Int. J . Cancer: 49, 246-253 (1991) 0 1991 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer

ESTABLISHMENT OF AN IL-2 INDEPENDENT, HUMAN T-CELL LINE POSSESSING ONLY THE p70 IL-2 RECEPTOR Gordon sTARKEBAUM'35,Thomas P. LOUGHRAN, JR.*, Cory A. WATERS3 and Francis w. RUSCETT14 'Department of Medicine, Seattle VA Medical Center and Department of Medicine, Division of Rheumatology, University of Washington, Seattle, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA; 3Seragen, Hopkinton, MA; and 4National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD, USA. A continuous cell line was established from the blood of a patient (HH) with an aggressive cutaneous T-cell leukemia/ lymphoma who lacked antibodies t o human T lymphotrophic virus, type 1. The immunophenotype of the cultured cells was CD2+, CD3+, CD4+, CD5+, CD8-, DR' and CD25- (Tac, IL-2 receptor a chain). Southern-blot hybridization analysis of 1-cell-receptor p chain DNA demonstrated the same rearrangement in freshly isolated blood cells and cultured cells, indicating that the cell line was derived from the patient's malignant clone. Since cultured T-cells grew in complete medium without added IL-2, we investigated whether HH cells could be producing and responding t o IL-2 in an autocrine fashion. However, no IL-2 was detectable in supernatant from the cell line, while antibodies t o IL-2, or t o the IL-2 receptor Q or f3 chains did not inhibit cell growth. In addition, no mRNA message for IL-2 was detectable in these cells. The results appear t o exclude an autocrine IL-2-dependentmechanism of cell growth for this T-cell line. Although cultured HH cells lacked detectable 11-2 receptor a chain, they did show increased proliferation t o exogenous IL-2. Binding studies with '251-IL-2demonstrated an intermediate affinity receptor for IL-2, & = 1.7n~,with 6400 binding sites per cell, suggesting the presence of an IL-2 receptor f3 chain. Consistent with these findings '251-IL-2cross-linking studies demonstrated a single receptor calculated t o be 75kDa. Also, the f3 chain of the IL-2 receptor was detected by immunofluorescence using specific monoclonal antibodies (MAbs). Nanomolar concentrations of an IL-2-diphteria toxin fusion protein inhibited cellular protein synthesis, an effect abrogated by native IL-2. These findings indicate that the IL-2 receptor @chain was functional. This novel mature T-cell line may be useful in studies of IL-2 receptor regulation and in analysis of the mechanism of T-cell leukemogenesis.

Malignancies of mature T cells are a heterogeneous group of generally chronic disorders which include adult T-cell leukemidlymphoma (ATLL), T-cell chronic lymphocytic leukemia (T-cell CLL), T-prolymphocytic leukemia and cutaneous T-cell lymphoma (Catovsky et a l . , 1982; Knowles, 1986). The latter designation includes Mycosis fungoides and the Skzary syndrome. ATLL has been of considerable interest since identification of the retrovirus human T-lymphotrophic virus type I (HTLV-I) as the likely causative agent for this disorder (Poiesz et a l . , 1980; Wong-Staal et a l . , 1985). The precise mechanism whereby HTLV-I infection causes ATLL is unknown. However, abnormal expression of IL-2 receptors on leukemic cells induced by the HTLV-I-encoded transcriptional activator protein, Tax, appears to play a key role in the cells' transformation (Maruyama et al., 1987; Siekevitz et al., 1987; Ruben et al., 1988). Cell lines established in vitro from ATLL patients, which are initially IL-Zdependent, frequently become independent of exogenous IL-2 (Popovic et a l . , 1983). It is unclear how such growth-factor independence arises in these T-cell lines, however. Initial studies indicated that HTLV-I positive T-cell lines generally failed to produce IL-2 in vitro (Arya et al., 1984). Subsequently, several T-cell lines established from ATLL patients have clearly been shown to produce and respond to IL-2, suggesting that autocrine growth through an IL-2/IL-2 receptor pathway was likely (Maruyama et al., 1987; Arima et al., 1986, 1987; Maeda et al., 1987). Also, T-cell lines from a patient with T-cell CLL not caused by HTLV-I

(Hori et a l . , 1887) as well as from a patient with lymphoma (Duprez et al., 1985) have been shown to grow in vitro through a similar autocrine IL-2/IL-2 receptor mechanism. Thus, T-cell lines established from such patients may provide important clues regarding the mechanism of malignant transformation. In this report we describe a CD4-positive T-cell line established from a patient with HTLV-I-negative T-cell leukemia/ lymphoma which possesses only the p75 IL-2 receptor. The cell line proliferates in the presence of complete medium alone, however, and does not secrete or require IL-2. Case report The patient was a 61-year-old white male admitted to the Seattle VA Medical Center in November 1986 with increasing skin rash and lymphadenopathy. He had had psoriasis for many years, which had been treated with topical steroids. Twelve months before admission he developed nodular ulcerating skin lesions, which were distinctly different from the psoriasis plaques. Five months before admission, biopsy of cervical and tonsillar lymph nodes revealed a lymphoma confirmed to be of T-cell origin by immunofluorescence. CT scans of the chest and abdomen showed only right para-tracheal nodes. Examination of the marrow was negative for lymphoma. Chemotherapy (CHOPP) was administered every 3 weeks, initially resulting in a decrease in adenopathy. However, adenopathy and skin lesions recurred without pruritis; skin biopsy showed patchy infiltration of malignant-appearing lymphocytes in the deep dermis and subcutaneous tissue without Pautrier's microabscesses. Immunochemical stains indicated that the malignant cells were T cells. The patient was admitted to the Seattle VA Medical Center for further evaluation. He had received a blood transfusion 8 years previously during stomach surgery. He had never travelled to the Far East or to the Caribbean but had been a prisoner of war in Germany in 1945. Physical examination revealed firm erythematosus nodules over most of his body, many with ulceration. Erythroderma was not present. Diffuse adenopathy , splenomegaly, unequal pupils and ptosis of the right eyelid were noted. The hematocrit was 33.5%, the WBC was 9,50O/p1 with 67% segs, 2% bands, 15% monocytes, and 11% lymphocytes, and the platelet count was 425,00O/pl. Serum from the patient contained no antibodies to HTLV-I by ELISA or by Western blot (personal communication Kathy Schreiber, ONCOGEN, Seattle). The patient was treated with Decadron and chemotherapy (MACOP) and antibiotics with some improvement over 5 weeks. However, lymphadenopathy and skin lesions worsened. His WBC increased to 40,0OO/pI inwith 55% abnormal-appearing lymphocytes. The Ca+ creased to 10.9 in spite of albumin of 2.7g/dl. The patient rapidly deteriorated and died on December 26, 1986.

+

5To whom correspondence and reprint requests should be sent, at the Arthritis Section (1 1l), VA Medical Center, 1660 So. Columbian Way, Seattle, WA 98108. ~~

Received: March 5, 1991 and in revised form May 3, 1991

HUMAN LEUKEMIC T-CELL LINE

METHODS

Cells and cell culture Mononuclear leukocytes (MNL) from the patient's heparinized blood were separated on Ficoll-Hypaque gradients and suspended at 0.1 X 106/ml in complete medium (RPMI 1640 with 10% fetal calf serum, both from GIBCO, Grand Island, NY) containing 1mM glutamine, 1mM penicillin and 1mM streptomycin. Initially the cells were cultured with recombinant IL-2 (rIL-2) 50 unitdm1 (various sources, primarily AmGen, Thousand Oaks, CA) at 37°C in a 5% CO, atmosphere and split once weekly. After the first month the cells were cultured in the same medium in the absence of IL-2. Cells were also cyropreserved in complete medium containing 10% DMSO and stored under liquid nitrogen. For some studies MNL were isolated in a similar fashion from normal donors and the cells were cultured in complete medium alone or in the presence of 1% PHA (Wellcome, Research Triangle Park, NC) . Proliferation of cells cultured in 96-well microtiter plates (Costar, Cambridge, MA) was measured by pulsing each with 1pCi of 3H-thymidine (ICN, Irvine, CA) for 4 hr. Cells were then collected on glass fiber filters using a cell harvester (Skatron, Sterling, VA). Filters were washed, dried and counted according to standard methods. The source of the antibodies used in cell culture experiments was as follows: rabbit anti IL-2: Genzyme (Boston, MA); MAb to the IL-2 receptor a chain: Interferon Sciences (New Brunswick, NJ); and MAb TU-27 to the IL-2 receptor p chain (Takeshita et al., 1989) kindly provided by Dr. K. Sugamura (Tohoku University School of Medicine, Sendai, Japan). IL-4 and rabbit antiserum to IL-4 were kindly provided by Dr. C. Smith (Immunex, Seattle, WA). Immunophenotyping Freshly isolated and cultured lymphocytes were analyzed for surface markers by indirect immunofluorescence and flow cytometry with the following panel of MAbs: Leu 5B (anti-CD2, pan-T-cells); Leu 4 (anti-CD3, mature T-cells): Leu 3A (antiCD4, T-helperhnducer cells); Leu 2A (anti-CD8, T-suppressor/cytotoxic cells; 10.2, anti-CD5); Leu 9, anti-( 1)7; antiHLA-DR, class-I1 MHC antigen; and anti-Tac, IL-2 receptor a chain (all from Becton Dickinson, Mountain View, CA). TU27 was used to identify the IL-2-receptor f3 chain. Cell-surface immunofluorescence was analyzed on a Coulter Epics-V flow cytometer (Coulter, Hialeah, FL) as previously described (Loughran et al., 1988).

247

normal MNL cultured in medium alone or with 1% PHA for 6 and 12 hr before harvest, then 20pg of total RNA from each sample were electrophoresed on a formaldehyde-agarose gel and blotted to nitrocellulose, Northern blots were hybridized to random-primed '*P-labelled cDNA probes. The following cDNA probes were used: pIL2R2 containing mucleotides 1-937 of the full-length IL-2 receptor cx chain cDNA (Leonard et a/., 1984); pTC6F-5 containing the full-length IL-2 cDNA (Clark et al., 1984); and pKS-IL-2R p which contains the full-length cDNA for IL-2 receptor p chain (Freimark and Robb, 1990), kindly supplied by Dr. B. Freimark (Dupont, Glenolden, PA). After overnight hybridization, the blots were washed successively 4 times for 5 rnin each at room temperature in 2 X SSC/O.l% SDS, once for 5 rnin in 0.1 X SSC/ 0.1% SDS, and once for 20 rnin at 55°C in 0.1 X SSC/O.I% SDS. After drying, the blots were exposed to X-ray film using an intensifying screen. Radiolabelled IL-2 binding assay HH cells were washed twice with RPMI 1640 medium and 10% FCS followed by a wash with acidified binding buffer (RPMI-1640, lmg/ml BSA, glutamine and 0.1% sodium azide acidified by gassing with CO,). One million cells were then incubated at 37°C for 30 min with approximately lOnM lZ5IIL-2 (sp. act. 39pWpg: NEN, Newton, MA) in 0.2ml suspension with constant agitation (Robb et al., 1981).These cells were then overlaid onto a 0.2 ml mixture of 80% silicone oil (Dexter Hysol 550 fluid; Dexter, Orleans, NY) and 20% paraffin oil (0-1 19, Fisher, Pittsburgh, PA), After a 20-min incubation at 37°C the tubes were centrifuged (8,500g for 90 sec) and the tips of the tubes containing the cell pellet were cut. The cell-bound and free radioactivity were determined by solid scintillation counting. The calculated values for the number of binding sites per cell were determined by Scatchard analysis of equilibrium binding data after subtraction of non-specific binding determined in the presence of 150-fold molar excess of unlabelled IL-2. The lower limit of detection for ligands was 50 binding sites per cell.

IL-2 cross-linking For these studies the cell line Hut-102 was included as a positive control. A total of 1 x lo7 HH or Hut cells was washed once with cold RPMI 1640 and 10% FCS and twice in cold acidified binding buffer and resuspended in 1 ml of binding buffer. Cells were then incubated with lo5 to lo6 cpm (2 pCi) 1251-IL-2(rHu, specific activity = 39 pCi/pg; NEN, Boston, MA). The tubes were rotated for 1 hr at 4°C. After incubation, cells were washed once in 1 ml binding buffer and Southern Blot hybridization analysis resuspended in 1 ml cross-linking buffer (PBS, PH 8.3, 10 mM Genomic DNA was extracted as previously described from MgCI,) in the presence of 0.5 mM disuccinimidyl suberate freshly isolated patient mononuclear cells or from cultured leu- (Pierce, Rockford, IL), which was dissolved in DMSO just kemic cells (Loughran et al., 1988). The DNA samples were before use. Cells were again rotated for 1 hr at 4°C. After then digested with restriction enzymes BamHI, EcoRI or incubation, cells were washed twice in cross-linking buffer and HindIII. Digested DNA was separated on 1.1% agarose gels centrifuged for 1 min at 8160g in an Eppendorf centrifuge. and transferred onto nitrocellulose filters by the method of Then the pellet was resuspended in 50 pi of lysis buffer (1OmM Southern. Filters were then hybridized to DNA probes 32P- TRIS-HCI, 5mM EDTA, 1% Triton X-100 and 0.15 M NaCI). labelled by nick-translation and visualized by autoradiography The pellet was incubated for 30 rnin in lysis buffer at 4"C, then (Loughran et al., 1988). The cDNA clone Jurkat p2 containing centrifuged at 816Og for 10 rnin at 4°C. Supernatant was rethe C and J regions of the T-cell-receptor p gene (Yoshikai et moved and 50 pl SDS-PAGE buffer were added (0.01%bromal., 1984) was kindly provided by Dr. T. Mak (Ontario Cancer phenol blue 10% glycerol, 5% 2-ME, 2.3% SDS, 0.625 M Institute, Toronto, Canada). The insert representing nucle- TRIS-HCl, PH 6.8). Samples were electrophoresed on a 7 to otides 100-870 was isolated on agarose gels and used as the 15% gradient polyacrylamide SDS gel, and developed by autoradiography . hybridization probe.

RNA isolation and Northern blotting Total RNA was isolated using guanidine hydrochloride (Chirgwin et a[., 1979) from 35 X 1U6 HH cells cultured in medium alone or with 50U/ml of IL-2 and from 80 X lo6

DAB4, IL-2 protein synthesis inhibition studies HH cells were washed once with RPMI 1640 medium supplemented with 2 5 m ~N-2-hydroxyethylpiperazine-Nt-2-ethanesulfonic acid (HEPES), PH 7.4 2 mM glutamine and 10%

248

STARKEBAUM ET AL.

fetal bovine serum, then seeded into 96-well V-bottom microtiter plates (Nunc, Rosklide, Denmark) at a concentration of lo5 per well in 100 ~1 of the same medium. Recombinant DAB,,$ IL-2 (Bacha et al., 1988) was added at varying con) medium. Cells cultured with centrations ( ~ O - ” M to 10 - h ~ in medium alone were included as a control. Human rIL-2 was added to certain cultures at a final concentration of 8.1 X ~O-’M. Subsequent to a 20-hr incubation at 37°C in a 5% C 0 2 atmosphere, the platcs were ccntrifuged for 5 min at 170g, then the medium was removed and rcplaced with 100 ~1 of leucinefree medium (DMEM Selcctamine, Gibco) containing 2.5 FCi/ ml [‘4C]-leueine (NEN). Cclls were then incubated at 37°C for 2 hr and harvested o n to glass fiber filters as described above. All determinations were performed in quadruplicate. RESULTS

Examination of blood obtained from the patient shortly before his death revealed abnormal lymphocytes with large nuclei and prominent nucleoli. Approximately 20% of these cells had deeply indented nuclci (Fig. 1). Histochemical and immunophenotyping of these cells showcd the following: TdT 0 9 , CD2 92%, 10.2 (CD5) 80%, CD3 0 % %CD4 87%, CD7 0%. CD8 0%, DR 79%, CD2S (Tac IL-2R a chain) 0%. Isolated mononuclear cells were initially cultured in the presence of 50 Uiml of recombinant human IL-2. When the cells werc first tested after 3 weeks in culture, however, they were found to bc IL-2-independent with a doubling time of 2.5 days; the cells were cultured thereafter in the absence of IL-2. Cyropreserved peripheral blood lymphocytes were also subsequently found to grow in the complete absence of IL-2. The immunophenotype of cultured cells was similar to that of uncultured cells except that 90% were CD3 . By electron microscopy most cultured cells showed large, convoluted nuclei (Fig. 2). To determine whether cultured HH cells represented the same clone as the malignant cells isolated from the patient‘s blood, DNA was cxtractcd from both populations and subjected to Southern blot hybridization analysis using the T-cellreceptor @chain probe. Analysis after BamHi and HindIII digestion revealed the same rearrangement of the C p 2 allele in both uncultured and cultured cclls (Fig. 3, arrow lanes 1.2. and lanes 4 3 ) . Analysis after digestion with HindIII, however,

FIGURE2 - Electron micrograph of cultured HH cell. Bar = 5 pm.

+

FIGURE1 stain. Bar

-

Leukemic cell in peripheral blood. Wright-Giemsa 10 prn.

FIGURE 3 - T-cell receptor p gene rearrangement studies. DNA was obtained from: uncultured HH cclls, lanes 1 and 4; cultured HH cells. lanes 2 and 5 ; normal neutrophils, lanes 3 and 6 (gem-line DNA). The arrows indicate the identical rearrangement involving the Cp2 allele which is present in both culturcd and uncultured HH cells. In addition, the cell line has deleted thc Cpl allele, indicated by loss of the 3.5-kb band in the HindIII digcst (lanc 5 ) .

also revealed that the ccll line had deleted the C p l allele as shown by loss of the 3.5-kb germ-line band (Fig. 3, lane 5). These findings indicated that thc ccll line was derived from the identical clone found in the patient’s blood but deletion of a portion of the T-cell receptor p gene had occurred in vitro. Immunofluorescence studies using antibodies to the a and p chains of the IL-2 receptor indicated that less than 5% of HH cells had detectable IL-2Ra chain but approximately 40% had detectable IL-2RP chain (Fig. 4a). Incubation of the cells with 50 Uiml of IL-2 for 7 days did not induce any change in the status of the IL-2 receptors (Fig. 4h). In equilibrium binding studies, ”jI-IL-2 bound to the cells with a single intermediate ) a calculated density of 6,400 bindaffinity (K, = 1 . 7 n ~with ing sites per cell (not shown). Finally, chemical cross-linking with Iz5I-IL-2 to H H cell-surface proteins followed by SDSPAGE and autoradiography revealed a single band of M, 90,000, indicating an IL-2 receptor of approximately 75,000 (Fig. 5a). In contrast, cross-linking M,. of I2’I-IL-2 to Hut-102 cells followed by SDS-PAGE revealed 2 bands of M, 90,000 and 68,000 (Fig. 5c). In the prcscnce of cxcess cold IL-2, no bands were seen with either ccll line (Fig. Sb,d). These results

249

HUMAN LEUKEMIC T-CELL LINE

Control

150 7-

a, 140

-r

A

10

50

cn c

0

a v)

a, [I:

Log Fluorescence

T

130

Log Fluorescence

FIGURE4 - Immunofluorescence and flow cytometry. ( a ) HH cells cultured in medium alone. (b) HH cells cultured with 50 p/ml IL-2.

1

5

IL-2 u/ml FIGURE6 - Response of HH cells to IL-2. The mean increase in proliferation k 1SD induced by IL-2 is shown for 4 separate experiments. 100-

.-c 5 80E 0 Cold IL-2

FIGURE5 - Iz5I-IL-2 cross-linking studies. Lane A, HH cells with medium alone; lane B, HH cells with 150-fold excess of unlabelled IL-2; lane C, Hut-102 cells with medium alone; lane D, Hut-102 cells with 150-fold excess of unlabelled IL-2. No 68-kDa band is seen with HH cells, indicating absence of p55 IL-2 receptor a chain.

indicate that only the IL-2 receptor p chain was detectable on HH cells. The response of the cell line to IL-2 was then examined. Addition of IL-2 induced a modest increase in cell proliferation in a dose-dependent fashion (Fig. 6 ) . Thus, 50U/ml of IL-2 increased 3H-thymidine uptake over baseline by 43 2 4.5% (mean 2 1 SD, n = 4, Fig. 6). To confirm that the IL-2 receptor on these cells was functional, the effect of the IL-2 diptheria toxin recombinant fusion protein, DAB,,,IL-2, on cellular protein synthesis was examined. Exposure of HH cells to DAB,,,IL-2 at a concentration of 2.3 X 1 0 - 8 ~for 20 hr resulted in 50% inhibition of protein synthesis (I&,) compared to that observed in untreated control cultures (Fig. 7). The inhibition was completely abolished by the addition of 8.1 X ~ O - ' M rIL-2, (Fig. 7), indicating that DAB,,,IL-2 uptake was mediated by an IL-2 receptor. To determine whether constitutive growth of HH cells could be dependent on endogenous IL-2, the effect of adding anti-

g

K 0 .--

2

60-

I

40-

s8

20-

c

01 10-10

I

I

I

10.0

10-9

DAB,,

10-7

I

10-6

IL-2 (M)

FIGURE7 - Inhibition of protein synthesis by DAB,,,IL-2. Incubation of HH cells in the presence of the hybrid toxin-fusion protein, DAB,,,IL-2 caused a dose-dependent inhibition of protein synthesis with an IC,, of 2.3 X ~ O - ' M (closed circles, mean k IsD). Adding rIL-2 (8 X 10- 'M) to the cultures completely blocked the effect of the IL-2-toxin (open circles).

bodies to IL-2 or to either the (Y or the p chain of the IL-2 receptor was examined. No inhibition of proliferation was seen with 20 p,g/ml of rabbit anti-IL-2 antibody (Table I), sufficient to neutralize 10 units/ml of IL-2 (Table 11). Furthermore, antibody to either IL-2 receptor (Y chain or p chain did not cause a significant decrease in cellular proliferation (Table I). Supernatant from the cells failed to stimulate either normal

250

STARKEBAUM E T A L .

TABLE I - EFFECT OF ANTIBODIES TO IL-2 AN? TO IL-2 RECEPTORS ON GROWTH OF HH CELLS

Conditions

'H-thymidine uptake, cpm (mean 2 1 SD)

Buffer Rabbit anti-IL-2 (20pgIml) Rabbit IgG (20pglml) Anti-Tac (1 pg/ml) Anti-p70 (1pg/ml) anti-p70 Anti-Tac Mouse IgGl (1kg/ml)

14,786 f 1,665 14,106 t 2,520 12,182 f 2,376 12,187 t 1,131 12,755 t 2,146 11,478 f 2,761 17,629 f 1,534

+

'5 x lo5 cells were cultured with reagent indicated for 24 hr, then pulsed with 'H-thymidine for 4 hr and harvested. The results are from 8 determinations obtained in 2 separate experiments.

PBMC or PHA-activated normal T-cells (Table 11). In contrast, normal T-cells were readily stimulated with either exogenous IL-2 or IL-4, effects which were attenuated or inhibited by their specific antibodies (Table 11). These results suggested that no free IL-2 or other lymphokine was being secreted by HH cells. To exclude the possibility that intracellular IL-2 could still be contributing to cell growth, possibly by reacting with an intracellular receptor, total RNA from HH cells was probed for IL-2. However, Northern analysis of RNA from the cell line cultured in either the presence or the absence of exogenous IL-2 revealed no IL-2 mRNA (Fig. 8a, lanes 1 and 2). IL-2 message was detected in RNA isolated from normal PBMC cultured for 6 and 12 hr with PHA (Fig. 8a, lanes 4 and 5). Furthermore, no IL-2Ra chain mRNA was detectable in HH cells (Fig. 8b, lanes 1 and 2) whereas IL-2RP chain mRNA was present (Fig. 8c, lanes 1 and 2). DISCUSSION

We have described a novel, CD4+ mature T-cell line established from the peripheral blood of a patient with an aggressive T-cell leukernidlymphoma. HH cells possessed the P chain of the IL-2 receptor complex but grew constitutively in complete medium containing no 1L-2. The clinico-pathologic diagnosis in this patient was cutaneous T-cell lymphoma with a terminal leukemic phase. The morphology of the circulating abnormal lymphocytes was consistent with that of SCzary cells although the absence of diffuse erythroderma and pruritis is atypical of SCzary syndrome (Wieselthier and Koh, 1990). On the other hand, the patient exhibited many features suggesting adult T-cell leukemidlymphoma

(ATLL) (Bunn, 1984; Neely, 1989) including ulcerating skin lesions, lymphadenopathy, splenomegaly , and progressive leukemia with circulating abnormal CD4+ lymphocytes, many of which had deeply indented nuclei. Also consistent with ATLL, was the development of hypercalcemia and a rapidly downhill course in spite of chemotherapy. Although the patient did not come from an area endemic for HTLV-I, he had received blood transfusions 8 years previously. His serum contained no antibodies to HTLV-I, however, and no HTLV-I proviral DNA sequences were detected in fresh or cultured cells by Southern analysis (D. Durnham, Fred Hutchinson Cancer Research Center, personal communication). Furthermore, no viral particles were seen by electron microscopy in cultured cells and attempts to transform normal T cells in vitro were unsuccessful ( G . Starkebaum, data not shown). Finally, both fresh and cultured HH cells lacked the IL-2 receptor a chain (Tac antigen), which is increased in the peripheral blood of ATLL patients and is seen in virtually all HTLV-I-positive T-cell lines (Uchiyama et al., 1985; Waldman et al., 1984). In contrast, T cells from patients with SCzary syndrome, who lack antibodies to HTLV-I, do not express Tac antigen (Waldman et al., 1984). These results strongly suggest that HTLV-I was not the etiologic agent in this case. Shimoyama et al. (1986) have described similar HTLV-I-negative cases of ATLL in Japan. On the other hand, Duggan et al. (1988) have described a patient with atypical Hodgkin's disease who lacked antibodies to HTLV-I and whose DNA lacked HTLV-I proviral sequences by conventional Southern blotting but did have HTLV-I sequences by the sensitive polymerase chain-reaction method. It will be of interest to evaluate this T-cell line for the presence of retroviral DNA sequences by this technique. Cultured cells of patient HH morphologically resembled those found in his peripheral blood and the immunophenotypes of both were nearly identical. The basis for the conversion of CD3- to CD3+ in culture is, however, unexplained. Additional strong evidence that the cultured cells were directly derived from the circulating malignant cells of the patient is the finding of an identical rearrangement of the T-cell receptor P-chain gene in both populations. These findings argue against the transformation and outgrowth of an unrelated T cell in vitro, as is commonly observed in HTLV-I-induced ATLL (Maeda et al., 1985). HH cells did not show detectable p55 IL-2 receptor a chain when anti-Tac antibodies were used. Nevertheless, HH cells showed a modest proliferative response to IL-2. Recent studies indicate that the IL-2 receptor exists as a heterodimer composed of a 55-kDa a chain, also known as the Tac antigen, and

TABLE I1 - EFFECT OF HH SUPERNATANT, IL-2 AND IL-4 ON NORMAL T CELLS'

'H-thvmidine uDtake (mean cpm -+

Conditions

Buffer only HH supernatant lop1 HH supernatant 50p1 HH supernatant 100pl IL-2 10U/ml 11-4 100ng/ml 11-2 10U/ml rabbit IgG (10pgirnl) IL-2 10U/ml + rabbit anti-IL-2 (10pg/ml) IL-4 100ng/rnl + rabbit serum IL-4 100ng/rnI + rabbit anti-IL-4 HH supernatant 50p1 + rabbit anti-IL-2 HH supernatant 50p1 + rabbit anti-IL-4 PHA 153. 2Oul

+

SEM,

n

=

4)

Preincubation with buffer

Preincubation with PHA

828 f 341 1,064 -t 320 1.205 -t 307 1;090 k 146 5,228 -t 177 2;867 f. 586 1078 3,312 2,665 f 964 3,845 t 349 1,146 f 469 1,151 2 58 1,578 t 410 40.034 f 1527

2,485 f 273 1,953 k 90 1,866 k 59 1,078 f 154 19,461 f 809 64,007 -C 5,358 19,871 f. 484 9,472 t 3,221 74,867 k 1,166 2,026 2 669 1,570 -C 53 1,808 f. 129 36.653 k 3887

*

'Normal mononuclear leukocytes were cultured for 5 days with either buffer or with PHA. After washing, I X 10' cellslwell were cultured for 2 days in the presence of the reagent indicated, then pulsed with 'H-thymidine and harvested as before.

HUMAN LEUKEMIC

FIGURE8 - Northern blot probed for ( a ) IL-2, (b) IL-2 receptor a chain, and (c) IL-2 receptor P-chain mRNA. Twenty micrograms of total RNA were loaded in each lane. Lane 1, HH cells cultured in medium alone; lane 2, HH cells cultured for 1 week in IL-2; 50U/ml; lane 3, freshly isolated normal PBMC; lane 4,normal PBMC stimulated with PHA for 6 hr; lane 5 , normal PBMC stimulated with PHA for 12 hr. In ( a ) the 900-bp IL-2 mRNA is detectable in lanes 4 and 5 only. In (b) IL-2 receptor a-chain mRNA is detectable only in lanes 4 and 5 . In (c) IL-2 receptor P-chain mRNA is present in lanes 1 , 2, 4 and 5 .

a 70- to 75-kDa P chain (Sharon et al., 1986; Tsudo et al., 1986; Teshigawa et al., 1987; Smith, 1988). Singly, the a chain binds IL-2 with only a low affinity with a dissociation . IL-2 receptor p chain has interconstant, K,, of 1 0 - 8 ~ The . however, mediate affinity for IL-2, K, = 1 0 - 9 ~ Together, the olP heterodimer exhibits high affinity for IL-2, K, = 1 0 - l ’ ~(Teshigawara et al., 1988; and Smith, 1988). Consistent with this model, studies with labelled IL-2 indicated that HH cells bound IL-2 with intermediate affinity. Additional

T-CELL LINE

25 1

evidence for the existence of IL-2 receptor P-chain molecules on the surface of HH cells was provided by IL-2 cross-linking studies which showed a single species calculated to be 75kDa. Finally, immunofluorescence studies with MAbs demonstrated the presence of IL-2 receptor p chain on these cells, and mRNA for this receptor, but not for the a chain, was seen on Northern blots. These findings are very similar to those observed with the subcloned human cell line YT-2C2 which lacks the IL-2 receptor a chain (Smith, 1988). YT cells bind IL-2 with a K, of 1.7nM, possess 20,000 IL-2 binding sites per cell and exhibit only the p75 IL-2 P-chain receptor in 1251-IL-2cross-linking studies (Smith, 1988). However, the YT line was derived from a patient with acute lymphoblastic leukemia, lacks T-cell markers such as CD3 or CD4 and exhibits many features characteristic of natural killer cells such as the presence of CD16 and CD57 (HNK-1) (Yodoi et al., 1985). The features of HH cells are compared to those of other selected human or primate leukemic T-cell lines in Table 111. Recent studies indicate that the IL-2 receptor P chain is essential for internalization of IL-2 and for signal transduction induced by IL-2 (Siege1 et al., 1987; Robb and Greene, 1987). In accordance with these findings, elucidation of the structure of the IL-2 receptor P chain by Hatakeyama et al. (1989) revealed an intracellular domain of 286 amino acids considerably longer than the 13 amino-acid intracytoplasmic domain of the IL-2 receptor a chain (Clark et al., 1984). The finding that HH cells proliferated in response to IL-2 while lacking the IL-2 receptor a chain suggested that their IL-2 receptor P chain was functional. Furthermore, in contrast to normal T cells (Smith and Cantrell, 1985), incubation of HH cells with IL-2 did not induce expression of IL-2 receptor a chain. To evaluate whether the P chain of HH cells’ IL-2 receptor could internalize IL-2, the effect of the recombinant diphteria toxin-IL-2 fusion protein DAB4,,IL-2 (Bacha et al., 1988) was examined. The data indicate that nanomolar concentrations of DAB4,,IL-2 inhibited protein synthesis by HH cells, an effect that was completely blocked by recombinant native IL-2. Similar effects of DAB4,,IL-2 are seen on YT-2C2 cells and on normal large granular lymphocytes (Waters et al., 1990) which also lack the IL-2 receptor a chain. Taken together, these findings provide strong evidence that the IL-2 receptor P chain on HH cells is functional. HH cells proliferated readily in complete medium alone. Since several T-cell lines have been reported to secrete IL-2 autonomously, allowing autocrine growth in vitro through engagement of their IL-2 receptors (Arima et al., 1986, 1987; Maedaetal., 1987; Hori etal., 1987; Duprezetal., 1985), we examined this possibility for HH cells. Supernatant from HH cells did not stimulate resting or PHA-activated normal peripheral blood lymphocytes, however, suggesting that IL-2 or other lymphokines were not being secreted. Also, antibodies to IL-2 failed to inhibit growth of the cells although the antibodies did abrogate the increase in proliferation due to exogenous IL-2. Additionally, mRNA for IL-2 was not detected in HH cells by Northern blotting. These studies were carried out since Dunbar et al. (1989) reported a cell line which synthesized intracellular IL-3, leading to autocrine cell growth by stimulating intracellular IL-3 receptors. Our findings, however, do not support an IL-2/IL-2-receptor-dependentautocrine pathway for in vitro growth of HH cells. The mechanism of malignant transformation of these cells is unknown and will require further investigation. In summary, we have established a novel, IL-2-independenk mature T-cell line from a patient with an aggressive cutaneous T-cell lymphoma who lacked antibodies to HTLV-I. The T-cell line was derived from the malignant clone in the patient’s blood and possessed only the IL-2 receptor p chain, which was

252

STARKEBAUM ET AL. TABLE 111 - SUMMARY OF SELECTED LEUKEMIC T-CELL LINES Characteristics

Line

Source'

HPB-ALL MOLT4 Jurkat Hut 102 MT- 1

ALL ALL ALL ATLL ATLL

IARC301 YT YT-2C2 MLA 144 HH

Lymphoma ALL Lymphoma CTCL

CDI

CD2

CD3

CD4

CD5

CD8

+ +

+ + + +

++

+

+

+

+ + -

-

NT

+ + +

+

+

-

+ + +

-

+ + +

-

-

N

-

-

+

+

+

-

-

-

-+

-

+

+

T

p75

+ -

+

1L-2R ps5

-

-

+

+ +--

-

+

++ + +

HTLV- 1

~

+

+ -

-

References

Martin et al., 1982 Martin et a / . , 1982 Martin et al., 1982 Robb and Greene, 1987 Martin et a / . , 1982; Robb and Greene, 1987 Duprez et at., 1985 Yodoi et al., 1985 Robb and Greene, 1987 Robb and Greene, 1987 Current line

'All are of human origin except MLA-144 which was derived from a gibbon T-cell lymphoma.

functional. This cell line may prove useful in studies of the IL-2 receptor and of T-cell leukemogenesis . ACKNOWLEDGEMENTS

We thank Mr. D. Webster for technical assistance, Dr. P.

Kidd for performing histochemical and immunophenotyping studies, Dr. J . Singer for many helpful suggestions, Ms. A. Kim for electron microscopy and Ms. B. Yancy for typing the manuscript. This work was supported by the Medical Research Service of the Department of Veterans Affairs and by American Cancer Society Grant CH457.

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