Effect of tumor promoters on human T cell leukemia/lymphoma virus (HTLV)-structural protein induction in adult T-cell leukemia cells

We assayed the capacity of tumor promoters to induce human T-cell leukemia/lymphoma virus (HTLV) structural proteins p19 and p24 from the HTLV genome-carrying adult T-cell leukemia (ATL) cell lines, MT-1 and KH-2Lo, and fresh ATL cells. Among the tested substances, 12-O-tetradecanoyl phorbol-13-acetate (TPA), 12-hexadecanoyl-phorbol-13-acetate (HPA) and teleocidin induced HTLV structural protein p19 and p24. This suggests that certain environmental substances, especially those known to be tumor promoters, may activate the HTLV-gene in ATL cells.     

Cell surface phenotypes and expression of viral antigens of various human cell lines carrying human T-cell leukemia virus

Human T-cell leukemia/lymphoma virus (HTLV)-carrying cells from various origins were characterized by cell surface markers and expression of HTLV antigens. Eight cell lines named TCL were obtained by transformation of peripheral blood leukocytes (PBL) of healthy donors or HTLV carriers in cocultures with HTLV-producer MT-2 cells. Nine cell lines named ILT were interleukin 2 (IL2)-dependent cell lines cloned from PBL of ATL patients and healthy HTLV-carriers. Tc-Kan9 cell line was also an IL2-dependent cell line clonally established from PBL culture stimulated with autologous TCLcells. Five cell lines named TL were established in vitro directly from PBL of an adult T-cell leukemia (ATL) patient and from ILT cells of an ATL patient and three HTLV-carriers, respectively, to grow autonomously without IL2. All the TCLs, ILTs, TLs and Tc-Kan9 possessed Leu-I antigen, a pan-T-cell marker. Leu3a antigen, a helper/inducer T-cell marker, was expressed on five of eight TCLs and all of the ILTs and TLs. Leu-2a, a cytotoxic/suppressor T-cell marker, was detected only on Tc-Kan9 but not others. Fresh ATL leukemic cells of patients had a helper/inducer T-cell marker. Ia, OKT9 and Tac antigens, markers for activated and differentiated T cells, were strongly expressed on all of the cell lines tested and fresh ATL leukemic cells were weakly positive for these antigens. Expression of HTLV antigens detected by mouse monoclonal antibodies and an ATL-patient serum varied among these cell lines. One TL, two ILTs and most of the fresh ATL leukemic cells did not express HTLV antigens on the cell surface. The other cell lines were all positive for the surface viral antigens. However, molecular species of antigens defined by radioimmunoprecipitation with an ATL-patient serum were not always identical among the cell lines. Molecular weights of polypeptides detectable in most of the cell lines were 62K, 46K, 40K, 24K, 21K and 19K which could never be detected in several control T-cell lines. 68K and 28K polypeptides were frequently detected in MT-2 and TCLs. GINI4, a mouse monoclonal antibody against HTLV core protein (p19) detected not only p19 in various cell lines but also p28, p29, p31 or p40 in certain cell lines tested. B-cell lines named LCL were established and cloned from PBL of two HTLV-carriers by EB-virus-induced transformation and they also expressed HTLV antigens, la, OKT9 and Tac antigens. Expression of Tac and HTLV antigens of fresh ATL leukemic cells were induced or enhanced after in vitro short-term cell cultivation with crude IL2.     

Infectious transmission of human T-cell leukemia virus to rabbits

A rabbit lymphoid cell line (Ra-1) was established by co-cultivation with a human T-cell line (MT-2) carrying human T-cell leukemia virus (HTLV). The Ra-1 cell line is chromosomally male and is persistently infected with HTLV. Ra-1 cells, with or without mitomycin C treatment, were inoculated intravenously (i.v.) into 3 female rabbits. All 3 animals responded with the production of antibodies to HTLV antigens. Lymphocytes from one of these seroconverters were cultured in the presence of T-cell growth factor (TCGF) and HTLV particles were detected in the TCGF-grown lymphocytes which were chromosomally female. Co-cultivation of lymphocytes from the 2 other seroconverters with lymphocytes from 2 anti-HTLV-negative healthy men gave rise to the establishment of an HTLV-producing T-cell line derived from each individual. Blood transfusion from one of the HTLV-infected rabbits into 2 female rabbits also resulted in the seroconversion of both recipients. An HTLV-carrying lymphoid cell line (Ra-2) was established from one of the transfusion-related seroconverters. The Ra-2 cell line was initially TCGF-dependent but later became TCGF-independent. There results indicate that HTLV can be transmitted to rabbits. These animals may provide a suitable model system for studying the mode of transmission and pathogenicity of HTLV.     

IL-2- and IL-2-R- independent proliferation of T-cell lines from adult T-cell leukemia/lymphoma patients

Human T-cell leukemia/lymphoma virus I (HTLV-I) is known to be associated with adult T-cell leukemia/lymphoma (ATL) as an etiological agent. The mechanism of leukemogenesis by HTLV, however, is still obscure. Two hypotheses have been proposed concerning abnormalities in IL-2 production and its receptor (Tac antigen) expression based on the experimental observations of IL-2-dependent ATL cell lines. In this study, we examine these hypotheses by using 3 leukemic T-cell lines from 3 Japanese patients with ATL. These cell lines were cultivated and established without addition of IL-2 to the culture medium. Cell-surface phenotype analysis by immunofluorescence with monoclonal antibodies (MAbs) and IL-2 binding assays revealed that one of the ATL cell lines, HPB-ATL-2, expresses only a minimal amount of IL-2 receptor (IL-2-R) on the cell surface and binds less radiolabelled human recombinant IL-2 than the other highly Tac-positive cell lines. Expression of Tac antigen in all ATL cell lines was not affected by IL-2, anti-Tac MAb or the tumor-promoter phorbol ester in the culture medium. The culture supernatant from these cell lines showed no IL-2 activity toward Con-A-stimulated human peripheral blood lymphocytes, and their growth was not affected by additional IL-2 in cultures. IL-2-independent growth and constitutive expression of its receptors on the cell surface were evident in our ATL cell lines. However, dense expression of IL-2 receptors was not essential for stimulation of leukemic proliferation of T cells by HTLV-I. Trans-activation of the PX40 gene product of HTLV-I for activation of IL-2-R gene might not be coincidentally associated with stimulation for cell proliferation.     

A glycoprotein antigen detected with new monoclonal antibodies on the surface of human lymphocytes infected with human T-cell leukemia virus type-I (HTLV-I)

We have prepared two new mouse monoclonal antibodies (MAbs) named TARM-34 (IgM) and TAG-34 (IgG1), that react with surface antigens of lines of human lymphocytes bearing a human T-cell leukemia virus type-I (HTLV-I). The characters of these antibodies are compared with those of anti-HTLV-1 gp21 MAb (TA-21, IgG1), anti-HTLV-I p19 MAb (GIN-14, IgG1) and human antibodies from patients with adult T-cell leukemia (ATL). An indirect membrane immunofluorescence assay showed that TARM-34, TAG-34 and TA-21 all reacted specifically with cell-surface antigens of HTLV-I-positive T- and B-cell lines and cultured peripheral blood lymphocytes from HTLV-I-infected adults. Radioimmunoassay showed that serum antibodies from the ATL patients interfered with the binding of TA-21 antibody to cells of the HTLV-I-positive T-cell line MT-2, but not with the bindings of TARM-34 and TAG-34 antibodies. TARM-34 and TAG-34 both precipitated a 34-kd glycoprotein (gP34), while TA-21 precipitated gp21 from a lysate of 3H-glucosamine-labelled MT-2 cells. TARM-34 and TAG-34 also precipitated the 34-kd protein from lysates of MT-2 and HUT 102 cells labelled with 125I- or 35S-cysteine. Interestingly, TARM-34 and TAG-34 also precipitated 35-kd protein from a lysate of other HTLV-I-positive cells (F-Taj cell line) derived from an ATL patient. TA-21 precipitated the 21-kd protein from the lysates of 35S-cysteine-labelled HTLV-IMT-2 virions, but TARM-34 and TAG-34 did not precipitate any protein from this lysate. TARM-34 lysed HTLV-I-bearing cells in the presence of rabbit complement. These results indicate that TARM-34 and TAG-34 both recognize a glycoprotein antigen that is expressed on the surface of HTLV-I-infected cells.     

Human T-cell leukemia virus type I: pseudotype neutralization of Japanese and American isolates with human and rabbit sera

CCC/2M, CCC/10Y and CCC/MT-2 cat kidney cells producing Japanese isolates of human T-cell leukemia virus type I (HTLVs) and HOS/PL human osteosarcoma cells producing an American isolate of HTLV were infected with vesicular stomatitis virus (VSV) to prepare VSV pseudotypes bearing envelope antigens of HTLVs. VSV propagated in CCC/2M cells contained plaque-forming fractions that were not neutralized by treatment with anti-VSV serum alone: VSV pseudotypes bearing envelope antigens of HTLV2M and CCC cat endogenous virus were formed by infection of CCC/2M cells with VSV. Japanese HTLV2M, HTLV10Y and HTLVMT-2 and American HTLVPL pseudotypes were neutralized by sera of Japanese, American and British patients with ATL. Each serum, including the serum of the patient from whom HTLV2M or HTLV10Y had been derived, gave similar antibody titers against Japanese and American HTLV pseudotypes. The HTLV pseudotypes were also neutralized by rabbit serum raised against HTLVMT-2. A rabbit antiserum against the C-terminal half of the HTLV env protein produced in E. coli also neutralized Japanese and American HTLV pseudotypes. Thus, VSV pseudotype analyses indicated that envelope antigens of HTLVs represent a single serotype worldwide. The env protein produced in E. coli may be used to raise neutralizing antibody against HTLVs.     

Antibodies against three purified structural proteins of the human type-C retrovirus,HTLV, in Japanese adult T-cell leukemia patients,healthy family members,and unrelated normals

The immunological relationship of human T-cell leukemia/lymphoma virus (HTLV) and the virus found in Japanese adult T-cell leukemia/lymphoma (ATL) was investigated in detail by testing the specific binding of serum antibodies from Japanese ATL patients and normal Japanese donors to the purified HTLV proteins p24, p19, and p15. Sera were prescreened for antibodies to p24. Of those positive, 67% of the ATL sera and 78% of the normal sera were further shown to have antibodies against p19. In both groups 17% had antibodies to p15. Generally, the average antibody titers were twice as high in ATL as in normal sera. Competition radioimmunoprecipitation assays done with various sera and involving HTLV-producing cells, virus-positive cells from a Japanese ATL patient, and virus-positive cultured T cells of one of his healthy family members as competing materials demonstrated no differences between the p24, p19, and p15 found in these cells. These results provide strong and detailed immunological evidence that the human retrovirus isolates first made from US patients with cutaneous T-cell malignancies, and those made later in Japanese ATL are either identical or very closely related strains of the same virus, HTLV, a finding verified in other detailed analyses of the HTLV genomes of the respective isolates. To date only HTLV-II, isolated from a US case of hairy-cell leukemia of a T-cell type, is a distinct additional human retrovirus class.     

In vitro infection of CD4+ T lymphocytes with HTLV-I generates immortalized cell lines coexpressing lymphoid and myeloid cell markers.

The human T cell leukemia/lymphoma virus (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL). CD4+ lymphocytes are the preferential targets of infection, even though other cell types can be infected in vitro by the virus. Although ATL cells show CD3 and CD4 surface markers, some ATL-derived cell lines were reported to express also myeloid antigens. In order to analyze possible phenotypic changes induced by HTLV-I after infection of human lymphocytes, CD4+ cells were isolated from peripheral blood of three healthy donors, by separation through immunomagnetic beads. CD4+ lymphocytes were then infected by coculture with irradiated HTLV-I producing MT-2 cells. The phenotypic profile of infected cells was studied by flow cytometric analysis using monoclonal antibodies against lymphoid (CD3, CD4, TCR alpha/beta) and myelomonocitic markers (CD13, CD14, CD15, CD33, CD34). The results show that HTLV-I immortalized cell lines coexpressed CD13, CD33 and lymphoid markers. No expression of CD14, CD15 and CD34 was observed. These data suggest that the presence of both myeloid and lymphoid phenotype in HTLV-I infected T cells is the results of an induction rather than a selection mechanism.     

Transformation of hamster spleen lymphocytes by human T-cell leukemia virus type I

Co-cultivation of spleen cells of Syrian golden hamsters with lethally irradiated MT-2 cells harboring human T-cell leukemia virus type I (HTLV-I) resulted in the establishment of lymphoid cell lines, HCT-1 and HCT-2, which exhibited the normal karyotype of golden hamsters. Cells of both the HCT-1 and HCT-2 lines lacked surface immunoglobulins and reacted with a monoclonal antibody (MAb) specific for hamster T cells. Some were positive for OKIa1. None of them expressed HTLV structural antigens (p19 and p24) or virus particles, but they contained HTLV-I proviral DNA monoclonally. By immunochemical analysis of the labelled cell antigens, sera from adult T-cell leukemia (ATL) patients reacted with the two polypeptides, p37 and p40, which may not be viral structural proteins and still remain to be characterized. HCT-1 and HCT-2 cells were transplantable into newborn hamsters, pre-treated with anti-hamster thymocyte serum and non-treated, respectively, producing diffuse malignant lymphoma. These findings indicated that HTLV-I not only immortalized but also transformed hamster T cells non-productively.     

In vitro susceptibility of different human T-cell subpopulations and resistance of large granular lymphocytes to HTLV-I infection

T4 subpopulation of T lymphocytes is the preferential target of infection with human T leukemia/lymphoma virus of subgroup I (HTLV-I). In this study we attempt to determine whether different T-cell subsets exhibit differences in susceptibility to virus infection. T cells from cord or peripheral blood were separated according to cell densities and T-cell surface markers by Percoll gradient and Sepharose anti-Fab immunoadsorbent affinity column (IAC), respectively. Separated T-cell subpopulations were infected with HTLV-I, by means of co-cultivation with irradiated virus producer cell lines (MT-2, TK). Percentages of HTLV-I-infected cells were assayed by immunofluorescence assay (IFA), using highly specific mouse monoclonal antibody (MAb) directed against HTLV-I p19 core protein. The results showed that different T-cell subpopulations separated either by Percoll or by IAC were susceptible to HTLV-I infection with the exception of large granular lymphocytes (LGL), which exhibit high cell-mediated natural cytotoxicity (CMNC). The susceptibility to HTLV-I infection of T cells with CMNC activity was further studied on established cell clones with LGL morphology. The results showed again that these cells were resistant to the virus infection. The present studies indicate that different T-cell subpopulations, irrespective of their size and of cell-surface markers, are susceptible to HTLV-I infection, with the exception of functionally mature LGL or of immortalized LGL clones.     

Human T-cell leukemia virus type I induces adult T-cell leukemia: from clinical aspects to molecular mechanisms.

BACKGROUND: Human T-cell leukemia virus type I (HTLV-I) is a causative virus of adult T-cell leukemia (ATL), HTLV-I-associated myelopathy/tropical spastic paraparesis, and HTLV-I-associated uveitis. ATL is a neoplastic disease of CD4-positive T lymphocytes that is characterized by pleomorphic tumor cells with hypersegmented nuclei, termed "flower cells." The mechanisms of leukemogenesis have not been fully clarified. METHODS: The authors reviewed the virological, clinical, and immunological features of HTLV-I and ATL and summarized recent findings on the oncogenic mechanisms of ATL and therapeutic advances. RESULTS: Multiple factors, such as viral genes, genetic and epigenetic alterations, and the host immune system, may be implicated in the leukemogenesis of ATL. Among them, viral genes, tax, and HBZ have been thought to play important roles. The prognosis of aggressive-type ATL remains poor, regardless of intensive chemotherapy. Effectiveness of allogeneic stem cell transplantation for ATL has been recently reported. CONCLUSIONS: Although the precise mechanism of leukemogenesis of ATL remains unclear, recent progress provides important clues in oncogenesis by HTLV-I. Future research should focus on the composition of novel therapeutic strategies, including prevention, based on the evidence in the leukemogenic mechanisms.     

In vitro infection of human B lymphocytes with adult T-cell leukemia virus

Experimental transmission of adult T-cell leukemia (ATL) virus (ATLV) into human B lymphocytes was attempted. Cocultivation of B-cell rich fraction of peripheral blood from a healthy adult with X-ray irradiated ATLV producer MT-2 cells resulted in the establishment of OKA(B) cell line co-infected with both Epstein-Barr virus (EBV) and ATLV. OKA(B) cells and its subclones contained: (1) B cell markers exclusively; (2) both EBV-specific antigen, EBNA and ATLV-specific antigen, ATLA detected by immunofluorescence test; (3) ATLV-specific polypeptides, p24 and p19; (4) ATLV-specific mRNA in ATLA-positive clones; (5) ATLV and EBV particles detectable by electron microscopy. These data clearly show that human B lymphocytes are susceptible to ATLV infection.     

Isolation of simian retroviruses closely related to human T-cell leukemia virus by establishment of lymphoid cell lines from various non-human primates

Simian retroviruses closely related to human T-cell leukemia virus (HTLV) were isolated by establishing virus-producing lymphoid cell lines from 7 species of non-human primates. By co-cultivation with human umbilical cord-blood cells and/or in the presence of interleukin-2, lymphoid cell lines were successfully established from the chimpanzee. African green monkey, pig-tailed macaque, red-faced macaque, Formosan monkey, Japanese monkey and bonnet monkey that had antibodies against HTLV antigens. These cell lines reacted with human sera of ATL patients and monoclonal antibodies against p19 and p24 of HTLV antigens. Cellular DNAs contained the provirus sequences homologous to HTLV-I by Southern blot hybridization. Moreover, they produced extracellular type-C virus particles and RNA-dependent DNA polymerase. All of these lymphoid line cells had Tac antigen, interleukin-2 receptor, and those of chimpanzee and red-faced macaque had helper/induced T-cell markers, while those derived from African green monkey had suppressor/cytotoxic T-cell markers. Furthermore, simian HTLV-related viruses of pig-tailed macaque, red-faced macaque and Japanese monkey were transmitted to human lymphocytes on co-cultivation.     

Differential methylation of class 1 histocompatibility antigen genes in T-cell lines derived from two different types of T-cell malignancies

We have previously shown that two human T-cell lines (HSB and 8402) derived from patients with childhood T-cell ALL (T-ALL) do not synthesize detectable mRNA for HLA-DR. The DR genes in both cell lines are hypermethylated relative to the same genes in T-cell lines infected with human T-cell leukemia virus (HTLV) and derived from patients with adult T-cell leukemia/lymphoma (ATL). These latter cell lines do express HLA-DR-mRNA, as well as HLA-DR surface antigens. We report here that the genes for HLA class I antigens are also highly methylated in the T-ALL T-cell lines relative to the same genes in the ATL T-cell lines, normal peripheral blood T cells, and autologous normal B-cell lines. In spite of substantial differences in the extent of methylation of class I-related genes, no obvious differences exist among these cell types in their levels of expression of HLA-A and -B antigens. The data clearly indicate, however, that the class I and class II components of the major histocompatability complex are unusually hypermethylated in several T-ALL-derived cell lines, while ATL T-cell lines do not substantially differ in this respect from normal peripheral blood T cells.     

Adult T-cell leukemia in two siblings. Acute crisis of smoldering disease in one patient

Two cases of human T-cell leukemia virus (HTLV)-positive adult T-cell leukemia (ATL) in brother and sister are presented. Six of 15 members of this family were seropositive for antibodies for ATL-associated antigens (ATLA). The sister of the ATL patient developed overt ATL after 5 years and 8 months of smoldering ATL. Immunologic examinations during the smoldering phase were normal except for negative skin tests for purified protein derivative. Factors leading to the induction of ATL among HTLV carriers remain to be studied.     

Location of human T-cell leukemia virus (HTLV) p19 antigen on virus-producing cells

Mouse monoclonal antibody to HTLV p19 was used to locate HTLV p19 on the surface of cells and virions by immunofluorescence microscopy (IFM) and immunoelectron microscopy (IEM). When HTLV-producing cells HUT 102 (B2 clone), MT-2 and strain A were used as target cells, HTLV p19 was detected on the surface of cells and virions as spots or small sectors by both IFM and IEM. Cells infected with animal type-C retroviruses, e.g., gibbon ape leukemia virus, simian sarcoma virus, feline leukemia virus, and Gross murine leukemia virus, were completely negative for HTLVp19 expression. Other human T cells not producing HTLV, including HUT78 and HSB2-0, immature or pre-T cells (Molt-3) derived from leukemia patients, and fresh peripheral blood T cells from healthy persons, were also negative. In addition, B cells including Rob-B, IM-9, Raji, and BT-1 did not react with the monoclonal antibody to HTLV p19. In the light of the presence of HTLV p19 in the periphery of acetone-fixed HTLV-producing cells as shown by IFM, it seems most likely that HTLV p19 is an internal antigen of HTLV with part of its structure protruding out of the viral and cell membrane. The monoclonal antibody to HTLV p19 did not lyse HTLV-producing cells in the presence of complement, as expected, because the antibody is an IgG1. Antibody-dependent cell-mediated cytotoxicity was also studied by the 51Cr-release assay. No cytotoxicity was observed. Although HTLV p19 does not contribute to the destruction of malignant T cells for treatment and/or virions for prophylaxis, this protein is an important marker for diagnosis of HTLV infection. The patterns of HTLV p19 expression described above were exactly the same for American HTLV-producing HUT 102 (B2 clone), for strain A cells and for Japanese HTLV-producing MT-2 cells. These results further substantiate the close relationship of the Japanese and American HTLV isolates.