Ectopic overexpression of c-mpl by retroviral-mediated gene transfer suppressed megakaryopoiesis but enhanced erythropoiesis in mice.

In this report, we tested whether ectopic overexpression of a cell surface receptor cDNA could be used to explore the physiological roles of that receptor. We generated c-mpl overexpressing animals by reconstituting mice with retroviral vector-transduced bone marrow (BM) cells. We observed that platelet counts in the c-mpl overexpressing mice failed to recover to normal levels and remained at <200 x 10(6)/mL post-transplantation, while platelet numbers in the control mice returned to > 800 x 10(6)/mL by 4 weeks post-transplantation. However, platelet counts in the c-mpl overexpressing mice could be stimulated to normal levels after administration of rhMGDF. No significant changes in peripheral leukocyte counts were observed, although the number of CFU-E, GM-CFC, and CFC-multi were reduced two- to threefold in the BM of the c-mpl overexpressing mice. In addition, enhanced erythropoiesis was observed in the c-mpl overexpressing mice. The mpl receptors on erythroid cells were functional as demonstrated by tyrosine-phosphorylation of mpl receptor on RBC and by in vitro erythroid colony-formation in response to MGDF stimulation, respectively. These results suggested that ectopically expressed mpl receptors competed for ligand in vivo leading to an insufficient amount of circulating thrombopoietin (Tpo) for the development of megakaryocytic lineage. These results further suggest that, in addition to sequestering circulating Tpo, overexpression of the mpl receptor on erythroid progenitors may directly contribute to enhanced erythropoiesis in vivo. Our studies demonstrate that ectopic overexpression of a receptor by retroviral-mediated gene transfer provides an approach to explore the biological roles of novel receptors.     

IRF-4 and c-Rel expression in antiviral-resistant adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a generally fatal malignancy. Most ATLL patients fare poorly with conventional chemotherapy; however, antiviral therapy with zidovudine (AZT) and interferon alpha (IFN-alpha) has produced long-term clinical remissions. We studied primary ATLL tumors and identified molecular features linked to sensitivity and resistance to antiviral therapy. Enhanced expression of the proto-oncogene c-Rel was noted in 9 of 27 tumors. Resistant tumors exhibited c-Rel (6 of 10; 60%) more often than did sensitive variants (1 of 9; 11%). This finding was independent of the disease form. Elevated expression of the putative c-Rel target, interferon regulatory factor-4 (IRF-4), was observed in 10 (91%) of 11 nonresponders and in all tested patients with c-Rel+ tumors and occurred in the absence of the HTLV-1 oncoprotein Tax. In contrast, tumors in complete responders did not express c-Rel or IRF-4. Gene rearrangement studies demonstrated the persistence of circulating T-cell clones in long-term survivors maintained on antiviral therapy. The expression of nuclear c-Rel and IRF-4 occurs in the absence of Tax in primary ATLL and is associated with antiviral resistance. These molecular features may help guide treatment. AZT and IFN-alpha is a suppressive rather than a curative regimen, and patients in clinical remission should remain on maintenance therapy indefinitely.     

Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo

Leukemogenic viruses like human T-lymphotropic virus and bovine leukemia virus (BLV) presumably persist in the host partly by latent integration of the provirus in a fraction of infected cells, leading to accumulative increase in the outgrowth of transformed cells. Furthermore, viral infection also correlates with a blockade of the apoptotic mechanisms concomitant with an apparent latency of the host cell. Conceptually, induction of viral or cellular gene expression could thus also be used as a therapeutic strategy against retroviral-associated leukemia. Here, we provide evidence that valproate, an inhibitor of deacetylases, activates BLV gene expression in transient transfection experiments and in short-term cultures of primary B-lymphocytes. In vivo, valproate injection into newly BLV-inoculated sheep did not abrogate primary infection. However, valproate treatment, in the absence of any other cytotoxic drug, was efficient for leukemia/lymphoma therapy in the sheep model leading to decreased lymphocyte numbers (respectively from 25.6, 35.7, and 46.5 x 10(3) cells per mm3 to 1.0, 10.6, and 24.3 x 10(3) cells per mm3 in three leukemic sheep) and tumor regression (from >700 cm3 to undetectable). The concept of a therapy that targets the expression of viral and cellular genes might be a promising treatment of adult T cell leukemia or tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy, diseases for which no satisfactory treatment exists so far.     

CD4- and CD56-positive T-cell line, MTA, established from natural killer-like T-cell leukemia/lymphoma

We have established a T-cell line, MTA, from the peripheral blasts of a patient with natural killer (NK)-like T-cell leukemia/lymphoma. The MTA cell displays a T-cell and NK-cell phenotype (CD2+, CD3+, CD4+, CD56+) identical to freshly isolated leukemic blasts from the patient. This cell line shows clonal T-cell receptor rearrangement and a distinguishable character by light microscopy with May-Grünwald Giemsa staining. G-banding analysis showed that the MTA cells had a karyotype of 94(4N), XXXX, add (1) (p36), del (5) (q14q23), add (17) (p11), add (19) (q13). However, unlike NK malignancy, we could not show a direct pathogenic role for Epstein-Barr virus (EBV) with EBV-encoded small RNA and polymerase chain reaction analysis. The MTA cell line is a novel cell line with which to study NK-like T-cell ontogenecity.     

EAPB0203, a member of the imidazoquinoxaline family, inhibits growth and induces caspase-dependent apoptosis in T-cell lymphomas and HTLV-I-associated adult T-cell leukemia/lymphoma

Imiquimod is an immune response modifier currently used as a topical treatment of genital warts, basal cell carcinoma, cutaneous metastasis of malignant melanoma, and vascular tumors. We developed more efficient killers from the same family of compounds that can induce apoptosis without the prominent pro-inflammatory response associated with imiquimod. Among these new products, tk;4EAPB0203, a member of the imidazo[1,2-a]quinoxalines, exhibits an important cytotoxic activity in vitro. HTLV-I-associated adult T-cell leukemia (ATL) and HTLV-I-negative peripheral T-cell lymphomas are associated with poor prognosis. Using potentially achievable concentrations of EAPB0203, we demonstrate inhibition of cell proliferation, G2/M cell- cycle arrest, and induction of apoptosis in HTLV-I-transformed and HTLV-I-negative malignant T cells and fresh ATL cells, whereas normal resting or activated T lymphocytes were resistant. EAPB0203 treatment significantly down-regulated the antiapoptotic proteins c-IAP-1 and Bcl-XL and resulted in a significant loss of mitochondrial membrane potential, cytoplasmic release of cytochrome c, and caspase-dependent apoptosis. Moreover, in HTLV-I-transformed cells only, EAPB0203 treatment stabilized p21 and p53 proteins but had no effect on NF-kappaB activation. These results support a potential therapeutic role for EAPB0203 in ATL and HTLV-I-negative T-cell lymphomas, either as a systemic or topical therapy for skin lesions.     

Detection of human T-cell leukemia/lymphoma virus, type II, in a patient with large granular lymphocyte leukemia.

We studied a patient with large granular lymphocyte (LGL) leukemia for evidence of human T-cell leukemia/lymphoma virus (HTLV) infection. Serum from this patient was positive for HTLV-I/II antibodies by enzyme-linked immunosorbent assay (ELISA) and was confirmed positive in Western blot and radioimmunoprecipitation assays. Results of a synthetic peptide-based ELISA showed that the seropositivity was caused by HTLV-II and not HTLV-I infection. Analyses of enzymatic amplification of DNA from bone marrow sections using the polymerase chain reaction (PCR) were positive for HTLV-II specific gag, pol, env, and pX gene sequences. Cloning and sequencing of amplified products showed that the HTLV-II pol and pX sequences in patient DNA differed from the sequences of 17 other HTLV-II isolates examined in our laboratory. HTLV infection may have a role in some patients in the pathogenesis of LGL leukemia.     

Transformation of human umbilical cord blood T cells by human T-cell leukemia/lymphoma virus.

Several isolates of human T-cell leukemia/lymphoma virus (HTLV) were transmitted to normal human T cells obtained from the umbilical cord blood of newborns. T cells from seven specimens were immortalized by infection with different HTLV isolates and their properties were compared with those of activated uninfected normal T cells grown in the presence of T-cell growth factor (TCGF) and with those of HTLV-positive neoplastic T-cell lines derived from patients with T-cell malignancies. The HTLV-infected cells generally belonged to a class of mature T cells (OKT4+ and Leu 3A+) and differed from the normal uninfected cells in that they could be propagated in culture indefinitely; possessed altered morphology, including convoluted nuclei and some bi- and multinucleated giant cells; formed large clumps in culture; demonstrated a diminished requirement for TCGF; had an increased density of TCGF receptors; often became completely independent of exogenous TCGF; and expressed HLA-DR determinants. These properties of the HTLV-infected cord blood T cells contrasted to those of uncultured cord blood T cells and of cord blood cells stimulated with mitogen and grown with TCGF but resembled the characteristics of T-cell lines established previously from patients with HTLV-associated T-cell malignancies. This in vitro system offers a unique opportunity to study the basic mechanism involved in abnormal growth and neoplastic transformation of a specific class of human T cells.     

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.     

Adult T-cell leukemia/lymphoma associated with HTLV-1 infection in a Brazilian adolescent.

We present the case of a 15-year-old patient infected with HTLV-1 who developed a cutaneous T-cell lymphoma, confirmed by histopathological and immunohistochemical examination, as well as clinically and hematologically confirmed leukemia. The patient died 3 months after initial presentation of the disease. The rarity of the disease in this age group justifies the present report.     

Long-term correction of phagocyte NADPH oxidase activity by retroviral-mediated gene transfer in murine X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited deficiency of the superoxide-generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in recurrent, severe bacterial and fungal infections. The X-linked form of this disorder (X-CGD) results from mutations in the X-linked gene for gp91(phox), the larger subunit of the oxidase flavocytochrome b(558). In this study, we used a murine model of X-CGD to examine the long-term function of retroviral vectors for expression of gp91(phox) based on the murine stem cell virus (MSCV) backbone. NADPH oxidase activity was reconstituted in neutrophils and macrophages for up to 18 to 24 months posttransplantation of transduced X-CGD bone marrow into lethally irradiated syngeneic X-CGD mice. Southern blot analysis and secondary transplant data showed proviral integration in multilineage repopulating cells. Although relatively small amounts of recombinant gp91(phox) (approximately 5% to 10% of wild-type levels) were detected in neutrophils after retroviral-mediated gene transfer, superoxide-generating activity was approximately 20% to 25% of wild-type mouse neutrophils. Expression of gp91(phox) is normally restricted to mature phagocytes. No obvious toxicity was observed in other hematopoietic lineages in transplant recipients, and provirus-marked cells were capable of reconstituting secondary transplant recipients, who also exhibited NADPH oxidase-positive neutrophils. MSCV-based vectors for long-term expression of gp91(phox) may be useful for gene therapy of human CGD targeted at hematopoietic stem cells.     

Infiltration of the lower respiratory tract by helper/inducer T lymphocytes in HTLV-1-associated adult T-cell leukemia/lymphoma.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATLL), a disorder in which peripheral blood and multiple organs are infiltrated by malignantly transformed T lymphocytes. We investigated the nature of pulmonary disease in a patient with serologic evidence of HTLV-1 infection. In this case, endobronchial biopsy specimens showed infiltration of the bronchial mucosa by pleomorphic cells exhibiting a high degree of nuclear irregularity. These cells were morphologically identical in appearance to malignant cells found in peripheral blood and infiltrating the dermis, expressed the OKT4/Leu3 phenotype and the receptor for interleukin 2, and, by analogy to gene rearrangement studies on leukemic blood cells, were monoclonal in origin. However, in situ hybridization of endobronchial biopsy specimens with full-length HTLV-1 probes failed to detect retroviral RNA or proviral DNA. These studies indicate that T lymphocytic involvement of the lower respiratory tract in HTLV-1-associated ATLL is characterized by expression of a malignant phenotype despite the inability to document actual cellular infection with this retrovirus by a molecular hybridization technique.     

Cre/loxP-mediated CTLA4IgG gene transfer induces clinically relevant immunosuppression via on-off gene recombination in vivo

OBJECTIVE: Transfer of the CTLA4IgG gene induces long-term and high levels of CTLA4IgG expression, which can result in generalized immunosuppression. In this study, we utilized Cre/loxP-mediated on-off switch recombination to eliminate transgene expression of CTLA4IgG following acceptance of murine cardiac allografts. METHODS: Fully MHC-mismatched hearts from BALB/c donor mice were transplanted into C3H/He recipient mice. Adenovirus-containing CTLA4IgG flanked between two loxP sites was administered via a recipient tail vein immediately after transplantation. Cre-recombinase gene was subsequently transferred at day 30 posttransplantation. RESULTS: Long-term allograft survival was observed in recipients that received the CTLA4IgG gene. Cre-mediated recombination reduced CTLA4IgG gene expression without any adverse effect on the graft survival. Secondary skin grafts of donor type and of third party were promptly rejected in the recipients that accepted cardiac allografts. In addition, the B cell response against ovalbumin was suppressed during high levels of serum CTLA4IgG, but recovered after Cre-mediated inactivation of CTLA4IgG gene. CONCLUSION: CTLA4IgG gene transfer promoted long-term survival of murine cardiac allografts; however, this was not sufficient to induce tolerance. Cre/loxP-mediated on-off switch recombination was useful to inactivate the CTLA4IgG gene so that recipients' immune responses against neoantigens were restored without an influence on the allograft survival. This system may open novel strategies to orchestrate clinically relevant immunosuppression.     

Induction of antigen-specific CD4+ T-cell anergy and deletion by in vivo viral gene transfer

Immune responses to the therapeutic gene product are a potentially serious complication in treatment of genetic disease by gene therapy. Induction and maintenance of immunologic hypo-responsiveness to the therapeutic antigen is therefore critical to the success of gene-based treatment of inherited protein deficiency. Here, we demonstrate induction of antigen-specific CD4+ T-cell tolerance to a secreted transgene product (ovalbumin, ova) in ova-specific T-cell receptor (TCR) transgenic mice by hepatic adeno-associated virus (AAV)-mediated gene transfer. Transduced mice maintained stable circulating ova levels without evidence of an immune response. Lymph node cells and splenocytes were hypo-responsive to ova as early as day 10 after gene transfer. Numbers of TCR+CD4+ cells were reduced in secondary lymphoid organs and in the thymus by 1 to 2 months after vector administration. The remaining TCR+CD4+ cell population was anergic to ova antigen in vitro and enriched for CD25+ cells. These data provide direct evidence that transgene expression following in vivo viral gene transfer can induce CD4+ T-cell tolerance to the transgene product, involving anergy and deletion mechanisms.