Restricted expression of human T-Cell leukemia-lymphoma virus (HTLV) in Transformed human umbilical cord blood lymphocytes

VIROLOGY

129, 51-64 (1983)

Restrict:ed Expression of Human T-Cell Leukemia-Lymphoma Virus (HTLV) in Transformed Human Umbilical Cord Blood Lymphocytes SYED Z. S.ALAHUDDIN,* GENOVEFFA

PHILLIP D. MARKHAM,? FLOSSIE WONG-STAAL,* FRANCHINI,* V. S. KALYANARAMAN,? AND ROBERT C. GALLO*,’

*L&m-atwy of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205, and f’Depwtment of Cell Biology, Lit&m Bionetics, Inc., Kensington, Maryland 20795 Received January

20, 1983; accepted May 6, 1983

The productive infection and transformation of fresh human lymphocytes by several HTLV isolates have recently been reported. We extend these observations here with the description of multiple immortalized, non-producer, human umbilical cord blood lymphocyte cultures developed by cocultivation or fusion of fresh cells with T cells cultured from leukemia-lymphoma patients. These transformed neonatal leukocytes exhibit morphological, cytochemical, and other phenotypic characteristics similar to those of other HTLVinfected cells but, in contrast to the usual productive infection seen, these cells contain only leow amounts of viral proteins and do not release virus particles. These cells contain at least one copy of HTLV proviral DNA/cell and transcribe viral RNA similar in size to virus-producing cells. Virus expression in these cultures was not enhanced by IUdR treatment. These cell cultures should be useful in studies of the regulation of viral expression in human cells and of the viral proteins and nucleic acids involved in T-cell immortalization and growth.

Wong-Staal, 1982); however, definitive evidence for a causal association was obtained only recently with the identification and isolation of human T-cell leukemialymphoma virus (HTLV) and the determination of its specific involvement with some adult T-cell malignancies (reviewed in Gallo and Wong-Staal, 1982; Essex, 1982; Gallo and Reitz, 1982). HTLV-I was first isolated and characterized in cultured cells from sporadic adult T-cell leukemia-lymphoma patients in the U. S. (Poiesz et al, 1980a, and 1981). Virus of the same type (HTLV-I) was isolated subsequently from adult T-cell leukemialymphoma patients living in various other parts of the world (Miyoshi et ah, 1981; Yoshida, et al., 1982; Gallo et cd., 1982; Popovic et al., 1983; Haynes et al., 1983). In addition, a closely related yet distinguishable subtype of HTLV (HTLV-II) was also identified in T cells established in cell culture from a patient with hairy cell leukemia (Kalyanaraman et al, 1982). Extensive characterization demonstrated that

INTRODUCTION

Infectious retroviruses are involved in naturally occuring leukemias and lymphomas in several animal species (Gardner, 1978; Jarrett. et ah, 1964; Essex et d, 19’77; Burny et ak, 1980; Ferrer et aZ., 1980; Kawakami and McDowell, 1980; and Gallo et al, 1978). The mechanisms by which these naturally occurring viruses cause leukemia are unknown but available data indicate that most do not carry transforming (one) genes as do acutely transforming retroviruses (e.g. ;seereview by Duesberg, 1979). Evidence obtained with avian leukosis virus, for example, suggests an indirect mechanism of leukemogenesis mediated through integration of proviral sequences near a cellular “one” gene (Hayward et al., 1981). The involvement of retroviruses in human neoplasia has been the subject of extensive research (reviewed in Gallo and r Author sent.

to whom requests for reprints

should be 51

0042.6822/83 $3.00 Copyright All rights

0 1983 by Academic Press. Inc. uf reproduction in any form reserved

SALAHUDDIN

52

neither type of HTLV is related to other known retroviruses (Reitz et ah, 1981; Kalyanaraman et ab, 1981; Robert-Guroff et aL, 1981; Rho et al, 1981; Oroszlan et ab, 1982). HTLV is infectious for human umbilical cord-blood T lymphocytes generally resulting in cell transformation, i.e., ability to grow without added T-cell growth factor (TCGF), and virus production (Miyoshi et al., 1981; Yamamoto et aL, 1982; Popovic et al., 1983; Markham et ai., 1983). In this communication we report that HTLV can also transform human umbilical cord-blood T lymphocytes without virus production when they are cocultivated or fused with some sources of HTLV. The immortalized cells contain the HTLV genome and synthesize viral RNA, but are restricted in their expression of viral structural proteins. These observations apparently represent a separation between viral replication and viral transforming functions and these procedures should be valuable for studies of virus-induced T-cell transformation and the regulation of HTLV expression by human T cells. MATERIALS

AND

METHODS

Virus and Cell Source Virus donor cells. CR-MB, a clone of cells isolated by Dr. Michiyoki Maeda in our laboratory from the first reported HTLVpositive human T-cell line (Poiesz et aL, 1980b), was further banded repeatedly on Percol gradients (Salahuddin et aL, 1982) to select cells with an increased level of virus production. One resulting cell culture was designated CRII. Another source of virus used in these studies was a TCGF-dependent T-cell culture initiated from adult T-cell lymphoma patient U.K. (Popovic et ab, 1983). Cells were grown in complete medium [RPM1 1640 or alpha medium supplemented with 1 mM glutamine and 20% heat-inactivated fetal calf serum (FCS)] with or without partially purified TCGF, Umbilical cord-blood mononuclear leukocytes. Leukocytes from freshly drawn, heparinized, human blood obtained from

ET AL

the umbilical cord of newborn children were banded on lymphocyte separation medium as previously described (Salahuddin et al., 1982). Mixed populations of mononuclear leukocytes recovered from the interphase were rinsed and resuspended in complete medium at a concentration of 1.5 X lo6 cells/ml for cocultivation or fusion experiments. Infection

of Cells

Coculture. CR11 cells were rinsed with media and treated with mitomycin-C (MMC, 100 pg/ml, Sigma Chemical Co., St. Louis, MO.) for 30,45, or 60 min at 37” and U.K. cells were rinsed and lethally irradiated (5000 R/12.5 min, Fixatron X-Ray Systems, Hewlett Packard Model 43804N). Following treatment, these virus donor cells were rinsed twice with medium and mixed at a 1:3 ratio with cord-blood mononuclear cells (5 X lo5 virus producing cells:1.5 X lo6 cord-blood cells) in a final volume of 5 ml complete medium. These cells were incubated at 37” in 5% COz atmosphere and were refed weekly by a total medium change. Actively growing cells were routinely subcultured at a concentration of l-2 X lo5 cells/ml. Cell fusion, CRII (1 X 106) cells and human cord-blood mononuclear cells (2 X 106) were mixed and pelleted by centrifugation at 400 9 for 10 min. Cells were then resuspended in 5 ml 50% solution of polyethylene glycol (PEG 6000, J. T. Baker, Phillipsburg, N. J.) in 0.15 iM NaCl and allowed to stand for 60 set at room temperature. Complete medium was then slowly added to a final volume of 18 ml. Cells were then pelleted, rinsed twice with complete media, and resuspended in 5 ml of complete medium. Cell cultures were incubated at 3’7” in 5% COZ atmosphere and were refed weekly by a total medium change. IUdR induction. Cells were incubated with 25 or 50 pg/ml IUdR in complete medium for 24 hr and samples taken at daily intervals for 8 days. The number of HTLV p19 and p24 positive cells was assessed by immunofluoresence assays. The level of intercellular HTLV p24 was determined by

TRANSFORMED,

NON-PRODUCER,

competition radioimmunoassay. Extracellular virus was detected as precipitable reverse transcriptase activity and by its ability to band in preformed 15-60% sucrose gradients. Designation of HTLV and transfkn-med cord-blood lymphocytes. Isolates of HTLV are designated by subtype (when known) and by the initials of the patient from whom virus was isolated, e.g., HTLV-Its (Poiesz et aZ., 1980a), or HTLV-IIMo (Kalyanaraman et ah, 1982). Immortalized cord-blood cells were assigned an abreviated designation. The numerator indicates the source of fresh cells and sample number and the denominator the initials of the leukemia patient whose cells were used as a source of virus, e.g., C63/CRu (cordblood No. Gtl/leukemic cell culture from donor CR). Characterization

of Cells

Cytogeneti’cs. Chromosome preparations were made according to standard procedures (Rowley and Potter, 1976; Salahuddin et al, 1982) Cells were fixed on slides, treated with trypsin, and stained with 2% Giemsa in Sorensen’s buffer, pH 6.8, after approximately 2 weeks of aging. Cytochemistry. Specific tests included staining for chloracetate esterase (Yam et al., 1971) and a-naphthyl acetate esterase (nonspecific esterase, Yam et al, 1971), acid phosphatase (Goldberg, 1964), myeloperoxidase (Kaplow, 1965), and, for neutral fats, phospholipids and lipoproteins with Sudan black (Sheehan and Storey, 1947). EBV antigens. Cells were tested for Epstein-Barr virus nuclear antigen (EBNA) by published procedures (Reedman and Klein, 1973). Rosette jkrmation. Leukocytes were tested for tlneir ability to form rosettes with sheep or bovine erythrocytes either directly (E :rosette), following treatment of erythrocytes with antibodies to sheep or bovine erythrocytes (EA rosettes), or after addition of C;-deficient mouse complement to the EA preparation (EAC rosettes), by published procedures (Jondal and Klein, 1973).

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53

Surface bound immunoglobulin (SIg). The presence of SIg was determined by direct procedures using FITC-labeled F(ab’), fragments of goat anti-human IgG, A, and M. Cell-speci$c mono&ma1 antibodies. Murine monoclonal antibodies reactive with human T lymphocytes and their subsets, e.g., helper/inducer and cytotoxic/suppressor, were used to characterize live cells as previously reported (Reinherz et al., 1979, Engleman et al, 1981). Histocompatibility antigens, locus D (HLA DR) (Grumet et al., 1980), and murine monoclonal antisera reactive with peripheral blood monocytes, OKM-1 (Breard et al., 1980), were also used. Positive cells were identified using FITC-labeled goat antimurine IgG, F(ab’)z-specific serum (TAG0 Inc., Burlingame, Calif.). OKT reagents were purchased from Ortho Pharmaceutical Corp., Raritan, New Jersey, and Leu reagents from Becton-Dickinson FACS Systems, Sunnyvale, California. Histocompatibility antigens (HLA). Microtoxicity analyses using typed allogenie sera and complement were performed as previously described (Mittal et al, 1968). Prepared HLA typing trays were obtained from National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland (Basic tray Y4 and extended tray W-9) and from Dr. P. Terasaki, University of California, Los Angeles, California (tray 25). Colony growth in semi-solid media. Cultured leukocytes were tested for their ability to grow as colonies in 0.3% agarose or 0.8% methylcellulose with or without addition of GM colony stimulating activity (GM-CSA). Conditioned medium from a SV-40-transformed human placental cell line (Ruscetti et ab, 1982) was used as a source of exogenous GM-CSA. Assays were performed routinely in duplicate as previously described (Salahuddin et al., 1982). T-cell growth factor (TCGF) cell receptors. Cells were assayed for the presence of TCGF receptors using the monoclonal antibody TAC as previously described (Leonard et al., 1982).

54

SALAHUDDIN

Terminal transferase (TdT). Cell lysates were analyzed for TdT activity as previously described (Sarin et al., 1974). Detection of Virus Reverse transcriptase. Proteins from cell culture fluids were concentrated by polyethylene glycol and assayed for viral reverse transcriptase activity using Mn2+ or Mg2+ as cations and dTIB. rA, as template primer. Cellular DNA polymerase activity was assessed using dTi5. dA, as template primer (Gallo et al, 1978). Electron microscopy and banding in sucrose. Thin sections of fixed and embedded cells were examined by electron microscopy for viral particles as previously described (Poiesz et aZ., 1980). Supernatant fluids from cell cultures labeled for 48 hr with [3H]uridine (25 &i/ml, 25 Ci/mmol) were layered over preformed 1560% sucrose gradients and centrifuged for 4 hr at 4” in a Beckman SW41 rotor at -200,OOOg. Fractions collected from the bottom of each gradient were assayed for precipitable radioactivity. ImmunoJuorescence. Fixed cells were analyzed for HTLV structural proteins using murine monoclonal antibody specific for HTLV p19 (Robert-Guroff et al. 1981) and goat anti-HTLV p24 (Kalyanaraman et al., 1981). Radioimmunoassay. Cell lysates were used to compete with the precipitation of lz51-labeled HTLV p24 by specific antisera as previously described (Kalyanaraman et al., 1981). DNA blot hybridization. High molecular weight cellular DNA was digested with restriction endonucleases and fractionated on 0.8% agarose gels by electrophoresis. Transfer of DNA to nitrocellulose filters was carried out as described by Southern (1975). Hybridization utilized 32P-labeled DNA fragments, containing predominately pol-env sequences derived from a cloned segment of the HTLV genome (Manzari et al, 1983). RNA blot hybridization. RNA was extracted from cells as described (Adams et al., 1977), denatured in a solution contain-

ET AL

ing 2.2 M formaldehyde and 50% (v/v) formamide at 60”, and 10 pg applied to each lane of a 1.2% agarose gel containing 2.2 M formaldehyde. Following electrophoresis at 30 V for 16 hr, RNA was transfered to a Genescreen membrane (New England Nuclear, Boston, Mass.) by electrophoresis at 10 V for 2 hr in 25 mM sodium phosphate buffer, pH 6.5. Hybridization of baked filters was performed according to the method of Thomas (1980) utilizing a HTLV long terminal repeat (LTR)-containing probe. RESULTS

Infection and Transfkwmation Lymphocytes

of Cord-Blood

HTLV-IcR, the first isolate of HTLV produced by a T-lymphocyte line, initiated from an adult T-cell lymphoma donor CR, is poorly infectious. Evidence for its transmission to other cells was obtained only by using T-lymphocyte cultures established from close relatives of CR (Ruscetti et al, 1983). In these experiments, the infected cells remained dependent on added TCGF for growth. Using a derivative of the Tcell line from CR (cell culture CRn) as a source of HTLV to infect fresh neonatal umbilical cord-blood leukocytes, nonproductively transformed cord-blood cells were obtained in one of five cord-blood samples attempted, using cocultivation procedures and in one experiment using cell-fusion techniques. Gne other nonproductively transformed cell culture C43/UK was obtained by cocultivation of cells from cord-blood number 43 with UK cells. These results were in contrast to the usual infection and productive transformation obtained using other isolates of HTLV, including many with UK (Popovic et al, 1983; Markham et al, 1983). Most cells in both HTLV-infected and uninfected cultures died within 2-3 weeks after infection before clumps of replicating cells eventually populated the cell cultures. In experiments involving PEG fusion, many multinucleated cells persisted in culture for several passages. Within 6-8 weeks, cells treated by either cocultivation

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or fusion became established in culture and have now been in culture for more than 18 months.

Characterization of Cells The transformed cord-blood cells have a lymphoid morphology indistinguishable from normal TCGF-dependent cells except for the increased incidence of cells containing lobulated nuclei (-1%) and multinucleation (-5%) (Fig. l), described for leukemic T-c’ell populations (Poiesz et aZ., 1980b). As summarized in Table 1, a high percentage of cells in each cell culture reacts with the T-cell specific antibody Leu 1, forms rosettes with sheep erythrocytes, reacts with E-rosette receptor antibody OKTll (datal not shown), and generally reacts with antibodies OKT 4 and Leu 2a, suggesting an inducer/helper T-cell phenotype. All cells were highly positive when tested with anti-HLA-DR, were generally negative with antibody OKT 3 (“Pan T”), and were consistently negative with OKT 6 (thymocytes) and OKM-1 (monocytes). Some cells also formed rosettes with antibody and complement-treated bovine erythrocytes (EAC rosettes), suggesting the presence of complement receptors. These cells d’o not contain B-cell markers, SIg or EBNA (data not shown). Cells from all cultures, including those from virus donors CRII and UK, were positive for acid phosphatase and nonspecific esterase (both with atypical., granular/globular, staining patterns) but did not react with granuloclorcyte markers, e.g., myeloperoxidase, acetate esterase, and Sudan black (data not shown). No terminal transferase activity was detected in cell lysates (data not shown). Cells from several of these cell cultures grew into colonies in 0.33% agarose without added G,M-CSA or TCGF (data not shown).

TCGF receptws A high percentage of cells in all cultures contain receptors for TCGF as detected by specific monoclonal antisera TAC. How-

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55

ever, no TCGF activity was detected in unconcentrated or lo- to 20-fold concentrated conditioned media, or as membrane-bound activity (personal communication, Drs. S. Lindner and J. Gootenberg).

Presence of HTLV Virus particles were not detected in sucrose gradients of supernatant fluids from [3H]uridine-labeled or unlabeled cells or by electron microscopic observation of cell sections. Also, cell-culture fluids concentrated 50- to 500-fold had no detectable reverse transcriptase activity and no biological effect was found using these cells for cocultivation with fresh cord-blood cells (data not shown). HTLV structural proteins p19 and p24 were not detected when cells were analyzed by indirect immunofluorescence procedures; however, sensitive competition radioimmunoassays performed on cell lysates detected low levels of HTLV p24 (2-27 ng/mg cell protein) in all cell cultures examined, as illustrated in Fig. 2 and Table 2. Expression of viral proteins and release of virus in cell cultures C63/CRn-2 and C43/UK were also tested following exposure to IUdR. No increase in viral expression was found using conditions where Balb/3T3 murine fibroblasts were readily induced to release virus (data not shown). The virus-negative transformed cell cultures appear to contain one or more copies of integrated HTLV provirus determined by comparison to the virus donor cells previously shown to have at least one DNA copy per cell. A representative DNA blot of C63/CRu-2 is shown in Fig. 3. A cloned segment of an integrated defective provirus of HTLV-IcR containing predominantly HTLV pal-env sequence was used as a probe (Manzari et al, 1983). This probe detected specific viral DNA bands in the original CR-M2 cells but not in control, uninfected cells. In some instances, portions of the genome appear to be amplified 20- to 30fold. When RNA from virus donor cells UK and transformed, virus producing, cordblood cells C43/UK were analyzed using a viral probe containing a portion of the 3’

56

SALAHUDDIN

ET AL.

FIG. 1. Wright-Giemsa-stained, transformed, cord-blood leukocytes. Transformed cord-blood kocyte culture C63/CRn-2 was pelleted in a cytocentrifuge, and stained with Wright-Giemsa described in Material and Methods. 2500X magnification.

leuas

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NON-PRODUCER, TABLE

57

T CELLS

1

IMMUNOLOGICAL CHARACTERISTICS OF HTLV-TRANSFORMED NON-PRODUCER UMBILICAL CORD-BLOOD LEUKOCYTES Monoclonal antibodies’

Resetting” Culture”

E

EA

EAC

OKT 3

Leo 1

OKT4

Leo 3a

OKTS

Leu2a

OKT 6

oHLA-DR

0 70 0 77 7 0

0 2 0 4 0 0

100 100 100 100 100 100

0 0 0 0 0 0

0 0

0 0

0 0

100 100

0 0

NT 0 35

NT 0 28

NT 0 0

NT 100 33

NT 0 22

OKM-1

Transformed cord-blood cells C63/CRit-1 C63/CR,t-2 C63/CR1i-3 C63/CR,i-4 C63/CRh-5 C43/UK

24 42 28 84 NTd 20

0 0 2 0 11 NT

45 87 87 0 63 NT

0 20 20 0 2 15

100 100 100 100 100 loo

loo 30 loo 100 10 loo

100 36 100 100 11 90

0 43 0 61 11 0

HTLV donor neoplastic T-cells CR,r UK

33 30

0 NT

80 NT

0 100

90 28

100 95

100 90

Other leukocytes Molt 4 PP-1 NPB

80 5 NT

0 18 NT

0 50 NT

NT 0 66

NT 0 53

NT 0 38

NT 0 36

a Fresh umbilical cord blood mononuclear cells from donor C63 were either cocultivated (l-3) or fused (4,5) with mitomyeinC-treated virus donor cells, CRt,. Cord-blood cells from donor C43 were cocultivated with the irradiated virus donor cells UK, as described in Materials and Methods. Molt 4, is a human T-cell line established from an acute lymphoid leukemia patient; PP-1, a human B-lymphoblast cell line initiated spontaneously from an adult normal donor; and NPB, fresh human mononuclear peripheral blood leukocytes prepared from a normal adult donor. *Cells were tested for ability to form rosettes spontaneously with: sheep erythrocytes, E, antibody-treated bovine erythrocytes, EA; and antibody and complement-treated bovine erythrocytes. EAC, as described in Materials and Methods. ‘Cells were tested for their reactivity with specific T lymphocytes and other monoclonal antibodies and detected by addition of FITC-specific antisera, as described in Materials and Methods. Data are percentage positive cells. d NT, not tested.

LTR sequences (Manzari et al, 1983), RNA species corresponding to the size of the HTLV genome (10 kb) and possibly to envelope mRNA (3.9 kb) were observed (Fig. 4A). In contrast, the transformed non-producer C63/CRn-2 cell line contained little or no genomic-sized viral mRNA. Both donor cells CRn and C63/CRn-2 contain an extra 3.7 kb species of RNA (Fig. 4B).

markers. As summarized in Table 3, all cell cultures initiated from the same cordblood donor cells, C63/CRn-1-5, have a common HLA haplotype distinct from the CRn cells used as a source of HTLV. C43/ UK is likewise distinct from UK cells. Detailed chromosomal analysis of representative cultures, e.g., C63/CRn-2, C63/CRn4, and C43/UK further substantiates their uniqueness. The virus donor cell culture Distinguishing Transformed Cord-Blood T CRn is -88% diploid, 46 XY, with 12% The major chromosomal Cells from Neoplastic T Cells Used as polyploidy. markers found in both diploid (Fig. 5A) a Source of HTLV and polyploid (data not shown) cells are: In addition to the restriction in expres(1) polymorphism of chromosome 16 sion of viral proteins, cells in these cord(16qh+) and (2) prominent satellite chroblood cultures were also distinguishable mosome segments associated with chrofrom virus dsonor cells by HLA haplotype mosomes 14 and 21. Cells from the coculand the presence of specific chromosome tivated cord-blood culture C63/CRn-2 were

SALAHUDDIN

ET AL.

one similar to CRn and the other similar to that in the cord-blood cells (data not shown). Also, as summarized in Table 3, C43/UK is easily distinguished from UK on the basis of HLA haplotype and the difference in sex chromosomes. DISCUSSION

0.01

0.1 Cellular

1 Protein

10 (pg)

FIG. 2. Competition radioimmunoassay for HTLV structural protein p24 in lysate of transformed cordblood cells. The precipitation of labeled HTLV p24 by specific antisera was competed by increasing concentration of lysed-cell proteins as described in Material and Methods and in Table 2. Cells examined include CRn, adult leukemia T-cell line used as a source of virus (0); cord-blood leukocyte transformed by cocultivation with CRn, C63/CRn1 (0), C63/CRu2 (IX), and C63/CRu-3 (A); and cord-blood leukocytes transformed by PEG-mediated cell fusion with CRu, C63/CRn-4 (m), and C63/CRn-5 (A).

Many recent isolates of HTLV can infect and productively transform fresh cordblood T cells (Popovic et al, 1983, Markham et aL, 1983); however, the first HTLV isolate, HTLV-Its, is poorly infectious posTABLE

PRESENCE OF HTLV GENOME AND STRUCTURAL PROTEINS IN TRANSFORMED NON-PRODUCER CORD-BLOOD LYMPHOCYTES

HTLV

enzyme and proteins* Immunofluorescence

Culture No. a

‘75% diploid, 46 XY, and 25% polyploid. Chromosomal markers found in these cells include: (1) prominent secondary constriction of chromosome 1 and 9 (9qh+) and (2) prominent satellite markers on chromosomes 21 and 22. Notably, C63/CRn-2 cells lacked the CRn marker 16qh+ and the prominent satellite on chromosome 14. The satellite on chromosome 21 and the Y chromosome in these cells were also distinguishable from those in CRn. The karyotype from a banded C63/CRn-2 diploid cell is shown in Fig. 5B. A fused culture C63/CRn-4 was predominately polyploid (>90%), having 82-92 chromosomes. These cells contain both identifiable CRn and cord-blood (C63) chromosomal markers and therefore appear to be hybrid cells. All cells examined contained, for example, one polymorphic chromosome 16 (16qh+) and the satellites of chromosome 21 detected in CRn in addition to chromosomal polymorphism not characteristic of CRn. These cells also contained two distinguishable Y chromosomes,

2

Reverse transscriptase Transformed

C63/CRn-1 C63/CR,,-2 C63/CRu-3 C63/CRn-4 C63/CRn-5 C43/UK

0 0 0 0 0 0

~24 PI9 (% positive cells) cord-blood
RIA for p24 (ndw cell protein) cells

0
6 6 10 <2 2 27

Virus donor cells ‘&I UK

+ +

90 80

90 80

4000 1360

n Cultures are described in Materials and Methods and in Table 1. b RT = reverse transcriptase activity in culture medium concentrated and collected from cultured cells tested as described in Materials and Methods. + = levels of enzymatic activity > twofold above background levels. Cultured cells were fixed and tested for the presence of HTLV structural protein by indirect immunofluoresence technique using monoclonal murine anti-HTLV p19 and goat anti-HTLV ~24, and cell lysates were tested for HTLV p24 by competition of radioimmunoassay, as described in Materials and Methods.

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FIG. 3. Presence of HTLV sequences in C63/CRn-2 DNA. 30 pg of cellular DNA were digested with different restriction endonucleases and analysed by blot hybridization as described in Materials and Methods. Hybridization was carried out with “P-labeled HTLV pal-env sequences. The sources of DNA are: (a-c) CR-M% (d-f) C63/CRn-2; and (g-i) normal human thymus. The restriction endonucleases used are: a, d, and g, EcoRI; b, e, and h, XbaI; and c, f, and i, Kpd.

sibly caused by low virus production or the existence of many defective particles (Ruscetti et al., 1983; and our personal observation). Using a subculture of the original cell line CRn, we have demonstrated that HTLV-Its, as well as virus produced by other leukemic T-cells, e.g., HTLV-Iu.x., can nonproductively transform fresh human umbilical cord-blood leukocytes. The transformed cells are lymphocytes with morphological and surface characteristics in common with those reported for fresh or cultured leukemic T cells and for HTLVinfected, cord-blood, and adult-blood cultures (Poiesz et al., 1980b; Miyoshi et al., 1981; Markham et al., 1983). As also reported for leukemic T cells, these transformed cord-blood cells do not contain detectable terminal transferase activity but are HLA-DR positive and stain with an atypical, granular/globular stain for nonspecific esterase and acid phosphatase (Pinkus et ak, 1979; Savage et ah, 1981). The newly established cell cultures were readily distinguishable from virus donor cells by HLA haplotype and specific chromosomal markers. The cord-blood cultures C63/CRn-4,-5, established by PEG-mediated cell fusion, appear to be composed

FIG. 4. Detection of HTLV mRNA in UK; C43/UK (A); and CRn, C63/CRn (B) cell lines. 10 pg of RNA prepared from the indicated cell lines were electrophoresed as described in Materials and Methods. Hybridization was carried out using a cloned 32P-labeled HTLV-LTR segment.

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TABLE

ET AL. 3

HLA HAPLOTYPE AND CHROMOSOMAL CHARACTERISTICS OF TRANSFORMED, NON-PRODUCER, CORD-BLOOD LYMPHOCYTE CULTURES HLA

Culture”

Chromosomal

haplotype* Transformed

C63/CFn-1,3,5

C63/CRn-2

C63/CR,,-4

C43/UK

BW4,6 (CW5)

75% diploid Polymorphic prominent

(46 XY), 25% polypoid cells in chromosome 9 (9qh+) and satellite on 21 and 22 chromosomes

80% Polypoid (82-91 XXYY) Two distinct chromosomes sets observed

Al (629) BW4 6 B8, BW12/44 ’ s

cells

Not tested

Al 6429) BW4 6 B8, BW12/44 ’ B8A;;f;;44

cord-blood

analysis”

xx

BW4,6 CW2

Virus donor cells UK

Al, A2 BW4.6 CW2,4 B5, BW35.53

XY

CR,,

AlO, AW30/31 BW4,6 CW2 B17, Bl8

88% diploid (46 XY) 12% polypoid Polymorphism of chromosome 16 (16qh+) prominent satellites on chromosomes 14 and 21

a Transformed cord-blood and virus donor cells are described in Materials and Methods and in Table 1. b The histocompatibility antigens (HLA) of specified cultures were determined by microtoxicity analysis as described in Materials and Methods. ’ Chromosomal preparations were made for analysis by standard procedures and were stained by trypsinGiemsa banding techniques as described in Materials and Methods.

of hybrid cells containing both CRII and cord-blood cell chromosomes. The HLA haplotype, however, corresponds only to that of the cord-blood leukocytes, suggesting, along with the restricted expression of HTLV provirus, that a dominant regulatory effect is exerted by the cordblood chromosomes. Analysis of diploid and polyploid cordblood cells for the presence and expression of the HTLV genome demonstrated that they both contain at least one copy of the HTLV genome and synthesize more than one viral RNA species. Expression of HTLV proteins in these cord-blood lymphocyte cultures is greatly restricted in all cases. They do not release detectable virus or reverse transcriptase activity and HTLV structural proteins p19 or p24 were not de-

tected by immunofluorescence procedures. Levels of HTLV ~24, lo- to loo-fold lower than in virus donor cells however, were detected by competition radioimmunoassay. Nucleic acid analysis of these cultures suggests the presence of at least one genome equivalent per cell. It is therefore likely that the low-level HTLV expression results from a restriction in all cells rather than from the presence of a very low percentage of virus-positive cells in the population. A difference was noted in the size of HTLV RNA in cells from two of the nonproductively transformed cord-blood cell cultures examined. One C43/UK contains genomic-sized RNA and the other C63/CRn-2 does not. This suggests a possible difference in the mechanism of virus restriction involved. In neither case was

FIG. 5. Karyotype of transformed cord-blood leukocytes and CRn. Cells were prepared for trypsinGiemsa banding as described in Material and Methods. The karyotypes shown are: from virus donor culture CRn (A); and diploid cell from culture C63/CRn-2 (B). 61

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this restriction overcome by conventional IUdR induction techniques. Both biochemical and biological evidence now strongly suggests a causal association between HTLV infection and development of at least some forms of malignant adult T-cell leukemia-lymphoma (reviewed in Gallo et al., 1982; Essex, 1982; Gallo and Wong-Staal, 1982). We provide in this report evidence which suggests that the “transforming” component of HTLV can be transferred to and expressed in normal T lymphocytes independent of viral replication. These cell cultures should provide systems for the study of proteins and nucleic acids involved in the immortalization of human T cells by HTLV and the cellular mechanisms involved in controlling expression of viral genes or in processing their products. ACKNOWLEDGMENTS We wish to acknowledge the expert assistance of Ms. Paula Berry and Ms. Helen Lebowitz (Litton Bionetics, Inc., Kensington, Md.) for chromosomal analysis, Dr. Beatrice Hahn (NCI, Bethesda, Md.) for DNA blot hybridization, Dr. P. Sarin (NCI, Bethesda, Md.) for terminal transferase tests, and Ms. Patie Porecha for excellent technical help. REFERENCES ADAMS, S. L., SOBEL, M. E., HOWARD, B. H., OLDEN, K., YAMADA, K. M., DE~ROMBRUGGHE, B., and PASTAN, I. (1977) Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Nat.

Acad.

Sci. USA 74, 3399-3403.

BREARD, J., REINHERZ, E. I., KUNG, P. C., GOLDSTEIN, G., and SCHLOSSMAN, S. F. (1980) A monoclonal antibody reactive with human peripheral blood monocytes. J. Immunol 124, 1943-1948. BURNY, A., BRUCK, C., CHANTRENNE, H., CLEUTER, Y., DEKEGEL,

D., GHYSDAEL,

J., KETTMAN,

R., LECLERQ,

M., LEUNEN, J., MAMMERICK, M., and PORTELELLE, D. (1980) Bovine leukemia virus: Molecular Biology and Epidemiology. In “Viral Oncology” (G. Klein, ed.), pp. 231-289. Raven Press, New York. DUESBERG, P. H. (1979) Transforming genes of retroviruses. Cold Spring Harbor Symp. &ant. Biol. 44, 13-30. ENGLEMAN, E. G., WARNKE, R., FIX, R. I., and LEVY, R. (1981) Studies of a human T-lymphocyte antigen

ET AL. recognized

by a monoclonal antibody. Proc. Nat. 78, 1’791-1795. ESSEX, M. (1982) Adult T-cell leukemia-lymphoma: Role of a human retrovirus. J. Nat. Cancer 1mt. 69, 981-988. ESSEX, M., COTTER, S. M., STEPHENSON, J. R., AARONSON,S. A., and HARDY, W. D. (1977) Leukemia, lymphoma, and fibrosarcoma of cats as models for similar disease of man. Cold Spring Harbor ConJ Cell Proliferatimz 4, 1197-1214. FERRER, J. F., CABRADILLA, C., and GUPTA, P. (1980) Bovine leukemia: A model for viral carcinogenesis. In “Viruses in Naturally Occurring Cancers” eds. (M. Essex, G. Todaro, and H. zun Hausen, eds.), pp. 887-900. Cold Spring Harbor, N. Y. GALLO, R. C., GALLAGHER, R., WONG-STAAL, F., AOKI, T., MARKHAM, P., SCHETTERS, H., RUSCETTI, F., VALERIO, M., WALLING, M., O’KEEFE, R., SAXINGER, W., :SMITH, R., GILLESPIE, D., and REITZ, M. (1978) Isolation and tissue distribution of type-C virus and viral components from a gibbon ape (Hyolates lar) with lymphocytic leukemia. Virology 84, 359373. GALLO, R. C., MANN, D., BRODER, S., RUSCETTI, F. W., MAEDA, M., KALYANARAMAN, V. S., ROBERT-GUROFF, M., and REITZ, M. (1982) Human T-cell leukemialymphoma virus (HTLV) is in T- but not B-lymphocytes from a patient with cutaneous T-cell leukemia. Proc. Nat. Acad. Sci. USA 79, 5680-5683. GALLO, R. C., and REITZ, M. (1982) Human retroviruses and adult T-cell leukemia-lymphoma. J. Nat. Cancer Inst. 69, 1209-1214. GALLO, R. C., and WONG-STAAL, F. (1982) Retroviruses as etiologic agents of some animal and human leukemias and lymphomas and as tests for elucidating the molecular mechanisms of leukemogenesis. Blood 60, 545-557. GARDNER, M. D. (1978) Type-C viruses of wild mice: Characterization and natural history of amphotropic, ecotropic, and xenotropic MuLV. Corn. Top. Acad.

Sci. USA

Microtriol.

Immunol.

79, 205-259.

GOLDBERG, A. F. (1964) Acid phosphatase activity in Auer rods. BZood 24, 305-308. GRUMET, F. C., CHARRON, D. J., FEUDLY, B. M., LEVY, R., and NESS, D. B. (1980) HLA-DR epitope region definition by use of monoclonal antibody probes. J. Immunol. 125, 2785-2789. HAYNES, B., MILLER, S., PALKER, T., MOORE, J., DUNN, P., BOLOGNESI, D., and METZGER, R. (1983) Identification of human T-cell leukemia virus in a Japanese patient with adult T-cell leukemia and cutaneous lymphomatous vesiculitis. Proc. Nat. Acad Sci. USA

80, 2054-2058.

HAYWARD, W. S., NEEL, B. G., and ASTRIN, S. M. (1981) Induction of lymphoid leukosis by avian leukosis virus: Activation of a cellular “one” gene by promoter insertion. Nature (Landon) 290.475-480.

TRANSFORMED,

NON-PRODUCER,

JARRETT, W. F. ML., MARTIN, W. B., CRIGHTON, D. W., DALTON, R. G., and STEWART, M. F. (1964) Leukemia

in the cat. Transmission experiments with leukemia (lymphosarcoma). Nature (London) 202, 566-567. JONDAL, M., and KLEIN, G. (1973) Surface markers on human B and T-lymphocytes. II. Presence of Epstein-Barr virus receptors on B-lymphocytes. J. Eq. Med 138,1365-13’78. KALYANARAMAN, V. S., SARNGATHARAN, M. G., ROBERT-GUROFF, M., MIYOSHI, I., BLAYNEY, D., GOLDE, D., and GALLO, IR. C. (1982) A new subtype of human T-cell leukemia virus (HTLV-II) associated with a T-cell variant of Hairy cell leukemia. Science 218, 571-573. KALYANARAMAN, ‘J. S., SARNGADHARAN, M. G., POIESZ, B. J., RUSCE~I, F. W., and GALLO, R. C. (1981) Immunological prloperties of a type-C retrovirus isolated from cultured human T-lymphoma cells and comparison to other mammalian retroviruses. J. Virology 38, 906-915. KAPLOW, L. A. (1965) Simplified myeloperoxidase stain using benzidine dihydrochloride. Blood 26,215-219. KAWAKAMI, T. G., and MCDOWELL. (1980) Factors regulating the onset of chronic myelogenous leukemia in gibbons. Cold Spring Harbor Con,f: Cell Proliferation. S., 719-727. LEONARD, W. J., DEPPER, J. M., UCHIYAMA, T., SMITH, K. A., WALDMAI~N, T. A., and GREENE, W. C. (1982) A monoclonal antibody that appears to recognize the receptor for human T-cell growth factor. Partial characterization of the receptor. Nature (London) 300,267-269.

MANZARI, V., WOI~G-STAAL, F., FRANCHINI, G., COLOMBINI, S., GELMANN, E. P., OROSZLAN, S., STAAL, S. P., and GALLO, R. C. (1983) Human T-cell leukemia-lymphoma virus (HTLV): Molecular cloning of an integrated defective provirus and flanking cellular sequences. Proc. Nat. Acad. Sci. USA 80, 1574-15'78.

MARKHAM, P. D., SALAHUDDIN, S. Z., KALYANARAMAN, V. S., POPOVIC,M., SARIN, P., and GALLO, R. C. (1983) Infection and transformation of fresh human umbilical cord blood cells by multiple sources of human T-cell leukemia-lymphoma virus (HTLV). Int. J. Cancer 31,413-420. MIITAL, K. K., MICKEY, M. R., SINGAL, D. P., and TERASAKI, P. I. (1968) Serotyping for homotransplantation. XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Z’ranqhntation 6, 913-927. MIYOSHI, I., KUBONICHI, I., YOSHIMOTO, S., AKAGI, T., OHISUK, Y., SHIA:AISHI, Y., NAGATA, K., and HINUMA, Y. (1981) Type-C virus particles in a cord T-cell line derived by cocultivating normal human cord leukocytes and human leukemic T-cells. Nature (Lrmdm)294

77'0-771.

OROSZLAN, S., ‘SA.RNGADHARAN, M. G., COPELAND, T. D., KALYANARAMAN, V. S., GILDEN, R. V., and

63

T CELLS

GALLO, R. C. (1982) Primary structure analysis of the major internal protein p24 of human type-C Tcell leukemia virus. Proc. Nat. Acad Sk USA 79, 1291-1294. PINKUS, G. S., HARGREAVES, H. K., MCLEOD, J. A., NADLER, L. M., ROSENTHAL, D. S., and SAID, J. W. (1979) cYNaphthy1 acetate esterase activity. A cytochemical marker for T-lymphocytes. Amer. J. Pa&l 97, 17-40. POIESZ, B. J., RUSCETTI, F. W., GAZDAR, F. A., BUNN, P. A., MINNA, J. D., and GALLO, R. C. (1980a) Detection and isolation of type-C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Nat. Acad. Sci. USA 77, 12, 7415-7419. POIESZ, B. J., RUSCETTI, F. W., MIER, J. W., WOODS, A. M., and GALLO, R. C. (1980b) T-cell lines established from human T-lymphocytic neoplasias by direct response to T-cell growth factor. Proc. Nut. Acad. Sti USA 11, 6815-6819. POIESZ, B. J., RUSCEWI, F. W., REITZ, M. S., KALYANARAMAN, V. S., and GALLO, R. C. (1981) Isolation of a new type-C retrovirus (HTLV) in primary cultured cells of a patient wth SCzary T-cell leukemia. Nature (L.ondon) 294, 268-271. POPOVIC, M., SARIN, P., ROBERT-GUROFF, M., KALYANARAMAN, V. S., MANN, D., MINOWADA, J., and GALLO, R. C. (1983) Isolation and transmission of human retrovirus (HTLV). Science 219, 856-859. REEDMAN, B. M., and KLINE, G. (1973) Cellular localization of an Epstein-Barr virus (EBV) associated complement fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer 11, 499-520. REINHERZ, E. L., KUNG, P. C., GOLDSTEIN, G., and SCHLOSSMAN, S. F. (1979) Further characterization of the human inducer T-cell subset defined by monoclonal antibody. J. Immunol. 123.2894-2896. REITZ, M. S., POIESZ, B. J., RUSCE~I, F. W., and GALLO, R. C. (1981) Characterization and distribution of nucleic acid sequences of a novel type-C retrovirus isolated from neoplastic human T-lymphocytes. Proc. Nat. Acad Sci. USA 78, 1887-1891. RHO, H. M., POIESZ, B. J., RUSCETTI, F. W., and GALLO, R. C. (1981) Characterization of the reverse transcriptase from a new retrovirus (HTLV) produced by a human cutaneous T-cell lymphoma cell line. Virology 112, 355-359. ROBERT-GUROFF, M., NAKAO, Y., NATAKE, K., ITO, Y., SLISKI,

A., and GALLO,

R. C. (1982)

Natural

anti-

bodies to human retrovirus HTLV in a cluster of Japanese patients with adult T-cell leukemia. Science 215.975-978. ROBERT-GUROFF, M., RUSCETPI, F. W., POSNER, L. E., POIESZ, B. J., and GALLO,

R. C. (1981)

Detection

of

the Human T-cell lymphoma virus p19 in cells of some patients with cutaneous T-cell lymphoma-

64

SALAHUDDIN

leukemia using a monoclonal antibody. J. Exp. Mea! 154, 19571964. ROWLEY, J. D., and POWER, D. (1976) Chromosomal bonding patterns in acute non-lymphocytic leukemia. Blood 47, 705-721. RUSCETTI, F. W., CHOU, J. Y., and GALLO, R. C. (1982) Human trophoblasts: Cellular source of colony stimulating activity in placental tissue. Blood 59, 1, 86-89. RUSCETX, F. W., ROBERT-GUROFF, M., CECCHERININELLI, L., MINOWADA, J., POPOVIC, M. and GALLO, R. C. (1983) Persistent in vitro infection by human T-cell leukemia-lymphoma virus (HTLV) of normal human T-lymphocytes from blood relatives of patients with HTLV associated mature T-cell neoplasia. Inf. J. Cumer 31, 171-180. SALAHUDDIN, S. Z., MARKHAM, P. D., and GALLO, R. C. (1982) Establishment of long-term monocyte suspension cultures from normal human peripheral blood. J. Exp. Med 155, 1842-1857. SARIN, P. S., ANDERSON, P. A., and Gwo, R. C. (1974) Terminal deoxynucleotidyltransferase in chronic myelogenous leukemia. J. BioL Chem. 249, 80518053. SAVAGE, R. A., VALENZUELA, R., and HOFFMAN, G. C.

ET AL. (1981) Acid phosphatase staining pattern as an indicator of T-cell acute leukemia. Amer. J. C&z. Pathol. 76, 760-764. SHEEHAN, H. L., and STOREY, G. W. (1947) An improved method of staining leukocyte granules with Sudan Black. J. Pathol. Bacterial. 59, 336-344. SOUTHERN, F. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol 98, 503-517. THOMAS, P. S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Nat. Acaa! Ski. USA 77, 5201-5205. YAM, L. T., LI, C. Y., and CROSBY, W. W. (1971) Cytochemical identification of monocytes and granulocytes. Amer. .I Pathol. 55. 283-290. YAMAMOTO, N., OKATA, M., KOYANAGI, Y., KANNAGI, M., and HINUMA, Y. (1982) Transformation of human leukocytes by cocultivation with an adult T-cell leukemia virus producer cell line. Science 217,737739. YOSHIDA, M., MIYOSHI, I., and HINUMA, Y. (1982) Isolation and characterization of retroviruses from cell lines of human adult T-cell leukemia and its implication in the disease. Prcx. Nat. Acad Sci. USA 79, 2031-2035.