Persistence of Infectious Friend Virus in Spleens of Mice After Spontaneous Recovery from Virus-Induced Erythroleukemia

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1 JOURNAL OF VIROLOGY, Dec. 1979, p X/79/12-832/6$2./ Vol. 32, No. 3 Persistence of Infectious Friend Virus in Spleens of Mice After Spontaneous Recovery from Virus-Induced Erythroleukemia BRUCE CHESEBRO,* MARSHALL BLOOM, KATHY WEHRLY, AND JANE NISHIO National Institute ofallergy and Infectious Diseases, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, Montana 5984 Received for publication 27 March 1979 Persistent infectious virus was detected in the majority of spleens of (C57BL/ 1 x A.BY)F1 mice after spontaneous recovery from Friend virus-induced erythroleukemia. The Friend murine leukemia helper virus (F-MuLV) was detected in titers up to 3 x 15 PFU/g of spleen. The defective spleen focus-forming virus (SFFV) was present in much lower titers and could be detected in cell-free spleen homogenates only after amplification of virus titer by growth of virus in vitro on SC1 cells. The incidence of cells producing F-MuLV alone in spleens after recovery from leukemia was.3 to.3%, and the incidence of cells producing both F-MuLV and SFFV was <.1 to.1%. In most recovered mouse spleens there appeared to be a selective reduction of SFFV relative to F-MuLV. Spontaneous recovery from erythroleukemia induced by the Friend leukemia virus (FV) complex (18, 19) occurs with a high incidence in certain mouse strains (1, 27). Many mouse genes including Fv-1, Fv-2, H-2 (Rfv-1 and Rfv- 2), and Rfv-3 have been found to influence this process (6, 7, 1, 27-29, 32). Although the mechanisms involved are not yet clearly understood, virus-specific cytotoxic T-lymphocytes appear in the spleen and bone marrow of mice shortly after recovery from splenomegaly (5). These immune effector cells may be responsible for elimination of the leukemia cells. We previously noted disappearance of the defective spleen focus-forming virus (SFFV) component of the FV complex from plasma and spleens of mice after spontaneous recovery from leukemia (4). However, we also observed discrepant elimination of SFFV compared to its nondefective helper murine leukemia virus (F- MuLV) in some strains of mice with persistent leukemia (4, 15). These findings led us to reexamine levels of both SFFV and F-MuLV in spleens of mice which had recovered from leukemic splenomegaly. The present results indicate that there is a high incidence of persistent F-MuLV in spleens of mice after recovery from leukemia. Furthermore, by using the more sensitive techniques of cocultivation of cells and palpation (1). Thirty to 9 days after FV injection, mice were bled and killed for analysis of spleen virus and spleen weight. Mice with spleens more than.5 g were considered leukemic, and those with spleens less than.4 g were considered recovered from leukemia. These limits are consistent with our previous experience following many mice with periodic cross-checking of spleen sie determined by palpation versus actual spleen weight. Although.4 g is more than the normal mouse spleen weight (.15 to.25 g), after regression of massive FV-induced splenomegaly many spleens never return to completely normal sie in spite of normal survival for over 1 year (1). Spleen cells were dissociated in phosphate-buffered balanced salt solution (4) to give a 1% suspension. After centrifugation (5 x g for 5 min) supernatant fluid was froen in portions at -7 C for analysis of virus, and cells were washed three times before inoculation into FV-susceptible mice or cocultivation with amplification of virus titers on monolayers in vitro, we detected or rescued SFFV from spleens SC1 cells in vitro. of most recovered nonleukemic mice. In vitro growth of SFFV. SC1 cells (22), obtained from J. Hartley, National Institutes of Health, Bethesda, Md., were seeded onto Corning 25-cm2 plastic MATERIALS AND METHODS tissue culture flasks (3 x 15 cells per flask) or 2.-cm2 Virus. Growth and preparation of stocks of the B- wells of Linbro TC-24 trays (5 x 14 cells per well) in tropic strain of Friend virus (3) were described pre- McCoy medium with 1% fetal calf serum. One day 832 viously (1). FV complex was assayed for focus-forming units (FFU) with the spleen focus assay (1). F- MuLV was assayed on mouse S+L- cells (2) as described preivously (8, 11). Titers were expressed as PFU. Mice. A.BY, C57BL/1OSn (B1), B1.A, and (BALB/c x A)F, mice were purchased from the Jackson Laboratory, Bar Harbor, Maine. Other F, hybrids and BALB.B mice were bred at the Rocky Mountain Laboratories, Hamilton, Mont. Leukemia induction. Mice were inoculated intravenously at 3 to 6 months of age with 15 or 1,5 spleen FFU of FV. Mice were followed for development of and recovery from FV leukemia by spleen

2 VOL. 32, 1979 later monolayers were treated for 2 min at 37C with 5 Ag of DEAE-dextran in phosphate-buffered balanced salt solution per ml, rinsed once with phosphatebuffered balanced salt solution, infected for 9 min with 1 ml (flasks) or.2 ml (wells) of a l/io dilution of a 1% cell-free spleen homogenate or with various numbers of washed spleen cells and then overlaid with medium. SC1 cells were passed twice weekly for four passages, and.5 ml of the supernatant fluid from the fourth passage was assayed for F-MuLV by S+L- assay and for the FV complex (SFFV plus F-MuLV) by intravenous (i.v.) inoculation into susceptible mice [(BALB/c x A)FI or BALB.B]. Mice were followed 45 days for development of obvious splenomegaly detectable by palpation. Although SFFV does not usually grow to titers greater than 14 FFU/ml in SC1 cells, we have found that the SFFV 5% infective dose (ID5) for SC1 cell cultures was similar to the SFFV ID5 in (BALB/c x A)FM mice when FV stocks obtained from mouse spleens 8 to 1 days after FV inoculation were tested (data not shown). The SC1 cultures were more sensitive than direct inoculation of mice when spleen homogenates from mice after recovery from leukemia were used (see below). RESUlLTS Persistent F-MuLV infection in spleens of recovered mice. F-MuLV titers in cell-free spleen suspensions from (BlO x A.BY)F1 and (B1O.A x A.BY)Fj mice which had recovered from FV-induced leukemia splenomegaly were compared to titers in spleen suspensions from leukemic mice 8 to 1 days after FV inoculation (Fig. 1). F-MuLV titers in leukemic mice ranged from 8 x 15 to 2 x 17 PFU/g of spleen. In contrast, in 37 of 113 recovered mice F-MuLV was not detectable (<2 PFU/g of spleen); however, in the remaining 7&recovered mice, F- MuLV ranged in titer from 3 x 12 to 4 x 15 PFU/g of spleen. Thus, persistent F-MuLV infection was found in 67% (76/113) of these mice after recovery from leukemia, although virus titers observed were usually 1- to 1,-fold lower than in leukemic mice. Detection of SFFV in spleens of recovered mice. Spleen homogenates from recovered mice which had detectable F-MuLV were also analyed for SFFV by inoculation into susceptible mice. SFFV was detected in only 2 of 55 mice tested (Table 1). However, all leukemic mice 8 to 1 days after FV inoculation had high titers of SFFV in their spleen homogenates (data not shown). We pursued the study of SFFV infection in (B1 x A.BY)F1 mice which had recovered from FV-induced splenomegaly by attempts to rescue SFFV from the spleen cells of these mice. Either cell-free spleen homogenates or washed live spleen cells were inoculated i.v. into susceptible recipient mice. None of the cell-free homogenates produced splenomegaly; however, 24 of 26 live spleen cell FRIEND VIRUS PERSISTENCE 833 inocula produced splenomegaly from 14 to 45 days after inoculation (Table 2, experiment 1). Some of the enlarged spleens from these positive recipients were then homogenied, and all caused typical SFFV spleen foci 9 days after inoculation into susceptible mice. It was clear that the splenomegaly which occurred after injection of spleen cells was not due to growth of the inoculated cells or to a graft versus host E U- IL cl LU < 2 3 Leukeemic ~~~~ ~ *-- Recovered ~ ~ ~~~~~ ~ ~~- - ~~: e- ; -;; * l;;;; FIG. 1. Spleen F-MuLV in leukemic mice 8 to 1 days after FV inoculation and in mice after spontaneous recovery from leukemic splenomegaly 3 to 9 days after FV inoculation. A total of 15 or 1,5 FFU were inoculated i.v. into (BJO x A.BY)F, and (BIOA x A.BY)F, mice. Data from both strains were indistinguishable and were pooled. Each point represents one mouse.

3 834 CHESEBRO ET AL. TABLE 1. Detection of SFFV in spleens of mice after spontaneous recovery from FV-induced leukemia Mouse strain No. with SFFV/totala (B1O x A.BY)F1 1/4 (BlO.A x A.BY)F, 1/15 'Values shown are the number of mice with SFFV detected in cell-free spleen homogenates/total number of mice tested (all were positive for F-MuLV). Ten percent spleen homogenates were diluted 1/1, and.5 ml was inoculated i.v. into (BALB/c x A)F1 mice, which were followed at least 6 weeks for development of splenomegaly. The minimum detectable concentration of SFFV by this method was 2 ID5/g of spleen = ID5o/.5 ml x 1 (dilution) x ml/g of spleen (i.e., 1% spleen suspension). TABLE 2. Detection of SFFV in spleen cells and cell-free spleen homogenates of recovered (BJO x A.BY)F1 mice 3 to 9 days after FV inoculation No. of SFFV-positive mice/total Cell-free Expt Spleen cells spleen homog- ExptSpleen cells ~enate Mice' SClb Micea SC1b 1 24/26 NTC /26 NT 2 NT 11/15 /15 4/ /16 NT /16 12/16 a Washed spleen cells (5 x 17) or.5 ml of a 1/1 dilution of a 1% spleen homogenate were injected i.v. into susceptible mice, which were observed 45 days for appearance of splenomegaly. bwashed spleen cells (16) or 1. ml of a 1/1 dilution of 1% spleen homogenate were added to DEAE-dextran-treated cultures of SC1 cells. These cultures were passaged twice weekly for four passages. To assay for SFFV, tissue culture supernatants were injected i.v. into susceptible mice which were followed 45 days for appearance of splenomegaly. Enlarged spleens from some positive mice were dissociated and assayed for capacity to produce typical FV spleen foci 9 days after inoculation into susceptible mice. c NT, Not tested. reaction because SFFV was detected with equal incidence in H-2 compatible and incompatible recipients, and the H-2 type of the spleen cells found in recipients with splenomegaly was that of the recipient, not the donor, cells (data not shown). These results indicated that either SFFV could be rescued from spleen cells of recovered nonleukemic mice or that inoculation of washed spleen cells was a more sensitive method for SFFV detection than inoculation of cell-free spleen homogenates. A total of 1 x 16 to 1 x 16 cells from thymus, mesenteric lymph nodes and bone marrow of these same mice were also inoculated into susceptible recipients; however SFFV was not J. VIROL. detected (data not shown). Thus, among the tissues tested, persistent SFFV infection appeared to be limited to the spleen. This apparent SFFV "rescue" from spleen cells was analyed further by attempting to rescue SFFV in vitro by infection of SC1 cells with spleen homogenates or live spleen cells from recovered nonleukemic mice. In experiment 2 (Table 2) SFFV was detected in live spleen cells from 11 of 15 recovered mice after cocultivation on SC1 cells. Furthermore, although SFFV was not detected after inoculation of mice with cellfree spleen homogenates, it was detected after in vitro infection of SC1 cells with 4 of 15 of these same spleen homogenates. This finding was confirmed in a third experiment with 16 spleens from recovered mice. SFFV was detected in the spleen homogenates of 12 out of 16 mice by infection of SC1 cells but in none of the 16 by direct inoculation into susceptible mice (Table 2). The occurrence of SFFV in spleens of mice which had recovered from splenomegaly appeared to be a frequent finding and could not be ascribed to a true rescue of latent SFFV, because SFFV could be detected in many cell-free homogenates. These results suggested that the amount of infectious SFFV was very low in spleen homogenates and required amplification in vitro in SC1 cells. Quantitation of F-MuLV and SFFV infectious centers in recovered mice. To determine the percentage of spleen cells releasing F- MuLV and SFFV in recovered mice, various numbers of spleen cells were plated on SCi monolayers which were then passaged four times and then assayed for F-MuLV and SFFV. The lowest number of spleen cells capable of infecting the SC1 cells was assumed to contain one infectious center. A total of 5 to 1% of cells from leukemic spleens were found to be infectious centers for both F-MuLV and SFFV (12; data not shown). In spleens of recovered mice the incidence of spleen cells infected with F- MuLV ranged from.3 to.3% (Fig. 2). These values were in close agreement with values obtained by direct plating of spleen cells into the S+L- infectious center assay (data not shown). The incidence of dually infected (SFFV plus F- MuLV) spleen cells was somewhat lower (.3 to.1%), and in 4 of 15 mice no SFFV was detected even after seeding 16 spleen cells (Fig. 2). The ratio of F-MuLV infected spleen cells to dually infected spleen cells was determined for each recovered mouse spleen and was compared to the ratio seen in six leukemic spleens (Fig. 3). In all leukemic spleens this ratio was 1, indicating all F-MuLV infected cells were dually infected. However, in 11 of 15 recovered spleens,

4 VOL. 32, 1979 V, LIu u _ vvv I* C9 SFFV * - FIG. 2. Incidence of F-MuLV and SFFV plus F- MuLV infectious centers in spleen cells from (B1O x A.BY)Fj mice after spontaneous recovery from FV leukemia. U) ui LI U) N1-1, I Loukemic Recovered 9 FIG. 3. Ratio of F-MuLV infectious centers to SFFV plus F-MuLV infectious centers in spleens of leukemic and recovered mice. S. ***. * FRIEND VIRUS PERSISTENCE 835 this ratio was greater than 1, ranging from 1 to 1,. This suggested that in these individuals 1- to 1,-fold more cells were producing F- MuLV alone than were producing both SFFV and F-MuLV. This agreed with the substantial titers of F-MuLV observed in the cell-free homogenates of these spleens (Fig. 1) compared to the barely detectable levels of SFFV observed (Tables 1 and 2). DISCUSSION (BlO x A.BY)F1 and (B1.A x A.BY)F1 congenic F1 hybrid mice, differing only for genes of the H-2 complex (1), had a high incidence of persistent infection with both components of the FV complex after spontaneous recovery from FV-induced erythroleukemia. In the spleen homogenates of most recovered mice F-MuLV was easily detected by direct plaque assay, although titers observed were decreased 1- to 15-fold compared to leukemic mice early after virus inoculation. In contrast, SFFV was usually detected in spleen homogenates from recovered mice only after amplification of virus titer by in vitro passage on SC1 cells before in vivo assay. I'hus, although both viruses appeared to persist in an infectious state, there was an apparent selective decrease in SFFV. The incidence of virus-producing spleen cells in recovered mice was low. A total of.3 to.3% of cells were scored as F-MuLV producers by infection of SC1 cells in vitro (Fig. 2), and.1 to.1% of cells appeared to produce both SFFV and F-MuLV as estimated by infection of SC1 cells in vitro (Fig. 2). The ratio of the percentage of cells detected producing F-MuLV alone to the percentage producing both SFFV and F-MuLV was high (1 to 1,) in many recovered mice (Fig. 3). This finding suggested either that cells producing both viruses were more efficiently eliminated than cells producing F-MuLV alone or that SFFV production was selectively decreased relative to F-MuLV production in some cells. Either possibility might indicate that selective recognition of cells producing SFFV occurred, possibly by means of SFFV-specific cell surface antigens (21, 25). The types of cells producing SFFV in spleens of mice after recovery from leukemia have not yet been identified. FV usually induces leukemia of primitive erythroid cells (9, 19). However, FVinduced nonerythroid leukemia cells (?lymphoid) containing rescuable SFFV have been observed (8, 9). In addition, production of SFFV by nontransformed fibroblasts and nonerythroid spleen cells has been noted in vitro (13, 16, 34). Therefore, many different cell types residing in the spleen remain possible sites of persistent infection. This point may be difficult to clarify

5 836 CHESEBRO ET AL. because the incidence of SFFV-producing cells in the spleens of recovered mice was so low. Although both F-MuLV and SFFV probably become integrated into host cell DNA as proviruses, the fact that we could detect both viruses in cell-free spleen homogenates indicated that they also persisted in an infectious state. Mechanisms of virus persistence such as antigenic shifts as seen with visna virus and equine infectious anemia virus (26, 31) or production of defective interfering particles (17, 35) are possible, but we have no evidence for either of these phenomena in this system yet. At present we feel that the most likely explanation for persistence is due to an interaction of the antiviral antibodies with virus-infected cells. Anti-FV antibodies can reduce or modulate the expression FV-induced cell surface antigens on leukemia cells (3, 14, 2, 33). This alteration of FV cell surface antigens appears to interfere with virus release from the cell perhaps due to dispersion of viral molecules necessary for virus budding (12, 38, 39). This possibility was supported by the observation that virus production was detected when spleen cells from mice after recovery from leukemia were transferred to nonimmune susceptible recipient mice (Table 2). It is also possible that antibody-induced dispersion of viral cell surface antigens could interfere with immune recognition and elimination of virusinfected cells by cytotoxic T-lymphocytes (23, 24), thus leading to persistence of infected cells. Persistence of FV was also found by Wheelock and co-workers (36, 37) after treatment of FVinfected DBA/2 mice with statalon. This system differed from ours in that over 5% of initial regressor mice relapsed over the course of 12 months. However, during remission, spleen cells could transfer leukemia to secondary recipients, whereas cell-free spleen supernatants could not. These authors interpreted these findings to be evidence for a state of dormant infection. In the mice we have studied it is not known whether the persistently infected cells are "dormant" leukemia cells (2). However, if these infected cells have undergone neoplastic transformation, it is unclear what is preventing them from proliferating freely yet at the same time allowing a few of them to persist. We have found that late FV leukemic spleen cells with reduced viral cell surface antigens from (BlO.A x A)F1 mice can proliferate in hyperimmunied (BlO.A x A.BY)F1 mice (manuscript in preparation). Therefore, one would expect that the residual leukemia cells in recovered spleens should also be able to expand in number even in the presence of anti-fv antibodies. Because relapse of leukemia is very rare (<5%) after recovery in these mice (1), it would appear that either the J. VIROL. residual virus-producing cells are not leukemia cells or, if they are leukemic cells, there exist additional unknown mechanisms which prevent their proliferation but do not kill them. ACKNOWLEDGMENTS We thank Helen Blahnik for preparation of the manuscript and James Wolfinbarger for technical assistance. LITERATURE CITED 1. Axelrad, A. A., and R. A. Steeves Assay for Friend leukemia virus: rapid quantitative method based on enumeration of macroscopic spleen foci in mice. Virology 24: Bassin, R. H., N. Tuttle, and P. J. Fischinger Rapid cell culture assay technique for murine leukemia viruses. Nature (London) 229: Boyse, E. A., E. Stockert, and L. J. Old Modification of the antigenic structure of the cell membrane by thymus-leukemia (TL) antibody. Proc. Natl. Acad. Sci. U.S.A. 58: Chesebro, B., and K. Wehrly Studies on the role of the host immune response in recovery from Friend virus leukemia. I. Antiviral and anti-leukemia cell antibodies. J. Exp. Med. 143: Chesebro, B., and K. Wehrly Studies on the role of the host immune response in recovery from Friend virus leukemia. II. Cell-mediated immunity. J. Exp. Med. 143: Chesebro, B., and K. Wehrly Rfv-1 and Rfv-2, two H-2-associated genes that influence recovery from Friend leukemia virus-induced splenomegaly. J. Immunol. 12: Chesebro, B., and K. Wehrly Identification of a non-h-2 gene (Rfv-3) influencing recovery from viremia and leukemia induced by Friend virus complex. Proc. Natl. Acad. Sci. U.S.A. 76: Chesebro, B., K. Wehrly, K. Chesebro, and J. Portis Characteriation of Ia8 antigen, Thy-1.2 antigen, complement receptors, and virus production in a group of murine leukemia virus-induced leukemia cell lines. J. Immunol. 117: Chesebro, B., K. Wehrly, and D. Housman Lack of erythroid characteristics in Ia-positive leukemia cell lines induced by Friend murine leukemia virus. J. Natl. Cancer Inst. 6: Chesebro, B., K. Wehrly, and J. Stimpfling Host genetic control of recovery from Friend leukemia virus-induced splenomegaly. Mapping of a gene within the major histocompatibility complex. J. Exp. Med. 14: Chesebro, B., K. Wehrly, K. Watson, and K. Chesebro Murine leukemia virus infectious centers are dependent on the rate of virus production by infected cells. Virology 84: Chesebro, B., K. Wehrly, D. Doig, and J. Nishio Antibody-induced modulation of Friend virus cell surface antigens decreases virus production by persistent erythroleukemia cells: influence of the Rfv-3 gene. Proc. Natl. Acad. Sci. U.S.A., in press. 13. Clarke, B. J., A. A. Axelrad, and D. Housman Friend spleen focus-forming virus production in vitro by a non-erythroid cell line. J. Natl. Cancer Inst. 57: Doig, D., and B. 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