HTLV-related markers in a hungarian patient with adult T-cell leukemia

Leukemia Research Vol. 16. No. 11, pp. 1125-1131, 1992.

0145-2126/92 $5.00 + .00 (~) 1992 Pergamon Press Ltd

Printed in Great Britain.

CASE REPORT H T L V - R E L A T E D M A R K E R S IN A H U N G A R I A N P A T I E N T W I T H A D U L T T-CELL L E U K E M I A JOLAN KISS, Bt~LA TELEK,* FERENC D. TOTH, LASZL0 REJT(), PI~TER SURANYIt and KALMAN RAK* Institute of Microbiology, *2nd Department of Medicine and t3rd Department of Medicine, University Medical School of Debrecen, Debrecen, Hungary

(Received 4 February 1992. Revision accepted 4 July 1992) Abstract--Monoclonal integration of DNA sequences related to, but not identical to HTLV-I provirus was detected in the peripheral blood lymphocytes of a Hungarian male suffering from ATL. The patient and his parents showed serological cross-reactivity with both HTLV-I and HTLV-II groupspecific antigens. Restriction enzyme analysis with EcoRI, PstI, BamHI, HindIII and SacI revealed structural similarity of the provirus integrated in the DNA of ATL cells to HTLV-I but not to HTLVII. Data suggest that this provirus and HTLV-I are similar to each other along gag and pol regions, but they are different in the env region.

Key words: HTLV-related proviral DNA, cross-reacting antibodies, ATL, familial cluster of carriers.

and serological analyses on specimens from this patient to investigate retroviral markers. D a t a suggest the presence of a retrovirus, which is similar to H T L V - I , although not identical.

INTRODUCTION THE first h u m a n retrovirus, associated with adult T-cell leukemia ( A T L ) was isolated at the end of the 1970s [1]. This virus was n a m e d h u m a n T-cell l e u k e m i a / l y m p h o m a virus type I ( H T L V - I ) . A second H T L V , k n o w n as H T L V type II ( H T L V - I I ) , has been isolated f r o m the transformed T cells of a patient with a hairy-cell leukemia [2]. Hints of the presence of other retroviruses related to H T L V s were indicated by serological cross-reactivities in tests for H T L V - I infection detection [3] and by cross-hybridization of neoplastic genomic D N A with H T L V - I probes [4]. A new h u m a n retrovirus, distantly related to H T L V - I , was isolated from a patient with cutaneous T-cell l y m p h o m a in the leukemic phase [5]. On the basis of biological data the virus was provisionally n a m e d H T L V - V . The patient described here developed an adult Tcell leukemia. We p e r f o r m e d molecular biological

PATIENT AND METHODS

Patient A 15-year-old male developed ATL in May 1990. On clinical examination small lymph nodes (less than 0.51.0 cm) were found in the cervical regions. Liver and spleen were enlarged to 8 and 4 cm under the respective costal margins. No skin lesions were noted. The serum calcium was 4.7 mmol/l. No osteolytic lesions, mediastinal mass, abdominal lymphadenopathy were observed by radiologic methods. The WBC count was 170 x 109/1with 90% abnormal lymphoid cells. These cells were pleomorphic with indented or lobulated nuclei. The leukemic cells were positive for CD2, CD3 and CD4, while they were negative for CD7 and CD8. Cytochemically acid phosphatase and beta-glucuronidase activity were detected in 75 and 60% of malignant lymphoid cells, respectively. The clinical course, hypercalcemia, the morphological and cytochemical feature of malignant cells and surface marker analysis confirmed the diagnosis of ATL.

Abbreviations: ATL, adult T-cell leukemia; HTLV, human T-cell leukemia/lymphoma virus; PBMC, peripheral blood mononuclear cells; IFA, immunofluorescence assay; Con-A, concavalin-A; FITC, fluorescein isothiocyanate; RIPA, radioimmunoprecipitation assay; LTR, long terminal repeat. Correspondence to: Dr Ferenc D. T6th, Department of Microbiology, University Medical School of Debrecen, P.O.B. 17, H-4012 Debrecen, Hungary.

Separation of peripheral blood mononuclear cells ( PBMC) Mononuclear cells consisting predominantly of lymphocytes were isolated from peripheral blood by FicollUromiro buoyant density gradient centrifugation by conventional technique. 1125

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Southern-blot hybridization

High molecular weight DNA was extracted from the cells by treating the cells with SDS-proteinase K followed by phenol extraction. The DNAs digested with restriction endonucleases EcoRI, PstI, BamHI, HindIII or SacI were applied to a 0.8% agarose gel and electrophoresed at 30 V overnight. Molecular sizes were determined with reference to phage DNA digested with HindIII. The transfer of DNA to Gene Screen (New England Nuclear) filters was performed according to Southern [6]. DNAs were probed with pMT-2 plasmid containing full length HTLV-I proviral DNA [7]. The pMT-2 probe was labeled with 32p by nick translation [8]. The filters were prehybridized overnight in 50% formamide, 3 × SSC, 5 x Denhardt solution, 0.5% SDS, 10% dextran sulfate and 200~tg/ml denaturated salmon sperm DNA. Hybridization was performed at 37°C for 24 h with 32p-labeled HTLV-I probe in the same hybridization buffer as described above. Filters were then washed in 3 x SSC, 0.5% SDS at 60°C or in 1 x SSC, 0.5% SDS at 65°C. Cells

The cell lines employed in this study were MT-2 and C344. MT-2 is a HTLV-I-producing cell line [9]. C3-44 cell line releases HTLV-II [10]. For control purposes Con-Astimulated normal human peripheral T lymphocytes [11[ were used. All cell cultures were maintained in RPMI-1640 medium containing 10% fetal calf serum and antibiotics. Assays for serum a n t i - H T L V antibiotics"

For indirect membrane immunofluorescence (IFA), 100 ~tl of sera diluted in PBS were incubated with 106 MT2 or C3-44 cells. After incubation for 20 min at 37°C the cells were washed three times. Then 100 ~tl of goat antihuman IgG conjugated with FITC was added. After incubation for 20 min at 37°C the cells were washed again. Control targets included Con-A-stimulated normal human peripheral T lymphocytes. For indirect cytoplasmic IFA, the target cells were fixed in acetone on slides. Twenty microliters of serum dilutions were applied to 2 × 105 cells. Samples were incubated for 30 min at 37°C. After washing, FITC-labeled anti-human IgG was added to the fixed cells for 30 min at 37°C. IFA titers reflect the serum dilution at which 50% of target cells fluoresced markedly. For radioimmunoprecipitation assay (RIPA), the experimental conditions described by Kurth et al. [12] were utilized. The HTLV-I p24 antigen was supplied by Dr R. C. Gallo (NCI, Bethesda, MD, U.S.A.) and labeled with 125I by the chloramine-T method [13]. Anti-p24 titers are defined as ng of viral protein precipitated by 10 ~tl of serum diluted 1 : 10.

RESULTS Figures 1 and 2 show the Southern-blot analysis of E c o R I digests under low and high stringency conditions, respectively. The MT-2 cell line was used as positive source for H T L V - I . P B M C from a healthy donor and Con-A-stimulated T lymphocytes of the same person served as negative controls. The H T L V I probe hybridized specifically, but only under low stringency conditions (3 x SSC at 60°C) with the

D N A s from both A T L and MT-2 cells (Fig. 1). U n d e r high stringency conditions (1 x SSC at 65°C), the same probe hybridized only with the D N A from MT-2 cells, but not with the A T L sample (Fig. 2). Restriction enzyme analysis with E c o R I revealed two bands in the D N A from MT-2 cells (approximately 9.0 and 5.0 kb) and only one band in the D N A from A T L cells (approximately 17.0 kb). For a more sensitive comparison, we examined D N A from MT-2 cells and the A T L sample by Southern hybridization, using the enzymes Pstl, B a m H I , Hind III and SacI, which excise characteristic internal fragments from within H T L V - I . Both proviral D N A sequences hybridized to the H T L V - I p r o b e at low stringency conditions. The proviral PstI fragments are shown in Fig. 3, PstI digestion of the D N A from the MT-2 cells generated the expected 3 internal fragments of 2.5, 1.8 and 1.2 kb [14]. Digestion of D N A from A T L cells gave rise to hybridizing fragments of 1.8 and 1.2 kb, whereas the 2.5 kb fragment was absent. The additional bands present in these samples represent junction fragments of viral and cellular flanking sequences arising from the integration of proviral copies at a particular integration site. Restriction enzyme analysis with B a m H I revealed the characteristic internal 1.0 kb fragment [15] in the D N A from MT-2 cells (Fig. 4). The provirus integrated in the D N A of the cells of the A T L patient was not cleaved by B a m H I within the proviral genome. H i n d l I I digestion of D N A from MT-2 cells yielded an internal hybridizing fragment of 2.0 kb characteristic for this variant of H T L V - I [16] as indicated in Fig. 5. Hind III digestion of D N A from the A T L sample gave rise to a hybridizing fragment of 5 . 0 k b , which is likely to represent internal proviral sequences [5], whereas the 8.0 and 16.0 kb fragments include flanking cellular sequences. As shown in Fig. 6, an 8.0 kb hybridizing fragment generated after digestion with SacI was found in D N A s from MT-2 cells and the A T L sample. Most H T L V - I genomes have a unique Sacl site in the L T R sequences [17]. Therefore, the observation of the 8.0 kb fragment is due to the presence of full proviral copies in both samples. Serum samples collected from the A T L patient, his close family members, 8 patients with mycosis fungoides (MF) and 10 healthy donors were investigated for antibodies reacting with H T L V antigens. G o a t polyclonal antibody to H T L V - I p24 ( f r o m R. C. Gallo) was used as a positive control. In m e m brane IFA experiment all sera were negative. In cytoplasmic I F A the patient's serum was found to have antibodies reacting with H T L V - I producing MT-2 cells and H T L V - I I producing C3-44 cells in dilution 1 : 32 and 1 : 16, respectively (Fig. 7). Out of

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FIG. 1. Hybridization of HTLV-I probe with genomic DNAs under low stringency conditions. D N A was taken as follows: lane 1, from normal PBMC; lane 2, from cells of A T L patient; lane 3, from MT-2 cells; lane 4, from ConA-stimulated T lymphocytes. FIG. 2. Hybridization of HTLV-I probe with genomic D N A s under high stringency conditions. D N A samples are the same as in Fig. 1. FI6.3. Southern-blot of genomic D N A s digested with PstI, and hybridized to the complete genome of HTLV-I (pMT2) under low stringency conditions. D N A samples: lane 1, normal PBMC; lane 2, MT-2 cells; lane 3, cells of ATL patient; lane 4, Con-A-stimulated T lymphocytes. FIG. 4. Southern-blot of genomic D N A s digested with BamHI and hybridized to pMT-2 under low stringency conditions. D N A samples are the same as in Fig. 3. 1127

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FIG. 5. Southern-blot of genomic DNAs digested with HindIII and hybridized to pMT-2 under low stringency conditions. D N A samples are the same as in Fig. 3. FIG. 6. Southern-blot of genomic DNAs digested with S a d and hybridized to pMT-2 under low stringency conditions. DNA samples are the same as in Fig. 3. FIG. 7. Immunofluorescence micrograph of acetone-fixed MT-2 cells reacted with serum from the ATL patient.

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H T L V - r e l a t e d sequences in a H unga ri a n case of A T L

1129

TABLE 1. DETECTION OF ANTIBODIES TO H T L V ANTIGENS IN SERUM SAMPLES

Source of serum

Antibody titers to HTLV detected By IFA On MT-2 cells On C3-44 cells Membrane Cytoplasm Membrane Cytoplasm

ATL patient Father Mother Brother MF patients Immune serum Healthy donors

<1 <1 <1 <1 <1 <1 <1

32 8 16 <1 <1 256 <1

his family members, the father and mother had also antibodies reacting with H T L V - I and H T L V - I I producing cells in cytoplasmic IFA (Table 1), whereas the brother proved to be seronegative. None of the sera from MF patients or healthy donors contained antibodies reacting with HTLV-I- or HTLV-IIinfected cells. The positive results were confirmed by anti-p24 R I P A , in which 5.2-18.6 ng of H T L V - I p24 were precipitated by 10 ~tl of sera diluted 1 : 10. DISCUSSION The current study documents the presence of D N A sequences related to, but not identical to H T V L - I provirus in a Hungarian patient suffering from ATL. In the first series of experiments the genomic DNAs were cleaved with the restriction endonuclease EcoRI that does not cut within the H T L V genome. Therefore, the number of bands detected in this analysis reflects that the MT-2 cell line contained two copies of the integrated H T L V - I , whereas A T L cells contained only one HTLV-I-related copy. The 5.0 kb EcoRI fragment in the D N A of MT-2 cells represents a defective H T L V - I genome. The plus provirus copy in the cultured cells is a consistent finding and may reflect reinsertion of the provirus in culture [18]. No common bands were found with EcoRI, indicating different integration sites of the sequences. The HTLV-I-related sequences were detected as one discrete band in digest of D N A from A T L cells with EcoRI. These data clearly indicate that the leukemic cells were monoclonal. The monoclonal expansion of cells harboring HTLV-I-related sequences suggests that these sequences are at least one of the causative agents leading to the development of this A T L case. The 2.5 and 1.8 kb PstI fragments are common to all H T L V - I proviruses analyzed [15, 19]. The restriction enzyme PstI cleaves the H T L V - I genome at several sites and produces three large internal fragments of 1.2 kb from the pol region, 1.8 kb from the gag region, and 2.5 kb from the env region [14].

<1 <1 <1 <1 <1 <1 <1

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By RIPA 18.6 5.2 6.3 <0.3 <0.3 146 <0.3

B a m H I cleaves H T L V - I within the 3' end of the p o l region and the env region [15]. The absence of the 1.0 kb B a m H I fragment and the 2.5 kb PstI fragment in the proviral D N A from A T L cells suggest that this provirus and HTLV-I are similar to each other along the gag and pol regions, but they are different in the env region. The provirus integrated in the D N A of the cells of the A T L patient appears to be close to HTLV-V, because restriction enzyme analysis with HindIII revealed a 5.0 kb internal fragment in both proviral DNAs [5], whereas the internal B a m H I 1.0kb band was not found in any of them [20]. However, the 2.5 kb PstI fragment, which is c o m m o n to both H T L V - I [15] and H T L V - V [20] was absent in the proviral D N A from A T L cells. This finding suggests that H T L V - V and the provirus integrated in the D N A of A T L cells are not identical. No structural similarity of the HTLV-like genome in A T L cells was found to HTLV-II. Restriction endonuclease HindIII does not cleave within the proviral genome of H T L V - I I , whereas E c o R I has two cleavage sites in the 5' half of it generating an internal 3.9 kb fragment [21]. B a m H I digestion of HTLV-II provirus produces a characteristic internal fragment of 3 . 6 k b [21,22]. Digestion of proviral D N A by PstI yields one large internal fragment of 2.4 kb from HTLV-IIMo, but no internal fragment from HTLV-IINR A [23]. SacI cleaves HTLV-IIMo within the LTRs and pol region to produce two internal fragments of 4.5 and 3.8 kb [24]. H T L V IINRA lacks the internal SacI site [23]. Our observations clearly indicate that the restriction endonuclease pattern of the proviral D N A of A T L cells is different from that of H T L V - I I proviruses. The gag gene-related group-specific polypeptides of H T L V are found in the cytoplasm of H T L V infected cells [25], whereas the type-specific env gene products are localized on the cell surface [26]. Hence, the reactivity of the patient's serum with HTLV-Iand HTLV-II-infected cells in cytoplasmic but not in membrane IFA indicates that the A T L patient did

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not have a g e n u i n e H T L V - I o r H T L V - I I infection. T h e H T L V - s p e c i f i c n a t u r e of the c r o s s - r e a c t i v i t y in I F A was c o n f i r m e d by R I P A s h o w i n g the p r e s e n c e of a n t i b o d i e s a g a i n s t t h e p24 p o l y p e p t i d e c o d e d for by the gag g e n e o f H T L V - I . F a m i l i a l clusters o f H T L V - I c a r r i e r s suggest two n a t u r a l t r a n s m i s s i o n r o u t e s . O n e is vertical transmission f r o m m o t h e r to child [27], a n d the o t h e r is t r a n s m i s s i o n b e t w e e n m e n a n d w o m e n [28]. P a r e n t s of the A T L p a t i e n t h a d no l y m p h a t i c p a t h o l o g y but their s e r a also c o n t a i n e d a n t i b o d i e s c r o s s - r e a c t i n g with g r o u p - s p e c i f i c a n t i g e n ( s ) o f H T L V s . This finding suggests the s a m e n a t u r a l r o u t e o f t r a n s m i s s i o n for this p u t a t i v e H T L V - l i k e a g e n t as t h o s e o b s e r v e d in g e n u i n e H T L V - I infection. In a d d i t i o n , o u r A T L p a t i e n t h a d no special risk f a c t o r s for infection with H T L V such as b l o o d t r a n s f u s i o n of i n t r a v e n o u s d r u g

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use.

In c o n c l u s i o n , the p r e s e n t results d o c u m e n t , for the first t i m e , the p r e s e n c e of a p r o v i r u s d i s t a n t l y r e l a t e d to the H T L V f a m i l y in a C e n t r a l E u r o p e a n A T L p a t i e n t . R e s u l t s of r e s t r i c t i o n e n z y m e analysis indicate that this p r o v i r u s is similar to H T L V - I a l o n g the gag a n d p o l r e g i o n s , b u t differs f r o m it in the eno region. It still r e m a i n s to clarify the r e l a t e d n e s s of these p r o v i r a l s e q u e n c e s to H T L V - V [5].

Acknowk'dgement--The authors are thankful to Dr R. C. Gallo for providing HTLV-infected cell lines, purified p24 polypeptide of HTLV-I and immune serum to HTLVI p24.

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