61

Journal of Immunological Methods, 142 (1991) 61-72 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100262F JIM 06030

Monoclonal antibodies defining distinct epitopes of the human IL-2 receptor/3 chain and their differential effects on IL-2 responses Kazuyuki O h b o 1, Toshikazu Takeshita 1, H i r o n o b u A s a o 1, Yumiyo Kurahayashi 1, Kotaro T a d a 1, Hisashi Mori 2, Masanori H a t a k e y a m a 2, Tadatsugu Taniguchi 2 and Kazuo Sugamura 1 1 Department of Microbiology, Tohoku University School of Medicine, Sendai and 2 Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan (Received 19 February 1991, accepted 29 April 1991)

We have established and characterized five new monoclonal antibodies (mAbs) which specifically immunoprecipitate the human interleukin-2 receptor/3 chain (IL-2R/3). One of them, TU30, recognizes the intracytoplasmic 'serine-rich region' of IL-2R/3 that is critical for IL-2 signal transduction. The others, TU12, TU21, TU23 and TU25, completely inhibit IL-2 binding, as does the previously characterized TU27. However, reciprocal binding competition assays show that the epitopes recognized by the individual mAbs are different from each other. The mAbs inhibit the growth of IL-2-dependent cells. The magnitude of their inhibitory effects is dependent on not only the affinities of the mAbs for IL-2R/3 but also upon the number of IL-2Ra subunits expressed on IL-2-dependent cells. These mAbs should be useful in studying the structure and function of the IL-2R. Key words: Interleukin-2 receptor; Monoclonal antibody; Epitope; Affinity; Interleukin-2-dependent cell growth

Introduction

Interleukin-2 (IL-2) is a growth or differentiation factor for certain lymphocytes and monocytes. IL-2 transduces a signal through a specific cell surface receptor which exists in 3 different

Correspondence to: K. Sugamura, Department of Microbiology, Tohoku University School of Medicine, 2-1 Seiryomachi, Sendai 980, Japan. Abbreviations: DSS, disuccinimidyl suberate; FACS, fluorescence-activated cell sorter; HTLV-I, human T-cell leukemia virus type I; Kd, dissociation constant; TdR, thymidine deoxyribose.

isoforms; low-affinity ( K d = 10 nM), intermediate-affinity ( K d -- 1 nM), and high-affinity (K d -10 pM) (Wang et al., 1987). The isoforms consist of either the a chain (P55), /3 chain (p75), or a complex of a and /3 chains, respectively (Sharon et al., 1986; Tsudo et al., 1986; Dukovich et al., 1987; Robb et al., 1987; Teshigawara et al., 1987). The /3 chain is thought to function in signal transduction since it contains a cytoplasmic domain large enough to encode a functional domain (Hatakeyama et al., 1989a, b), and expression of the /3 chain is necessary for responsiveness to IL-2 (Siegel et al., 1987; Tsudo et al., 1987; Ishii et al., 1988). The a chain lacks a signal transduction domain (Leonard et al., 1984; Nikaido et al.,

62 1984). Transfection with a /3 chain gene cDNA enabled otherwise non-responsive lymphoid cell lines to bind and internalize IL-2, and transduce growth signal to the cytoplasm (Hatakeyama et al., 1989b). However, non-lymphoid cell lines transfected with the/3 chain cDNA failed to bind IL-2 (Minamoto et al., 1990; Tsudo et al., 1990). Therefore, certain lymphoid specific component(s), or modification of the /3 chain, may be required for the expression of functional IL-2R/3. We previously characterized two mAbs specific for IL-2R/3 (Suzuki et al., 1989; Takeshita et al., 1989). Using these mAbs we have recently demonstrated that binding of IL-2 to cells rapidly activates tyrosine kinase activity (Asao et al., 1990). The typical consensus sequence found in tyrosine kinases including several growth factor receptors is not found in the/3 chain gene (Hunter et al., 1985; Hatakeyama et al., 1989a). However, we also showed that immunoprecipitation with the mAbs precipitated a third molecule, p64, which is distinct from the a and /3 molecules (Takeshita et al., 1990). These observations suggest that IL-2R/3 might associate with molecules other than IL-2Ra, such as a tyrosine kinase and/or p64 to form a functional IL-2R for signal transduction. To further investigate IL-2R complex formation and signal transduction we report here characterization of five new mAbs specific for different epitopes of the IL-2R/3 molecule.

Materials and methods

Cell lines The cell lines used were four human IL-2Rpositive T cell lines carrying HTLV-I; MT-1, MT2, ILT-Mat and TL-Mor, two non-human IL-2Rpositive T cell lines; gibbon ape MLA144 and murine CTLL-2, and one human IL-2R-negative cell line; HL-60 (Takeshita et al., 1989). YTU14, which expressed IL-2R/3 but little of IL-2Ra, is a subline of natural killer-like YT cell line (Yodoi et al., 1985) and BAF-BO3, expressed murine IL-2Ra, a murine IL-3-dependent pro-B cell line (Hatakeyama et al., 1989b). ILT-Mat is a human IL-2-dependent cell line which was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, antibi-

otics and 1 nM human recombinant IL-2 (obtained from Shionogi Pharm. Co., Osaka, Japan). CTLL-2 was maintained in the IL-2-containing medium with 50 /zM 2-mercaptoethanol (ME). BAF-BO3 was maintained in RPMI 1640 medium supplemented with 10% FCS, 50/zM 2-ME, and 20% (v/v) conditioned medium from the WEHI3B cell line (Hatakeyama et al., 1989b). F-7, ST-7, H-4, S-25 and A-15 cell lines are transformants of BAF-BO3 cells, which were introduced with wild type or various deletion mutants of cDNA encoding human IL-2R/3 (Hatakeyama et al., 1989b). In detail, F-7 was introduced with pLCKR/3, an expression plasmid containing the entire protein coding region of human IL-2R/3. ST-7 was introduced with pLCKR/3-ST, retaining only the coding region of 27 amino acids of the intracytoplasmic domain of IL-2R/3. H-4, A-15, and $25 were introduced with pLCKR/3-H, pLCKR/3-A and pLCKR/3-S, respectively, which lacked 147 amino acids from the carboxyl terminus, the internal acidic region (a.a. 313-382) and the serine-rich region (a.a. 267-322), respectively (Hatakeyama et al., 1989b). The other cell lines were maintained in RPMI1640 medium containing 10% FCS without IL-2.

Monoclonal antibodies mAbs used were two anti-human IL-2R/3 mAbs; TU27 (IgG1) (Takeshita et al., 1989) and T U l l (IgG1) (Suzuki et al., 1989), H-31 (IgG1) specific for human IL-2Ra (Tanaka et al., 1985), and a control mAb, PAR3 (IgG1) specific for human parvovirus (Yaegashi et al., 1989). H-31 completely inhibits IL-2 binding to IL-2Ra and recognizes IL-2 binding pockets of IL-2Ra (Tanaka et al., 1985; Robb et al., 1988). mAbs were purified from mouse ascites with Affi-Gel protein A MAPS II Kit (Bio-Rad Laboratories, Richmond, CA). Preparation of hybridomas producing rnAbs The IL-2R/3 molecules were purified from lysates of MT-2 cells by TU27 mAb-affinity column chromatography. B A L B / c mice were immunized with the purified/3 molecules emulsified in complete or incomplete Freund's adjuvant at weekly intervals. The sensitized spleen cells were

63 fused with a mouse myeloma cell line, SP2/0Agl4, and hybridoma cell clones were obtained as previously described (Sugamura et al., 1984). mAbs produced by hybridomas were screened by inhibition assays of ~25I-labeled IL-2 binding or by sandwich radioimmunoassays with 125I-labeled TU27 as described previously (Suzuki et al., 1989; Takeshita et al., 1989). In the inhibition assays, 3 hybridomas were established; TU21, TU23 and TU25, and in the sandwich radioimmunoassays, two hybridomas, TU12 and TU30 were established. The class of all mAbs was IgG1.

Antibody binding assay and antibody binding competition assay The purified mAbs were labeled with Na125I by the chloramine T method, and their binding assays for various cell lines were performed as previously described (Fujii et al., 1986). In the Scatchard plots analysis of antibody binding, 1 x 106 cells/well were incubated with serially diluted 125I-mAb for 1.5 h at 4°C. Then, the cell suspension was layered on 850 /zl of sucrose cushion [RPMI 1640 medium containing 1 M sucrose and 0.1% bovine serum albumine (BSA)] and centrifuged at 10,000 × g for 3 min at 4 ° C. Cell-bound, and free radioactivity were measured separately and non-specific binding was determined in the presence of 10/xM of the unlabeled mAb. The binding sites of mAbs were calculated from Scatchard plots analysis and their specific radioactivity; TU27(4.4 × 106 d p m / p m o l ) , TUll(3.6 x 106 dpm/pmol), TU21(1.3 × 106 dpm/pmol), TU23(1.5 x 106 dpm/pmol), and TU25(4.7 x 106 dpm/pmol). Because TU12 lost its binding ability after iodination, the binding assay of TU12 was done by indirect radioimmunoassay as previously described (Tanaka et al., 1986), and its number of binding sites was estimated. Antibody binding competition assays were performed as follows. 1 x 106 MT-2 cells were preincubated in 50 ~1 of 400 nM unlabeled mAbs for 1 h at 4°C, and 25 /~I of 3 nM 125I-mAbs was added and further incubated for 1.5 h at 4 ° C. The cells were then washed 4 times with 0.5% BSA-PBS and the cell-bound radioactivity was measured in a gamma counter.

Radioimmunoprecipitation Cells were surface-labeled with 1 mCi of Na125I by using the iodination reagent (IODO-GEN Pierce Chemical Co., Rockford, IL) and solubilized in the lysis buffer as previously described (Takeshita et al., 1989). The cell lysates were used for sequential immunoprecipitation with TU27 and various mAbs as follows. The lysates were preabsorbed with TU27 or PAR3 and protein A-Sepharose beads (Pharmacia Fine Chemicals, Piscatawat, NJ) coupled with anti-mouse IgG for 4 h at 4 ° C. Subsequently, the preabsorbed lysates were immunoprecipitated with various mAbs and anti-mouse IgG-protein A-Sepharose beads. In the case of an experiment to define the epitope of TU30, the iodinated cell lysates were preabsorbed with protein A-Sepharose beads and immunoprecipitated with TU30- or TU27-coupied anti-mouse IgG-protein A-Sepharose beads. The resultant immunoprecipitates were analyzed by 10% sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) as previously described (Takeshita et al., 1989).

Immunofluorescence staining of permeabilized cells Flow cytometric analysis of permeabilized cells was performed as previously reported (Anderson et al., 1989). YTU14 cells were stabilized by 0.01% formaldehyde in PBS for 20 min on ice. After washing with PBS, the cells were treated with 20 txg/ml of purified digitonin (SIGMA, St. Louis, MO) for 10 min on ice. After washing out "the digitonin, they were incubated with mAbs for 30 min on ice. Subsequently, they were washed with 1% BSA-PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (MBL, Nagoya. Japan) for 30 min on ice. After washing with 1% BSA-PBS, the cells were resuspended in PBS containing 2.5% paraformaldehyde and analyzed by FACScan flow cytometry (Becton Dickinson Immunocytometry System, Inc., Mountain View, CA) as described elsewhere (Ohashi et al., 1989).

Blocking of IL-2 binding with mAbs 1 x 106 MT-2 cells were washed 3 times in PBS, then preincubated with 670 nM of mAbs in binding buffer for 1 h at 4 ° C, and then incubated with serially diluted 125I-IL-2 for 1 h at 4 ° C. The

64

cell suspension was layered on 850/xl of sucrose cushion and centrifuged at 10,000 × g for 3 min at 4 ° C. The 125I-IL-2 in the supernatant and cell precipitate were counted separately. The ~25I-IL-2 bindings were analyzed by Scatchard plots as previously described (Fujii et al., 1986).

Chemical crosslinking with 125I-IL-2 YTU14 cells were affinity-labeled with 2 nM of 125I-IL-2. The cells were preincubated with 2 /zM mAbs for 1 h at 4 ° C and then incubated with 125I-IL-2 for 1 h at 4°C. After centrifugation, the cells were resuspended in 5 ml of PBS containing 1 mM MgCI 2, pH 8.3. The cells were then treated with a chemical crosslinker, disuccinimidyl suberate (DSS, Pierce Chemical Co.) (10 mg/ml) at 4 °C for 20 min and then the reaction was stopped by the addition of 5 ml of 25 mM Tris-HCl (pH 7.4) containing 140 mM NaCI and 1 mM EDTA. The DSS treated cells were solubilized and analyzed by 7.5% SDS-PAGE as previously described (Sharon et al., 1986).

3H-thymidine (3H-TdR) incorporation assay Peripheral blood leukocytes (PBL) were obtained from heparinized peripheral blood of a normal donor by Ficoll-Conray gradient centrifugation. PBL were stimulated with phytohemagglutinin (PHA) for 24 h, and then cultured for 7 days with the IL-2-containing medium. The cells were washed 3 times with PBS and preincubated for'6 h in the medium without IL-2 before use in 3H-TdR incorporation assays. 100-/zl aliquots of the cell suspension (2 × 105/ml) were distributed

into wells of microtiter plates and preincubated with 50 ~1 of various mAbs (50/zg IgG/ml) for 30 min at 37 o C. Subsequently, 50 /zl of serially diluted IL-2 was added and incubated at 37 °C for 48 h. During the last 4 h incubation, 1/xCi of 3H-TdR was added to each well. The incorporated 3H-TdR was determined and mean values of triplicate counts were recorded as described elsewhere (Ishii et al., 1987). 3H-TdR incorporation assays of ILT-Mat cells were done using the same procedure.

Results

Specificity of monoclonal antibodies We isolated five hybridomas specific for IL2R/3 by immunization with affinity purified IL2Rfl protein. We examined the ability of the mAbs to bind the surface of various cell lines by radioimmunoassay. Among them, four mAbs, TU12, TU21, TU23 and TU25, reacted only with cell lines positive for human IL-2R/3, including MT-2, ILT-Mat, TL-Mor and YTU14, but not with cell lines negative for human IL-2R/3, such as HL-60, MT-1 (human IL-2Ra-positive), and CTLL-2 (murine IL-2Ra- and fl-positive) (data not shown). Three out of four mAbs, TU12, TU21 and TU25, bound to both human cells and gibbon ape MLA 144 cells expressing IL-2Rfl, whereas TU23 did not bind to MLA144 cells. The number of binding sites for these mAbs were very similar to that found with TU27 and T U l l (Table I). The other mAb, TU30, failed to bind to all cell lines

TABLE I BINDING SITES OF VARIOUS mAbs Cell line MT-2 YTU14 ILT-Mat TL-Mor MLA144

Binding of mAbs (sites/cell) TU27

TUll

TU12 a

TU21

TU23

TU25

TU30

4,400 11,000 1,700 3,900 1,600

5,100 12,000 1,900 4,400 1,800

4,700 8,500 1,600 4,000 1,500

4,300 8,500 1,800 3,700 1,400

4,100 10,200 1,400 3,200 NS

4,600 11,000 1,600 3,800 1,500

NS b NS NS NS NS

The binding sites of TU12 were calculated in an indirect radioimmunoassay of TU12 in comparison with TU27, because TU12 lost the binding ability after iodination. b Not significant. a

65 Preclear

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Fig. 1. Sequential immunoprecipitation of mAbs. 125I-labeled MT-2 cell lysates were precleared with PAR3 (lane 1, 2, 3, 4, 9, 10, 13, 14) and TU27 (lane 5, 6, 7, 8, 11, 12, 15, 16). They were then precipitated by PAR3 (lane 1, 5), TU27 (lane 2, 6), T U l l (lane 3, 7), TU12 (lane 4, 8), TU21 (lane 9, 11), TU23 (lane 10, 12), TU25 (lane 13, 15), and TU30 (lane 14, 16). The immunoprecipitates were analyzed by 10% SDS-PAGE.

tested, even those positive for IL-2R/3, implying that the TU30 may bind to intracellular or transmembrane domain of IL-2R/3. Radioimmunoprecipitation with these mAbs w e r e c a r r i e d o u t to c o n f i r m t h e i r specificity. T h e lysates w e r e p r e p a r e d f r o m s u r f a c e - r a d i o l a b e l e d

MT-2, precleared with either TU27 or PAR3, a n d t h e n p r e c i p i t a t e d w i t h t h e five m A b s a n d positive controls, TU27 and TUll. All the mAbs including TU30 specifically precipitated a cell surface molecule with 73-79 kDa molecular w e i g h t t h a t c o u l d b e p r e c l e a r e d w i t h T U 2 7 (Fig.

TABLE II RECIPROCAL COMPETITIVE BINDINGS OF mAb Competitor mAbs

Competitive binding of 125I-labeled mAbs (%) a H31

TU27

TU25

TU21

TU23

TU 11

TU30

H31 TU27 TU25 TUI2 TU21 TU23 TU11

0 96 104 93 91 96 101

102 0 2 88 2 3 86

108 0 0 83 - 2 4 90

110 0 1 5 0 5 102

107 - 9 - 7 - 6 - 9 0 98

104 69 69 87 93 100 0

ND b ND ND ND ND ND ND

a % of competitive binding of mAbs bound 125I-mAb with excess competitor mAb-bound 125

125 I-mAb

with excess cold mAb

bound I-mAb with excess PAR3 mAb-bound 125I-mAb with excess cold mAb b Not detected

×100,

66

1). These results indicate that all the five mAbs recognize the human IL-2R/3 molecule.

TU3OmAb

TU25mAb

a

b

Extracytoplasmic epitopes defined by mAbs Epitopes for the mAbs were examined by reciprocal competitive binding assays. 125I-labeled mAbs were separately incubated with MT-2 cells in the presence of cold excess competitor mAbs. Reciprocal binding inhibition was observed between TU27, TU21, TU23 and TU25 (Table II). These results indicate that the four mAbs bind to same epitope or epitopes close each other. However, the binding competition assay with TU12 distinguished TU21 and TU23 from TU27 and TU25, because competitor TU12 blocked bindings of 125I-TU21 and 125I-TU23 but not of 1251TU27 and 125I-TU25. In addition, the difference in the reactivity to MLA144 cells demonstrated that the epitopes for TU21 and TU23 were distinct. None of the mAbs blocked the binding of T U l l reciprocally. Therefore, we have isolated mAbs specific for at least five different epitopes on IL-2R/3 including TU30.

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Log. F l u o r e s c e n c e intensity Fig. 2. Flow cytometric analysis of TU30 and TU25. Digitonin permeabilized YTU14 cells (a, b) and untreated YTU14 cells (c, d) were incubated with TU30 (a, c) or TU25 (b, d) [solid lines] and control PAR3 (dotted line), and subsequently stained with FITC-conjugated goat anti-mouse IgG and analyzed by FACScan.

Epitope for TU30 As suggested above, it is possible that the epitope for TU30 is present in the intracytoplasmic or transmembrane region of IL-2R/3. To examine this possibility, we permeabilized YTU14 cells to mAbs with digitonin, and performed indirect immunofluorescence staining with TU30. After the cell membrane is permeabilized by digitonin treatment, mAbs can penetrate into the cytosol of the treated cells (Fiskum et al., 1980). TU30 clearly stained the digitonin-treated cells but not untreated cells, whereas TU25 stained both types of ceils, confirming that TU30 reacts with an epitope located in the cytoplasmic or transmembrane portion of IL-2R/3 (Fig. 2). The staining of permeabilized cells was less intense than that seen on intact ceils with TU25. The weaker staining was probably due to less efficient in staining the digitonin treated molecules (Anderson et al., 1989). To further define the epitope recognized by TU30, we immunoprecipitated the IL-2R from cell lines expressing deletion mutants of the IL2R/3 chain gene. H-4, A-15, S-25, and ST-7 have deletions in the carboxyl-terminal half of the cy-

toplasmic region (a.a. 379-525), the internal acidic region (a.a. 313-382), the serine-rich region (a.a. 267-322), and almost all of the cytoplasmic region (a.a. 267-525), respectively. Both TU30 and TU27 immunoprecipitated 75 kDa, 59 kDa, and 67 kDa molecules from F-7, H-4 and A-15 cells corresponding to the predicted molecular weights of the transfected /3-chain gene products. However, TU30 failed to immunoprecipitate specific molecules from ST-7 and S-25 although TU27 immunoprecipitated the expected 47 kDa and 69 kDa molecules respectively (Fig. 3). Thus, we concluded that TU30 recognizes an intracytoplasmic epitope overlapping the serine-rich region (a.a. 267-322) of IL-2R/3.

Effects of mAbs on IL-2 binding Four mAbs reacting with the cell surface, TU12, TU21, TU23 and TU25, were examined for their effects on IL-2 binding to the receptor by Scatchard plots analysis. Similar to previous results obtained with TU27, the four mAbs blocked IL-2 binding to the high-affinity receptor of MT-2 cells but not to the low-affinity receptor

67 F-7 I

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Fig. 3. Immunoprecipitationof various deletion mutants of IL-2R/3. 1251-labeledcell lysates from transformants were immunoprecipitated with TU30 (lane 1, 3, 5, 7, 9, 11) or TU27 (lane 2, 4, 6, 8, 10, 12). The immunoprecipitates were analyzed by 10% SDS-PAGE.

(Fig. 4) (Takeshita et al., 1989). Like T U l l (Suzuki et al., 1989), TU30 did not affect the IL-2 bindings to either type of the receptor (Fig. 4). Similar results were obtained by chemical crosslinking assays using YTU14 ceils which express IL-2R/3 but little IL-2Ra. TU12, TU21, TU23 and TU25 inhibited IL-2 binding to the IL-2R/3 on YTU14 cells, similar to TU27 whereas T U l l failed to show an inhibitory effect (Fig. 5 and data not shown). The reciprocal experiment demonstrated that the binding of TU12, TU21, TU23 and TU25 was inhibited by IL-2 (data not shown).

Effects of mAbs on IL-2-dependent cell growth We have previously shown that in spite of the complete inhibition of IL-2 binding to the high affinity receptor by TU27, TU27 scarcely inhibits IL-2 dependent cell growth (Takeshita et al., 1989). We examined the effects of the newly characterized mAbs on IL-2-dependent cell

growth. 3H-TdR incorporation was measured in the presence of 670 nM of mAbs and IL-2 at various concentrations up to 450 pM. TU21 and TU27 scarcely inhibited IL-2-dependent cell growth of ILT-Mat cells at IL-2 concentrations from 450 pM to 6 pM (Fig. 6). TU25 inhibited 3H-TdR incorporation at low concentrations of IL-2; 70% inhibition at 17 pM and 84% inhibition at 6 pM of IL-2 (Fig. 6). The difference in effects between TU27 and TU25 may be due to their affinity for IL-2R/3. To clarify this, the binding affinities of TU21, TU25 and TU27 were calculated by Scatchard plots analysis. The affinities of TU21, TU25 and TU27 were determined to be 1.7 nM, 0.5 nM, and 1.5 nM, respectively (Fig. 7). The three-fold difference in affinities between TU25 and the others probably explains the difference of their inhibitory effects on 3H-TdR incorporation of ILT-Mat cells. The same difference of growth inhibition between TU27 and TU25

68

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high affinity IL-2R, but only 13,600 sites/cell of IL-2Ra. IL-2-dependent cell growth of PHA-PBL was significantly inhibited by not only TU25 but also by TU21 and TU27. TU21 and TU27 showed 55% inhibition at 17 pM IL-2 concentrations (Fig. 8). However, ILT-Mat cells showed only 20% inhibition with the same mAbs, at the same concentration of IL-2 (Fig. 6). Furthermore, even at the presence of 450 pM IL-2 at which ILT-Mat cells failed to be inhibited by TU25, TU21, and TU27, TU21 and TU27 inhibited PHA-PBL growth by 12% (Fig. 8). TU25, which shows higher affinity to IL-2Rfl than TU27 and TU21, inhibited growth by more than 90% at IL-2 concentrations of 17 pM, and 51% at 450 pM in PHA-PBL (Fig. 8). TU12 and TU23 showed similar in-

Bound IL-2 molecules per cell ( x l O "4 )

Fig. 4. Effects of mAbs on Scatchard plots analysis of 12sI-IL-2 binding. MT-2 cells were pretreated with TU12 (©), TU21 ([]), TU23 (zx), TU25 (e), TU30 ( • ) and PAR3 ( • ) . Subsequently, they were incubated with 125I-IL-2 for 1 h at 4°C. Bindings of 125I-IL-2 were analyzed by Scatchard plots.

was observed in the case of PHA-stimulated PBL (Fig. 8). Preliminary observations have suggested that IL-2-dependent cell growth of PBL is significantly inhibited by TU27 when PBL are assayed 7 days after activation with PHA, whereas a little inhibition is seen with only 3-day activation. The major difference between 7-day and 3-day cultured cells is the amount of IL-2Ra chain expressed on the cell surface. IL-2R/3 expression is relatively constant. Expression of the IL-2Ra peaks 3 days after stimulation and decreases later. These observations imply that the amount of IL-2Ra expressed may influence the ability of mAbs to inhibit IL-2-dependent cell growth. In order to examine the effect of IL-2Ra expression on growth inhibition by anti-IL-2R/3 mAbs, we compared growth inhibition between 7-day PHAstimulated PBL, which express relatively low levels of IL-2Ra, and ILT-Mat cells, which express relatively high levels of IL-2Ra. ILT-Mat cells expressed 2,400 sites/cell of high-affinity IL-2R, and 129,000 sites/cell of IL-2Ra. 7-day PHA stimulated PBL cells expressed 1,700 sites/cell of

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Fig. 5. Chemical crosslinking with 125I-IL-2. YTU14 cells were pretreated with 2/zM of PAR3 (a), TU12 (b), TU23 (c), TU27 (d) and T U l l (e), and then chemically crosslinked with 2 nM of 125I-IL-2. These cell lysates were analyzed by 7.5% SDSPAGE.

69 6 ILT-Mat

i

2

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5

4"

0 0

t ,, 0

I 6

I 17

I 50

I 150

I 450

IL-2 (pM)

Fig. 6. Effects of mAbs on 3H-TdR incorporation of IL-2-dependent ILT-Matcells. IL-2-inducedincorporation of 3H-TdR was assayed for IL-2-dependent ILT-Mat cells in the presence of 50/zg/ml of PAR3 (e), TUll (B), TU21 (•), TU25 (×), TU27 (n), H-31 (©), and combination of TU25 and H-31 (zx). IL-2 concentrations ranged from 0 to 450 pM. The standard errors were alwaysless than 10% of the mean value.

150 450

IL-2 (pM)

Fig. 8. Effects of mAbs on 3H-TdR incorporation of PHAstimulated PBL. IL-2-induced incorporation of 3H-TdR was assayed for PHA-PBL in the presence of 5 0 / z g / m l of PAR3

(e), TUll (B), TU21 (*), TU25 (×), TU27 (n), H-31 (o), and combination of TU25 and H-31 (zx), similarly to Fig.6. The standard errors were always less than 10% of the mean value. .

hibitory effects to TU27 in both ILT-Mat and P H A - P B L (data not shown). The combination of TU25 and H-31, which inhibited the assembly of IL-2 to I L - 2 R a in comparison with the examination of TU25 only, induced the complete inhibition of 3H-TdR incorporation of both ILT-Mat and PHA-PBL. This effect was observed at any IL-2 concentrations tested (Figs. 6 and 8). T U l l showed no effect on both type of IL-2-dependent cell growth.

0.2

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Bound Ab molecules per cell ( x l 0 -3 )

Fig. 7. Scatchard plots of antibody binding. MT-2 cells were incubated with 125I-labeled TU21 ( • ) , TU25 (0), and TU27 (o). Free and cell-bound 125I-labeled antibodies were measured and analyzed by Scatehard plots.

We report here the characterization of 5 new mAbs TU12, TU21 TU23, TU25, and TU30 specific for the human IL-2R/3. The 5 mAbs recognize distinct epitopes on IL-2R/3. All of the mAbs reacted with the extracellular portion of IL-2R/3, except for TU30 which apparently recognizes the intracellular domain of the receptor. TU25 could not be distinguished from the previously characterized TU27 (Takeshita et al., 1989), but its affinity was 3-fold higher than that of TU27. Several groups have reported crosslinking and

70 immunoprecipitation studies that have identified a 70 kDa protein in addition to the IL-2Ra (55 kDa) and IL-2R/3 (75 kDa) chains that can be immunoprecipitated in IL-2/IL-2R complexes (Tanaka et al., 1987; Fujii et al., 1988). The relationship between the molecules has not been determined. All of our mAbs immunoprecipitated only one molecule, IL-2R/3, with a molecular weight of 75 kDa. None of our mAbs immunoprecipitated a 70 kDa molecule demonstrating that the 70 kDa and 75 kDa species are antigenically distinct from one another. Both members may be functional components of the IL-2R. We have previously demonstrated that a tyrosine kinase and a cell surface molecule, p64, may be involved in the high affinity receptor (Asao et al., 1990; Takeshita et al., 1990). We do not know whether the 70 kDa molecule is identical to p64 at present, but clearly the IL-2R is a more complicated complex than initially thought. Fully functional IL-2R may consist of other molecules in addition to a and/3 chains. Our mAbs specific for the IL-2R/3 chain should be useful in the analysis of the fine structure and function of the IL-2R. We believe that TU30 recognizes the intracellular domain, or transmembrane portion, of the IL-2R/3 chain by two criteria. First, TU30 stained YTU14 cells only after membrane permeabilization with digitonin. Secondly, immunoprecipitation from lysates of cells expressing deletion mutants of IL-2R/3, suggested that the epitope for TU30 is in the cytoplasmic domain of IL-2R/3. Specifically TU30 failed to immunoprecipitate IL-2R/3 from cells expressing constructs lacking the 'serine-rich region'. Our previous results indicate that the IL-2Rfl chain may associate with a tyrosine kinase (Asao et al., 1990). The serine-rich region has been shown to be essential for IL-2 receptor signal transduction (Hatakeyama et al., 1989b). Thus, TU30 may bind to the domain responsible for 1L-2R signal transduction. Monoclonal antibodies TU12, TU21, TU23 and TU25 inhibit binding of IL-2 to the IL-2R/3 chain. Transfection experiments have shown that IL2R/3 can bind IL-2 only when expressed in lymphoid cells (Hatakeyama et al., 1989b; Minamoto et al., 1990; Tsudo et al., 1990), whereas IL-2Ra can bind IL-2 when expressed in lymphoid or

non-lymphoid cells (Leonard et al., 1984; Nikaido et al., 1984; Hatakeyama et al., 1985). These results indicate that there may be lymphoidspecific component(s) in addition to IL-2Ra necessary for IL-2 binding to IL-2R/3. Thus it is possible that TU12, TU21, TU23, and TU25 recognize epitopes essential for the formation of the IL-2 binding pocket of the IL-2R/3 chain, or alternatively they may inhibit association of IL2R/3 to the putative component(s) necessary for endowing IL-2 binding. The component(s) may be the coprecipitated protein, p64, or a tyrosine kinase, or the other lymphoid-specific molecules. One would predict that mAbs that completely block IL-2 binding to high-affinity IL-2R would completely block IL-2-dependent cell growth. However, like our previous experiments with TU27, we found with 4 new mAbs, that IL-2-dependent cell growth was only slightly inhibited by the presence of saturating amounts of mAbs (Takeshita et al., 1989). The unexpected result might be explained by the large difference in affinity of IL-2 for the high affinity IL-2R versus the affinity of the mAbs for the receptor. Since the growth inhibition studies were performed at 37°C, IL-2Rfl molecules as well as other molecules would be constantly synthesized, resulting in the association of pre-existed IL-2Ra with newly synthesized IL-2R/3. Because the affinity of IL-2 for both IL-2Ra and/3 (25 pM) is much higher than TU27 for the receptor (1.5 nM), IL-2 may compete out for binding, be internalized, and transduce a signal. The association of the newly synthesized IL-2R/3 molecules with IL-2Ra occurs more frequently in cells expressing a larger amount, than a small amount, of IL-2Ra. Therefore, cells expressing a large amount of IL-2Ra might be resistant to the inhibitory effect of anti-IL-2Rfl mAbs. Consistent with the model, we demonstrated that in the presence of excess anti-IL-2R/3 mAb, the inhibition of PHA stimulated PBL which express 104 IL-2Ra sites/cell was greater than ILT-Mat cells that express 105 sites/cell. Furthermore, consistent with the model, there was a correlation between the affinity of the mAbs for the receptor and the inhibitory effect. Antibody with a high affinity like TU25 showed the greatest effect. This may explain the functional defect in ATL

71

cells which express abnormally high levels of IL2Ra. Such a model might explain how abnormal expression of IL-2Ra may be involved in development of the leukemic cells even though IL-2Rt~ can not transduce signals to cytoplasms.

Acknowledgements We thank Dr. J. Yodoi for providing us with YTU14 ceils and Dr. D. Ross for critical reading of this manuscript. This work was supported in part by Grants-in Aid for Scientific Research and Cancer Research from the Ministry of Education, Science and Culture, and by a grant from the Takeda Science Foundation.

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