Spontaneous regression of friend virus-induced erythroleukemia—VIII. Humoral immune reactivity in regressed and leukemic mice

Leukemia Research Vo[. 5 N o I, p p 41 to 55, 1981 Printed in Great Britain

0145-2126/81/010041-14502.00/0 © 1981 Pergamon Press Lld

SPONTANEOUS REGRESSION OF FRIEND VIRUS-INDUCED ERYTHROLEUKEMIA--VIII. HUMORAL IMMUNE REACTIVITY IN REGRESSED AND LEUKEMIC MICE DAVID L. HINES, MICHAEL DIETZ, JOHN MARCELLETTI and PHILIP FURMANSKI Department of Biology, Michigan Cancer Foundation, 110 East Warren, Detroit, MI 48201 U.S.A. (Received 16 June 1980. Accepted 6 October 1980) Abstract--Mice infected with the RFV strain of the Friend murine leukemia virus complex (FV) develop an erythroleukemia that spontaneously regresses. Sera from regressed mice contain virus neutralizing activity and cytolytic activity for leukemic spleen cells and RFV-indueed erythroleukemia cell lines. The virus neutralizing antibody in regressed sera is directed against antigens on viral gp70 and/or p15(E), while the cytolytic antibody is directed against antigens contained on gp70. p12 and possibly pl5(E). Sera from 24% of regressed mice contain only neutralizing activity, 17°.~ contain only cytolytic activity, 26~o contain both and 33% contain neither activity. The failure to detect anti-RFV or cytotoxic antibody in one third of regressed mice suggests that humoral immunity does not play an important role in causing regression. Additionally, there was no correlation between the presence of cytolytic reactivity in the sera of viremic, leukemic mice and subsequent regression of the disease. However, neutralizing activity in regressed mice was associated with the absence of leukemia recurrence. Regressed mice with neutralizing titers of I: 80 or greater rarely redevelop leukemia, either spontaneously, or following reinoculation with RFV. Key Words: Regressing Friend virus, erythroleukemia, virus neutralization, cytotoxic antibody.

INTRODUCTION INFECTION of susceptible mice with conventional strains of Friend virus (FV) leads to a progressive leukemia characterized by massive splenomegaly, immunosuppression, viremia and death [9, 14]. The regressing strain of Friend virus (RFV) is a specific, stable variant which induces an erythroleukemia initially indistinguishable from that induced by FV, but which spontaneously regresses in about 50~o of the infected animals [34, 35]. Regression is characterized by a return to a histopathologically and virologically normal or near-normal state [34, 38]. The detection of anti-virus and anti-leukemia cell immune reactivity in regressed animals and the dependence of regression on normal thymocyte [10, i6] and macrophage function [28, 29] have led to the suggestion that regression of RFV-induced erythroleukemia is an immunologically mediated event. Potent cell-mediated anti-virus immunity, as measured by the indirect macrophage migration inhibition assay, can be detected in all spontaneously regressed animals [23]. The consistent appearance and magnitude of the cell-mediated response in regressor leukemic mice, i.e. those leukemic mice infected with RFV that will subsequently regress, as well as in fully regressed mice, suggests that this component of the immune system is an important factor in recovery from the disease. Earlier studies of humoral immunity in RFV infected animals demonstrated that mice also produce antibody to virus antigens. Reactivity was detectable by complement fixation [36], neutralization of virus leukemogenic activity [5, 37] and complement-mediated Abbreviations: RFV. regressing strain of Friend virus: FV. Friend virus complex; SFFV, spleen focus forming virus: PFU. plaque forming unit; LSC. leukemic spleen cells. 4I

42

DAVID L. HINES et al.

lysis of leukemic spleen cells [15, 21]. The neutralizing activity was shown to have both type and group specific components [17] and to be associated with the immunoglobulin fraction of regressed sera [11] and not with the oncornavirus inactivating factor present in normal mouse serum [27,31]. Another feature of RFV-induced disease is that approximately half of the animals in which leukemia has spontaneously regressed subsequently redevelop an erythroleukemia that is histopathologically identical to the initial disease [35]. The ability to prolong the disease-free period by active immunotherapy of regressed mice suggests that maintenance of the regressed state is also immunologically mediated [20]. We have investigated the role of virus neutralizing and leukemia cell cytotoxic antibodies during both recovery from the initial leukemic state and prevention of leukemia recurrence. We report here on the specificity of the antibodies in regressed sera and their distribution in individual RFV infected and regressed animals. The data show that humoral immune reactivity is not an essential factor in the process of regression. However, virus neutralizing antibody appears to be important in maintaining the regressed state and preventing leukemia recurrence. METHODS AND MATERIALS Viruses

RFV was prepared as cell-free spleen homogenates (205/0 w/v in phosphate buffered saline) from leukemic mice as described previously [33] and stored at - 7 0 ° C in sealed ampules. Unless otherwise specified weanling mice were inoculated intraperitoneally with 0.5 ml of virus containing 100 ID~o'S. One IDs0 produces leukemia in 50~ of weanling Swiss mice within 25 days. RFV preparations were titrated by the XC plaque assay adapted to 16 mm multiwell dishes [8] with titers expressed in plaque forming units (PFU). The defective spleen focus forming virus (SFFV) was titered by the spleen focus assay [2] with titers expressed in spleen focus forming units (SFFU). An RFV pseudotype of the Kirsten sarcoma virus (KiSV) was used in virus neutralization assays. A culture of nonproducer K-NIH cells I l l was superinfected with 500 XC PFU of RFV. The culture supernatant was harvested 7-14 days later, titered for focus-forming activity and stored at -70°C. 12SI-labeled virus for use in radioimmunoprecipitation was prepared as described [17]. Mice

Random-bred Swiss/ICR and inbred N/PLCR mice were from colonies maintained at the Michigan Cancer Foundation. Mice were checked biweekly for leukemia development and regression by spleen palpation, which we have previously shown to be an accurate indicator of leukemic status [5, 33, 34]. Mice were considered leukemic when spleen weight exceeded 0.5 g and regressed when subsequent to the development of leukemia the spleen weight decreased below 0.5 g. Cells

Leukemic cell suspensions were obtained from the enlarged spleens of mice 12-14 days after inoculation with RFV. Pooled spleens were teased with forceps in RPMI 1630 medium containin$ 10% fetal calf serum (fcs) and 5.0 x I0 -s M 2 mercaptocthanol (complete RPMI 1630 medium). The cells were pelleted and resuspended in 5.0 ml of ice-cold 0.17 M NH4CI for 5 rain to lyse erythrocytes and then 5.0 ml of complete RPMI 1630 was added followed by centrifugation at 400 0. The resuspended cells were then used in cytotoxicity assays (see below). Leukemic spleen cells (LSC) consistently gave low and variable levels of virus-specific cytotoxicity when used as target cells, regardless of the potency of the antiserum used. This was probably due to the heterogeneity of the spleen cell population. To obtain a more consistent target cell population two erythroblast cell lines were derived from RFV-infected spleen cells. These cell lines were both used routinely for detection of cytolytic reactivity in regressed serum. No sera were found which were reactive with leukemic spleen cells and not one of the cell lines. The DI erythroleukemia cell line was established from a local tumor arising after the fifth subcutaneous trocar implant of tissue from the leukemic spleen of an RFV-infected inbred N/PLCR mouse 60 days after virus inoculation. DI cells grow as lightly-adherent or nonadherent oval or fusiform cells and are routinely cultured in RPMI 1640, containing 10% fcs and 5.0 x I0 -s M mercaptoethanol. The uncloned cells exhibit a low ( < 1~/0) level of benzidine positive staining which can be increased by exposure to l% dimethylsulfoxide (DMSO) and other compounds known to induce differentiation of Friend erythroleukemia cell lines [unpublished observations]. The DI P3 cell line was derived from the DI line by three rapid cycles of passage in vivo. In each cycle, a subcutaneous tumor in a syngeneic host was dissociated and the tumor cells grown in vitro in suspension culture only 7 to l0 days. DIP3 cells do not constitutively produce, nor are they inducible for hemoglobin

Antibody in regression of leukemia

43

synthesis detectable by benzidine staining. They are more tumorogenic than D1 cells (LDso'S are 10 and 105 cells respectively) and exhibit a high rate of metastasis in vivo. Neither DI nor DIP3 cells produce infectious murine leukemia virus. Antisera All mouse sera were collected from the retro-orbital sinus. Collection of serum from animals for use in prospective studies was limited to 0.2 ml of blood measured in 0.1 ml capillary tubes. Monospecific goat antisera to Rauscher virus-derived gp70, p30, p15, p12 and pl0 were obtained through the Office of Program Resources and Logistics, Virus Cancer Program, National Cancer Institute. Rabbit antisera to Rauscher virus p15(E) were obtained from Dr. E. Fleissner, Memorial Sloan Kettering Cancer Center and Dr. S. Oroszlan, Frederick Cancer Research Center. Neutralization assay Assays of serum neutralizing activity were carried out as previously described [173. Briefly, the RFV (KiSV) pseudotype virus, diluted to 400 ffu/ml, was incubated for 1 h at 37°C with dilutions of serum. Following incubation, 0.1 ml aliquots were inoculated onto l04 polybrene-treated NIH/3T3 cells in 16 mm wells of plastic dishes (Costar). On day 7 post-infection the cells were fixed, stained with Giemsa and loci of transformation counted microscopically. For neutralization assays with complement, freshly reconstituted, lyophilized guinea pig serum (Colorado Serum Co.) was added to the virus suspension to a final concentration of 5~o, before admixture with the antiserum. Cytotoxicity assays Assays for complement-dependent serum cytotoxicity were performed using either a modification of the 5~Cr release method originally described by Wigzell [46] or a modification of the microtiter method of Terasaki [30]. The 5~Cr release assay was performed in plastic 10 x 75 mm tubes (Falcon, No. 2038). 100 #1 of 5~Cr-labeled target cells (D1, D1P3, or LSC) were added to tubes containing 100BI of test serum diluted with RPMI 1630 + 10~o fcs. The cells and serum were incubated at 4°C for 30rain and then 100~1 of rabbit complement (normal rabbit serum adsorbed with DIP3 cells and diluted 1:10) was added. The cells, serum and complement were incubated at 37°C in a shaking water bath for 90 rain. After this incubation, 0.7 ml of ice-cold PBS was added, the cells were pelleted by centrifugation at 200 g and 0.7 ml of the supernatant withdrawn. Both the supernatant and cell pellet were counted for 5~Cr radioactivity. The total radioactivity in the supernatant fraction was calculated as the counts/rain in the measured supernatant aliquot divided by 0.7. Controls contained, in a final volume of 0.3 ml, either labeled cells alone, labeled cells plus serum alone or labeled ceils plus complement alone. Maximum release of 51Cr in the presence of an antiserum lytic to all of the target cells was obtained using unadsorbed rabbit anti-mouse thymocyte serum and complement. All assays were done at least in duplicate. Percent specific release was calculated by the formula

=lOOx

X-C -

-

M-C'

where X is the soluble counts/min in the experimental group, C is the soluble counts/mln in the complement control and M is the soluble counts/min in the maximum release control. Cytotoxicity assays using the microtiter method were performed in microtest 1 plates (Falcon, No. 3034). This method was used only for screening for antibody or when only very limited quantities of serum were available. All quantitative assays were performed using the 51Cr release method. In the microtiter procedure all mouse sera were tested at a 1 : 10 dilution. Briefly, 5/d of serum diluted with RPMI 1630 + fcs were added to each well of the microtest plate. This was followed by 5/~l of unlabeled target cells (2 x 105 viable cells/ml). After incubation of the cells and serum at 37°C for 45 min, one/~l of adsorbed rabbit complement was added and the cells incubated for an additional 60 min at 37°C. The individual wells were scored for the approximate percent of viable cells using an inverted phase contrast microscope. The controls used were the same as those described for the SlCr release assay. Radioimmunoprecipitation

Virus proteins were immunoprecipitated from 1251-labeled preparations and identified by SDS-PAGE as follows: the virus suspension was diluted with TNE (0.01 M Tris, 0.15 M NaC1 and 0.002 M EDTA, pH 7.4) containing 0.2°Jo NP4o, 100 ~M phenylmethylsulfonyl fluoride and I mg/ml bovine serum albumin. Normal goat serum was added (2 #l) and the mixture incubated for l h at 4°C, followed by the addition of 20/~l of 10~o suspension of Staphylococcus aureus, Cowan strain I, prepared as described by Kessler [25]. After incubation at 4°C for 30 rain. the bacteria were removed by centrifugation (2000g, 20 rain) and the supernatant used for immunoprecipitation. Two microlitres of the appropriate monospecific antiserum or mouse serum was added to 200 ~l of the labeled virus and the mixture incubated at 4°C for 1 h. In some experiments, secondary antisera {rabbit anti-mouse immunoglobulins) were also added. Staphylococcus organisms were then added, incubated and removed, as above. The pellet containing the bacteria and antigen-antibody complexes was then resuspended in 0.5 ml TNE containing 0.SYo NP40 and 0.02% Na azide. The suspension was layered over l ml of a solution of TNE containing 5°/Osucrose, 0.5?0 Na deoxycholate and 3~o NP40. Following centrifugation (2000 g, 10 minl, the pellet was washed with TNE + 0.5°,o NP,o and drained. The complexes were dissociated with

44

DAVID L. HINES et al.

0.01 M phosphate buffer, pH 7.4, 20/0 SDS and 2°/o 2-mercaptoethanol at 100~C for 3 min and the bacteria removed by centrifugation (2000 g, 10 min). The supernatant was then removed and virus proteins were analyzed by SDS-PAGE in 15~o acrylamide, 15 cm gels prepared according to the method of Lae'mmli [26]. The gels were electrophoresed at 20 mA/gel for about 16 h. After drying, the gels were analyzed by autoradiography using intensifying screens. Protein standards (t2~I-labeled bovine serum albumin, ovalbumin and RNase) were included with each gel run for determination of molecular weights. Macrophage infectious center assay The quantitation of virus infection of macrophages was performed as previously described [28].

RESULTS Characterization of anti-virus humoral immune reactivity in reyressed mice To determine which viral components react with regressed sera during neutralization. we compared regressed sera with monospecific antisera against each of the major oncorTABLE I. NEUTRALIZATIONOF RFV BY REGRESSED MOUSESERUM AND MONOSPECIFIC ANT1SERATO VIRALPOLYPEPTIDES 50~o Viral neutralization titer Without complement With complement added

Antiserum Regressed mouse serum Anti-gp70 Anti-pl5(E) Anti-p30 Anti-p 15 Anti-pl2 Anti-10

240 650 70 Neg.* Neg. Neg. Neg.

500 1500 600 Neg. Neg. Neg. Neg.

*Neg., no neutralization at the lowest dilution tested, 1:40.

navirus peptides in neutralization assays both in the presence and absence of complement. In the absence of complement significant neutralization was obtained only with regressed sera and anti-gp70 serum (Table 1). Antisera against p30, p15, p12 or pl0 were negative. In the presence of complement neutralizing activity of regressed sera and anti-gp70 serum was increased 2 to 4-fold (Table 1), but antisera against the gag gene products (p30, p15, p12, pl0 1-43-1)were still negative. Anti-pl5(E), which has some neuA

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FIG. 1. Cytotoxicity of pooled regressed mouse sera and monospecific goat antisera to leukemic spleen cells and DI and DIP3 leukemia cell lines. (A) cytotoxicity by pooled regressed mouse serum; (B) cytotoxicity by anti-pl5; (C) cytotoxicity by anti-gp70; (D) cytotoxicity by anti-pl2. Symbols: leukemic spleen cells as target cells (A); DI cells as targets (0); D1 P3 as targets (©).

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FI(;. 2. SDS polyacrylamide gel electrophoresis of immunoprecipitated '251-labeled RFV components. (A) Control and standard sera; a, anti-gp70; b, anti-p30; c, anti-pl5; d, anti-pl2; e, anti-pl0; f, anti-pl5(E); g, normal rabbit serum; h, normal mouse serum; i, pooled regressed mouse serum; j, hyperimmune mouse serum to RFV; k, hyperimmune mouse serum to RFV leukemic cells; 1 protein molecular weight standards. (B) Individual regressed mouse sera.

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A n t i b o d y in regression of l e u k e m i a

47

tralizing activity against RFV in the absence of complement, exhibited a 9-fold increase in neutralizing titer in the presence of complement, (Table 1). Complement alone had no effect on RFV, nor did heat inactivated complement (56°C, 30 min) either in the presence or absence of anti-pl5(E). These results confirm previous findings in our own and other laboratories on the neutralization of FV strains with monospecific antisera [13, 17, 42]. Sera from mice in which erythroleukemia has regressed also contain antibodies which, in the presence of complement, lyse spleen cells from RFV leukemic mice. Anti-leukemia cell cytotoxicity was quantitated using the 51Cr release cytotoxicity assay. Pooled regressed serum was titered for complement-dependent cytotoxicity against RFV leukemic spleen cells and the two erythroleukemia cell lines (D1 and D1P3) derived from an RFV-infected spleen (Fig. 1A). The cytotoxicity of regressed sera for leukemic spleen cells and D1P3 cells was very similar while reactivity against D1 cells was significantly greater. Adsorption of regressed sera with leukemic spleen cells or density gradient purified RFV removed all reactivity to D1 and D1P3 cells [unpublished observations]. Regressed sera were not cytotoxic to spleen cells, bone marrow cells or erythrocytes from normal mice nor to any cells in the absence of complement. Normal sera were not cytotoxic to any of the target cells used. To identify the virus components on the surface of RFV infected cells which can serve as targets for cytotoxicity, leukemic spleen cells, D1 cells and D1P3 cells were tested for susceptibility to lysis by antisera to the major viral polypeptides. Leukemic spleen cells and the erythroleukemia cell lines were killed by anti-gp70 (Fig. 1C), anti-pl5 (Fig. 1B) and anti-P12 (Fig. 1D), but not by anti-p30 or anti-pl0 (data not shown). D1 and D1P3 cells were also highly susceptible to killing by anti-pl5(E) antisera as detected in the microtiter test for cytotoxicity (data not shown). Reactivity of leukemic spleen cells with anti-pl 5(E) was not determined. The results described above, comparing virus neutralization and leukemic cell cytotoxicity of regressed serum with monospecific antisera to viral polypeptides, suggested that the predominant reactivity in regressed serum was directed against determinants on gp70 and to a lesser extent possibly p15, p12 and pl5(E). The reactivity of regressed serum with RFV proteins was demonstrated more directly by immunoprecipitation of dissociated l zsI-labeled virus proteins followed by SDS-PAGE to identify the components precipitated. Controls included normal mouse serum, monospecific antisera to gp70, p30, p15, p12, pl0 and pl5(E) and hyperimmune mouse anti-RFV antisera. As shown in Fig. 2, the major reactivity was against gp70, but in some sera reactivity was also detected against pl5(E) and p12.

Distribution of humoral immune reactivity in regressed mice To determine if there is an association between the presence of antibody and leukemia regression we examined humoral reactivity in individual regressed mice. Serum samples were collected 2 to 4 weeks after regression from 219 mice. Virus neutralization, both in the presence and absence of complement, and cytotoxicity to both the cell lines were assayed. Both cell lines were used as target cells since they differ in susceptibility to antibodies present in regressed sera (Fig. 1t. The data are shown in Table 2. TABLE 2. DISTRIBUTION OF HUMORAL REACTIVITY IN REGRESSED MICE

Group I II II1 IV

Virus neutralizing antibody

Leukemia cell c y t o t o x i c antibody

N u m b e r of mice

~/o

-+ -+

--+ +

75 52 38 54

34 24 17 25

48

DAvm L. HINESet al.

A mouse was considered reactive if either assay for neutralization (with or without complement) was positive or if cytotoxicity was detected to either D 1 or D 1P3 cells. One hundred and forty-four of the mice were reactive by these criteria (679/0) with substantial numbers of mice showing either cytotoxicity (179/0) or neutralization (249/0) only, demonstrating the independence of these two reactivities. Seventy-five sera (33~o) had no reactivity detectable in either assay. Radioimmunoprecipitation was used to further analyze the reactivity in individual regressed sera (Fig. 2B). All sera that exhibited high neutralizing and/or cytotoxic activity precipitated gp70. Some sera, in addition, precipitated p l 2 or p l 5(E). However, there was no distinct correlation between the amount or the pattern of viral proteins precipitated and the antibody activity detected in the sera. For example, in Fig. 2B, the sera in lanes b and f were strongly reactive in both the neutralization and cytotoxicity assays while the sera in lanes c and e were reactive in only neutralization and cytotoxity, respectively. Sera with no detectable antibody reactivity generally showed little to no specific precipitation of viral proteins. The failure to detect antibody in the sera of about one third of regressed mice does not appear to be related to the time of sampling,. Weekly sequential samples were tested from both reactive and unreactive individual mice, beginning 1 week after regression. No unreactive animals became reactive, although reactivity was diminished in some previously positive animals just prior to leukemia recurrence. Additionally, no regressed serum found to be negative in virus neutralization or cytotoxicity against D1 or D1P3 cells was positive in cytotoxicity assays using spleen cells from leukemic mice. These sera were also unreactive in immunofluorescence assays for detection of cytoplasmic MuLV antigens or complement-dependent lysis of RFV-infected NIH/3T3 cells or SFFVinfected nonproducer NIH/3T3 cells [-3, 44]. These cells express an SFFV-specific cell surface neoantigen [-19] and viral p15 [3-] and were highly reactive in assays of cellmediated immunity in regressed mice [23-]. The negative sera were also unreactive in assays of immunoprecipitation of radiolabeled RFV proteins. Taken together these results strongly imply that the 75 unreactive sera shown in Table 2 contain no appreciable amount of unbound anti-RFV antibody. The possibility that these sera contain anti-RFV antibody in the form of antigen-antibody complexes cannot be excluded and is currently under investigation. Presence of humoral immune reactivity in leukemic mice

The observation that virus neutralizing and leukemia cell cytotoxic activities can be present independently in individual regressed sera suggested that these assays detect antibodies reactive with different RFV-related antigens. Additional evidence for this came from analysis of sera from RFV leukemic mice. Serum was collected from 23 individual RFV infected mice at 26 or 33 days after virus infection, a time when all the animals were leukemic (spleen weight greater than 0.5 g) but just prior to the time regression would be expected to occur. The sera were analyzed for virus neutralization and leukemia cell cytotoxicity as was done for regressed sera. Plasma from the same mice was assayed for the presence of circulating infectious virus using the XC plaque assay, and resident peritoneal marcophages were tested for productive infection with virus. Subsequent regression of erythroleukemia can be accurately predicted in individual leukemic mice by determining whether their peritoneal macrophages are infected with virus [-28]. As shown in Table 3, the spleen weights for this group of animals ranged from 0.69 to 2.35 g. Eleven of twenty-one of the mice had uninfected peritoneal macrophages and, therefore, would have been expected to undergo subsequent spontaneous regression of their erythroleukemia. Seven of the eleven mice with uninfected macrophages also had

Ar.tibody in regression of l e u k e m i a

49

TABLE 3. ANTI-VIRAL AND ANTI-LEUKEMIA CELL HUMORAL REACTIVITY OF R F V LEUKEMIC MICE FOUR WEEKS AFTER VIRUS INFECTION

Spleen weight Mouse No.

*

(g)

Infected peritoneal macrophages

Plasma* virus

Serum titer for R F V neutrafizationt

S e r u m cytotoxicity to l e u k e m i a cells$

1

1.35

--

--

--

+ +

2

1.55

+

+

--

+ + +

3

1.07

--

+

--

--

4

1.24

+

+

--

+ +

5

1.90

+

+

--

+

6 7

1.85 1.20

+ +

+ N.D.§

---

+ + + --

8

2.35

--

+

--

+ +

9

1.31

--

+

--

+++

10

0.92

--

--

--

+ +

11

1.95

--

--

--

+ +

12

1.77

+

+

--

+ + +

13

1.70

+

+

--

+ +

14

1.50

--

--

--

+ + +

15

1.69

N.D.

+

--

+ + + +

16

1.70

+

+

--

17 18

1.54

N.D.

+

--

--

1.10

+

+

--

+++

19

0.75

--

--

80

+ + + +

20

1.23

--

+

--

+ +

21

2.21

+

+

--

+ +

22

1.19

--

--

20

+ + + +

23

0.69

--

--

--

+ + + +

. negative,

< 100 XC PFU

per ml plasma.

t--, negative, < 1:20. ++--, < 1 0 % specific cytotoxicity; + + +. between

50

+ , between 10 and 2 5 ~ specific cytotoxicity; + + , b e t w e e n 2 5 and 5 0 ~ ; and 75~o; + + + + > 7 5 Y o specific cytotoxicity.

§N.D., not tested,

little circulating infectious virus, (less than 100 XC PFU per ml of plasma) and thus may have already been undergoing leukemia regression. Only two of the mice had neutralization titers of 20 or greater. Titers of neutralization of RFV less than 20 are not significant because normal mouse serum often nonspecifically neutralizes RFV to this extent. Both of these neutralization-positive mice also had uninfected peritoneal macrophages and low plasma virus titers. However, the other 5 mice with uninfected macrophages and low plasma virus titers and 4 additional mice with uninfected macrophages (regressors) had no detectable virus neutralizing activity. In contrast to neutralization, antibody cytotoxic to either the D1 or D1P3 leukemia cell lines was detected in 20 of 23 leukemic animals in this particular experiment. This cytotoxicity was complement dependent and specific for leukemia cells. The presence of cytotoxicity against either cell line was not correlated with any other parameter measured in these animals. High levels of cytotoxicity were observed both in animals having greater than 2000 PFU of virus per ml of plasma and infected peritoneal macrophages, as well as in mice with low plasma virus titers and uninfected macrophages. Thus, the anti-leukemia cell cytotoxicity in the serum of leukemic animals did not correlate with predicted subsequent regression of the disease.

Role of humoral reactivity in maintenance of the regressed state Mice in which erythroleukemia has spontaneously regressed may redevelop a pathologically identical disease between 1 and 3 months after regression. Leukemia usually recurs in about 50% of the regressed animals. We have previously shown that a conserva-

50

DAVID L. HINES et aL

tive protocol of combined specific and nonspecific active immunotherapy of RFV regressed mice prolongs the period of regression [203. We therefore determined whether virus neutralizing and cytotoxic immune responses in regressed mice might have a role in preventing leukemia recurrence. The ability to detect and quantitate the heterogeneous humoral immune responses to virus-related antigens using small serum samples allowed us to directly investigate leukemia recurrence as a function of humoral reactivity in individual regressed mice. Twentythree mice in which leukemia had spontaneously regressed were bled weekly from the retro-orbital sinus beginning 1 week after regression. The sera were tested for cytotoxicity to both D1 and D1P3 leukemia cell lines, for neutralization of RFV and in selected cases by radioimmunoprecipitation of l:5I-labeled RFV. The results of these assays were then correlated with spontaneous leukemia recurrence. All animals were followed for 6 months or until death. Seven of the animals had high neutralizing activity (defined for these experiments as a titer greater than 1:80), 12 had high cytotoxic antibody levels (greater than 50~o specific TABLE 4. LEUKEMIA RECURRENCE AS A FUNCTION OF HUMORAL REACTIVITY IN REGRESSED MICE

Cytotoxic antibody Neutralizing antibody Neither

Disease-free

Recurred

3 7 0

9 0 4

P = 0.0003. Fisher's exact test.

cytotoxicity at a 1 : 10 serum dilution) and 4 had neither. Table 4 shows leukemia recurrence in these three groups of animals. The overall incidence of recurrence was 13 of 23 or 56.5~. The presence of high titered virus neutralizing activity was clearly correlated with maintenance of the disease-free state. In contrast, the absence of detectable humoral reactivity was always associated with recurrence of disease. Mice which had predominantly cytotoxic antibody in their serum were found in both the disease-free (25~o) and the leukemia recurrence (75~o) groups. Analysis by radioimmunoprecipitation of selected serum samples from these mice showed that strong reactivity to viral gp70 and in some instances additional weaker reactivity to pl5(E) or p12, was associated with the diseasefree state (data not shown).

Resistance of regressed mice to leukemia following reinoculation with virus A group of 67 regressed mice and an age-matched group of normal animals, were inoculated with 100 SFFU of RFV within 1 month after regression and observed for 2 months for leukemia redevelopment. A second matched control group of 62 regressed mice was left uninoculated to determine the incidence of spontaneous leukemia redevelopment. As shown in Fig. 3, the incidence of leukemia development in RFV challenged regressed mice, although greater than in the uninoculated regressed controls, was significantly less (p < 0.0005, Chi square test) than in the age-matched normal control animals. The relationship between the presence of high titered virus neutralizing activity in regressed mice and refractoriness to redevelopment of leukemia following RFV challenge was investigated. Mice which had been regressed for at least 6-8 weeks were used because we expected them to have a low incidence of spontaneous leukemia recurrence and high virus neutralizing titers. The mice were test bled and 2 days later one group of 11 regressed animals was inoculated with 100 SFFU of RFV, along with control groups of age-matched normal animals which had or had not been bled. The other group of nine

51

Antibody in regression of leukemia IOO

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25

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6

12

18

24

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~0

36

60

CHALLENGE

FIG. 3. Leukemia redevelopment following reinoculation of RFV into regressed mice: regressed mice (n = 64) reinoculated with 100 S F F U of RFV (O); normal age-matched mice (n = 48) inoculated with 100 S F F U of RFV (e); unchallenged regressed mice (X) (n = 62).

regressed mice was not rechallenged with virus. Leukemia development in these animals is shown in Fig. 4. As expected, all of the control animals were overtly leukemic by day 20 after virus challenge, but only three of the eleven challenged regressed mice became leukemic and only two of the nine unchallenged regressed mice spontaneously redeveloped leukemia by day 25. Of the seven challenged regressed mice with a neutralization titer of 1:80 or greater, only one (14~o) redeveloped leukemia. Thus among this selected group of regressed mice there was a low incidence of leukemia redevelopment following challenge with RFV. In contrast, in a large randomly-selected group of regressed mice challenged with the same dose of RFV 45% of the animals were leukemic by day 25

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IS

~

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I

25

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DAYS POST CHALLENGE FIG. 4. Leukemia development following reinoculation of RFV into regressed mice of tested humoral immune status: regressed mice (n = 11) bled and then inoculated with RFV (O); regressed mice (n = 10) bled only (x); normal age-matched control mice (n = 9) bled and then inoculated with RFV (e); normal age-matched control mice (n = 10) inoculated with RFV only

(E2).

52

DAVID L. HINES et al.

(Fig. 3). We conclude that the presence in regressed mice of high titers of virus neutralizing activity (> 1:80) is associated with the maintenance of the disease-free state and the protection from redevelopment of leukemia induced by challenge with RFV.

DISCUSSION We have previously reported that the spontaneous regression of RFV-induced erythroleukemia is dependent on the presence of an intact immune system [10, 16, 28, 29]. To determine what specific components of the host's immunological system are involved in regression, we are testing regressed and leukemic mice for various anti-virus and antileukemia cell responses. We consider that if any particular immune response is causally related to regression, it should be observed in all regressed and leukemic regressor mice. We have shown that anti-virus cell-mediated immunity, detected in the macrophage migration inhibition assay, is highly correlated with regression [23]. In this report we show that although virus neutralizing and leukemia cell cytotoxic antibodies are detectable in regressed mice, their presence is not correlated with regression. One-third of regressed mice had no detectable humoral activity. Similar antibodies were detected in some overtly leukemic mice, but they were also not correlated with subsequent regression of the disease. In contrast, we found a strong correlation between the presence of virus neutralizing antibody and maintenance of the disease-free state. No animals with high titers of neutralizing antibody experienced a recurrence of their disease within the period of observation. By comparison, leukemia normally recurs in 30-50~ of unselected regressed mice. The presence of high titers of cytotoxic antibody did not protect mice from disease recurrence. All regressed mice exhibit very low levels of infectious virus [ 11, 34] and a histopathologically normal spleen size and architecture [38]. Presumably, most of the productively infected leukemic erythroblasts have been eliminated in regressed mice and replaced with uninfected or non-virus expressing cells. This sequence has been established for macrophage progenitor cells during regression [28]. Redevelopment of leukemia in regressed mice would then involve reinitiation of infection of normal erythroblastic cells. The virus responsible might be derived from low levels of infectious virus released by a small number of productively infected cells remaining in these mice [6]. In this event, neutralizing antibody could prevent disease recurrence by interference with the infection process. The correlation of neutralizing antibody with the lack of spontaneous redevelopment of leukemia and resistance to rechallenge with virus is in accord with this possibility. A salient finding in this study is the absence of detectable humoral reactivity against virus or leukemia cells in many of the regressed mice. However, we cannot exclude the possibility that these regressed mice produce some antibody, but that it is bound by free antigen present in the animals. This possibility is currently under study. The specificity of the humoral response in regressed mice has been analyzed by immunoprecipitation of disrupted virus. Sera that contain neutralizing and/or cytotoxic antibodies, precipitate gp70. Some sera precipitate, in addition, pl2 or pl5(E), but there is no correlation between precipitation of these proteins and the type of humoral reactivity detected. Monospecific, xenogeneic antisera against viral gp70 neutralizes virus [42]. Antisera against pl5(E) will also neutralize virus, but only in the presence of complement [13, 17]. Addition of complement to anti-gp70 sera increases the apparent neutralization titer 2 to 3-fold. Addition of complement to most regressed sera also increases neutralization 2 to 3-fold and is probably a function of reactivity against gp70. Occasionally, the apparent

Antibody in regression of leukemia

53

neutralization titer of serum from a regressed mouse is considerably increased by complement and this may reflect the presence of anti-pl5(E) reactivity. Cytotoxicity against leukemic cells is obtained with xenogeneic antisera against gp70, p12 and in this study, p15 and pl5(E). However, the reactivity of the anti-pl5(E) antisera used has not been shown to be entirely virus specific [see also 24]. In addition, antisera against p15 have not generally been reported to be cytotoxic [22, 24, 41] and thus the activity we observed was probably due to reactivity against p12 in the sera used (see Fig. 2A). We conclude, therefore, that the bulk of the humoral reactivity in regressed mice can be accounted for by antibodies against viral gp70. These antibodies can cause both virus neutralization and cytotoxicity. The independence of these two activities in regressed mice suggests that the antigenic sites on the gp70 molecule responsible for neutralization and cytotoxicity are not necessarily identical [see also 32]. Reactivity against gp70 is also an important component of cell-mediated immunity detectable in regressed mice [23]. Our results show that neutralizing antibody is not necessary to cause spontaneous regression of RFV-induced leukemia. Moreover, we conclude that cytotoxic antibody is neither necessary nor sufficient to cause regression, based on the observation that high titers are detected in many leukemic mice that will not regress (progressors). These findings do not exclude the possibility that humoral antibody may play some role in reactive mice and may be associated with recovery from leukemia in other systems. Anti-Friend virus antibody has been implicated in statolon-induced recovery from Friend erythroleukemia [4, 18, 45] and in the spontaneous recovery from leukemia in mice with certain specific genotypes [6, 12]. In addition, Sch~er et al. 1-39,40] have demonstrated that xenogeneic antibody against viral gp70, administered to leukemic mice 1 to 2 weeks after infection with FV, can induce recovery from the disease and that host production of antibody against gp70, detectable by both neutralization and cytotoxicity, is associated with this treatment. Acknowledqements--We thank Dr. Marvin A. Rich for his guidance and advice and Clifford Longley, Christopher Bolles and Susan Fouchey for their excellent technical assistance. This study was supported by grant CA-14100 from the National Cancer Institute and an institutional grant to the Michigan Cancer Foundation from the United Foundation of Greater Detroit.

REFERENCES 1. AARONSONS. A. & ROWE W. P. (1970) Nonproducer clones of murine sarcoma virus transformed Balb/3T3 cells. Virology 42, 9-19. 2. AXELRADA. & STEEVESR. A. (1964) Assay for Friend leukemia virus: rapid quantitation method based on enumeration of macroscopic spleen loci in mice. Virology 24, 513. 3. BERYSTEIY A.. MA~ T. W. & STEPHE~SON J. R. (1977) The Friend virus genome: evidence for the stable association of MuLV sequences and sequences involved in erythroleukemia transformation. Cell 12, 287-294. 4. CALLAnAN R. M , MARX P. A. & WHEELOCK E. F. (1979) Group-specific cytolytic antibody directed against the major glycoprotein (gp70) of murine leukemia viruses in serum of mice with dormant FLV infections. Virology 97, 55-67. 5. CERNV J., ESSEX M., RICH M, A. & HA~DV W. D. (1975) Expression of virus associated antigens and immune cell functions during spontaneous regression of Friend viral murine leukemia. Int. J. Cancer 15, 351-365. 6. CHESEBROB. & WEHRLY K. (1976) Studies on the role of the host immune response in recovery from Friend virus leukemia. I. Antiviral and antileukemia cell antibodies. J. exp, Med. 143, 73-84. 7. CHESEBROB., BLOOMM., WEHRLY K. & NISHIO J. (1979) Persistence of infectious Friend virus in spleens of mice after spontaneous recovery from virus-induced erythroleukemia. 3. Virol. 32, 832-837. 8. CROKER B. P.. DEVILLANO B. C,, JENSE~ F. C., LERNER R. A. & DIXON F. J. (1974) Immunosensitivity and oncogenicity of murine leukemia viruses. I. Induction of immunologic disease and lymphoma in (Balb/c X NZB) mice by Scripps leukemia virus. J. exp. Med. 140, 1028-1048. 9. DE~T P. (1972) Immunodepression by oncogenic viruses. Prog. med. Virol. 14. 1-35.

54

DAVID L. HINES et al.

I0. DIVTZ M., FURMANSKI P., CLYMER R. & RICH M. A. (1976) Effects of thymectomy and antithymocyte serum on spontaneous regression of Friend virus induced erythroleukemia. J. natn. Cancer Inst. 57, 91 95. 11. DIETZ M., LONGLEY C., FOUCHEV S. P., HALL L., RWH M. A. & FURMANSKI P. [1977) Spontaneous regression of Friend virus-induced erythroleukemia. II. Regression of Friend routine leukemia virus induced lymphocytic leukemia. J. nam. Cancer Inst. 59, 957-961. 12. DOIT D. & CHESEaRO B. (1979) Anti-Friend virus antibody is associated with recovery from viremia and loss of viral leukemia cell-surface antigens in leukemic mice. J. exp. Med. 150, 10-19, 13. FISCHINGER P. J., SCH.~ER W. & BOLOGNesl D. P. (1976) Neutralization of homologous and hetero[ogous oncornaviruses by antisera against the pl5(E) and gp71 polypeptides of Friend murine leukemia virus. Virolooy 71, 169-184. 14. FRIENO C. (1957) Cell-free transmission of a disease having the character of a leukemia. J. exp. Med. 105. 307-318. 15. FURMANSKI P., GOODENOW R., BALDWIN J., CLYMER R. & RICH M. A. (1975) Immune status of mice in which leukemia has spontaneously regressed. Fedn Proc. Fedn Am. Socs exp. Blot. 34. 1973. 16. FURMANSKIP., DIETZ M., FOUCHEY S., HALL L., CLYMER R. & RICH M. A. (1979) Spontaneous regression of Friend murine leukemia virus-induced erythroleukemia. IV. Effects of radiation and athymia on leukemia regression in mice. J. natn. Cancer Inst. 63, 449-454. 17. FURMANSKXP., LONGLEYC., BOLLESC. S., HINES D. L. & DIETZ M. (1980) Spontaneous regression of Friend virus induced erythroleukemia. VI. Structural and antigenic differences between the regressing and conventional strains of virus. J. Virol. 33, 1083-I096. 18. GENOVESlE. V., MARX P. A. & WHEELOCK E. F. (1977) Antigenic modulation of Friend virus erythroleukemic cells in vitro by serum from mice with dormant erythroleukemia. J. exp. Med. 146, 520-534, 19. GILLIS S., RUSCETTI S. K , GILLIS A, E., TROXLER D. H., SCOLNICK E. M. & SMITH K. A. 11979) The spleen focus-forming virus (SFFV)-specific neoantigen shares cross-reactive determinants with mink cell focus inducing (MCF) virus gp70. Virology 96, 421-428, 20. HINES D. L., DtETZ M., RICH M. A. & FURMANSKI P. (1978a) Active immunotherapy of Friend virusinduced erythroleukemia and its spontaneous regression. Cancer lmmun, lmmunother. 5, 11-16. 21. HINES D. L., DIETZ M., MARCELLETTIJ,, Rlcrt M. A. & FURMANSKI P. (1978b) Involvement of the immune system in the spontaneous regression of Friend virus induced erythroleukemia. In Advances in Comparatire Leukemia Research, 1977 (BENTVELZEN P., HILGERS J. and YOHN D. S., Eds.), p. 233. Elsevier/NorthHolland, Amsterdam. 22. HUNSMANNG., CLAVIEZ M., MOENNIG H. S. & SCH.~FER W. (1976) Properties of mouse leukemia viruses X. Occurrence of viral structural antigens on the cell surface as revealed by a cytotoxicity test. Virology 69, 157-168. 23. JOHNSON C. S,, FOUCHEY S. P. & FURMANSKI P. (1980) Spontaneous regression of Friend virus-induced erythroleukemia. V. Cell-mediated anti-virus immunity in regressed and leukemic mice. J. HatH. Cancer Inst. (in press). 24. KENDE M., S~SS B., HINO S., DtYNArlOE R. M. & KELLO~F G. J. (1978) Type-C virus structural antigens on the surface of the infected cell as determined by a humoral cytotoxicity assay. Int. J. Cancer 21, 194--203. 25. KesSLER S. W. (1975) Rapid isolation of antigens from cells with a Staphylococcal protein-A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. hnmun. 115. 1617-1624. 26, LAEMMLIV. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 226, 680-685. 27. LEVV J. A., IHLE J. N., OLESZKO O. & BARNES R. D. (1975) Virus-specific neutralization by a soluble non-immunoglobulin factor found naturally in normal mouse sera. Proe. HatH. Acad. Sci. U.S.A. 72, 5071-5075. 28. MARCELLETT1J. & FURMANSKI P, (1979a) Spontaneous regression of Friend virus-induced erythroleukemia. III. The role of macrophages in regression. J. lmmun. 120, 1-8. 29. MARCELL~'r! J. & FURMANSKI P. (1979b) Infection of macrophages with Friend virus: Relationship to the spontaneous regression of viral erythroleukemia. Cell 16, 649-659. 30. MtTTAL K. K., Mtcr~v M. R., SINCAL D. P. & TERASAm P. I. (1968) Serotyping for homotransplantation. XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 6, 913-927. 31. MONTELAROR. C., FISCHINGER P. J., LARRICK S. B., DUNLOP N. M., 1HLE J. N., FRANK H., SCH~FER W. & BOLOGNESl D. P. (1979) Further characterization of the oncornavirus inactivating factor in normal mouse serum. Virolo#)' 9g, 20-34. 32. NOWINSKI R. C., EMERY S- & LEDBETTERJ. (1978) Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells. J. Virol. 26, 805-812. 33. RICH M. A., GELDNER J. & MEVERS P. (1965) Studies on murine leukemia. J. Ham. Cancer Inst. 35, 523-536. 34. RICH M. A,, SIEGLER R., KARL S. & CLYMER R. (1969a) Spontaneous regression of virus induced murine leukemia. I. Host-virus system. J. natn. Cancer Inst. 42, 559-570. 35. Rtcrl M. A., CLVMER R. & KARL S. (1969b) Spontaneous regression of virus induced murine leukemia. II. Influence of environmental factors. J. HatH. Cancer Inst. 42, 571-577. 36. RICH M. A., KARL S. & CLVMER R. (1971) Complement fixation in conventional and regressing virus induced routine leukemia. J. lmmun. 1tl6, 1488-1492. 37. RICH M. A. & CLYMER R. (1971) Host response in spontaneous regression of murine leukemia. Cancer Res. 31,803-807.

Antibody in regression of leukemia

55

38. Russo I., Russo J., BALDWIN J. & RICH M. A. (1976) Histopathology of leukemia regression in virus induced murine leukemia, Am. J. Path. 85, 73-80. 39. SCH~FERW., SCHWARZ H., THIEL H. J., WECKER E. & BOLOGNESID. P. (1976) Properties of mouse leukemia viruses. XIII. Serum therapy of virus-induced murine leukemias. Virology 75, 401-418, 40. SCH,~FERW., BOLOGNESlD. P., DE NORONKA F., FISCHINGER P. J., HUNSMANN G., IHLE J. N., MOENNING V.. SCHWARZ H. & THIEL H. J. 11977) Immunoprophylaxis and therapy of C-type oncornaviral disease in mice and cats. Med. microbiol. Immunol. 164, 217-229. 41. SCHNEIDER J. & HU~SMANN G. (1978) Surface expression of murine leukemia virus structural polypeptides on host cells and the virion. Int. J. Cancer 22, 204-213. 42. S'rEEVES R. A., STRAND M. & AUGUST .I.T. (1974) Structural proteins of mammalian oncogenic RNA viruses. Murine leukemia virus neutralization by antisera prepared against purified envelope glycoprotein. d. Virol. 14, 187-189. 43. STEPHENSON J. R,. DEVARE S. G. & REYNOLDS F. H. (1978) Translational products of type C RNA tumor viruses. Adv. Cancer Res. 27, 1-53. 44. TROXLER D. H., PARKS W. P,, VASS W. C. & SCOLNICK E. M. (1977) Isolation of a fibroblast nonproducer cell line containing the Friend strain of the spleen focus-forming virus. Virology 76, 602-615. 45. WHEELOCK E. F., TOZ S. T., CAROLINE N. L., SIBAL L. R.. FINK M. A., BEVERLEY P. C. L. & ALLISON A. C. (1972) Suppression of established Friend virus leukemia by statolon. IV. Role of humoral antibody in the development of a dormant infection. J. hath. Cancer Inst, 48, 665~73. 46. WIGZELL H. (1965) Quantitative titrations of mouse H-2 antibodies using 5~Cr-labeled target cells. Transplantation 3, 423-431.