Adult T-cell leukemia (ATL) develops in a small proportion of human T-cell lymphotrophic virus-I infected individuals. The leukemia consists of an overabundance of activated T cells, which are characterized by the expression of CD25, or IL-2Rα, on their cell surface. Presently,there is not an accepted curative therapy for ATL. We developed an in vivo model of ATL in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice by introducing cells from an ATL patient (MET-1) into the mice. The leukemic cells proliferated in these mice that lack functional T, B, and natural killer (NK) cells. The MET-1 leukemic cells could be monitored by measurements of both serum soluble Tac (IL-2Rα) and soluble humanβ 2-microglobulin (β2μ) by ELISA. The disease progressed to death in the mice after ∼4–6 weeks. The mice developed grossly enlarged spleens and a leukemia involving ATL cells that retained the phenotype and the T-cell receptor rearrangement and human T-cell lymphotrophic virus-I integration pattern of the patient’s ATL leukemia cells. This model is of value for testing the efficacy of novel therapeutic agents for ATL. The administration of humanized anti-Tac (HAT), murine anti-Tac (MAT), and 7G7/B6, all of which target IL-2Rα, significantly delayed the progression of the leukemia and prolonged the survival of the tumor-bearing mice. In particular, HAT induced complete remissions in 4 of 19 mice and partial remissions in the remainder. It appears that the antibodies act by a mechanism that had not been anticipated. The prevailing view is that antibodies to the IL-2Rα receptor have their effective action by blocking the interaction of IL-2 with its growth factor receptor,thereby inducing cytokine deprivation apoptosis. However, although both HAT and MAT block the binding of IL-2 to IL-2Rα of the high affinity receptor, the 7G7/B6 monoclonal antibody binds to a different epitope on the IL-2Rα receptor, one that is not involved in IL-2 binding. This suggested that the antibodies provide an effective therapy by a mechanism other than induction of cytokine deprivation. In accord with this view, the MET-1 cells obtained from the spleens of leukemic mice did not produce IL-2, nor did they express IL-2 mRNA as assessed by reverse transcription-PCR. Another possible conventional mechanism of action involves complement-mediated killing. However,although MAT and 7G7/B6 fix rabbit complement, HAT does not do so. Furthermore, in the presence of NOD/SCID mouse serum, there was no complement-mediated lysis of MET-1 cells. In addition, the antibodies did not manifest antibody-dependent cellular cytotoxicity with NOD/SCID splenocytes that virtually lack NK cells as the effector cells as assessed in an in vitro chromium-release assay. However,in contrast to the efficacy of intact HAT, the F(ab′)2version of this antibody was not effective in prolonging the survival of mice injected with MET-1 ATL cells. In conclusion, in our murine model of ATL, monoclonal antibodies, HAT, MAT, and 7G7/B6,appear to delay progression of the leukemia by a mechanism of action that is different from the accepted mechanism of IL-2 deprivation leading to cell death. We consider two alternatives: the first, antibody-dependent cellular cytotoxicity mediated by FcRI- or FcRIII-expressing cells other than NK cells, such as monocytes or polymorphonuclear leukocytes. The second alternative we consider involves direct induction of apoptosis by the anti-IL-2R antibodies in vivo. It has been shown that the IL-2R is a critical element in the peripheral self-tolerance T-cell suicide mechanism involved in the phenomenon of activation-induced cell death.

ATL2 was first identified in Japan in 1977 (1, 2). Common findings for patients with ATL include peripheral lymph node enlargement,hepatomegaly, splenomegaly, hypercalcemia, and skin lesions. At the present time, there is no accepted curative therapy for ATL, and the patients progress to death with a median survival duration of 9 months in acute ATL and 24 months in chronic ATL (3). The causative agent of ATL is HTLV-I (4). HTLV-I and ATL are both endemic in similar regions of the world, i.e.,southwestern Japan, the Caribbean basin, and parts of central Africa.

HTLV-I was the first retrovirus to be associated with a human malignancy (4, 5, 6, 7, 8). There is monoclonal integration of HTLV-I in the DNA of ATL cells, and patients with ATL have antibodies to HTLV-I (7). One of the genes encoded by HTLV-I, tax, is expressed in the early phases of ATL. taxis responsible for the transactivation of the expression of many viral and cellular genes, including those of IL-2 and the IL-2Rα,Tac, the private receptor that is IL-2-specific (9). The malignant cells associated with all phases of ATL express abnormally high numbers of IL-2Rα (3–11,000 receptors/cell; Refs.9, 10, 11). The up-regulation of these cellular genes by tax could be responsible for the proliferation of ATL cells in the premalignant and early leukemic phases of the disease (12, 13). However, neither IL-2 mRNA nor IL-2 protein is expressed in most cases of acute ATL, and thus autocrine stimulation by IL-2/IL-2R interaction is not observed in most stages of the disease(14). Therefore, there could be different subsets of ATL patients that would respond to different strategies to achieve effective therapy.

The conventional therapies [i.e., multidrug chemotherapies or zidovudine (AZT) and IFN α] do not appear to prolong life in patients with ATL, although there is some benefit with AZT/IFN therapy (15). We have targeted the IL-2Rα expressed by ATL cells using a murine monoclonal antibody, MAT. Six of 19 treated patients had a partial (4 patients) or complete (2 patients) remission(16). Our goal was to develop a preclinical in vivo murine model of the ATL disease, such that new agents could be tested before conducting patient trials. To establish this model, cells from a patient (MET-1) with ATL were injected i.p. into NOD/SCID mice. The human leukemic cells were able to proliferate in these mice that lack functional B, T, and NK cells(17). The injected mice developed leukemia distributed widely in the tissues, similar to the leukemia in the patients. The cells manifested a phenotype (CD3 dim, CD4±,CD25+, CD7) and a molecular integration pattern of HTLV-I and the TCR-βgene rearrangement as assessed by Southern analysis that were identical to those of ATL cells immediately ex vivo from this patient MET-1. The ATL cells infiltrated the spleen, which could then be used to serially transfer the leukemia to other mice. In addition, the disease was monitored by measurement of the plasma levels of the soluble form of human IL-2Rα or β2μby ELISA. The MET-1 ATL cells obtained ex vivo from the patient and those harvested from the mouse spleens did not express IL-2 mRNA.

The observation that IL-2Rα is not expressed by normal resting cells,but is expressed by ATL cells, provided the rationale for the use of mAbs directed toward IL-2Rα as a treatment modality. Malignant T cells of ATL patients express 3,100–11,400 IL-2Rα receptors per cell (11). Therefore, antibodies to IL-2Rα(CD25), such as HAT, MAT, and 7G7/B6 (non-IL-2 blocking) were evaluated in the MET-1 model for their efficacy in the therapy of ATL. Each of the antibodies inhibited the progression of the leukemia and prolonged the survival of the mice. Because there was no IL-2 mRNA expression or IL-2 production by the leukemic cells, the prevailing view of the mechanism for anti-Tac action (i.e., the blockade of IL-2 interaction with its growth factor receptor IL-2Rα with consequent cytokine deprivation cell death) was not supported by the data. Furthermore, we present evidence against complement fixation or ADCC involving NK cells as the mode of effective therapeutic mAb action. However, we demonstrated that F(ab′)2 HAT in contrast to intact antibody was not effective in this model. We consider Fc-dependent antibody-mediated cytotoxicity involving FcRI or-III-expressing cells, such as monocytes or polymorphonuclear leukocytes, as one operative mechanism of action of these mAbs in this model and, by inference, in the IL-2-independent phase of ATL in patients.

Patient Leukemic Cell Preparation.

Twenty-four ml of heparinized blood were collected from a patient(MET-1) with acute ATL. The PBMCs were separated by centrifuging the blood through lymphocyte separation medium (Organon-Teknika). After separation, the PBMC layer was washed with PBS, then subjected to ACK lysing buffer (10-548E; BioWhittaker) for 3 min to eliminate remaining RBCs, yielding 2 × 109viable PBMCs as determined by trypan blue staining. The PBMCs were then negatively selected using antibodies to CD7 (Sigma Chemical Co.) and CD20 (Coulter) after the antibodies had been dialyzed 48 h against PBS and sterile-filtered. The antibody-selected cells were then removed using magnetic beads coated with goat antimouse antibody (Dynal USA,Lake Success, NY). The purified PBMCs were then injected i.p. into NOD/SCID mice (Jackson Laboratories, Bar Harbor, ME).

ATL Transfer.

Both the spleen, if enlarged, or the s.c. tumor from the injection site were ground between two frosted ends of microscope slides and then dispersed through a cell strainer (Falcon 2340) into a 50-l centrifuge tube. The tube was centrifuged at 4°C for 5 min at 1200 rpm. The cells were then resuspended in 5–10 ml of ACK lysing buffer and maintained at room temperature for 5 min. The cells were then centrifuged for 5 min at 1200 rpm and resuspended in RPMI 1640. The cells were counted using a hemacytometer, and media was added to yield the appropriate cell concentration for injection (20 × 106 cells/0.5 ml).

Phenotypic Analysis of ATL Cells.

Before transfer, the leukemic cells isolated from mouse spleens containing ATL cells were analyzed by flow cytometry to insure that the cells being transferred were pure MET-1 ATL cells. Cells for phenotyping were washed in FACS staining buffer (100 ml of PBS, 1 g of BSA, and 1 ml of a 10% solution of NaN3). One million cells/well were stained with FITC-labeled antihuman CD2, CD3, CD4, CD7, CD8, CD20, CD25, CD28, and class I HLA for 30–45 min on ice in the dark. After staining with antibodies and washing with buffer, 100 μl of a 1:20 dilution of 0.1 mg/ml propidium iodide was added to each well as a stain to confirm viability. After a wash, the viable cells were then enumerated on a Becton Dickinson FACSort Flow Cytometer. The phenotypic cell subtypes were expressed as a percentage of live cells.

Mouse Model of ATL.

Female NOD/SCID mice (pathogen-free) were purchased from the Jackson Laboratory at the age of 4–6 weeks. The mice were used in studies at the age of 4–12 weeks. Leukemia was established by i.p. injection of either 15–20 × 106 viably frozen ATL-NOD/SCID spleen cells or the same number of splenocytes from NOD/SCID mice with a high (>400,000 pg/ml) soluble serum IL-2Rαlevels (sIL-2Rα). Therapy experiments were performed on these mice when their sIL-2Rα levels were >1000 pg/ml serum, i.e.,∼10–14 days after tumor inoculation.

Measurement of sIL-2Rα and Solubleβ 2-Microglobulin by ELISA.

Throughout the therapy experiments, the serum concentrations of the soluble released IL-2Rα chain (Tac) and solubleβ 2 μ were measured in the mice using ELISA kits purchased from R&D Systems (soluble Tac; solubleβ 2μ). Mice were anesthetized with metofane(A.J. Buck, Mundelein, IL) for retro-orbital bleeding. The blood was collected into Microtainer serum separator tubes (Becton Dickinson). The tubes were centrifuged at high speed for 4 min in an Eppendorf centrifuge and the serum was collected. The ELISAs were performed as indicated in the manufacturer’s kit inserts.

Monoclonal Antibodies.

MAT was produced as described previously (9, 18) by fusion of NS-1 mouse myeloma cells with spleen cells of mice that had been immunized with a cell line derived from a patient with ATL. Large quantities of the antibody were produced by inoculating hybridoma cells into the peritoneal cavity of BALB/c mice and then purifying the mouse IgG2a anti-Tac from the resulting ascites by DEAE cellulose chromatography. The final antibody preparation was in saline at pH 7.4 in a concentration of 2 mg/ml HAT (Daclizumab, Zenapax) was obtained from Hoffmann-La Roche (Nutley, NJ). 7G7/B6 is a mouse IgG2a,κ mAb directed toward an epitope of the IL2-Rα peptide other than that identified by anti-Tac (19). The 7G7/B6 was purified from supernatants of a hybridoma (American Type Culture Collection) using ImmunoPure Protein A columns (Pierce). For the F(ab′)2-HAT studies, the antibody fragments were generated by a procedure described previously, with the exception that a monospecific HAT F(ab′)2, rather than a bispecific agent, was generated (20). A homodimer-forming“zipper” peptide was generated with the Jun zipper linked to the HAT F(ab′) portion of the genetically engineered HAT by gene fusion. The HAT F(ab′-Jun)2 homodimers were expressed in the mouse myeloma cell line NS0. The resulting end products were >95% F(ab′-Jun)2 homodimers as confirmed by acrylamide gel analysis. Humanized Mikβ1 (HuMikβ1), an antibody that is directed to the IL-2 and IL-15 binding sites of the IL-2/15Rβ subunit, was obtained from Hoffmann-La Roche. Human IgG,the negative control antibody, was purified from the serum of a normal volunteer by DEAE cellulose chromatography. The HuMikβ1 F(ab′-Fos)2 antibody fragments were generated as described previously, with the exception that a monospecific HuMikβ1 F(ab′)2, rather than a bispecific agent, was generated.

Comparison of the Avidity of the HAT F(ab′-Jun)2Homodimers with Intact HAT.

The avidity of the intact HAT monoclonal antibody was compared with that of HAT F(ab′-Jun)2 by two FACS assays. In the first assay, CD25+ HuT-102 line cells were incubated with serially diluted HAT or HAT F(ab′-Jun)2, washed and stained with FITC-conjugated goat antihuman κ antibodies. The potency of each antibody in shifting the mean channel fluorescence was measured. In the second assay, a fixed concentration of FITC-conjugated HAT F(ab′-Jun)2 was incubated with serially diluted HAT or HAT F(ab′-Jun)2. After incubation and washing, the mean channel fluorescence at each competitor antibody was measured. In both cases, HAT F(ab′-Jun)2 appeared to have an avidity for HuT-102 cells ∼2-fold higher than that of HAT(data not shown).

Treatment with Antibodies.

For the complete antibody studies, 100 μg/mouse in a volume of 0.2 ml was injected once per week for 4 weeks by i.v. injection in the tail vein. In addition to HAT, MAT, and 7G7/B6, the control treatments at the same protein concentrations included groups injected with the vehicle (PBS), normal human IgG (HuIgG), or HuMikβ1. The leukemic cells expressed the IL-2/15Rβ peptide identified by HuMikβ1 as well as the IL-2Rα chain. Therefore, the HuMikβ1 antibody was a control for an effect mediated by any specific binding of a mAb to the cells. In light of the short survival of F(ab′-Jun)2-HAT, this antibody fragment was injected twice daily for 14 days at a molar equivalent dose to that of weekly administered HAT (6.67 × 10−10 mol; 66.7 μg/mouse/day F(ab′)2-HAT). During this latter study involving F(ab′)2-HAT, the PBS and HAT were administered once per week for the same 2-week period. Throughout the studies, the leukemic progression was monitored by ELISA for sIL-2Rα andβ 2μ in the serum as well as by Kaplan Meier analysis of the survival of the mice.

Statistics.

The serum levels of sIL-2Rα and β2μ at different time points for the different treatment groups were analyzed for statistical significance using StatView (Abacus Concepts, Berkeley,CA). The tests used included ANOVA for significant differences between groups, and the Fisher’s F procedure for post-hoc comparisons to determine P. In terms of the mouse survival plots, StatView was used to generate Kaplan-Meier cumulative survival plots.

IL-2-induced Proliferation of Kit-225 (K6) Cells.

Kit 225 (K6) cells were cultured in RPMI (+ 10% FCS) without IL-2 for 3 days before the assay. Two × 104 cells/well in triplicate in a 96-well microtiter plate were incubated for 3 days at 37°C with a range of IL-2 concentrations (1.56, 3.13, 6.25, 12.5, and 25 units) in the presence or absence of HAT, MAT, or 7G7/B6 (4 μg/well). Six h before harvest, 1 μCi/well of [3H]thymidine was added to each well. The data are presented as the proportion of IL-2-induced proliferation blocked by the antibody.

RT-PCR for IL-2 Message.

RNA was isolated from the spleens of NOD/SCID mice with a high tumor burden as determined by sIL-2Rα ELISA. The isolation of total RNA was performed as described in the PURESCRIPT RNA isolation kit manual(Gentra Systems, Inc., Minneapolis, MN). The Advantage RT-for-PCR Kit(CLONTECH Laboratories, Inc., Palo Alto, CA) was used to make cDNA from the RNA. One μl of oligo-dT primers was added to 1 μg of RNA from each sample (3 MET-1 NOD/SCID mouse spleens, SP-2/Tac cells, and a positive control, Jurkat cells stimulated with PHA/PMA, and Jurkat cells stimulated with Ionomycin/PMA). These mixtures were heated at 70°C for 2 min. Then a mixture of reaction buffer, dNTPs, RNase inhibitor, and Moloney murine leukemia virus reverse transcriptase was added while the tubes were on ice. The thermal cycles were as follows:(a) 42°C for 1 h; and (b) 94°C for 5 min. diethyl pyrocarbonate water was then added and the cDNAs stored at−70°C. The cDNAs were used for the PCR reaction with primers for IL-2 or G3PDH (CLONTECH). The thermal cycles for the reaction with primers were as follows: (a) 1 cycle of 94°C, 4 min;58°C, 45 s; 72°C, 1 min; (b) 30 cycles of 94°C, 1 min; 58°C, 45 s; 72°C, 1 min; (c) hold at 72°C for 5 min; and (d) hold at 4°C. The PCR products were then run at 100 volts on a 1% agarose gel (0.5 g agarose + 50 ml 1 × Tris acetate buffer dissolved, then 5μl ethidium bromide).

ADCC.

The capacity of the various antibodies to function in ADCC was measured by a chromium release assay. MET-1-NOD/SCID spleen cells were labeled with 400 μCi/107 cells of chromium-51(Amersham, Piscataway, NJ) by incubation at 37°C for 1–2 h. The cells were washed three times and aliquoted into a flat-bottomed 96-well microtiter plate (1 × 104cells/well). The antibodies were added at 1 μg/ml to each of the appropriate wells. Effector cells were spleen cells from either CB17-SCID (the parental strain of NOD/SCID mice) or naïve NOD/SCID mice at effector:target ratios of 0:1, 5:1, 10:1, 30:1, 60:1,100:1 or 200:1. After 4 h of incubation at 37°C, the plates were centrifuged at 1200 rpm for 2 min. Plates were harvested using the Skatron harvesting system (Skatron Instruments, Inc.), and the supernatants were counted in a gamma counter. The data were plotted as the percentage specific lysis {% SL = [(experimental cpm − spontaneous cpm/maximum cpm − spontaneous cpm)] × 100}.

Complement Fixation.

A viability assay using trypan blue was used to determine whether the unmodified antibodies to IL-2Rα could fix complement as their mechanism of action. HuT-102 cells (1 × 105 cells/well), which are from an HTLV-I infected T-cell line typically used as a prototype ATL in vitro, were incubated with the unmodified antibodies (1 μg/ml)on ice for 1 h. The cells were washed, and 200 μl/well of a source of complement were added for 1–2 h at 37°C. The assay positive control serum source was Rabbit Complement MA (Accurate Chemical & Scientific, Westbury, NY) at a 1:5 dilution. The experimental serum source was serum from naïve NOD/SCID mice at a 1:5 dilution. The data are plotted as the percentage of cells that remain viable as assessed by trypan blue exclusion.

ATL is a rare but aggressive leukemia of mature T cells which currently has no standard curative therapy. In contrast to resting normal T cells that do not express IL-2Rα in meaningful quantities,activated T cells express large numbers of the IL-2Rα subunit of the multisubunit high-affinity receptor for IL-2 (Tac, IL-2Rα), this receptor provides a target for rationally designed therapies to target the cells involved in the leukemia. We developed an in vivomodel of ATL to test the efficacy of potential therapies including IL-2R-directed approaches for the disease.

Establishment of a Murine Model of ATL.

Peripheral blood lymphocytes from a patient (MET-1) with ATL were used to develop a model of ATL in mice. NOD/SCID mice were used to establish this model because the mice lack functional B, T, and NK cells. Once the initial patient cells were established in NOD/SCID mice, the mice were used to expand and serially transfer the cells to other NOD/SCID mice for studies. After the i.p. injection of 20 × 106 MET-1 leukemic cells, there was wide tissue distribution of human CD3- and CD25-expressing leukemic cells with marked infiltration of the lungs, spleen, liver, kidney, and lymph nodes and moderate infiltration of the bone marrow. The spleens of the mice increased to a size of ∼3 cm × 1 cm by 4–6 weeks after tumor cell injection. At that time the peripheral WBC counts included more than 10,000/mm3 leukemic lymphocytes. This therapeutic model appears to be clinically meaningful because the cells that proliferate in the mice were characterized and shown by several methods to be identical to the actual leukemic MET-1 cells obtained ex vivo. Each person with ATL has a certain pattern of HTLV-I integration and TCR-β gene rearrangement that can be used as a “fingerprint” of the leukemic cells. By Southern blot analysis, we were able to show that the molecular markers(HTLV-I integration pattern and TCRβ gene rearrangement)present in ex vivo leukemic cells were conserved after the transfer to the mouse model (data not shown). A second method of characterization was based upon the fact that the original cells from MET-1 had a distinct phenotype elucidated by FACS analysis: CD3 dim,CD7-negative, and CD25-positive. Therefore, before the transfer of cells, they were analyzed for several surface markers by flow cytometry. The typical phenotype of MET-1 cells from the mice is as follows: CD2+, CD3 dim,CD4±, CD7,CD8, CD20,CD25+, CD28+, and HLA class I+ (Table 1). Thus we view the cells in the murine model as the counterpart of the patient’s circulating ATL cells. Lastly, it is well known that the soluble portion of the IL-2Rα is cleaved and can be found circulating in serum from patients with ATL. Human IL-2Rα as well as soluble humanβ 2μ could be detected and quantified in the serum from MET-1 ATL-inoculated mice by an ELISA methodology. The levels of the soluble IL-2Rα (Tac) and β2μincreased as the disease progressed in the mice (Fig. 1). The soluble IL-2Rα reached levels of 500,000 to 1,000,000 pg/ml immediately before death. Mice typically survived 6–10 weeks after leukemic cell inoculation. The ELISAs for sIL-2Rα and β2μ provided a good measure of the progression of the disease throughout therapy studies.

Treatment of ATL Using Unmodified Antibodies Directed toward IL-2Ra.

Soluble IL-2Rα and β2μ were indicative of the tumor load of ATL in our mouse model. When the levels of sIL-2Rαreached 1000 pg/ml, we concluded that the leukemic cells were proliferating in the mice. At this point the mice were entered into a therapeutic study. In the trial, groups of 8–10 mice each were treated with PBS, IgG, HAT, MAT, 7G7/B6, or HuMikβ1. The study was performed on six occasions. In each study, the antibodies at 100 μg in 200 μl were injected i.v. one time per week for 4 weeks. The HAT, MAT, and 7G7/B6 antibodies, all of which are directed toward the IL-2Rα on the leukemic cells, had a therapeutic effect as seen by their effect on the serum levels of sIL-2Rα or β2μ (Fig. 2). In comparison with the serum concentration of IL-2Rα in the PBS control mice, on day 28 there was a significant reduction of sIL-2Rα in the HAT-(P < 0.01), MAT- (P < 0.01), or 7G7/B6- (P < 0.01) treated animals compared with the PBS-treated controls. Similarly, the humanβ 2μ levels were significantly reduced(P < 0.01) for the same groups. Furthermore,there was a significant (P < 0.01)prolongation of the survival of the mice treated with HAT, MAT, or 7G7/B6 as compared with the PBS control (Fig. 3). The median survival duration of the control group (PBS) was 37 days. All of the mice in this group as well as the IgG and HuMikβ1 groups had died by day 58 of the study. In contrast, 60% of the 7G7/B6-treated and all of the MAT- and HAT-treated mice were alive at that time. Comparable results were observed when the study was repeated five additional times. The graph shown in Fig. 3 is typical of the six comparable studies performed. An additional prolongation of survival was observed when mice were treated continuously weekly with HAT until death. In particular, 90% of the mice remained alive at 110 days after tumor inoculation in this group,compared with 60% in mice that had been treated with HAT for only 4 weeks (data not shown).

Mode of Action.

Because HAT, MAT, and 7G7/B6 have a therapeutic effect in our murine model of ATL, this raises a question concerning their mechanism of action. Traditionally, it has been presumed that HAT and MAT act by blocking the interaction of IL-2 with its receptor, thereby inducing cytokine deprivation apoptotic cell death. However, 7G7/B6 binds to a different IL-2Rα epitope than that identified by HAT, one which is not involved in the binding of IL-2. Furthermore, as shown in Fig. 4, 7G7/B6 does not block IL-2-induced proliferation. This suggested that such a blockade of the IL-2 action was not the mechanism of antibody action. In strong support of this view, the ATL cells obtained from the mouse spleen did not express IL-2 mRNA as assessed by RT-PCR (Fig. 5),excluding the presence of an IL-2/IL-2R autocrine loop. Furthermore,there was no evidence of human IL-2 protein in the serum of these AJ-ATL-bearing mice as assessed by an IL-2-specific ELISA (data not shown). Moreover, as is true for human cells in general, MET-1 cells did not proliferate in response to murine IL-2 (data not shown). Therefore, there appears to be a mechanism of effective therapeutic action for these antibodies other than the blockade of IL-2/IL-2R interaction. An alternative theoretical mechanism of action could involve complement fixation. The assay used to test for this action was a viability assay using trypan blue exclusion in the presence of the antibodies and a serum source. The assay positive control involved the use of rabbit serum (1:5 dilution). With this as the complement source,MAT and 7G7/B6 were associated with ∼50% death of the target HuT-102 cells. The other antibodies used, including HAT, did not fix rabbit complement. Furthermore, when naïve NOD/SCID mice serum was used as the potential complement source, none of the antibodies were able to fix complement (data not shown). This was expected because it had been reported previously that NOD/SCID mice are deficient in complement-mediated killing (17).

Another possibility is that the antibodies could theoretically act through NK cell-mediated ADCC. To address the issue of NK-mediated ADCC, we used a chromium-release assay to measure the ability of the antibodies, in conjunction with splenic effector cells, to cause lysis of the MET-1-NOD/SCID cells. To validate the ADCC assay, we used CB17-SCID mice spleens (four to eight spleens combined) as the effector cells. These mice are reported to have functional NK cells, therefore,the antibodies had the best chance to demonstrate any ADCC capacity. In this system, the rank of antibody ability to cause specific lysis was as follows: 7G7/B6 (30.2% at 200:1) > HAT (17% at 200:1) > MAT (9.7% at 200:1), with PBS, IgG, and HuMikβ1 not yielding any specific lysis (Fig. 6,B). To define the role of NK-mediated ADCC in the MET-1 model, the effector cells analyzed were from naïve NOD/SCID mice spleens because these mice were the ones used in the in vivo model of antibody therapy for ATL. Using the same targets (MET-1 NOD/SCID splenocytes containing ATL cells) and the same antibodies, but different effectors (NOD/SCID splenocytes), the results were very different. None of the antibodies caused specific lysis in this ADCC system (Fig. 6 A). This observation is in accord with the reported virtual lack of NK-cell activity in NOD/SCID mice.

A more definitive way of testing for any Fc-mediated cellular cytotoxicity is to evaluate in vivo an antibody fragment which cannot perform either complement fixation or Fc-mediated killing. The F(ab′)2 fragment of HAT that lacks the Fc segment required for complement-mediated cytotoxicity as well as other Fc-mediated cytotoxicity was evaluated for therapeutic efficacy in the murine model of ATL. The fragments of HAT lacking the Fc segment used in the present studies were F(ab′)2 HAT generated by linking the genetically engineered F(ab)′ portion of HAT to the Jun zipper by gene fusion. The resulting end products were F(ab′-Jun)2 homodimers as confirmed by SDS polyacrylamide gel analysis (Fig. 7). Furthermore, as noted in “Materials and Methods,” the avidity of the F(ab′-Jun)2 homodimer for the IL-2Rαreceptor on cells was at least as great as that of the intact mAb. In vivo survival of F(ab′- Jun)2 HAT antibodies were evaluated in 10 mice using the F(ab′-Jun)2 homodimers iodinated by iodogen. The t1/2 of the terminal exponential of decline of the radiolabeled F(ab′-Jun)2 HAT in these mice was approximately 7 h, which contrasts with the 3.5–4 days observed with intact anti-Tac antibodies (data not shown). For the efficacy studies, the F(ab′-Jun)2 anti-Tac fragment was administered at 100 μg 2 × a day for 14 days in light of the short survival of F(ab′)2observed in the mice. In parallel, 100-μg doses of intact HAT were administered weekly for the same 2-week period. However, it must be noted that the short survival of the fragments may alter the exposure and binding of these fragments so that the IL-2Rα is not occupied to the same extent and time as is true with the intact antibody. There was a significant increase (P < 0.01) of the survival of mice with MET-1 tumors that were treated with intact HAT when compared with the PBS controls (Fig. 8). In contrast, the F(ab′-Jun)2 HAT was ineffective in prolonging the survival of the tumor-bearing mice, with no significant increase(P > 0.35) in survival of the tumor bearing mice. Thus an FcR requiring action of the HAT must be considered for at least one of the modes of action of HAT, MAT, and 7G7/B6 in the MET-1 mouse model and, by inference, in select human patients with ATL where the malignant cells do not express IL-2 mRNA nor produce IL-2 protein,yet undergo remissions after administration of HAT.

ATL is a malignancy of mature lymphocytes caused by the retrovirus HTLV-I. A total of 854 patients with HTLV-I antibody-positive ATL newly diagnosed from 1983–1987 were analyzed for prognostic factors and survival after combination chemotherapy by Shimoyama and the Japanese Lymphoma Study Group (3). The median survival duration and projected 2- and 4-year survival rates of all patients were 10 months and 28% and 12%, respectively. The authors of this study concluded,“The various chemotherapies thus far developed have not increased significantly the survival of patients with ATL.” In light of the disappointing results using conventional combination chemotherapy, new approaches to the treatment of ATL are required. Research and evaluation of such novel agents would be facilitated by the availability of a murine model of ATL. Kondo et al.(21) reported the establishment of a model of in vivo cell proliferation of ATL cells using SCID mice. Such mice were treated with a daily injection of human IL-2 as well as with an anti-murine IL-2/IL-15Rβ mAb, the latter to eliminate NK cells. In subsequent studies, Imada et al.(22) succeeded in the serial transplantation of ATL cells from a patient into comparably treated SCID mice. In these studies, nonleukemic T cells immortalized by HTLV-I did not survive in the mice, but rather the cells that could be passaged in mice were shown by Southern blot hybridization to have the same rearrangement pattern of human TCRβ as those of the leukemic ATL cells of the patients. No expression of IL-2 mRNA by the tumor cells proliferating in the mice could be detected,and thus an IL-2-mediated autocrine mechanism was not involved in the neoplastic growth of the HTLV-I infected cells in this SCID mouse model. Our MET-1 ATL leukemic model parallels that of Imada and coworkers in a number of features. In both models there was infiltration of leukemic cells into a variety of organs including the lungs, liver, and spleen. Furthermore, the TCRβ gene rearrangement was identical between the cells ex vivo from the patient and those serially transplantable in the mice. The leukemic cells in both studies did not express IL-2 mRNA. Moreover, in both the model of Imada et al.(22) and in our own, the cells could not be grown as a cell line in vitro but could be passaged serially in immunodeficient mice. This observation suggests that host factors in vivo are required for the continued proliferation and survival of the ATL cells.

Finally, the MET-1 ATL model presents many features that parallel those observed in patients with adult T-cell leukemia and thus represents a valuable model to evaluate the tissue toxicity and efficacy of therapeutic agents directed toward the treatment of ATL. We are evaluating an array of such agents including antibodies directed toward the multiple elements of the IL-2R system, unmodified as well as armed with toxins and α- and β-emitting radionuclides. Furthermore, the MET-1 ATL model is being used to test Jak3 inhibitors, rapamycin analogues and NFκB inhibitors. During the present trial, we made the observation that antibodies directed toward IL-2Rα provided meaningful therapy for ATL in this model. This was an unanticipated observation in light of the fact that the MET-1 ATL cells neither produced nor required IL-2 for their survival.

The dominant target of our anti-ATL therapeutic program has been the IL-2Rα, a subunit identified by the anti-Tac mAb(23, 24, 25). The scientific basis for this approach is that resting normal cells, including the normal cells of patients with leukemia, do not express IL-2Rα in contrast with its expression by the leukemic cells of patients with HTLV-I-associated ATL. Furthermore,IL-2Rα is constitutively expressed by select malignant cells of patients with cutaneous T-cell lymphoma, hairy cell B-cell leukemia,and the Reed Sternberg cells of Hodgkin’s disease (26). Moreover, IL-2Rα is expressed by the abnormal T cells in a variety of autoimmune disorders as well as on those T cells involved in organ allograft rejection (26). To exploit this difference in IL-2Rα expression, we performed therapeutic trials with the mAb anti-Tac that blocks the binding of IL-2 to IL-2Rα. On the basis of successful clinical trials, the humanized form of this antibody has been approved by the Food and Drug Administration for use to prevent renal organ allograft rejection and has shown efficacy in the treatment of such autoimmune disorders as tropical spastic paraparesis and T-cell mediated uveitis (23, 27, 28, 29). In addition, approximately one-third of ATL patients treated with MAT developed a partial or complete remission (24). In those cases where an effective therapeutic response was observed, it had been presumed that the antibody mediated its effective response by preventing IL-2 from interacting with its growth factor receptor, thereby leading to cytokine deprivation apoptosis. Support for this mechanism in select cases is provided by the observation that the leukemic cells of a proportion (≥15–20%) of patients with ATL are in the autocrine IL-2/IL-2R phase of their disease (12, 13, 14). In such patients, predominantly those with the smoldering and chronic stages of ATL, we have demonstrated spontaneous proliferation of their ex vivo leukemic cells. This spontaneous proliferation could be inhibited by anti-Tac in the in vitro culture. These observations in these ATL patients parallel the ones observed previously in patients with tropical spastic paraparesis/HTLV-I associated myelopathy (30). Although anti-Tac blockade of IL-2 interaction with IL-2Rα may play a role in the frequent therapeutic responses observed in patients with the early stages of ATL, during the disease progression the malignant cells advance to a phase where, although they continue to express IL-2Rα, they no longer produce nor require IL-2 for their proliferation and survival(30). Nevertheless, the signaling pathway involving Jak1 and Jak3 as well as Stat 5 remains activated (31, 32). A number of factors underlie this IL-2-independent action. HTLV-I tax transactivates IL-15, which acts on the private IL-15Rα and on IL-2/IL-15β and γc shared with IL-2 (33). In addition,in IL-2-independent cell lines, there is a loss of SHP-1, the phosphatase that normally inactivates Jak3 (34). It had been presumed that ATL in its IL-2-independent phase would no longer respond to unmodified antibodies such as HAT that are directed to the private IL-2-specific receptor IL-2Rα. However, in conflict with this prediction, IL-2Rα directed mAbs provided effective therapy in the MET-1 ATL model by a mechanism other than the blockade of IL-2/IL-2Rαinteraction. In particular, in each of six separate trials, the 7G7/B6 antibody that binds to an epitope on the IL-2Rα receptor that is not involved IL-2 binding provided effective therapy. Furthermore, the MET-1 cells obtained ex vivo from the spleens of leukemic mice did not express IL-2 mRNA as assessed by RT-PCR, nor did they release IL-2 into the media during short-term culture. In clinical parallel with this observation in the murine model, we have noted at least one patient who responded to high-dose HAT therapy, although his leukemic cells did not proliferate spontaneously ex vivo nor did they produce IL-2 mRNA as assessed by RT-PCR.

We searched for other conventional mechanisms of action to explain the efficacy of IL-2Rα-directed antibodies in the MET-1 ATL model. Complement-mediated cytotoxicity appears to be excluded by the observation that HAT did not fix rabbit complement, and in the presence of NOD/SCID mouse serum that is complement-poor there was no complement-mediated lysis of MET-1 cells with any of the three anti-IL-2Rα antibodies examined. Classical ADCC mediated by NK cells also does not appear to be a likely mode of action. The NOD/SCID mice were chosen for the transfer of ATL cells because they virtually lacked functional NK cells. Furthermore, the three antibodies directed toward IL-2Rα did not manifest ADCC with NOD/SCID splenocytes as effector cells as assessed in a chromium-release assay. Although classical NK-mediated ADCC does not appear to be a dominant factor in the action of the anti-IL-2Rα antibodies in the MET-1 ATL model, the immunoglobulin-Fc receptor does appear to be one of the elements involved in the action of the antibodies. In contrast to the efficacy of intact HAT, the F(ab′)2 version of this antibody was ineffective in the present study in prolonging the survival of the mice injected with MET-1 ATL cells. In these studies,we treated the mice with the F(ab′)2 version of the antibody twice a day for 14 days, whereas the HAT in this experiment was used weekly for 2 weeks. The more frequent administration of the F(ab′)2 version was used to compensate for the short (7 h versus 77 h) terminal t1/2 of the F(ab′)2 fragment as compared with that of the intact antibody. However, because of the wide-spread distribution of leukemic cells and their small number in the body at the time of therapy, we cannot be assured that the exposure and binding of the fragments to IL-2Rα on the tumor cells is equivalent to that provided by the intact HAT mAb. The effective action of the intact antibody may involve the FcγRIII present on monocytes and macrophages in the NOD/SCID mice. This conclusion is in accord with the results of Clynes et al.(35, 36). The FcR common γchain-deficient mice (FcRγ−/−) they generated that lack the activation FcγRs I and III did not manifest passive or active protection against pulmonary metastases in the syngeneic B16 melanoma mouse model. Furthermore, such FcγRIII-deficient mice did not manifest the efficacy in tumor models observed in wild-type mice when anti-HER2/neu (Trastuzumab, Herceptin)or anti-CD20 (Rituximab) antibodies were evaluated. Thus Clynes et al.(35, 36) have proposed that although multiple mechanisms have been suggested for the ability of antitumor antibodies to mediate their effects in vivo, there is a dominant and necessary role for FcγR-dependent binding for in vivo activity (35, 36).

A final alternative mechanism, for which we have no evidence,that might contribute to the efficacy of these IL-2Rα-directed mAbs is the induction of apoptotic death through the IL-2/IL-2R-associated pathway that is involved in AICD. Normally the apoptotic cell death of mature peripheral T cells occurs after persistent T-cell receptor stimulation (37, 38, 39). After this stimulation,IL-2 is produced and interacts with IL-2R, ultimately placing the cell in cycle. The triggering of these cycling cells through the CD3-TcR pathway leads to the expression of fas ligand or tumor necrosis factor-α that are effector molecules associated with T cell suicide. IL-2 and IL-2Rα appear to play critical roles in this AICD process, in that mice made deficient in IL-2 or IL-2Rα by gene targeting manifest lymphocytosis, hyperγglobulinemia, and autoimmune hemolytic anemia. We reason that IL-2-specific AICD might be associated with IL-2Rα expression, and that the addition of the three mAbs to IL-2Rα may have induced AICD-related cell death in the MET-1 ATL T cells.

In summary, in our murine model of ATL the mAbs HAT, MAT, and 7G7/B6 appear to delay the progression of leukemia by a mechanism of action other than the blockade of IL-2/IL-2Rα interaction. The one clear mechanism is FcR-mediated death by FcRIII-expressing cells with monocytes or granulocytes as the potential effector cells. We also consider induction of AICD by anti-IL-2Rα antibodies in vivo as a contributing factor.

Fig. 1.

sIL-2Rα and β2 μ serum levels increased as ATL progressed in MET-1 mice. NOD/SCID mice were injected (i.p.) with transfer MET-1 spleen cells. Serum collections from the mice were taken at 7, 14, 21, 28, 35, and 49 days posttransfer. The blood was drawn by retro-orbital bleeding during anesthesia with metofane. ELISAs for sIL-2Rα and β2 μ were performed to follow the ATL progression. The data presented represent 23 mice for days 7 and 15, 15 mice for days 21 and 35, and 7 mice for days 14 and 28. The values are presented as pg/ml for sIL-2Rα and ng/ml for β2 μ.

Fig. 1.

sIL-2Rα and β2 μ serum levels increased as ATL progressed in MET-1 mice. NOD/SCID mice were injected (i.p.) with transfer MET-1 spleen cells. Serum collections from the mice were taken at 7, 14, 21, 28, 35, and 49 days posttransfer. The blood was drawn by retro-orbital bleeding during anesthesia with metofane. ELISAs for sIL-2Rα and β2 μ were performed to follow the ATL progression. The data presented represent 23 mice for days 7 and 15, 15 mice for days 21 and 35, and 7 mice for days 14 and 28. The values are presented as pg/ml for sIL-2Rα and ng/ml for β2 μ.

Close modal
Fig. 2.

Inhibition of the growth of MET-1 ATL cells in NOD/SCID mice by HAT, MAT, and 7G7/B6. MET-1 NOD/SCID spleen cells were transferred into 48 mice. Once the mice developed sIL-2Rα levels of 1000 pg/ml, therapy with various antibodies was initiated. The groups(eight mice/group) included those receiving PBS, IgG, HuMikβ1, HAT,MAT, and 7G7/B6. The mice were injected once per week for 4 weeks with the antibodies. The data represent the sIL-2Rα levels in pg/ml(A) and the β2 μ data in ng/ml (B). The HAT, MAT, and 7G7/B6-treated animals had significantly decreased values compared with the PBS control and the IgG-negative control groups (∗∗ = P < 0.01).

Fig. 2.

Inhibition of the growth of MET-1 ATL cells in NOD/SCID mice by HAT, MAT, and 7G7/B6. MET-1 NOD/SCID spleen cells were transferred into 48 mice. Once the mice developed sIL-2Rα levels of 1000 pg/ml, therapy with various antibodies was initiated. The groups(eight mice/group) included those receiving PBS, IgG, HuMikβ1, HAT,MAT, and 7G7/B6. The mice were injected once per week for 4 weeks with the antibodies. The data represent the sIL-2Rα levels in pg/ml(A) and the β2 μ data in ng/ml (B). The HAT, MAT, and 7G7/B6-treated animals had significantly decreased values compared with the PBS control and the IgG-negative control groups (∗∗ = P < 0.01).

Close modal
Fig. 3.

Kaplan-Meier survival plot of MET-1 NOD/SCID Mice. MET-1 NOD/SCID mice spleen cells were transferred to 48 mice. Once the mice had sIL-2Rα levels of 1000 pg/ml, therapy with various antibodies was initiated. The groups (eight mice/group) included PBS,IgG, HuMikβ1, HAT, MAT, and 7G7/B6. The mice were injected once per week for 4 weeks. Event-free survival was followed out to 75 days. HAT,MAT, and 7G7/B6 treatment prolonged the life of MET-1 NOD/SCID mice(P < 0.01).

Fig. 3.

Kaplan-Meier survival plot of MET-1 NOD/SCID Mice. MET-1 NOD/SCID mice spleen cells were transferred to 48 mice. Once the mice had sIL-2Rα levels of 1000 pg/ml, therapy with various antibodies was initiated. The groups (eight mice/group) included PBS,IgG, HuMikβ1, HAT, MAT, and 7G7/B6. The mice were injected once per week for 4 weeks. Event-free survival was followed out to 75 days. HAT,MAT, and 7G7/B6 treatment prolonged the life of MET-1 NOD/SCID mice(P < 0.01).

Close modal
Fig. 4.

The 7G7/B6 that binds to IL-2Rα does not inhibit IL-2-induced proliferation. Kit-225 (K6) cells were cultured in RPMI + 10% FCS with IL-2 for 3 days before the assay. The cells in triplicate in a 96-well microtiter plate were incubated for 3 days at 37°C with a range of IL-2 concentrations in the presence or absence of HAT, MAT, or 7G7/B6 (4 μg/well). Six h before harvest, 1μCi/well of [3H]thymidine was added to each well. The data presented are the percentage inhibition of IL-2-induced proliferation assessed by thymidine uptake in the presence or absence of the antibodies.

Fig. 4.

The 7G7/B6 that binds to IL-2Rα does not inhibit IL-2-induced proliferation. Kit-225 (K6) cells were cultured in RPMI + 10% FCS with IL-2 for 3 days before the assay. The cells in triplicate in a 96-well microtiter plate were incubated for 3 days at 37°C with a range of IL-2 concentrations in the presence or absence of HAT, MAT, or 7G7/B6 (4 μg/well). Six h before harvest, 1μCi/well of [3H]thymidine was added to each well. The data presented are the percentage inhibition of IL-2-induced proliferation assessed by thymidine uptake in the presence or absence of the antibodies.

Close modal
Fig. 5.

RT-PCR for IL-2 message. RNA from the spleens of three MET-1 NOD/SCID mice with high serum levels of sIL-2Rα SP-2/Tac cells(negative control) and stimulated Jurkat cells (PHA/PMA or ionamycin/PMA; positive control) was isolated. cDNA was made from the RNA samples and PCR for IL-2 message was performed. Message for G3PDH was used as the internal control. There was no detectable IL-2 message in the negative control (SP-2/Tac) or in the MET-1 NOD/SCID mouse-spleen samples. However, there was message for IL-2 in the stimulated Jurkat cells and in the primer positive control. All samples had comparable levels of G3PDH message.

Fig. 5.

RT-PCR for IL-2 message. RNA from the spleens of three MET-1 NOD/SCID mice with high serum levels of sIL-2Rα SP-2/Tac cells(negative control) and stimulated Jurkat cells (PHA/PMA or ionamycin/PMA; positive control) was isolated. cDNA was made from the RNA samples and PCR for IL-2 message was performed. Message for G3PDH was used as the internal control. There was no detectable IL-2 message in the negative control (SP-2/Tac) or in the MET-1 NOD/SCID mouse-spleen samples. However, there was message for IL-2 in the stimulated Jurkat cells and in the primer positive control. All samples had comparable levels of G3PDH message.

Close modal
Fig. 6.

A, ADCC. The capacity of various antibodies to function in ADCC was measured in a chromium release assay using MET-1 NOD/SCID spleen cells labeled with chromium-51. The antibodies were added at 37°C for 1–2 h at 1 μg/ml at various effector:target ratios and were assayed after 4 h of incubation for specific lysis. None of the antibodies caused specific lysis in this ADCC system. B, the study was identical to that described in 6A with the exception that CB17-SCID mouse spleens in the absence of MET-1 were used as the effector cells. The rank of the ability of the antibody to cause specific lysis was as follows:7G7/B6 (30.2% at 200:1) > HAT (17% at 200:1) > MAT (9.7% at 200:1),with PBS, IgG, and HuMikβ1 not yielding any specific lysis.

Fig. 6.

A, ADCC. The capacity of various antibodies to function in ADCC was measured in a chromium release assay using MET-1 NOD/SCID spleen cells labeled with chromium-51. The antibodies were added at 37°C for 1–2 h at 1 μg/ml at various effector:target ratios and were assayed after 4 h of incubation for specific lysis. None of the antibodies caused specific lysis in this ADCC system. B, the study was identical to that described in 6A with the exception that CB17-SCID mouse spleens in the absence of MET-1 were used as the effector cells. The rank of the ability of the antibody to cause specific lysis was as follows:7G7/B6 (30.2% at 200:1) > HAT (17% at 200:1) > MAT (9.7% at 200:1),with PBS, IgG, and HuMikβ1 not yielding any specific lysis.

Close modal
Fig. 7.

Acrylamide gel analysis of the F(ab′-Jun)2HAT. F(ab′)2 antibodies to IL2R. The protein preparations were electrophoresed on a 4% to 20% acrylamide gradient Tris glycine gel (Novex). Lane 1, intact HAT; Lane 2, non-reducing SDS PAGE HAT F(ab′-Jun)2;Lane 3, HuMikβ1 F(ab′-fos)2.

Fig. 7.

Acrylamide gel analysis of the F(ab′-Jun)2HAT. F(ab′)2 antibodies to IL2R. The protein preparations were electrophoresed on a 4% to 20% acrylamide gradient Tris glycine gel (Novex). Lane 1, intact HAT; Lane 2, non-reducing SDS PAGE HAT F(ab′-Jun)2;Lane 3, HuMikβ1 F(ab′-fos)2.

Close modal
Fig. 8.

Kaplan-Meier survival plot of AJ-NOD/SCID mice treated with HAT and F(ab′)2 HAT for 2 weeks. There was a significant increase (P < 0.01) in the survival of mice with MET-1 tumors that were treated with intact HAT weekly for 2 weeks. In contrast, the F(ab′-Jun)2 was ineffective in prolonging the survival of the tumor-bearing mice with no significant increase (P > 0.35) in the survival of the mice as compared with those in the groups treated with PBS or with IgG.

Fig. 8.

Kaplan-Meier survival plot of AJ-NOD/SCID mice treated with HAT and F(ab′)2 HAT for 2 weeks. There was a significant increase (P < 0.01) in the survival of mice with MET-1 tumors that were treated with intact HAT weekly for 2 weeks. In contrast, the F(ab′-Jun)2 was ineffective in prolonging the survival of the tumor-bearing mice with no significant increase (P > 0.35) in the survival of the mice as compared with those in the groups treated with PBS or with IgG.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

The abbreviations used are: ATL, adult T-cell leukemia; HTLV-I, human T-cell lymphotrophic virus-I; IL-2, interleukin 2; IL-2Rα, IL-2 receptor α; NK, natural killer; TCR, T-cell receptor; PBMC, peripheral blood mononuclear cell; FACS,fluorescence-activated cell sorting; ADCC, antibody-dependent cellular cytotoxicity; AICD, activation-induced cell death; HAT, humanized anti-Tac; mAb, monoclonal antibody; MAT, murine anti-Tac; NOD/SCID,non-obese diabetic/severe combined immunodeficient; RT-PCR, reverse transcription-PCR.

Table 1

Phenotype of MET-1 ATL cells from the NOD/SCID mouse model and from the patient MET-1

Cells for phenotyping were washed in FACS staining buffer (100 ml PBS,1 gm BSA, and 1 ml of a 10% solution of NaN3). One million cells/well were stained with FITC-labeled antihuman CD2, CD3, CD4, CD7,CD8, CD20, CD25, CD28, and class I HLA for 30–45 min on ice in the dark. After a wash, the viable cells were then enumerated on a Becton Dickinson FACSort Flow Cytometer. The phenotypic cell subtypes were expressed as a percentage of live cells.

Cell surface marker% positive% positive
MET-1-NOD/SCIDMET-1-patient ex vivo
CD2 99 96 
CD3 50 95 
CD4 46 15 
CD7 
CD8 NDa 
CD20 0.3 
CD25 98 95 
CD28 99 ND 
HLA 100 ND 
Cell surface marker% positive% positive
MET-1-NOD/SCIDMET-1-patient ex vivo
CD2 99 96 
CD3 50 95 
CD4 46 15 
CD7 
CD8 NDa 
CD20 0.3 
CD25 98 95 
CD28 99 ND 
HLA 100 ND 
a

ND, not done.

We thank Barbara Holmlund for her excellent editorial assistance.

1
Takatsuki K., Uchiyama T., Sagawa K., Yodoi J. Adult T-cell Leukemia in Japan
73
Amsterdam Excerpta Medica Amsterdam  
1977
.
2
Uchiyama T., Yodoi J., Sagawa K., Takatsuki K., Uchino H. Adult T-cell leukemia: clinical and hematologic features of 16 cases.
Blood
,
50
:
481
-492,  
1977
.
3
Shimoyama M. Diagnostic criteria and classification of clinical subtypes of adult T-cell leukaemia-lymphoma.
A report from the Lymphoma Study Group (1984–87). Br. J. Haematol.
,
79
:
428
-437,  
1991
.
4
Poiesz B. J., Ruscetti F. W., Gazdar A. F., Bunn P. A., Minna J. D., Gallo R. C. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma.
Proc. Natl. Acad. Sci. USA
,
77
:
7415
-7419,  
1980
.
5
Tajima K., Tominaga S., Shimizu H., Suchi T. A hypothesis on the etiology of adult T-cell leukemia/lymphoma.
Gann.
,
72
:
684
-691,  
1981
.
6
Catovsky D., Greaves M. F., Rose M., Galton D. A., Goolden A. W., McCluskey D. R., White J. M., Lampert I., Bourikas G., Ireland R., Brownell A. I., Bridges J. M., Blattner W. A., Gallo R. C. Adult T-cell lymphoma-leukaemia in Blacks from the West Indies.
Lancet
,
1
:
639
-643,  
1982
.
7
Blattner W. A., Kalyanaraman V. S., Robert-Guroff M., Lister T. A., Galton D. A., Sarin P. S., Crawford M. H., Catovsky D., Greaves M., Gallo R. C. The human type-C retrovirus, HTLV, in Blacks from the Caribbean region, and relationship to adult T-cell leukemia/lymphoma.
Int. J. Cancer
,
30
:
257
-264,  
1982
.
8
Seiki M., Hattori S., Yoshida M. Human adult T-cell leukemia virus: molecular cloning of the provirus DNA and the unique terminal structure.
Proc. Natl. Acad. Sci. USA
,
79
:
6899
-6902,  
1982
.
9
Uchiyama T., Broder S., Waldmann T. A. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells.
I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J. Immunol.
,
126
:
1393
-1397,  
1981
.
10
Waldmann T. A., Greene W. C., Sarin P. S., Saxinger C., Blayney D. W., Blattner W. A., Goldman C. K., Bongiovanni K., Sharrow S., Depper J. M., Leonard W., Uchiyama T., Gallo R. C. Functional and phenotypic comparison of human T cell leukemia/lymphoma virus positive adult T cell leukemia with human T cell leukemia/lymphoma virus negative Sézary leukemia, and their distinction using anti-Tac.
Monoclonal antibody identifying the human receptor for T cell growth factor. J. Clin. Investig.
,
73
:
1711
-1718,  
1984
.
11
Uchiyama T., Hori T., Tsudo M., Wano Y., Umadome H., Tamori S., Yodoi J., Maeda M., Sawami H., Uchino H. Interleukin-2 receptor (Tac antigen) expressed on adult T cell leukemia cells.
J. Clin. Investig.
,
76
:
446
-453,  
1985
.
12
Maeda M., Arima N., Daitoku Y., Kashihara M., Okamoto H., Uchiyama T., Shirono K., Matsuoka M., Hattori T., Takatsuki K., Ikuta K., Shimizu A., Honjo T., Yodoi J. Evidence for the interleukin-2 dependent expansion of leukemic cells in adult T cell leukemia.
Blood
,
70
:
1407
-1411,  
1987
.
13
Arima N., Daitoku Y., Ohgaki S., Fukumori J., Tanaka H., Yamamoto Y., Fujimoto K., Onoue K. Autocrine growth of interleukin 2-producing leukemic cells in a patient with adult T cell leukemia.
Blood
,
68
:
779
-782,  
1986
.
14
Tendler C. L., Greenberg S. J., Blattner W. A., Manns A., Murphy E., Fleisher T., Hanchard B., Morgan O., Burton J. D., Nelson D. L., Waldmann T. A. Transactivation of interleukin 2 and its receptor induces immune activation in human T-cell lymphotropic virus type I-associated myelopathy: pathogenic implications and a rationale for immunotherapy.
Proc. Natl. Acad. Sci. USA
,
87
:
5218
-5222,  
1990
.
15
Gill P. S., Harrington W. J., Kaplan M. H., Ribeiro R. C., Bennett J. M., Liebman H. A., Bernstein-Singer M., Espina B. M., Cabral L., Allen S., Kornblau S., Pike M. C., Levine A. M. Treatment of adult T-cell leukemia-lymphoma with a combination of interferon alfa and zidovudine.
N. Engl. J. Med.
,
332
:
1744
-1748,  
1995
.
16
Waldmann T. A., Goldman C. K., Bongiovanni K. F., Sharrow S. O., Davey M. P., Cease K. B., Greenberg S. J., Longo D. L. Therapy of patients with human T-cell lymphotrophic virus I-induced adult T-cell leukemia with anti-Tac, a monoclonal antibody to the receptor for interleukin-2.
Blood
,
72
:
1805
-1816,  
1988
.
17
Shultz L. D., Schweitzer P. A., Christianson S. W., Gott B., Schweitzer I. B., Tennent B., McKenna S., Mobraaten L., Rajan T. V., Greiner D. L., Leiter E. H. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.
J. Immunol.
,
154
:
180
-191,  
1995
.
18
Uchiyama T., Nelson D. L., Fleisher T. A., Waldmann T. A. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. II. Expression of Tac antigen on activated cytotoxic killer T cells, suppressor cells, and on one of two types of helper T cells.
J. Immunol.
,
126
:
1398
-1403,  
1981
.
19
Rubin L. A., Kurman C. C., Biddison W. E., Goldman N. D., Nelson D. L. A monoclonal antibody 7G7/B6, binds to an epitope on the human interleukin-2 (IL-2) receptor that is distinct from that recognized by IL-2 or anti-Tac.
Hybridoma
,
4
:
91
-102,  
1985
.
20
Tso J. Y., Wang S. L., Levin W., Hakimi J. Preparation of a bispecific F(ab′)2 targeted to the human IL-2 receptor.
J. Hematother.
,
4
:
3893
-3894,  
1995
.
21
Kondo A., Imada K., Hattori T., Yamabe H., Tanaka T., Miyasaka M., Okuma M., Uchiyama T. A model of in vivo cell proliferation of adult T-cell leukemia.
Blood
,
82
:
2501
-2509,  
1993
.
22
Imada K., Takaori-Kondo A., Sawada H., Imura A., Kawamata S., Okuma M., Uchiyama T. Serial transplantation of adult T cell leukemia cells into severe combined immunodeficient mice.
Jpn. J. Cancer Res.
,
87
:
887
-892,  
1996
.
23
Waldmann T. A., O’Shea J. The use of antibodies against the IL-2 receptor in transplantation.
Curr. Opin. Immunol.
,
10
:
507
-512,  
1998
.
24
Waldmann T. A., White J. D., Goldman C. K., Top L., Grant A., Bamford R., Roessler E., Horak I. D., Zaknoen S., Kasten-Sportès C., England R., Horak E., Mishra B., Dipre M., Hale P., Fleisher T. A., Junghans R. P., Jaffe E. S., Nelson D. L. The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-induced adult T-cell leukemia.
Blood
,
82
:
1701
-1712,  
1993
.
25
Waldmann T. A., White J. D., Carrasquillo J. A., Reynolds J. C., Paik C. H., Gansow O. A., Brechbiel M. W., Jaffe E. S., Fleisher T. A., Goldman C. K., Top L. E., Bamford R. N., Zaknoen S., Roessler E., Kasten-Sportès C., England R., Litou H., Johnson J. A., Jackson-White T., Manns A., Hanchard B., Junghans R. P., Nelson D. L. Radioimmunotherapy of interleukin-2R α-expressing adult T-cell leukemia with Yttrium-90-labeled anti-Tac.
Blood
,
86
:
4063
-4075,  
1995
.
26
Waldmann T. A. Immune receptors: targets for therapy of leukemia/lymphoma, autoimmune diseases and for the prevention of allograft rejection.
Annu. Rev. Immunol.
,
10
:
675
-704,  
1992
.
27
Vincenti F., Kirkman R., Light S., Bumgardner G., Pescovitz M., Halloran P., Neylan J., Wilkinson A., Ekberg H., Gaston R., Backman L., Burdick J. Interleukin-2-receptor blockade with daclizumab to prevent acute rejection in renal transplantation.
Daclizumab Triple Therapy Study Group. N. Engl. J. Med.
,
338
:
161
-165,  
1998
.
28
Lehky T. J., Levin M., Kubota R., Bamford R. N., Flerlage A. N., Soldan S. S., Leist T. P., Xavier A., White J. D., Brown M., Fleisher T. A., Top L. E., Light S., McFarland H. F., Waldmann T. A., Jacobson S. Reduction in HTLV-I proviral load and spontaneous lymphoproliferation in HTLV-I-associated myelopathy/tropical spastic paraparesis patients treated with humanized anti-Tac.
Ann. Neurol.
,
44
:
942
-947,  
1998
.
29
Nussenblatt R. B., Fortin E., Schiffman R., Rizzo L., Smith J., Van Veldhuisen P., Sran P., Yaffe A., Goldman C. K., Waldmann T. A., Whitcup S. M. Treatment of noninfectious intermediate and posterior uveitis with the humanized anti-Tac mAb: a Phase I/II clinical trial.
Proc. Natl. Acad. Sci. USA
,
96
:
7462
-7466,  
1999
.
30
Tendler C. L., Greenberg S. J., Burton J. D., Danielpour D., Kim S. J., Blattner W. A., Manns A., Waldmann T. A. Cytokine induction in HTLV-I associated myelopathy and adult T-cell leukemia: alternate molecular mechanisms underlying retroviral pathogenesis.
J. Cell Biochem.
,
46
:
302
-311,  
1991
.
31
Migone T. S., Lin J. X., Cereseto A., Mulloy J. C., O’Shea J. J., Franchini G., Leonard W. J. Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I.
Science
,
269
:
79
-81,  
1995
.
32
Xu X., Kang S. H., Heidenreich O., Okerholm M., O’Shea J. J., Nerenberg M. I. Constitutive activation of different Jak tyrosine kinases in human T cell leukemia virus type 1 (HTLV-1) tax protein or virus-transformed cells.
J. Clin. Investig.
,
96
:
1548
-1555,  
1995
.
33
Azimi N., Brown K., Bamford R. N., Tagaya Y., Siebenlist U., Waldmann T. A. Human T cell lymphotropic virus type I Tax protein trans-activates interleukin 15 gene transcription through an NF-κB site.
Proc. Natl. Acad. Sci. USA
,
95
:
2452
-2457,  
1998
.
34
Migone T. S., Cacalano N. A., Taylor N., Yi T., Waldmann T. A., Johnston J. A. Recruitment of SH2-containing protein tyrosine phosphatase SHP-1 to the interleukin 2 receptor; loss of SHP-1 expression in human T-lymphotropic virus type I-transformed T cells.
Proc. Natl. Acad. Sci. USA
,
95
:
3845
-3850,  
1998
.
35
Clynes R., Takechi Y., Moroi Y., Houghton A., Ravetch J. V. Fc receptors are required in passive and active immunity to melanoma.
Proc. Natl. Acad. Sci. USA
,
95
:
652
-656,  
1998
.
36
Clynes R. A., Towers T. L., Presta L. G., Ravetch J. V. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets.
Nat. Med.
,
6
:
443
-446,  
2000
.
37
Lenardo M. J. Fas and the art of lymphocyte maintenance.
J. Exp. Med.
,
183
:
721
-724,  
1996
.
38
Wang R., Murphy K. M., Loh D. Y., Weaver C., Russell J. H. Differential activation of antigen-stimulated suicide and cytokine production pathways in CD4+ T cells is regulated by the antigen-presenting cell.
J. Immunol.
,
150
:
3832
-3842,  
1993
.
39
Refaeli Y., Van Parijs L., London C. A., Tschopp J., Abbas A. K. Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis.
Immunity
,
8
:
615
-623,  
1998
.