Human T -Cell Leukemia-Lymphoma Virus (HTLV): A Progress Report
R. C. Gallo, M. Popovic, P. Sarin, M. S. Reitz, Jr., V. S. Kalyanaraman, T. Aoki, M. G. Sarngadharan, and F. Wong-Staal    Hämatol. Bluttransf. Vol 28

A. Introduction

Since type-C retroviruses are known to be involved in naturally occurring leukemias of many animal species [26], a similar viral etiology has been sought in human leukemias. Some of the animal models provide important insight for consideration of human leukemias. For example, while most virus-induced animal leukemias and lymphomas are associated with abundant virus production in the tumor cells, bovine leukemia virus (EL V), the causative agent of bovine leukemias, was not detected until the leukemic cells were cultured in vitro (see review by Miller and Van Der Maaten [13]). This brings out the importance of long-term culture of the appropriate target cells for virus detection and isolation. In 1976, our laboratory reported the discovery of a factor termed T-cell growth factor (TCGF) [ 15]. Following interaction with an antigen, different subsets of mature T cells respond by making and releasing TCG F or making a receptor to TCG F. The TCG F binds to the receptor-bearing T cells and induces cell growth. Addition of exogenous TCGF can maintain growth of activated mature T cells for long periods [6, 26]. When TCGF was added to T cells obtained from patients with mature T -cell leukemias and lymphomas, some cells directly responded without prior activation in vitro [18]. Some of those samples released a retrovirus which we call human T -cell leukemia-Iymphoma virus (HTL V) ([19, 20]; Popovic et al., in preparation). The morphology of HTL V is typically type c. Figure 1 shows an electron micrograph of some HTL V particles. Like all retroviruses, HTL V contains reverse transcriptase, has a high molecular weight RNA genome, and buds from cell membranes. It is distinct from all other known animal retroviruses [9, 16, 22, 23] and to date is the only unequivocal human retrovirus. (The retrovirus later isolated independently in Japan [14, 31] and called ATLV is, in fact, HTL V.) Furthermore, it is specifically associated with certain forms of human leukemia and lymphoma [4]. Here we wish to describe some of the new isolates of HTL V and report on some recent findings on the nature and distribution of HTL V and its transmission to and biological effects on normal T -lymphocytes in vitro.

B. Identification of New HTL V Isolates in Established Cell Lines
    Derived from Patients with T -Cell Leukemias/Lymphomas

Cell lines derived from patients with leukemia/lymphomas of mature T cells from geographically different parts of the world have been established in culture in the presence of TCGF as described previously for normal mature human T cells [ 15, 26] and neoplastic mature T cells [ 18 ]. These cell lines were analyzed for HTL V by (a) competition radioimmunoprecipitation assay (RIPA) for the major core protein p24 [9], (b) indirect immune fluorescence assay (IF A) using highly specific monoclonal antibody for another HTLV antigen, pl9 [24], (c) reverse transcriptase activity in the culture fluids, and (d) electron microscopy. In addition to the positive cell lines CR [19] and MB [20] published earlier, seven of eight recently established T -cell lines fully expressed HTL V (Table I) and one showed partial expression, These patients include four individuals from the United States, one from Israel, and three from a single Japanese family from the northwest part of the Honshu Island in Japan. In this family, the patient SK with acute T -cell lymphoma (A TL) and both his parents are virus positive. The father (MK) is clinically healthy and the mother has persistent lymphocytosis, which is considered to be a preleukemic state. All cell lines have karyotype and HLA patterns that match those of the donors, and the HLA profiles are different for all eight cell lines. In addition, a T -cell line established by Golde and colleagues from a patient (MO) with hairy cell leukemia [27] was positive vor HTL V p 19 and p24 [II]. We propose to designate each virus isolate with a subscript of the patient's initials, e.g., HTL V CR, HTL V MB, etc. All isolates except HTL V MO are highly related to each other as assayed by competitive radioimmunoassay with p24 (Fig. 2) and hybridization of viral cDNA to mRNA of the producer cell lines (Fig. 3). By these assays, the virus of Japanese A TL is indistinguishable from the prototype HTL V as exemplified by the earlier isolates HTL V CR and HTL V MB. On the other hand, HTL V MO competes poorly in the p24 assay (Fig.2), and nucleic acid sequence homology with HTL V CR was detected only under very nonstringent hybridization conditions ( our unpublished data). Therefore, this virus may form a distinct subgroup in the HTLV family. We propose to group them as HTLV-IcR, etc. versus HTLV-IIMo.

Fig. I. Electron microscopic examination of
the cellline MB, showing extracellular and
budding HTL V particles. Bar represents 90 µ

Table I. Expression of new HTL V isolates in T -cell lines
derived from patients with adult T -cellleukemia/lymphomas

a. Detected by competitive radioimmunoprecipitation
assay (RIPA) in cell extract

b. Indirect immunofluorescence assay (IFA)

c. Reverse transcriptase activity (RTA) in culture
fluids was measured with (dT)15(rA)n

d. Celllines were derived from peripheral blood (PE) or bone marrow (EM) of different patients as follows. (a) MJ from PB of a 50-yearold white male with mycosis fungoides, from Boston, Massachusetts; (b) UK from PB of a 45-year-old white male with diffuse histiocytic lymphoma, from Jerusalem, Israel; (c) MI from PB of a 32-year-old black female with T -cell lymphosarcoma cell leukemia, from Granada, West Indies; (d) WA from BM ofa 24-year-old black male with diffuse mixed lymphoma, from Augusta, Georgia; (e) PL from PB of a 27-year-old black female with T -cell diffuse mixed lymphoma, from Ovita, Florida; (I) SK from PE ofa 21-year-old male with adult T -cell leukemia, from Akita perlecture, Japan; (g) TK from PB of a 45-yearold female (mother of patient SK) who has 7% abnormal cells in her blood, from Akita perfecture, Japan; and (h) HK from PE of a 49year-old male (father of patient SK) who is normal, from Akita perfecture, Japan.

Fig.2A-C. Homologous and heterologous competition radioimmunoassays of HTL v p24. Assays were carried out as described [9] using l25I-labeled HTL V CR p24 and a limiting dilution of hyperimmune rabbit antibody to HTLV CR or sera from patients MO and MJ. A Competition RIA using rabbit anti-HTL V CR. B Competition RIA using MO serum. C Competition RIA using MJ serum. virus extracts used for competition were: black circle - black circle, HTLV CR; white circle- white circle , HTLV MO; x-x, Mason Pfizer monkey virus; white triangle- white triangle, bovine leukemia virus; white square - white square , Rauscher murine leukemia: black triangle - black triangle, simian sarcoma virus

Fig.3A-F. Relationship of different HTL V isolates by RNA hybridization. ³H-HTL V (R cDNA was synthesized by calf thymus DNA primer [22] and hybridized to cellular RNA from different HTLVpositive cell lines. Results with the controls with poly(A) + RNA from CR- T (HUT 102) and CR-B cells are superimposed on each panel for comparison with the others

Fig.4. HTL V proviral DNA in T- but not B-celllines of patient CR, ³H-HTL V cDNA was annealed to cellular DNA from CR-T lymphocytes (black circle), CR-B lymphocytes (black triangle), and PHA-stimulated normal human peripheral blood T -lymphocytes (black square)

C. HTLV Provirus in Neoplastic T Cells: Evidence for Exogenous Infection

We had reported earlier that HTL V sequences are present in the infected cells and not in normal uninfected human cells [22], suggesting that HTL V is not an endogenous human virus. In the case of the patient CR, we also had the opportunity to find out whether he was infected pre- or post-zygotically [5]. Several T -cell lines, some clonal derivatives of these lines and a B-cellline have been established from CR. These cells were shown to have originated from the same individual by HLA typing. HTL V proviral DNA was detected in some but not all of the independently established T -cell lines of CR and not in the B cells. An example of the DNA hybridization kinetics is shown in Fig. 4. Furthermore, the surface phenotype OKT3-, OKT4+, and OKT8appears to correlate with the presence of HTL V. These results indicate that HTL V was acquired by CR by horizontal transmission and suggest that only a subtype of T cells is the target for HTL V infection.
Recently, molecularly cloned sequences representing the 5' and 3' ends of HTL V have been obtained in our laboratory [12 a]. These clones have been used as probes for Southern hybridization of fresh leukemic DNA from patients with HTL V -positive diseases [30]. These revealed one or few copies of HTL V integrated at a site which is unique for a given patient but varies from patient to patient. DNA from normal people did not contain hybridizing sequences. A similar observation has been made by others [31 ]. These results suggest that the infected cells are of clonal origin, so infection must have occurred prior to disease development. This feature is also found in animal leukemia-Iymphomas induced by chronic leukemia retroviruses.

D. Clinical Features of HTL V -Positive Diseases

Seroepidemiological studies have identified HTL V -positive patients from many regions of the world with at least three major areas that appear to be endemic: Southwestern Japan [4, 8, 9,25], the Caribbean [I], centraI South America (see also Blattner et al., this volume), but only sporadically in the United States [21 ]. Similar clinical features are found in the diseases associated with these areas, i.e., Japanese adult T -cellleukemia (ATL) and T-cell lymphosarcoma cell leukemia (T- LCL ) in the West Indian Blacks from the Caribbean. Both are represented by an aggressive course an frequent association with lymphadenopathy, hypercalcemia, hepatosplenomegaly, and cutaneous manifestations [2, 28]. The tumor cells are all mature, lack terminal deoxynucleotidyl transferase and express differentiated functions. Typing with monoclonal antibodies as well as functional studies showed that the cells may be either of the helper-inducer or suppressor-cytotoxic phenotype. Histologically, the cells are pleomorphic, often with highly convoluted nuclei. Almost all patients with A TL and T-LCL are HTLV positive. These observations led to the hypothesis that HTL V is associated with a subtype of adult T -cell malignancy which may include an aggressive form of cutaneous T -cell lymphoma (CTCL) found in patients CR and MB. In fact, the presence of HTL V may be of practical importance in disease classification. However, at least two HTL V -positive patients have relatively benign diseases: MJ with Sezary syndrome and MO with T -cell hairy cell leukemia. It should be noted, however, that at least the virus in MO is significantly different from the prototype HTL V.

E. Infection and Transformation of Human Cord Blood T Cells by HTL V In Vitro

Seven of the HTL V isolates described above have been successfully transmitted into fresh human cord blood T cells by cocultivation (Popovic et al., in preparation). The virus-positive neoplasic cells used as donors were first treated with mitomycin-C or X-irradiation before cocultivation with recipient cord blood cells. After 4 weeks, assays for T -cell markers, HTL V, karyotype, and HLA-typing were performed. As shown in Table 2, all recipient cord blood are mature T cells, positive for HTL V provirus, and express various levels of HTL V antigens (pI9, p24, and RT). Karyotype and HLA typing consistently matched the recipient cells. Since cord blood T cells from the same donors were consistently negative for HTL V markers and the plasma from their cord blood were also negative for HTL V antibodies, we conclude that the virus was transmitted from HTL V -producing neoplastic T -cell lines into cord blood recipient T cells.
To characterize further whether a target for HTL V could represent a certain subset of mature T cells, phenotypes of HTL Vproducing cells were analyzed by a series of monoclonal antibodies specific for helper/ inducer and suppressor/cytotoxic T cells. We found that a majority of HTL V -producing T -cell lines consistently exhibited only helper-inducer phenotype. Two established T -cell lines, SK and TK, both from Japanese patients and two HTL V -infected cord blood T cells (C 1 and C5) revealed "double" phenotype. However, none of the T -cell lines exhibited pure suppressor/cytotoxic phenotype. Unlike HTL V-infected cord blood T cells, PHAstimulated cells (control) consist of 70% helper/inducer and 30% of T cells with suppressor/cytotoxic phenotype. Thus, these data from T -cell phenotype characterization of HTL V -infected T cells again suggest that a certain subset of mature T -cells is the target for HTL V. HTL V infection studies with cord blood cells deprived ofT-cell population with helper/ind ucer of suppressor / cytotoxic phenotype are currently being carried out.
HTL V -infected cord blood T cells differ from mitogen-stimulated cord blood T cells in several growth properties and cell surface characteristics, the infected cells resembling more the neoplastic cells transformed in vivo by HTL V (see Sarin et al., this volume for details). The most striking feature of HTL V -infected cord blood T cells is their potential for indefinite growth as shown in Fig.5. In contrast, mitogenstimulated cord blood T cells from the same patients consistently exhibited growth "crises" after 1 month in culture, even in the continued presence ofTCGF. Furthermore, the infected cells, like the neoplastic cells, had the tendency to form clumps in culture. When analyzed by electron microscopy, the cells were seen to have convoluted nuclei (not shown) while the mitogen-stimulated cells did not. Another important and reproducible difference is the decrease in requirement for TCGF by the infected cells. In fact, some of the infected cells are completely independent of exogenous TCGF (see Sarin et al., this volume). Other changes of the infected cells include alteration in their HLA profile and expression of receptors for transferrin, TCGF, and HAA (human-activated lymphocyte antigen detected by monoclonal antibodies) in a high percentage of cells. The data indicate that HTL V is also capable of causing morphological transformation of cord blood T cells in vitro (see Sarin et al., this volume).

Table 2. Transmission of HTLV into human cord blood T cells

Fig.5. Growth curves of uninfected and HTL V -infected human cord blood T cells in vitro. Left panel, mitogen-stimulated cord blood T cells. Right panel, HTL V -infected cord blood T cells. C6/W A and C7/TK cell lines are primary cocultures (for details see Table 2). C5/MJ cells were obtained in three successive transmissions of HTL V MJ isolate into cord blood cells

F. Possible Molecular Mechanism of Transformation by HTL V

As mentioned earlier, analysis of HTL V positive leukemic T cells showed that the cells are of clonal origin with respect to the provirus integration sites. In animal systems monoclonality has also been shown to be a common feature of leukemias induced by retroviruses which are chronic leukemia viruses but not those induced by retroviruses which are acute leukemia viruses. Consequently, in spite of its high efficiency to transform T cells in vitro, HTL V probably does not carryon onc gene. Several chronic leukemia viruses are known to induce leukemia by activating cellular onc genes (myc in B-cell lymphomas and erb in erythroleukemias) ([7]; Kung, personal communication) by integrating in the proximity of these genes. Activation of thcse genes is brought about by providing either a viral promotor or viral nucleotide sequences dubbed ""enhancer" [12, 17], the real function of which is still unknown. Since HTL V specifically transforms mature T cells, it is likely to affect expression of genes that are important in T -cell proliferation. A model has been proposed for the mechanism of leukemogenesis by HTL V [4]. Briefly, the HTL V envelope protein interacts with the population ofT cells normally designed to make TCGF receptors, mimicking an antigen stimulation of blastogenesis. These cells then synthesize receptors for TCGF. Simultaneously, the HTLV provirus integrates in the vicinity of the TCGF gene or a gene that exerts a pleiotropic effect on TCGF expression and activates this gene either hy direct promotion or enhancement. The production ofTCGF by a cell bearing a TCGF receptor may result in autostimulation and increased cell proliferation. As an approach to study the gene(s) activated by HTL V infection, we have recently identified and isolated a gene that is expressed at high levels in all HTL V -positive neoplastic T cells and in normal cord blood T cells after infection with HTLV hut not the uninfected counterparts [12 b]. Study of the expression pattern of this gene in uninfcctcd human hematopoietic cells suggests that its expression may be linked to TCGF production. Experiments are in progress to determine if HTL V integrates at a preferred locus in the human chromosome and affects transcription of specific cellular genes in the vicinity, including this gene in question.


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29. Wong-Staal F, Gallo RC (to be published) Retroviruses and leukemia. In: Gunz F, Henderson E (eds) Leukemia. Grune and Stratton, New York

30. Wong-Staal F, Hahn B, Manzari V, Colombini S, Franchini G, Gelmann EP, Gallo RC (to be published) A survey of human leukemias for sequences of a human retrovirus, HTLV Nature

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