitation as above. 1251immunoprecipitates were analyzed by SDS-PAGE. Dried gels were exposed to Kodak X-Omat R-film at -70” with a Kodak Lanex intensifying screen.

134

CHESEBRO

Virus neutralization. 0.15 ml containing 150 to 300 PFU of virus was mixed with 0.15 ml hybridoma culture supernatant or antibody dilution. A l/8 dilution of fresh frozen guinea pig serum (0.1 ml) was added for complement. After 1 hr at 37”, the mixture was diluted with 1.1 ml cold PBBS and 0.5 ml was assayed in duplicate on appropriate target cells. For F-MuLV the mouse S+L- assay with D56 cells was used (Bassin et al., 1971) and for FMCF virus CCL64 mink lung cells were used. Immunoglobulin isotype. Ig class of antibodies produced by hybridoma cultures was determined by gel diffusion in 1% agarose of 5- to lo-fold concentrated culture supernatant fluid. Rabbit antisera specific for mouse Ig classes were obtained from Litton Bionetics, Kensington, Maryland. RESULTS

Immunovecipitation of Viral Proteins Monoclonal Antibodies

by

As an approach to characterization of the mouse humoral immune response to Friend virus, we have obtained a group of hybridomas which have reactivity for Friend erythroleukemia cells. These hybridomas were derived from (C57BL/ 10 X A.BY)F1 or C57BL/lO mice inoculated with FV-B 10 to 30 days previously. Initialiy culture supernatants from wells containing colonies of fused cells were screened by indirect membrane immunofluorescence (MF) for ability to bind to Y5’7 erythroleukemia cells. Positive cultures were cloned by limiting dilution. Tissue culture supernatants from 16 clones reactive with Y57 cells were characterized. In addition, one clone (372) was characterized which did not react with Y57 cell surface antigens but did react with purified intact Friend virus in a polyvinyl tray binding assay (Nowinski et al., 1979). The antibodies produced by these hybridoma cultures were monoclonal and could be distinguished from one another by their pattern of L chain migration on SDSPAGE (Fig. 1). In order to determine the specificity of reactivity of these antibodies,

ET AL.

supernatant fluids were analyzed by immunoprecipitation of radiolabeled Friend virus proteins. Sucrose gradient purified virions were lysed in NP40 detergent and labeled with lz51. From this lysate, proteins ~10, ~12, ~15, ~30, and gp70 could be immunoprecipitated by specific goat antisera and identified on SDS-polyacrylamide slab gels (Fig. 2). p15E was not detected, presumably due to its lack of iodinated tyrosines (Lostrom et al., 1979). Supernatants from two hybridomas (34 and 257) precipitated FV ~15, and supernatants from nine hybridomas (59, 48, 55, 273,350, 66,47, 372, and 307) immunoprecipitated FV gp70 (Fig. 2). No hybridomas specific for ~10, ~12, or ~30 were found. The p15 precipitated by 34 and 257 could be precleared from the lysate by prior precipitation with the goat anti-p15 serum (data not shown). Thus, these comigrating bands appeared to be identical. The same was true for the anti-gp70 serum and the gp70 precipitated by monoclonal anti-gp70 supernatants. All antibodies were also tested by immunoprecipitation using [35S]methioninelabeled FV-infected SC-l cells (Fig. 3). The anti-p15 monoclonal antibodies (34 and 257) precipitated gag proteins, p42 and pr65. These proteins were also precipitated by goat anti-p15 and anti-p12 sera. Antibody from hybridoma 372 precipitated p15E and some pr90”““, but little or no gp70. Therefore, it appeared to have anti-pl5E specificity. The detection of some gp70 precipitated by this antibody from iodinated virions (Fig. 2) was probably due to a small amount of residual disulfide linkage between virion gp70 and p15E (Montelaro et al., 1978). When [35S]methionine labeled cell lysates were immunoprecipitated with individual anti-gp70 hybridoma supernatants, pr90”“’ and gp70 proteins were poorly visualized. However, when two different anti-gp70 hybridoma supernatants were mixed, precipitation was strongly potentiated (Fig. 3). This would appear to be due to the ability of different hybridomas to bind at different sites on the polypeptide and thus increase the overall binding af-

MONOCLONAL

mo P

ANTIBODIES

TO FRIEND

LEUKEMIA

CELLS

135

USE

chains

chains

FIG. 1. Coomassie blue stain of SDS-PAGE analysis (307) hybridoma supernatants were precipitated with mouse Ig serum. In these tracks monoclonal L chain ogeneous rabbit L chain bands, and in the case of 307 gamma chain band. Other hybridomas were precipitated chain bands are visible. In contrast, tracks containing diffuse H and L chain bands. The band labeled X above and appeared to be derived from the SA.

finity of polypeptide to antibody complex. Hybridomas 48 and 55 did not complement each other in this way and thus appeared to recognize the same antigenic site on pr90”“” and gp70. The L chains of these two hybridoma antibodies had identical mobility in SDS-PAGE, whereas in most cases different monoclonal antibodies could easily be distinguished from each other by this criterion (Fig. 1). Therefore, in spite of the fact that these two hybridomas were derived from different culture wells after the fusion procedure, they appeared to be identical. In contrast to the goat anti-gp70, none of the hybridoma anti-gp70 antibodies precipitated gp55, the SFFV env protein (Dresler et al., 19’79; Ruscetti et al., 1979).

of immunoprecipitates. IgA (47) and IgM Staphylococcus azwtxs (SA) plus rabbit antibands can be seen within the diffuse hetera G chain band can be seen above the rabbit with SA alone, and monoclonal y and L goat anti-Rauscher leukemia virus sera have the H (y) bands was seen in all precipitates

Furthermore, the hybridoma antibodies did not bind to NRK cells nonproductively infected with SFFV in the absence of FMuLV (Table l), suggesting that these antibodies did not recognize any antigens common to both F-MuLV gp70 and SFFV gp55. Six IgM monoclonal antibodies (46, 50, 109,111,112, and 121) were negative in all immunoprecipitations attempted against lz51-labeled FV, 35S-labeled FV-infected SC-1 cells, or 35S-labeled Y57 cells (data not shown). It was not clear whether the appropriate cellular antigen(s) had not been labeled effectively or whether the affinity of the IgM antibody combining sites was too low for binding of free soluble polypeptides in the lysates.

136

CHESEBRO

ET

AL.

p30

pl5. p12. PI0 FIG. 2. lzI-labeled FV lysates were immunoprecipitated with natants (0.5 to 1.0 ml) or with goat antisera to Rauseher leukemia were analyzed on SDS-PAGE slab gels by autoradiography.

Membrane Cells

Fluorescence

of Virus-Infected

In order to analyze the viral specificities of the monoclonal antibodies, hybridoma supernatants were tested in indirect membrane fluorescence (MF) against a panel of leukemia cells and tissue culture cell lines infected with various MuLVs. The results indicated that the hybridomas fell into four groups (Table 1). The first group consisted of two hybridomas, 34 and 273, which appeared to have the Friend-Moloney-Rauscher (FMR) pattern of specificity because they reacted with FV, FMuLV, R-MuLV, and M-MuLV-infected NIH-3T3 or SC-1 cells and also with several FV-induced erythroleukemias, with the M-MuLV-induced leukemia, YAC, and with the B/T-L virus-induced leukemia,

hybridoma tissue culture supervirus proteins (2 ~1). Precipitates

A13. It was of interest to note that these two “anti-FMR” hybridomas recognized different viral proteins, ~15 and gp70. The second group of hybridomas (257, 48, 350, 59, 66, 47, and 307) reacted with FV erythroleukemias and FV, F-MuLV, and R-MuLV FV-infected cells but not with M-MuLV-infected cells or with MMuLV or B/T-L-induced leukemia cells. Thus, these antibodies were restricted to Friend and Rauscher type specificity. This group included one anti-p15 and six antigp70 clones. One anti-gp70 clone, 350, also reacted with F-MCF infected cells. The third group consisted of a single hybridoma, 372, which reacted with FMR and AKR ecotropic MuLVs as well as with three MCF viruses, F-MCF, M-MCF, and AKR-13MCF. This hybridoma recognized the p15E protein and therefore this pattern of viral

MONOCLONAL

ANTIBODIES

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FIG. 3. [%]Methionine-labeled lysates of SC-l cells infected with FV were immunoprecipitated with antisera (2X) or hybridoma culture supernatants (0.5 to 1.0 ml). Precipitation of env gene products from this lysate was amplified when two nonidentical anti-gp70 monoclonal antibodies were mixed. Neither the monoclonal antibodies nor the mouse antisera were able to immunoprecipitate the SFFV protein, gp55. Antibody 372 was also reacted with lysate in the presence of 0.05 M Z-mercaptoethanol to dissociate disulfide-linked gp70 and p15E proteins.

cross-reactivity was not unexpected as it has previously been seen with some monoclonal anti-pl5E antibodies (Lostrom et al., 1979). It was surprising that the availability of p15E at the cell surface appeared to vary among different cell lines. In membrane fluorescence tests with hybridoma 372, Y57, and YAC cells were negative, as were virusinfected. NIH/3T3 cells, however, infected SC-l cells were positive with all five viruses tested (Table 1). The fourth group of hybridomas consisted of six clones (46, 50, 109, 111, 112, and 121) which reacted only with FV erythroleukemia cells and did not react with any lymphoid leukemia cells or virusinfected nonleukemic cells (Table l), nor did they bind to purified Friend virus in the tray assay (data not shown).

Virus Neutralization tibodies

by Monoclonal

An-

Hybridoma culture supernatants and ascites were tested for ability to neutralize F-MuLV and FMCF virus infectivity in the presence or absence of guinea pig complement. Anti-gp70 hybridomas 48, 307, 350, 55, 273, 47, and 66 were effective at neutralization of F-MuLV. However, these worked only in the presence of complement (Table 2). One other anti-gp70 hybridoma antibody, 59, did not neutralize virus. However, it appeared that this antibody recognized a gp70 determinant which was not freely accessible on intact viruses since it bound poorly to intact virus in the polyvinyl tray assay (Nowinski et al., 1979) (data not shown). Antibodies

138

CHESEBRO

from hybridomas 350 (anti-gp70) and 372(anti-p15E) neutralized FMCF virus. However, the monoclonal anti-pl5E failed to neutralize F-MuLV. This was analogous to previous findings with rabbit anti-FMuLV p15E serum (Fischinger et al., 1976).

Comparison of Cytotoxicity and Membrane Fluorescence Using Erythroleukemia Cell Targets In order to investigate whether both p15 and gp’70 viral cell surface antigens detected by MF were suitable for antibody plus complement-mediated cytolysis of erythroleukemia cells, six hybridomas from complement-fixing immunoglobulin classes were studied. Ascites fluid produced by the hybridomas was titered in membrane fluorescence and complementmediated cytotoxicity assays using Y57 cells (Fig. 4). All six ascites fluids had very high titers in MF, giving 50% positive fluorescent cells at dilutions of lop3 to 10e5. However, cytotoxicity results were much more varied. The cytotoxicity titer for hybridoma 350 coincided with the MF titer. Hybridomas 307, 34, and 48 had reduced cytotoxicity compared to MF, and hybridomas 273 and 257 were totally negative for cytotoxicity. These results indicated that both gp70 and p15 could function as appropriate antigenic targets for lysis of these erythroleukemia cells by mouse antibodies. However, certain mouse antibodies (273 and 257) with specificity for gp70 or p15 were not cytotoxic in spite of the fact that they bound efficiently to the cell surfaces and belonged to the complementfixing IgGza class. In fact the three IgGza anti-gp70 hybridomas, 350,48, and 273, all differed in their relative efficiency of cell lysis compared to cell binding. As determined by their ability to potentiate each other in immunoprecipitation (Fig. 3), these three anti-gp7Os all recognized different antigens on the gp70 molecule. Therefore, it appeared that the lack of lysis by certain anti-gp70 hybridomas might be due to unfavorable steric relationships of the Fc portions of antibody molecules once bound to the cell surface antigens. The possibility of a noncomplement-fixing subclass within the IgGz, class

ET AL.

seemed unlikely since the noncytolytic hybridoma 273 was capable of fixing complement after reaction with virus in a neutralization test (Table 2). DISCUSSION

The hybridomas described in this paper were all obtained from H-2b’b mice from 10 to 30 days after inoculation with FV and represent a portion of the repertoire of anti-erythroleukemia cell antibodies produced during recovery from FV leukemia. Eight of the 17 clones characterized had anti-gp70 activity, two were anti-p15, and one was anti-pl5E. Seven of the antigp70 hybridomas and one of the anti-p15 hybridomas reacted only with proteins of Friend or Rauscher viruses and not with Moloney leukemia virus, which is the other member of the serologically related FMR group (Old et al., 1964). This was surprising because most antisera raised against one FMR virus have strong cross-reactions with the other two viruses. However, competition radioimmunoassays have indicated that the proteins of the helper viruses of the Friend and Rauscher virus complexes differed partially from those of M-MuLV (Fischinger et al., 1978; Collins and Chesebro, 1980b). The fact that most of our anti-gp70 and anti-p15 hybridomas were directed to the unique portions of Friend and Rauscher proteins suggested that the predominant antiviral response to FV-induced erythroleukemia in these mouse strains was type-specific rather than cross-reactive for the entire FMR group or for other unrelated MuLV. Two hybridomas (273 and 34) had significant cross-reactivity with both MMuLV-infected fibroblasts and YAC leukemia cells induced by M-MuLV. Thus, these antibodies appeared to have the typical “FMR” reactivity. Interestingly, one of these was anti-p15 in specificity and the other was anti-gp70. Thus, the “FMR” pattern of cross-reactivity was associated with both p15 and gp70 proteins. This could explain the apparent discrepancy in data from other groups on this issue. Anti“FMR” cross-reactive antibodies immunoprecipitate (Nowinski et al., 1978) and cocap (Bismuth et al., 1978) with cell sur-

W,, WA. I&, w*. Mb. kG2, lg.4 W

wa

kM Id Id kM kM IgM

251 4x 55 350 59 66 47 307

372

46 50 109 111 112 121

? ? ? ? 1 1

p15E

PI5 gP70 gp79 gp70 gp70 BP70 gp76 gp70

PI5 gP70

+ + + + + +

+ + + + + + + +

+ +

Y57d FV

-

-

-

-

-

+

-

-

-

+ +

Al3 B/T-L

+ +

YAC M-MuLV

Leukemia cells”

-

-

-

-

-

-

-

-

-

EL-4 ?

IMMUNOFLUORESCENCE

-

-

+

+ + + + + + + +

+ +

FV

-

+

+ +

+ + + +

+ +

F-MuLV

-

-

+

+ +

+ + +

+ +

R-MuLV

+

-

-

+ +

M-MuLV

cellsh

ANTIBODIES

SC-l and NIH/3T3

OF HYBRIDOMA

1

-

+

-

-

AKR2a

TESTED

-

-

-

-

-

-

+

-

+ -

-

+

+

+

F-MCF

-

-

+

-

-

+

M-MCF

-

+

-

-

-

-

-

-

-

BALB-IU-ltxeno)

Mink lung cells’

CELLS

AKR-13.MCF

VIRUS-INFECTED

SFFV

NRK cells

AGAINST

-

-

-

-

-

+

4070tampho)

’ MuLV belived to have induced the leukemia is shown under the cell line. EL4 is a carcinogen-induced leukemia; however, xenotropic and dual-tropic (MCF) MuLV have been isolated from these cells (data not shown). ‘All five viruses were tested on both SC-l and NIH/3T3 cells. Uninfected cells were negative. All antibodies except 372 gave similar results on both cell types. 372 was positive on SC-l cells infected with all five viruses tested, but was negative on similarly infected NIH/3T3 cells. ‘Uninfected mink lung cells were negative. “Identical results were obtained when erythroleukemia cell lines YA97 and AA41 were used.

wzs W,,

34 273

Hybridoma

Ig class

Viral protein specificity

INDIRECT

TABLE

E

w

s

E

s

E

3

2 s

3

B

8

5 z

F

z

s

CHESEBRO

140

ET AL.

TABLE NEUTRALIZATION

OF F-MuLV

AND FMCF

VIRUS

2

BY HYBRIDOMA

ANTIBODIES

Neutralization

F-MuLV

PLUS COMPLEMENT titer&

FMCF Culture supernatant

Culture supernatant

Hybridoma

Specificity

Ascites

34 257

P15 P15

0

0

ntb

0

0

372

p15E

nt

0

1

48 307 350 55 273 47 66

gp70 gp70 gp70 gp70 gp70 gp70 gp70 gp70

10,000 10,000 100 nt nt nt nt nt

10 100 0 1 1 1 0 1

0 nt 1 nt 0 nt nt nt

46 50

? ?

109 111 112 121

? ? ? ?

nt nt nt nt nt nt

0 0 0 0 0 0

nt nt nt nt nt nt

59

0

Q Reciprocal of highest dilution giving >50% decrease in F-MuLV PFU on mouse S+L- cells or FMCF foci on mink lung cells. Ten-fold dilutions were tested starting at undiluted tissue culture supernatant or l/l0 diluted ascites fluid. 0 indicates fluid was negative at all dilutions tested. * nt = not tested.

face gp70. However, Strand et al. (1974) noted that cytotoxic anti-“FMR” activity was blocked by Rauscher ~15. Based on our hybridoma data, both gp70 and p15 contain antigens which show cross-reactivity within the FMR group of viruses. Thus, the discrepancies previously noted were most likely due to variability in amounts of antibodies to different viral proteins in the different antisera tested. The identity of the cell surface molecule detected by our anti-p15 clones has not been accurately determined. Both these clones and goat anti-p15 and anti-p12 sera immunoprecipitated two or more pl5-containing molecules from [35S]methioninelabeled cell lysates (Fig. 2). Our preliminary data suggest that a 93,000 dalton protein can be immunoprecipitated from surface-labeled erythroleukemia cells by anti-

gag sera (Collins and Chesebro, manuscript in preparation). This protein would appear to be analogous to the 94,000 dalton gag cell surface polyprotein seen on other MuLV-infected cells (Tung et al., 1976; Buetti and Diggelmann, 1980). The data were somewhat confusing regarding which viral antigens of this protein were exposed at the cell surface. Goat anti-p12 sera (Collins et al., 1980) and monoclonal mouse anti-p15 antibodies reacted with erythroleukemia cells in MF and cytotoxicity assays. However, goat anti-p15 serum was negative in both tests, even though this serum and the mouse monoclonal anti-p15 both precipitated the same ~15 molecules from lZ51-labeled Friend virus, as detected by sequential precipitation tests (data not shown). It is difficult to explain these findings, and we can only speculate that the

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.

I I : 1 I

A’ 2 .’ I

I ‘I

: 1

7

I

?. I , I

/ /’

/ _’

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goat anti-p15 serum does not contain antibodies to the p15 antigenic determinants which were exposed on the leukemia cell surfaces and detected by both mouse antip15 clones. It was somewhat surprising to note that none of our anti-gp70 hybridomas recognized the SFFV-induced defective env gene protein, gp55 (Dresler et al., 1979). Genetic and biochemical evidence indicated that Friend gp70 and SFFV gp55 have some regions of identity, however, gp55 appeared more closely related to Friend MCF gp’70 (Ruscetti et al., 1979). Goat anti-R-MuLV gp70 serum could immunoprecipitate both proteins, but this could be due to separate populations of non-cross-reacting anti-ecotropic gp70 and anti-MCF gp70 antibodies in this serum. The lack of reactivity of any mouse antigp70 monoclonal antibodies with gp55 suggested that the common regions of these proteins were poorly immunogenic in the mouse strains tested. This was consistent with our observation that (BlO X A.BY)F1 mouse anti-FV sera that had anti-gp70 activity also failed to immunoprecipitate gp55 (Fig. 2). Risser (1979) has found that Fv-2”” mice express an alloantigen on normal erythroid stem cells which is crossreactive with an antigen induced by SFFV in erythroleukemia cells. If these antigens were located on gp55, this might explain the lack of antibodies to this protein in antisera or hybridomas derived from (BlO X A.BY)F1 mice (Fv-2”“). With this in mind, we have looked for anti-gp55 hybridomas in C57BL/lO mice, which are not tolerant to the antigen described by Risser, however, to date none has been detected. Most of the anti-gp70 monoclonal antibodies and the anti-pl5E antibody were effective in complemert-mediated virus neuralization tests (Table 2). Hybridoma 59 was an exception in that it did not neutralize F-MuLV, although it effectively precipitated gp70 from viral lysates. Furthermore, it bound very well to purified gp70 in polyvinyl trays, but was poorly bound to intact virus in these same trays (data not shown). Thus, this hybridoma appeared to recognize an antigen to gp70

ET

AL.

which was partially hidden when this molecule was displayed on the virion or cell surface. The neutralization which we observed with monoclonal anti-gp70 and anti-pl5E antibodies differed from complement-mediated virus lysis observed ta be induced by monoclonal anti-pl5E antibodies but not by anti-gp70 antibodies (Orozlan and Nowinski, 1980). This difference would appear to indicate that complement can amplify virus neutralization by mechanisms other than virus lysis. One possibility might be that virions covered with antibody-complement complexes are unable to interact with cell surface receptors for viral gp70, thus preventing virus infection. The binding affinity of the monoclonal antibodies appeared to be low compared to conventional polyvalent antisera. Although the monoclonal antibodies bound strongly to multivalent antigens such as cell and virion membranes, they were less effective at reacting with polypeptides in solutions. For example, none of the antigp70 hybridomas bound sufficient ‘251-labeled gp70 to be detectable in a conventional radioimmunoassay, however, they did bind sufficient lz51-labeled gp70 from iodinated viral lysates to be identified in SDS-PAGE. This weak binding was in part due to the fact that each hybridoma probably recognized only a single antigenie determinant on each protein and thus each protein was bound only at a single point, in contrast to the multipoint attachment possible in most polyvalent antisera. When nonidentical anti-gp70 hybridomas were mixed immunoprecipitation of gp70 containing protein (pr90”““) was enhanced (Fig. 3). In addition to the anti-gp70, anti-p15 and anti-pl5E hybridomas, six other hybridomas were found which had strong reactivity with erythroleukemia cells. These antibodies failed to react with FVinfected fibroblasts or lymphoid leukemia cells induced by other MuLVs. Unfortunately, all these antibodies were of the IgM class and thus probably had a low binding affinity per combining site. Therefore, although cell binding and lysis were very strong on a polymeric antigenic

MONOCLONAL

ANTIBODIES

TO FRIEND

structure such as an intact cell surface, these hybridomas failed to immunoprecipitate any labeled molecules from detergent lysates of [35S]methionine or lz51-labeled cells. This type of IgM hybridoma was quite prevalent in many of our fusion experiments, and in the earlier experiments where we used antibody plus complementmediated cytotoxicity against erythroleukemia targets these hybridomas were preferentially detected. It is possible that some of these clones might recognize determinants characteristic of the erythroid differentiation line, rather than virus-induced antigens.’ ACKNOWLEDGMENTS The paring

authors thank the manuscript.

Mrs.

Helen

Blahnik

for

pre-

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CIKES, M., FRIBERG, JR., S., and KLEIN, G. (1973). Progressive loss of H-2 antigens with concomitant increase of cell surface antigen(s) determined by Moloney leukemia virus in cultured murine lymphomas. J. Nat. Cancer Inst. 50,347-362. CLOYD, M. W., HARTLEY, J. W., and ROWE, W. P. (19’79). Cell-surface antigens associated with recombinant mink cell focus-inducing murine leukemia viruses. J. Exp. Med. 149, 702-712. COLLINS, J. K., and CHESEBRO, B. (1980a). Spontaneous cessation of Friend murine leukemia virus production by leukemia cell line Y57: overgrowth by nonproducer cells. J. Nat. Cancer Inst. 64,11531159. COLLINS, J. K., and CHESEBRO, B. (1980b). Synthesis, processing and cell surface expression of Friend and xenotropic murine leukemia virus gp70 antigens on Friend virus-induced erythroleukemia cell clones. J. Immunol. 125,1325-1331. COLLINS, J. K., BRITT, W. J., and CHESEBRO, B. (1980). Cytotoxic T lymphocyte recognition of gp70 on Friend virus-induced erythroleukemia cell clones. J. Immunol. 125,1318-1324. DOIG, D., and CHESEBRO, B. (1979). Anti-Friend virus antibody is associated with recovery from viremia and loss of viral leukemia cell-surface antigens in leukemic mice. Identification of Rfv-3 as a gene locus influencing antibody production. J. Exp. Med. 150,10-19. DRESLER, S., RUTA, M., MURRAY, M. J., and KABAT, D. (1979). Glycoprotein encoded by the Friend spleen focus-forming virus. J. Viral. 30, 564-575. FISCHINGER, P. J., SCHAFER, W., and BOLOGNESI, D. P. (1976). Neutralization of homologous and heterologous oncornaviruses by antisera against the p15(E) and gp71 polypeptides of Friend murine leukemia virus. Virology 71, 169-184. FISCHINGER, P. J., FRANKEL, A. E., ELDER, J. H., LERNER, R. A., IHLE, J. N., and BOLOGNESI, D. P. (1978). Biological, immunological, and biochemical evidence the HIX virus is a recombinant between Moloney leukemia virus and a murine xenotropic C type virus. Virology 90,241-254. KOHLER, G., and MILSTEIN, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256,495-497. KOHLER, G., and MILSTEIN, C. (1976). Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6, 511519. LOSTROM, M. E., STONE, M. R., TAM, M., BURNETTE, W. N., PINTER, A., and NOWINSKI, R. C. (1979). Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p15(E) and gp70 envelope proteins. Virology 98, 336-350. MARKWELL, M. A. K., and Fox, C. F. (1978). Surfacespecific iodination of membrane proteins of viruses

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and eucaryotic cells using 1,3,4,6-tetrachloro-3a, Gal-diphenylglycouril. Biochemistry 17, 4807-4817. MONTELARO, R. C., SULLIVAN, S. J., and BOLOGNESI, D. P. (1978). An analysis of type-C retrovirus polypeptides and their associations in the virion. Virology 84, 19-31. NOWINSKI, R. C., EMERY, S., and LEDBETTER, J. (1978). Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells. J Viral. 26, 805-812. NOWINSKI, R. C., LOSTROM, M. E., TAM, M. R., STONE, M. R., and BURNETTE, W. N. (1979). The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of ecotropic murine leukemia viruses. Virology 93, 111-126. OLD, L. J., BOYSE, E. A., and STOCKERT, E. (1964). Typing of mouse leukemias by serological methods. Nature (London) 201,777-779. OROZLAN, S., and NOWINSKI, R. C. (1980). Lysis of retroviruses with monoclonal antibodies against viral envelope proteins. Virology 101, 296-299. PORTE, J. L., and MCATEE, F. J. (1981). Dissociation of H-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line. Immunogenetics 12, 101-115.

ET AL. RISSER, R. (1979). Friend erythroleukemia antigen. A viral antigen specified by spleen focus-forming virus and differentiation antigens controlled by the Fv-2 locus. J. Exp. Med. 149, 1152-1167. RUSCETTI, S. K., LINEMEYER, D., FEILD, J., TROXLER, D., and SCOLNICK, E. M. (1979). Characterization of a protein found in cells infected with the spleen focusing-forming virus that shares immunological cross-reactivity with the gp70 found in mink cell focus-inducing virus particles. J. Viral. 30,787-798. STRAND, M., WILSNACK, R., and AUGUST, J. T. (1974). Structural proteins of mammalian oncogenic RNA viruses: immunological characterization of the ~15 polypeptide of Rauscher murine virus. J. Viral. 14, 1575-1583. TROXLER, D. H., BOYARS, J. K., PARKS, W. P., and SCOLNICK, E. M. (1977). Friend strain of spleen focus-forming virus: A recombinant between mouse type C ecotropic viral sequences and sequences related to xenotropic virus. J. Viral. 22, 361-372. TUNG, J.-S., YOSHIKI, T., and FLEISSNER, E. (1976). A core polyprotein of murine leukemia virus on the surface of mouse leukemia cells. Cell 9, 573-578.