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.     

Characterization of T cells immortalized by Tax1 of human T-cell leukemia virus type 1

Peripheral blood T cells were immortalized in vitro by introduction of the Tax1 gene of human T-cell leukemia virus type 1 (HTLV-1) with a retroviral vector and were characterized for transformation-associated markers. Long-term observation showed that these Tax1-immortalized T cells eventually exhibited very similar features that were characteristic of HTLV-1-immortalized T cells, ie, increased expression of egr-1, c-fos, IL-2R alpha, and Lyn and decreased expression of Lck and cell-surface CD3 antigen. Among these changes, an increase in the expression of Lyn and a decrease in the expression of Lck and cell- surface CD3 antigen were observed only in Tax1-immortalized T cells after long-term culture. The expression level of Tax1 protein did not differ significantly between early and late passage of cells, and the cellular clonality was found to be the same by the analysis of the retroviral vector integration site and the T-cell receptor beta-chain gene rearrangement pattern. These changes in the expression of Lyn, Lck, and cell-surface CD3 antigen probably resulted from indirect effects of Tax1 that appeared after extended culture.     

Frequent clonal proliferation of human T-cell leukemia virus type 1 (HTLV-1)-infected T cells in HTLV-1-associated myelopathy (HAM-TSP).

Human T-cell leukemia virus type 1 (HTLV-1) integrates its proviruses into random sites in host chromosomal DNA. Random integration of the proviruses was observed in asymptomatic HTLV-1 carriers and patients with HTLV-1-associated myelopathy (HAM/TSP). However, clonal integration has been reported in patients with adult T-cell leukemia (ATL), including that in the smoldering, chronic, and acute states, indicating clonal expansion of infected cells. In this study, we found that about 20% of HAM/TSP patients and their seropositive family members harbored subpopulation(s) of clonally proliferated cells infected with HTLV-1, although they still maintained randomly infected cells as a major population. These clones were stable during examination periods of 4 months to 3 years. However, these carriers or HAM/TSP patients did not show any significant indication of ATL. This extremely high frequency of clonal expansion of HTLV-1-infected cells indicates that some clones of HTLV-1-infected cells have a tendency to proliferate more efficiently than the other population without malignant transformation.     

Hematopoietic progenitor cells from patients with adult T-cell leukemia- lymphoma are not infected with human T-cell leukemia virus type 1

The in vivo host range of human T-cell leukemia virus type 1 (HTLV-1) has not been definitively established. To determine if hematopoietic stem cells from patients with adult T-cell leukemia-lymphoma (ATL) are infected with HTLV-1, we used a clonogenic progenitor assay followed by the polymerase chain reaction for the detection of HTLV-1 DNA. In vitro growth characteristics of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells among nonadherent T-cell-depleted bone marrow (BM) mononuclear cells (NA-T-MNCs) from 10 patients with ATL was not significantly different from those of HTLV-1-seronegative controls (P = .20); numbers of colonies per 1 x 10(5) NA-T-MNCs were 34.9 +/- 7.6 for CFU-GM and 39.0 +/- 12.5 for BFU-E in patients with ATL, whereas those were 32.1 +/- 9.5 for CFU-GM and 41.4 +/- 12.7 for BFU-E in normal controls. HTLV-1 DNA was not detected in individual colonies formed by CD34+ cells from any of the patients. Similarly HTLV-1 DNA was not detected in 1 x 10(3) CD34+ cells sorted on a fluorescence-activated cell sorter (FACS) from six patients with ATL studied. In contrast, HTLV-1 DNA was detected in BM mononuclear cells from all patients. These observations clearly indicate that hematopoietic progenitor cells from patients with ATL are normal in their colony-forming capacity and that CD34+ cells from patients with ATL are not infected with HTLV-1 in vivo.     

Frequent lack of antibodies against human T-cell leukemia virus type I gag proteins p24 or p19 in sera of patients with adult T-cell leukemia

Summary Specificities of antibodies against human T-cell leukemia virus type I (HTLV-I) in sera of 27 patients with adult T-cell leukemia (ATL) were studied. All sera were positive for HTLV-I antigens by immunofluorescence assay using cells expressing HTLV-I antigens; sera from five ATL patients, however, did not react or hardly reacted with p19 or p24 gag proteins of HTLV-I upon immunoprecipitation assay. Therefore, the relationships among antibody specificities against HTLV-I, the proviral structures of HTLV-I genomes in leukemic cells, and the expression of viral antigens by leukemic cells after cultivation in vitro for a few days were examined. Analyses of the genomic structures of the proviruses revealed deletions in at least seven cases. However, we could not detect deletions in the proviral genomes of four out of the five ATL patients who lacked antibodies against gag proteins. Furthermore, expression of p19 and p24 was detected in these patients' peripheral blood lymphocytes (PBL) cultured in vitro for a few days. Thus, some ATL patients could not or could hardly raise antibodies against gag proteins, although they harbored complete HTLV-I genomes and their PBL expressed gag proteins in vitro. All patients harboring deleted proviruses, so far tested, raised antibodies not only against viral proteins that should be encoded by the integrated proviruses, but also against viral proteins that should be encoded by the deleted regions. Antibodies against viral proteins were detected also in sera of ATL patients whose PBL did not express viral proteins after in vitro cultivation. Specificities of antibodies against viral proteins in ATL patients could not be predicted by the structures of proviruses in leukemic cells or by expression of viral proteins in vitro. Immune responses to HTLV-I antigens were weak or lost in some ATL patients.Abbreviations used HTLV-I human T-cell leukemia virus type I - ATL adult T-cell leukemia - PBL peripheral blood lymphocytes - PAGE Polyacrylamide gel electrophoresis - kb kilobases Supported in part by a grant-in-aid from the Ministry of Health and Welfare of Japan for a comprehensive 10-year strategy for cancer control, and a grant-in-aid from the Ministry of Education, Science and Culture of Japan     

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.     

Human T lymphotropic virus type-I and adult T-cell leukemia in Japan

HTLV-I is the first retrovirus to be associated directly with human malignancy. In ATL-endemic areas, the rate of HTLV-I carriers is high. Both HTLV-I and ATL have been shown to be endemic in some regions of the world, especially in southwest Japan, the Caribbean islands, South Americas, and parts of Central Africa. Antibodies against HTLV-I have been found in over one million individuals, and more than 700 cases of ATL have been diagnosed each year in Japan alone. The cumulative incidence of ATL among HTLV-I carriers in Japan is estimated at 2.5% (3-5% in males, 1-2% in females). In endemic areas, HTLV-I Ab were found in the sera of 6 to 37 percent of healthy adults over 40 years of age. This clustering is thought to be due to the limited transmission of virus between socially isolated populations. The diagnostic criteria for HTLV-I associated ATL have been defined as follows. 1) Histologically and/or cytologically proven lymphoid malignancy with T cell antigens. 2) Abnormal T-lymphocytes present in the peripheral blood, except in the lymphoma type. 3) Serum specimens for all patients with ATL have HTLV-I Ab. 4) Demonstration of clonality of HTLV-I proviral DNA is a definite diagnosis of ATL. ATL shows diverse clinical features but can be divided into four subtypes: acute, chronic, smoldering, and lymphoma type. The pattern of HTLV-I transmission is through one of three different modes. Infected mothers can transmit the virus to newborns mainly via breast milk. The virus also can be transmitted from male to female by sexual intercourse, and through blood transfusion. Chemotherapy is not effective; the acute and lymphoma types have a poor prognosis. ATL is generally treated with curative intent using combination chemotherapy, although long-term success has been very limited. Unfortunately that advance did not translate into an improvement in the overall survival; the median remain 10 months. In contrast, smoldering ATL, or some cases of chronic ATL, may have a more protracted natural course, which may be compromised by aggressive chemotherapy. Alternative strategies for both acute and chronic forms are clearly needed. After infection of HTLV-I, there is a long latent period before onset of ATL. Analyses by PCR showed that clearly proliferation occurred in intermediate state or even carriers with high virus load. Such clonal proliferation might be preleukemic stage, which suggested that carriers with high virus load should be risk group to have ATL.     

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.     

In vitro infection of human macrophages with human T-cell leukemia virus type 1

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.     

Stable expression of the tax gene of type I human T-cell leukemia virus in human T cells activates specific cellular genes involved in growth.

Stable expression of the 40-kDa transactivator protein (Tax) from the type I human T-cell leukemia virus (HTLV-I) in Jurkat T cells leads to the activation and sustained expression of certain cellular genes that are transiently induced during normal T-cell growth. Cellular genes induced by Tax include those encoding the alpha subunit of the high-affinity interleukin 2 receptor (Tac), interleukin 2, and granulocyte/macrophage colony-stimulating factor. Tax induction of the interleukin 2 gene is synergistically amplified by mitogens that augment cytoplasmic levels of calcium. These changes in the pattern of cellular gene expression reflect a specific action of Tax, as they are undetectable in isogenically matched control cell lines expressing antisense tax cDNA. The spectrum of cellular genes regulated by Tax appears to be restricted: several other T-cell genes, either inducibly or constitutively expressed, are unaffected by this viral protein. These cell lines constitutively expressing Tax provide valuable reagents to explore the molecular basis for Tax action and to delineate the full spectrum of cellular genes regulated by this retroviral gene product.     

Foxp3 expression on normal and leukemic CD4+CD25+ T cells implicated in human T-cell leukemia virus type-1 is inconsistent with Treg cells

Foxp3 is a master gene of Treg cells, a novel subset of CD4⁺ T cells primarily expressing CD25. We describe here different features in Foxp3 expression profile between normal and leukemic CD4⁺CD25⁺ T cells, using peripheral blood samples from healthy controls (HCs), human T-cell leukemia virus type-1 (HTLV-1)-infected asymptomatic carriers (ACs), patients with adult T-cell leukemia (ATL), and various hematopoietic cell lines. The majority of CD4⁺CD25⁺ T cells in HCs were positive for Foxp3, but not all CD4⁺CD25⁺ T cells in ACs were positive, indicating that Foxp3 expression is not always linked to CD25 expression in normal T cells. Leukemic (ATL) T cells constitutively expressing CD25 were characteristic of heterogeneous Foxp3 expression, such as intra- and inter-case heterogeneity in intensity, inconsistency with CD25 expression, and a discrepancy in the mRNA and its protein expression. Surprisingly, a discernible amount of Foxp3 mRNA was detectable even in most cell lines without CD25 expression, a small fraction of which was positive for the Foxp3 proteins. The subcellular localization of Foxp3 in HTLV-1-infected cell lines was mainly cytoplasmic, different from that of primary ATL cells. These findings indicate that Foxp3 has two facets: essential Treg identity and molecular mimicry secondary to tumorigenesis. Conclusively, Foxp3 in normal T cells, but not mRNA, is basically potent at discriminating a subset of Treg cells from CD25⁺ T-cell populations, whereas the modulation of Foxp3 expression in leukemic T cells could be implicated in oncogenesis and has a potentially useful clinical role.