Dissociation of interleukin-2-mediated cell proliferation and interleukin-2 receptor upregulation in adult T-cell leukemia cells

Leukemia Research Vol. IlL No. 12, p p 1447-I453, 1986. Printed in Great Britain.

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

DISSOCIATION OF INTERLEUKIN-2-MEDIATED CELL P R O L I F E R A T I O N AND INTERLEUKIN-2 R E C E P T O R U P R E G U L A T I O N IN A D U L T T-CELL L E U K E M I A CELLS* TOSHIYUKI HORI, TAKASHI UCHIYAMA, HIROSHI UMADOME, SHIGEKI TAMORI, MITSURU TSUDO, KOICHI ARAKI and HARUTO UCHINO The First Division of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto, Japan and The Second Division of Internal Medicine, School of Medicine, University of the Ryukyus, Okinawa, Japan (Received 10 June 1986) Abstract--We studied cell proliferation and interleukin-2 (IL-2) receptor upregulation mediated by exogenous IL-2 in leukemic ceils from adult T-cell leukemia (ATL) patients to characterize some aspects of abnormal IL-2 receptor expression in ATL. Leukemic cells from 7 ATL patients examined showed no or very poor proliferative response to IL-2 though they expressed IL-2 receptors without any stimulation. In contrast, ATL leukemic cells cultured with IL-2 for 2 days expressed about twice as many IL-2 receptors as those of cells cultured without IL-2. Thus, in ATL leukemic cells, there seems to be a dissociation between the signal(s) for two different effects mediated by IL-2, cell proliferation and IL-2 receptor upregulation. Key words: Adult T-cell leukemia, IL-2, IL-2 receptor.

INTRODUCTION ADULT T-cell leukemia (ATL) [1-3] has been regarded as one of the best models for studying the leukemogenesis in man since a close association between ATL and human T lymphotropic retrovirus or human T-cell leukemia/lymphoma virus type I (HTLV-I) [4] was demonstrated [5-7]. It is accepted that HTLV-I is the primary causative agent of ATL. However, only a small population of HTLV-I-infected individuals develop leukemia, and the precise mechanism of that process remains unclear. We previously reported that leukemic cells from ATL patients (ATL cells) express interleukin-2 (IL-2) * Supported in part by grants from the Ministry of Education, Science and Culture, Japan, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and from the Yamanouchi Foundation for Research on Metabolic Disorders. Abbreviations: ATL, adult T-cell leukemia; HTLV-I, human T-cell leukemia/lymphoma virus type I; IL-2, interleukin-2; Ag, antigen; A TLA, ATL-associated antigen; PHA, phytohemagglutinin FCS, fetal calf serum; SSC, standard saline citrate. Correspondence to: Dr Takashi Uchiyama, The First Division of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606, Japan.

receptor/Tac antigen (Ag) recognized by anti-Tac monoclonal antibody [8, 9], which may be involved in their neoplastic growth, considering the essential role of the IL-2/IL-2 receptor system in normal T-cell proliferation. Subsequently, we also found that most ATL cells do not proliferate in response to IL-2 despite the expression of a certain amount of high affinity receptors [10]. Recent studies revealed that under appropriate conditions, exogenous IL-2 upregulates the expression of IL-2 receptor on T cells as well as induces cell proliferation [11-14]. In other words, the signal(s) for at least two different effects are transmitted through the IL-2 receptor. In the present study, we investigated whether IL-2 regulates the expression of IL-2 receptor on ATL cells to characterize some aspects of the abnormal signal transmission through the IL-2 receptor in ATL cells.

MATERIALS AND METHODS Patients Seven ATL patients with a high white blood cell count (>39 x 109/1) were examined. The age of the patients ranged from 24 to 64. The diagnosis of ATE was made by accepted criteria for clinical features, hematological characteristics and laboratory findings. Sera from all patients were positive for anti-ATLA antibody [5]4 and the monoclonal HTLV-I proviral

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TOSHIYUKI HORI et al.

TABLE 1. PATIENTSWITH ATL Patient

Age

Sex

WBC (× 109/1)

Anti-ATLA antibody

HTLV-1 provirus

MS YS DM "IT HA SH KI

24 35 61 62 55 64 57

M M M F M F M

77 250 130 40 230 50 39

+ + + + + + +

+ + NT NT NT + NT

NT, not tested.

DNA integration in leukemic cells was demonstrated in all 3 cases examined (Table 1).

Cell separation and culture Peripheral blood mononuclear cells were separated from heparinized blood from ATL patients and normal volunteers by Ficoll-Conray density gradient centrifugation. Normal peripheral blood mononuclear cells, which had been cultured with 0.1% phytohemagglutinin-P (PHA-P) (DIFCO Laboratory, Detroit, MI) for 7 days, were washed and further incubated without PHA-P for 24 h. They were used in the present studies as normal activated T cells (normal PHA-blasts). ATL leukemic cells and PHA-blasts were cultured in RPMI 1640 medium (Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) containing 10% fetal calf serum (FCS) (GIBCO, Grand Island, NY), 20 Ixg/ml tobramycin, with or without 1 U/ml recombinant human IL-2 (kindly supplied from Takeda Chemical Industries Inc., Osaka, Japan) at 37°C for 2 days, and subjected to the analysis of IL-2 receptor expression. Proliferative response to IL-2 ATL leukemic ceils and normal PHA-blasts were studied for the proliferative response to IL-2. One hundred thousand cells were cultured in 200 Ixl of RPMI 1640 medium containing 10% FCS and 20 Ixg/ml Tobramycin in the presence of various concentrations of IL-2 in a 96 well microtiter plate (Falcon Labware, Oxnard, CA) at 37°C in a humid atmosphere with 5% CO2 for 72 h. The cultured cells were pulsed with 0.5 IxCi/ well [3H]thymidine (2 Ci/mol, Amersham Corp., Arlington Heights, IL) for the last 6 h, followed by precipitation onto glass fiber filters, and the radioactivity was counted by a liquid scintillation counter. Flow cytofluorometric analysis of the IL-2 receptor Two to five hundred thousand cells were incubated with a saturating amount of anti-Tac antibody at 4°C for 30 min, washed twice with Hanks' balanced salt solution containing I mg/ml bovine serum albumin and 1 mg/ml sodium azide, and then incubated with fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat anti-mouse IgG antibody (Cappel Laboratories, Cochranville, PA) at 4°C for 30 min. Control staining was performed with normal BALB/c mouse serum instead of anti-Tac antibody. Washed and resuspended cells were subjected to cytofluorometric analysis using Spectrum III (Ortho Diagnostic System, Westwood, MA). Radiolabeled IL-2 binding assay Radiolabeled IL-2 binding assay was performed according

to the method described previously [9]. In brief, recombinant human IL-2 was radiolabeled with Na[125I] (16.9 mCi/txg of iodine, Amersham Corp.) by the chloramine T method. Specific radioactivity of radiolabeled IL-2 was 7000-9000 cpm/ ng and the effective bindability was >90%. Cells were extensively washed and incubated at 37°C in IL-2 free RPMI 1640 medium containing 10% FCS for 60 min to promote dissociation or degradation of endogenous or exogenous bound IL-2. Serial dilutions of radiolabeled IL-2 and 2-5 × 10 6 cells in a total volume of 500 ~tl of RPMI 1640 medium containing 25 mM Hepes, pH 7.4, 10 mg/ml bovine serum albumin, 1 mg/ ml sodium azide were incubated at 37°C for 40 min. Then, 200 ~tl of the cell suspension in duplicate was centrifuged through a layer of a mixture of 20% olive oil (Nakarai Chemicals Ltd, Kyoto, Japan) and 80% Dim-butyl phthalate (Nakarai Chemicals Ltd). The tips of the tubes containing the cell pellet were cut off and the radioactivity was counted in a gamma counter. Non-specific binding was measured by incubating cells with radiolabeled IL-2 in the presence of a 250fold excess of unlabeled IL-2. The number of IL-2 binding sites and the affinity were estimated by Scatchard analysis.

Radiolabeled anti-Tac antibody binding assay Radiolabeled anti-Tac antibody binding assay was performed as previously described [9]. Cytoplasmic dot hybridization Cytoplasmic dot hybridization was performed according to the method described by White et al. [15]. In brief, 5 x 106 ceils were washed with PBS and resuspended in 45 ~d cold 10 mM Tris (pH 7.0) and 1.0 mM EDTA. They were then lysed by adding 5 ~1 of 5% Nonidet P-40 and 2.5 ~tl of 200 mM vanadyl ribonucleoside complex (Bethesda Research Laboratories Life Technologies Inc., MD) with 5 min of mixing on ice. Following pelleting of nuclei (15,000 x g, 2 min), 50 ~tl of the supernatant was transferred to a sterile 1.5 ml tube containing 30 ~tl of 20× standard saline citrate (SSC: 0.15 M NaCI/0.015 M trisodium citrate) and 20 ~tl of 37% (w/w) formaldehyde and the mixture was incubated at 60°C for 15 min. The samples serially diluted with 15× SSCwere then applied with suction to a 4-mm diameter spot in a nitrocellulose sheet BA85 (0.45 ~tm pore size) (Schleicher & Schull Inc., Dassel, F.R.G.) supported by S&S #470 paper employing a 96-hole Minifold apparatus. The nitrocellulose sheet was baked at 80°C for 6 h. The Sau 3A fragment of the Tac-2 clone was used as the IL-2 receptor cDNA probe [16]. Prehybridization of the sheet, nick translation of the probe, hybridization, and autoradiography were performed as described [17].

IL-2 receptor upregulation in ATL cells

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TABLE2. PROLIFERATIVERESPONSETO IL-2 [3H]TdR uptake (counts/min ± S.D.) Patient

Medium

IL-2 1 U/ml

MS YS DM TT HA SH KI Normal PHA-blasts (n = 7)

762 ± 248 146 ± 24 150 ± 19 1531 ± 852 251 ± 90 41 ± 3 8694 ± 627 250 ± 137

2713 ± 149 211 ± 67 1039 ± 41 132 ± 64 428 ± 19 597 ± 123 12,283 ± 625 38,179 ± 18,577

RESULTS

Proliferative response to IL-2 To ascertain the results in our previous report, we studied the proliferative response of the leukemic cells from 7 A T L patients to exogenous IL-2. Leukemic cells from all A T L patients examined in the present study showed no or very poor proliferative response to IL-2 compared with that of normal PHA-blasts (Table 2). Also in the presence of higher concentrations of IL-2 (up to 125 U / m l , data not shown), leukemic cells did not proliferate.

Flow cytofluorometric analysis of IL-2 receptor Leukemic cells from all ATL patients examined expressed IL-2 receptor detected by anti-Tac antibody immediately after the separation of the cells. We then studied the effect of exogenous IL-2 on the expression of IL-2 receptor in A T L cells. After 2 days of culture, there was no significant difference in cell number or/ and viability between the culture of ATL cells with and without IL-2. In 6 of 7 cases an augmentation of IL-2 receptor expression was observed after culture in medium alone. Most A T L cells cultured with IL-2

~

150

KI

PHA-blasts

expressed about twice as many IL-2 receptors, indicated by the mean fluorescence intensity, as those of cells cultured without IL-2. The magnitude of IL-2-mediated IL-2 receptor upregulation observed in ATL cells was comparable to that of normal PHA-blasts (Fig. 1). No significant IL-2 receptor upregulation by IL-2 was detected when ATL cells were cultured in the presence of 10 ~tM cycloheximide (data in 2 cases examined, not

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10 Free IL-2(nM)

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FIG. 1. Changes in IL-2 receptor (Tac antigen) expression in ATL leukemic cells (left) and normal PHA blasts (right). Cells were cultured with or without 1 U/ml IL-2 for 2 days and the mean fluorescence intensity of IL-2 receptor stained with antiTac antibody was-measured.

10,0oo Bound IL-2(molecules/cell)

FIG. 2. (A) Radiolabeled IL-2 binding assay of 2-day-cultured ATL leukemic cells from patient MS. Cells were cultured in medium alone (0) or medium containing 1 U/ml IL-2 (A) for 2 days. (B) A Scatchard plot of the radiolabeled IL-2 binding data shown in (A).

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TOSHIYUKIHORI et al. TABLE 3. NUMBER OF ANTI-TAc AND IL-2 BINDING SITES

IL-2 (sites/cell) Patient MS YS DM

Condition Fresh Medium IL-2 Fresh Medium IL-2 Fresh Medium IL-2

Anti-Tac (sites/cell)

High affinity

Low affinity

2800 8800 22,000 2200 14,000 22,000 2400 11,900 27,300

130 500 300 180 1000 500

2300 10,000 30,000 1600 37,000 58,000

shown) indicating that de novo protein synthesis is necessary for IL-2 receptor upregulation by IL-2. Radiolabeled IL-2 and radiolabeled anti-Tac antibody binding assay To investigate more quantitatively the changes in IL2 receptor expression, leukemic cells from two patients were analysed by radiolabeled IL-2 binding assay. In patient MS, Scatchard analysis revealed that leukemic cells cultured in medium alone displayed 500 receptors/ cell with high affinity (Kd: 8 pM) and 10,000 receptors/ cell with low affinity (Kd: 6 nM). In contrast, leukemic cells cultured with IL-2 displayed 300 receptors/ceU with high affinity and 30,000 receptors/cell with low affinity (Fig. 2). Radiolabeled IL-2 binding assay performed in another case (YS) gave similar results. Radiolabeled anti-Tac antibody binding assay performed in three patients revealed that the number of anti-Tac antibody binding sites/cell was comparable to the total number of IL-2 binding sites/cell and proportional to the mean fluorescence intensity of IL-2 receptor in flow cytofluorometry (Table 3). Thus, also in ATL cells, IL2 upregulated the total expression of IL-2 receptor by mainly increasing the number of low affinity receptors. Cytoplasmic dot hybridization In order to study whether IL-2 upregulates IL-2 receptor expression at the m R N A level, we isolated the cytoplasm from leukemic cells at various times of culture with or without IL-2 and performed cytoplasmic dot hybridization in 2 patients. As shown in Fig. 3 (represented by patient SH), the cytoplasmic IL-2 receptor m R N A increased after a short term culture in medium alone. In addition, IL-2-mediated augmentation of IL-2 receptor m R N A was detected within 48 h, and it became maximum after 72 h.

DISCUSSION In the present study, we demonstrated that exogenous IL-2 augmented IL-2 receptor expression in leukemic cells from A T L patients although IL-2 did not induce proliferation of these cells. The IL-2-mediated IL-2 receptor upregulation was mainly due to an increase of low affinity receptors and required de nooo protein

synthesis preceded by an increase of IL-2 receptor mRNA. It is unlikely that IL-2-mediated IL-2 receptor upregulation was attributed to contaminated normal lymphocytes because cells separated from ATL patients with a high white blood cell count contained a very small population of normal lymphocytes, and normal resting T cells express a negligible amount of IL-2 receptors even after 2 days of culture with IL-2 (unpublished data). Another possibility is that an apparent IL2 receptor upregulation might be observed if a minor population of leukemic cells that expressed a large amount of IL-2 receptors proliferated selectively in the presence of IL-2. Such selection or change of population seems unlikely because leukemic cells examined in the present study did not proliferate in response to IL-2 and there was no significant difference in cell number or/and viability between the culture with and without IL-2. In normal activated T cells, it was reported that IL-2 itself upregulates IL-2 receptor expression under appropriate conditions [11-13]. Subsequently, it was also demonstrated that IL-2 augments IL-2 receptor expression at the transcriptional level [14]. Thus, the signal(s) for two different effects, cell proliferation and IL-2 receptor upregulation, appear to be generated by IL-2 binding to IL-2 receptor expressed on the cell membrane. The precise mechanism and pathway of the transduction and transmission of the signal(s) to the cell nucleus remain unclear. We demonstrated in this study that the signal(s) for one effect could be transmitted through IL-2 receptor in dissociation with that for the other effect in ATL cells. The mechanism of the poor proliferative response to IL-2 in ATL cells is to be clarified. It may be attributed to unknown intra-cellular conditions rather than the number of high affinity IL-2 receptors because cultured leukemic cells that expressed a sufficient amount of high affinity IL-2 receptors (>1000 receptors/cell) did not proliferate in response to IL-2 (unpublished data). In contrast to A T L cells, leukemic cells from an HTLV-I-negative T-CLL patient, which expressed a small amount of IL-2 receptors, showed good proliferative response to IL-2 as well as IL-2-mediated IL-2 receptor upregulation [17]. Thus, the poor proliferative response to IL-2 despite IL-2 receptor expression seems to be a characteristic feature of ATL leukemic cells.

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o

FIG. 3. Cytoplasmic dot hybridization. Cytoplasm was prepared from fresh or cultured leukemic cells (patient SH) and from fresh or PHA-activated normal peripheral blood lymphocytes. Leukemic cells were cultured with or without 1 U/ml IL-2 for up to 3 days. Normal lymphocytes were cultured with 0.1% PHA-P for 20 h. Serial dilutions of each sample from 4 x 105 cells were applied to a nitrocellulose sheet, which was then hybridized with 32p-labeled Sau-3A fragment of IL-2 receptor cDNA and autoradiographed.

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IL-2 receptor upregulation in ATL cells H o w e v e r , leukemic cells f r o m a few A T L patients were recently found to proliferate in response to IL-2 and could be maintained for a long term with recombinant IL-2. Cell lines established from these patients were d e m o n s t r a t e d to be the same clone as the primary leukemic cells by S o u t h e r n hybridization analysis of Tcell receptor beta chain gene r e a r r a n g e m e n t [19]. W e need further studies on the difference between most A T L cases and the rare atypical A T L cases in regard to IL-2 receptor signal transmission, in order to elucidate the role of the I L - 2 / I L - 2 receptor system in A T L leukemogenesis.

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8. Uchiyama T., Broder S. & Waldmann T. A. (1981) A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of antiTac monoclonal antibody and distribution of Tac(+) cells. J. lmmun. 126, 1393. 9. Hattori T., Uchiyama T., Toibana T., Takatsuki K. & Uchino H. (1981) Surface phenotype of Japanese adult Tcell leukemia cells characterized by monoclonal antibodies. Blood 58, 645. 10. Uchiyama T., Hori T., Tsudo M., Wano Y., Umadome H., Tamori S., Yodoi J., Maeda M., Sawami H. & Uchino H. (1985) Interleukin-2 receptor (Tac antigen) expressed on adult T-cell leukemia cells. J. clin. Inoest. 76, 446. 11. Reem G. H. & Yeh N. H. (1984) Interleukin 2 regulates expression of its receptor and synthesis of gamma interferon by human T lymphocytes. Science, N.Y. 225, 429. 12. Welte K., Andreeff M., Platzer E., Holloway K., Rubin B. R., Moore M. A. & Mertelsmann R. (1984) Interleukin 2 regulates the expression of Tac antigen on peripheral blood T lymphocytes. J. exp. Med. 160, 1390. 13. Smith K. A. & Cantrell D. A. (1985) Interleukin 2 regulates its own receptors. Proc. natn. Acad. Sci. U.S.A. 82, 864. 14. Depper J. M., Leonard W. J., Drogula C., Kronke M., Waldmann T. A. & Greene W. C. (1985) Interleukin 2 (IL-2) augments transcription of the IL-2 receptor gene. Proc. natn. Acad. Sci. U.S.A. 82, 4230. 15. White B. A. & Bancroft F. C. (1982) Cytoplasmic dot hybridization. J. biol. Chem. 257, 8569. 16. Nikaido T., Shimizu A., Ishida N., Sabe H., Teshigawara K., Maeda M., Uchiyama T., Yodoi J. & Honjo T. (1984) Molecular cloning of cDNA encoding human interleukin2 receptor. Nature, Lond. 311, 631. 17. Honjo T., Obata M., Yamawaki-Kataoka Y., Kataoka T., Kawakami T., Takahashi N. & Mano Y. (1979) Cloning and complete nucleotide sequence of mouse immunoglobulin 1 chain gene. Cell 18, 559. 18. Tsudo M., Uchiyama T., Umadome H., Wano Y., Hori T., Tamori S., Uchino H., Kita K., Chiba S., Mitsutani S. & Nesumi N. (1986) Expression of interleukin-2 receptor on T-CLL cells and their response to interleukin-2. Blood 67, 316. 19. Maeda M., Shimizu A., Ikuta K., Okamoto H., Kashihara M., Uchiyama T., Honjo T. & Yodoi J. (1985) Origin of human T-lymphotropic virus 1-positive T cell lines in adult T-cell leukemia. J. exp. Med. 162, 2169