H U M A N G E N E T H E R A P Y 2:301-306 (1991) Mary Ann Liebert, Inc., Publishers

I L - 6 P r o d u c t i o n b y Retrovirus P a c k a g i n g Cells a n d Bone Marrow

Cultured

Cells

M.P.W. EINERHAND, T.A. BAKX, and D. VALERIO

ABSTRACT Retrovirus integration into the host cell genome occurs most efficiently in replicating cells. In agreement with this notion, it w a s observed that the efficiency with which hemopoietic stem cells ( H S C ) can be transduced is greatly enhanced w h e n the hemopoietic growth factor ( H G F ) interleukin 3 (IL-3) is added to co-cultures of bone m a r r o w cells with retrovirus-producing cells. T h e H G F IL-6, which enhances the IL-3-induced formation of blast cell colonies in vitro, is also believed to improve the transduction of H S C . Because IL-6 can be produced by a n u m b e r of different cell types, w e investigated whether IL-6 w a s present in the culture supernatant of retrovirus packaging cells and bone m a r r o w cells. W e found that the six retrovirus packaging cells tested produce large amounts of IL-6. Bone m a r r o w cells cultured with IL-la and IL-3 also m a k e IL-6, and, following co-cultivation of both cell types, the concentration of IL-6 in the m e d i u m is even u p to 10-fold higher than the s u m of the concentrations obtained w h e n both are cultured separately. Considering that IL-6 is produced in large amounts during co-cultivations, w e believe that its effect on the transduction of H S C cannot be measured by adding extra growth factor to the co-culture m e d i u m .

co-cultivated with virus-producing cell lines for a period of 2-5 days (reviewed in Chang and Johnson, 1989; Einerhand and Valerio, A major goal of investigators at present is to increase the 1992). Since division of the target cell is required for efficiency of gene transfer into murine hematopoietic pluri- efficient integration of retroviruses (Weiss et aL, 1984), copotent stem cells as a step toward human gene therapy via cultivations are usually performed in the presence of HGFs bone marrow. The optimization of the growth medium by known to act on primitive HSC. In the mouse system, the addition of the appropriate growth factors is one of the presence of IL-3 during co-cultivation greatly facilitates the important variables in this process. Einerhand et al. evalu- efficiency with which H S C can become transduced (Eglitis et al., 1985; Lemischka et aL, 1986; Lim et aL, 1989). Most ate the influence of several of the critical growth factors. laboratories also add IL-la to their co-culture medium because it stimulates the proliferation of colony-forming units on the spleen (CFU-S) in short-term cultures in a synergistic fashion with IL-3 (Belmont et aL, 1988; Valerio et aL, 1989; Jordan and Lemischka, 1990; Jordan etaL, 1990; Moore etaL, 1990; INTRODUCTION Van Beusechem et aL, 1990). Recently, it was discovered that the H G F IL-6 in combination with IL-3 enhances the formation Maximizing the efficiency with which HSC can of beblast cell colonies (an in vitro assay for multipotent hemopoigenetically modified is of major interest for the devel- etic progenitor cells with self-renewing potential), apparently opment of gene therapy protocols for hematological disorders. by reducing the period during which these cells reside in G0 The most efficient method of gene transfer into H S C is by (Ikebuchi et aL, 1987; Koike et aL, 1988). IL-6 is therefore means of retroviral vector technology where bone marrow is considered to be a growth factor that could enhance gene trans-

OVERVIEW S U M M A R Y

Department of Gene Therapy, Institute of Applied Radiobiology and Immunology TNO, 2280 H V Rijswijk, the Netherlands.

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302 fer into HSC. A study performed on the efficiency with which CFU-S could be transduced by a retroviral vector revealed that 40-80% of the CFU-S were transduced when both IL-3 and IL-6 were present in the co-culture medium, whereas 30-37% of the CFU-S were transduced in the presence of only IL-3 (Bodine et aL, 1989). Therefore, IL-6 has been included in the co-culture medium by adding the recombinant growth factor (Bodine et aL, 1989) or by transfection of an IL-6 expression vector into retrovirus-producing packaging cells to enable the continuous production of IL-6 (Bodine et aL, 1990). Since a large number of different cell types, including fibroblasts and mononuclear cells residing in the circulating blood or the bone marrow can make IL-6 (Weissenbach et aL, 1980; Aarden et aL, 1987;Cayphas67tf/., 1987; Van Snick, 1990), we investigated whether IL-6 was produced by fibroblastoid retrovirus packaging cells or bone marrow cells during culture. W e found that all commonly used packaging cells produce large amounts of IL-6. Bone marrow cells cultured with IL-la and IL-3 also produce IL-6, but to a lesser extent. When both cell types were co-cultivated in the presence of IL-la and IL-3, the production of IL-6 increased further and reached levels that are saturating for blast cell colony-forming cells (Koike et aL, 1988). The production of IL-6 during co-cultivation could be induced by IL-la and by IL-3. The presence of both factors in the medium of co-cultivations resulted in the highest concentration of IL-6. Due to the large amounts of IL-6 present also at the start of the co-culture, we conclude that its effect on the transduction of H S C can not be studied by adding extra IL-6.

E I N E R H A N D E T AL. streptomycin and 100 units (u)/ml penicillin. The B9 cell line (Aarden et aL, 1987) was maintained in RPMI-1640 containing 1 0 % heat-inactivated FCS, 100 |xg/ml streptomycin, 100 U/ml penicillin, and 50 U/ml recombinant hIL-6. B9 assay for IL-6 The assay was carried out essentially as described by Helle et aL (1988). Briefly, the B 9 cells were washed in IL-6-free medium and plated 5,000 cells per 200-(xl culture medium in the presence of the samples to be tested. Proliferation was measured by adding 7.4 kBq [3H]thymidine ([3H]dThd) (74 GBq/mmole) after 64 hr of culture. Four hours later the cells were harvested and washed, and the incorporated radioactivity was measured. Samples for the mIL-6 inhibition assay were titrated by three-fold dilutions; the other samples were titrated by 10-fold dilutions. All samples were tested in triplicate and related to a standard batch of IL-6. The concentration of IL-6 that gave rise to half-maximal [3H]dThd incorporation was designated 1 U/ml. The murine IL-6 inhibition assay was performed by titrating the inactivating m A b 6b4 (Vink etaL, 1988) into approximately 1 U/ml of the samples to be tested. Bone marrow harvest and co-cultivation

Bone marrow from 7-week-old BCBA mice was harvested by flushing the femora and tibiae with HEPES-buffered (10 m M , p H 6.7) Hanks' balanced salt solution (HH). Most of the erythrocytes and granulocytes were removed by running the bone marrow cells through a discontinuous metrizamide gradient (Visser et aL, 1984). The top fraction was washed once with MATERIALS A N D M E T H O D S H H to remove the metrizamide. The retrovirus packaging cells or the POC-1 cells were seeded 1 day prior to the co-cultivation Reagents such that they were approximately 7 0 % confluent on the folRecombinant murine (m) IL-6 (Simpson et aL, 1988) and the lowing day. Prior to the co-cultivation the feeder cells were mIL-6 inactivating monoclonal antibody (mAb) 6b4 (Vink et irradiated (20 Gy, 0.70 Gy/min l37Cs gamma-ray). The coaL, 1988) were kind gifts from Prof. J. van Snick (Ludwig cultures were initiated by replacing half of the culture medium Institute for Cancer Research, Brussels). Recombinant human by fresh culture medium containing in final concentrations, (h) IL-6 (Brakenhoff et aL, 1987) was a generous gift from Dr. bone marrow top-fraction cells (1 X 106/ml), polybrene (0.4 L. Aarden (Central Laboratory of the Netherlands Red Cross (xg/ml), and the recombinant growth factors hIL-la (10 U/ml) blood transfusion service, Amsterdam). and/or mIL-3 (500 U/ml). Cell-free supernatants were harvested by spinning (7 min, 500 x g, rt) the culture medium and subsequently filtering the supernatant (0.22 \xm, Millipore). All Animals samples were stored at — 20°C until use. BCBA (C57BL/KaLwRij x CBA/BrARij)F, mice were bred and maintained under specified pathogen-free conditions RESULTS in our Institute.

A growth factor that stimulates the IL-6 dependent cell line B 9 is produced by (co-)cultures of bone marrow cells and retrovirus packaging cells The retrovirus packaging cell lines i|j-2 (Mann et aL, 1983), PA317 (Miller and Buttimore, 1986), G P + E-86 (Markowitz To examine whether IL-6 is produced during co-cultivation et aL, 1988b), G P + envMA\2 (Markowitz et aL, 1988a). of bone marrow with retrovirus packaging cell lines, we started i|/-CRE and i|i-CRIP (Danos and Mulligan, 1988), and the co-cultures of bone marrow with the frequently used ecotropic POC-1 cell line (Van Beusechem et aL, 1990), a subclone packaging cell lines i|/-2, G P + E-86 and v|i-CRE and the amderived from i|/-CRIP which produces the retroviral vector photropic packaging cell lines PA317, G P + envAM\2, and L#AL(AMo + PyFlOl) containing an h adenosine deaminase i|i-CRIP. Supernatants were harvested after 3 days of culture (ADA)-cDNA gene, were maintained in high glucose (4.5 and tested on the IL-6 dependent cell line B9. A growth factor grams/liter) alpha-modified Delbecco's essential medium con- with activity on B9 cells was produced in all co-cultures. The taining 1 0 % heat-inactivated fetal calf serum (FCS), 100 |xg/ml levels ranged from 4 x 103 U/ml for the co-culture with PA317 Cell lines

IL-6 PRODUCTION BY PACKAGING CELLS A N D BONE M A R R O W

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cells to 4 x 107 U/ml in the co-culture with GP + E-86 cells (Fig. 1). To determine whether this activity was produced by the bone marrow cells or by the packaging cells, supernatants were harvested from parallel monocultures of bone marrow or retrovirus packaging cell lines. Titration of these supernatants on B9 cells revealed that both sources produced this activity, although the levels clearly differed (Fig. 1). The culture with only bone marrow cells contained 103 U/ml. The levels produced by the packaging cells ranged from 5 x 102 U/ml for PA317 cells to 4 x 106 U/ml for G P + E-86 cells. Supernatant from a control culture with only the co-culture medium (i.e., with IL-la, IL-3, and Polybrene) did not contain a stimulatory activity on B9 cells (i.e., < 20 U/ml; Fig. 1).

concentrations of m A b , recombinant mIL-6 was likewise inactivated (Fig. 2D). Human IL-6 is 4 2 % homologous to mIL-6 (Van Snick, 1990), but it is still active on murine cells. Its specific activity on B9 cells is, however, 5- to 10-fold lower than that of mIL-6 (Fig. 2E). As a control for the murine specificity of the inhibition reaction, we therefore performed an inhibition assay on 1 U/ml hIL-6. As expected, no inhibition was detected even at a concentration of 5 fxg/ml m A b 6b4, thereby demonstrating the specificity of the inhibition test for mIL-6 (Fig. 2D). Since murine B9 cells can, upon prolonged culture, become independent from added IL-6 due to endogenous production of mIL-6 (not shown), we verified the absence of endogenous mIL-6 by an inhibition assay on B9 cells to which no IL-6 was added. In this titration of the antibody the The B9 activity produced by packaging cells and bone [3H]dThd incorporation of the B9 cells was only marginally influenced (Fig. 2D). Therefore, we conclude that murine bone marrow is inhibited by mIL-6-inactivating antibodies marrow cells as well as the fibroblastoid retrovirus packaging To obtain additional evidence for the presence of mIL-6 in produce mIL-6 when co-cultured or cultured separately in cells the culture supernatants, we performed an inhibition assay with the presence of IL-la, IL-3, and polybrene. the mIL-6-inactivating monoclonal antibody (mAb) 6b4. The m A b was diluted into a concentration of approximately 1 U/ml The mIL-6 concentration in the medium of co-cultures of test sample or recombinant growth factor. The supernatants increases markedly after 1 day of culture tested in this assay were derived from i|;-CRE and i|;-CRIP, From the above it is evident that large amounts of mIL-6 cultured alone or with bone marrow cells, and from a culture of were produced during a 72-hr co-cultivation with IL-la, IL-3, bone marrow cells alone. In all of these supernatants, the stimand Polybrene. To study the production of IL-6 during coulatory activity was blocked by the addition of the m A b at cultivation, we measured its presence in the supernatants of concentrations of 0.15 (xg/ml to 5 |xg/ml (Fig. 2A-C). At these co-cultivations at different time points. For this assay we used a subclone derived from i|;-CRIP called POC-1, which produces a hADA-cDNA-containing retrovirus. The first co-culture was stopped immediately after seeding the bone marrow cells and 108n thus contains only IL-6 that is produced by POC-1 cells during 107the overnight incubation in the absence of HGFs. This sample 1 contained 2 x 103 U/ml mIL-6 (Fig. 3), which proves that IL-6 t 106is also produced by the retrovirus packaging cells in the absence 3 of IL-la, IL-3, and Polybrene. During the first 24 hr of cor^ § 105- • • cultivation, the levels of mIL-6 increased slightly (Fig. 3). *" After this period the concentration increased 7- to 30-fold. This S 10J P" I• • • increase could be caused by an increase in the number of IL-6