Eur. J. Immunol. 1990.20: 1125-1129

Interleukin 2 production by a marmoset B cell line

1125

Lawrence H. Brento, Toshio Miyawaki, Michael P. Everson and Joseph L. Butler

Interleukin 2 production by a marmoset B cell line*

Division of Developmental and Clinical Immunology, Departments of Pediatrics and Medicine, University of Alabama, Birmingham

Several B cell lines constitutively secreting interleukin (IL) 2 were derived from the Epstein-Barr virus-positive marmoset B cell line, B95-8. A representative line, KRC-18, was cloned by limiting dilution and found to be 40% surface IgM+, 60% cytoplasmic IgM+, > 95% DR+,weaklyTac+ and devoid of Tcell, monocyte and NK cell surface antigens. Supernatant from KRC-18 cells supported the long-term growth of an IL 2-dependent murine Tcell line, HT-2, and contained 7-8 unitslml of IL 2 activity when compared to recombinant IL 2. The supernatant was fractionated by Sephadex G-75 gel filtration, and maximal proliferation of HT-2 cells was supported by the 20-22-kDa column fraction. The proliferative response of HT-2 cells to KRC-18 supernatant was inhibited by monoclonal antibodies to human IL 2 or the murine IL 2 receptor in a dose-dependent manner, suggesting that the KRC-18 IL 2 has epitopes that are similar to human IL 2, and that its activity is mediated through binding to the IL 2 receptor of the target cell 1ine.When KRC-18 cells were analyzed for cytoplasmic IL 2, > 90% of the cells contained intracellular IL 2 in amounts equivalent to, or greater than, mitogen-activated Tcells. These data indicate that certain B lineage cell lines are capable of IL 2 synthesis and secretion.

reported [lo],the accepted cell source of IL 2 is theTcell. In this report, we demonstrate the constitutive production of I L 2 by an EBV+ marmoset B cell line and suggest its addition to the growing list of lymphokines produced by B cells.

1 Introduction

The primary function of B cells is to produce specific antibodies in response to antigenic challenges. Other functions include antigen presentation and lymphokine production [l]. For example, production of an IL 1-like activity by EBV-positive and -negative B cell lines has been demonstrated [2]. Although this activity has an apparent 2 Materials and methods molecular mass (25-30 kDa) higher than monocyte-derived IL 1, its biological effects are similar. An E B V B cell line 2.1 Cell lines has also been shown to produce a B cell differentiation factor (BCDF) with biological properties similar to T KRC-18 is an EBV-transformed marmoset B cell line cell-derived BCDF [3]. Low molecular mass (20-30 kDa) B derived by LD cloning of B95-8 cells. Its species origin was cell growth factors (BCGF) have been demonstrated in SN determined by chromosomal analysis. KRC-18 is 40% from mitogen-stimulated normal human B cells [4], EBV+ sIgM+, 60% cytoplasmic IgM+, > 95% DR+,weakly Tac+ B cell lines and EBV- neoplastic B cells [5,6]. Similarly, the and devoid of all T cell, monocyte and NK cell surface EBV+ B cell line, Namalva, has been used as a source of antigens. This cell line secretes approximately 2 yg/ml of both high molecular mass BCGF (50 kDa) and IFN-y [7]. IgM as determined by ELISA. HT-2 is an IL 2-dependent Several B cell lines have been shown not only to produce, murine T cell line used to measure IL 2 activity. but also to respond to, these growth factors in an autocrine fashion with increased proliferation [6, 81. 2.2 Media IL 2 plays a critical role in the growth and differentiation of Tcells, and there is increasing evidence that it may serve a All cell cultures were performed in RPh4I 1640 (Flow similar function for B cells [9]. Although IL 2 production by Laboratories, McLean,VA) supplemented with 10% heata stimulated murine B cell lymphoma line has been inactivated FCS (Flow Laboratories), 2 m~ L-glutamine, 5 x 10-5 M 2 - W , 100 IU/ml of penicillin, 100 yg/ml of streptomycin and 0.25 pg/ml of amphotericin B. [I 68921 ~~

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* This work has been supported by NIH Grants CA 16673, 2.3 Reagents CA13148 and AM 03555 and by an Arthritis Foundation Arthritis Pediatric Center Grant. Recipient of an Arthritis Foundation Fellowship. Correspondence: Lawrence Brent, Division of Rheumatology, Hahnemann University, MS 117, Broad and Vine Streets, Philadelphia, PA 19102, USA Abbreviations: BCDF B cell differentiation factor BCGF B cell growth factor 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

Recombinant human IL 2 (rIL 2; 5 x lo6 U/mg), used as a standard source of IL 2 in all assays, was a gift from Dr. Arabella Tilden (University of Alabama, Birmingham, AL). Anti-human IL 2 mAb KNT-1 [11] and L-61-d (both IgGI) were used in IL 2 inhibition assays. The CT3 mAb is directed against the chicken TcR and was used as an isotype-matched, irrelevant control antibody (a gift from Joseph Sowder, University of Alabama, Birmingham, AL). 0014-2980/90/0505-1125$02.50/0

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Eur. J. Immunol. 1990. 20: 1125-1129

L. H. Brent,T. Miyawaki, M. €! Everson and J. L. Butler

An anti-murine IL 2R mAb, AMT 13 (Boehringer Mannheim Biochemicals, Indianapolis, IN), of the IgGza isotype was used to block the I L 2 R on HT-2 cells. Anti-Leu-1 (Becton Dickinson, Mountain View, CA) was used as an isotype-matched, irrelevant control mAb.

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2.4 IL 2 assays Biological assays were performed to compare known concentrations of rIL 2 to various dilutions of KRC-18 SN [12]. HT-2 cells, 5 x lo3 well, were cultured in 0.1 ml of media in 96-well flat-bottom microtiter plates (Costar, Cambridge, MA). The cultures were incubated for 24 h at 37°C in 95% humidified air with 5% COz. Each well was pulsed with 1 pCi (= 37 kBq) of [3H]dThd (Amersham, Arlington Heights, IL) for the last 8 h of the culture period, and [3H]dThd incorporation was determined using standard liquid scintillation techniques. Anti-human IL 2 and control mAb were added at the initiation of cell culture in the amounts indicated. Anti-murine IL 2R and control mAb (20 pg) were incubated with 2 x lo5 HT-2 cells for 20 min at 4°C. The cells were then washed twice in RPMI 1640, resuspended in medium and cultured in microtiter plates as described above. Each assay was performed in triplicate, and the data are reported as cpm f SEM.

2.5 Molecular mass determination KRC-18 culture SN (300 pl) was collected and concentrated 40-fold using an Amicon YMlO ultrafiltration membrane (Amicon Corp., Danvers, MA). Concentrated SN (4 ml) was applied to a Sephadex G-75 (Pharmacia Fine Chemicals, Piscataway, NJ) gel filtration column (1.6 X 60 cm; bed volume 120 ml; flow rate 7.4 cm/h) and eluted at 4 "C with PBS, pH 7.4. Two-milliliter fractions were collected, filter sterilized and assayed for IL 2 activity. Column calibrations were determined using thyroglobulin, y-globulin, OVA, myoglobin and vitamin B12 with molecular mass of 670, 158, 44, 17 and 1.35 kDa, respectively (Bio-Rad Laboratories, Richmond, CA). Elution patterns were measured with a Uvicord S spectrophotometer (A277) (LKB, Gaithersburg, MD).

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Figure 1. IL 2 activity in the SN from the KRC-18 clone. HT-2 cells (5 x 10'lwell) were cultured in 0.1 ml of medium in 96-well flat-bottom microtiter plates. [3H]dThd incorporation was determined after 24 h of culture. (A) rIL2. (B) KRC-18 SN. Data shown are from one of five experiments with similar results.

incorporation by HT-2 cells cultured with serial dilutions of rIL 2 or KRC-18 SN. The dose-response curves are strikingly similar and suggest that the KRC-18 B cell line constitutively secretes detectable amounts (7-8 U/ml) of IL 2 activity. 3.2 Molecular mass determination of KRC-18 IL 2 activity

Concentrated KRC-18 culture SN was applied to a Sephadex G-75 gel filtration column, and eluted fractions were 35 30

2.6 Cytoplasmic staining for IL 2 KRC-18 cells and normal human blood Tcells, activated for 72 h with PHA (5 pglml) and PMA (10 ng/ml), were used. The cells were then harvested, washed and cytocentrifuged onto glass slides. The slides were fixed and stained with KNT-1 or CT3 mAb. Next, they were washed with PBS and counterstained with goat anti-mouse Ig conjugated with fluorescein (Cappel Worthington, Malvern, PA). Cytoplasmic staining was analyzed by fluorescence microscopy.

3 Results 3.1 IL 2 activity produced by KRC-18 SN from the EBV+ marmoset B cell line, KRC-18, was tested for IL 2 activity using the IL 2-dependent, murine T cell line, HT-2. Fig.-l depicts the pattern of [3H]dThd

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F r a c t i o n Number Figure 2. Sephadex (3-75 gel filtration chromatography of KRC-18 SN. Concentrated KRC-18 SN was fractionated over a Sephadex (3-75 gel filtration column. Excluded (V,,) and total (V,) column volumes and molecular mass markers are indicated by the open and closed arrows, respectively (described in Sect. 2.5). Fractions of 2 ml were collected and tested for IL 2 activity by measuring the proliferative response of HT-2 cells ( 0 )as previously described. Protein concentration was determined by measuring absorption at 277 nm (0). Data are from one of three experiments with similar results.

Eur. J. Immunol. 1990.20: 1125-1129

Interleukin 2 production by a marmoset B cell line

tested for I L 2 activity. Fig. 2 indicates that peak IL 2 activity was present in the 20-22-kDa fraction (fraction number 37). This molecular mass range is similar to that reported for human IL2 [13]. We also compared the molecular mass of the IL 2 activity present in KRC-18 SN to that secreted by the h4LA-144 gibbonTcell line [14] and found that the peak activity of MLA-144 IL 2 waspresent in the same fraction as KRC-18 IL 2 (data not shown).

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3.3 Anti-IL 2 mAb blocks KRC-18 IL 2 activity An anti-human I L 2 mAb of the IgGl isotype, KNT-1, inhibited HT-2 proliferation induced by rIL 2 or KRC-18 IL 2 activity in a dose-dependent manner (Fig. 3). Another anti-IL 2 antibody, L-61-d, gave similar results (data not shown). In contrast, an isotype-matched, irrelevant control mAb, CT3, had no effect on IL 2-induced HT-2 proliferation.These data demonstrate that KRC-18 IL 2 activity can be blocked by anti-human IL 2 mAb and suggest that the IL 2 activity produced by KRC-18 has epitopes similar to those found on human IL 2.

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The activities of both rIL 2 and KRC-18 IL 2 could be inhibited by AMT 13, an anti-murine IL 2R mAb (Fig. 4). HT-2 cells preincubated with AMT 13 did not respond to either rIL 2 or KRC-18 SN. Anisotype-matched, irrelevant (A)

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titer Figure 4. Anti-murine IL 2R mAb inhibits proliferation of HT-2 cells in response to KRC-18 SN. The anti-murine IL 2R mAb, AMT 13 (IgGZa), was compared with an isotype-matched, irrelevant control mAb, anti-Leu-1. HT-2 cells (2 x lo5)were incubated with 20 p g h l of mAb for 20 min at 4"C,washed twice and cultured in microtiter plates in an IL 2 assay. Data shown are from one of five experiments with similar results.

control mAb, anti-Leu-1, did not inhibit the proliferative response of HT-2 to either source of IL2. These results suggest that KRC-18 IL 2 activity induces HT-2 proliferation by binding to IL2R on the HT-2 cell surface in a manner similar to rIL 2. 3.5 KRC-18 cells contain cytoplasmic IL 2

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To document the presence of intracellular IL 2 in KRC-18 cells, cytocentrifuge preparations were made and stained with either the anti-IL 2 mAb, KNT-1, or CT3 as a negative control mAb. The presence of cytoplasmic IL 2 was analyzed in both KRC-18 cells and mitogen-activated human blood T cells. Approximately 50% of the activated T cells stained positively with KNT-1 (Fig. 5 ) , and the brightest staining was observed in the area of the golgi apparatus. In contrast, >90% of KRC-18 cells stained positive for cytoplasmic I L 2 and showed a more diffuse staining pattern than that observed in activated T cells.

4 Discussion 0

25

50

100

200

Fg/ml Figure 3. Anti-IL 2 mAb inhibits the proliferation of HT-2 cells in response to KRC-18 SN. IL 2 assay was performed as previously described. KNT-1 is an IgGl mAb directed against IL 2. CT3 is an isotype-matched, irrelevant control mAb. (A) rIL 2. (B) KRC-18 SN. Data shown are from one of five experiments with similar results.

B cells have been shown to produce several lymphokines whose activities correspond to IL 1 [2], BCGF [4-81 and BCDF [3]. In this report, we document the constitutive production of IL 2 by an EBV+ marmoset B cell line.The B lineage cell line expresses IgM on its surface, secretes IgM into the culture SN and possesses no surface antigens specific for other cell lineages. The IL 2 activity produced

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Eur. J. Irnmunol. 1990. 20: 1125-1129

L. H. Brent,T. Miyawaki, M. F! Everson and J. L. Butler

(A)

(6)

Figure 5. KRC-18 cells contain cytoplasmic IL 2. (A) Normal human T cells activated with PHA and PMA and stained with KNT-1. (B) Normal human T cells activated with PHA and PMA and stained with CT3. (C) KRC-18 cells stained with KNT-1. (D) KRC-18 cells stained with CT3.

by KRC-18 cells is similar to IL2 produced by normal mitogen-activated T lymphocytes in that its molecular mass is 20-22 kDa, and its biological activity is inhibited by mAb directed against IL 2 and the IL 2R. More than 20 human EBV+ and EBV- B cell lines were screened for IL2 production using the HT-2 cell assay system. These cell lines represented various stages of B cell development, from pre-B cells (acute lymphocytic leukemia cell lines) to plasma cells (myeloma cell lines). Several EBV+ B cell lines were also studied to assess whether mitogenic stimulation (PMA) could induce IL 2 production. Similarly, normal human blood B cells were studied to determine whether mitogenic stimulation (anti-IgM antibodies, Staphylococcus uureuS Cowens strain 1 or PMA) could induce IL 2 production. No IL 2 activity could be detected in any of the culture SN using a biological assay, and it is possible that normal B cells and most B cell lines are not able to produce IL 2 under any circumstances. However, we have not ruled out the possibility that small amounts of IL 2 are in fact being produced by these cells and are subsequently being adsorbed from the medium by the increased number and affinity of IL2R present on mitogen-stimulated normal B cells [9] and EBV+ B cell lines [15]. It is feasible to search for IL 2 mRNA as an indication of IL 2 production in instances where autocrine utilization of the protein may prevent its detection in culture SN using biological assays.To this end, we searched for IL2 message in KRC-18 cells; however, we were unsuccessful in our attempts to hybridize human probes (IL 2, Cp and JH) to marmoset RNA or DNA. The EBV+ parent cell line of KRC-18, B95-8, produces very little IL2 activity (0.0014.002 U/ml). After LD cloning, low levels of IL 2 activity were detected in 70% of the clones, and 10% produced significant amounts of IL 2

activity. IL 2 production was not enhanced by mitogenic stimulation with PMA or other B cell mitogens. Addition of mAb to IL2 or IL2R did not affect the growth characteristics of the B95-8 clones, suggesting that IL 2 is not directly responsible for the growth of these cells. We have no direct evidence as to why this particular B cell line produces IL 2 activity. Possibly, the EBVgenome is inserted near the IL2 gene resulting in its transcription and ultimately IL 2 production. B cell-derived lymphokines may be important in the regulation of B lymphocyte proliferation and differentiation. The production of low molecular mass BCGF and BCDF [ l , 3-81 would suggest that B cells are capable of regulating, at least in part, their own growth and differentiation by antigen nonspecific mechanisms. The production of IL 1activity by B cells [2] adds the further possibility of regulation of T cell activation. The identification of a marmoset B cell line producing IL 2 suggests that B cells may have the potential to participate in IL 2-mediated T and B cell activation and differentiation in this animal system. Received June 13, 1988; in final revised form January 15, 1990.

5 References Matsushima, K., Kuang,Y.-D., Tosato, G., Hopkins, S. J. and Oppenheim, J. J., Cell. Immunol. 1985. 94: 406. Mastushima, K.,Tosato, G . , Benjamin, D. and Oppenheim, J. J., Cell. Immunol. 1985. 94: 418. Yoshizaki, K., Nakagawa, T., Fukunaga, K., Tseng, L. T., Yamamura,Y. and Kishimoto, T., J. Imrnunol. 132: 2948. Jurgensen, C. H., Ambrus, Jr., J. L. and Fauci, E S., J. Immunol. 1986. 136: 4542.

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5 Gordon, J., Ley, S. C., Melamed, M. D., English, L. S. and Hughes-Jones, N. C., Nature 1984. 310: 145. 6 Gordon, J., Aman, P., Rosen, A., Emberg, I., Ehlin-Henriksson, B. and Klein, G., Znt. J. Cancer 1985. 35: 251. 7 Ambrus, J. L., Jr. and Fauci, A. S., J. Clin. Invest. 1985. 75: 732. 8 Gordon, J., Ley, S. C., Melamed, M. D., Aman, P. and Hughes-Jones, N. C., J. Exp. Med. 1984. 159: 1554. 9 Muraguchi, A., Kehrl, J. H., Longo, D. L.,Volkman, D. J., Smith, K. A. and Fauci, A. S., J. Exp. Med. 1985. 161: 181. 10 Maino,V C. and Pace, D. R., J. Immunol. 1985. 134: 2419.

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11 Sato, H., Natsuume-sakai, S., Miyawaki,T. ,Taga, K., Hatano, M. and Taniguchi, N., J. Biol. Resp. Mod. 1986. 5: 191. 12 Gillis, S., Ferm, M. M., Qu,W. and Smith, K. A., J. Immunol. 1978. 120: 2027. 13 Stadler, B. M. and Oppenheim, J. J., Lymphokines 1982. 6: 117. 14 Rabin, H., Hopkins 111, R. F., Ruscetti, F.W., Neubauer, R. H., Brown, R. L. and Kawakami,T. G., J. Immunol. 1981. 127: 1852. 15 Polke, C. F., Lowenthal, J. W., Roth, S. A., Rohwer, I?, MacDonald, H. R. and Kalden, J. F., Eur. J. Immunol. 1986. 16: 146.