Two types of defective human T-lymphotropic virus type I provirus in adult T-cell leukemia

Adult T-cell leukemia (ATL), an aggressive neoplasm of mature helper T cells, is etiologically linked with human T lymphotropic virus type I (HTLV-1). After infection, HTLV-I randomly integrates its provirus into chromosomal DNA. Since ATL is the clonal proliferation of HTLV-I- infected T lymphocytes, molecular methods facilitate the detection of clonal integration of HTLV-I provirus in ATL cells. Using Southern blot analyses and long polymerase chain reaction (PCR) we examined HTLV-I provirus in 72 cases of ATL, of various clinical subtypes. Southern blot analyses revealed that ATL cells in 18 cases had only one long terminal repeat (LTR). Long PCR with LTR primers showed bands shorter than for the complete virus (7.7 kb) or no bands in ATL cells with defective virus. Thus, defective virus was evident in 40 of 72 cases (56%). Two types of defective virus were identified: the first type (type 1) defective virus retained both LTRs and lacked internal sequences, which were mainly the 5' region of provirus, such as gag and pol. Type 1 defective virus was found in 43% of all defective viruses. The second form (type 2) of defective virus had only one LTR, and 5'- LTR was preferentially deleted. This type of defective virus was more frequently detected in cases of acute and lymphoma-type ATL (21/54 cases) than in the chronic type (1/18 cases). The high frequency of this defective virus in the aggressive form of ATL suggests that it may be caused by the genetic instability of HTLV-I provirus, and cells with this defective virus are selected because they escape from immune surveillance systems.     

Thrombocytopenic purpura in a carrier of human T-cell leukemia virus type I

Thrombocytopenic purpura developed in a 64-year-old woman seropositive for human T-cell leukemia virus type I (HTLV-I). She had no underlying disorders and HTLV-I is suggested as a possible cause of her thrombocytopenia.     

Defective human T-lymphotrophic virus type I provirus in T-cell prolymphocytic leukaemia

T-cell prolymphocytic leukaemia (T-PLL) is a rare form of post-thymic T-cell neoplasm, the aetiology of which remains unknown. We examined human T-lymphotropic virus (HTLV) provirus in five HTLV-I/II seronegative patients with T-PLL. Southern blotting did not show monoclonal integration of the HTLV-I genome in any of the DNA samples. However, two of the five DNA samples contained an HTLV-I tax sequence. Other sets of oligonucleotide primers for HTLV-I gag, pol, env and LTR regions were all negative. HTLV-I tax gene expression and p40tax antibody were not detected in samples from cases with HTLV-I tax sequence. Our findings suggest that there may be alternative mechanisms involved in HTLV-associated leukaemogenesis, in which HTLV-I genome insertion triggers T-PLL but the deletion of various regions of the integrated provirus subsequently prevents active replication and the expression of the virus.     

Monoclonal integration of human T-cell leukemia provirus in all primary tumors of adult T-cell leukemia suggests causative role of human T-cell leukemia virus in the disease.

The genome of human T-cell leukemia virus (HTLV) was surveyed in fresh tumor cells of 163 patients with lymphoma and leukemia from the southwest part of Japan where adult T-cell leukemia (ATL) is endemic. Leukemic cells of all 88 cases of ATL tested so far were found to contain the provirus genome and also found to be monoclonal with respect to the integration site of provirus genome. In most cases of ATL, leukemic cells contained one or two copies of the complete HTLV provirus genome, and it was shown that the single species of HTLV with a fully determined sequence is typical in ATL. Some cases of T-cell malignancies, diagnosed as chronic lymphocytic leukemia or non-Hodgkin lymphoma, also had the provirus genome in their tumor cells, whereas some cases with the same diagnosis did not. No cases of other types of lymphoma or leukemia contained the provirus genome in their tumor cells. Monoclonal integration of the HTLV provirus genome in all primary tumor cells of ATL not only indicates that HTLV directly interacts with target cells, which become leukemic, and that integration of the provirus genome is a prerequisite for development of ATL and possibly other related diseases but also indicates that the virus is not associated with other types of lymphoma or leukemia.     

A novel diagnostic method of adult T-cell leukemia: monoclonal integration of human T-cell lymphotropic virus type I provirus DNA detected by inverse polymerase chain reaction

Adult T-cell leukemia (ATL) is neoplasm of the mature helper T lymphocytes and human T-cell lymphotropic virus type-I (HTLV-I) has been shown to be causative virus of ATL. Because HTLV-I integrates its provirus randomly into host chromosomal DNA, monoclonal integration of HTLV-I provirus indicates the clonal proliferation of HTLV-I-infected cells. Therefore, demonstration of clonality of HTLV-I proviral DNA is essential to diagnosis of ATL. Southern blot analysis was used for this purpose. We developed the novel method using inverse polymerase chain reaction (IPCR) to detect the clonality of HTLV-I proviral DNA. This method identified the clonality in all ATL cases. Diagnosis could be made within 3 days using this method. It enabled us to detect specifically the presence of minimal numbers of ATL cells with high sensitivity. It also identified the monoclonal or oligoclonal proliferations of HTLV-I-infected cells in HTLV-I carriers and the intermediate state, in which no clonality could be shown by conventional Southern blot analyses. This finding indicated that even HTLV-I carriers had monoclonal proliferation of HTLV-I-infected cells without any symptoms. This novel method is shown to be useful for the diagnosis of ATL and provides information on the natural course of HTLV- I infection.     

Gastric lymphoma associated with human T-cell leukemia virus type I

A 41-year-old woman presented with a gastric lymphoma. A total gastrectomy was performed, and the tumor was found to consist of T cells of the helper/inducer (E+, Leu-1+, Leu-2a-, Leu-3a+) phenotype. The patient was seropositive for T-cell leukemia virus type I, and the tumor cells contained the proviral genome.     

Oligoclonal proliferation of human T-cell leukaemia virus type 1 bearing T cells in adult T-cell leukaemia/lymphoma without deletion of the 3' provirus integration sites

We report a new case of an asymptomatic carrier with a deletion of a 3' HTLV-1 integration site. We further investigated whether these 3' deletions of flanking sequences may explain the oligoclonal pattern of HTLV-1 replication, evidenced by inverse PCR (IPCR) analysis of tumourous samples from patients with adult T-cell leukaemia (ATLL). 48 HTLV-1 3' integration sites, derived from tumourous DNA of five ATLL patients were sequenced. One dominant flanking sequence was obtained in the four samples harbouring a unique band after Southern-blotting. In one sample, which harboured two signals after Southern-blotting, IPCR amplification of diluted tumourous DNA revealed that these two sequences corresponded to one clone harbouring two integrated proviruses rather than to two distinct cellular clones, a result consistent with superinfection of the tumourous sample. In addition to integration sites corresponding to malignant clones, two to six oligoclonal forms were sequenced in four samples. No flanking sequence homology was found between clones derived from each patient, indicating that integration sites deletion in the vicinity of the provirus is a rare event in ATLL. The oligoclonal pattern of HTLV-1 replication in ATLL may result from clonal expansion of non-malignant HTLV-1-bearing clones within the sample and partly from HTLV-1 superinfection of monoclonal tumour cells.     

Virion-associated trans-regulatory protein of human T-cell leukemia virus type I.

Western blot analysis of HTLV-I virus particles from HUT-102 cells revealed a 40-kD protein strongly reactive with Tax-specific rabbit antisera. This protein subsequently was isolated from density gradient purified virions by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), purified from comigrating Gag and human cellular proteins by reversed-phase high-performance liquid chromatography (HPLC) and identified as the tax-encoded gene product by amino acid composition analysis. Among extracellular virions from five HTLV-I producing cell lines, only those from HUT-102 and C10MJ cells contained a detectable Tax protein, although all cells expressed Tax mRNA and protein intracellularly. To investigate the diagnostic implications of virion-associated Tax protein, sera from HTLV-I-infected individuals were compared on HUT-102 and MT-2 virus Western blots. The seroprevalence of antibodies to Tax, but not Gag or Env proteins, was substantially higher among adult T-cell leukemia and tropical spastic paraparesis patients using HUT-102 viral proteins. Thus, immunoassays utilizing HUT-102 virus are most sensitive for detection of Tax-reactive antibodies.     

Adult T-cell leukemia/lymphoma not associated with human T-cell leukemia virus type I.

We describe five patients with adult T-cell leukemia/lymphoma (ATL) with neither integration of human T-cell leukemia virus type I (HTLV-I) into their leukemia cells nor anti-HTLV-I antibody in their sera. These findings indicate that HTLV-I may not have been involved in leukemogenesis in these patients. The clinicohematological, cytopathological, and immunological features of HTLV-I-negative ATL were exactly the same as those of HTLV-I-associated ATL. Leukemia cells with pleomorphic nuclei, generalized lymphadenopathy, hepatosplenomegaly, skin lesions, hypercalcemia, and elevated lactate dehydrogenase levels, all of which are characteristic features of typical ATL, were also seen in these patients with HTLV-I-negative ATL. Leukemia cells expressed T3, T4, and pan-T-cell antigens in three cases, and T3 and pan-T-cell antigens in two. All five patients had lived in ATL-nonendemic areas. The finding of HTLV-I-negative ATL suggests that factor(s) other than HTLV-I infection may be involved in ATL leukemogenesis.     

Establishment of human T-cell leukemia virus type I T-cell lymphomas in severe combined immunodeficient mice

Human T-cell leukemia virus type I (HTLV-I) is recognized as the etiologic agent of adult T-cell leukemia (ATL), a disease endemic in certain regions of southeastern Japan, Africa, and the Caribbean basin. Although HTLV-I can immortalize T lymphocytes in culture, factors leading to tumor progression after HTLV-I infection remain elusive. Previous attempts to propagate the ATL tumor cells in animals have been unsuccessful. Severe combined immunodeficient (SCID) mice have previously been used to support the survival of human lymphoid cell populations when inoculated with human peripheral blood lymphocytes (PBL). SCID mice were injected intraperitoneally with PBL from patients diagnosed with ATL, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), or from asymptomatic HTLV-I-seropositive patients. Many of these mice become persistently infected with HTLV-I. Furthermore, after human reconstitution was established in these mice, HTLV-I-infected cells displayed a proliferative advantage over uninfected human cells. Lymphoblastic lymphomas of human origin developed in animals injected with PBL from two ATL patients. The tumor cells represented outgrowth of the original ATL leukemic clone in that they had monoclonal or oligoclonal integrations of the HTLV-I provirus identical to the leukemic clone and predominantly expressed the cell surface markers, CD4 and CD25. In contrast, cell lines derived by HTLV immortalization of T cells in vitro did not persist or form tumors when inoculated into SCID mice, indicating differences between in vitro immortalized cells and ATL leukemic cells. This system represents the first small animal model to study HTLV-I tumorigenesis in vivo.     

Kaposi's sarcoma in human T-cell leukemia virus type I-associated adult T-cell leukemia

Kaposi's sarcoma (KS) developed in a patient with human T-cell leukemia virus type I (HTLV-I)-associated adult T-cell leukemia who was treated with a short-term course of monoclonal antibody immunotherapy. The presentation was transient and temporally related to the underlying clinical course. The association of KS in an HTLV-I infected, but not human immunodeficiency virus (HIV)-infected, individual should alert investigators to the occurrence of KS in retroviral-associated diseases other than acquired immunodeficiency disease syndrome. Recognition of the similarities and differences between HTLV-I and HIV infections may provide insights concerning the angiopathogenesis of KS.     

Infection of human endothelial cells by human T-cell leukemia virus type I.

The effects of the human T-cell leukemia virus type I (HTLV-I) on cultured human endothelial cells were evaluated. Coculture of endothelial monolayers with either irradiated HTLV-producing lymphocytes or cell-free virus resulted in the production of multinucleated syncytia. The development of syncytia was inhibited by sera from patients with adult T-cell leukemia/lymphoma (ATLL). HTLV antigens were present on endothelial syncytia passaged in culture for greater than 3 months as detected by an anti-p19 monoclonal antibody, which detects a core protein of HTLV-I, and by ATLL sera. Moreover, these HTLV-infected endothelial cells were then able to infect and transform normal cord blood T lymphocytes with HTLV. These studies demonstrate that human endothelial cells are susceptible to productive HTLV-I infection in vitro and may have relevance for the spectrum of human disease associated with this family of retroviruses.     

CD30-positive lymphoma in human T-cell leukaemia virus type 1 carrier without monoclonal integration of HTLV-1

Summary. CD30, Ki-1 antigen, an activated T-cell antigen, is a member of the nerve growth factor receptor family. This antigen is expressed on the lymphoma cells of some adult T-cell leukaemia/lymphoma (ATL/L) patients and some patients with Epstein-Barr virus infection. CD30-positive large cell cutaneous T-cell lymphomas occasionally integrate a defective HTLV-1 provirus. We describe here an HTLV-1 carrier who developed Ki-1 lymphoma with no evidence of monoclonal integration of the HTLV-1 proviral sequence.     

Development of leukemia in mice transgenic for the tax gene of human T-cell leukemia virus type I.

The human T-cell leukemia virus type I Tax protein trans-activates several cellular genes implicated in T-cell replication and activation. To investigate its leukemogenic potential, Tax was targeted to the mature T-lymphocyte compartment in transgenic mice by using the human granzyme B promoter. These mice developed large granular lymphocytic leukemia, demonstrating that expression of Tax in the lymphocyte compartment is sufficient for the development of leukemia. Furthermore, these observations suggest that human T-cell leukemia virus infection may be involved in the development of large granular lymphocytic leukemia.     

Progressive spastic paraparesis associated with human T-cell leukemia virus type I (HTLV-I).

Patients with progressive spastic paraparesis (PSP), commonly middle-aged women, are distributed throughout the country of Chile. During the three years from 1987 to 1990, we collected 83 cases of PSP from among 225 patients with various neurological diseases. The clinical picture was of a bilateral pyramidal syndrome, with sensory deficits in only 15.5% of the cases, and a slow illness progression in the majority of them. In patients with PSP, antibody to human T-cell leukemia virus type I (HTLV-I) was analyzed by enzyme linked immunosorbent assay (ELISA) and confirmed by western blot analysis. Forty-five (54.2%) patients were anti-HTLV-I antibody positive in cerebrospinal fluid (CSF) and peripheral blood. Among them, 2 patients had leukemia/lymphoma and one had Sj?gren syndrome. In the laboratory study of seropositive PSP, mononuclear pleocytosis was found in 35.7%; there was an abnormal increase of the IgG index in 66.6% and an increase in CD2 in blood and CSF, and CD4 in blood. A delayed latency of somatosensory evoked potentials was observed in 90.9%. The neuropsychological study revealed a WAIS with a mean verbal IQ of 80.7 and a mean performance IQ of 84.8. The most impaired items were digit symbol and digit span. Seven subjects (18.9%) with anti-HTLV-I antibody were found among 37 relatives from 19 anti-HTLV-I positive cases of PSP.