HYBRIDOMA Volume 10, Number 6, 1991 Mary Ann Liebert, Inc., Publishers

Histocompatible Miniature Pig (d/d Haplotype): Generation of Hybridomas Secreting A or M Monoclonal Antibody BERTRAND KAEFFER, ELISABETH BOTTREAU, DOMINIQUE MARCON, MICHEL OLIVIER, ISABELLE LANTIER, and HENRI SALMON Laboratoire de

Pathologie Infectieuse

et

Immunologie INRA,

37380

Nouzilly,

France

ABSTRACT Two Hypoxanthine/Aminopterin/Thymidine-sensitive cell sublines (L142 and L231) have been derived from independent lymphoblastoid cell lines of B lineage. After propagation for more than 100 population doublings (1 year) in culture, these cells still retained a doubling time between 19 to 20 hours, near diploidy and relatively low (L142) and high (L231) secretion rate of M immunoglobulins. Near diploid hybrid cells were easily generated with leukocytes from the spleen, the gut lamina propria or the mesenteric lymph nodes of pigs immunized against the transmissible gastroenteritis virus. Both the tumor sublines and the B cells were derived from histocompatible miniature pigs (d/d haplotype). Demonstration of fusion between the tumor sublines and B-cells was supported by the selection of hybridomas making the antigen-specific heavy (alpha isotype) and light chains from the B cell parent as well as the heavy and light chains of the lymphoblastoid parent. Moreover, some hybridomas were found to secrete only class A (dimeric) or class M immunoglobulins (0.2-10 u.g/ml). Forty hybridomas secreted antibodies reactive in a virus-enzyme-linked cell immunoassay against cell-bound antigens and two were found to produce an antibody active only against the infected cell monolayer. Construction of intraspecies hybridoma can be used to perpetuate lymphocyte subsets useful for the study of the porcine immune system. INTRODUCTION

Somatic cell fusion with a tumor cell line to perpetuate individual lymphocytes in tissue culture has been developed by Köhler & Milstein [1] for mouse B cells allowing the establishment of continuous cell lines producing monoclonal antibody (MAb) of defined specificity (hybridomas). The hybridoma technology has been rapidly improved in Balb/c mouse to generate fusion partners which do not secrete immunoglobulins and retain a high proliferation rate [2, 3]. Application of this technology to other species has been limited in part by the tumor cell lines available. Generation of intraspecies hybridomas have been demonstrated in rat [4], human [5, 6] and bovine [7]. In the pig, construction of interspecies hybridoma producing monoclonal antibody of defined specificity is well documented [8, 9, 10]. Since 1976, strains of miniature pigs homozygous for a different alíele of the major histocompatibility locus have been developed [11]. From the peripheral blood mononuclear cells of an histocompatible boar (d/d haplotype), we have established lymphoblastoid cell lines of B and T lineage producing retrovirus [12]. The growth properties and karyotype analyses of these lines suggest that they might be used as partners for fusion with B cells from the corresponding pig strain. The generation of intraspecies hybridomas might be valuable not only to produce monoclonal antibodies but also to study the pig immune system and the mechanism of oncogenesis involved in the lymphocyte transformation. We have chosen to immunize pigs against the transmissible gastroenteritis virus because this coronavirus is the ethiologic agent of a highly contagious enteric disease of swine under investigation in our laboratory for humoral [13] and local [14] immune responses. Fusions have been performed with leukocytes from the spleen, the gut lamina propria or

731

the mesenteric lymph nodes with the aim of perpetuating different B lymphocyte subsets. Hybrid cell cultures were screened for IgG, IgM or IgA production by a monoclonal antibody-capture-ELISA [12] and for any antibody activity against cellular or viral antigens by a virus enzyme-linked cell

immunoassay (VELCIA) [15].

We report the selection and properties of two aminopterin-sensitive sublines of B lineage (L142 and L231). Hybrid cells were easily generated and some identified as fusion products by showing that after cloning they secrete both the parent sublines M immunoglobulin and new immunoglobulin. Hybridomas were shown to secrete only class A or class M immunoglobulin with an antibody activity in VELCIA.

MATERIALS and METHODS Cell lines and selection of tumor fusion partners All cells were maintained mycoplasma-free [16]. The swine-testis (ST) cell line [17] was used in VELCIA. Myeloma P3X63Ag-8 653 of murine origin [18], donated by Dr. Wahib Mahana (INRA-Nouzilly, France), was used as control for its HAT-sensitivity. The L142 and L231 lines are available for research use from the Laboratoire de Pathologie Infectieuse et Immunologie of the Institut National de la Recherche Agronomique. Parental cell lines LI4 and L23 can be obtained from the European Collection of Animal Cell Cultures, Portón Down, Salisbury, UK. Growth medium used to cultivate lymphoblastoid cells was RPMI 1640 supplemented with 10% fetal calf serum, ImM pyruvate, 2mM L-glutamine, 50 |iM 2-ß-mercaptoethanol and 0.5% penicillin-streptomycin. A cell survival selection in 6-thioguanine supplemented medium [19] was used to obtain HATsensitive sublines from lymphoblastoid cell lines of B (L14, L23 and L52) and T (L35 and L45) lineage [12]. Briefly, samples often million lymphoblastoid cells were selected in a growth medium containing 10, 20 or 30 (ig of 6-thioguanine per ml renewed every 3 days for 3 weeks. Then, clusters of 20 to 50 surviving cells were recovered with a Pasteur pipette with the aid of an inverted microscope. Each cluster was transferred to a Costar P24 well and cultivated in growth medium supplemented with 5 (ig of 6-thioguanine per ml renewed twice a week. Characterization of cell lines Metaphase chromosomes were revealed by buffered-Giemsa staining as reported elsewhere [12] and referred to as solid stained metaphase. Fifty metaphases per cell line were counted using an Olympus microscope (x 1300 magnification). Karyotypic analyses of L142 and L231 sublines were made on photographs of solid stained metaphases what allowing detection of numeric abnormalities and gross structural abnormalities [20]. Chromosomes of 10 metaphase preparations per subline were classified using swine chromosome standard classification [21]. Only one preparation per subline is

presented.

Cellular morphology was observed on cytocentrifuged cell preparations following MayGriinwald Giemsa staining and photographs were taken in phase contrast microscopy under oil with an

Olympus microscope.

Panel of murine monoclonal antibodies against pip immunoglobulin heavy and light chains During screening and isotypic analyses, we used MAb specific to heavy chains of mû (28.4.1 MAb donated by Dr Bianchi, Lelystad, NL and K513D MAb donated by Dr Stevens, University of Bristol, UK), gamma ( 23.7.1. MAb from Dr Bianchi), alpha (27.9.1. MAb from Dr Bianchi and K61 1B4 M Ab donated by Dr Bourne, University of Bristol, UK) isotypes and to light chains (K139 3 El MAb from Dr Stevens). The properties of the MAb received from Dr Bianchi have been described [22]. In our hands, the anti-light chains specificity of MAb K139 3 El was demonstrated by its reactivity with all immunoglobulins both in a monoclonal antibody-capture-ELISA and in immunofluorescence labelling on living B cells. By western blotting1, this MAb binds to a protein of molecular weight of 24 kDa. Immunization procedures of the histocompatible miniature boars (d/d haplotype) The herd bred at Nouzilly since July 1985 was originally provided by Dr Sachs (N.I.H., Bethesda, MD). We chose to immunize 2 boars (referred as 1 and 2 in this paper) because they were already hyperimmunized against transmissible gastroenteritis (TGE) virus and displayed seroneutralizing titers up to or equal to 64. The boars were respectively 3.5 and 2.5 years old at autopsy. During previous experiments, they were hyperimmunized against ovalbumin by multiple intravenous injections. To boost boar 1, we used supernatant of tissue-culture-adapted viral strain referred to as Nouzilly 188-SG [23]. Boar 1 was given per os 108 Plaque Forming Units (PFU) of the virus, 70 '

Stokes, C. R„ Stevens, K. and Bourne, F. J. (1987) EEC meeting, New Methods in Animal Cell Culture and Cell Bank, 24-27 May, Seillac, France. 732

and 63 days before fusion. An intramuscular booster was done 56 days before fusion with 10° PFU. A final intraperitoneal injection with 10° PFU was done 3 days before fusion. To boost boar 2, we used a crude intestinal sample of the virulent GEP-II strain of transmissible gastroenteritis virus prepared as in [23]. Boar 2 was given per os viral preparation 4 days before fusion. These immunization procedures were chosen with the aim to elicit an immune response against the virus involving IgA-secreting plasma cells. Source and preparation of leukocytes for fusion Spleen was aseptically stored in ELY medium (ELAC medium from Flow Laboratories supplemented with 0.1% yeast extract (Difco)) and minced before isolation of splenocytes. Cell suspensions were passed through gauze and red-blood cells were lysed using NH4CI 0.155 M, pH 7.4 (2-5 min on ice). This is referred to as crude cell preparation. Thereafter, leukocytes were rinsed three-times in ELY medium and separated on Ficoll (Pharmacia, n° 17-0400-01 used as follows : d 1.09, 15 min, 2000 x g, room temperature). Lamina propria and mesenteric lymph nodes were gently minced and cells were recovered on a Ficoll gradient using a standard procedure [24]. Cell viability was assessed by the trypan blue dye exclusion test. Both the crude cell preparation and the purified leukocyte preparation were used for fusion provided that the cell viability was at least 75%. Cell fusion and cloning The main procedure was derived from Harlow & Lane [25]. Briefly, 4 x 10' leukocytes were mixed in warm medium with 10' cells of L231 or L142 sublines (viability: 90-98%) and pelleted at 200 x g, 10 min. Supernatants were discarded and 1 ml polyethylene glycol 1500 (PEG 50%, w/v, Boehringer-Mannheim, 783641) was added dropwise on the pellet with gentle stirring for 1 min. Over 20 min, warm growth medium was added dropwise to dilute out the fusogen. Cells were pelleted (200 x g, 10 min), resuspended in warm HAT (5 x 10"^ M hypoxanthine, 2 x 10"-* M aminopterin, 8 x 10~4 M thymidine, Sigma, H-0262) growth medium and distributed either in P96 (50 (il/well, Falcon, 3072) or in P24 (500 ul/well, Linbro, 7603305) microplates, at a density of 2 x 10^ cells (either L142 or L231) per ml. Microplates had been prepared the day before fusion and were preconditioned with HAT growth medium (50 or 500(0.1 for P96 or P24 respectively) by storage in an humidified incubator (37^, 5% CO2). No feeder-cells were used to sustain growth of hybrids or clones. Twenty days after fusion, the HAT growth medium was progressively replaced by HT (5 x 10"3 M hypoxanthine, 8 x 10"4 M thymidine, Sigma, H-0137) growth medium. Modified steps in this procedure involved the fusogen (1 ml PEG 1500 (45%) + 5% DMSO) and the handling of cells following fusion : the fused cells were distributed immediately or left aside for 2 or 4 hours at 37*0 in growth medium before adding to plates. =

Screening procedure From six days after fusion, the growth of hybrids was monitored and supernatants were periodically sampled and assayed by VELCIA to detect antibodies reactive against viral or cellular antigens as well as by a MAb-capture-ELISA to determine the isotypes of the secretedimmunoglobulins. The virus enzyme-linked cell immunoassay [15] was performed on infected or mock-infected ST cell monolayers. We did not find any significant difference between cell monolayers infected by the Purdue [26] or the Nouzilly 188-SG [23] TGE virus strains. Briefly, 2 x 105 ST cells per ml were distributed in P96 microplates 3 days before infection with TGE virus (multiplicity of infection of 1) or mock-infection. Seventeen hours later, the cell monolayers were fixed in 80% acetone at 20^ for 20 min. Microplates were washed and stored in PBS with azide (0.02%) at 4^. Before use the microplates were washed using a standard procedure: 2 times gently with tap water and 2 times with a 5 min incubation steps in a washing solution (isotonic solution containing 0.9% NaCl and 0.05% Tween 20). A short blocking step was done during 15 min by incubating with 100 ul / well of a PBS buffer containing 0.05% Tween 20 and 5% skim-milk powder. Twofold dilutions of cell supernatants were incubated for 90 min in blocking buffer. Following a washing cycle, peroxidase labelled-MAb K139 3 El against pig immunoglobulin light chains (1/1000 dilution in washing buffer: PBS containing 0.05% Tween 20) was incubated for 90 min. Reactions were developed for 60 min at 37^ with 2, 2' azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid serving as the chromogen (Sigma). Reaction was stopped with a 10% sodium-dodecyl-sulfate solution (20 (il per well). Antibodythe ratio positive hybridomas were selected as reported in a murine system [27], after calculating between the OD^q^ of wells with virus and mock-infected cell monolayers incubated with or without hybridoma supernatants. Wells with ratio > 1.25 were considered to be antibody-positive. The MAb capture-ELISA was performed to detect the presence of G, M or A immunoglobulins in cell-culture supernatants [12]. Microplates (Nunc, 4.42404) were coated overnight at 4^ with monoclonal antibodies specific to pig immunoglobulin isotypes (50 (il of a 1/250 dilution of anti-

733

gamma, alpha and mû isotypes). Two-fold dilutions of cell supernatants were incubated for 90 min in PBS buffer containing 0.05% Tween 20 and 5% skim-milk powder. Following three washing cycles, peroxidase labelled-MAb K139 3 El against pig immunoglobulin light chains (1/1000 dilution in washing buffer) was incubated for 90 min. Reactions were developed for 60 min at 37^ with 2, 2' azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid serving as the chromogen (Sigma). Quantitative analysis of immunoglobulins in the cell culture supernatant was performed by this ELISA in triplicate by serial dilution of sample in washing buffer using porcine IgM, IgA and IgG preparations of known concentrations as reference. Results obtained by these two assays were checked on tenfold concentrates of hybridoma supernatants (P1.2, P1.3, P1.6, S2.ll, S2.1, S2.5, S2.20, S2.2, S2.3 and S2.4) prepared by ammonium sulfate precipitation (50%, v/v) and desalted by passage through a disposable gel filtration column (Sephadex G-25, Pharmacia Fine Chemicals, Uppsala, Sweden). Selection and cultivation of hybridomas Following fusion, cells were cloned twice by limiting dilution in growth medium and called hybridoma. Each hybridoma was coded S, P or M depending on the organ of origin (spleen, lamina propria, or mesenteric lymph nodes respectively), 1 and 2 after sublines L142 and L231 and was given an arbitrary number. For example hybridoma S2.1 came from a fusion between Splenocytes and L231 cells numbered arbitrarily I. Periodically, samples of cells were stored in liquid nitrogen in a freezing-medium : fetal bovine serum (Jacques Boy, Reims, France), 95% and dimethylsulfoxide, 5% (tissue culture quality, Sigma). Mortality rates observed after thawing were between 10 to 30%. Separation of the molecular forms of pig monoclonal antibody by High performance liquid chromatography (HPLC) As we have no monoclonal antibody against the immunoglobulin joining piece, cell culture supernatants (500 u.1) of L142, L231 cells and of 5 hybridomas (S2.1, S2.20, S2.35, P1.2 and P2.6) were fractioned on an HPLC gel filtration column (type TSK3000 SW, LKB) in order to separate pentameric, dimeric and monomeric immunoglobulin molecules. Twenty-five fractions (1 ml/fraction) were collected over 30 min in PBS buffer. A MAb capture-ELISA was performed on each fraction to detect porcine immunoglobulins. Isotypes were detected in reference to HPLCpurified porcine pentameric IgM, dimeric IgA (from sow milk), IgG and a serum sample of a sow. Results obtained in HPLC for L142, S2.20 and PI.2 were checked by ultracentrifugation, in a sucrose gradient (5-20%), of preparations previously concentrated by ammonium sulfate precipitation

heavy chains of

(data not shown).

RESULTS

Origin and properties of the fusion partners Two lymphoblastoid cell lines (L14 and L23) were used successfully to select for HATsensitive cell sublines. Doses of 20 and 30 (ig of 6-thioguanine did not give satisfactory results as only one mutant cell was selected from L14 with a doubling time of 41 hours. However two sublines, named L142 and L231 after their parental cell lines L14 and L23 respectively, were recovered after cultivation in a growth medium containing 10 u.g of 6-thioguanine per ml. Doubling times of both sublines were of 19 to 20 hours. Following 3 days in HAT medium, we found 0.38% of surviving cells both for L231 and LI42. Comparative experiments performed with murine myeloma P3X63Ag-8 653 yielded similar results. No surviving cells were found following 20 days of culture with HAT medium. Results concerning doubling times and aminopterin-sensitivity were obtained with both sublines on experiments carried on cell samples of 10 and 70 generations (2 and 8 months respectively after mutant propagation). Samples of these sublines were maintained 12 months (up to 100 doublings) in growth medium containing 5 (ig 6-thioguanine per ml. For fusions, we have used L142 and L231 cells passaged 10 times in culture. To avoid any risk of overgrowth by L142 and L231 cell revenants for aminopterin-sensitivity, hybridomas were maintained in HAT medium 20 days after fusion. Thereafter, hybrid cells were passaged in a HT containing medium before being cloned and grown in growth-medium. Chromosome enumerations were done periodically to check for any genetic drift. For the L142 subline, chromosome mean numbers were of 38.2 (± 1.28) at generation 3 and of 37.8 (± 1.0) at generation 15. These data are not significantly different. The presence of a double minute chromosome was reported in 84% and in 96% of the metaphase preparations enumerated at generation 3 and 15 (some cells containing 2 to 3 copies). The double minute chromosomes were not included in the calculation of means chromosome numbers. Concerning L231 subline, chromosome mean numbers were of 39.3 (± 1.20) at generation 3 and of 40.8 (± 0.78) at generation 20. We did not find any double minute chromosomes during the enumeration of the metaphase chromosomes fromL231 cells. Solid-stained metaphase analyses for LI42 as well as forL231 subline (Figure 1)

734

Subline L142

Subline L231

3 n

4

gM.M»

14 II

K :*^

6

II

** 10 M

13

7

11

12

14

10

XY

11

12

XY

II || •••»•»!•

"

15

II Ü M

16

iê

Double minute chromosome located near the Y

17 ••

18 ••

13

14

III!

15

1

18 17 A| Aft

Unclassified

(arrow) FIGURE 1. Karyotypic analyses of L142 and L231 fusion partners made 3 generations after fusion on solid-stained metaphase preparation. On this simple morphological analysis, some additional chromosome found in L231 metaphase preparations were difficult to classified and referred to as unclassified.

revealed that the gross structural morphology of typical pig chromosomes was preserved. Monosomy of chromosome pair 18 described as typical of L14 cell line [12] was not evidenced for LI42 solidstained metaphases. The presence of chromosomes X and Y was always reported both for LI42 and L231 sublines.We cannot identify the presence of tumor cell genetic material in the hybrid cells except for dramatic numeric abnormalities or for the presence of a double minute chromosome in hybrids constructed with LI42 cells. Morphologies of the lymphoblastoid cells are displayed in Figure 2. The cells had a typical lymphoblast appearence. Cells of L231 presented cytoplasmic extrusions and an heterogeneous size compared to LI42 cells. Cells of L231 were secreting between 1 to 3 (ig IgM / ml corresponding to L23 rate. Cells of LI42 until 10 generations in culture were secreting very low levels of IgM (at the lower limit of our detection system which is 0.2 u.g/ml) like parental LI4 cells but were secreting 0.5 to 1 (ig IgM / ml by generation 70. We performed an HPLC size fractionation on the L231 cell culture supernatant. A capture ELISA was performed using anti-mû chain 28.4.1 MAb. Presence of immunoglobulin molecules was demonstrated in the molecular weight range corresponding to pentameric molecules. These results have been confirmed for LI42 and L231 supernatants concentrated tenfold by ammonium sulfate precipitation, ultracentrifuged in a sucrose gradient (5-20%), fractionated and analysed by the MAb capture-ELISA. The antibody specificity of both molecules is unknown. No

735

antibody binding activity was evidenced in

VELCIA for

cell lines.

immunoglobulins produced by

the

parental

In conclusion, at the time of fusion only L231 cells were secreting detectable amounts of freeimmunoglobulin in cell culture. The L142 subline might be valuable as a fusion partner devoid of immunoglobulin genes but may need frequent cloning to avoid any genetic drift. Fusion between L231 cells and boar 1 splenocvtes We did not find any significant difference in quality and quantity of the hybrids obtained using the different fusion procedures described in Material and Methods (between 25 to 35% antibodypositive wells according to the fusion procedure). The hybrid cells did not seem to be dilutionsensitive as cells grew well in P24 plates (around 30 colonies/well) compared with growth in P96 (1 to 5 colonies/well) microplates. Six days after fusion, cells were growing actively in HAT-medium and the cell viability was of 80%. Of 1056 wells screened at day 6 in VELCIA, we found that 267 were antibody-positive. We chose to keep 43 positive wells for propagation in 25 crrr flasks. Of 43 cell populations, we cloned 5 secreting antibody as determined in VELCIA (ratio up or equal to 1.5 against either infected and mock-infected cell monolayers or only the infected one) to select for hybridomas producing antibody with a predefined specificity in VELCIA or on the immunoglobulinisotype secreted (IgM, IgA or IgG). A total of 66 hybridomas were generated and characterized. None of the MAb secreted by these hybridomas were found to be strongly reactive against the infected cell monolayers (ratio up to or equal to 3) and non-reactive against the mock-infected cell monolayer. Results are summarized in Table 1.

TABLE 1. Yield of antibody-positive wells in VELCIA 6 days after fusion between L231 cells from different origins. Source of B-cells Fusion partner Number of

antibodypositive wells 6 days after fusion

Total number of wells

or

L142 cells and B

Number of wells cloned

by limiting dilution

spleen

L231

267

mesenteric lymph nodes

L231

12

96"'

8

lamina propria

L231

40

96**'

9

L142

22

96**'

10

*

1056*

5'

96 wells in P24 and 960 wells in P96

"

3 from P24 wells and 2 from P96 wells P24 wells

"*

The fusion procedure could be simplified for the next fusions. We chose a fusion step with 1 ml PEG 1500 (50%) and cultivation in P24 which was found more convenient for our facilities. Mutant cell L142 was introduced to test the fusogenic properties of a low-secreting cell line and to trace the presence of double minute chromosomes in hybrid cells. Fusion between L231 or L142 cells and boar 2 leukocytes of the lamina propria or the mesenteric

lymph nodes

The gut lamina propria leukocytes fused with L231 cells were distributed in 96 wells. Forty wells were found antibody-positive in VELCIA. We chose 8 cell lines secreting antibody against the Purdue virus-infected cell monolayer for cloning. A total of 38 hybridomas have been obtained. The lamina propria leukocytes fused with L142 cells were distributed in 96 wells. Twenty-two wells were found antibody-positive in VELCIA. We chose 10 wells with an antibody-activity against the Purdue virus-infected cell monolayer for cloning. A total of 49 hybridomas were obtained. The mesenteric lymph node leukocytes fused with L231 cells were distributed in 96 wells. Twelve wells were found

736

Morphologies of the fusion partners (L142, L231) and of two typical IgA producing hybridomas (S2.1, P2.6). May-Grünwald Giemsa staining of cytocentrifuged cell preparations, the photographs were taken at x 1300 magnification. FIGURE 2.

antibody-positive in VELCIA. We chose 9 wells with an antibody-activity against the Purdue virusinfected cell monolayer for cloning. A total of 37 hybridomas were obtained. Hybridomas were readily obtained in every fusion except for the fusion between L142 cells and leukocytes from the mesenteric lymph nodes. For all combinations, the hybridomas were secreting MAb strongly reactive 737

Fusion

:

L231

x

Fusion

:

L231

x

cells

spleen

Fusion

Fusion

mesenteric node

L231

:

:

L142

lamina

x

x

lamina

lymph

40

propria

propria

30 20 10 —i-í-1—r

alpha

mû

alpha +

mû

Isotype

no

secretion

of the

alpha

heavy chain

mû

alpha +

mû

no

secretion

secreted

FIGURE 3. Distribution of the number of hybrids, generated after a fusion between L231 cells and boar 1 splenocytes and after a fusion between L231 or L142 cells and boar 2 leukocytes of the lamina propria or the mesenteric lymp nodes, combined in a stacked column graph with the isotype of the heavy chain secreted (whole column) and the antibody-positive activity in VELCIA (dark pattern).

against the infected cell monolayers (ratio up or equal 3) and non-reactive against the mock-infected cell monolayer. Results are summarized in Table 1. In conclusion, the anatomical origin of the individual lymphocytes did not seem to be important for a successful fusion. Of 288 wells distributed and screened 6 days after fusion, 74 wells were antibody-positive against the mock-infected ST cell monolayer and 6 were antibody-positive against the TGE virus-infected ST cell monolayer. Stability of the hybridomas and properties of the pig monoclonal antibodies Figure 3 shows the results obtained in VELCIA combined with the heavy chain isotype determination for 189 hybridomas. Hybridomas were found to produce immunoglobulins with an antibody activity attributed to the heavy and light chains of the parental B cells. We were able to demonstrate that the heavy chains were only of alpha and mû isotypes. The rates of immunoglobulin synthesis were estimated to be between 1 to 10 (lg/ml depending on the hybridoma. During cloning, supernatants of hybrid cells were screened for immunoglobulin-secretion of mû, alpha and gamma heavy chain isotypes. Two MAb against mû heavy chain were available (MAb 28.4.1. and K513D). The IgM molecules produced by the hybridomas were always recognized by MAb 28.4.1 and only by MAb K513D in one cell population during screening. We did not isolate any hybridomas producing gamma heavy chain. Only two cell culture supernatants during screening were found with a low level of gamma heavy chains (0.5 (ig / ml). In both cases, we were unable to clone the putative hybrid cells. Forty-three hybrid cells were found devoid of immunoglobulin production. In addition, supernatants of five hybrids were found with a binding activity in VELCIA and were found devoid of 738

any molecules recognized by the panel of murine MAb directed against mû, alpha or gamma heavy chains although recognized by the murine MAb anti-light chains (K139 3 El). We chose representative hybridomas to check any change in the growth rate, the capacity to secrete immunoglobulins (quantity, isotype, antibody activity) and the plo'idy during long-term cultivation (6 months, 50 generations). Ten hybridomas were maintained in culture over 50 generations (6 months) without any significant loss of proliferation rate (24 hours) and immunoglobulin synthesis. Antibody activity was preserved when it was active. Twenty-one hybridomas were karyotyped for metaphase enumeration and observation of chromosome morphology in solid-stained metaphase preparations. As shown in Table 2, whatever the origin of parental cells, the hybridomas were near-diploid with rare occurence of trisomy and for PI.3 hybrid, only 20% of the cells harbored a double minute chromosome after fusion with L142 cells (Table 2). TABLE 2. Chromosome mean number of 21 hybridomas (secreting IgA, IgM or both; with or without activity in VELCIA) and corresponding fusion partners in comparison with normal pig lymphocytes. Hybridomas were in culture for 5 generations and fusion partners for 3 generations after fusion. Source of B-cel!s

spleen

Fusion

partner

L231

Cell

'

S2.1 S2.21 S2.22 S2.33

S2.4 S2.35 S2.11 S2.5 S2.20 mesenteric

lymph

L231

M2.5 M2.7

nodes

M2.10

M2.25 lamina

propria

L231

lamina

propria

L142

P2.7 P2.2

P2.12 P1.36

P1.24 P1.6 P1.14 P1.3 Blood

None

L231

Normal blood

None

None

L142

Mean

(± standard deviation) (+ 0.56) (± 0.43) (±0.51) 37.9 (± 0.65) 38.0 (+ 0.45) 38.2 (± 0.49) 38.9 (± 0.43) 39.0 (± 0.28) 39.2 (± 0.78) 38.0 (± 0.53) 38.1 (+ 0.38) 38.1 (±0.71) 38.2 (± 0.51) 37.6 (± 0.75) 38.0 (± 0.44) 38.1 (± 0.48) 37.8 (±0.51) 37.8 (± 0.63) 37.9 (± 0.56) 38.0 (± 0.39) 38.3 (± 0.78) 39.3 (± 1.20) 38.2 (± 1.28) 37.9 (± 0.44) 37.9 37.9 37.9

"

**'

lymphocyte metaphases enumerated except for L142, L231 and normal lymphocytes double minute chromosomes were found in 20% of the metaphases *** double minute chromosomes were found in 84% of the metaphases *

25

(50 metaphases)

**

Morphologies of 2 hybridomas (P2.6 and S2.1) are shown in Figure 2. The cells had a lymphoblast appearance and were apparently metabolically active as illustrated by numerous vacuoles in the cytoplasm of P2.6 cells. Evidence of dimeric and pentameric immunoglobulins Two IgM producing hybridomas (S2.35 and PI.2) were found to secrete pentameric molecules

following HPLC size fractionation of supernatant samples and immunoglobulin detection by a MAb capture-ELISA (Figure 4). The presence of dimeric molecules was demonstrated for IgA producing P2.6 and S2.1 even if the distribution of S2.1 fractions shown in Figure 4 is slightly different from corresponding IgA control suggesting a mixture of dimeric and monomeric molecules. The presence of dimeric IgA was demonstrated in the supernatant of PI.2 hybridoma which secreted both alpha and mû heavy chains. Surprisingly, many molecules produced by S2.20 and classified by isotypic analysis as IgM have a molecular weight lower than the monomeric control IgG from purified fraction or sow sera (Figure 4). Following ultracentrifugation in sucrose gradient of concentrated preparation, we confirmed these results and found that S2.20 produced low amounts of pentameric IgM and a molecule with a 739

Standard IgM ( ) in comparison with IgMsecretlng hybridoma ( y )

Standard

IgA (

) in

comparison with IgAsecreting hybridoma ( • ) .

Hybridoma

Hybridoma S2.35

P2.6

1.2

0.6

0.0

«

M

1—'—I— —I— —I

Hybridoma S2.20

Hybridoma

S2.1

1.2

0.6

Q

O 0.0

Hybridoma secreting mû ( ) and alpha (•) heavy chains

Hybridoma

Serum

sample

:

IgM (A), IgA (•) Sow serum

P1.2

and

IgG ( * )

1.2

A

0.6

*

—£*%»—

0.0 0

5'

10

15

20

25

0

- —i—i—i— —r 10 15 5

20

25

Fractions FIGURE 4. Identification of pentameric, dimeric and monomeric pig immunoglobulins present in cell culture supernatants of two IgM-secreting hybridomas (S2.35, S2.20), two IgA-secreting hybridomas (P2.6, S2.1) and one hybridoma (P1.2) secreting both heavy chain of mû and alpha isotypes. Standard immunoglobulin preparations and a sow serum have been included for reference. Samples were size fractionated by HPLC. Immunoglobulins A or M contained in each fraction were captured by a MAb to mû (28.4.1) or by a MAb to alpha (K61) heavy chains and detected by a MAb against light chains (Kl 39 3 El) coupled with horse-radish peroxidase. molecular weight lower than IgG, harboring light chain epitope recognized by K139 3 El MAb and heavy chain epitope recognized by 28.4.1 MAb. Specificity of the monoclonal antibodies obtained We found 40 hybridomas secreting antibodies reactive against a ST cell monolayer infected or mock-infected with Purdue strain of TGE virus. These antibodies were found directed against cellbound antigens of the ST cells because they did not cross-react against murine 3T3 cells in parallel cell immunoassays nor against proteins of the growth medium. Two monoclonal antibodies were found weakly reactive against the virus-infected cell monolayers. Results of VELCIA are summarized in Figure 3. Supernatants of 10 hybridomas were concentrated by ammonium sulfate precipitation 740

and used to ascertain the MAb specificity. We found that the hybridomas producing a MAb active in VELCIA were generated following fusion with both sublines and with leukocytes from every organ. Hybridomas producing IgA were even selected following a fusion performed with splenocytes of a boar immunized per os and systemically. We conclude that LI42 and L231 cells can be used as fusion partners to immortalize B cells secreting either IgM or IgA. DISCUSSION Two mutant sublines of B lineage (L142 and L231) were rendered stably aminopterin-sensitive, propagated for 100 generations after fusion with swine leukocytes and retained doubling between 19 to 20 hours. Fusions were performed with leukocytes of 2 different boars. Using L231 or L142 cells as the tumor cell line and leukocytes from spleen, gut lamina propria or mesenteric lymph nodes, we have been able to derive hybridomas making monoclonal antibody of IgA or IgM isotypes. In pigs as well as in other mammals, the Hypoxanthine phosphoribosyl transferase (HPRT) locus has been located on the X chromosome [28]. Inactivation of the HPRT locus by point mutation or gene methylation leading to enzyme deficiency have been studied for 6-thioguanine-resistant lymphoblast mutants [29]. Higher HPRT mutation frequency has been reported in embryonal carcinoma cells chronically infected by a retrovirus as the result of retroviral insertion in the locus [30]. We have been able to select the L142 and L231 cells which can double in a relatively short period in a medium supplemented with 5 p_g 6-thioguanine per ml and remain near-diploid. As reported for human lymphoblast mutants [19], we did not demonstrate any gross chromosomal abnormality nor any loss of the X chromosome except for the occurence of a double minute chromosome associated with LI42 genetic material (Figure 1). We have yet to analyse the HPRT locus of L142 and L231 sublines to check for its physical integrity and to demonstrate that the HPRT deficiency is the result of a definitive impairment of the corresponding locus or an epigenetic phenomenon leading to the production of a defective HPRT enzyme with a very low activity. Karyotypes of 21 hybrids secreting different immunoglobulins (either IgM, IgA or both with or without any activity in VELCIA) were found nearly diploid (Table 2). No double minute chromosome was found in hybridomas resulting from fusion between L142 and B cells except for

PI.3

(Table 2). Double minute chromosomes

are known to be either associated with DNA with multi-drug resistance or c-myc oncogene amplification [31, 32]. We think that the double minute chromosomes present in L142 cells correlate with 6-thioguanine resistance of the fusion partner and that they are progressively lost from the hybrid genome in absence of the drug selective pressure. The quasi-diploidy of the tumor cells and some hybridomas are maintained over numerous generations suggests that the cells retain the accuracy of chromosome disjunction at mitosis and that following fusion, the hybrids loss chromosomes at random to restore a stable diploidy retaining the cellular or viral genes involved in oncogenesis and an active HPRT gene. These results are similar to the situation found in human for cell lines transformed by Epstein-Barr virus. The Epstein-Barr virus is known to be associated with diploid lymphoblastoid cell lines [19] and has been used to immortalize B lymphocyte cell lines producing specific antibody [33]. The selection of HPRT deficient variants of human EBV-transformed cell lines has been proposed as a means for constructing more efficiently human B cell hybridomas producing immunoglobulin [34]. In the pig, immortalization of the available lymphoblastoid cell lines has been putatively attributed to either some herpes virus of swine closely related to Epstein-Barr virus [35] or to retrovirus as retrovirus-like-particles have been found associated with these lines [36, 12]. From coculture experiments2 between our tumor fusion partners and splenocytes of boar 1, we know that viral transformation could occur by transduction during fusion with L231 cells but at a low frequency which does not explain the relatively easy recovery of hybridomas. The infection of splenocytes with 0.2 pm filtered tumor cell culture supernatants did not lead to the selection of transformed leukocytes suggesting either that the putative oncogenic virus is produced at a very low level or that the oncogenesis is an integration dependent event. The construction of intratypic hybrids might be valuable in selecting new cell phenotype over-expressing the viral genes as crosses between such cells are well known to produce a great variety of phenotypes [37]. In addition, these tumor sublines pave the way to generate intertypic hybrids which might be used to identify the group of chromosomes bearing the oncogenes. Nevertheless the capacity of the fusion partners to shed retrovirus particles precludes their use for prophylactic or therapeutic purposes. In our hands the generation of hybrid cells was easy and was not dependent on strict physicochemical handling conditions. In the absence of any well-defined chromosome marker, the demonstration that fusion did occur between lymphoblastoid sublines and B cells is supported by the

amplification correlating

2

Kaeffer, B., Bottreau, E. and Phan-Thanh, L. (1990) poster P26-005, VHIth International Congress of Virology, 26-31 August, Berlin, Germany.

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hybridomas producing immunoglobulin heavy chain of both alpha and mû isotypes Some hybridomas, like PI.2 presented in Figure 4, were producing IgA active in VELCIA coded by the B cell genome and IgM inactive in VELCIA putatively coded by the tumor cell genome. In addition, we have been able to select 30 hybridomas producing IgA among which 10 were producing immunoglobulins with activity in VELCIA. The HPLC size fractionation performed on two IgA producing hybridomas shows that these molecules can be dimeric (Figure 4). We consider that these 10 hybridomas are expressing only the heavy and light chains of the parent B cell following either a chromosome loss of the tumor cell or a shut-off of the immunoglobulin gene from the parental tumor cell. It could be argued that the hybridomas secreting both IgA and IgM originate from in vitro switch recombinations leading to the progressive selection of a population of pure IgA secreting B cells or of a mixture of two populations secreting either IgA or IgM. We think this hypothesis is unlikely because stable clones secreting either IgA or an irreducible co-secretion of IgA and IgM arose from multiple cloning and recloning. In the future, these hybridomas constitute unique and valuable tools to study the regulation of IgA synthesis in pig B cells isolated from different organs or tissues. Analysis of the type M immunoglobulins produced by 97 hybridomas is more difficult as the immunoglobulin coded by the tumor cell genome cannot be distinguished by a simple monoclonal antibody analysis. Concerning the size fractionation in HPLC of IgM, we found pentameric molecules for monoclonal antibodies produced by S2.35 and PI.2 (Figure 4). The secretion of M immunoglobulin by sublines LI42 and L231 is a disadvantage of our fusion partner. The selection of 43 hybridomas which were found devoid of any VELCIA activity and without any detectable secretion of immunoglobulin in cell supernatant might be a source of interesting fusion partners to select for HPRT mutants producing neither free-virus particles nor selection of

(Figure 3).

immunoglobulins. The selection of hybridomas producing IgA and IgM without any IgG-producing hybridoma might be due either to the maturation of our fusion partners, to the immunization procedures or to the screening assay for the production of immunoglobulins. New fusions involving immunization of pigs with viral or bacterial antigens are under way to take into account both the maturation of our fusion partners allowing immortalization of only a subset of committed B cells and any class-restricted antibody responses similar to the IgG2a restriction of murine antibodies elicited by viral infections [38]. The MAb capture-ELISA which has been used to screen for immunoglobulin-secreting hybridomas is performed only with four monoclonal antibodies specific to mû, alpha and gamma heavy chains. The total number of immunoglobulin-secreting hybrids could be considerably higher. This observation seems to be supported by the selection of 5 hybridomas on a total of 194 with an activity in VELCIA and which were found negative in the isotype screening assay. Our immunization procedures were performed on animals already hyperimmunized with crude antigenic preparations of different origins and we have used only one screening assay (VELCIA) to select for antiviral MAb. With these conditions,

we

think that it was easier to select for MAb directed

against ST cell antigens [39] or cell-bound contaminants like mycoplasma which could escape the detection by Hoechst staining technique. We demonstrated that 40 MAb were specifically directed against ST cell antigens. Two MAb were found weakly reactive against the virus-infected cell monolayers. None of these 42 monoclonal antibodies neutralized the TGE virus infectivity in tissue culture (data not shown). The selection of a monoclonal antibody of IgA isotype specific of the TGE virus should be reconsidered to include in the screening procedure a virus neutralization testing and an ELISA realized with purified TGE virus. In

conclusion, the L142 and L231 lines

are

convenient fusion partners

to

immortalize B cells

secreting IgM or IgA which might help to develop the hybridoma technology in pig. Minipigs are proposed as an animal model for studies of transplantation biology because the major histocompatibility complex of this animal exhibits extensive similarities to its human counterpart in both structure and function [40]. The cell lines that we have established will be useful for the study of oncogenic viruses and cancer in a pig strain with a well-defined histocompatibility pattern [12]. ACKNOWLEDGMENTS We thank Mr. Ph. Bernardet and Mr. R. Delaunay for help in pig rearing and autopsies, Dr. L. Phan-Thanh for help in HPLC, Dr. To Long Thanh and Dr. Ph. Velge for fruitful discussion. This work was supported in part by an EEC contract : BAP-0178-F.

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accepted

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