Phenotype characteristics of human B cells studied by Epstein-Barr virus infection I. Immunoglobulin expression on human lymphoblastoid cells established from various lymphoid cells

hnmunoloKv Letters, 8 (1984) 335 341

Elsevier Imlet 528

PHENOTYPE CHARACTERISTICS OF H U M A N B CELLS STUDIED BY EPSTEIN-BARR VIRUS INFECTION I. IMMUNOGLOBULIN EXPRESSION ON H U M A N LYMPHOBLASTOID CELLS ESTABLISHED F R O M VARIOUS LYMPHOID CELLS Toshio K U D O j, 2 and Takehiko T A C H 1 B A N A 2 tSerological Section, School of Medical Sciences, and 2Department of Immunology, Research Institute.for Tuberculosis and Cancer, Tohoku University, Sendai 980, Japan

(Received 21 September 1984) (Accepted 24 September 1984)

1. Summary

2. Introduction

Forty-seven lymphoblastoid cell lines were established from human fetal lymphoid tissues, cord blood lymphocytes (CBL) and adult peripheral blood lymphocytes (PBL) by Epstein-Barr virus (EBV) infection. Their surface immunoglobulin (slg), intracytoplasmic immunoglobulin (clg) expression, and immunoglobulin (Ig) content in the culture supernatant were tested. Expression of slgM, slgG and slgA were predominant on fetus-derived cell lines, while slgD was the most prominent on CBLderived cells. Though clg expression did not vary between cell lines of different origin, lg content in the culture supernatant differed greatly. Fetus- and CBL-derived cells secreted IgM exclusively, but PBLderived cells secreted not only IgM, but also IgG and IgA abundantly. These results indicate that the lymphoblastoid cells established by EBV infection reflect the Ig phenotype of the cell from which they originated.

Epstein-Barr virus (EBV) transforms human B cells and establishes lymphoblastoid cell lines [1, 2]. Most EBV-infected B lines secrete Igs. Studies on the lg classes produced by the lymphoblastoid cell lines have revealed variations [3-5]. A possible explanation for these differences is that the cells of the infection event differ in terms of differentiation stage. If this is the case, the following criteria should be satisfied; the demonstration that (1) B lymphocytes with EBV receptor have distinctive phenotypes at very early stages of B cell differentiation, and (2) that certain markers are maintained on the lymphoblastoid cells after transformation with EBV. These viewpoints motivated our experiments in which B cells from three different stages of development were transformed with EBV. As the most immature B cell source, liver, spleen, bone marrow and appendix lymphoid cells from fetus were chosen. Though fetal liver of 16-wk gestation contained less than I% slg positive cells, after EBV infection the cell lines were established. The second source of B cells was cord blood lymphocytes. Adult peripheral blood lymphocytes (PBL) were used for EBV infection as mature B cells. The Ig expression of the established lymphoblastoid lines was found to be similar to those of the target infection.

Key words: lg expression - human lymphoblastoidcells - B cell differentiation EBV infection Address of correspondence: Toshio Kudo, School of Medical Sciences, Tohoku University, 2-! Seiryo-machi,Sendai 980, Japan

0163 24t~ / 84 / $3.00 © ElsevierScience Publishers B.V.

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3. Materials and Methods

3.1. Cell fines Transforming virus was obtained from the supernatants of an EBV-producing cell line (B95-8 cells) [6]. The supernatant was passed through a 0.22 ~m millipore filter and stored at -70°C in aliquots until use. TCDs0 of the supernatant was about 104 according to Moss and Pope [7]. Fetal liver, spleen, bone marrow and appendix were aseptically taken from a 16- to 24-wk-old fetus. The lymphoid tissues were minced by scissors and suspended in RPMI-1640 (GIBCO) with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 #g/ml). The cell suspension containing small solid tissues was transferred to Petri dishes (Falcon 3002). Blood mononuclear cells were isolated from heparinized cord blood and blood from healthy adults (22-35 yr old) by isopycnic centrifugation on Lymphoprep (Nyegaad, Oslow), according to Boyum [8]. The cells were suspended at 106 cells/ml in R P M I 1640 supplemented with 10% FBS, penicillin and streptomycin. The cell suspension (4.5 ml) was mixed with EBV (0.5 ml) in a Petri dish and cultured at 37°C in 5% CO 2 incubator. One-third or one-half of the medium was changed twice a week. Morphological transformation was observed within 10 days and lines were established usually within a month. The lines were maintained in RPMI-1640 enriched with 10% FBS using a plastic flask (Falcon 3014) and were tested for their Ig expression within 2 mth after establishment. EBV-infection was assessed by the presence of EBV-associated nuclear antigen (EBNA) in the cells, according to Reedman and Klein [9]. 3.2. Surface lg detection Direct membrane immunofluorescence was used for slg detection. The lymphoblastoid cells, after being washed three times with PBS containing NaN 3 (0.1%), were adjusted to 5 × 105 cells per test tube and packed by centrifugation. The packed cells were mixed with 50 #1 of FITC conjugated anti-human Ig heavy chain (#, % a, 6; Behringwerke, MarburgLahn) at a 1 : 5 dilution and followed by incubation at 37°C for 30 min, and washing in PBS containing 0.1% NaN 3 3 times [10]. The membrane immuno336

fluorescence on the cells was observed using the Chiyoda transmitted light fluorescence microscope (FM-200A). slgD (6)-positive cells could not be detected by the above microscope, but by the Nikon epi-illumination fluorescence microscope (XY-EF) using a B excitation system. The presence of exogenous cytophilic Ig was checked by treating cells with the buffer of pH 4.0 [11]. The percentage of slg-positive cells was obtained from the observation of more than 300 cells in each cell line. 3.3. lntracytoplasmic lg detection The cells were deposited on a glass slide (Matsunami, No. 1 for fluorescence microscopy), dried and fixed in acetone at room temperature for 10 min. Thereafter the slides were stained with FITC antihuman Ig (1 : 5 dilution) at 37 °C for 30 min in a humid chamber, followed by washing in PBS. The slides were examined under the fluorescence microscope (Chiyoda FM-200A) [12]. A minimum of 300 cells from each sample was counted.

3.4. Ig content in the culture supernatant Ig secreted into supernatant was measured by an enzyme-linked sandwich immunoassay [13, 14]. Every IgG fraction from goat antisera (anti-human #, 7, c~, 6; MBL Co., Nagoya, Japan) was prepared by ammonium sulfate precipitation method [15]. The goat lgG was coupled with glass beads (6.8-7.0 mm in diameter, Sanyo-kasei Chemical Industries, Kyoto, Japan) by the method of Robinson [16]. lgGcoupled glass beads were stored in PBS containing 1% bovine serum albumin (BSA) and 0.1% NaN 3 at 4°C until use. A single IgG-coupled glass bead was incubated with a series of appropriately diluted culture supernatant (0.5 ml) overnight at 4°C in the test tube (15 × 105 mm). Horseradish peroxidase conjugated goat anti-human Ig, of which conjugation was performed by Nakane's method [17] with a slight modification, was prepared in a volume of 0.5 ml of PBS with 1% BSA and was added to the glass bead, which had been prewashed in PBS. This was then incubated at 37 °C for 90 min with agitation. Each glass bead was washed in PBS extensively (5 times) and transferred to another test tube to avoid the enzyme activity being nonspecifically absorbed on the wall of the tube. The enzyme activity bound to glass

beads measured by the method of Joyce et al. [18] with a slight modification. Two ml of 0.04% o-phenylene diamine/0.03% H202 in 0.1 M citric acid/0.2 M NazHPO4 buffer (pH 4.8) were added to all test tubes which were then incubated in the dark for 30 min with agitation. Sulfuric acid (6 N, 0.5 ml) was added to stop the reaction and the optical density of the supernatant was measured at 492 nm on UVIDEC-60 (Nihon Bunko Medical, Tokyo, Japan). A standard curve was made by using Ig standards (MBL Co.) diluted to concentrations from 50 to 250 ng/ml in PBS.

Table 1 Surface immunoglobulin expression on the lymphoblastoid cells established from various cell sources Cell origin

Fetal lymphoid cellsa

Cord blood lymphocytes

Adult PBL

Ig class No. of cell lines

Range (%)

Mean_+SD (%)

lgM lgG lgA IgD

10 10 10 4

43.4 7.5 19.4 2.9 11.1 0.6 10.3-1.2

23.7±11.9 b 9.4_+ 5.3 c 5.6_+ 3.1 d 5.1_+ 3.9

IgM lgG lgA IgD

21 21 21 7

3.2 3.5 3.0 23.3

1.95:1.3 e 1.2± 1.0 0.6± 0.9 15.6± 4.8 f

lgM IgG lgA lgD

16 16 16 5

0.0 0.0 0.0 9.7

4.0-0.0 3.0 0.0 1.0 0.0 6.3 1.0

1.0± 0.5± 0.2± 2.4±

1.2 0.9 0.4 2.3

a Fetal lymphoid cells including 3 spleen, 3 liver, 3 bone marrow and 1 appendix. b P<0.01, compared with surface igM of cord blood derived, and adult PBL derived cells. c P<0.01, compared with surface IgG of cord blood derived, and adult PBL derived cells. d P<0.01, compared with surface lgA of cord blood derived, and adult PBL derived cells. e P < 0 . 0 5 , compared with surface lgM of adult PBL derived cells. f P<0.01, compared with surface lgD of fetus derived cells and adult PBL derived ceils. Surface IgM, lgG and IgA were tested by a transmitted light fluorescence microscope. Surface IgD was tested by an epi-illumination fluorescence microscope.

4. Results 4. I. slg expression In the light microscope the lymphoblastoid lines established from various sources had a similar morphology irrespective of origin. All lines were EBNA positive. The percentage of Ig positive cells differed depending upon their origin (Table 1). In fetus-derived cells, sIgM, sIgG and sIgA-positive cells were significantly higher than CBL- and PBL-derived cells (P<0.05). In CBL-derived cells, sIgD-positive cells were more numerous than those derived from fetus and PBL (P<0.05). The absorbing capacity for FITC-labeled antihuman IgM (#) serum of the different cells varied considerably (Fig. I). The lymphoblastoid cells ( 107) derived from fetal liver absorbed almost completely the anti-# activity, but PBL-derived cells did not. This result was also assessed by photographs of the stained cells. Fetus- and PBL-derived cells were stained by FITC-conjugated anti-# and photographed with Kodak Ektachrome 400 film under the same conditions. Clear membrane fluorescence could be seen on the fetus, but was very faint on the PBL-derived cells (Fig. 2). The sIg expression was not significantly influenced by acidic buffer (data not shown). These results ruled out the possibility of exogenous cytophil100

~

75

~

50

o

o-,

,]5

215

25

S'.O 7~ 16.0 Cell number xlO 6

Fig. I. Demonstration of surface lgM on lymphoblastoid cells by antibody absorption test. FITC labelled rabbit anti-human # (1 : 15 diluted, 0.1 ml) was absorbed with either adult PBL derived cells (FuPBL, O - - O ) , or fetal liver derived cells (FLV-6, o---o) at 4°C, 60 min. The absorbed antiserum (0.05 ml) was tested for the residual antibody activity on Daudi cells (5 × 105 cells).

337

Table 2 lntracytoplasmic immunoglobulin-positive cells Cell origin Fetal lymphoid cells

Cord blood lymphocytes

Adult PBL

Fig. 2. Fluorescence photographs showing the markedly different surface lgM expression on the fetal liver derived cells (left) and adult PBL derived lymphoblastoid cells (right). Fetal liver derived cells (FLV-6) were stained with FITC labelled anti-# (left) as in the text and adult PBL derived cells (FuPBL) stained similarly (right). Both cell lines were photographed under the same conditions using a Chiyoda fluorescence microscope (Ektachrome 400 film). ic antibodies attached to cell surface Fc receptors. W i t h a transmitted light fluorescence m i c r o s c o p e s l g D - p o s i t i v e cells could not be identified. But, with a m o r e sensitive epi-illumination fluorescence microscope a considerable n u m b e r of s l g D - p o s i t i v e cells were detected.

Ig class No. of Range cell lines (%)

Mean+SD (%)

lgM lgG IgA IgD

10 10 10 5

96.0 21.0 22.2 1.0

8.9 0.0 0.0 0.3

36.5+32.3 8.7_+ 8.0 11.2+ 6.6 0.5_+ 0.3

IgM IgG IgA lgD

7 7 7 8

84.0 15.6 10.0 2.5

0.4 0.0 0.0 1.0

25.7+34.6 8.5+ 5.3 4.4+ 4.1 1.5+ 0.5

IgM IgG lgA lgD

I1 I1 I1 5

20.5 I 1.0 15.0 0.6

0.0 0.0 0.0 0.0

9.5_+ 6.0 2.0_+ 3.5 4.2+ 5.3 0.2+_ 0.3

w h e n the cell n u m b e r was between 106/ml and 2 × [06/ml (Table 3). T h e I g M content in the supernatant of all lines was over 10 # g / m l . In contrast high a m o u n t of IgG was present only in P B L - d e r i v e d cell cultures. Less t h a n 100 n g / m l of IgG was detected in fetus- and C B L - d e r i v e d cultures. IgA showed similar results. P B L - d e r i v e d cells secreted a considerable a m o u n t of IgA, while fetus- and C B L - d e r i v e d cells only a small amount. M o r e than 190 n g / m l of I g D was detected in the supernatant of C B L - d e r i v e d cells, while a smaller a m o u n t (100-150 n g / m l ) and a m u c h less a m o u n t of I g D were measured in the supernatants of fetus- and P B L - d e r i v e d cells, respectively.

5. Discussion 4.2. clg expression C o m p a r i s o n of the cIg expression s h o w e d a wide range of distribution of the frequency of positive cells (Table 2). In a l m o s t every cell line, the m o s t p r o m i n e n t class was I g M . T h e differences did not relate to the origin of the lines. 4.3. Ig classes secreted in the culture supernatant T h e Ig content in the culture supernatant of the cell lines was the highest 6 days after subculture, 338

T h r e e groups of h u m a n l y m p h o i d cells at various differentiation stages were chosen for EBV infection. T h e l y m p h o b l a s t o i d cells established by EBV were tested for the expression of slg and cIg and for Ig secretion (Table 4). T h e expression of sIgM, sIgG and sIgA of the fetus-derived cells was strongest. T h e expression of s I g D gave a different pattern. It was strongest on the C B L - d e r i v e d cells. T h e m e a n percentage of s I g D

Table 3 Immunoglobulin content in the culture supernatant Cell origin Fetal lymphoid cells

Cord blood lymphocytes

Adult PBL

lg class

No. of cell lines

Range (ng/ml)

MeaniSD (ng/ml)

IgM IgG lgA lgD

5 5 5 5

37,000 12,000 50 0 6 0 150 100

22,050± 10,958 11+ 21 2_+ 3 115+ 22

IgM lgG lgA IgD

6 6 6 6

30,000 15,000 100 0 I1 0 1,200 190

22,450+ 5,128 22+ 39 2_+ 4 569_+ 366~

IgM IgG IgA lgD

6 6 6 6

135,000 13,000 25,000 9 , 0 0 0 3,100 200 50 5

52,420_+42,587 18,667±11,911 1,913+ 1,022 17+ 17

P<0.05, compared with lgD of fetal lymphoid cells, and P<0.01, compared with lgD of adult PBL-derived cells. was 1 5 . 6 + 4 . 8 % (SD). s l g D could be detected only with the highly sensitive epi-illumination system [19]. The strong s l g D expression o n the CBL-derived cells is in accordance with their origin. M o r e slgD Table 4 Immunoglobulin expression of the lymphoblastoid cells (summary) Cell origin

Fetal lymphoid cells

Cord blood lymphocytes

Adult PBL

Ig Cell class surface Ig

lntracytoplasmic Ig

Secreted Ig

+

++

lgM lgG lgA IgD

++ + + +

IgM lgG IgA lgD

+

+

++

± ± ++

+ + ±

+

±

+

+ + _+

+ +

lgM lgG lgA lgD

+ +

++ ++ +

+4 : Strongly positive, +: definitely positive, ±: weakly positive, : negligible.

positive cells have been observed in CBLs t h a n in adult PBEs [20]. T h u s this characteristic is preserved o n the transformed cells. Fetal l y m p h o i d cells a n d CBLs are not primed with external antigens. The fetal liver a n d spleen cells are at the stage of rapid hematopoiesis a n d contained the most i m m a t u r e B cells. Adult PBLs contain m e m o r y a n d m a t u r e cells. Even t h o u g h the m o r p h o l o g y of the l y m p h o b l a s t o i d cells from different sources was similar [21], it is quite feasible that both fetus- a n d CBL-derived cells secrete only IgM. O n the other hand, PBL-derived cells secrete IgM, IgG a n d IgA. In the course of B cell differentiation, r e a r r a n g e m e n t of lg genes occurs. Ig expression changes from cell m e m b r a n e b o u n d form to secretory form a n d the classes switch from l g M to I~G or IgA. T a k i n g these facts into account, we interpret our results as follows (Fig. 3): cells transformed by EBV make a "short range shift" to the direction of terminal cells (plasma cell). T h o u g h pre-B type cell lines were established from b o n e m a r r o w cells of X-linked h y p o g a m m a g l o b u l i n e m i a patients [22], there is no direct evidence that EBV infects pre-B cells [23]. The authors tentatively n a m e d the target cells in fetal l y m p h o i d p o p u l a t i o n y o u n g B. F o r convenience, we n a m e d cord blood B cells virgin B cells and adult 339

Memory B I

Pn.-B

Y~,ng B

Virgio B

I

(())

~r,~o.o~!

;-'lasmaDlaSI

~

P,~ma c~[

0_.0_0, # Fetal B cell

I EB virus infection Cord brood B cell

) )

Adult peripheral B cell

Fig. 3. A diagram of human B cell differentiationcomposed from the present data and data obtained by other investigators,demonstrating "short range shift" of B cell immunoglobulinexpression followed by EBV transformation.

oped from cord blood spontaneously produced a b u n d a n t interferon without induction with Sendal virus, whereas lymphoblastoid cells from adult PBLs did not. These results are valuable for studying h u m a n B cell differentiation, because EBV selects B cells a m o n g the mixed population of hematopoietic cells such as fetal liver in which lymphoid cells with EBV receptors are in minority and the permanent lines reflect the character of the original B cells.

Acknowledgements PBL B cells memory B cells. The lymphoblastoid cells established from fetal liver or spleen (young B) showed the character of early plasmablast cells which still have slg and secrete IgM. Adult PBL (memory B) derived lymphoblastoid cells express the phenotype of late plasmablast which has faint slg and secretes not only IgM but also IgG and IgA. Steel et al. [4] reported that immunoglobulin was secreted by h u m a n lymphoblastoid cell lines derived from either adult PBLs or CBLs. Our results obtained by a more reliable method than hemagglutination inhibition technique were consistent with theirs concerning IgM, IgG and IgA secretion. They speculated the possibility that there was the preferential transformation of the cells already secreting Ig in vivo. However, the present understanding is that EBV is a polyclonal B cell activator [3]. Pereira et al. [24] measured the lg content in the supernatant of EBV-stimulated cells from various lymphoid cells over a 4-wk culture and their results were similar to ours. There was a difference concerning the IgD content in supernatant of the cells of cord blood origin, because of the differences in culture between both. Pereira et al. used the culture supernatant in early period after EBV-infection with changing a half volume of the medium twice weekly. While we used the supernatant of the cell lines cultured for 6 days without changing the medium. It is likely that IgD secretion of the lymphoblastoid lines depends on slgD expression of the cells of origin and CBL-derived lines secrete more IgD than the ones derived from other sources. We have reported that alpha-interferon production by the lymphoblastoid lines depends on the maturation stage of their origin [25]. Most lines devel340

This work was supported in part by a Grant-inAid for Cancer Research (5 18) from the Ministry of Health and Welfare, Japan. We thank Mr. J. F. Morris for preparation of the manuscript and Ms. C. Arai for expert secretarial support.

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