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experiments with IL-7 virus-infected cells resulted in >70% growth inhibition. Thus ...... Bender, M. A., T. D. Palmer, R. E. Gelinas, and A. D. Miller. 1987. Evidence ...
Vol. 11, No. 3

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1991, p. 1590-1597 0270-7306/91/031590-08$02.00/0 Copyright ) 1991, American Society for Microbiology

Interleukin-7 Retroviruses Transform Pre-B Cells by an Autocrine Mechanism Not Evident in Abelson Murine Leukemia Virus Transformants ROBERT W. OVERELL,* LAURETTA CLARK, DAVID LYNCH, RITA JERZY, ANN SCHMIERER, KAREN E. WEISSER, ANTHONY E. NAMEN, AND RAYMOND G. GOODWIN Immunex Corporation, Seattle, Washington 98101 Received 28 August 1990/Accepted 18 December 1990

In this study, we have constructed retroviral vectors expressing the interleukin-7 (IL-7) cDNA and have used infection with these retroviruses to express this cytokine endogenously in an IL-7-dependent pre-B-cell line. Infection with IL-7 retroviruses, but not with a control retrovirus, resulted in the conversion of the cells to IL-7 independence. The frequency at which this occurred, together with data on vector expression levels, indicated that secondary events were required for factor independence in this system. Southern analysis showed that the IL-7-dependent clones harbored unrearranged copies of the vector proviruses. The factor-independent cells produced variable quantities of IL-7 as measured by an IL-7-specific bioassay, and their proliferation could be substantially inhibited by a neutralizing antibody directed against IL-7, indicating that a classical autocrine mechanism was responsible for their transformation. These IL-7-independent cells were tumorigenic, in contrast to the parental IL-7-dependent cells or those infected with a control vector. These results showed that IL-7 could participate in the malignant transformation of pre-B cells. However, neither of two Abelson murine leukemia virus (A-MuLV)-transformed pre-B-cell lines expressed detectable IL-7 mRNA, at a level of sensitivity corresponding to less than one molecule of mRNA per cell. Moreover, the proliferation of the A-MuLV transformants was unaffected by addition of the IL-7 antisera under conditions in which parallel experiments with IL-7 virus-infected cells resulted in >70% growth inhibition. Thus, transformation of pre-B cells by A-MuLV was not associated with a demonstrable autocrine loop of IL-7 synthesis. These results show that IL-7 can participate in the malignant transformation of pre-B cells and suggest studies aimed at assessing the role of autocrine production of IL-7 in the generation of human leukemias and lymphomas.

transform pre-B cells (4), can confer growth factor independence on IL-3-dependent cells (11, 35), and there is evidence to indicate that this occurs by an autocrine mechanism involving IL-3 (17). IL-7 is a hematopoietic growth factor originally purified on the basis of its ability to induce the proliferation of bone marrow-derived pre-B cells grown in Whitlock-Witte cultures (30, 42). Subsequent studies using purified recombinant IL-7 (29) have shown that this cytokine is a potent mitogen for pro- and pre-B cells, although no proliferative activity for mature surface immunoglobulin-positive (sIg+) B cells has been demonstrable (28a). IL-7 is also active in promoting the growth of T lymphocytes (28). High-affinity plasma membrane receptors for IL-7 are found on lymphoid cells and also on a wide range of other somatic cell types (34). The IL-7 receptor belongs to a superfamily of hematopoietin receptors which share common structural motifs (16, 19). The full spectrum of biological activities of IL-7 on hematopoietic and other cell types that express IL-7 receptors has yet to be determined. The potent mitogenic activity of IL-7 suggested that it might be capable of functioning in an autocrine manner to provide early lymphoid cells with a capacity for autonomous or neoplastic growth. This study was undertaken to determine whether autocrine production of IL-7 could lead to the malignant transformation of pre-B cells. To this end, we have constructed retroviral vectors carrying the IL-7 cDNA and have shown that they confer growth autonomy on an immortal, IL-7-dependent pre-B-cell line. In contrast to the parental cells, the infected, IL-7-independent cells were acutely tumorigenic in syngeneic hosts. However, neither of

The proliferation of hematopoietic cells is controlled by a family of polypeptide growth factors. These cytokines, through their binding to high-affinity plasma membrane receptors, act at different levels in the hierarchy of the hematopoietic compartment to control the rates of production of hematopoietic and lymphoid cells (27). In accordance with the autocrine hypothesis of carcinogenesis (13, 39), there is substantial evidence that dysregulation of the genes encoding hematopoietic growth factors can lead to the generation of human hematopoietic malignancies. Documented examples of autogenous growth factor production being involved in human leukemias and lymphomas include granulocyte-macrophage and granulocyte colony-stimulating factor (GM- and G-CSF) in some cases of acute myelogenous leukemia (8, 47) and interleukin-2 (IL-2) in adult T-cell leukemia (3). Additionally, the hybridoma-plasmacytoma growth factor IL-6 has been shown to be produced in an autocrine or paracrine fashion in many human multiple myelomas (22). Autocrine production of IL-2, IL-3, colonystimulating factor 1, and chicken myelomonocytic growth factor has also been observed in hematopoietic malignancies in animal models (2, 5, 14, 17). In most of these studies, the proliferation of the tumor cells was shown to be inhibited by neutralizing antibodies against the cytokine which they produced, indicating that the tumor cells exhibited classical autocrine growth characteristics (2, 3, 5, 22, 47). In addition, Abelson murine leukemia virus (A-MuLV), which has been most extensively characterized on the basis of its ability to *

Corresponding author. 1590

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A

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2

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;-ra100 cells, using an inverted microscope. A-MuLV-transformed pre-B cells. Supernatant from A-MuLV-producing 3T3 cells was used to infect bone marrow cells from young adult mice. The A-MuLV supernatant had a titer of 5 x 105 focus forming units per ml, measured on NIH 3T3 cells as previously described (32), and conferred expression of immunoprecipitable P160gag-abl protein (31a). Infected bone marrow cells were assayed for transformation in agar as previously described (36). Colonies plucked from agar could be readily propagated as rapidly growing cell lines in medium without added IL-7. Analysis of vector structure and expression infected cells. Genomic DNA from infected 2b cells was isolated by the in-gel procedure (44). Each lane for Southern analysis contained DNA isolated from 106 cells. Southern and Northern (RNA) analyses were carried out as previously described (32). The gag-specific probe was a BsmI-BstEII fragment derived from the gag region of MoMLV. For RNA analysis, total cell RNA was extracted and poly(A)+ mRNA was selected by oligo(dT)-cellulose chromatography as previously described (25). The antisense U3-specific riboprobe used to detect vector-specific RNAs was generated by SP-6 RNA polymerase transcription of pGEM2-U3. This vector was constructed by inserting the HpaII fragment of the MoMLV U3 region from U3-Ml3mp9 (12) into pGEM2 (Promega, Madison, Wis.). For analysis of IL-7 mRNA levels, poly(A)+ mRNA was loaded onto agarose-formaldehyde gels in parallel with a series of dilutions of sense IL-7 RNA standards (1,600 bp) which were transcribed in vitro from the 1046 cDNA (29). The standards were loaded at 10, 5, and 2.5 pg per lane,

which corresponded to approximately 0.5, 0.25, and 0.125 molecules of mRNA per cell, based on maximal estimates of the amount of poly(A)+ RNA per A-MuLV-transformed pre-B cell (0.5 pg) and the number of cell equivalents of RNA from the A-MuLV-transformed cells loaded on the gel [10

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MOL. CELL. BIOL. TABLE 2. Induction of IL-7 independence in infected pre-B cells Wells growth positive/

TABLE 1. Plating efficiencies of 2b cells infected with retroviral vectors Infecting vector

No selection Mock LSNL

L.IL-7SN G418 selection Mock LSNL

L.IL-7SN

Infecting vector

Growth-positive wells/24 wells seeded ata: 10,000

24 24 24

3,000

1,000

24 24 24

300

24 24 24

0

0

0

0

24 10

21 9

8 3

0 0

30

24 22 24

IL_7a

3

18 4 8 -

Figures represent numbers of 2b cells seeded

per 0.2-ml well; 24 wells scored for each group. All cells were seeded in the presence of IL-7. -, Groups not done. The vectors used were produced by clones of t2 cells and had titers of 1.3 x 106 and 4 x 105 G418r CFU/ml for LSNL and L.IL-7SN, respectively. on NIH 3T3 cells. a

wells seeded without

were

Lg, or 2 x 107 cell equivalents of poly(A)+ RNA]. Northern hybridization was carried out with riboprobes as previously described (33). IL-7 cDNA probes were generated from a Dral-EcoRI fragment of the 1046 murine IL-7 cDNA (29). IL-7 production and neutralization assays. Infected 2b cells were assayed for IL-7 production by seeding them at 105 cells per ml in 2b medium without IL-7 and allowing them to condition the medium for 4 days. Cell-free supernatants were then collected and assayed for IL-7 levels, using the 2b proliferation assay. The effect of M25, a neutralizing antibody to IL-7, on the original 2b cells and the infected IL-7-independent clone 2b/7SN.1 was also assessed by using this proliferation assay. The assay conditions were identical to those described previously (30) for the quantitative determination of IL-7. Various concentrations of M25 were tested for their ability to inhibit the IL-7-mediated stimulation of the 2b and 2b/7SN.1 cell lines. Tumorigenicity assay. BALB/cAnN mice (Charles River, Kensington, Md.) were either subjected to 500 R of wholebody irradiation to inhibit the ability to generate a primary immune response or left unirradiated. Groups of irradiated and unirradiated mice (five mice per group) were challenged with 106 cells via intradermal injection. The presence of actively growing tumors was evaluated in all groups 25 days after tumor challenge.

RESULTS The IL-7-dependent pre-B-cell line 2b (30) was infected with a control vector, LSNL (32), expressing the neo gene only, or with the L.IL-7SN retroviral vector, coexpressing the neo gene and the murine IL-7 cDNA (Fig. 1B). Following cocultivation with virus-producing tl2 cells, 2b cells were plated at limiting dilutions in the presence and absence of G418 in medium containing IL-7. Under these growth conditions, the 2b cells exhibited a high cloning efficiency and were infectable with these ecotropic retroviral vectors (Table 1). The infection frequencies of the 2b cells were 0.1 to 1%, as judged from the relative plating efficiencies of the cells in G418, and were higher for the LSNL vector, consistent with the higher titer of this virus (Table 1). Infected 2b cells were also assayed for their ability to grow in the absence of IL-7 after infection with LSNL, L.IL-7SN, and L.IL-7. The structure of the L.IL-7 vector is shown in Fig. 1B. This vector carried the IL-7 cDNA but lacked a selectable marker gene (Fig. 1B). In the absence of IL-7, no

LSNL ..... L.IL-7SN ..... L.IL-7 .....

0/384 4/288 15/384

a All cells assayed were seeded at 5,000 cells per well without added IL-7.

outgrowth of cells was observed with use of 2b cells infected with the control LSNL vector (Table 2), nor has spontaneous IL-7 independence ever been observed with this cell line in several other experiments (data not shown). In contrast, IL-7 independence was consistently induced in the 2b cells infected with the L.IL-7SN or L.IL-7 virus (Table 2). The frequency of induction of IL-7 independence was approximately 1 in 4 x 105 cocultivated 2b cells with L-IL-7SN and 1 in 1.3 x 105 with the L.IL-7 retrovirus. Comparison of the frequencies of drug resistance and IL-7 independence in the L.IL-7SN*42-cocultivated 2b populations indicated that only a small fraction of the cells which were infected by the criterion of drug resistance were IL-7 independent. Parallel selections for G418 resistance and IL-7 independence with the same cocultivated 2b populations indicated that less than 1% of the infected cells were factor independent in this assay (Tables 1 and 2). Seven clones selected for IL-7 independence were isolated (one from the L.IL-7SN group and six from the L.IL-7 group). Of these, two of the L.IL-7-infected clones failed to grow after transfer from the initial wells. The other five clones expanded rapidly in the absence of IL-7. These were propagated over more than five passages without added IL-7 and were therefore stably IL-7 independent (data not shown). Southern analysis of the proviral structure in infected 2b cells showed that both the L.IL-7 and the L.IL-7SN vectors had been transmitted by retroviral infection without undergoing gross deletions or rearrangements (Fig. 1A). This was the case whether the infected 2b cells were selected for IL-7 independence with either vector (Fig. 1A, lanes 1 and 7 to 10) or for G418 resistance with the L.IL-7SN vector (Fig. 1A, lanes 2 to 6). All of the infected cells assayed were isolated from wells seeded at limiting cell dilutions. Their clonality was confirmed by Southern analysis with a restriction enzyme which cut once in the provirus, which indicated that the clones analyzed contained single proviral insertions with junction fragments of different sizes (data not shown). Flow cytometry showed that the surface phenotype of the IL-7-independent pre-B cells was similar (B220- sIg+) to that of the parental 2b cells (Fig. 2 and data not shown). The IL-7-independent clones were assayed for levels of secretion of IL-7 into the culture medium. The clones produced variable quantities of IL-7 (Table 3), as measured by the ability of the culture supernatants to support the proliferation of uninfected 2b cells (30). The cells were dependent upon the IL-7 which they produced, since their proliferation could be substantially inhibited by the addition to the cultures of the IL-7 neutralizing antibody M25 (Fig. 3A). At the highest concentration of antibody used, the growth of the cells was inhibited by approximately 75%, and the inhibition was specific, since it was reversed by the addition of exogenously added IL-7 (Fig. 3A). 2b cells and their infected derivatives were injected subcutaneously into BALB/c mice. None of 38 animals injected

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B220

I% I

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FIG. 2. Surface phenotypes of 2b cells and infected derivatives as determined by flow cytometry. 2b, Starting 2b cells; 2b/7SN.1, clone of 2b cells made IL-7 independent by infection with the L.IL-7SN vector; 2b/7SN.lT, cells from a tumor induced by the 2b/7SN.1 cells. The dashed lines represent background staining oberved with either cells alone [for the anti-immunoglobulin reagent, which was directly fluorescein isothiocyanate conjugated F(Ab')2 (Cappel)] or fluorescein isothiocyanate-conjugated anti-rat kappa (second-step reagent for the 2C2 antibody [9] used to detect the B220 cell surface antigen).

with naive 2b cells or the LSNL-infected cells gave rise to tumors, whereas all of the 2b clones rendered factor independent by infection with the IL-7 retroviruses generated large (>2.5-cm diameter) tumors at the site of injection by day 25 (Table 4). Histological examination indicated that the tumors were composed of cells with a lymphoid morphology. The proportion of tumor-bearing animals varied between the groups injected with the different IL-7-independent clones (Table 4). No clear correlation was evident between the levels of IL-7 produced by the infected clones in vitro and their tumorigenicity, as assessed by the proportion of tumor-bearing animals by day 25 (Tables 3 and 4). A tumor induced by injection of clone 2b/7SN.1 was excised and disaggregated, and the resulting single-cell suspension placed in liquid culture without IL-7. These cells, termed 2b/7SN.1T, were found to be of the same surface phenotype as the 2b/7SN.1 cells which were injected into the animal (Fig. 2). Moreover, Southern analysis of the parental 2b/ 7SN.1 and the 2b/7SN.1T tumor cells showed that they had proviral junction fragments of the same size hybridizing with the neo probe (data not shown), indicating that it was the injected cells that had given rise to the tumor. The tumor cells and the parental cells expressed similar levels of vector-specific transcripts, as judged by RNA blot analysis using a probe specific for the U3 region of MoMLV (Fig. 4A, lanes 2 and 3). The position of the major transcript on the blot corresponded to the size of the spliced vector mRNA TABLE 3. Production of IL-7 by pre-B cells infected with IL-7 retroviruses Cell clone

Infected with (vector):

Cells (106)/mla

U of IL-7/ml

2b/7.1 2b/7.2 2b/7.3 2b/7.4

L.IL-7 L.IL-7 L.IL-7 L.IL-7

2.0 1.2 1.8 1.4

14 57 3