Natural resistance in mice against Friend leukemia cells. I. Studies with in vitro passaged interferon-sensitive and interferon-resistant cell clones

Anti-lymphoma natural resistance (NR) has been detected in DBA/2 mice inoculated intravenously (iv) with syngeneic Friend leukemia cells (FLC). Interferon-sensitive 745 or interferon-resistant 3Cl-8 clones, passaged in vitro and exhibiting "low" tumorigenicity in syngeneic DBA/2 mice, were used. NR, measured as rapid clearance of radiolabeled cells from lung and liver of recipient mice, was age-dependent, was boosted by host pretreatment with polyinosinic-polycytidylic (poly I:C) acid or Friend leukemia virus, and was decreased by mice pretreatment with cyclophosphamide or i-carrageenan. Treatment of "target" FLC with interferon suppressed the susceptibility of 745 FLC, but not that of 3Cl-8 FLC to host's NR. These data suggest that the "low" in vivo tumorigenicity of in vitro passaged FLC is, at least in part, due to host's NR directed against target structures associated with leukemia cells.     

Natural resistance against hematopoietic cells in lethally-irradiated mice infected with Friend leukemia virus

Summary The influence of in vivo infection with the polycythemic substrain of Friend leukemia virus on noninducible (natural) resistance against allogeneic normal or malignant grafts was studied in lethally irradiated mice. Parallel studies were performed on the NK system in the same experimental conditions. The results indicate that FLV-P infection of mice with full (DBA/2) vs partial (BALB/c and CD2F1) susceptibility did not suppress their in vivo natural resistance against bone marrow or El-4 leukemia cells. On the other hand, a decline in NK activity paralleled the progression of leukemic disease in the more susceptible DBA/2 hosts. Abbreviations used: FLV-P, N-tropic polycythemic substrain of Friend Leukemia Virus Complex; NR, natural resistance; NR in vivo, natural resistance against normal or malignant hemopoietic grafts occurring in vivo in lethally irradiated mice; NK, natural killer; (¹²⁵I)IUdR, ¹²⁵I-labeled 5-iodio-2-deoxyuridine; IV, intravenous     

Isolation and properties of DMSO resistant variant clones of Friend leukemia cells.

DMSO resistant clones have been isolated from the inducible Friend leukemia cell line 5-86 both from unmutagenized cultures and following EMS mutagenesis. All the clones can grow in the presence of 1.8% DMSO and are non-inducible or poorly inducible for hemoglobin synthesis by DMSO as well as by other known inducers of Friend leukemia (FL) cells differentiation like hemin, hypoxanthine, hexamethylene bisacetamide. The clones are also defective for the expression of other properties of differentiating Friend cells like agglutinability by plant lectins and expression of the surface protein glycophorin. Some of the clones show an impaired ability to form tumors in vivo. These resistant clones might be useful for a genetic analysis of the differentiation process of Friend leukemia cells.     

Isolation of paralysis-inducing murine leukemia viruses from Friend virus passaged in rats.

Four clones of murine leukemia viruses (PVC-111, PVC-211, PVC-321, and PVC-441) were isolated from a paralyzed Fischer rat which had been infected with rat-passaged Friend leukemia virus. PVC-211 and PVC-321 viruses induced hind leg paralysis in rats and killed them within 1 month, and PVC-441 did so within 2 months after infection, whereas PVC-111 did not within 4 months. PVC-321 and PVC-441 but not PVC-111 virus grew well in brain and spinal cord media. The viral antigens were found often in glia cells and rarely in neurons of the rats infected with each of these PVC viruses. All of the PVC viruses induced neuronal degeneration but neither inflammation nor leukemic infiltration in the spinal cord. The isolated viruses were all ecotropic and NB-tropic. Age dependency of the susceptibility of rats to paralysis induction was observed.     

Effect of mouse interferon alpha/beta on the expression of H-2 (class I) antigens and on the levels of 2'-5' oligoadenylate synthetase activity in interferon-sensitive and interferon-resistant Friend leukemia cell tumors in mice

DBA/2 mice were injected intraperitoneally (i.p.) with interferon-sensitive 745 or interferon-resistant 3C1-8 Friend erythroleukemia cells (FLC) and then injected i.p. with mouse interferon alpha/beta. Interferon enhanced the expression of histocompatibility (H-2) antigens on individual 745 FLC within the peritoneum, but did not alter the expression of H-2 antigens on individual 3C1-8 FLC. Likewise, interferon treatment resulted in an increase in the level of 2'-5' oligo-adenylate (2-5A) synthetase activity in 745 FLC, but did not affect the level of activity in 3C1-8 FLC. These results provide evidence that the phenotype of interferon sensitivity or resistance of FLC does not change within the peritoneum. An incidental finding was that the basal level of 2-5A synthetase activity of in vivo passaged 745 cells was greater than that of 3C1-8 FLC. The finding that injection of mice bearing 745 FLC with antibody to mouse interferon alpha/beta reduced the level of 2-5A synthetase activity in these cells, but did not alter the level of 2-5A activity in 3C1-8 FLC, suggests that endogenous interferon in the peritoneum may have been the responsible factor.     

Interference established in mice by infection with Friend murine leukemia virus.

Retroviral interference is manifested in chronically infected cells as a decrease in susceptibility to superinfection by virions using the same cellular receptor. The pattern of interference reflects the cellular receptor specificity of the chronically infecting retrovirus and is mediated by the viral envelope glycoprotein, which is postulated to bind competitively all cellular receptors available for viral attachment. We established retroviral interference in mice by infecting them with Friend murine leukemia virus and them measured susceptibility to superinfection by challenging the mice with the erythroproliferative spleen focus-forming virus. Infection of approximately 10% of nucleated splenocytes rendered mice 1% as susceptible to superinfection as untreated controls. The magnitude of this effect was the same in mice incapable of producing neutralizing antibodies or genetically deficient for T cells. The results indicated that retroviral interference in vivo was established rapidly with infection of a fraction of the host cell population and that the decrease in susceptibility to superinfection occurred without a detectable contribution by immunologic factors.     

Natural killer cell suppression of Friend virus-induced preleukemic hemopoietic stem cells.

To determine whether hemopoietic cells infected with Friend polycythemia-inducing spleen focus-forming virus (SFFVp) are conserved or suppressed via natural surveillance in leukemia-resistant adult mice, we engrafted C57BL/6 recipients with isologous transgenic (donor origin marker) or natural killer (NK) cell-deficient B6 beige marrow cells exposed to SFFVp in vitro. Both groups of primary recipients were viremic and nonleukemic. Spleen cells from primary SFFVp-infected chimeras were engrafted into irradiated leukemia-susceptible secondary recipients to reveal dormant leukemia and grew as tumors of donor origin in 8 of 38 (21%) and 33 of 47 (70%) instances, respectively. Treatment of marrow donors and recipients with anti-asialo GM1 serum resulted in the depression of NK cell activity and the rapid development of dormant leukemia. We conclude that NK cells are an effective surveillance mechanism able to suppress SFFVp-induced preleukemic stem cells.     

Type II collagen-reactive T cell clones from mice with collagen-induced arthritis

Certain strains of mice develop a symmetrical polyarthritis after immunization with type II collagen. The incidence of arthritis after such immunization is variable. To study the arthritogenic potential of T cells reactive with type II collagen, we isolated draining lymph node cells from mice that had developed arthritis after immunization with bovine type II collagen. From these immune lymph node cells we were able to clone T cells reactive with type II collagen. Two separate sets of T cell clones were isolated. The first set reacted with either native bovine or native chick type II collagen, but did not react with type I collagen. The second set of T cell clones reacted with bovine type II collagen, but did not respond to either native chick type II collagen or type I collagen. These clones will be tested for their influence on the development of arthritis in vivo.     

Induction of immune resistance against L1210 lymphatic leukemia in mice after lethal irradiation and reconstruction with fetal liver cells.

The immunohematopoietic potential of syngeneic fetal liver cells (SFLC) was examined and compared with syngeneic bone marrow cells (SBMC). SFLC generated about 3 times less 12th-day spleen colonies (CFU-S) than adult SBMC did. To test the SFLC ability for reconstitution of the immune system, mice were lethally total body irradiated (TBI) and transplanted i.v. with 3 x 10(7) SFLC or 1 x 10(7) SBMC. Thus, injected hematopoietic cells contained the same number of CFU-S. On days 28, 35, 42, and 49 after transplantation the mice were injected i.p. with 10(6) immunogenic L1210-Maf cells (L1210 leukemia cells treated in vitro with mafosfamide for inhibition of their growth in vivo) to test the ability for generation of immune response against L1210 leukemia. On day 56 the animals were challenged with 10(3) L1210 leukemia cells. Strong resistance against the leukemia was induced in TBI + SFLC and TBI + SBMC mice, suggesting that the SFLC similarly as SBMC are able to reconstitute immune system of the TBI host.     

Natural killer cell mediated missing-self recognition can protect mice from primary chronic myeloid leukemia in vivo

Background

Natural Killer (NK) cells are thought to protect from residual leukemic cells in patients receiving stem cell transplantation. However, multiple retrospective analyses of patient data have yielded conflicting conclusions regarding a putative role of NK cells and the essential NK cell recognition events mediating a protective effect against leukemia. Further, a NK cell mediated protective effect against primary leukemia in vivo has not been shown directly.

Methodology/Principal Findings

Here we addressed whether NK cells have the potential to control chronic myeloid leukemia (CML) arising based on the transplantation of BCR-ABL1 oncogene expressing primary bone marrow precursor cells into lethally irradiated recipient mice. These analyses identified missing-self recognition as the only NK cell-mediated recognition strategy, which is able to significantly protect from the development of CML disease in vivo.

Conclusion

Our data provide a proof of principle that NK cells can control primary leukemic cells in vivo. Since the presence of NK cells reduced the abundance of leukemia propagating cancer stem cells, the data raise the possibility that NK cell recognition has the potential to cure CML, which may be difficult using small molecule BCR-ABL1 inhibitors. Finally, our findings validate approaches to treat leukemia using antibody-based blockade of self-specific inhibitory MHC class I receptors.     

Natural cytotoxicity against mouse hepatitis virus-infected cells. II. A cytotoxic effector cell with a B lymphocyte phenotype

The effector cell in mouse spleen which mediates natural cytotoxicity against mouse hepatitis virus (MHV)-infected target cells was characterized. The target cells were MHV-infected BALB/c 3T3, and the assay time was 3 hr. The effector cell, designated virus killer (VK) cell for the purpose of discussion, had the following phenotype: lymphocyte morphology, plastic-nonadherent, nylon wool-adherent, nonphagocytic, cyclophosphamide-sensitive; by antibody plus complement (C) depletion studies, it was asialo GM1-, NK 1.2 alloantigen-negative, Thy-1.2-, Lyt-5-, and macrophage antigen-negative; by rosetting techniques, it was Fc receptor-positive and surface Fab+; by flow cytometry (FACS) analysis, it was Lyt-2-, MAC-1-, Ia+, IgG (gamma)+, IgM (mu)+, IgD (delta)+, and B cell lineage antibody B-220+. NK cells, measured for cytotoxicity on YAC-1 cells, were similarly tested and were found to differ from the VK cell in the following properties: nylon wool-nonadherent, asialo GM1+, NK alloantigen-positive, Lyt-5+, surface Fab-, MAC-1+, Ia-, IgG-, IgM-, IgD-, and B-220-. The VK effector cell had a phenotype highly distinguishable from NK cells, effectors most commonly associated with antiviral natural cytotoxicity. The VK cell had a phenotype identical to that of a B lymphocyte and was identified as such. Although the effector cells displayed cell surface antibody, the antibody did not appear to be involved in lysis, because lysis could not be blocked by F(ab)'2 directed against Fab, mu, or delta. Cytotoxicity was more likely associated with recognition of the B lymphocyte surface by the MHV glycoprotein E2, as shown in the accompanying companion paper. This is the first demonstration that natural cytotoxicity can be mediated by B lymphocytes.     

Heteroduplex analysis of molecular clones of the pathogenic Friend virus complex: Friend murine leukemia virus, Friend mink cell focus-forming virus, and the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus.

The pathogenic Friend virus complex is of considerable interest in that, although members of this group are genetically related, they differ markedly in biochemical and biological properties. Heteroduplex mapping of molecular clones of the Friend virus complex, which includes the replication-competent ecotropic Friend murine leukemia virus (F-MuLV) and mink cell focus-forming virus (F-MCF) and replication-defective polycythemia- and anemia-inducing strains of spleen focus-forming virus (SFFVp and SFFVa, respectively), was employed to provide insight into the molecular basis of their relationships. In heteroduplexes of F-MuLV X F-MCF, a major substitution of 0.89 kilobases in the env gene of F-MCF was discerned. Heteroduplexes of SFFVp X F-MuLV or F-MCF and SFFVa X F-MuLV or F-MCF showed several major deletions in the pol gene region and a single major deletion in the 3' half of the env gene region of SFFVp and SFFVa. A major substitution of 0.89 kilobases was mapped to the 5' end of the env deletion of SFFVp and SFFVa in heteroduplexes with F-MuLV, similar to that seen in F-MuLV X F-MCF heteroduplexes. In contrast, this env gene region was totally homologous in F-MCF X SFFVp or SFFVa and SFFVp X SFFVa heteroduplexes. Our results suggest that (i) both SFFVp and SFFVa lack part of the env gene at its 3' end, corresponding to the p15(E) coding region, (ii) major deletions occur in the pol and env genes which account for the replication defectiveness of SFFVp and SFFVa, (iii) minor substitutions occur in the gag gene region of SFFVa that are not present in SFFVp, F-MuLV, or F-MCF, (iv) a major substitution exists in the gp70 region of the env gene between F-MuLV and F-MCF that probably accounts for the differences in their host range specificities, (v) this substitution in F-MCF is identical to the gp70 part of the gp52 coding region of SFFVp and SFFVa, and (vi) heteroduplexes to F-MCF show unambiguously that no additional large substitutions are present in SFFVp or SFFVa that could account for differences in their leukemogenicity.     

Protection of A/He mice by a Friend virus pseudotype against challenge with Friend virus.

Prior inoculation of 7-wk-old A/He mice with the Graffi pseudotype of Friend virus protected the animals against subsequent challenge with Friend virus. Graffi leukemia virus itself did not induce protection, probably because it failed to replicate in these mice.     

Haemoglobin synthesis in inducible, uninducible and hybrid Friend cell clones

In clone 707 of the Friend virus-induced erythroleukaemic cell line less than 1% of the cells stain detectably for haemoglobin with benzidine. On treatment with 2% dimethylsulphoxide (DMSO) the fraction of staining cells increases to 70–80%. Line Fw has a similar origin but does not respond to DMSO although up to 7–8% of these cells stain for haemoglobin when they are grown in intraperitoneal perfusion chambers. Evidence is presented that clone 707 does not contain sub-populations of non-inducible cells nor does the Fw line contain a sub-population of inducible cells. A BUdR-resistant derivative of clone 707, designated clone 707B2/7, was isolated and shown to be incapable of incorporating ³H-thymidine. A thioguanine-resistant derivative of line Fw, designated clone FwT6/4, was also isolated and shown to be incapable of incorporating ³H-hypoxanthine. Hybrids were prepared by fusing cells of clone 707B2/7 and clone FwT6/4 in the presence of inactivated Sendai virus and selecting the hybrids in HAT medium. The properties of parental and hybrid cells were studied. The hybrids contain chromosomes from both parents and can be induced with DMSO to form an increased fraction of haemoglobinized cells.     

Tissue-specific replication of Friend and Moloney murine leukemia viruses in infected mice.

We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.     

Effect of rifamycin and tilorone derivatives on Friend virus leukemia in mice.

Several derivatives of rifamycin, and analogs of the tilorone-fluoranthene group were tested for inhibition of splenic enlargement in Friend virus leukemia. At least three members of the rifamycin group caused significant inhibition (31-49%) as did at least three members of the tilorone group (32-48%). These six compounds are among those found by others (6, 7) to be most inhibitory in vitro to the RNA-directed DNA polymerase of oncornaviruses. However our studies do not furnish direct evidence for or against a role of inhibition of the viral enzyme in the suppression of splenomegaly. None of the agents was as effective as methotrexate, which caused 90-92% inhibition. The activity of five of the agents was reduced, rather than enhanced by the injection of adjuvants (M. butyricum and pertussis vaccine). Three of the agents had a subtractive, rather than an additive effect on the inhibition caused by methotrexate alone.