human b cell lines can be triggered to secrete an interleukin 2-like molecule

CELLULAR IMMUNOLOGY 121,30-48 (1989)

Human B Cell Lines Can Be Triggered to Secrete an lnterleukin 2-like Molecule

DAVIDBENJAMIN,*DANIELP.HARTMANN,~LEONARDS.BAZAR,S ROBERT J. JACOBSON,#IANDMICHAELS. GILMORES

*Saint Francis Research Institute, Oncology Section, Lymphokine Research Laboratory, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190; tDepartment ofPathology, Georgetown University Hospital, Washington, D.C. 20007; *Division ofHematology, Georgetown University Hospital, Washington, D.C. 20007; and §Department of Immunology and Microbiology,

University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190

Received September 22, 1988; accepted February 10, I989

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt’s and non-Burkitt’s types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 r&ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 ( 100%) normal lymphoblastoid cell lines, but in only l/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt’s cell lines. IL-2 secretion was not detected in control supematants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS- 1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tat monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti&c monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation. 0 1989 Academic Press, Inc.

INTRODUCTION

The most thoroughly characterized lymphokine to date is interleukin 2 (IL-2), a T lymphocyte product which serves as an obligatory factor for the proliferation of antigen- or mitogen-stimulated T lymphocytes (reviewed in Ref. (1)). IL-2 has also been shown to exert numerous effects, such as stimulating differentiation of precursor cells into lymphokine-activated killer (LAK) cells (2), and influencing the secretion

’ Abbreviations used: IL, interleukin; IF’N, interferon; PMA, 4-P-phorbol- 12-myristate- 13-acetate; T = 10, teleocidin (10 rug/ml); Ig, immunoglobulin.

30

000%8749/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

HUMAN B CELL INTERLEUKIN 2 ACTIVITY 31

TABLE I

Effect of Teleocidin on IL2 Secretion: Day 2

Cell type Origin Cell line

IL-2 activity Chromo- @J/ml)

EBV somal status translocation Control T= 10

American

Tumor cell lines Burkitt’s (N = 14) (N= 19)

African Burkitt’s (N= 5)

Normal lympho- Infectious blastoid cell mononucleosis lines (N = 6) cells (N = 3)

EBV-transformed cord blood cells (N= 3)

EW36 JD38 CA46 JLP(C) 119 ST486 DS179 JD39 MCI 16

JLP(C) DW6 RR124 PA682PB PA682PE- 1 PA682BM-2

RAJI DAUDI HRI+Z NAMALVA AK778

IM-B182 IM-1178 IM-El82

CB23 CB33 CB34

-

+ + + + + + + + + + + + + + + + +

8;14 8;14 8;14 8;14 8;14 8;14 8;14 8;14

8;14 8:14 -

8;22 8;22 8;22

8;14 8;14 8;14 8;14 8;14 -

- - -

<l <l <I <l <I <I il il

<l <1 <l <l <I <1

<I <I <l <l <1

<l <I <l

<1 <I il

30 43 14 53 <I

283 162 117

<1 <I 100 <l <I <1

<l <1 <I <l <l

22 42 59

174 39

180

Note. Kinetic studies over 1 to 7 days showed that the maximum secretion of IL-2 was within 24-48 hr. The data included in this table represent results obtained on Day 2 only; T = 10, teleocidin 10 rig/ml; -, negative; +, positive.

of other lymphokines, including interferon-y (IFN--/), tumor necrosis factor (TNF), B cell growth factor (BCGF), and B cell differentiation factor (BCDF) (3-5). There is some controversy as to the role of IL-2 in regulating B cell activation, proliferation, and differentiation (6- 12). Several reports suggested that B cell responsiveness to IL- 2 correlates with the expression of high affinity IL-2 receptors (6, 1 l- 13). More recent studies, however, questioned whether the presence of IL-2 receptors on B cells is a prerequisite for the effect of IL-2 since B lymphocytes with very little concomitant IL-2 receptor expression can proliferate and differentiate in vivo into immunoglobulin (Ig)-secreting cells ( 14, 15). Moreover, to stimulate the growth and differentiation of B cells in vitro, the response often requires the use of very high, essentially nonphys- iologic levels of IL-2 (16, 17). Thus, although IL-2 appears to support B cell activation, proliferation, and differentiation in vitro, many questions remain concerning its physiologic role in regulating B cell growth and development in viva. In addition to responding to IL2 stimulation, recent isolated reports have suggested that mu&e B

32 BENJAMIN ET AL.

cell lines can synthesize and release an IL-2-like molecule in vitro. Maino and Pace ( 18) described the secretion of IL-2 by the murine B cell tumor cell line 2PK-3, following activation with an uncharacterized, high MW non-Ig component of hyperim- mune sera. The MW and isoelectric point of this IL-2-like molecule were similar to IL-2 derived from the T cell lymphoma cell line EL-4G 12. Like T cell-derived IL-2, B cell-derived IL-2 activity supported the proliferation of the two IL-2 dependent cell lines, CTLL-2 and HT-2. In another report, Parodi et al. ( 19) described the ability of a supernatant derived from an EBV-negative human B cell culture to support the proliferation of CTLL-2 cells. Taira et al. (20) demonstrated that the murine B cell lymphoma cell line A20.2J and murine splenic B cells produced an active material which supported the proliferation of CTLL-2 cells, following stimulation with both calcium ionophore A23 187 and 4-P-phorbol- 12-myristate- 13-acetate (PMA). This lymphokine was deduced to be IL-2 and not B cell-stimulating factor- 1 (BSF- 1) since the proliferation of CTLL-2 cells in the presence of this B cell IL-2 was inhibited by anti-IL-2 but not by anti-BSF- 1. Furthermore, following activation with A23 187 and PMA, IL-2 mRNA was detected in both A20.2J cell line and splenic B cells. In the most recent study, Walker et al. (2 1) showed that the murine B cell tumor cell lines 2PK-3 and LlOA2J synthesize and release IL-2 after stimulation with selected poly- clonal activators such as Staphylococcus aureus. This IL-2 activity was inhibited with anti-IL-2 monoclonal antibodies.

These four reports strongly support the prospect that human B cells are capable of secreting IL-2 as well. This possibility is underscored by the results of recent studies that human B cells secrete their own lymphokines, including B-BCDF (22) IIN-7 (23), TNFP (24), interleukin 1 (IL-l) (25), and B-BCGF (26), and express receptors for IL- 1 (27), BCGF (28) BCDF (29), and TNF (30).

In the present manuscript we report that human B cell lines express tat antigen and secrete soluble IL-2 receptors without being activated and that following stimulation with the new tumor promoter, teleocidin, they are triggered to secrete an IL-2- like molecule. We also provide evidence that B cell IL-2 shares common biological activities with T cell IL-2 and discuss the possible role of B cell IL-2 in T cell activation and proliferation.

MATERIALS AND METHODS

Cell Lines

The study included 19 tumor cell lines derived from patients with undifferentiated lymphoma of Burkitt’s and non-Burkitt’s types and 6 normal lymphoblastoid cell lines (Table 1). Of the 6 normal lymphoblastoid cell lines, 3 lines were obtained spon- taneously from peripheral lymphocytes of patients with infectious mononucleosis (IM) and 3 cord blood (CB) lymphocyte cell lines were transformed with EBV. Meth- ods of derivation and documentation of the tumor origin of these cell lines have been previously reported (3 1). All cell lines were screened by a panel of B and T cell surface markers (HLA-DR, Leu 10, BA- 1, B-l, B-2, J-5, OKT- 10, OKT-3, OKT-11, and surface Ig) and were positive for B lymphocyte markers only.

Induction of IL-2 Secretion

Cells obtained on the fourth day following subculture were resuspended in RPM1 medium plus 10% FCS, at 5 X lo5 cells/ml, and incubated at 37°C 5% C02, for 1 to


7 days in the presence or absence of teleocidin ( 10 rig/ml). Supernatants were assayed for the ability to support the growth of CTLG2, a murine IL-2-dependent cytotoxic cell line, and IL-2 activity was calculated as previously described (32,33).

Characterization of the Biologic Activity of IL-2

I. Neutralization ofIL-2 by anti-IL-2 monoclonal antibody (DMS-I). The methods used for preparation of DMS-1 from ascitic fluid were adapted and modified from those reported by Smith et al. (33). Supernatants obtained from control and teleocidin-activated cells were incubated in 96-well microtiter plates with serial twofold dilutions of DMS- 1. Following 4 hr of incubation, CTLL-2 cells (5 X 1 O3 cells/well) were added, and cultures were further incubated for 24 hr at 37°C 5% C02. Six hours before harvesting, [3H]TdR (1 &/well) was added, and its incorporation was determined by liquid scintillation counting. The neutralizing effect of DMS-1 on IL-2 activity was expressed as the percentage of inhibition of [3H]TdR uptake in the presence of DMS- 1 compared with control.

II. El&ion of IL-2 following its adsorption to CTLL-2 cells. Further evidence that the factor-secreted postactivation with teleocidin had IL-2 activity was provided by measuring its specific adsorption to CTLL-2 cells. The methodology used was adapted from that described by Farrar and Hilfiker (34). Briefly, CTLL-2 cells (2 X lo6 ml) were washed twice in ice-cold RPM1 1640 supplemented with 5% FCS. Then, supematant derived from control or teleocidin-treated cells was added to the cell pellet, and following resuspension the pellet was kept on ice for 45 min. The cells were then centrifuged at 300g for 10 min in the cold; the supematant was carefully removed and the cell pellet was washed three times (3OOg, 10 min) in 1 ml of ice-cold Dulbecoo’s PBS (DMEM; GIBCO). To the cell pellet, 0.5 ml of 0.1 Mglycine-HCL buffer (pH 2.3) was added. The supernatant containing the eluted IL-2 was collected by centrifugation (3OOg, 10 min) and neutralized with 1 MTris-HCL (pH 8.2). Elu- ates obtained from control and teleocidin-activated cells were kept at 4°C and were tested simultaneously for their ability to support the growth of CTLL-2 cells.

III. Molecular weight ofB cell IL-2: a. MWof eluted B cell IL-2. To determine the MW of B cell IL-2, three eluates (one control and two cell lines) were studied. An aliquot of either the supernatant or the eluate from the CTLL-2 cells was applied to 11% SDS-PAGE. The gels were then uniformly sliced at 2-mm intervals, and the protein bands were eluted from the gel slices at 4°C. Following 16 hr of extensive dialysis, the eluates were assayed for the ability to support the growth of CTLL-2 cells as previously described.

b. MW of purified B cell IL-2. Since microheterogenity in the size of B cell IL-2 occurred in eluates obtained from both JD38 and IM-E 182, purification of the active protein was attempted. The JD38 cell line was grown to 1 liter in 10% FCS to the optimal density of 1 X 1 O6 cells/ml. The cells were harvested, activated with teleocidin ( 10 rig/ml), and incubated in serum-free medium at 37°C 5% CO2 , for 72 hr. The supematant was then brought to 80% saturation with ammonium sulfate, and the precipitate was dialyzed and clarified by centrifugation. The resulting extract (5 ml) was applied to a 2.5 X 90-cm Sephacryl S-200 column, and fractions with maximum IL-2 activity were concentrated and applied to an SDS-PAGE. Since an identical pattern of B cell IL-2 microheterogenity was observed in chromatographically separated fractions, Western blot analysis was performed using previously described

34 BENJAMIN ET AL.

methods (35, 36). Briefly, following electrophoresis, the protein bands were trans- ferred to a nitrocellulose sheet and DMS- 1 diluted 1:200 in PBS, 0.5% Tween 20, was then added. Antibody binding occurred overnight at 4°C with occasional agitation. The blot was washed and biotinylated goat anti-mouse IgG (BRL, Gaithersburg, MD) was incubated with the nitrocellulose blot for 2 hr at room temperature. Then, the blot was washed and incubated with 1: 1000 diluted streptavidin-horseradish peroxi- dase conjugate (BRL, Gaitherburg, MD). After 2 hr at room temperature, the blot was washed and incubated for 30 min at room temperature with the following substrate solution: 0.5 mg/ml4-chloro- 1 -naphthol, 0.0 15% Hz02 in TBS (20 mM Tris-Cl, pH 7.4, 250 mJ4 NaCl.). The substrate solution was decanted, and the nitrocellulose sheet was washed with deionized HzO. A photograph of the blot was taken while the blot was still wet for better color contrast.

IV. Eflect of anti-tat monoclonal antibody on B cell IL-2 ability to support T cell proliferation. Peripheral blood cells obtained from normal donors were stimulated with PMA and grown for 15 days in the presence of IL-2 (Electronucleonics Inc., Silver Spring, MD). Utilizing these IL-2-dependent long term cultured human T lymphocytes as indicator cells (37), the IL-2 activity was quantitated by comparison of dilution curves of sample and standard IL-2 preparations. Serially diluted supernatants (1: 1 to 1:2048) obtained from teleocidin-activated IM-E 182 cells were incubated in 96 microtiter plates with 1.5 X lo5 T cells/well. Following 20 hr of incubation, 1 PCi of [3H]TdR was added to the cultures. The plates were incubated for an additional 6 hr and harvested as previously described. Samples containing RPM1 1640 with or without teleocidin ( 10 rig/ml) were used as controls. In parallel experiments, activated T cells were incubated with anti-tat monoclonal antibody ( 1 O-3 to 1 Op6 dilution) for 30 min at room temperature, followed by the addition of the serially diluted IM-E 182 supematant. The ability of anti-tat to block B cell IL-2 activity was compared with its effect on blocking the activity of T cell IL-2.

Quantitation ofIL-2 Membrane Receptor (Tat) and Soluble IL-2 Receptor

The tat antigen was studied by flow cytometry using an anti-tat monoclonal antibody as previously described (6). The soluble IL-2 receptor level was determined with a sandwich enzyme immunoassay (Cellfree, T cell Sciences, Inc., Cambridge MA), following manufacturer instructions.

Ig Secretion

Ig secretion (IgM, IgG, IgA) was measured using the ELISA method as previously described (38).

Lymphokine Assays

In addition to IL-2, supernatants derived from control and teleocidin-treated cells were studied for BCGF, IL- 1, and IFN--/ as previously described (23,39). For BCDF activity, the CESS cell line was used (40,41). TNF activity was determined using the standard crystal violet assay with actinomycin D and the L929 fibroblast cell line (42).


RNA PuriJication

RNA was purified from three cell lines: IM-E 182, JD38, and DS 179. For RNA purification, approximately 1 X lo9 cells (control and teleocidin-treated) were harvested at 3,6, 12, and 24 hr. As a control, RNA was also purified from the MLA- 144 cell line under identical conditions. The SDS, phenol/chloroform, DNAse method (43, 44) and the guanidinium thiocyanate, phenol/chloroform, method (45) were both used. The latter method minimized the opportunity for RNA degradation by endogenous RNases by eliminating steps where chaotropic agents were absent. Ra- dioactive probes were prepared by incorporating 32P-dATP (NEN, Glenolden, PA; 3000 Ci/mmol) into the IL-2 encoding recombinant plasmid pTCGF-5 (kindly provided by Dr. Susha Arya) (46) and the 700 bp IL-2 specific probe sequence (Oncor, Gaithersburg, MD) using a nick translation kit (BRL, Gaithersburg, MD) according to manufacturers specifications. Nick-translated probes were obtained with specific activities of 5 X 1 O7 to 1 X 10’ dpm/pg. Additionally, an oligonucleotide probe com- plementary to nucleotides 487-501 of the IL-2 structural gene (46) 5’-AGT CAG TGT TGA GAT-3’) was synthesized on an Applied Biosystems Model 380B DNA synthesizer (Foster City, CA), prepared at The Molecular Biology Resource Facility of The Saint Francis Hospital of Tulsa Medical Research Institute (Oklahoma City, OK). This oligonucleotide was substituted for the hexanucleotide mixture provided in a random primer labeling kit (Amersham, Arlington Heights, IL) and was used to label PstI digested, denatured pTCGF-5 DNA otherwise according to the manufacturers recommendations. Using this method, probes with specific activities of approximately 5 X 10’ dpm/pg were obtained.

For maximum sensitivity, 10 pg of RNA prepared as described above was assayed by dot blot hybridization (47) using Gene Screen Plus (DuPont) according to manufacturers recommendations. High and low stringency hybridization (50% deionized formamide, 42 and 24°C respectively) and washing (65 and 45°C) conditions were used in alternate experiments. Hybridization signals were detected on XAR-5 (Ko- dak) film after 24 hr to 14-day exposures at -70°C using Cronex (DuPont) intensify- ing screens.

RESULTS

Efect of Teleocidin on IL-2 Secretion

Teleocidin, the tumor promoter used in this study, is known to share similar biological activities with PMA and binds to the same specific cell surface receptors as PMA (48). However, in contrast to PMA which mimics IL-2 activity in the CTLL-2 assay (49), no such interference was observed with teleocidin. Teleocidin (10 rig/ml) triggered the secretion of IL-2 activity in seven of eight (87%) EBV-negative lymphoma cell lines of American origin and in six of six ( 100%) normal lymphoblastoid B cell lines, but in only one of six (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on five of five (0%) African Burkitt’s lines (Table 1). Kinetic studies over 1 to 7 days demonstrated that the optimal induction of IL-2 was obtained within 24-48 hr following activation with teleocidin. IL-2 secretion was not detected in any of the control supernatants.

Correlation of EB V Status and Karyotype with Induction of IL-2 Secretion

The secretion of IL-2 activity correlated with EBV: 7 of 8 EBV-negative American lymphoma cell lines were triggered to secrete IL-2, whereas only one (KIS124) of

36 BENJAMIN ET AL.

TABLE 2

Correlation of EBV Status and Karyotype with Induction of IL-2 Secretion

Cell line number Source

EBV status

Karyotype t(8;14)

or t(8;22) Induction of

IL-2

Group A Group B Group C

N=8 American - + 7/8 (87%) N= 10 American and African + + o/10 (0%) N= 1 American (KK14) + - N=6 Normal + - 7/7 (100%)

lymphoblastoid

the 11 EBV-positive lymphoma cell lines (6 American and 5 African cell lines) was triggered (Table 1). These results suggest that EBV may suppress IL-2 synthesis. Sup- pression at an early stage in synthesis is in line with the observation that human T leukemia virus I (HTLV-I) exerts inhibitory effects on the transduction of the IL-2 signal (50). Triggering of IL-2 secretion in the 6 EBV normal lymphoblastoid cell lines, however, indicates that in addition to EBV, other factors may also play a role in IL-2 secretion. Correlating IL-2 secretion with EBV status and karyotype showed that the common denominator between the 6 normal lymphoblastoid cell lines and the exceptional EBV-positive tumor cell line (KK124) is the normal karyotype (Table 1). Therefore, IL-2 secretion appears to be linked to both EBV status and karyotype (Table 2): IL-2 secretion occurred in EBV-negative cell lines with abnormal karyotype (8; 14 or 8;22 chromosomal translocation) [Group A: (-) and (+)] or in EBV- positive cell lines with normal karyotype [Group C: (+) and (-)I. Tumor cells with abnormal karyotype and positive for the EBV genome were not triggered to secrete IL-2 [Group B: (+) and (+)I.

Correlation of IgM Induction with IL-2 Secretion and Relationship to BCDF, BCGF, IL-l, and IFN--, The secretion of IL-2 also correlated with the induction in IgM secretion (Table 3).

Teleocidin induced IgM secretion in the IgM secretor cell lines, but did not trigger de novo synthesis of IgM in IgM-nonsecretor cell lines. No Ig subclass switch (IgG and/ or IgA) was detected in any of the IgM secretor cell lines. The induction in IgM secretion was also accompanied by morphological changes which were consistent with differentiation toward plasma cells, similar to those we previously observed with PMA (38). These results suggest that B cell IL-2 plays a functional role in B cell differentiation, or alternatively, that teleocidin triggers BCDF, in addition to IL-2 activity. When 12 supernatants (control and teleocidin-treated cells) were tested for BCDF activity, the lymphokine was found to be triggered in eight cell lines (Table 4). However, BCDF secretion did not correlate with the secretion of IgM. Supernatants obtained from controls and teleocidin-activated cells were also tested for the possible presence of several other B cell lymphokines, such as B-BCGF, IL-l, IFN-7, and TNF. Like BCDF, no distinct overlapping pattern of reactivities between these B cell lymphokines and IL-2 secretion was found (Table 4).

Neutralization of IL-2 by DMS-1 As shown in Table 5, the maximum inhibition of control IL-2 by DMS- 1 did not

exceed 30%. This result was expected considering (i) the high binding affinity of IL-2

HUMAN B CELL INTERLEUKIN 2 ACTIVITY 3-l

TABLE 3

Correlation between IgM Induction and IL-2 Secretion

Cell line

IgM secretion IL-2 secretion

Control T= 10 T= 10

Category I JD38 CA46 JLP(C)ll9 DS179 JD39 MCI 16 KK124 DW6 ST486

Category II PA682BM-2 PA682PE-1 PA682PB NAMALVA AK778

Category III EW36 JLP(C) RAJI HRI+Z DAUDI

NS NS NS NS NS

4t 5t 3t 5t 5t 2t 4t 3t 3t

No change No change No change No change No change

NS NS NS NS NS

S S S S S S S

NS NS

NS NS NS NS NS

S NS NS NS NS

Note. The cell lines were grouped into three categories: Group I includes 9 IgM secretor cell lines, in which teleocidin induced two- to fivefold increase in IgM secretion (range: 200-900 r&ml). In 7/9 cell lines IL-2 secretion was triggered. Group II includes five IgM secretor cell lines (range: 300-6000 rig/ml). Teleocidin did not induce IgM secretion and did not trigger IL-2 secretion in these cell lines. Group III includes five IgM nonsecretor cell lines. Teleocidin did not trigger IgM secretion in S/5 cell lines. IL-2 secretion was triggered in only l/5 cell lines. S, secretor; NS, nonsecretor; t, stimulation index; T = 10, teleocidin 10 rig/ml.

to its receptor (33) and (ii) the markedly lower afhnity of the monoclonal antibody (5 1). A similar level of inhibition was observed for IL-2 secreted by the Jurkat T cell line and by the MC1 16 B cell line. In JD39 and IM-E182, however, 93 and 58% inhibition, respectively, was demonstrated.

Elution of IL-2 following Its Adsorption to CTLL-2 Cells Additional evidence that the factor-secreted postactivation with teleocidin had IL-

2 activity was provided by measuring its specific adsorption to CTLL-2 cells. Follow- ing elution, IL-2 activity was recovered from four of four eluates derived from supernatants of cell lines triggered with teleocidin. Serial twofold dilutions (1:2 + 1:64) were tested in 96-well microtiter plates, and the results obtained at 1:4 dilution are presented in Table 6. Eluates derived from unstimulated cell lines had no IL-2 activity and eluates of sham-eluted CTLG2 cells yielded background activity only.

Determination of the MW of B Cell IL-2 and Its Recognition by Monoclonal Anti-IL- 2 B cell IL-2 obtained from eluates derived from IM-El82 and JD38 cell lines

showed microheterogenity in size with MW corresponding to 18.5, 14.5, and 13 kDa.

38 BENJAMIN ET AL.

TABLE 4

Effect of Teleocidin on Lymphokine Secretion in Human B Cell Lines

BCDF rig/ml IL-2 U/ml IL- 1 U/ml IFN-7 U/ml TNF U/ml &m

Cell line C T=lO C T=lO C T=lO C T=lO C T= 10 -

EW36 - JD39 - JD38 - DS179 - JLP(C)ll9 - KK124 - CB23 - IM1178 - CA46 - MC1 16 - CB33 - CB34 - IM-B182 - IM-El82 - JLP(C) - ST486 - DW6 - PA682PB - PA682PE-1 - PA682BM-2 - AK778 - HR1+2 - RAJI - DAUDI - NAMALVA -

30 162 43

283 53

100 39 42 14

117 174 180 22 59 -

- - - - - - - - - - - - - - - - - 30 25 56 18 41 21 45 17 51 15 67 8 80

12 32 10 20 15 16 24 25 32 32 - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - -

103 - - - - - - - - - -

- - - - - - - - - - - - - - - - - - 54 62 - - - - -

- 249 - 68 - -

580 785 -

- - - - - - - - - -

- 319 184 146 215 680 - 73

150 169 88

293 -

- - - - - - - - - - - - - - - - - - - - - - - - -

- 120 ND 166 ND ND 124 12

ND 101 136 35

ND ND ND ND - -

- - -

- ND ND ND

13 ND ND

Note. c, control; T = 10, teleocidin 10 rig/ml; -, not detected; ND, not determined; t, increase in lymphokine activity following stimulation with teleocidin.

TABLE 5

% Inhibition of [3H]TdR from supernatants

DMS-I Human dilution IL-2 Jurkat

MC1 16 JD39 IM-El82 (T = 10) (T = 10) (T = 10)

I:2 30 25 1:3 11 40 58 1:4 26 12 1:7 38 69 42 1:13 32 80 23 1:16 18 6 1:27 21 93 17 I:53 19 80 3 1:64 0 2

Note. The neutralizing effect of DMS-1 on IL-2 activity is expressed as the percentage of inhibition of [3H]TdR uptake in the presence of DMS-1 compared with control. Supematants at 1:4 dilution were incubated with various concentrations of DMS-I. Whereas the inhibitory effect is dose dependent and is optimal with the lowest dilution in MC1 16 and IM-El82 cell lines, in the JD39 cell line, optimal inhibition was detected at DMS- 1 dilution of 1:27.


TABLE 6

Elution of IL-2 following Its Adsorption to CTLL-2 Cells

Source of IL-2 Initial activity

W/ml) Eluted activity

W/ml) % Recovery

Human IL-2

Rat IL-2

JLP(C)I 19 MC1 16 JD39 CB23 RPM1 medium

(sham activity)

213 200 73

324 47 14

45 35 78 162 42 26 78 59 76 45 33 73

<l <l

Since this microheterogenity may have been attributable to contaminating membrane proteins extracted from the CTLL-2 cells during elution, JD38 cell line IL- 2 was partially purified. Chromatographic fractions eluted from a Sephacryl S-200 column, containing maximum IL-2 activity (Fig. l), were concentrated and separated

0.9 -

0.6 -

0.7 - E

g 0.6 - (Y z 8 0.5 - c

3 4 OA-

I 0.3 -

0.2 -

66KD UKD 30KD 3OKD 15.3KD 0.1 KD 5SKD

I I 1 I I I c h

Fraction Number

FIG. 1. JD38 cell line was grown to 1 liter in serum-free medium for 72 hr. Following precipitation and dialysis, the extract (5 ml) was applied to Sephacryl S-200. All fractions were tested for IL2 activity.

BENJAMIN ET AL.

43.6 25.7 KD- KD,+

16.4 KD+

14.3 KD+ 12.3 KD+

6.2 KD -

3.0 KD-,

FIG. 2. Fractions with maximum IL-2 activity obtained from the Sephacryl S-200 column were pooled, concentrated, and applied to SDS-PAGE. Lane A, fractions 50-59; lane B, fractions 60-65; lane C, fractions 66-70; lane D, fractions 7 l-80. IL-2 activity in these fractions was compared with purified recombinant IL-2.

by SDS-PAGE. As shown in Fig. 2, B cell IL-2 microheterogenity was detected again. Western blot analysis of the resolved protein bands with DMS- 1 revealed, however, a single band of MW 13.5 kDa, compared with MW of 13 kDa for the rIL-2 (Fig. 3). Aside from similarity in size and activity, these results demonstrated that B cell IL-2 and rIL-2 share identity in the domain recognized by monoclonal anti-IL-2.

Anti-Tat Monoclonal Antibody Partially Blocks B Cell IL-2 Activity

The effect of serially diluted supernatant ( 1: 1 to 1: 16) obtained from the teleocidin- stimulated IM-El82 cell line on activated human T cells is shown in Table 7. IM- E 182 optimal proliferation was at 1: 1 dilution. Anti-tat at all dilutions (even down to

A B C D

14.3 KD 13 KD

6.2 KD

FIG. 3. Western blot analysis of B cell IL-2 (lane B) and recombinant IL-2 (lane A). Lanes C and D included control fractions which did not contain IL-2.


TABLE 7

Effect of Anti-tat on B Cell IL-2 (IM-E 182 Cell Line)

Dilution

RPM1 +

T= 10

IM-El82 T= 10 @pm) 1o-3

IM-E 182 (T = 10) + anti-tat

1O-4 1o-5 1om6

I:1 949 24,922 13,724 14,210 16,047 16,474 (45%) (33%) (36%) (36%)

1:2 423 4015 2,650 2,730 3,003 3,590 (44%) (33%) (26%) (11%)

I:4 220 919 633 691 645 778 (32%) (25%) (30%) (16%)

1:8 129 229 406 362 285 193 (0%) (0%) (0%) (12%)

1:16 85 171 240 171 195 118 (0%) (0%) (0%) (31%)

10-6) inhibited [3H]TdR incorporation into target cells. In contrast to the progressive inhibition of T cell IL-2 demonstrated with increasing amounts of anti-tat and the maximum inhibitory effect of anti-tat observed using T cell IL-2 obtained even at low dilutions (data not shown), the maximum inhibition of B cell IL-2 by anti-tat did not exceed 45%. RPM1 medium containing teleocidin only and/or anti-tat had no proliferative effect.

B Cell IL-2 Receptor: Constitutive Expression and Secretion

Six of the 11 cell lines studied express membrane receptors for IL-2 detected by anti-tat monoclonal antibody (Table 8). In studying the supernatants (control and teleocidin-treated) for the presence of soluble IL-2 receptors (Table 9), changes in receptor reactivities were found following activation with teleocidin.

TABLE 8

Effect of Teleocidin on IL-2 Receptors (Quantitated with Anti-tat)

% Positive cells

Cell lihe Control T= 10

JD38 79 78 KK124 6 13 CA46 21 22 JLP(C) 119 16 26 DS17d 0 0 JD39 0 17 ST488 0 0 DW6 35 34 Namalva 24 22 Daudi 0 0 CB23 0 0

42 BENJAMIN ET AL.

TABLE 9

Effect of Teleocidin on Soluble IL-2 Receptor Reactivity (U/ml)

EBV status Cell line Control Teleocidin ( 10 w/ml)

American cell lines Negative

American cell lines Positive

African cell lines Positive

Normal lymphoblastoid cell lines

Positive

Note. ND. not determined.

EW36 4 4 JD38 65 134 CA46 175 136 JLP(C) 119 15 66 ST486 8 14 DS179 12 12 JD39 144 393 MC1 16 4 10

JLP(C) 13 23 DW6 26 57 KK124 15 148 PA682PB ND ND PA682PE-1 ND ND PA682BM-2 65 55

RAJI 0 4 DAUDI 7 7 HR1+2 10 10 NAMALVA 10 20 AK778 10 26

CB33 2 12 CB23 I1 24 CB34 6 15 IM-B182 2 13 IMl178 2 27 IM-El82 54 7

IL-2 mRNA Assay

Dot blot assays of RNA purified from IM-E 182 and DS 179 cell lines failed to yield a detectable signal on autoradiograms, although IL-2 mRNA was detected in dot blot hybridizations of MLA 144-derived RNA. Serially diluted, denatured plasmid pTCGF-5 was detectable at levels of 60 pg. Using low stringency conditions, hybridization signals above background levels were also not obtained. The lack of detectable hybridization between probes labeled by two methods and target RNA purified by two methods suggests that the IL-2 mRNA may be in extremely low abundance in the cell lines tested. Alternately, the IL-2 activity observed to be elaborated by these cell lines may result from a peptide encoded by a divergent or unrelated nucleotide sequence such that hybridization does not occur with the authentic IL-2 probes used under the conditions tested.

DISCUSSION

In the present study we demonstrate that human B cell lines express tat antigen and secrete soluble IL-2 receptors without being activated and can be triggered to

HUMAN B CELL INTERLEUIUN 2 ACTIVITY 43

secrete a lymphokine which supports the proliferation of both CTLL-2 cells and activated T lymphocytes. Unlike the published reports of B cell IL- 1 and B-BCGF, there have been only few reports on B cell IL-2, mostly confined to the murine system (l&20,2 1). Although the data obtained on murine B cell IL-2 strongly support the secretion of IL-2 by human B cells as suggested by this study, other reports on bio- chemical variants of T cell IL-2 (52,53) and the recent identification of p40, a mouse glycoprotein distinct from IL-2 which is capable of supporting the growth of helper T cell lines (54), raise the question whether human B cell IL-2 described in this study is a true IL-2.

Neutralization of IL-2 by DMS- 1 monoclonal antibody, elution of IL-2 following its adsorption to CTLL-2 cells, and determination of its MW are all consistent with the secretion of authentic IL-2 by the B cell lines tested. Further evidence that this lymphokine is not a factor which mimics IL-2 activity in the CTLL-2 essay was provided by (i) its ability to support the proliferation of activated T lymphocytes and (ii) blocking of this activity by anti-tat monoclonal antibody. These in vitro studies indicate that both B cell IL-2 and T cell IL-2 recognize and bind to similar IL-2 receptors on T cells, and that under certain conditions, B cell IL-2 may be capable of influencing T cell proliferation in vivo. The basis for the observed reduced ability of B cell IL- 2 to overcome the inhibitory activity of anti-tat compared with that of T cell IL-2, however, is not clear. Whether these differences are due to the differential binding activity of B cell IL-2 and T cell IL-2 to IL-2 receptors on T cells remains to be determined.

Whereas mRNA IL-2 was detected in murine B cells (20), the lack of detection of mRNA IL-2 in both untreated and teleocidin-activated cells is of special interest, and the following possibilities are raised: (i) Human B cells synthesize pro-IL-2, and post- translational modification induced by teleocidin prompted the conversion of the molecule to an active B cell IL-2. These results are consistent with the report of Kup- per et al. (53) that murine keratinocytes secrete a factor which supports the growth of the helper T cell-derived IL-Zdependent cell line HT-2, but not of CTLL-2. Since this substance, kerotinocyte-derived growth factor (KTGF), has the same biochemi- cal properties of T cell IL-2, this raised the possibility that keratinocytes both synthesize conventional IL-2 and modify it post-translationally differently from T cells. This mechanism, i.e., that teleocidin modulates a biologically inactive IL-2 precursor, is different from that shown for murine B cell IL-2 where de ylovo synthesis of mRNA was induced following activation with A23 187 and PMA (20). (ii) IL-2 mRNA occurs in extremely low abundance in induced B cell lines. This prospect is supported by the recent observation that IL-2 mRNA is turned over rapidly in T lymphoblasts, apparently resulting from degradation by a specific inducible RNase (55). Therefore, variations in the level of IL-2 mRNA synthesized by B and T cells or differences in the intracellular levels of this inducible RNase may account for the detectability of IL-2 mRNA in T cells, but not B cells. Alternatively, IL-2 may be produced by a subpopulation of induced cells, effectively diluting the levels of IL-2 mRNA con- tained in total RNA extracts. Enrichment methods such as oligo dT affinity chroma- tography or polymerase chain reaction may enhance the detectability of B cell IL-2 mRNA by standard hybridization methods. (iii) The IL-2 synthesized by B cells is genetically distinct from that synthesized by T cells. Although structural domains on T cell and B cell IL-2 were found to share sufficient identity to interact with the tat receptor and a monoclonal antibody, differences in the primary amino acid sequence

44 BENJAMIN ET AL.

or the redundant genetic code for possible alternate genes encoding IL-2 expressed by different cell types may exist. Thus, as has been observed for IL-l and TNF, a family of IL-2 species may occur.

The mechanism(s) involved in triggering B cell IL-2 by human B cells is unknown. One possibility tested was that teleocidin exerts its effect on IL-2 secretion via triggering of other lymphokines. To study this two-signal model, supernatants obtained from control and teleocidin-activated cells were studied for IL-l, BCGF, BCDF, TNF, and IFN-7 activities. Since monocyte IL- 1 is known to be a costimulant for the release of IL-2 from mitogen-activated T cells (56, 57) and is required for the induction of IL-2 receptors on both T (56) and B cells (58) and since some of the B cell lines express IL-2 receptors, we initially tested the 25 cell lines for IL-1 activity. Of the total 20 cell lines that secrete IL-2 or IL- 1, only 6 cell lines secrete both lymphokines. In 8 of 14 cell lines, however, the induction of IL-2 was not accompanied by IL-l. This raises the following possibilities: (i) In contrast to T cells, B cells can be triggered to secrete IL-2 without being activated by IL- 1. These results are in line with new immunological models questioning the role of monocyte-derived IL-l in the activation of T cell IL-2 (59). (ii) B cells secrete IL-1 different from monocyte IL-l. Several recent reports demonstrated that human B cell-derived IL- 1 is different from IL- 1 (Y and IL- l@ (60-62). Purification to homogeneity and NH2 terminal amino acid sequence of B cell IL-l derived from the lymphoma cell line PA682BM-2 (60) and from the EBV-transformed B cell line 3B6 (6 1) demonstrated two novel species of B cell IL- 1 s. Thus, the sequential events of IL- 1 mediating IL-2 secretion shown in the T lymphokine cascade may not apply for B cell IL- 1. Similarly, IL-2 secretion was not accompanied by IFN-7 secretion, and did not correlate with the secretion of TNF or BCDF. With BCGF secretion, B cell IL-2 seemed to exert some synergistic effect on B cell proliferation (39).

Since teleocidin triggered the cell lines to secrete TNF via down-regulation of TNFa! receptors (30), an alternate approach for elucidating the mechanism of IL-2 secretion by B cells was to correlate IL-2 receptor modulation with IL-2 secretion. The constitutive expression of tat antigen and soluble IL-2 receptors in cell lines which were not triggered to secrete IL-2 and the lack of dramatic changes in tat level, following activation with teleocidin, suggested that as for T cell IL-2 (63) the signals required for B cell IL-2 synthesis and for tat expression are not identical and that the induction of tat antigen and IL-2 are not obligately linked. Alternatively, B cell IL-2 secretion is mediated via other IL-2 receptors. Recent reports of a novel p70/75 non- tat IL-2-binding peptide, in addition to p55 tat protein (64) suggest a multichain model in which high affinity receptors are expressed when both tat and p70/75 IL- 2-binding peptides are present and associated in a receptor complex (65). Current radiolabeling studies in our laboratory already indicate that several B cell lines in which IL-2 secretion was triggered express the cz subunit (~55 tat) or the p subunit (p70/75) of the IL-2 receptor, and that the number of receptor sites per cell is lo- to 1 OO-fold less compared to that found on T cells. Teleocidin up-regulated the number of receptor sites per cell in several cell lines and induced de ~OVO synthesis of IL-2 receptors in other cell lines (unpublished data). Upon completion of these studies, it will be possible to determine whether the effect of teleocidin on IL-2 secretion by B cells is mediated via up-regulation of IL-2 receptors, and what role, if any, the IL-2 receptor(s) plays in B cell IL-2 secretion.


The observation that IL-2 secretion is linked to both EBV status and karyotype is of special interest. Recently it has been shown that the long terminal repeating (LTR) sequence for the human IL2 gene is very similar in sequence homology with the LTR sequence of HTLV-I. Thus, if the IL-2 promoter regions are provided within the inserted HTLV-I LTR sequence, the virus could play some role in modulating IL-2 expression in infected cells (66). Moreover, transformation of T cells by HTLV- I has been found to involve a transacting transcriptional factor protein (tat-I) that not only regulates viral gene expression but also is able to produce inappropriate activation of the genes encoding the tat antigen and IL-2 (67). Human immunodeficiency virus-l (HIV-l) has also been shown to produce marked, but reversible, inhibition of IL-2 gene expression in CD4+ T lymphocytes (68). These reports, combined with our data, suggest that EBV might also play a role in triggering B cell IL-2 and in modulating IL-2 gene expression. However, detection of IL-2 secretion in most of the EBV- negative cell lines and correlation of IL-2 secretion with both karyotype and the EBV genome indicate more complex interactions than mere influence by EBV. We hy- pothesize that similarly to tat-1 and tat-II, the transactivator gene products encoded by HTLV-I and HTLV-II (66, 67) IL-2 secretion by B cells is influenced and regulated by several complex feedback mechanisms, in which both the EBV genome and IL-2 gene expression are involved.

Whereas the significance of the linkage to EBV and karyotype is not known, the correlation between the induction of IL-2 and the increase of IgM synthesis suggests that B cell IL-2, similarly to T cell IL-2, plays a functional role in B cell differentiation and immunoregulation. The results presented in this study are suggestive of one (or more) of the following mechanisms: (i) Teleocidin triggers B cells to secrete both IL- 2 and IgM. (ii) Teleocidin triggers some B cells to secrete IL-2, which then activates another B cell population to differentiate into Ig-secreting cells. This is in line with recent observations which suggested that IL-2 transiently up-regulated the expression of IL-2 receptors, via which it induced the terminal growth and differentiation of activated B cells into plasma cells (69). (iii) The IL-2 triggered in the surface Ig+ B cells subsequently activates the same cells to differentiate into Ig+-secreting cells via surface Ig. (iv) Teleocidin triggers, in addition to IL-2, a differentiating factor similar to BCDF secreted by T cells or to B-BCDF (22). Quantitating the number of IgM- secreting cells, with the reverse-hemolytic plaque assay, we previously estimated that only 1% of the cells secrete the total amount of IgM detected in the supernatant (70). This might apply to IL-2 secretion as well. Thus, to test these possibilities, several cell lines have been cloned, and IgM, IL-2, and B-BCDF secretion are being studied at the single cell level.

The potential ability to trigger B cells to secrete IL-2 in both the murine system and the human system may provide new insights into the complexity of B cell:T cell immunoregulation. In addition to potential autoregulation of B cell growth and differentiation suggested in our study, a second possible function of B cell IL-2 relates to the B cell ability to influence T lymphocytes. Human B cell lines activated by EBV have been shown to induce suppressor T cells from autologous lymphocytes, which can in turn effect mitogen-stimulated B cell Ig production (7 1). Antigen-stimulated memory B cells have also been shown to modulate feedback suppression of Ig re- sponses (72). Thus, it is possible to speculate that B cell IL-2 may play a role in regulating the response of both helper and suppressor T lymphocytes to antigen or mitogen stimulation, and if T cells which express IL-2 receptors are presented by B cell IL-2,

46 BENJAMIN ET AL.

antigen-stimulated T cells might be activated and proliferate. This should also apply to B cell populations which express IL-2 receptors but do not secrete IL-2. Thus, B cell IL-2 may influence the proliferation, growth, and development of both T and B cells. Furthermore, IL-2 receptor-bearing precursor cells for natural killer or LAK cells could possibly be activated in the vicinity of antigen-stimulated B cells.

Finally, it is important to note that our results to date have been obtained with human B cell lines. It is possible that these cell lines have lost the normal regulatory controls which would prohibit the expression of IL-2 molecular species by normal B lymphocytes and thus do not represent normal B cell physiology. We suggest that normal human B cells, or a subset of B cells, are capable of secreting IL-2 under certain circumstances. If this hypothesis is valid, the ability of B cells to play an important role in B cell:T cell immunoregulation, in conjunction with the possible influence on LAK precursors, might have future implications as to the potential role of B cell IL-2 in cancer therapy.

ACKNOWLEDGMENTS

We thank Dr. Hirota Fujiki and Dr. Takashi Sugimura for providing us with teleocidin; Dr. Kendall Smith for the DMS- 1 hybridoma; Dr. Eugene Koren for preparation of DMS- I monoclonal antibody; Dr. David Volkman for the CESS cell line; Dr. Susha Arya and Dr. Warner Greene for the cDNA probes for mRNA; Dr. Kenneth Jackson for preparation of the oligonucleotide probe, Dr. Thomas Waldmann for anti-tat monoclonal antibody; and special thanks to Mrs. Tommie Howard for excellent secretarial as- sistance.

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human b cell lines can be triggered to secrete an interleukin 2-like molecule

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