Role of marrow stromal cells in the establishment of a transformed lymphoblastic B-cell line from a normal human subject

Leukemia Research Vol. 10, No. 10, pp. 1209-1219. 1986. Printed in Great Britain.

0145-2126/86 $3.00 + .00 © 1986 Pergamon Journals Ltd.

ROLE OF M A R R O W STROMAL CELLS IN THE ESTABLISHMENT OF A TRANSFORMED LYMPHOBLASTIC B-CELL LINE FROM A NORMAL H U M A N SUBJECT HARINDER S. JUNEJA,* SIRINIVASANRAJARAMAN,t KEITH M. RAMSEY* and FREDERICK F. B. ELDER~: The Mary Jeane Kempner Thorne Pryce-Jones Memorial Laboratory for Hematologic Research, Department of Internal Medicine*, and Departments of Pathologyt and Pediatrics:~, University of Texas Medical Branch, Galveston, TX 77550, U.S.A.

(Received 14 June 1985. Revision accepted 15 April 1986) Abstract--A monoclonal human B-lymphoblastoid cell line (UTMB-460) arose spontaneously from the bone marrow of a normal healthy woman who was seropositive for an EB-virus infection. Chromosomally, the UTMB-460 cells are near tetraploid, with a specific translocation (8;9) (pll.2; p24), and have surface IgMk. The UTMB-460 cells are resistant to killing in vitro by spontaneous and rlFNo~2 and rlL-2 stimulated NK cells from the patient and other normal subjects, but are killed by lymphokine activated killer cells. The index patient has not developed leukemia/lymphoma during the follow-up interval of 22 months. The growth of UTMB-460 cells is supported by undefined growth factors in FCS and by BCGF in the absence of FCS. rlL-2 stimulates DNA synthesis by UTMB-460 cells. The UTMB-460 cells were adherent to the normal MSC in the primary culture and show specific heterotypic adherence to normal MSC when compared to skin fibroblasts. In addition, 6/6 normal marrow stromal cells and 4/6 normal skin fibroblasts induced growth of colonies from UTMB-460 cells. These data suggest that MSC interacted with the transformed cells (UTMB-460) in vitro and played a critical role in the establishment of the UTMB-460 cell line.

Key words: Marrow stromal cells, human, lymphoblastic leukemia, B lymphocytes, malignancy, transformation, Epstein-Barr virus, immune-surveillance against malignant cells.

INTRODUCTION THE IMPORTANCE of the marrow microenvironment in normal hematopoiesis has become apparent over the last two decades. Genetically anemic mice (S1/SId) fail to benefit from infusion of suspensions of tissue cells from histocompatible, normal fetal liver or adult

Abbreviations: ADCC antibody dependent cellular cytotoxicity; EA, early antigen, diffuse (D) and restricted (R); EBV, Epstein-Barr virus; EBNA, Epstein-Barr nuclear antigen; ELISA, enzyme-linked immunosorbentassays; FCS, fetal calf serum; rlFNot2, recombinant interferon-alpha-2; rlL-2, recombinant interleukin-2; IMDM, Iscove's modified Dulbecco's medium; LAK, lymphokine activated killing; MEM, Eagle's minimum essential medium; MSC, marrow stromal cells; MSCcm, marrow stromal cell condition medium; NK, natural killer; PBM, peripheral blood mononuc|ear cells; UTMB-460, lymphoblastic B cell line; UTMB-460c,~, UTMB460 cell conditioned medium; VCA, viral capsid antigen. Correspondence to: Dr Harinder S. Juneja, Division of Hematology-Oncology, Rm 4.164, R-E65, The University of Texas Medical Branch, Galveston, TX 77550, U.S.A.

marrow, or a suspension of intact spleen cells, even though the stem cells in the S1/S1d mice are functionally normal when infused into anemic W / W v mice, a strain with defective stem cells and a normal marrow microenvironment [1]. Several reports suggest that murine marrow stromal cells (MSC) cultured in vitro for 3--4 weeks and the 'adherent' layer of the murine tong-term marrow cultures (LTMC), support proliferation and differentiation of a variety of hematopoietic progenitors [2-4]. In addition, human marrow stromal cells also support granulopoiesis in vitro but have either a stimulatory or inhibitory effect on erythropoiesis [5-8]. We have developed a transformed lymphoid B-cell line (UTMB-460) from a normal healthy young female donor. We report its genetic, phenotypic, and morphologic characteristics, as well as its susceptibility for lysis by cytotoxic peripheral blood lymphocytes, and the results of an extensive virologic and immunologic evaluation of the healthy donor. The data presented indicate that human MSC supported the growth and proliferation of UTMB-460 in vitro and may have played an important role in the establishment of the cell line in vitro.

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H . S . JUNEJA et al.

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MATER I ALS AND M E T H O D S Development and maintenance of the transformed B-cell line (UTMB-460) A sternal bone marrow aspirate was drawn from a 33-yr-old normal healthy white female undergoing tubal ligation after informed consent was obtained in writing. The aspirate was obtained as a source of normal MSC. Cultures for growing MSC were initiated in triplicate with 5 × 106 marrow buffy coat cells/25 cm 2 tissue-culture flask in 5 ml of Eagle's minimum essential medium (MEM), supplemented with 15% fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT) [8]. Three weeks later a confluent monolayer of adherent MSC was growing in each of the three flasks along with mononuclear hematopoietic cells. The hematopoietic cells were sparse and adherent to the MSC. After the first passage, the round hematopoietic cells continued to adhere to the MSC and grew in suspension culture in the same flasks. Eight weeks after the primary culture, the hematopoietic cells growing in the suspension cultures were present in numbers adequate for characterization and have since been passaged 1:4 in MEM with 15% FCS, twice weekly in vitro, in the absence of MSC. Clinical follow-up of the index case The index case has been followed closely in the clinic. Bilateral iliac bone-marrow biopsies and aspirates were done at 3 months, and the marrow was examined for presence of tetraploid cells by a flow cytometric technique (see methods below) and cytogenetic analysis. Peripheral blood was obtained at 3 and 15 months, and the following tests were done: routine blood chemistries; serum protein electrophoresis; quantitative immunoglobulin; cytogenetic analysis; ploidy-by flow cytometry; percentage of T and B cells; T-cell subsets; and lymphocyte function tests. Viral serology Serum antibodies to Epstein-Barr (EB) viral capsid antigen (VCA) were measured by indirect immunofluorescence [9] and to EBNA and components of early antigen, diffuse and restricted [EA(D) and EA(R)], by ELISA techniques [10]. The presence of viral antigens on UTMB-460, and on the patients' peripheral blood cells, was also tested by indirect immunfluorescence by the same techniques [9]. Cytotoxicity assays Natural killer cell (NK) activities and augmentation by recombinant interferon alpha-2 (rIFNa¢2) (Schering) and purified interleukin-2 (IL-2), of both the patient's and normal controls' peripheral blood mononuclear cells (PBM), were assessed against mycoplasma-free 51Cr-labelled K562 cells and UTMB-460 cells [11]. ADCC against UTMB-460 was assessed in parallel, after preincubation of 5aCr-labelled UTMB-460, with a 1:200 dilution of the patient's heat-inactivated serum (collected at the 15-month visit). Lymphokine-activated killing (LAK) was measured against the UTMB-460 cells after a 72h incubation of PBM with 25 U/ml of purified IL-2 [12]. All cytotoxicity data were converted to lytic units (LU/107) at 20% cytotoxicity by linear regression analyses [13], and significance (p < 0.05) was determined by Student's t-test. Characterization of UTMB-460 Morphology. The cells were examined after staining with

the standard battery of cytochemical stains used to classify acute leukemias [14]. They were also fixed, sectioned, and examined under a transmission electron microscope by previously described methodology [15]. lmmunocytochemical studies. Immunocytochemical studies were performed on air-dried and acetone-fixed cytospin preparations of UTMB-460 cells by the Avidin-Biotin-Peroxidase-Complex method of Hsu et al. [16]. Each monoclonal antibody was used at the concentration that gave optimal labelling and minimal background staining (1-10 ~tg/ml). The following antibodies were used OKT3, OKTs, OKTH, OKIa (Ortho Diagnostic Systems Inc., Raritan, NJ), Leu 3a + 3b, Leu M1, Leu 1 lb, monoclonal antihuman IgD, IgM, kappa and lambda and anti IL-2 receptor antibody (Beckton Dickinson Monoclonal Center, Inc., Mountainview, CA). In addition, UTMB-460 cells cultured for 72 h in the presence of 10 NIH units of rlL-2 were examined for the presence of IL-2 receptors using the anti IL-2 receptor antibody. Rosetting with sheep and ox red blood cells. The percentage of UTMB-460 that formed rosettes with sheep and ox redblood-cells was assessed on two occasions by standard techniques. Chromosomal analysis and DNA profile. Cells were harvested for chromosome analysis by conventional methods following a 1-h treatment with Colcemid (Gibco). Trypsin Gbanding was done by the method of Seabright et al. [17]. Cytogenetic studies were performed and the DNA profile was developed on UTMB-460 cells in continuous culture at 8 weeks, 8 months, and 18 months after the initial primary MSC cultures were established. DNA distribution histograms obtained after the cells were stained with propidium iodide were used to monitor the DNA content [18]. Effect of recombinant IL-2 and B-cell growth factor, rlL-2 was obtained from Biogen (Cambridge, MA) lot #NP5004N09Y. B-cell growth factor (BCGF) was supplied by Cytokine Tech International (Buffalo, NY), lot #15030. The BCGF was free of IL-1 and IL-2 activity in standard biological assays performed by Cytokine Tech International. UTMB-460 cells were cultured in Isocove's modified Dulbecco's medium (IMDM) or IMDM in the presence or absence of 1% or 10% fetal calf serum in 96 well plates (Flow Laboratories, Inc., McLean, VA). Influence of rlL-2 (1 NIH unit and 10 NIH units) on DNA synthesis were determined under optimal (10% FCS) and suboptimal conditions (0% FCS). DNA synthesis was measured during the last 6 h of a 48 h culture pulsing the cells with 0.5 ~tCi of (3H) thymidine (specific activity 80 Ci/ mM, ICN Pharmaceuticals, Irvine, CA). Incorporation of 3H thymidine was measured by standard liquid scintillation counting techniques after harvesting by a cell harvester. In concurrent experiments effect of 1 and 10 NIH units of rlL-2 on DNA synthesis of a well defined IL-2 dependent murine natural killer cell line [19] was also determined. The results are expressed as counts/min under test and control culture conditions. Results were analysed using analysis of variance procedures from the statistical package, SPSS [20]. Effect of BCGF on the proliferation of UTMB-460 cells was determined by generating growth curves under suboptimal conditions of growth (0 and 1% FCS) in the presence or absence of 10% BCGF. Cultures were initiated with 5000 ceils per well in 500 ~tl of culture medium in 24-well coStar plates (Falcon, Oxnard, CA). BCGF (10%) was added to half the cultures. The cultures were fed by removing half the media

Marrow stromal cells induce growth of a human lymphoblastic B-cell line and cells every 2-4 days over a period of 15 days. Cell counts were done at times indicated in Fig. 2. Cumulative cell numbers per well were calculated using the formulae: x 2n where, x is the number of cells per well and n = number of times the cultures had been subdivided 1:2.

Interactions of UTMB-460 with MSC and skin fibroblasts MSC and skin fibroblast (SF) cultures. Bone marrow was obtained (following informed consent) from six healthy women at the time they were undergoing tubal ligation. The MSC were cultured as described above and were used either directly from the primary cultures or from any culture between the second and the tenth passage. Skin fibroblasts (SF) were cultured under similar conditions from 3-mm skin biopsy specimens taken from the forearm of 7 different healthy donors (4 men and 3 women) and were used in experiments done from the 6tb to 18th passage. Co-cultivation studies. Using a bilayer system, UTMB-460 cells were co-cultured with MSC or SF in 24 well tissue culture plates [21]. Two to 5 × 103 UTMB-460 cells were co-cultured with one of several concentrations of MSC or SF (1 x 104 to 20 X 1 0 4 per ml). Simultaneous controls were set up. Colony counts were done on day 14 by means of an inverted microscope (40x). Using the semisolid culture technique the ability of MSC conditioned medium and medium conditioned by UTMB460 cells for 72 h to support clonal growth of UTMB-460 was tested at several concentrations, i.e. 1-10%.

Heterotypic adherence to normal MSC and normal SF. Replicate cultures of MSC and SF were prepared in 35-ram tissue culture dishes. The radiolabelled (51Cr) UTMB-460 cells were added in varying numbers onto confluent monolayers of two normal MSC or one SF. After 2 h the floating cells were removed and the dishes were gently rinsed 3-4 times with MEM to remove all nonadherent cells. The adherent cells were lysed with 1 ml of 1N sodium hydroxide and radioactive counts were determined in a gamma scintillation spectrometer (Packard Instrument Co, Downers Grove, IL). The radioactive counts in the adherent layers was taken as a semiquantitative measure of heterotypic adherence of 5~Cr labelled UTMB-460 cells to MSC or SF. Adherence to four other MSC and three other SF were determined semiquantitatively on a single concentration of (2-3 x 105) radiolabelled UTMB-460 cells.

RESULTS

Characteristics o f U T M B - 4 6 0 cell line T h e cells w e r e 15-20 ~xm in size, with a nuclear cytoplasmic ratio of 3:5 to 4:5. T h e nuclei w e r e i n d e n t e d or c o n v o l u t e d and c o n t a i n e d 0-2 nucleoli. The cells were positive for periodic-acid Schiff's reaction and acid phosp h a t a s e and negative on assay with S u d a n black, myelop e r o x i d a s e , and nonspecific and specific esterases. O n e l e c t r o n microscopy, the cells w e r e r o u n d or ovoid, and

18000-

UTMB 460

NK CELLS

12000-

T T

IL-2 UNITS ADDED P%IOU F--IIU I~= IOU

n tj ,

I I i I

6000-

I

0

i 10

FCS

(7o)

1211

i 0

10

FCS (%)

FIG. 1. Effect of rIL-2 on DNA synthesis by UTMB-460 and by an IL-2 dependent cell line (NK cell), is shown. Each bar represents mean CPM (-4- 1 S.D.) of the experiment done in triplicate. NK cells were rlL-2 dependent at both 0% FCS (p < 0.01, F = 33) and 10% FCS (p <0.01, F = 190). DNA synthesis in UTMB-460 cells was significantly increased by IL-2 under suboptimal conditions (0% FCS) ( p < 0 . 0 1 ; F = 10.12). Under optimal culture conditions (10% FCS), IL2 did not enhance DNA synthesis of UTMB-460 cells.

1212

H.S. JUNEJAet al.

1#'

/

lo', lo ~,

~

D N A synthesis in NK cells was clearly IL-2 dependent. In 4 separate experiments rIL-2 stimulated the D N A synthesis by UTMB-460 ceils 6-7 fold in the absence of fetal calf serum. In the presence of optimal concentrations of fetal calf serum (10%), however, rlL2 did not enhance D N A synthesis by UTMB-460 cells (Fig. 1). B C G F significantly enhanced the growth of UTMB-460 cells in cultures containing 1% fetal calf serum~ More importantly, the growth of UTMB-460 cells was supported by B C G F under serum free conditions (Fig. 2). The cells have been maintained in continuous culture in I M D M supplemented only with B C G F (10%) for greater than one month.

10 s,

~, lo", 1031 ~t 102'

lO' o

lO°

)

5 10 15 DAYS

o

5 10 I'5 DAYS

FIG. 2. Growth curves were generated starting with 5000 cells/ well in the presence or absence of 1% FCS. BCFG (10%) was added to half the cultures. Cumulative cell numbers (mean -+ 1 S.D.) of UTMB-460 cells/well from two experiments are shown. FCS 1%, (ll) FCS 1% + 10% BCGF (El), FCS 0% (O), FCS 0% + 10% BCGF (C)). In both experiments the cumulative cell numbers in the presence of 1% FCS with 10% BCGF were significantly higher than in cultures supplemented with 1% FCS only (left panel, p < 0.01, F = 390; right panel, p < 0.01, F = 397). UTMB-460 cells proliferated actively in IMDM supplemented with 10% BCGF in the absence of FCS. had large oval convoluted nuclei with indentations. A nuclear cytoplasmic ratio of 1:1 was determined and two to three small nucleoli were observed in juxtaposition to the nuclear membrane. The cytoplasm exhibited several mitochondria, some polyribosomes and a few strands of rough endoplasmic reticulum. On immunocytochemical studies, the cells expressed monotypic surface IgMk. No T-cell or monocytic lineage-specific antigens were expressed. Ox red-cell receptors were expressed by 56 --- 2% of the cells, while less than 1% of the cells had sheep RBC receptors. The cells were near tetraploid (modal number 94; range 90--98), with a translocation (8;9) ( p l l . 2 ; p 2 4 ) occurring prior to tetraploidization. On flow cytometric studies, the cells had tetraploid D N A content at 8 weeks, as well as after 8 and 18 months in culture.

Profile o f the index case The index patient is free of leukemia/lymphoma 22 months after the initial bone marrow. No abnormal hematopoietic cells grew on repeat MSC cultures 3 months after the initial study. The D N A ploidy and cytogenetic studies on her bone marrow and blood collected at this time were normal. A complete physical examination, including a detailed head and neck exam, was negative, as were the chest X-rays, and abdominal ultrasound examination for spleen size. Serum chemistries, serum protein electrophoresis, and quantitative immunoglobulins were normal. The T:B ratio, and T4:Ts ratio was within normal limits; in addition, a normal number of monocytes and natural killer cells (approx. 10% labelled by Leu l l b ) were present. The peripheral blood B cells expressed SIgM and SIgD, with a r:;t ratio of 3:1. Serologic studies (Table 1) indicate that the patient had an active Epstein-Barr virus (EBV) infection at the time of the initial bone marrow aspiration and still had evidence of a persistent or resolving EBV infection 15 months later. Both the UTMB-460 cell line and the PBM cells from the patient contained no detectable E B N A , V C A or early-antigens, E A ( R ) and E A ( D ) . The NK-cell activity of the patient's PBM against K562 targets was found to be significantly depressed, in comparison with normal controls (Table 2). In addition, there was little augmentation of NK in vitro by rlFNo:2 or IL-2. In contrast, UTMB-460 cells were resistant both to NK cell activity among patients own PBM, or PBM of normal controls (n = 10), and to NK cell activity after augmentation with r-IFNo:2 and IL-2. After a 72h incubation of PBM, however, from the patient or

TABLE 1. E P S T E I N - B A R R VIRUS SEROLOGY OF THE INDEX PATIENT

Antibody detection assays* Indirect Immunofluorescence* Viral capsid antigen IgM IgG 9/83 (3 months)t 9/84 (15 months)t

<20 <20

640 640

EBNA

ELISA EA-D

EA-R

320 320

320 40

80 80

* Reciprocal of the highest dilution of serum eliciting a positive reaction. The antibody titers are considered positive at the following dilutions: Antibodies to the viral capsid antigen; IgM >/1:20; IgG/> 1:640. EBNA/> 1:20, early antigen (EA), restricted (R) and diffuse (D) I> 1:20. t The serum samples were obtained from the index case 3 and 15 months after the initial marrow from which UTMB-460 cell line was established.

FIG. 3. UTMB-460 cells are adherent to the cell surface of the two marrow stromal cells (MSC) and their cytoplasmic extensions. The UTMB-460 cells do not attach to the intervening plastic surface. (Final magnification, x200.)

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Marrow stromal cells induce growth of a human lymphoblastic B-cell line

1215

TABLE 2. NATURALKILLER CELL ACTIVITYAGAINSTK562 AND UTMB-460 TARGETS Cytotoxicity LU/107. Medium

K562 Targets +rIFNol

+rIL-2

Control

82

110

130

Patient

(p < 0.05) 15

(p < 0.05) 21

(p < 0.05) 35

Med

UTMB-460 Targets +rIFNo~ +rIL-2

Controlt (n = 10)

<1

<1

<1

Patient

<1

<1

<1

* Mean lytic units per 107 cells at 20% cytotoxicity. t Control PBM were obtained from healthy subjects donating blood to the UTMB blood bank. f r o m n o r m a l c o n t r o l s with IL-2, l y m p h o k i n e - a c t i v a t e d killing ( L A K ) against t h e 460-cell line could b e d e m o n s t r a t e d ( T a b l e 3).

Interactions between UTMB-460 and MSC (or SF) Selective heterotypic adherence to MSC. T h e h e t e r o typic a d h e r e n c e to t h e M S C b e g a n within 10 min, with m a x i m u m a d h e r e n c e at a b o u t 1 h. T h e U T M B - 4 6 0 cells a d h e r e d to t h e surface of t h e a d h e r e n t M S C (Fig. 3). T h e r a d i o a c t i v e c o u n t s in t h e a d h e r e n t ceils w e r e prop o r t i o n a l to the n u m b e r of U T M B - 4 6 0 cells a d d e d . T h e s e m i q u a n t i t a t i v e m e a s u r e m e n t of t h e h e t e r o t y p i c a d h e r e n c e of U T M B - 4 6 0 cells to M S C a n d SF r e v e a l e d

t h a t a d h e r e n c e of U T M B - 4 6 0 to M S C is 1.5-4.0 times g r e a t e r t h a n to SF (Fig. 4). Co-culture studies. No s p o n t a n e o u s U T M B - 4 6 0 colony f o r m a t i o n was o b s e r v e d in 10 of t h e 12 (six e a c h with M S C a n d S F ) co-culture e x p e r i m e n t s . In o n e e x p e r i m e n t with M S C a n d in 2 e x p e r i m e n t s with SF, colonies of U T M B - 4 6 0 cells f o r m e d s p o n t a n e o u s l y . All six M S C t h a t were t e s t e d i n d u c e d g r o w t h of colonies f r o m U T M B - 4 6 0 cells at day 14. N o t a b l y , the M S C o b t a i n e d f r o m t h e i n d e x p a t i e n t , 3 m o n t h s after the initial m a r r o w was d r a w n , i n d u c e d colony f o r m a t i o n b y U T M B - 4 6 0 cells by day 7. F o u r of t h e 6 n o r m a l SF t h a t were t e s t e d s u p p o r t e d clonal g r o w t h of U T M B - 4 6 0

TABLE 3. NATURAL KILLER AND LYMPHOKINE ACTIVATED KILLING OF UTMB-460 CELL LINE Cytotoxicity LU/10 7. NK

Control Patient

LAK

Med

+rlFNtr

+rlL-2

<1 <1

<1 <1

<1 <1

Controlt Patientt

Med

+rlFNtr

+rlL-2

10 8

10 10

10 10

* Lytic unit per 107 cells at 20% cytotoxicity. t Killing of UTMB-460 is significantly higher (p < 0.05) by LAK over NK.

TABLE 4. INDUCTION OF CLONAL GROWTH OF U T M B - 4 6 0 CELLS BY NORMAL M S C

Pearson correlationt coefficient Experiment number l*(a) (b) 2 3 4 5 6

0 0 76 ± 0 0 16± 6~

10 4 1 1 1

13 - 2 395 - 39 0 4± 1 5± 1 0 19 -+ 10

Number of MSC (x 10 3) 50 100 77 ± 10 262 - 20 7± 3 36 ± 5 26 ± 2 5- 1 88 -+ 15

150 -+ 4 TNTC 64 --- 11 93 -+ 8 -20 ± 3 41 --- 6

200

r

p

110 -+ 4 TNTC 76 -+ 5 230 ± 2[) 97 --- 16 273 ± 1I 0

0.7561 0.8564 0.9186 0.9304 0.8635 0.9187 0.3858

0.001 0.001 0.001 0.001 0.001 0.001 0.121

The numbers in the table represent the mean (-+ 1 S.D.) of UTMB-460 cell colonies induced by normal MSC. In experiments 1-3, 5000 UTMB-460 cells were co-cultured with MSC. In experiments 4--6, 2000 UTMB-460 ceils were co-cultured with the MSC. In the absence of MSC, spontaneous colony formation at day 14 was 0 to 6 +-- 1 in 5 experiments, i.e. expts 2-6, and 76 -+ 4 in expt. 1. * MSC in expt 1 was obtained from the index case 3 months after the initial sample. (a) 460-cell colonies at day 7; (b) 460-cell colonies at day 14. For expts 2-6 the numbers represent colonies at day 14. No colonies were noted on day 7 in expts 2-6. The MSC were used at 10th passage in expt 2. TNTC = >2000 colonies. t Pearson correlation coefficient between the number of UTMB-460 colonies and the number of MSC in each experiment.

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H . S . JUNEJAet al. cells. A linear correlation was observed between the n u m b e r (104 to 2 × 105) of M S C (or SF) in the underlayer and the U T M B - 4 6 0 colonies formed (Tables 4 and 5). W h e n a d o s e - r e s p o n s e curve was obtained with different n u m b e r of U T M B - 4 6 0 cells, with a fixed n u m b e r of M S C or SF (1 x 105/ml) (2 experiments with MSC; and 1 experiment with SF), a linear correlation was also observed (r = 0.5191;p = 0.001). Such a linear correlation suggests that the colonies of the U T M B - 4 6 0 cells in the co-cultures originated from single cells (that is, they were clonal in origin). R e p r e s e n t a t i v e U T M B 460 colonies were r e m o v e d from the cultures and shown to have the same cytochemical and phenotypic characteristics as the U T M B - 4 6 0 cells in suspension culture. In separate experiments 41 U T M B - 4 6 0 colonies were plucked with a micropipette and transferred to a micro well (1 colony/well) containing I M D M with 10% FCS. A f t e r 7 days 37 of 41 colonies were noted to be actively proliferating. This indicates that the colonies originated from U T M B - 4 6 0 cells were capable of extensive clonal expansion. N e i t h e r MSCcm nor UTMB-460cm supported growth of U T M B - 4 6 0 in semisolid medium.

"I"

W

n a

10~00

DISCUSSION s~

+0

15

20

25

UTMB

460

30

3s

40

1xi041

FIG. 4. Semiquantitative determination of heterotypic adherence of UTMB-460 cell to MSC and SF: The radioactivity (DPM/dish) is proportional to the number of 51Cr labelled UTMB-460 cells added to each dish. Each data point is a mean +- 1 S.D. of studies done in triplicate. Note that the UTMB-460 show greater adherence to MSC ( • and ~-) than to the SF (D). Control (lI). Pearson coefficient correlation for relationship between the number of radiolabelled UTMB460 ceils added to the dishes, and the radioactivity in the adherent cells was as follows: for the two MSC ( ( • ) r = 0.9807, p <0.001; (~r) r = 0.9957, p <0.001) and for the SF (0) r = 0.9739, p < 0.001; control (11) r = 0.8663, p < 0.001,

AND

CONCLUSIONS

The ability of the U T M B - 4 6 0 cells to form colonies in semisolid media, the expression of monotypic surface IgMk, and the presence of a specific chromosomal translocation (8;9) p11.2; p24) suggests that U T M B 460 is a transformed lymphoblastic B-cell line. The transformed lymphoblastic B-cell line (UTMB-460) was grown from b o n e m a r r o w stromal cell cultures from a healthy w o m a n with serologic evidence of an asymptomatic E B V infection. Subsequent serologic analyses were consistent with the presence of an asymptomatic persistent or resolving E B V infection [22-24]. A l t h o u g h our assays r e v e a l e d no V C A , E B N A or EA-antigens on the surface of the U T M B - 4 6 0 cells, these tests do not exclude the presence of E B V - g e n o m e incorporated within cellular D N A .

T A B L E 5. INDUCTION OF CLONAL GROWTH OF

UTMB-460 BY NORMAL SKIN FIBROBLASTS

Number of SF* (xl03) Experiment number 1

2 3 4 5 (a) (b) 6

0

10

50

100

200

6---1

0

0

0

0

0 1"--1 6 +- 1 0 439 "-. 28 439 - 28

0 0 58 - 9 3 +- 1 801 "-. 73 887 --. 76

0 2---1 139 +- 9 4--- 1 790 --- 53 1187 --- 56

0 27"--5 280 "-. 16 7--- 1 1092 --. 96 1187 +- 56

0 93"--2 360 +- 40 21 "--2 1242 --- 63 1260 - 101

* Only SF used in expt 5 induced colonies on day 7 and 14. In expts 1--4 and 6 colony formation was noted only on day 14. A significant number of UTMB-460 colonies were formed spontaneously in expts 5 and 6. The SF in expts 5 and 6 increased the UTMB 460 colony formation about 3 fold over the spontaneous colony formation. In expts 1-4 spontaneous colony formation was poor. Two SF did not induce any U T M B - 4 6 0 colony formation even at day 14. One SF induced UTMB-460 colony formation at both days 7 and 14 (expt 5). Pearson correlation coefficient between the number of UTMB-460 colonies formed and the number of SF co-cultured with UTMB-460 cells was uncomputable for expts 1 and 2. The correlation coefficient for expts 3-6 was r = 0.8663, p = 0.001.

Marrow stromal cells induce growth of a human lymphoblastic B-cell line A large proportion of PBM are infected with the EBV in infectious mononucleosis, and establishment of lymphoid B-cell lines from the PBM of patients following an acute E B V infection is easily reproducible [25, 26]. This generally requires the depletion of T lymphocytes that suppress the spontaneous outgrowth of EBV transformed B cells [27], however, or the use of cyclosporin A in the culture systems [28, 29]. Thus, the development of UTMB-460 cell line from the unmanipulated bone marrow of a EBV-seropositive (IgG) normal healthy woman is an intriguing observation. Cross-contamination of the cultures in the laboratory by malignant cells can be ruled out, as no other Bcell lines were being cultured in the laboratory. The presence of four X-chromosomes in the near tetraploid karyotype of UTMB-460 cells confirms that the cells originated from a female. It is highly unlikely that B lymphoid cells in three different flasks underwent a simultaneous and identical in-vitro transformation. We therefore contend that the UTMB-460 cells grown in vitro were present in the bone marrow aspirated from the index patient. UTMB-460 cell line differs from other reports of EB virus-transformed cell lines, in that UTMB-460 is a transformed lymphoid B-cell line of clonal origin, as indicated by cytogenic studies and by the presence of a single light chain on the surface of the UTMB-460 cells. Most in-vitro EB virus transformed cell lines, and even lymphomas related to EBV infections are polyclonal in origin [24, 30, 31]. The in-vitro growth of UTMB-460 from the bone marrow of our patient is analogous to the in-vitro growth of malignant cells from bone marrow in patients with Burkitt's and undifferentiated lymphomas and small cell lung cancer, whose malignancies by all accepted criteria were in complete remission [32-34]. Our index case, however, did not have leukemia/lymphoma at the time of the initial marrow sampling. The patient may develop a leukemia or a lymphoma in the future, but 22 months after the UTMB-460 cells were grown from her marrow she is still free of a lymphoid malignancy. Several explanations are possible: first, these cells were unstable and failed to gain a proliferative advantage in vivo, or second the patient's immune surveillance efficiently contained or successfully eradicated the transformed cells in vivo [35, 36]. The UTMB-460 cells are resistant to lysis by unstimulated circulating NK cells, as well as IFNo~2 and rIL-2-stimulated NK cells, but are killed in vitro by lymphokine-activated killer cells. Also, the patient's PBM exhibited poor A D C C against UTMB460 cells. Cytotoxic activity of the macrophage system toward UTMB-460 cells was not tested and may have played a role in controlling the in-vivo growth of these malignant cells [37]. Our data demonstrating L A K but not NK or A D C C against UTMB-460 may, however, suggest an in-vivo role for L A K as proposed by Grimm et al. [12]. The limited in-vitro test performed do not elucidate the exact immune-surveillance mechanisms that have contained the growth of the transformed cells in vivo; nonetheless, these mechanisms have been successful in containing or eradicating the transformed cells in vivo. Patients undergoing a bone marrow transplantation may develop a monoclonal or a polyclonal

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lymphoma as early as two months or as late as two years after the bone marrow transplantation [30]. In immunesuppressed marrow recipients who develop the lymphoma within two months, it is possible that malignant lymphoid-B cells in a normal subject donating bone marrow could be engrafted to a marrow recipient. In fact, a recent abstract [38] reports the transfer of a transformed lymphoid-B cells from a healthy donor to a marrow recipient. Interestingly, as with our index case, both the marrow transplant recipient and the donor were able to eradicate the malignant cells from their bone marrow over a period of five months. These observations provide support for the existence of an active and effective immune surveillance against transformed cells in vivo. The growth of UTMB-460 cells in vitro is supported by undefined growth factors in FCS and by B C G F in the absence of fetal calf serum, rIL-2 stimulates D N A synthesis under suboptimal conditions of growth. The effect of rIL-2 is not receptor mediated as the cells lack the rIL-2 receptor. The effects of rIL-2 and B C G F on UTMB-460 cells are reminiscent of effects of these defined growth factors on human B lymphocytes activated in vitro or in vivo [39, 40]. The cell line will be useful to define the cellular mechanism of actions of IL2 and B C G F on human B lymphocytes. The observation of the selective heterotypic adherence of the UTMB-460 cells to the MSC obtained from the index case and five other normal female subjects, along with induction of clonal growth of UTMB-460 cells by the MSC in semisolid medium, suggests that the MSC provided an essential in vitro microenvironment for the development of this transformed cell line. Several reports in the murine system support this concept. It has been observed that the marrow adherent cells played a critical role in establishment of a cell line from rat leukemic cells [41]. In the first two weeks of culture, there was massive leukemic cell death. A growing population associated with an adherent cell layer, however, constantly survived and proliferated. It has also been demonstrated that marrow stem cells (CFU-S, CFU-C) and mouse lymphoid leukemic cell lines can be maintained better in vitro in the presence of adherent cells from the bone marrow than adherent cells of other organs [42,43]. Both B and T lymphocytes are hematopoietic cells and it is, therefore, not surprising that MSC would support invitro growth of cells of malignant human T lymphoblastic cell-line (CEM), as well as cells obtained from patients with leukemia and lymphoma [21, 44]. Our data would suggest that the use of marrow, instead of blood, as a source of leukemic cells, would increase the efficiency of establishment of human leukemic cell lines in vitro as the MSC growing in the culture would provide an essential in-vitro microenvironment for support of the leukemic stem cells. The presence of primitive stem cells in the adherent layer of both murine and human LTMC, and the necessity of the adherent layer to the viability of the LTMC [4, 5, 45], suggests that the interaction between the 'adherent' cells and the primitive hematopoietic stem cell is critical to normal hematopoiesis in vitro. There is a striking

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H.S. JUNEJAet al.

similarity between the 'cobble-stone' areas seen in the human and murine LTMC [4, 45] and the heterotypic adherence of UTMB-460 cells to MSC. We speculate that the human MSC may play an essential role in fostering and harboring normal hematopoietic stem cells during the process of leukemogenesis in vivo, much like the murine marrow stroma does during the development of leukemia in murine long-term marrow cultures infected with a helper-independent Friend leukemia virus [46]. In addition, the adherence of leukemic stem cells to the MSC may be the mechanism by which leukemic cells 'home' to the bone marrow and the MSC may induce proliferation of the leukemic stem cells in vivo. We are currently characterizing the exact nature of the human MSC involved in the interactions with the malignant human T- and B-lymphoblastic ceils.

Acknowledgements--Expert technical assistance was provided by Mr Sang Lee, Tom Peskuric and Mrs Maitryee Sahu. Dr J. A. Hokanson of the UTMB Cancer Center performed the statistical analyses. Dr T. Yamaguchi's advice on experiments with IL-2 is highly appreciated. Ms Mary McChesney typed the manuscript. This project was supported by Clinical Research Center, RR-73, Division of Research Resources, National Institutes of Health.

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