Vol. 26, No. 3
JOUJRNAL OF VIROLOGY, June 1978, p. 805-812 0022-538X/78/0026-0805$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Identification of an FMR Cell Surface Antigen Associated with Murine Leukemia Virus-Infected Cells ROBERT C. NOWINSKI,* SANDRA EMERY, AND JEFFREY LEDBETTER Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 Received for publication 27 December 1977
FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virusinfected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp7O, p15(E), and p12(E) from detergent-disrupted preparations of [3H]leucinelabeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp7O is directed against typespecific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted '25I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelope protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product. Infection of mouse cells with murine leukemia virus (MuLV) results in the induction of virusspecific cell surface antigens. Two serologically distinct antigenic systems have been identified: the FMR antigens (16) are found on the surface of cells that are infected with exogenous MuLV [Friend, (F)MuLV; Moloney, (M)MuLV; and Rauscher, (R)MuLV], whereas the G antigens (17) are found on the surface of cells that are infected with endogenous or Gross type MuLV. A serological distinction between these viruses. can be demonstrated by neutralization of viral infectivity (1,11) and by radioimmunoassay with viral envelope proteins (21). In general, viruses of the FMR group tend to be strongly antigenic in mice. As a result, transplants of virus-infected tumors or leukemias often are rejected in syngeneic mice (16). Hyperimmunization of mice that have regressed FMR leukemias with increasing inocula of virus-infected cells leads to the production of cytotoxic FMR typing antisera. These antisera react with cells infected by FMR viruses, but they do not react with normal tissues from mice of high or low leukemic strains, or with tumors or leukemias that occur spontaneously in mice (16). In contrast, viruses of the G group tend to be weakly antigenic in mice, and antisera cannot be prepared by syngeneic immunization. Instead, antisera against G antigens are made by immunization of C57BL/6 mice with the AKR leuke-
mia K36 (17). These antisera, although produced by allogeneic immunization, are cytotoxic for histocompatible C57BL/6 leukemias that are induced by Gross passage A virus. Tissues from mice of diverse genetic backgrounds can be examined for G antigens by absorption tests with these typing antisera. In such tests, G antigens are found on the surface of normal lymphoid tissues and spontaneous leukemias of mice of high leukemic strains. These antigens are not found, however, on the surface of normal tissues of mice of low leukemic strains, and they occur in only low amounts on the surface of FMR virus-induced leukemias (17). FMR typing antisera react in immune electron microscopy with both the envelope of budding viruses and with regions of the cell surface that do not contain virions (12). Since these FMR antisera also neutralize the infectivity of FMR viruses (1, 20), it is possible that these antigens represent viral envelope proteins. However, studies from three laboratories suggest that two different populations of antibodies in FMR antisera are responsible for neutralization and cytotoxicity (2-5, 18, 20, 21). To date, this question has not. been resolved by biochemical analysis. We describe here an immunochemical analysis of FMR-specific viral and cell surface antigens. These studies demonstrated that FMR antisera reacted strongly with type-specific antigens of the gp70 envelope protein in virions
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and on the surface of virus-infected cells. These antisera did not react with any other FMR-specific cell surface proteins. MATERIALS AND METHODS Animals and cells. BALB/c mice were purchased from the Jackson Laboratories. FBL3 and RBL5 were C57BL/6 leukemias that were induced by (F)MuLV and (R)MuLV, respectively. LST'RA was a BALB/c leukemia that was induced by (M)MuLV. These leukemias were kindly provided by Alex Fefer, Fred Hutchinson Cancer Research Center, and have been maintained by serial transplantation of ascites cells in C57BL/6 mice or by growth in vitro. EdG2 was a C57BL/6 leukemia that was induced by Gross passage A virus; it was maintained by transplantation of spleen cells in C57BL/6 mice or by growth in vitro. Primary Friend virus-infected spleen cells [referred to as (F)MuLV leukemia cells] were obtained from BALB/c mice 10 to 14 days after intravenous inoculation of NB-tropic (F)MuLV. K36 was a transplanted ascites variant that was derived from a spontaneous AKR leukemia. Viruses. All viruses were purified by density-gradient centrifugation. (F)MuLV and (M)MuLV were obtained from tissue culture fluids of FLB3 and LSTRA cells. (R)MuLV was obtained from tissue culture fluids of chronically infected JLSV-9 cells. (A)MuLV was obtained from tissue culture fluids of the AKR pIl embryo fibroblast cell lines. Proteins of these viruses were radiolabeled by growth of the virusinfected cells overnight in culture medium supplemented with ['H]leucine (20 ,iCi/ml of culture fluid). For absorption studies, (R)MuLV and (A)MuLV were obtained from the National Cancer Institute as 1,000fold concentrates from tissue culture fluids. Antisera. FMR antisera were prepared in mice by immunization with virus or virus-infected cells. FMR(NNB) antiserum was prepared by immunization of BALB/c mice with (F)MuLV; mice were initially inoculated with N-tropic (F)MuLV and then hyperimmunized with NB-tropic (F)MuLV. FMR(RCSB) antiserum was prepared by immunization of BALB.B (H-2") congenic mice with the syngeneic (F)MuLVinduced BALB.B RCSB leukemia. FMR(NNB) and FMR(RCSB) antisera were kindly provided by F. Lilly, Albert Einstein College of Medicine. FMR murine sarcoma virus (MSV) antiserum was obtained from BALB/c mice that had regressed transplants of syngeneic MSV-induced sarcomas. FMR(FBL3) antiserum was prepared in C57BL/6 mice by immunization with C57BL/6 FBL3 leukemia cells. FMR(MSV) and FMR(FBL3) antisera were kindly provided by Joseph Brown and A. Fefer of the Fred Hutchinson Cancer Research Center. Antisera against G antigen were prepared by immunization of C57BL/6 mice with the AKR K36 leukemia. Goat antisera against the purified proteins gp7O, p30, p15, p12, and plO of (R)MuLV were obtained from the National Cancer Institute. Goat antiserum against the purified p15 protein of (A)MuLV was kindly provided by E. Fleissner (Sloan-Kettering Institute). Rabbit antiserum against purified p15(E) protein of (R)MuLV was kindly provided by S. Oroszlan (Frederick Cancer Center). Immune precipitation of viral proteins. [1Hl-
J. VIROL.
leucine-labeled MuLV was solubilized on ice by treatment for 30 min with 0.5% Nonidet P-40 (NP-40) in phosphate-buffered saline (PBS), pH 7.4. Aggregated viral proteins and nondisrupted viral components were then removed by centrifugation at 100,000 x g for 20 min. Radioimmune precipitation (RIP) assays contained 50 Al of virus extract (105 cpm) and 2 pl of antiserum in a total volume of 200 Al of PBS buffer containing 0.5% NP-40. Reactions were terminated after a 1-h incubation at 4°C by addition of 0.5 mg of Staphylococcus aureus (7). After a 30-min incubation, the bacteria and their bound immune complexes were removed from the reaction mixture by centrifugation through a cushion of 5% sucrose (in PBS) containing 3% NP-40. The bacteria were then washed three additional times in PBS with 0.5% NP-40 and then treated with sodium dodecyl sulfate-containing sample buffer. RIP reactions were analyzed by polyacrylamide gel electrophoresis in 10% slab gels in the presence of sodium dodecyl sulfate (8). Immune precipitation of cell extracts. Radiolabeling of cell membranes by the [2'I]lactoperoxidase method was performed by the method of Vitetta et al. (23). Briefly, 5 x 107 viable cells were suspended in 1 ml of PBS containing 100 yg of lactoperoxidase and 3 mCi of 12'1; this reaction mixture was activated by the addition of two pulses of 0.06% H202 at 5-min intervals. The entire labeling procedure was performed on ice with continuous shaking; viability of cell suspensions (determined by trypan blue exclusion) before and after radiolabeling was >95%. The labeled cells were then disrupted with gentle Vortex mixing in 0.5% NP-40 for 30 min at 4°C; nonsolubilized cellular structures were removed by centrifugation at 100,000 x g for 45 min. The supernatant containing the solubilized membrane proteins was frozen at -20°C in small portions for future analysis. Conditions for the RIP assays that were performed with these cell extracts were similar to those with viral proteins (see above), with the exception that the RIP assays with cell extracts contained 106 to 10' cpm of input antigen. Complement-dependent cytotoxicity. Assays for complement-dependent cytotoxicity were performed by the 5"Cr release method. Briefly, 5 x 10" viable cells in 500 Al of RPMI 1640 medium containing 15% fetal calf serum were labeled with 250 ,uCi of 5'Cr by incubation at 37°C for 1 h with frequent, gentle agitation. The assay was performed in the wells of a microtiter plate (Falcon Microtest II); each well of the plate contained (i) 104 "Cr-labeled cells in 50 ,ul of diluent, (ii) 50 ,ul of diluted antiserum, and (iii) 50 ,ul of rabbit serum (diluted 12-fold) as a complement source. Rabbit sera used as the source of complement were initially selected for low toxicity on mouse thymocytes and were then absorbed in the presence of EDTA with pooled normal lymphoid tissues. The diluent for all procedures was RPMI 1640 medium containing 15% fetal calf serum. The plates were incubated at 37°C for 45 min, and the reaction was stopped by the addition of 100 Al of cold diluent to each well. Intact cells were removed from the reaction mixture by centrifugation at 1,000 rpm for 10 min in a refrigerated centrifuge, and a constant volume of each of the supernatants was removed for the determination of the amount of "Cr released. Calculations were made as
VOL. 26, 1978
FMR CELL SURFACE ANTIGEN
follows: percent specific 5'Cr release = (experimental release) - (control release)/(maximum release) (control release). Maximum 5'Cr release was determined by three cycles of freeze-thawing of the radiolabeled cells. Control release was the highest value that was scored in either of the three controls that were performed with each experiment: (i) spontaneous release (no antibody or complement present), (ii) complement release (no antibody present), and (iii) antibody release (no complement present). Results of experiments in which the 5"Cr release of any of the controls exceeded 10% of the maximum release value were excluded.
RESULTS Cytotoxic assays for FMR and G cell surface antigens. The reactions of different FMR and G antisera with virus-induced leukemias was determined by cytotoxic reactions with 5'Cr-labeled target cells. FMR(RCSB) antiserum showed a high degree of type specificity for the] FMR target cell. This antiserum was cytotoxic for the (F)MuLV leukemia, but not for EdG2 (Fig. 1). In contrast, the FMR(NNB) antiserum was cytotoxic for cells of both the (F)MuLV
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contained antibodies that reacted predominantly with FMR-specific antigens, and (ii) the FMR(NNB) antiserum contained two populations of antibodies, one which reacted specifi-
c
cally wth FMR antigens and one which reacted with antigens that were common to the (F)MuLV and EdG2 cells. Antisera prepared against G-cell surface antigens (C57BL/6 anti-AKR K36) were cytotoxic for EdG2 cells (Fig. 1). It was not possible, however, to assess by direct cytotoxic tests the specificity of these antisera on the (F)MuLV leukemia, since alloantibodies present in these sera reacted with BALB/c cells. As a result, further analyses of these sera were performed by absorption tests. The cytotoxic reaction of the G typing antiserum on EdG2 was completely 100 A
B
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leukemia and EdG2. As a result of the difference in activity of these antisera on leukemia cells, the specificity of these reactions was examined further by absorp20tion tests. In these studies (Fig. 2), the cytotoxicity of the FMR(RCSB) and the FMR(NNB) antisera for the (F)MuLV leukemia was found 0 to be absorbed completely by both nonlabeled lo 20 40 80160 10 20 40 80 160 Serum dilution target cells [(F)MuLV leukemia] and by cells of FIG. 1. (F)MuLV Direct cytotoxic assays with FMR and G the (R)MuLV-induced C57BL/6 RBL5 leuke- antisera. (A)5'and leukemia cellswith C57BL/6 meantisera mia. In contrast, cytotoxicity of these Cr and tested EdG2 cells (B) were radiolabeled was not absorbed by the E&G2 leukemia or by in cytotoxic assays with: FMR(RCSB) antiserum normal C57BL/6 spleen cells. It was concluded, (0), FMR(NNB) antiserum (l); and C57BL/6 antitherefore, that (i) the FMR(RCSB) antiserum AKR K36 serum (A).
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Serum dilution ( reciprocol) FIG. 2. Cytotoxic absorption tests demonstrating the FMR and G antigenic systems. (F)MuLV leukemia cells were radiolabeled with 5Cr and tested in cytotoxic assays with FMR(NNB) antiserum (A) and FMR(RCSB) antiserum (B); C57BL/6 E.3G2 cells were radiolabeled with 51Cr and tested in cytotoxic assays with C57BL/6 anti-AKR K36 antiserum (C). Reactions of unabsorbed sera are shown by the dashed lines. Antisera were each absorbed at 1/20 dilution with 10O nonlabeled cells and then tested for residual activity on "Cr-labeled cells. Cells used for absorption included: (F)MuLV leukemia cells (O); (R)MuLV-induced leukemia C57BL/6 RBL5 (-); C57BL/6 normal spleen (A); and C57BL/6 EPG2(0).
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absorbed by nonlabeled target cells (E6G2), but only partially by the (F)MuLV and (R)MuLV leukemias (Fig. 2). Thus, the G antigen was expressed in high levels on the E6G2 cell, but only partially on the (F)MuLV and (R)MuLV leukemia cells. The cytotoxicity of the G antiserum was not absorbed by normal C57BL/6 spleen cells (Fig. 2). Absorption of cytotoxic antibodies by purified viruses. The relationship of the FMR cell surface antigen to the proteins of MuLV was explored by cytotoxic absorption tests with density-gradient purified viruses. Preparations of (R)MuLV and (A)MuLV were frozen and thawed five times (to disrupt the viral envelope) and then used for absorption of FMR typing antisera. The cytotoxicity of both FMR(RCSB) and FMR(NNB) antisera for the (F)MuLV leukemia was absorbed completely by (R)MuLV (Fig. 3). These cytotoxic reactions were not absorbed by (A)MuLV. Immune precipitation of MuLV proteins by FMR and G antiserum. RIP reactions of FMR(NNB) and G antisera with FMR MuLV and (A)MuLV are shown in Fig. 4. FMR(NNB) antiserum precipitated the gp7O, p15(E), and P12(E) proteins from each of the FMR viruses [(F)MuLV, (M)MuLV, (R)MuLV], as well as from the (A)MuLV. G antiserum, on the other hand, precipitated primarily p30 protein from each of these viruses. The G antiserum also reacted to a lesser extent with the gp7O and
p15(E) proteins of each of these viruses. RIP reactions of (F)MuLV and (A)MuLV with several different FMR antisera [FMR(NNB), FMR(RCSB), and FMR(MSV)] and with the natural immune serum of an I strain mouse are shown in Fig. 5. These antisera reacted primarily with the gp7O, p15(E), and p12(E) proteins of the viruses. Although additional minor reactions were observed with p30 and plO, these reactions varied from one experiment to another and were considered to be the result of nonspecific precipitation. Quantitative differences in the reactions of mouse sera with FMR and (A)MuLV (Fig. 5) presumably reflected the titer of type-specific antibodies that were present in these sera. In this regard, the FMR(NNB) antiserum reacted equally well with the gp7O and p15(E) proteins of (F)MuLV and (A)MuLV, whereas the FMR(RCSB) antiserum reacted preferentially with the gp7O, p15(E), and p12(E) proteins of the (F)MuLV. The FMR(MSV) antiserum reacted equally well with the p15(E), p12(E), and gp7O proteins of (F)MuLV and (A)MuLV, whereas the I serum reacted preferentially with the p15(E) and gp7O proteins of (A)MuLV. Further analysis of these reactions was therefore performed by absorption tests with purified viruses of the FMR and G groups. The FMR(NNB) antiserum strongly precipitated gp7O, p15(E), and p12(E) proteins from (F)MuLV (Fig. 6). Absorption of this antiserum with (A)MuLV removed reactivity against the p15(E) and p12(E) proteins, but did not influ100- A -B ence the reaction of this antiserum with the gp7O protein. [By densitometric measurements, this 80absorption resulted in a >99% reduction of the .. ' . p15(E)/p12(E) reaction; under these same coni *N cS \ % % there was retention of 86% of the gp7O ditions, 6 60 -\ %Xs , reaction.] Absorption of the same pool of \ \FMR(NNB) antiserum with (R)MuLV com%% \\ 4 40\ pletely removed reactivity with the gp7O, p15(E), % and p12(E) proteins of (F)MuLV. The results of absorption tests with G antisera 20 also are presented in Fig. 6. G antiserum primarily precipitated p30 protein from (A)MuLV. Absorption of this antiserum with (A)MuLV completely removed the reaction of this antiseSerum dilution (reciprocal) rum with (A)MuLV, whereas absorption with FIG. 3. Cytotoxic absorption ofFMR antisera with (R)MuLV resulted in a considerable, but not FMR and AKR MuL V. (F)MuL V leukemia cells were complete, loss of precipitating activity against radiolabeled with 5'Cr and tested in cytotoxic assays p30 protein. with FMR(NNB) antiserum (A) and FMR(RCSB) It was concluded from these studies that the antiserum (B). Reactions of unabsorbed sera are FMR antisera reacted primarily with the gp7O, shown by the dashed lines. Antisera were each ab- p15(E), and p12(E) proteins of MuLV. Reactions sorbed at 1/40 dilution with 100 jug of density-gra- with the pl 2(E) proteproteins ins dient purified (R)MuLV (-) or (A)MuLV (CJ). Virus with the p15(E) and p12(E) were based based preparations were initially frozen and thawed five exclusively on the detection of group-specific times to partially disrupt the viral envelope and per- antigens. In contrast, the reactions of FMR anmit accessibility of antibody to the internal structural tisera with gp7O were based on the detection of proteins. type-specific and group-specific antigens. The
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FIG. 4. Immune precipitation of MuLVproteins by FMR and G typing antisera. FMR(NNB) and C57BL/6 anti-AKR K36 serum were reacted in RIP reactions with NP-40-disrupted ['Hileucine-labeled MuLV. (1) (A)MuLV; (2) (A)MuLV tested in RIP with FMR (left panel) or G (right panel) antisera; (3) (F)MuLV; (4) (F)MuL V tested in RIP with FMR (left panel) and G (right panel) antisera; (5) (M)MuL V; (6) (M)MuL V tested in RIP with FMR (left panel) and G (right panel) antisera; (7) (R)MuL V; (8) (R)MuL V reacted in RIP with FMR (left panel) and G (right panel) antisera.
FMR(RCSB) antiserum showed a predominant reaction with FMR-specific antigens of gp7O. These studies also confirmed the findings of cytotoxic assays, suggesting that antibodies to gp7O protein may be responsible for the typespecific cytotoxic activity of FMR antisera. Immune precipitation of FMR and G cell surface antigens. To determine the presence of viral proteins on the surface of the FMR typing cell, RIP reactions were performed with NP-40 lysates of ['25I]lactoperoxidase-labeled (F)MuLV leukemia cells. Immune precipitation reactions presented in Fig. 7 demonstrated that four different FMR antisera [FMR(NNB), FMR(RCSB), FMR(MSV), and FMR(FBL3)] reacted primarily with a 70,000-dalton protein on the surface of (F)MuLV leukemia cells. Immune precipitation studies with NP-40 lysates of [3H]glucosamine-labeled (F)MuLV leukemia cells (data not shown) confirmed these findings. The 70,000-dalton protein that was precipitated by the FMR antisera coincided in migration with the gp70 that was precipitated by goat anti-gp7O serum. Although the mouse antisera also precipitated smaller amounts of lower-molecularweight proteins, the total patterns of reaction of these sera were qualitatively and quantitatively similar to that observed with anti-gp7O serum. A similar reaction with gp7O was observed with G antiserum and with the serum from a normal I strain mouse. In addition, the G antiserum precipitated an 85,000-dalton protein from the cell extract. This protein coincided in migration with an 85,000-dalton protein that was precipitated by goat anti-p30 serum. This 85,000dalton protein has been previously demonstrated (8, 9, 18, 21) to be one of the glycosylated polyprotein precursors of the internal structural
proteins (p30, p15, p12, and plO) of MuLV. Reactions between ['25I]lactoperoxidase-labeled EdG2 cells and mouse antisera are presented in Fig. 8. FMR antisera [FMR(NNB), FMR(RCSB), and FMR(MSV)] precipitated only gp7O protein from this cell extract; these sera did not react with the glycosylated polyproteins or with other viral proteins that were present on the surface of these cells. In contrast, antisera prepared against G antigen reacted with gp7O and with two additional proteins of 85,000 and 95,000 daltons. The 85,000- and 95,000-dalton proteins corresponded in migration to the two glycosylated polyproteins that were precipitated by anti-p30 serum. A comparison of the relative activities of FMR antisera with lysates of different cell types demonstrated that the FMR(RCSB), FMR(MSV), and FMR(FBL3) antisera reacted to a greater extent with the gp7O from (F)MuLV leukemia cells than with the gp7O from EdG2. In contrast, the FMR(NNB) antiserum reacted equally well with the gp7O from (F)MuLV and EdG2 leukemia cells. This was consistent with the known distribution of type- and group-reactive antibodies in these antisera. In line with these findings, the natural immune serum of the I strain mouse was found to react preferentially with the gp7O of EdG2 cells. It was concluded from these studies that the (F)MuLV and EdG2 leukemia cells contained on their cell surface the viral envelope protein gp7O and the glycosylated polyprotein precursors of the internal structural proteins of MuLV. FMR antisera reacted with the gp7O proteins of these cells, but not with the glycosylated core polyproteins. In contrast, the G antiserum reacted with both gp7O and the glycosylated polyproteins.
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communication) and to be cytotoxic for virusinfected cells. In absorption tests the cytotoxic activity of FMR antisera was removed by disrupted density-gradient purified FMR MuLV and by FMR MuLV-infected cells, but not by purified endogenous MuLV or by cells infected with endogenous viruses. These findings pro____ * ~.XQvided evidence for a link between the FMR cell surface antigens and FMR viral proteins. This possibility was strengthened by the observation that FMR antisera contained antibodies against
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FIu .6. Absorption analysis of FMR and G antisera. ['Hjleucine-labeled NP-40-disrupted preparations were reacted in RIP assays with mouse antisera. (1) nontreated (A)MuLV; (2) RIP of (A)MuLV with C57BL/6 anti-AKR K36 serum; (3) RIP of (A)MuLV with C57BL/6 anti-AKR K36 serum that was absorbed with 100 MLg of (A)MuLV; (4) RIP of (A)MuLV with C57BL/6 anti-AKR K36 serum that was absorbed with 100 Mg of (R)MuLV; (5) nontreated (F)MuL V; (6) RIP of (F)MuL V with FMR(NNB) antiserum; (7) RIP of (F)MuLV with FMR(NNB) antiserum that was absorbed with 100 Ag of (A)MuL V; (8) RIP of (F)MuLV with FMR(NNB) antiserum that was absorbed with 100Mg of (R)MuLV.
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FIG. 5. Immune precipitation of proteins from MuL V by mouse antisera. ['H]leucine-labeled NP40-disrupted preparations of (F)MuL V and (A)MuL V were reacted in RIP assays with: (1) FMR(NNB) antiserum; (2) FMR(RCSB) antiserum; (3) FMR(MSV) antiserum; (4) I strain normal serum. Track (5) contains the nontreated virus control.
DISCUSSION
In efforts to identify the FMR cell surface antigen, we have analyzed the reactivity of a variet of antisera of antisera that.were that were prepared prepared in in mice variety against FMR MuLV or FMR MuLV-infected cells. These antisera were known to neutralize the infectivity of FMR viruses (F. Lilly, personal mice
FIG. 7. Immune precipitation of proteins from (F)MuL V leukemia cells by mouse antisera. (F)MuL V leukemia cells were surface labeled in vitro with 125I by the lactoperoxidase method and then lysed in NP40-containing buffer RIP reactions were performed with: (1)FMR(NNB) antiserum; (2) FMR(RCSB) antiserum; (3) FMR(MSV) antiserum; (4) I strain nor-
mal serum; (5) C57BL/6 anti-AKR K36 serum; (6) FMR(FBL3) antiserum; (7) anti-(R)MuLV gp7O serum; and (8) anti- (R)MuL V p30 serum. Track (9) contains '25I-labeled marker proteins gp70 and p30.
FMR CELL SURFACE ANTIGEN
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onstrated by cytotoxic absorption tests and by immune precipitation assays with extracts of radiolabeled cells. In most instances, the FMRinfected cells produced FMR-specific products in excess of the products of endogenous MuLV. These findings agree with those of Old et al.
W ~~~~~~~(17). 4W ~nTheidentification of the FMR cell surface antigen as gp70 protein is in conflict with the findings of other investigators (2-4, 18, 20). Two possible explanations for these conflicts could include: (i) the FMR antisera used by different investigators are dissimilar, and (ii) there are multiple FMR cell surface antigens. A discussion FIG. 8. Immune precipitation of proteins from of each of these possibilities is presented below: (i) We described here that FMR antisera conC57BL/6EdG2 cells by mouse antisera. NP-40 lysates of "5'I surface-labeled E3G2 cells were reacted in RIP tained various ratios of antibodies that were assays with: (1) FMR(NNB) antiserum; (2) directed against antigens of the envelope proFMR(RCSB) antiserum; (3) FMR(MSV) antiserum; teins of both FMR MuLV and endogenous (4) I strain normal serum; (5) C57BL/6 anti-AKR K36 MuLV. The occurrence of antibodies in these serum; (6) anti-(R)MuLV gp70 serum; and (7) anti- antisera that reacted with endogenous MuLV (R)MuLVp3O serum. Track (8) contains '25Ilabeled was attributed to the fact that (a) the mice used marker proteins gp 70 and p30. to generate the FMR antisera were of strains that naturally developed an immune response to the viral envelope proteins gp70, p15(E), and endogenous viruses (13), and (b) the FMR cells p12(E). In absorption tests with viruses of the used for immunization contained products of FMR and G serotypes, it was found that the endogenous MuLV on their cell surface. The immune response to p15(E) and p12(E) was presence of several distinct populations of antidirected against group-specific antigens, and as body [directed against group- and type-specific such, it was possible to exclude these proteins as antigens of gp70, p15(E), and p12(E)] in FMR candidates for the FMR antigen. Instead, evi- antisera, as well as the differential expression of dence from these absorption tests suggested that endogenous MuLV in FMR cells, might be exgp7O was the sole FMR-specific viral protein pected to cause complications in the analysis of that was immunologically identified by the the FMR antigen. It can be envisaged that under mouse. This conclusion was substantiated by the certain circumstances the cytotoxic activity of demonstration that FMR antisera precipitated FMR antisera for selected target cells could be only gp7O from the membrane of FMR MuLV- the result of reactions with gp70 antigens of endogenous MuLV, rather than with antigens of infected cells. Similar analysis with G typing antiserum led FMR MuLV. Or alternatively, the FMR antisera to the conclusion that this serum detected anti- may react to a major extent with p15(E) and genic determinants of the major core protein p12(E) proteins, rather than with gp70. In this (p30) of endogenous MuLV (14). The G anti- regard, we have previously shown (14, 24) that genic determinants of p30 protein were repre- a variety of alloantisera that were prepared in sented on the cell surface in the form of two C57BL/6 and BALB/c mice against H-2 and Ia serologically distinct glycosylated polyproteins antigens contained high titers of contaminating (9, 19). The 95,000-dalton polyprotein contained antibodies that were directed against endogeantigens of p30, p12, and plO, whereas the nous MuLV. As a consequence of these contam85,000-dalton polyprotein contained antigens of inating anti-viral antibodies, the H-2 and Ia alp30 and p12, but not plO. Similar findings have loantisera were anomalously cytotoxic for syngeneic tumor and leukemia cells that expressed been described by Snyder et al. (19). Cells infected with endogenous MuLV con- antigens of endogenous MuLV on their cell surtained gp7O and the two glycosylated core poly- face. (ii) Studies by Steeves (20) demonstrated that proteins as constituents of their membranes. These cells did not contain FMR-specific anti- the virus neutralizing activity of FMR antisera gens on their cell surface. In contrast, cells in- was independent of their cytotoxic activity. fected with FMR MuLV contained the protein These results have been confirmed by Freedman products of endogenous MuLV, as well as FMR- et al. (4). The apparent conflict between these specific proteins, on their cell surface. This dual findings and those presented here could be exexpression of FMR and G antigens on the cell plained by the presence of two antigenically surface of FMR MuLV-infected cells was dem- distinct sites on the gp7O protein, one of these _
812
NOWINSKI, EMERY, AND LEDBETTER
sites being responsible for the binding of neutralizing antibody, and the other for the, binding antibody.A related hypothess ofofcytotoxic cytotoxic antibody. A related hypothesis.as has been presented by Lilly and Steeves (11). These conjectures are supported by the studies of Ihle et al. (6) in which it was found that gp7O protein had a different antigenic configuration in the envelope of the virion than it had in virus-free regions of the cell membrane. These authors (6) demonstrated by immune electromicroscopy anti-gp7O that both ootn goat goat antl-gp7u serum and ana mouse natnatural immune serum reacted with the budding virus, whereas only the goat anti-gp7O serum reacted with virus-free regions of the cell surface.
J. VIROL.
3. Fenyo, E. M., and G. Klein. 1976. Independence of the Moloney virus induced surface antigen (MCSA) in immunoand membrane associatedcellvirion selected lymphoma sublines. antigens Nature (London) 260:355-356. 4. Freedman, H. A., F. Lilly, and R. A. Steeves. 1975. Antigenic properties ofcultured tumor lines derived from spleens of Friend virus-infectedcellBALB/c and
BALB/c-H-2b mice. J. Exp. Med. 142:1365-1376.
5. Friedman, M., F. Lilly, and S. G. Nathenson. 1974.
Cell surface antigen induced by Friend murine leukemia virus is also in the virion. J. Virol. 14:1126-1131. 6. IhIe, J. N., J. C. Lee, J. Longstreth, and M. G. Hanna. serum mouse 1976. In R. L. Crowell (ed.), Tumor virus infections and immunity, p. 197-203. University Park Press, Baltimore. 7. Kessler, S. W. 1975. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbant: of interaction of antibody-antigen comrecently (Ja foundaparameters nave recently rouna (J. LedbelJeaDetIn addition, we have plexes with protein A. J. Immunol. 115:1617-1624. ter, R. Nowinski, and R. Eisenman, unpublished 8. Laemmli, U. K. 1970. Cleavage of structural proteins data) that the glycosylated polyprotein of the during the assembly of the head of bacteriophage T4.
MuLV internal structural proteins resides on the cell surface in two distinct configurations and that each of these configurations has a unique antigenic activity. Heterogeneity in FMR cell surface antigens strated by of hashetalsoberoen also beendeity demonstrated by the the findings findings of Klein and coworkers (2, 3, 18). These investigators found that the (M)MuLV-associated cell surface antigen (MCSA) was a high-molecularweight glycoprotein (>110,000 daltons) that was weight glycoprotein not a structural component of MuLV. Furthermore, these authors have also shown that mouse antisera prepared against MCSA could not be absorbed ()MuLV or (R)M LV or by purabsorbed byby (R)MuLV pur(M)MuLV. ified gp7O. On this basis, MCSA can be clearly distinguished from the FMR cell surface antigens that we have described here. In addition, it should be stressed that in our studies with several different FMR antisera we have not been able to detect a high-molecular-weight antigen comparable to MCSA. Since our immune precipitation assays have been performed both with glucosmine*abele andwitf glucosamine-labeled and with I251 surface-labeled cells, we have concluded that either our FMR antisera do not contain antibodies against MCSA, or that MCSA is not expressed on the sufface of primary virus-induced leukesurface of primary Friend Friend virus-induced leuke-
mia cells. These discrepancies should be resolved in the near future upon the immunochemical identification of MCSA. ACKNOWLEDGMENTS
These studies were supported by Public Health Service grant CA 18074 from the National Cancer Institute and by Public Health Service contract CP 61009. LITERATURE CITED 1. Eckner, R. J., and R. A. Steeves. 1972. A classification of the murine leukemia viruses: neutralization of pseudotypes of Friend spleen focus-forming virus by type specific murine antisera. J. Exp. Med. 136:832-884. 2. Fenyo, E. M., G. Grundner, and E. Klein. 1974. Virusassociated surface antigens on L cells and Moloney lymphoma cells. J. Natl. Cancer Inst. 52:743-751.
9.
Nature (London) 227:680-685. Ledbetter, J., and R. C. Nowinski.
1977. Identification of the Gross cell surface antigen associated with murine
leukemia virus-infected cells. J. Virol. 23:315-322.
10. Ledbetter, J., R. C. Nowinski, and S. Emery. 1977. Viral proteins expressed on the surface of murine leukemia cells. J. Virol. 22:65-73.
1. Lilly, F., and R. Steeves. 1974. Antigens of murine leukemia viruses. Biochim. Biophys. Acta 355:105-118. 12. Micheel, B., and D. Bierwolf. 1969. Demonstration of Graffi virus-induced surface antigens of leukemia cells by indirect immunoferritin technique. Exp. Cell Res. 13. Nowinski, R. C., S. L. Kaehler, and R. R. Burgess. 1974. Immune response in the mouse to endogenous leukemia viruses. Cold Spring Harbor Symp. Quant.
~~~~~~~54:268-271.
Biol. 39:1123-1128. 14. Nowinski, R. C., and P. A. Klein. 1975. Anomalous
reactions of mouse alloantisera with cultured tumor cells. II. Cytotoxicity is caused by antibodies to leuke-
mia viruses. J. Immunol. 115:1261-1268.
R. C., and A. Watson. 1976. Immune re15- Nowinski, sponse of the mouse to the major core protein (p30) of
ecotropic leukemia viruses. J. Immunol. 117:693-696.
16. Old, L. J., E. A. Boyse, and E. Stockert. 1964. Typing of mouse leukemias by serological methods. Nature
(London) 201:777-779. (Gross) leukemia antigen. Cancer Res. 25:813-819.
17. Old, L. J., E. A. Boyse, and E. Stockert. 1965. The G
18. Siegert, W., E. M. Fenyo, and G. Klein. 1977. Separation of the Moloney leukemia virus-determinal cell surface antigen (MCSA) from known virion proteins associated with the cell membrane. Int. J. Cancer 20:75-82.
19. Snyder, H. W., Jr., E. Stockert, and E. Fleissner. 1977. Characterization of molecular species carrying Gross cell surface antigen. J. Virol. 23:302-314. 20. Steeves, R. A. 1968. Cellular antigen of Friend virus induced leukemias. Cancer Res. 28:338-342. 21. Strand, M., and J. T. August. 1975. Structural proteins of RNA tumor viruses as probes for viral gene expression. Cold Spring Harbor Symp. Quant. Biol. 39:1109-1116. 22. Tung, J. S., T. Yoshiki, and E. Fleissner. 1976. A core polyprotein of murine leukemia virus on the surface of mouse leukemia cells. Cell 9:573-578. 23. Vitetta, E. S., S. Baur, and J. Uhr. 1971. Cell surface immunoglobulin. Isolation and characterization of immunoglobulin from mouse splenic lymphocytes. J. Exp. Med. 134:242-264. 24. Wettstein, P. J., R. Krammer, R. C. Nowinski, C. S. David, J. A. Frelinger, and D. C. Shreffler. 1976. A cautionary note regarding Ia and H-2 typing of murine lymphoid tumors. Immunogenetics 3:507-516.
JOUJRNAL OF VIROLOGY, June 1978, p. 805-812 0022-538X/78/0026-0805$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Identification of an FMR Cell Surface Antigen Associated with Murine Leukemia Virus-Infected Cells ROBERT C. NOWINSKI,* SANDRA EMERY, AND JEFFREY LEDBETTER Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 Received for publication 27 December 1977
FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virusinfected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp7O, p15(E), and p12(E) from detergent-disrupted preparations of [3H]leucinelabeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp7O is directed against typespecific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted '25I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelope protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product. Infection of mouse cells with murine leukemia virus (MuLV) results in the induction of virusspecific cell surface antigens. Two serologically distinct antigenic systems have been identified: the FMR antigens (16) are found on the surface of cells that are infected with exogenous MuLV [Friend, (F)MuLV; Moloney, (M)MuLV; and Rauscher, (R)MuLV], whereas the G antigens (17) are found on the surface of cells that are infected with endogenous or Gross type MuLV. A serological distinction between these viruses. can be demonstrated by neutralization of viral infectivity (1,11) and by radioimmunoassay with viral envelope proteins (21). In general, viruses of the FMR group tend to be strongly antigenic in mice. As a result, transplants of virus-infected tumors or leukemias often are rejected in syngeneic mice (16). Hyperimmunization of mice that have regressed FMR leukemias with increasing inocula of virus-infected cells leads to the production of cytotoxic FMR typing antisera. These antisera react with cells infected by FMR viruses, but they do not react with normal tissues from mice of high or low leukemic strains, or with tumors or leukemias that occur spontaneously in mice (16). In contrast, viruses of the G group tend to be weakly antigenic in mice, and antisera cannot be prepared by syngeneic immunization. Instead, antisera against G antigens are made by immunization of C57BL/6 mice with the AKR leuke-
mia K36 (17). These antisera, although produced by allogeneic immunization, are cytotoxic for histocompatible C57BL/6 leukemias that are induced by Gross passage A virus. Tissues from mice of diverse genetic backgrounds can be examined for G antigens by absorption tests with these typing antisera. In such tests, G antigens are found on the surface of normal lymphoid tissues and spontaneous leukemias of mice of high leukemic strains. These antigens are not found, however, on the surface of normal tissues of mice of low leukemic strains, and they occur in only low amounts on the surface of FMR virus-induced leukemias (17). FMR typing antisera react in immune electron microscopy with both the envelope of budding viruses and with regions of the cell surface that do not contain virions (12). Since these FMR antisera also neutralize the infectivity of FMR viruses (1, 20), it is possible that these antigens represent viral envelope proteins. However, studies from three laboratories suggest that two different populations of antibodies in FMR antisera are responsible for neutralization and cytotoxicity (2-5, 18, 20, 21). To date, this question has not. been resolved by biochemical analysis. We describe here an immunochemical analysis of FMR-specific viral and cell surface antigens. These studies demonstrated that FMR antisera reacted strongly with type-specific antigens of the gp70 envelope protein in virions
805
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NOWINSKI, EMERY, AND LEDBETTER
and on the surface of virus-infected cells. These antisera did not react with any other FMR-specific cell surface proteins. MATERIALS AND METHODS Animals and cells. BALB/c mice were purchased from the Jackson Laboratories. FBL3 and RBL5 were C57BL/6 leukemias that were induced by (F)MuLV and (R)MuLV, respectively. LST'RA was a BALB/c leukemia that was induced by (M)MuLV. These leukemias were kindly provided by Alex Fefer, Fred Hutchinson Cancer Research Center, and have been maintained by serial transplantation of ascites cells in C57BL/6 mice or by growth in vitro. EdG2 was a C57BL/6 leukemia that was induced by Gross passage A virus; it was maintained by transplantation of spleen cells in C57BL/6 mice or by growth in vitro. Primary Friend virus-infected spleen cells [referred to as (F)MuLV leukemia cells] were obtained from BALB/c mice 10 to 14 days after intravenous inoculation of NB-tropic (F)MuLV. K36 was a transplanted ascites variant that was derived from a spontaneous AKR leukemia. Viruses. All viruses were purified by density-gradient centrifugation. (F)MuLV and (M)MuLV were obtained from tissue culture fluids of FLB3 and LSTRA cells. (R)MuLV was obtained from tissue culture fluids of chronically infected JLSV-9 cells. (A)MuLV was obtained from tissue culture fluids of the AKR pIl embryo fibroblast cell lines. Proteins of these viruses were radiolabeled by growth of the virusinfected cells overnight in culture medium supplemented with ['H]leucine (20 ,iCi/ml of culture fluid). For absorption studies, (R)MuLV and (A)MuLV were obtained from the National Cancer Institute as 1,000fold concentrates from tissue culture fluids. Antisera. FMR antisera were prepared in mice by immunization with virus or virus-infected cells. FMR(NNB) antiserum was prepared by immunization of BALB/c mice with (F)MuLV; mice were initially inoculated with N-tropic (F)MuLV and then hyperimmunized with NB-tropic (F)MuLV. FMR(RCSB) antiserum was prepared by immunization of BALB.B (H-2") congenic mice with the syngeneic (F)MuLVinduced BALB.B RCSB leukemia. FMR(NNB) and FMR(RCSB) antisera were kindly provided by F. Lilly, Albert Einstein College of Medicine. FMR murine sarcoma virus (MSV) antiserum was obtained from BALB/c mice that had regressed transplants of syngeneic MSV-induced sarcomas. FMR(FBL3) antiserum was prepared in C57BL/6 mice by immunization with C57BL/6 FBL3 leukemia cells. FMR(MSV) and FMR(FBL3) antisera were kindly provided by Joseph Brown and A. Fefer of the Fred Hutchinson Cancer Research Center. Antisera against G antigen were prepared by immunization of C57BL/6 mice with the AKR K36 leukemia. Goat antisera against the purified proteins gp7O, p30, p15, p12, and plO of (R)MuLV were obtained from the National Cancer Institute. Goat antiserum against the purified p15 protein of (A)MuLV was kindly provided by E. Fleissner (Sloan-Kettering Institute). Rabbit antiserum against purified p15(E) protein of (R)MuLV was kindly provided by S. Oroszlan (Frederick Cancer Center). Immune precipitation of viral proteins. [1Hl-
J. VIROL.
leucine-labeled MuLV was solubilized on ice by treatment for 30 min with 0.5% Nonidet P-40 (NP-40) in phosphate-buffered saline (PBS), pH 7.4. Aggregated viral proteins and nondisrupted viral components were then removed by centrifugation at 100,000 x g for 20 min. Radioimmune precipitation (RIP) assays contained 50 Al of virus extract (105 cpm) and 2 pl of antiserum in a total volume of 200 Al of PBS buffer containing 0.5% NP-40. Reactions were terminated after a 1-h incubation at 4°C by addition of 0.5 mg of Staphylococcus aureus (7). After a 30-min incubation, the bacteria and their bound immune complexes were removed from the reaction mixture by centrifugation through a cushion of 5% sucrose (in PBS) containing 3% NP-40. The bacteria were then washed three additional times in PBS with 0.5% NP-40 and then treated with sodium dodecyl sulfate-containing sample buffer. RIP reactions were analyzed by polyacrylamide gel electrophoresis in 10% slab gels in the presence of sodium dodecyl sulfate (8). Immune precipitation of cell extracts. Radiolabeling of cell membranes by the [2'I]lactoperoxidase method was performed by the method of Vitetta et al. (23). Briefly, 5 x 107 viable cells were suspended in 1 ml of PBS containing 100 yg of lactoperoxidase and 3 mCi of 12'1; this reaction mixture was activated by the addition of two pulses of 0.06% H202 at 5-min intervals. The entire labeling procedure was performed on ice with continuous shaking; viability of cell suspensions (determined by trypan blue exclusion) before and after radiolabeling was >95%. The labeled cells were then disrupted with gentle Vortex mixing in 0.5% NP-40 for 30 min at 4°C; nonsolubilized cellular structures were removed by centrifugation at 100,000 x g for 45 min. The supernatant containing the solubilized membrane proteins was frozen at -20°C in small portions for future analysis. Conditions for the RIP assays that were performed with these cell extracts were similar to those with viral proteins (see above), with the exception that the RIP assays with cell extracts contained 106 to 10' cpm of input antigen. Complement-dependent cytotoxicity. Assays for complement-dependent cytotoxicity were performed by the 5"Cr release method. Briefly, 5 x 10" viable cells in 500 Al of RPMI 1640 medium containing 15% fetal calf serum were labeled with 250 ,uCi of 5'Cr by incubation at 37°C for 1 h with frequent, gentle agitation. The assay was performed in the wells of a microtiter plate (Falcon Microtest II); each well of the plate contained (i) 104 "Cr-labeled cells in 50 ,ul of diluent, (ii) 50 ,ul of diluted antiserum, and (iii) 50 ,ul of rabbit serum (diluted 12-fold) as a complement source. Rabbit sera used as the source of complement were initially selected for low toxicity on mouse thymocytes and were then absorbed in the presence of EDTA with pooled normal lymphoid tissues. The diluent for all procedures was RPMI 1640 medium containing 15% fetal calf serum. The plates were incubated at 37°C for 45 min, and the reaction was stopped by the addition of 100 Al of cold diluent to each well. Intact cells were removed from the reaction mixture by centrifugation at 1,000 rpm for 10 min in a refrigerated centrifuge, and a constant volume of each of the supernatants was removed for the determination of the amount of "Cr released. Calculations were made as
VOL. 26, 1978
FMR CELL SURFACE ANTIGEN
follows: percent specific 5'Cr release = (experimental release) - (control release)/(maximum release) (control release). Maximum 5'Cr release was determined by three cycles of freeze-thawing of the radiolabeled cells. Control release was the highest value that was scored in either of the three controls that were performed with each experiment: (i) spontaneous release (no antibody or complement present), (ii) complement release (no antibody present), and (iii) antibody release (no complement present). Results of experiments in which the 5"Cr release of any of the controls exceeded 10% of the maximum release value were excluded.
RESULTS Cytotoxic assays for FMR and G cell surface antigens. The reactions of different FMR and G antisera with virus-induced leukemias was determined by cytotoxic reactions with 5'Cr-labeled target cells. FMR(RCSB) antiserum showed a high degree of type specificity for the] FMR target cell. This antiserum was cytotoxic for the (F)MuLV leukemia, but not for EdG2 (Fig. 1). In contrast, the FMR(NNB) antiserum was cytotoxic for cells of both the (F)MuLV
807
contained antibodies that reacted predominantly with FMR-specific antigens, and (ii) the FMR(NNB) antiserum contained two populations of antibodies, one which reacted specifi-
c
cally wth FMR antigens and one which reacted with antigens that were common to the (F)MuLV and EdG2 cells. Antisera prepared against G-cell surface antigens (C57BL/6 anti-AKR K36) were cytotoxic for EdG2 cells (Fig. 1). It was not possible, however, to assess by direct cytotoxic tests the specificity of these antisera on the (F)MuLV leukemia, since alloantibodies present in these sera reacted with BALB/c cells. As a result, further analyses of these sera were performed by absorption tests. The cytotoxic reaction of the G typing antiserum on EdG2 was completely 100 A
B
80 60-
leukemia and EdG2. As a result of the difference in activity of these antisera on leukemia cells, the specificity of these reactions was examined further by absorp20tion tests. In these studies (Fig. 2), the cytotoxicity of the FMR(RCSB) and the FMR(NNB) antisera for the (F)MuLV leukemia was found 0 to be absorbed completely by both nonlabeled lo 20 40 80160 10 20 40 80 160 Serum dilution target cells [(F)MuLV leukemia] and by cells of FIG. 1. (F)MuLV Direct cytotoxic assays with FMR and G the (R)MuLV-induced C57BL/6 RBL5 leuke- antisera. (A)5'and leukemia cellswith C57BL/6 meantisera mia. In contrast, cytotoxicity of these Cr and tested EdG2 cells (B) were radiolabeled was not absorbed by the E&G2 leukemia or by in cytotoxic assays with: FMR(RCSB) antiserum normal C57BL/6 spleen cells. It was concluded, (0), FMR(NNB) antiserum (l); and C57BL/6 antitherefore, that (i) the FMR(RCSB) antiserum AKR K36 serum (A).
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64040 80 160 320 640 60 160 320 640 1280
Serum dilution ( reciprocol) FIG. 2. Cytotoxic absorption tests demonstrating the FMR and G antigenic systems. (F)MuLV leukemia cells were radiolabeled with 5Cr and tested in cytotoxic assays with FMR(NNB) antiserum (A) and FMR(RCSB) antiserum (B); C57BL/6 E.3G2 cells were radiolabeled with 51Cr and tested in cytotoxic assays with C57BL/6 anti-AKR K36 antiserum (C). Reactions of unabsorbed sera are shown by the dashed lines. Antisera were each absorbed at 1/20 dilution with 10O nonlabeled cells and then tested for residual activity on "Cr-labeled cells. Cells used for absorption included: (F)MuLV leukemia cells (O); (R)MuLV-induced leukemia C57BL/6 RBL5 (-); C57BL/6 normal spleen (A); and C57BL/6 EPG2(0).
808
NOWINSKI, EMERY, AND LEDBETTER
J. VIROL.
absorbed by nonlabeled target cells (E6G2), but only partially by the (F)MuLV and (R)MuLV leukemias (Fig. 2). Thus, the G antigen was expressed in high levels on the E6G2 cell, but only partially on the (F)MuLV and (R)MuLV leukemia cells. The cytotoxicity of the G antiserum was not absorbed by normal C57BL/6 spleen cells (Fig. 2). Absorption of cytotoxic antibodies by purified viruses. The relationship of the FMR cell surface antigen to the proteins of MuLV was explored by cytotoxic absorption tests with density-gradient purified viruses. Preparations of (R)MuLV and (A)MuLV were frozen and thawed five times (to disrupt the viral envelope) and then used for absorption of FMR typing antisera. The cytotoxicity of both FMR(RCSB) and FMR(NNB) antisera for the (F)MuLV leukemia was absorbed completely by (R)MuLV (Fig. 3). These cytotoxic reactions were not absorbed by (A)MuLV. Immune precipitation of MuLV proteins by FMR and G antiserum. RIP reactions of FMR(NNB) and G antisera with FMR MuLV and (A)MuLV are shown in Fig. 4. FMR(NNB) antiserum precipitated the gp7O, p15(E), and P12(E) proteins from each of the FMR viruses [(F)MuLV, (M)MuLV, (R)MuLV], as well as from the (A)MuLV. G antiserum, on the other hand, precipitated primarily p30 protein from each of these viruses. The G antiserum also reacted to a lesser extent with the gp7O and
p15(E) proteins of each of these viruses. RIP reactions of (F)MuLV and (A)MuLV with several different FMR antisera [FMR(NNB), FMR(RCSB), and FMR(MSV)] and with the natural immune serum of an I strain mouse are shown in Fig. 5. These antisera reacted primarily with the gp7O, p15(E), and p12(E) proteins of the viruses. Although additional minor reactions were observed with p30 and plO, these reactions varied from one experiment to another and were considered to be the result of nonspecific precipitation. Quantitative differences in the reactions of mouse sera with FMR and (A)MuLV (Fig. 5) presumably reflected the titer of type-specific antibodies that were present in these sera. In this regard, the FMR(NNB) antiserum reacted equally well with the gp7O and p15(E) proteins of (F)MuLV and (A)MuLV, whereas the FMR(RCSB) antiserum reacted preferentially with the gp7O, p15(E), and p12(E) proteins of the (F)MuLV. The FMR(MSV) antiserum reacted equally well with the p15(E), p12(E), and gp7O proteins of (F)MuLV and (A)MuLV, whereas the I serum reacted preferentially with the p15(E) and gp7O proteins of (A)MuLV. Further analysis of these reactions was therefore performed by absorption tests with purified viruses of the FMR and G groups. The FMR(NNB) antiserum strongly precipitated gp7O, p15(E), and p12(E) proteins from (F)MuLV (Fig. 6). Absorption of this antiserum with (A)MuLV removed reactivity against the p15(E) and p12(E) proteins, but did not influ100- A -B ence the reaction of this antiserum with the gp7O protein. [By densitometric measurements, this 80absorption resulted in a >99% reduction of the .. ' . p15(E)/p12(E) reaction; under these same coni *N cS \ % % there was retention of 86% of the gp7O ditions, 6 60 -\ %Xs , reaction.] Absorption of the same pool of \ \FMR(NNB) antiserum with (R)MuLV com%% \\ 4 40\ pletely removed reactivity with the gp7O, p15(E), % and p12(E) proteins of (F)MuLV. The results of absorption tests with G antisera 20 also are presented in Fig. 6. G antiserum primarily precipitated p30 protein from (A)MuLV. Absorption of this antiserum with (A)MuLV completely removed the reaction of this antiseSerum dilution (reciprocal) rum with (A)MuLV, whereas absorption with FIG. 3. Cytotoxic absorption ofFMR antisera with (R)MuLV resulted in a considerable, but not FMR and AKR MuL V. (F)MuL V leukemia cells were complete, loss of precipitating activity against radiolabeled with 5'Cr and tested in cytotoxic assays p30 protein. with FMR(NNB) antiserum (A) and FMR(RCSB) It was concluded from these studies that the antiserum (B). Reactions of unabsorbed sera are FMR antisera reacted primarily with the gp7O, shown by the dashed lines. Antisera were each ab- p15(E), and p12(E) proteins of MuLV. Reactions sorbed at 1/40 dilution with 100 jug of density-gra- with the pl 2(E) proteproteins ins dient purified (R)MuLV (-) or (A)MuLV (CJ). Virus with the p15(E) and p12(E) were based based preparations were initially frozen and thawed five exclusively on the detection of group-specific times to partially disrupt the viral envelope and per- antigens. In contrast, the reactions of FMR anmit accessibility of antibody to the internal structural tisera with gp7O were based on the detection of proteins. type-specific and group-specific antigens. The
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VOL. 26, 1978
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FIG. 4. Immune precipitation of MuLVproteins by FMR and G typing antisera. FMR(NNB) and C57BL/6 anti-AKR K36 serum were reacted in RIP reactions with NP-40-disrupted ['Hileucine-labeled MuLV. (1) (A)MuLV; (2) (A)MuLV tested in RIP with FMR (left panel) or G (right panel) antisera; (3) (F)MuLV; (4) (F)MuL V tested in RIP with FMR (left panel) and G (right panel) antisera; (5) (M)MuL V; (6) (M)MuL V tested in RIP with FMR (left panel) and G (right panel) antisera; (7) (R)MuL V; (8) (R)MuL V reacted in RIP with FMR (left panel) and G (right panel) antisera.
FMR(RCSB) antiserum showed a predominant reaction with FMR-specific antigens of gp7O. These studies also confirmed the findings of cytotoxic assays, suggesting that antibodies to gp7O protein may be responsible for the typespecific cytotoxic activity of FMR antisera. Immune precipitation of FMR and G cell surface antigens. To determine the presence of viral proteins on the surface of the FMR typing cell, RIP reactions were performed with NP-40 lysates of ['25I]lactoperoxidase-labeled (F)MuLV leukemia cells. Immune precipitation reactions presented in Fig. 7 demonstrated that four different FMR antisera [FMR(NNB), FMR(RCSB), FMR(MSV), and FMR(FBL3)] reacted primarily with a 70,000-dalton protein on the surface of (F)MuLV leukemia cells. Immune precipitation studies with NP-40 lysates of [3H]glucosamine-labeled (F)MuLV leukemia cells (data not shown) confirmed these findings. The 70,000-dalton protein that was precipitated by the FMR antisera coincided in migration with the gp70 that was precipitated by goat anti-gp7O serum. Although the mouse antisera also precipitated smaller amounts of lower-molecularweight proteins, the total patterns of reaction of these sera were qualitatively and quantitatively similar to that observed with anti-gp7O serum. A similar reaction with gp7O was observed with G antiserum and with the serum from a normal I strain mouse. In addition, the G antiserum precipitated an 85,000-dalton protein from the cell extract. This protein coincided in migration with an 85,000-dalton protein that was precipitated by goat anti-p30 serum. This 85,000dalton protein has been previously demonstrated (8, 9, 18, 21) to be one of the glycosylated polyprotein precursors of the internal structural
proteins (p30, p15, p12, and plO) of MuLV. Reactions between ['25I]lactoperoxidase-labeled EdG2 cells and mouse antisera are presented in Fig. 8. FMR antisera [FMR(NNB), FMR(RCSB), and FMR(MSV)] precipitated only gp7O protein from this cell extract; these sera did not react with the glycosylated polyproteins or with other viral proteins that were present on the surface of these cells. In contrast, antisera prepared against G antigen reacted with gp7O and with two additional proteins of 85,000 and 95,000 daltons. The 85,000- and 95,000-dalton proteins corresponded in migration to the two glycosylated polyproteins that were precipitated by anti-p30 serum. A comparison of the relative activities of FMR antisera with lysates of different cell types demonstrated that the FMR(RCSB), FMR(MSV), and FMR(FBL3) antisera reacted to a greater extent with the gp7O from (F)MuLV leukemia cells than with the gp7O from EdG2. In contrast, the FMR(NNB) antiserum reacted equally well with the gp7O from (F)MuLV and EdG2 leukemia cells. This was consistent with the known distribution of type- and group-reactive antibodies in these antisera. In line with these findings, the natural immune serum of the I strain mouse was found to react preferentially with the gp7O of EdG2 cells. It was concluded from these studies that the (F)MuLV and EdG2 leukemia cells contained on their cell surface the viral envelope protein gp7O and the glycosylated polyprotein precursors of the internal structural proteins of MuLV. FMR antisera reacted with the gp7O proteins of these cells, but not with the glycosylated core polyproteins. In contrast, the G antiserum reacted with both gp7O and the glycosylated polyproteins.
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communication) and to be cytotoxic for virusinfected cells. In absorption tests the cytotoxic activity of FMR antisera was removed by disrupted density-gradient purified FMR MuLV and by FMR MuLV-infected cells, but not by purified endogenous MuLV or by cells infected with endogenous viruses. These findings pro____ * ~.XQvided evidence for a link between the FMR cell surface antigens and FMR viral proteins. This possibility was strengthened by the observation that FMR antisera contained antibodies against
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FIu .6. Absorption analysis of FMR and G antisera. ['Hjleucine-labeled NP-40-disrupted preparations were reacted in RIP assays with mouse antisera. (1) nontreated (A)MuLV; (2) RIP of (A)MuLV with C57BL/6 anti-AKR K36 serum; (3) RIP of (A)MuLV with C57BL/6 anti-AKR K36 serum that was absorbed with 100 MLg of (A)MuLV; (4) RIP of (A)MuLV with C57BL/6 anti-AKR K36 serum that was absorbed with 100 Mg of (R)MuLV; (5) nontreated (F)MuL V; (6) RIP of (F)MuL V with FMR(NNB) antiserum; (7) RIP of (F)MuLV with FMR(NNB) antiserum that was absorbed with 100 Ag of (A)MuL V; (8) RIP of (F)MuLV with FMR(NNB) antiserum that was absorbed with 100Mg of (R)MuLV.
_-
See_ _
FIG. 5. Immune precipitation of proteins from MuL V by mouse antisera. ['H]leucine-labeled NP40-disrupted preparations of (F)MuL V and (A)MuL V were reacted in RIP assays with: (1) FMR(NNB) antiserum; (2) FMR(RCSB) antiserum; (3) FMR(MSV) antiserum; (4) I strain normal serum. Track (5) contains the nontreated virus control.
DISCUSSION
In efforts to identify the FMR cell surface antigen, we have analyzed the reactivity of a variet of antisera of antisera that.were that were prepared prepared in in mice variety against FMR MuLV or FMR MuLV-infected cells. These antisera were known to neutralize the infectivity of FMR viruses (F. Lilly, personal mice
FIG. 7. Immune precipitation of proteins from (F)MuL V leukemia cells by mouse antisera. (F)MuL V leukemia cells were surface labeled in vitro with 125I by the lactoperoxidase method and then lysed in NP40-containing buffer RIP reactions were performed with: (1)FMR(NNB) antiserum; (2) FMR(RCSB) antiserum; (3) FMR(MSV) antiserum; (4) I strain nor-
mal serum; (5) C57BL/6 anti-AKR K36 serum; (6) FMR(FBL3) antiserum; (7) anti-(R)MuLV gp7O serum; and (8) anti- (R)MuL V p30 serum. Track (9) contains '25I-labeled marker proteins gp70 and p30.
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onstrated by cytotoxic absorption tests and by immune precipitation assays with extracts of radiolabeled cells. In most instances, the FMRinfected cells produced FMR-specific products in excess of the products of endogenous MuLV. These findings agree with those of Old et al.
W ~~~~~~~(17). 4W ~nTheidentification of the FMR cell surface antigen as gp70 protein is in conflict with the findings of other investigators (2-4, 18, 20). Two possible explanations for these conflicts could include: (i) the FMR antisera used by different investigators are dissimilar, and (ii) there are multiple FMR cell surface antigens. A discussion FIG. 8. Immune precipitation of proteins from of each of these possibilities is presented below: (i) We described here that FMR antisera conC57BL/6EdG2 cells by mouse antisera. NP-40 lysates of "5'I surface-labeled E3G2 cells were reacted in RIP tained various ratios of antibodies that were assays with: (1) FMR(NNB) antiserum; (2) directed against antigens of the envelope proFMR(RCSB) antiserum; (3) FMR(MSV) antiserum; teins of both FMR MuLV and endogenous (4) I strain normal serum; (5) C57BL/6 anti-AKR K36 MuLV. The occurrence of antibodies in these serum; (6) anti-(R)MuLV gp70 serum; and (7) anti- antisera that reacted with endogenous MuLV (R)MuLVp3O serum. Track (8) contains '25Ilabeled was attributed to the fact that (a) the mice used marker proteins gp 70 and p30. to generate the FMR antisera were of strains that naturally developed an immune response to the viral envelope proteins gp70, p15(E), and endogenous viruses (13), and (b) the FMR cells p12(E). In absorption tests with viruses of the used for immunization contained products of FMR and G serotypes, it was found that the endogenous MuLV on their cell surface. The immune response to p15(E) and p12(E) was presence of several distinct populations of antidirected against group-specific antigens, and as body [directed against group- and type-specific such, it was possible to exclude these proteins as antigens of gp70, p15(E), and p12(E)] in FMR candidates for the FMR antigen. Instead, evi- antisera, as well as the differential expression of dence from these absorption tests suggested that endogenous MuLV in FMR cells, might be exgp7O was the sole FMR-specific viral protein pected to cause complications in the analysis of that was immunologically identified by the the FMR antigen. It can be envisaged that under mouse. This conclusion was substantiated by the certain circumstances the cytotoxic activity of demonstration that FMR antisera precipitated FMR antisera for selected target cells could be only gp7O from the membrane of FMR MuLV- the result of reactions with gp70 antigens of endogenous MuLV, rather than with antigens of infected cells. Similar analysis with G typing antiserum led FMR MuLV. Or alternatively, the FMR antisera to the conclusion that this serum detected anti- may react to a major extent with p15(E) and genic determinants of the major core protein p12(E) proteins, rather than with gp70. In this (p30) of endogenous MuLV (14). The G anti- regard, we have previously shown (14, 24) that genic determinants of p30 protein were repre- a variety of alloantisera that were prepared in sented on the cell surface in the form of two C57BL/6 and BALB/c mice against H-2 and Ia serologically distinct glycosylated polyproteins antigens contained high titers of contaminating (9, 19). The 95,000-dalton polyprotein contained antibodies that were directed against endogeantigens of p30, p12, and plO, whereas the nous MuLV. As a consequence of these contam85,000-dalton polyprotein contained antigens of inating anti-viral antibodies, the H-2 and Ia alp30 and p12, but not plO. Similar findings have loantisera were anomalously cytotoxic for syngeneic tumor and leukemia cells that expressed been described by Snyder et al. (19). Cells infected with endogenous MuLV con- antigens of endogenous MuLV on their cell surtained gp7O and the two glycosylated core poly- face. (ii) Studies by Steeves (20) demonstrated that proteins as constituents of their membranes. These cells did not contain FMR-specific anti- the virus neutralizing activity of FMR antisera gens on their cell surface. In contrast, cells in- was independent of their cytotoxic activity. fected with FMR MuLV contained the protein These results have been confirmed by Freedman products of endogenous MuLV, as well as FMR- et al. (4). The apparent conflict between these specific proteins, on their cell surface. This dual findings and those presented here could be exexpression of FMR and G antigens on the cell plained by the presence of two antigenically surface of FMR MuLV-infected cells was dem- distinct sites on the gp7O protein, one of these _
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NOWINSKI, EMERY, AND LEDBETTER
sites being responsible for the binding of neutralizing antibody, and the other for the, binding antibody.A related hypothess ofofcytotoxic cytotoxic antibody. A related hypothesis.as has been presented by Lilly and Steeves (11). These conjectures are supported by the studies of Ihle et al. (6) in which it was found that gp7O protein had a different antigenic configuration in the envelope of the virion than it had in virus-free regions of the cell membrane. These authors (6) demonstrated by immune electromicroscopy anti-gp7O that both ootn goat goat antl-gp7u serum and ana mouse natnatural immune serum reacted with the budding virus, whereas only the goat anti-gp7O serum reacted with virus-free regions of the cell surface.
J. VIROL.
3. Fenyo, E. M., and G. Klein. 1976. Independence of the Moloney virus induced surface antigen (MCSA) in immunoand membrane associatedcellvirion selected lymphoma sublines. antigens Nature (London) 260:355-356. 4. Freedman, H. A., F. Lilly, and R. A. Steeves. 1975. Antigenic properties ofcultured tumor lines derived from spleens of Friend virus-infectedcellBALB/c and
BALB/c-H-2b mice. J. Exp. Med. 142:1365-1376.
5. Friedman, M., F. Lilly, and S. G. Nathenson. 1974.
Cell surface antigen induced by Friend murine leukemia virus is also in the virion. J. Virol. 14:1126-1131. 6. IhIe, J. N., J. C. Lee, J. Longstreth, and M. G. Hanna. serum mouse 1976. In R. L. Crowell (ed.), Tumor virus infections and immunity, p. 197-203. University Park Press, Baltimore. 7. Kessler, S. W. 1975. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbant: of interaction of antibody-antigen comrecently (Ja foundaparameters nave recently rouna (J. LedbelJeaDetIn addition, we have plexes with protein A. J. Immunol. 115:1617-1624. ter, R. Nowinski, and R. Eisenman, unpublished 8. Laemmli, U. K. 1970. Cleavage of structural proteins data) that the glycosylated polyprotein of the during the assembly of the head of bacteriophage T4.
MuLV internal structural proteins resides on the cell surface in two distinct configurations and that each of these configurations has a unique antigenic activity. Heterogeneity in FMR cell surface antigens strated by of hashetalsoberoen also beendeity demonstrated by the the findings findings of Klein and coworkers (2, 3, 18). These investigators found that the (M)MuLV-associated cell surface antigen (MCSA) was a high-molecularweight glycoprotein (>110,000 daltons) that was weight glycoprotein not a structural component of MuLV. Furthermore, these authors have also shown that mouse antisera prepared against MCSA could not be absorbed ()MuLV or (R)M LV or by purabsorbed byby (R)MuLV pur(M)MuLV. ified gp7O. On this basis, MCSA can be clearly distinguished from the FMR cell surface antigens that we have described here. In addition, it should be stressed that in our studies with several different FMR antisera we have not been able to detect a high-molecular-weight antigen comparable to MCSA. Since our immune precipitation assays have been performed both with glucosmine*abele andwitf glucosamine-labeled and with I251 surface-labeled cells, we have concluded that either our FMR antisera do not contain antibodies against MCSA, or that MCSA is not expressed on the sufface of primary virus-induced leukesurface of primary Friend Friend virus-induced leuke-
mia cells. These discrepancies should be resolved in the near future upon the immunochemical identification of MCSA. ACKNOWLEDGMENTS
These studies were supported by Public Health Service grant CA 18074 from the National Cancer Institute and by Public Health Service contract CP 61009. LITERATURE CITED 1. Eckner, R. J., and R. A. Steeves. 1972. A classification of the murine leukemia viruses: neutralization of pseudotypes of Friend spleen focus-forming virus by type specific murine antisera. J. Exp. Med. 136:832-884. 2. Fenyo, E. M., G. Grundner, and E. Klein. 1974. Virusassociated surface antigens on L cells and Moloney lymphoma cells. J. Natl. Cancer Inst. 52:743-751.
9.
Nature (London) 227:680-685. Ledbetter, J., and R. C. Nowinski.
1977. Identification of the Gross cell surface antigen associated with murine
leukemia virus-infected cells. J. Virol. 23:315-322.
10. Ledbetter, J., R. C. Nowinski, and S. Emery. 1977. Viral proteins expressed on the surface of murine leukemia cells. J. Virol. 22:65-73.
1. Lilly, F., and R. Steeves. 1974. Antigens of murine leukemia viruses. Biochim. Biophys. Acta 355:105-118. 12. Micheel, B., and D. Bierwolf. 1969. Demonstration of Graffi virus-induced surface antigens of leukemia cells by indirect immunoferritin technique. Exp. Cell Res. 13. Nowinski, R. C., S. L. Kaehler, and R. R. Burgess. 1974. Immune response in the mouse to endogenous leukemia viruses. Cold Spring Harbor Symp. Quant.
~~~~~~~54:268-271.
Biol. 39:1123-1128. 14. Nowinski, R. C., and P. A. Klein. 1975. Anomalous
reactions of mouse alloantisera with cultured tumor cells. II. Cytotoxicity is caused by antibodies to leuke-
mia viruses. J. Immunol. 115:1261-1268.
R. C., and A. Watson. 1976. Immune re15- Nowinski, sponse of the mouse to the major core protein (p30) of
ecotropic leukemia viruses. J. Immunol. 117:693-696.
16. Old, L. J., E. A. Boyse, and E. Stockert. 1964. Typing of mouse leukemias by serological methods. Nature
(London) 201:777-779. (Gross) leukemia antigen. Cancer Res. 25:813-819.
17. Old, L. J., E. A. Boyse, and E. Stockert. 1965. The G
18. Siegert, W., E. M. Fenyo, and G. Klein. 1977. Separation of the Moloney leukemia virus-determinal cell surface antigen (MCSA) from known virion proteins associated with the cell membrane. Int. J. Cancer 20:75-82.
19. Snyder, H. W., Jr., E. Stockert, and E. Fleissner. 1977. Characterization of molecular species carrying Gross cell surface antigen. J. Virol. 23:302-314. 20. Steeves, R. A. 1968. Cellular antigen of Friend virus induced leukemias. Cancer Res. 28:338-342. 21. Strand, M., and J. T. August. 1975. Structural proteins of RNA tumor viruses as probes for viral gene expression. Cold Spring Harbor Symp. Quant. Biol. 39:1109-1116. 22. Tung, J. S., T. Yoshiki, and E. Fleissner. 1976. A core polyprotein of murine leukemia virus on the surface of mouse leukemia cells. Cell 9:573-578. 23. Vitetta, E. S., S. Baur, and J. Uhr. 1971. Cell surface immunoglobulin. Isolation and characterization of immunoglobulin from mouse splenic lymphocytes. J. Exp. Med. 134:242-264. 24. Wettstein, P. J., R. Krammer, R. C. Nowinski, C. S. David, J. A. Frelinger, and D. C. Shreffler. 1976. A cautionary note regarding Ia and H-2 typing of murine lymphoid tumors. Immunogenetics 3:507-516.
