Avian Pathology (1999) 28, 171±186

Duck lymphocytes. VIII. T-lymphoblastoid cell lines from reticuloendotheliosis virus-induced tumours Sarah W. S. Chan 1 , Yuki Bando 1,3 , G. W. Warr 2 , Darlene L. Middleton2 and D. A. Higgins1,* 1

Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, and 2Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, S.C. 29425± 2211, USA

The T strain of reticuloendotheliosis virus (REV-T) obtained, along with the helper chicken syncytia virus (CSV), from the CSO4 cell line was highly oncogenic and rapidly fatal in ducks. Tumours were mainly seen in spleen, but neoplastic cells were observed microscopically in many organs. In vitro REV transformation of duck lymphocytes failed to yield stable cell lines, so cells from organs (blood, bone marrow, spleen, lymph node, bursa of Fabricius) of infected birds were used to establish cell lines. Some of these cell lines have been cloned. The success rates of establishment and cloning were increased if cells were cultured in a range of media containing different supplements; however, medium containing 5% foetal calf serum (FCS) and 5% duck serum was generally most ef® cacious for initial establishment, while spent medium from the parental line supplemented with a further 20% FCS gave best results for cloning. Cloned cell lines had the morphology of lymphoblastoid cells, with irregular nuclei and diffuse chromatin. Analysis of mRNA extracted from these cell lines showed that the uncloned lines were strongly expressing the b chain of the T cell antigen receptor (TCR) and weakly expressing immunoglobulin (Ig) polypeptides [l light chain and m , y , y (D Fc) and a heavy chains in various proportions], suggesting the presence of T and B cells. The cloned cell lines that could be classi® ed were TCR b 1 ve T cells. This is the ® rst report of the establishment, cloning and partial characterization of duck lymphoblastoid cell lines.

Introduction The immune system of the duck (Anas platyrhynchos) displays unique characteristics. The predominant serum immunoglobulin (Ig) is IgY(D Fc), a truncated form of IgY lacking the Cu 3 and Cu 4 domains, and probably functionally defective with regard to complement ® xation, opsonization and tissue sensitization (Higgins & Warr, 1993). This molecule arises due to the occurrence of a cleavage site associated with a small exon encoding only the two COOH-terminal amino acids of the u (D Fc) heavy (H) chain, and positioned in the intron between the exons encoding the Cu 2 and Cu 3 domains (Magor et al., 1992, 1994a). The duck also possesses a secretory Ig resembling IgA, which is not expressed and produced until about 3 3

weeks of age (Ng & Higgins, 1986; Magor et al., 1998); this delayed production is probably related to the fact that the a gene is in reverse orientation in the IgH locus (Magor et al., 1999). Hence, questions arise about the control of the expression of Ig isotypes and isoforms in the duck. First, how is the expression of the IgY and IgY(D Fc) isoforms controlled? Are these Igs secreted by different cells or by the same cells, and, if the latter, in what order (Higgins & Warr, 1993; Bando & Higgins, 1996)? Second, how is expression of the a locus controlled? Does IgA production require correction of the inversion of the a gene and is such correction under the control of a population of T H cells speci® c for IgA? With regard to the biology of duck T cells, it is

Present address: Department of Immunology, Juntendo University, 2±8±4 Hongo, Bunkyo-Ku, Tokyo, Japan. * To whom correspondence should be addressed. Tel: 1 852 2855 4870, Fax: 1 852 2855 8284, E-mail: [email protected] Received 13 July 1998. Accepted 2 November 1998. 0307-9457/99/020171-16

Ó

1999 Houghton Trust Ltd

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noteworthy that there is, as yet, no con® rmed phenotypic marker for these cells. In the absence of techniques for identifying duck T cells, it has been dif® cult to create speci® c typing reagents, such as monoclonal antibodies (MAbs). MAbs raised against chicken T cell surface antigens do not react with duck lymphocytes (Bertram et al., 1996; D. A. Higgins, unpublished observations). A rabbit polyclonal antibody against the « chain of human CD3 (Mason et al., 1989; Jones et al., 1993) reacts with a determinant on the internal aspect of the cell membrane of a population of duck lymphocytes believed to be T cells (Bertram et al., 1996), but this is of limited value in functional studies of duck lymphocytes as cells must be ® xed and permeabilized to allow staining. It remains unknown whether ducks have the three major functional subsets of T cells that occur in chickens (Chen et al., 1994). One experimental approach towards enhancing our understanding of the anatidine immune system would be to develop duck lymphoblastoid cell lines representing different stages of T and B cell differentiation. These could be used to study gene rearrangements and control mechanisms, and as antigens for the production of MAbs. Here, we describe the outcome of experimental infection of ducks with the T strain of reticuloendotheliosis virus (REV-T), use of the tumours arising for establishment of duck lymphoblastoid cell lines, and preliminary characterization of some of the cell lines so derived. Materials and Methods Ducks The Super M2 hybrid strain of white Pekin (Cherry Valley Farms Ltd, Rothwell, Lincoln, UK) was used throughout. Fertile eggs were transported by air to Hong Kong, where ducks were hatched and raised in a laboratory animal house. They were fed a commercial ration, which was monitored to ensure minimal contamination with a¯ atoxins. The birds received no vaccinations or medication.

Culture media Unsupplemented tissue culture medium was Roswell Park M emorial Institute medium 1640 (RPMI; Gibco BRL, Grand Island, NY) containing 5 mM L -glutamine, buffered with 20 mM HEPES and 23 mM NaHCO 3 and containing 100 units benzylpenicillin and 100 m g dihydrostreptomycin per ml. Supplements included: a pool of normal adult duck serum (PDS); foetal calf serum (FCS; Gibco or HyClone, Logan, Utah); 2-mercaptoethanol (2-ME); sodium pyruvate (SP); and tryptose phosphate broth (TPB). Modi® ed Hahn (M H) medium (Hahn et al., 1977) was RPMI containing 8% FCS, 10% PDS, 5% TPB, 1 mM SP and 10 mM 2-ME. Phytohaemagglutinin (PHA) and E. coli lipopolysaccharide (LPS) (both from Sigma, St Louis, Mo) were dissolved in RPMI and used at ® nal concentrations of 5 and 1 m g/ml, respectively. Cyclosporin A (CyA; a gift from Sandoz Pharmaceuticals Ltd, Hong Kong) was dissolved in ethanol at 50 m g/ml and used at a ® nal concentration of 0.5 m g/ml. Media were sterilized by ® ltration (0.22m m membrane, 25952±1L, Corning, NY).

and the helper chicken syncytia virus (CSV), was kindly provided by Dr H. Bose. It was maintained with regular splitting/passage in RPMI/10% FCS. For production of infectious inocula, supernatant ¯ uids were collected, immediately chilled in an ice bath, clari® ed by centrifugation (31,300 g av , 5 min, 4 ° C), and ® ltered through a membrane with 0.22-m m pore size (Schleicher and Scheull, Dassel, Germany). Supernates were held on ice until inoculation and injected, undiluted, into ducks or embryos within 25 min of initial harvest.

Inoculation Embryos received REV supernate into the allantoic sac via a small hole made in the shell above the air sac, 0.1 to 0.2 ml/embryo. Ducklings up to 14 days of age received 0.25 to 1.5 ml REV supernate/bird by the intraperitoneal route (i.p.). This was achieved by holding the bird vertical (i.e. head up) and injecting into a space caudal to, and left of, the gizzard. Older ducks received virus by the intravenous route (i.v.), 1 to 2 ml into the brachial vein. In the course of our studies we infected a total of 40 duckling embryos, 130 ducklings 1 to 14 days of age, 14 ducks 4 weeks of age, 20 ducks 6 weeks of age and 10 ducks 8 weeks of age.

Histopathology A group (n 5 14) of 6-week-old ducks were infected with 1 ml CSO4 supernate i.v. One or two birds were killed each day for 10 days, and uninfected control birds were examined on the day of infection and on day 10. Organ blocks from bone marrow, spleen, cervical lymph node, bursa of Fabricius, pancreas, harderian gland, liver, kidney, myocardium and lung were ® xed in neutral buffered formalin, embedded in paraf® n wax, cut at 5 m m, and stained with haematoxylin and eosin (HE).

Cell preparation and culture Lymphocytes were prepared from blood and organs other than bursa of Fabricius by centrifugation over Ficoll-paque (F/P; Pharmacia); (Higgins & Chung, 1986; Higgins & Teoh, 1988). Cell suspensions from bursa of Fabricius were cultured without F/P centrifugation. Cells were initially cultured (37.4 °C, 5% CO 2 ) at about 10 7 cells/ml in 24 well trays, 1 ml/well, with subsequent expansion into six-well trays and ¯ asks (see Results for details of media preferences).

Cloning Cells were counted using a Coulter Counter, model ZM (Coulter Electronics Limited, Luton, Bedfordshire, UK), adjusted as previously described (Higgins & Ko, 1995). Cloning was performed in 96-well trays with cells seeded at 1 cell/well. The medium requirements for cloning are described in the Results. Cultures were monitored daily by light microscopy (Fluovert, Leitz Wetzlar GmbH, Germany) to ensure that clones selected were the progeny of single cells.

Electron microscopy (EM ) Cells were washed in RPMI, then resuspended and ® xed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 to 8 ° C for 15 min. Cell pellets were formed by centrifugation at 400 g for 10 min. The pellets were then further ® xed with glutaraldehyde for 60 min. The pellets were washed with 0.1 M sodium cacodylate buffer containing 0.1 M sucrose and post-® xed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. They were then dehydrated through graded ethanol and embedded in Epon 812 (Fluka Chemie AG, Buchs, Switzerland). Ultra-thin sections were cut, and stained with uranyl acetate and lead citrate. They were examined under a JEOL 100SX transmission electron microscope at 80 kV. Cytochemistry

REV The chicken cell line CSO4, which constitutively produces REV-T

Cytosmears (Cytospin 3, Shandon, Pittsburgh, PA) of cell lines were stained for leukocyte acid phosphatase (ACP) (with and without the

Duck lymphoblastoid cell lines

173

Table 1. Primers used in reverse transcription Code

Sequence

Size

Speci® city

G-413

59 TCT GAA TTC TCG AGT CGA CAT C(T)17 39

39m er

G-630 G-754

59 ATA GCA GGA GCT GGC GGT GTC39 59 GTA ACA GGT GCT GTC GGC GTC39

21mer 21mer

Poly(A) tail [actin, u , u (D Fc), l , TCRb ] m (primes in C4) a (primes in C4)

Table 2. Primers used for polymerase chain reaction Code

Sequence

Size

Speci® city

Product size

G-773 G-784

5© CCC TAC AGG AAC TCC AGC3© 5© GTT GGC TTG GAG GAC TCG3©

18mer 18mer

m

447 bp

G-779 G-799

5© CTG CTG GGT CCA AGT CAC3© 5© CTA CAT CAG CCA AAA CGC3©

18mer 18mer a

397 bp

G-246 G-247

5© CCT TCG TGG ACC ACC AT G3© 5© CCT ACA TCT TCA CCT TCC3©

18mer 18mer u

268 bp

G-1283 G-1284

5© GGT GCG TCG CCG GAG GTG AAC CAA3© 5© GGA GGA CAA CAA AGG TGG TCA GAA3©

24mer 24mer

u (D Fc)

361 bp

G-314 G-348

5© CAG GTA GCT GCT GGC CAT3© 5© GCC AGC CCA AGG TGT CTC3©

18mer 18mer l

318 bp

G-1026 G-1027

5© TAT GGA TCC AGA AGA GCA AAG CCA CAC TGG TAT3© 5© ATA AGC TTG CAT CAT ATG TCC AT C TTC CTC TT3©

33mer 32mer

TCRb

463 bp

G-480 G-482

5© AA(TC) GGI GA(AG) AA(AG) ATG ACI CA(AG) AT (TCA) ATG TT3© 5© TTI (CG)(AT)(AGT) ATC CAC AT(TC) TG(TC) TG(CA) AAI GT3©

29mer 26mer

Actin

750 bp

addition of L -tartaric acid), non-speci® c esterase (NSE), chloracetate esterase (CAE) and leukocyte myeloperoxidase (MPO) by standard techniques.

RNA extraction Total RNA was prepared from duck organs using guanidinium isothiocyanate, followed by extraction with phenol-chloroform (Chomczynski & Sacchi, 1987), and was stored as a precipitate in 70% ethyl alcohol at 2 70 ° C.

Primers and probes The primers used in reverse transcription (RT) and polymerase chain reaction (PCR) are listed in Tables 1 and 2. Oligonucleotides were prepared by the MUSC Nucleic Acid Synthesis Facility. Gene-speci® c primers were used for RT of a and m , because priming from the poly(A) tail was, in these cases, inef® cient. The cloned cDNAs on which our primers and probes for Ig H and L chains and actin were based have been described (Magor et al., 1992, 1994a,b, 1998; GenBank accession numbers: m , U27213; u , X65219; u (D Fc), X65218; a , U27222; l , X82069). Probes and primers for the duck TCR b chain

Figure 1. 1a: livers of uninfected (right) and REV-infected (8 days post-infection) 50-day-old ducks. 1b: spleens of uninfected (right) and REV-infected (8 days post-infection) 50-day-old ducks.

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Figure 2. Spleens of 5-day-old ducklings; bottom right (arrowed) is an uninfected control, others were infected with REV as embryos at 24 days of incubation.

were derived from a truncated clone (GenBank accession number AF068228) obtained from a l ZAPII cDNA library by hybridization with a probe for the chicken TCR b (Tjoelker et al., 1990), kindly supplied by Dr W. T. McCormack. For hybridization techniques, probes generated in PCR were puri® ed by gel electrophoresis and samples were labelled with 32 P-deoxycytidine triphosphate (Amersham International plc, Amersham, Buckinghamshire, UK) by random priming (Feinberg & Vogelstein, 1983) to a speci® c activity of , 1 3 106 cpm/m g.

Reverse-transcriptase polymerase chain reaction (RT-PCR) RNA was dissolved in RNAase-free water and to 5 to 10 m l were added: 1 m l primer (at 0.2 m g/m l), 10 m l ® rst strand buffer (BRL, Gaithersburg, MD; 5 3 concentration), 2 m l RNAsin (80 units), 1 m l dithiothreitol (0.1 M), 3 m l 10 mM deoxynucleoside triphosphate (dNTPs), 1 m l M oloney murine leukaemia virus RT (BRL, 200 units/ m l) and water to 50 m l. The mixture was incubated at 37 ° C for 60 to 90 min. The resulting cDNA was precipitated and washed with ethyl alcohol, air dried and redissolved in 100 m l TE. For PCR, 5 m l cDNA was used in a total reaction volume of 50 m l containing proprietary

buffer, 4 mM MgCl 2 , 0.1 m g of each primer, 0.3 mM dNTPs and 2 units of Taq polymerase. The reaction consisted of 30 cycles, each of 1 min at 94 ° C, 2 min at 50 ° C and 3 min at 72 ° C. Northern blot hybridization Total RNA was separated under denaturing conditions in 1% agarose gels (20 m g/lane), blotted to Hybond membranes (Amersham International) and cross-linked by ultra-violet light (Stratalinker, Stratagene, La Jolla, CA). The blots were hybridized for 16 h in 50% formamide, 5 3 SSPE (1 3 SSPE is 150 mM NaCl, 10 mM NaH 2 PO 4 , 1 mM EDTA, pH 7.4), 2 3 Denhardt’ s reagent (0.004% Ficoll 400, 0.04% polyvinyl-pyrrolidone, 0.04% BSA, 1% SDS and 100 m g low molecular weight, denatured salmon sperm DNA; all reagents from Sigma Chemical Company, St Louis, MO). Blots were washed twice for 20 min at high stringency (0.1 3 SSPE, 0.1% SDS at 60° C). Exposure to X-ray ® lm was at ±80° C. Southern blot hybridization DNA derived from RT-PCR, and dissolved in TE, was electrophoresed in 0.8% agarose gels. After depurination and denaturation,

Duck lymphoblastoid cell lines

175

Figure 3. 3a: histopathology of spleen of a 46-day-old duck, 4 days after infection with REV (Bar 5 200 m m); invasion by lymphoblastoid cells (arrows) is apparent. 3b: liver of 52-day-old duck, 10 days after infection with REV (Bar 5 40 m m); the normal architecture of the liver is replaced by tumour cells.

176

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Figure 4. 4a: histopathology of bursa of Fabricius of 52-day-old control duck; the normal anatomy of the bursal follicles, with distinct cortex and medulla, is apparent (Bar 5 200 m m). 4b: bursa of 46-day-old duck, 4 days after infection with REV (Bar 5 200 m m); the bursal follicles are atrophic. 4c: bursa of 52-day-old duck, 10 days after infection with REV; the atrophic follicles have been in® ltrated by tumour cells (Bar 5 200 m m).

Duck lymphoblastoid cell lines

177

Table 3. Establishment of cell lines from lymphoid organs of ducklings after infection with REV Number of cell lines established from

Day

Duck numbers

Blood

Spleen

2 4 5

1,2 3,4 5±7

0 0 0

0 0 0

6 7 8 9

8±10 11±13 14±16 17±19

0 0 0 0

0 0 0 1 (19;i)

Lymph node

Bone marrow

Bursa of Fabricius

0 0 3 (5;a±d,i) (6;i) (7;i) 0 1 (13;a±d,i) 2 (16;a±d) 3 (17;b,f,i) (19;b)

ND 0 0

ND ND 0

2 (10;a,b) 0 0 0

1 (9;a,i) 0 1 (16;a±d) 3 (17;a±d,i) (18;a±d,i)

Nineteen ducks, 14 days old, were infected with 1.5 ml of CSO4 cell line supernatant i.p.. Ducks were killed 2 to 9 days post-infection, as indicated. Cell suspensions were prepared from organs, as described in Materials and Methods, and cultured in a variety of media. Data in parentheses indicate the num bers of the ducks yielding cell lines and the medium preferences of the cell lines for initial establishment: a, 5% PDS; b, 10% PDS; c, 15% PDS; d, 20% PDS; e, 5% FCS; f, 10% FCS; g, 15% FCS; h, 20% FCS; i, 5% PDS 1 5% FCS.

the DNA was transferred to Nytran membranes (Schleicher and Schuell). After hybridization for 18 h at 65 ° C, blots were washed twice for 15 min at high stringency (Magor et al., 1992). Exposure to X-ray ® lm was for 5 days at 2

80 °C.

ically abnormal. There was no difference in the timing or the appearance of the pathology observed in different ages of ducks. Histopathology

Results Mortalities due to REV The clinical effects of REV on experimentally infected ducks was the same in all ages of birds examined. Mortalities ® rst occurred 7 to 8 days after inoculation, with 100% mortality by 11 to 12 days after inoculation. Birds appeared healthy until within 18 h of death. Thereafter, they became listless, ceased eating and eventually became recumbent shortly before dying. Gross pathology Autopsy of birds of all ages revealed, 6 to 12 days after infection, greatly enlarged livers, which were usually pale and friable (Figure 1a). Rarely, livers also showed red nodular tumours. Spleens were consistently enlarged, but the gross appearance varied (Figures 1b and 2). Some affected spleens were dark red, ® lled with thick blood-stained ¯ uid, and contained several large (4 to 10 mm in diameter) nodular tumours. Others were pale in colour, and contained many small (0.5 to 2 mm) nodular tumours. In most birds, by 7 or 8 days after infection, the heart was also enlarged, with a pale myocardium; rarely, tumours were found in the heart wall. Enlargement of the cervical lymph nodes, usually with congestion, and nodular tumours in the head of the pancreas, were recorded rarely. The bursa of Fabricius was not macroscop-

The earliest change, seen from about 2 to 4 days, was an in® ltration of homogeneous large lymphoid cells, which occurred in areas of the spleen and lymph nodes, but also affected the lymphoid follicles of lung, kidney and harderian gland (Figure 3). From 3 to 6 days most organs (including spleen, harderian gland, lymph node, bursa, pancreas, liver and myocardium) showed destruction and disorganization of normal architecture, with apoptosis and necrosis. This was particularly apparent in liver, bursa of Fabricius (Figure 4) and harderian gland. Tumour cell in® ltration occurred in all organs from 4 to 6 days and thereafter became increasingly generalized. Within the tumours the predominant malignant cell was a small round lymphoid cell having a pleomorphic nucleus with one or more nucleoli and moderate deep-staining cytoplasm. Cells having a darkly stained nucleus with clumped chromatin were also present. Apoptosis and mitosis were frequent. The tumour in® ltrates also contained scattered heterophils and macrophages and there was mild extravasation of red blood cells (Figures 3 and 4). Establishment of cell lines Numerous attempts, using MH medium, RPMI 1 10% PDS and RPMI 1 10% FCS, were made to transform blood, spleen, lymph node and bone marrow cells in vitro. Cells did not survive beyond 10 days of culture. Co-stimulation of cells

B2

Organ

Blood

S2 S7 S19

BM10

LN5 LN6 LN7 LN13 LN16 LN17 LN19

BU1 BU3 BU9 BU10

Spleen

Bone marrow

Lym ph node

Bursa

B3

Uncloned lines

B3/6

B2/4

B2/2 B2/3

B2/1

Clones

B2/3a B2/3b B2/3c B2/3d B2/3e B2/3f B2/3g B2/3h B2/3i

B2/1c B2/1d

Subclones

2

1

2

1

1

1

1

1

1

1 1

2

2

2

2

1

2

1

1

1

2

1

1 1

1

1

1

1

1 1

2 NT 2 2 2 2 2 2 2 2 1

1

2

2

2

2

1 NT 1 1 1 1 1 1 1 1 1

1

1

1

1

1

2

2

1 1

ACP 1

ACP

T

2

1

2

2

2

1

2

1

1

1

1

2

1

2

2

2 NT 2 2 2 2 2 2 2 1 1

2

2

1

2

2

2

1

NSE

Cytochemistry

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1 NT 1 1 1 1 1 1 1 1 1

1

1

1

1

1

1

1

CAE

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2 NT 2 2 2 2 2 2 2 2 2

2

2

2

2

2

2

2

MPO

1 NT

1

1

1 NT 1 2 NT 1

1

1

2

2

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

TcRb

Table 4. List of REV-induced duck cell lines, with cytochemical and molecular characteristics.

2 NT

2

2

1 NT 2 2 NT 2

2

2

2

1

1

2

1

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

1

l

2 NT

2

2

2 NT 2 2 NT 2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

m

NT NT 2 NT

2 NT 2 2 NT 2

2

2

2

1

2

2

1

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

1

u

m RNA expression

NT NT 2 NT

2 NT 2 2 NT 2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

u (D Fc)

2

1 NT

2

2

2 NT 2 2 NT 2

2

2

1

1

2

1

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

1

a

178 S. W. S. Chan et al.

BU18/1 BU18/2 BU18/3 BU18/4 BU18/5 BU18/6 BU18/7 BU18/8 BU18/9 BU18/10 BU18/11 BU18/12 BU18/13 BU18/14 BU18/15 BU18/16 BU18/17 BU18/18 BU18/19 BU18/21 BU18/23 BU18/24 BU18/25 BU18/26 BU18/27 BU18/30 BU18/31 BU18/32 BU18/33 BU18/34

NT, not tested.

BU35 BU3198 EBU198 EBU498 EBU1298 EBU1598 EBU1698

BU14 BU16 BU17 BU18

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2 1

1

1

1 1

1

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

1

2

2

1

2

1

2

1

1

2

1

2

1

1

1

2

1

1

2

2

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

1 NT 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 NT NT 1 NT NT NT NT

2 NT 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 NT NT 2 NT NT NT NT

2 NT 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 NT NT 2 NT NT NT NT NT NT 1 2 2 2 2 2 2 2 2 2 2 NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT 2 NT NT NT NT

NT NT 1 2 2 2 2 2 2 2 2 2 2 NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT 2 NT NT NT NT

2 NT 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 NT NT 2 NT NT NT NT

Duck lymphoblastoid cell lines 179

180

S. W. S. Chan et al.

Figure 5. Transmission electron micrographs (TEM) of duck cell lines. 5a: bursa line 17 showing morphology consistent with lymphoblastoid cells (Bar 5 5 m m). 5b: cloned blood line B2/3h (Bar 5 1.5 m m); virus particles budding from the cell membrane were seen in all cell lines.

Duck lymphoblastoid cell lines

181

Figure 6. M olecular analysis of uncloned cell lines from blood (B2, B3), spleen (S2, S7) and lymph node (LN). 6a: products of RT-PCR with primers for TCRb and actin; size of the products (bp) is shown; control used was a spleen cDNA. 6b: Southern blots of RT-PCR products against probes for Ig heavy [m , u , u (D Fc), a ] and light (l ) chains; size of the products (bp) is shown.

182

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Figure 7. Analysis of RT-PCR products from uncloned bursa cell lines 9, 17 and 18 (control was a spleen cDNA) using primers for actin (standard), TCRb , Ig L (l ) chains, and Ig H chains m , u , u (D Fc) and a .

(with PHA or LPS, or by allogeneic stimulation by co-culturing cells from two or more birds), or the addition of CyA, did not enhance the establishment of cell lines from cultures transformed with REV in vitro. We therefore attempted to propagate cells collected from organs of infected birds. Preliminary experiments used lymphocytes collected from blood and cervical lymph nodes of ducks 5 to 8 days after infection and employed MH medium. The success rate was , 5%. Most cultures, albeit appearing transformed, died after 2 to 4 weeks and again the success rate was not enhanced by stimulation with PHA, LPS or allogeneic cells, or the addition of CyA. Analysis of the effects of the components of MH medium, based on omission of selected components, not only failed to demonstrate any clear requirement, but also showed that, based on morphology and longevity of the cell lines, 2-ME inhibited the establishment and survival of duck cell lines. However, using MH as the primary medium, several useful parental cell lines were established: blood lines B2, B3; spleen lines S2, S7; lymph node line LN. A broader range of media was then investigated, utilizing various proportions of FCS and PDS in RPMI (typically 5% PDS 1 5% FCS, 5, 10, 15 and 20% PDS, and 5, 10, 15 and 20% FCS were used),

and experiments were expanded to include bursa of Fabricius. One such experiment is summarized in Table 3. Although different cell preparations would establish in different ranges of media, RPMI containing 5% PDS and 5% FCS was superior to RPMI supplemented with PDS or FCS alone (at concentrations of 5 to 20%), with about 70% of our bursa cell lines being established in this medium. Cell lines were more readily established as the infection progressed, with best results at 8 to 10 days after infection. The success of establishing cell lines was not related to the age of the ducks at infection; even the use of organs from ducklings infected as embryos did not enhance the ef® ciency of the establishment of cell lines. After initial establishment, cell lines were expanded from 24-well trays into ¯ asks and were weaned into medium consisting of RPMI 1 20% FCS; lower concentrations of FCS did not support long-term growth. All our current lines have been repeatedly passaged in this medium, in some cases for up to 3 years. Cloning As with primary establishment of cell lines, a wide range of media and conditions were investigated.

Duck lymphoblastoid cell lines

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Morphology of cell lines Cell lines examined by EM had morphology characteristic of lymphoblastoid cells and were seen to be producing REV particles (Figure 5); the infectivity of ® ltered cell-line supernatant ¯ uids was con® rmed by inoculation into normal ducks which developed lesions in the same pattern as those birds receiving CSO4 supernatant ¯ uid (data not shown). Cytochemistry All cell lines stained strongly positive for ACP, which was only partially blocked by L -tartaric acid, and all cell lines failed to stain for MPO. All lines stained for CAE, but in some cases weakly. The cell lines were mostly negative for NSE; those that did stain contained only a few positive cells (Table 4). Molecular characterization of cell lines

Figure 8. RT-PCR products of cloned blood cell lines using primers for TCRb and actin, and of a spleen cDNA library using primers for Ig m chains, TCRb and actin. All blood cell lines that gave reactions were positive for TCRb ; these lines were also tested for their expression of Ig mRNAs, but were invariably negative.

Best results were obtained using spent medium from the parent cell line supplemented with a further 25% FCS, with cells seeded to 96-well trays at 1 cell/well. Nevertheless, some parental cell lines have not yet been cloned. Among lines that have been cloned, the yield (frequency of wells with clonal growth) was about 15%. In general, the amenability of parent cell lines to cloning increased as the cell lines got older.

The occurrence of T or B lymphocytes in the uncloned cell lines was investigated by assaying for the presence of mRNA encoding the constant regions of duck TCRb , and of duck Ig L (l ) and H [m , u , u (D Fc) and a ] chains, by RT-PCR using primers speci® c for each of these messages. It was apparent, from ethidium bromide stained gels of the PCR products (Figure 6), that TCRb was expressed in all the parental cell lines, in some cases (e.g. those derived from blood) very strongly. Ig mRNA was not consistently evident in these cultures by ethidium bromide staining, but Southern blot transfer of the PCR products, followed by hybridization with 32 P-labelled probes (Figure 6), revealed that all parental cultures were expressing some Ig polypeptides (Table 4). The cultures containing the greatest range of Ig isotypes were among those from bursa (Figure 7). The same approach to the cloned cell lines indicated that most were T cells expressing the TCRb chain, while the remainder could not be characterized with the probes available (Figure 8 and Table 4). Discussion REV-T was highly oncogenic and invariably fatal in the ducks used. The doses of virus used revealed no differences in susceptibility of ducks of different ages, but it is possible that age resistance would have been observed had we used threshold infectious doses of virus. It is surprising that natural infection of ducks (e.g. Li et al., 1983) is reported so rarely, presumably a re¯ ection of the inef® ciency of horizontal transmission. Using cells taken from organs of ducks infected with REV-T we have been able to establish lymphoblastoid cell lines. The media requirement

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for the establishment of cell lines from REVinduced tumours was critical, but the optimum medium depended on the bird and organ. The most commonly successful medium was RPMI containing 5% PDS and 5% FCS, while RPMI supplemented with 5 to 15% PDS alone also supported the initial establishment of more cell lines than did RPMI supplemented with FCS alone. With the establishment of chicken cell lines transformed by Marek’ s disease virus, several components of the Hahn medium cocktail (Hahn et al., 1977) were bene® cial, but 2-ME was the most critical (Calnek et al., 1978); no comparable requirements were observed with duck cells and, indeed, 2-ME had an inhibitory or toxic effect on duck cells. Following establishment and a period of expansion in the primary medium, the duck cell lines were readily weaned into FCS. Cloning also had fastidious requirements of serum concentration, and these requirements also varied between the organ origins of the cells and the individual lines. However, as with the primary cell cultures, a range of conditions usually yielded a formula that was successful, and many primary lines established have subsequently been cloned. These new lines considerably expand the previous limited range of established duck cell lines (Nazerian, 1987). The majority of our cell lines cloned from REV-transformed duck organs, and all of those that could be classi® ed, appear to be of the a /b T cell lineage. In the chicken, g /d T cells occupy about 40% of the T cell population of blood (Chen et al., 1994), and bone marrow T cell lines transformed by REV contain the a /b and g /d T cell subpopulations in proportions similar to those in normal marrow. If the distribution of subsets in the duck parallels that in the chicken, and if the duck a /b and g /d subsets are comparably susceptible to the REV, it would seem likely that some of our unclassi® ed cell lines belong to the duck g /d T cell lineage, but were unrecognized as such in the absence of relevant probes. The dif® culty of detection of message for Ig in the primary cultures of blood, lymph node and spleen, and our failure to clone B-cell lines from these parent cultures, indicates that the frequency of B-lineage cells is very low. Our failure to clone B cells from bursa cell lines shown to be expressing multiple Ig isotypes also con® rms the indication of the mRNA analysis that B cells were few in the primary cultures. However, in view of our extensive attempts at cloning, it is also likely that duck B cells have stringent growth requirements, which might include the need for growth factors derived from T cells. The failure to generate lines of B cells was disappointing in view of our primary objective of studying rearrangement events in the Ig H chain locus during transcription of the various Ig isotypes and isoforms, and also against the back-

ground of the known oncogenicity of REV for chicken B cells. REVs include replication defective strains, which are usually highly oncogenic, and non-defective strains, which are only weakly oncogenic. The replication-defective REV-T contains the oncogene v-rel (Wong & Lai, 1981), the viral homologue of the cellular proto-oncogene c-rel, and is often associated with the non-defective (or helper) strain A (Rev-A) or CSV which supplies help for those functions deleted from REV-T (Chen et al., 1981; Rice et al., 1982). However, different laboratories have reported a wide range of target cells for these viruses in the chicken, hence both putative T- (Barth et al., 1990; Schat et al., 1992; Marmor et al., 1993) and B-cell (Keller et al., 1979; Nazerian et al., 1982; Barth & Humphries, 1988a,b; Chen et al., 1988; Zhang et al., 1989, 1991) lines have been described, though it seems likely that all uncloned REV-induced-cell lines contain T and B cells of various maturities (Beug et al., 1981; Shibuya et al., 1982). Among the B-cell lines derived, some have been classi® ed as ª earlyº , based on limited or no Ig gene rearrangements, while others have productive gene rearrangements and are secreting IgM. It seems likely, therefore, that duck B-cell lines useful for studies of Ig gene rearrangements will ultimately be obtained, either by improving our cloning techniques as applied to current and future parental cell lines or by using different combinations of REVs. In the meantime, the establishment of cloned lines of duck T cells will facilitate several aspects of studies of T-cell biology in this species. We have already used these cells to stimulate the production of mouse MAbs speci® c for duck T cells. The characterization of these antibodies will be reported elsewhere. Acknowledgements This work was supported by grants from the Hong Kong Research Grants Council (HKU 405/94M, HKU 421/96M and HKU 7261/ 98M), Cherry Valley Farms Ltd, USDA (NRICGP 9602942), and the Health Sciences Foundation of MUSC. The authors would like to thank S. K. Lau for photographs and Judy Tse for secretarial assistance.

References Bando, Y. & Higgins, D.A. (1996). Duck lymphoid organs: their contribution to the ontogeny of IgM and IgY. Immunology, 89, 8±12. Barth, C.F. & Humphries E.H. (1988a). A nonimmunosuppressive helper virus allows high ef® ciency induction of B cell lymphomas by reticuloendotheliosis virus strain T. Journal of Experimental M edicine, 167, 89±108. Barth, C.F. & Humphries, E.H. (1988b). Expression of v-rel induces mature B-cell lines that re¯ ect the diversity of avian immunoglobulin heavy- and light-chain rearrangements. M olecular and Cellular Biology, 8, 5358±5368. Barth, C.F., Ewert, D.L., Olson, W .C. & Humphries, E.H. (1990). Reticuloendotheliosis virus REV-T(REV-A)-induced neoplasia: development of tumours within the T-lymphoid and myeloid lineages. Journal of Virology, 64, 6054±6062. Bertram, E.M., Wilkinson, R.G., Lee, B.A., Jilbert, A.R. & Kotlarski, I. (1996). Identi® cation of duck T lymphocytes using an anti-human

Duck lymphoblastoid cell lines T cell (CD3) antiserum. Veterinary Immunology and Immunopathology, 51, 353±363. Beug, H., MuÈ ller, H., Grieser, S., Doederlein, G. & Graf, T. (1981). Hematopoietic cells transformed in vitro by REV T avian reticuloendotheliosis virus express characteristics of very immature lymphoid cells. Virology, 115, 295±309. Calnek, B.W., Murthy, K.K. & Schat, K.A. (1978). Establishment of Marek’ s disease lymphoblastoid cell lines from transplantable versus primary lymphomas. International Journal of Cancer, 21, 100±107. Chen, C.-L.H., GoÈ bel, T.W .F., Kubota, T. & Cooper, M.D. (1994) T cell development in the chicken. Poultry Science, 73, 1012±1018. Chen, I.S.Y., Mak, T.W., O’ Rear, J.J. & Temin, H.M. (1981). Characteristics of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning. Journal of Virology, 33, 1058±1073. Chen, L., Lim, M.Y., Bose, H. & Bishop, J.M . (1988). Rearrangements of chicken immunoglobulin genes in lymphoid cells transformed by the avian retroviral oncogene v-rel. Proceedings of the National Academy of Sciences, USA, 85, 549±553. Chomczynski, O.P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162, 156±159. Feinberg, A.P. & Vogelstein, B. (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high speci® c activity. Analytical Biochemistry, 132, 6±13. Hahn, E.C., Ramos, L. & Kenyon, A.J. (1977). Lymphoproliferative diseases of fowl: JM-V leukemic lymphoblasts in cell culture: brief communication. Journal of the National Cancer Institute, 59, 267±271. Higgins, D.A. & Chung, S.H. (1986). Duck lymphocytes. I. Puri® cation and preliminary observations on surface markers. Journal of Immunological M ethods, 86, 231±238. Higgins, D.A. & Ko, W .K.W. (1995). Duck lymphocytes. VII. Selection of sub-populations using lectin coated magnetic beads. Veterinary Immunology and Immunopathology, 44, 181±195. Higgins, D.A. & Teoh, C.S.H . (1988). Duck lymphocytes. II. Culture conditions for optimum transformation response to phytohaemagglutinin. Journal of Immunological M ethods, 106, 135±145. Higgins, D.A. & Warr, G.W. (1993). Duck immunoglobulins: structure, functions and molecular genetics. Avian Pathology, 22, 211±236. Jones, M., Cordell, J.L., Beyers, A.D., Tse, A.G.D . & Mason, D.Y. (1993). Detection of T and B cells in many animal species using cross-reactive anti-peptide antibodies. Journal of Immunology, 150, 5429±5435. Keller, L.H., Rufner, R. & Sevoian, M. (1979). Isolation and development of a reticuloendotheliosis virus-transformed lymphoblastoid cell line from chicken spleen cells. Infection and Immunity, 25, 694±701. Li, J., Calnek, B.W ., Schat, K.A. & Graham, D.L. (1983). Pathogenesis of reticuloendotheliosis virus infection in ducks. Avian Diseases, 27, 1090±1105. Magor, K.E., Warr, G.W., Middleton, D., Wilson, M.R. & Higgins, D.A. (1992). Structural relationship between the two IgY of the duck (Anas platyrhynchos): molecular genetic evidence. Journal of Immunology, 149, 2627±2633. Magor, K.E., Higgins, D.A., Middleton, D.L. & Warr, G.W. (1994a). One gene encodes the heavy chains for three different forms of IgY in the duck. Journal of Immunology 153, 5549±5555. Magor, K.E., Higgins, D.A., Middleton, D.L. & Warr, G.W. (1994b). cDNA sequence and organization of the immunoglobulin light chain gene of the duck (Anas platyrhynchos). Developmental and Comparative Immunology 18, 526±531. Magor, K.E., Warr, G.W., Bando, Y., Middleton, D.L. & Higgins, D.A. (1998). Secretory immune system of the duck (Anas platyrhynchos). Identi® cation and expression of the genes encoding IgA and IgM heavy chains. European Journal of Immunology 28, 1063±1068. Magor, K.E., Higgins, D.A., Middleton, D.L. and W arr, G.W . (1999). Opposite orientation of the a and u constant region genes in the immunoglobulin heavy chain locus of the duck. Immunogenetics, 49, in press.

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Marmor, M.D., Benatar, T. & Ratcliffe, M.J. (1993). Retroviral transformation in vitro of chicken T cells expressing either a or b g /d T cell receptors by reticuloendotheliosis virus strain T. Journal of Experimental M edicine, 177, 647±656. Mason, D.Y., Cordell, J., Brown, M., Pallesen, G., Ralfkiaer, E., Rothbard, J., Crumpton, M. & Gatter, K.C. (1989). Detection of T cells in paraf® n wax embedded tissue using antibodies against a peptide sequence from the CD3 antigen. Journal of Clinical Pathology, 42, 1194±1200. Nazerian, K. (1987). An updated list of avian cell lines and transplantable tumours. Avian Pathology, 16, 527±544. Nazerian, K., Witter, R.L., Crittenden, L.B., Noori-Dalloii, M.R. & Kung, H.J. (1982). An IgM-producing B lymphoblastoid cell line established from lymphomas induced by a non-defective reticuloendotheliosis virus. Journal of General Virology, 58, 351±360. Ng, P.L.K. & Higgins, D.A. (1986). Bile immunoglobulin of the duck (Anas platyrhynchos). Preliminary characterization and ontogeny. Immunology, 58, 323±327. Rice, N.R., Hiebsch, R.R., Gonda, M.A., Bose, H.R. & Gilden, R.V. (1982). Genome of reticuloendotheliosis virus: characterization by use of cloned proviral DNA. Journal of Virology, 42, 237±252. Schat, K.A., Pratt, W.D., Morgan, R., Weinstock, D. & Calnek, B.W. (1992). Stable transformation of reticuloendotheliosis virustransformed lymphoblastoid cell lines. Avian Diseases, 36, 342±439. Shibuya, T.T ., Chen, I., Howatson, A. & M ak T.W. (1982). Morphological, immunological, and biochemical analyses of chicken spleen cells transformed in vitro by reticuloendotheliosis virus strain T. Cancer Research, 42, 2722±2728. Tjoelker, L.W ., Carlson, L.M., Lee, K., Lahti, J., McCormack, W.T ., Leiden, J.M., Chen, C.-L.H., Cooper, M .D. & Thompson, C.B. (1990). Evolutionary conservation of antigen recognition: the chicken T cell receptor b chain. Proceedings of the National Academy of Sciences, USA, 87, 7856±7860. Wong, T.C. & Lai, M.M.C. (1981). Avian reticuloendotheliosis virus contains a new class of oncogene of turkey origin. Virology, 111, 289±293. Zhang, J.Y., Bargmann, W. & Bose, H.R. (1989). Rearrangement and diversi® cation of immunoglobulin light-chain genes in lymphoid cells transformed by reticuloendotheliosis virus. M olecular and Cellular Biology, 9, 4970±4976. Zhang, J.Y., Olson, W., Ewert, D., Bargmann, W. & Bose, H.R. (1991). The v-rel oncogene of avian reticuloendotheliosis virus transforms immature and mature lymphoid cells of the B cell lineage in vitro. Virology, 183, 457±466.

REÂSUME Lym phocytes du canard. VIII. Les ligneÂes cellulaires lym phoblastiques T obtenues aÁ partir de tum eurs induites par le virus de la reÂticulo-endotheÂliose La souche T du virus de la reÂticulo-endotheÂliose (REV-T) obtenue avec le virus syncitium helper du poulet (CSV) aÁ partir de la ligne e cellulaire CSO4 est treÁ s oncogeÂnique et entraõ à ne rapidement la mort des canards. Les tumeurs ont eÂteÂprincipalement observeÂes au niveau de la rate, par contre des cellules neÂoplasiques ont eÂteÂobserveÂes en microscopie au niveau de nombreux organes. In vitro la transformation des lymphocytes du canard par le REV n’ a pas permis l’ obtention d’ une ligneÂe cellulaire stable, aussi les cellules des organes (sang, moelle osseuse, rate, formations lymphoõ È des, bourse de Fabricius) de poulets infecteÂs ont-elles e teÂutiliseÂes pour l’ eÂtablissement de ligne es. Quelques unes de ces ligne es cellulaires ont e teÂcloneÂes. Le taux de reÂussite de l’ eÂtablissement et du clonage a eÂt eÂplus important quand les cellules ont eÂteÂcultiveÂes dans des milieux des suppleÂments en proportion diffeÂrente; le milieu contenant 5% de seÂrum de veau foetal (FCS) et 5% de seÂrum de canard a e teÂen geÂneÂral le plus ef® cace aÁ l’ eÂtablissement initial, alors que le milieu de survie de la ligne e parentale, suppleÂmenteÂavec 20% FCS, a donneÂles meilleurs reÂsultats pour le clonage. Les ligneÂes cellulaires cloneÂes ont preÂsenteÂla meà me morphologie que celle des cellules lymphoblastiques avec des noyauz irreÂguliers et de la chromatine diffuse. L’ analyse de l’ ARNm extrait de ces ligneÂs cellulaires a montreÂque les ligneÂes non cloneÂes expri-

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maient de facË on intense, la chaõ à ne b correspondant au reÂcepteur antigeÁne des cellules T (TCR) et de facË on plus faible, les polypeptides correspondant aÁ l’ immunoglonbuline (Ig) (l de la chõ à ne leÂgeÁ re et m , u , u (D Fc) et a des chai à nes lourdes dans des proportions variables), suggeÂrant la preÂsence de cellules B et T. Les ligneÂes cellulaires cloneÂes qui ont pu eà tre classeÂes, ont e teÂles cellules T TCR b 1 ve. Ceci est le premier rapport de l’ eÂtablissement, du clonage et de la caracteÂrisation partielle des ligneÂes cellulaires lymphoblastiques du canard.

Die klonierten Zellinien, die klassi® ziert werden konnten, waren TCR-b-positive T-Zellen. Dies ist der erste Bericht uÈ ber die Etablierung, Klonierung und partielle Charakterisierung von lymphoblastoiden Enten-Zellinien.

ZUSAMMENFASSUNG

]La cepa T del virus de la retriculoendoteliosis (REV-T), obtenida con la ayuda del virus sincitial del pollo (CSV) en la lõ nea celular CSO4, fue altamente oncogeÂnica y de curso fatal en patos. Los tumores se localizaban principalmente en el bazo pero se pudieron observar ce lulas neoplaÂsicas en otros muchos oÂrganos. Mediante el REV no se consiguioÂla transformacioÂn in vitro de linfocitos de pato en lõ neas celulares estables, por lo tanto se utilizaron ce lulas de oÂrganos (sangre, meÂdula oÂsea, bazo, ganglio linfaÂtico, bolsa de Fabricio) de aves infectadas para establecer lõ neas celulares. Algunas de estas lõ neas celulares han sido clonadas. El õ  ndice de eÂxito en el establecimiento y clonacioÂn de lõ neas celulares se incrementaba si las ce lulas eran cultivadas en un rango de medio con diferentes complementos; sin embargo un medio con un 5% de suero fetal de ternero (FCS) y un 5% de suero de pato fue el maÂs uÂtil para el establecimiento inicial, mientras que un medio obtenido a partir de la lõ nea parental y suplementado con un 20% maÂs de FCS dio mejores resultados para la clonacioÂn. Las lõ neas celulares clonadas presentaban una morfologõ  a de ce lulas linfoblastoides, con un nuÂcleo irregular y cromatina difusa. El anaÂlisis del ARN m extraido de estas lõ neas celulares mostroÂque las lõ neas no clonadas expresaban intensamente la cadena b del receptor antigeÂnico de las ceÂlulas T (TCR) y deÂbilmente polipeÂptidos de inmunoglobulinas (Ig) (cadena ligera l y m u , u (D Fc) y cadenas pesadas a en diferentes proporciones), siendo sugestivo de la presencia de ceÂlulas T y B. Las lõ neas celulares clonadas que pudieron ser clasi® cadas fueron ceÂlulas T TCR b 1 ve. Este es el primer trabajo de establecimiento, clonacioÂn y caracterizacioÂn parcial de lõ neas celulares linfoblastoides de pato.

Enten-Lymphozyten. VIII. T-lym phoblastoide Reticuloendotheliosevirus-induzierten Tum oren

Zellinien

aus

Der Stamm T des Retikuloendotheliosevirus (REV-T), der zugleich mit dem HuÈ hner-Synzytialvirus (CSV) als Helfervirus aus der Zellinie CSO4 isoliert wurde, war bei Enten hochpathogen und rasch toÈ dlich. Tumoren wurden hauptsaÈ chlich in der Milz gesehen, aber neoplastische Zellen wurden in vielen Organen mikroskopisch festgestellt. Die REV-Transformation von Enten-Lymphozyten in vitro ergab keine stabilen Zellinien; daher wurden Zellen aus Organen (Blut, Knochenmark, Milz, Lymphknoten, Bursa Fabricii) in® zierter Enten verwendet, um Zellinien zu etablieren. Einige dieser Zellinien wurden geklont. Die Erfolgsraten der Etablierung und Klonierung waren erhoÈ ht, wenn die Zellen in einer Kollektion von Medien mit unterschiedlichen ZusaÈ tzen kultiviert wurden; Medium mit 5% fetalem KaÈ lberserum (FKS) und 5% Entenserum war allerdings fuÈr die anfaÈ ngliche Etablierung am wirksamsten, waÈ hrend beim Klonieren die besten Resultate mit gebrauchtem Medium von der Elternlinie mit weiteren 20% FKS erzielt wurden. Die klonierten Zellinien hatten die Morphologie von lymphoblastoiden Zellen, mit unregelmaÈ û igen Kernen und diffusem Chromatin. Die Analyse der aus diesen Zellinien extrahierten mRNA zeigte, daû die nicht-klonierten Linien die b-Kette des T-Zell-Antigenrezptors (TCR) stark exprimierten und die Immunglobulin (Ig)-Polypeptide (leichte l-Kette und schwere Ketten m, u, u (DFC) und a in unterschiedlichen Proportionen) schwach exprimierten, was auf das Vorhandensein von T- und B-Zellen schlieû en lieû .

RESUME N Linfocitos de pato. VIII. LõÂ neas celulares linfoblastoides t obtenidas a partir de tum ores inducidos por virus