Prabal Banerjee

Adult T-cell leukemia/lymphoma development in HTLV-1–infected humanized SCID mice

The molecular and genetic factors induced by human T-lymphotropic virus type-1 (HTLV-1) that initiate adult T-cell leukemia/lymphoma (ATLL) remain unclear, in part from the lack of an animal model that accurately recapitulates leukemogenesis. HTLV-1-infected humanized nonobese diabetic severe combined immunodeficiency (HU-NOD/SCID) mice were generated by inoculation of NOD/SCID mice with CD34(+) hematopoietic progenitor and stem cells (CD34(+) HP/HSCs) infected ex vivo with HTLV-1. HTLV-1-HU-NOD/SCID mice exclusively developed CD4(+) T-cell lymphomas with characteristics similar to ATLL and elevated proliferation of infected human stem cells (CD34(+)CD38(-)) in the bone marrow were observed in mice developing malignancies. Purified CD34(+) HP/HSCs from HTLV-1-infected patient peripheral blood mononuclear cells revealed proviral integrations suggesting viral infection of human bone marrow-derived stem cells. NOD/SCID mice reconstituted with CD34(+) HP/HSCs transduced with a lentivirus vector expressing the HTLV-1 oncoprotein (Tax1) also developed CD4(+) lymphomas. The recapitulation of a CD4(+) T-cell lymphoma in HU-NOD/SCID mice suggests that HSCs provide a viral reservoir in vivo and act as cellular targets for cell transformation in humans. This animal model of ATLL will provide an important tool for the identification of molecular and cellular events that control the initiation and progression of the lymphoma and potential therapeutic targets to block tumor development.

Induction of Cell Cycle Arrest by Human T-Cell Lymphotropic Virus Type 1 Tax in Hematopoietic Progenitor (CD34+) Cells: Modulation of p21cip1/waf1 and p27kip1 Expression

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia, an aggressive CD4+ malignancy. Although HTLV-2 is highly homologous to HTLV-1, infection with HTLV-2 has not been associated with lymphoproliferative disorders. Lentivirus-mediated transduction of CD34+ cells with HTLV-1 Tax (Tax1) induced G0/G1 cell cycle arrest and resulted in the concomitant suppression of multilineage hematopoiesis in vitro. Tax1 induced transcriptional upregulation of the cdk inhibitors p21cip1/waf1 (p21) and p27kip1 (p27), and marked suppression of hematopoiesis in immature (CD34+/CD38−) hematopoietic progenitor cells in comparison to CD34+/CD38+ cells. HTLV-1 infection of CD34+ cells also induced p21 and p27 expression. Tax1 also protected CD34+ cells from serum withdrawal-mediated apoptosis. In contrast, HTLV-2 Tax (Tax2) did not detectably alter p21 or p27 gene expression, failed to induce cell cycle arrest, failed to suppress hematopoiesis in CD34+ cells, and did not protect cells from programmed cell death. A Tax2/Tax1 chimera encoding the C-terminal 53 amino acids of Tax1 fused to Tax2 (Tax221) displayed a phenotype in CD34+ cells similar to that of Tax1, suggesting that unique domains encoded within the C terminus of Tax1 may account for the phenotypes displayed in human hematopoietic progenitor cells. These remarkable differences in the activities of Tax1 and Tax2 in CD34+ hematopoietic progenitor cells may underlie the sharp differences observed in the pathogenesis resulting from infection with HTLV-1 and HTLV-2.

Hematopoietic stem cells and retroviral infection

Retroviral induced malignancies serve as ideal models to help us better understand the molecular mechanisms associated with the initiation and progression of leukemogenesis. Numerous retroviruses including AEV, FLV, M-MuLV and HTLV-1 have the ability to infect hematopoietic stem and progenitor cells, resulting in the deregulation of normal hematopoiesis and the development of leukemia/lymphoma. Research over the last few decades has elucidated similarities between retroviral-induced leukemogenesis, initiated by deregulation of innate hematopoietic stem cell traits, and the cancer stem cell hypothesis. Ongoing research in some of these models may provide a better understanding of the processes of normal hematopoiesis and cancer stem cells. Research on retroviral induced leukemias and lymphomas may identify the molecular events which trigger the initial cellular transformation and subsequent maintenance of hematologic malignancies, including the generation of cancer stem cells. This review focuses on the role of retroviral infection in hematopoietic stem cells and the initiation, maintenance and progression of hematological malignancies.

Proinflammatory Cytokine Gene Induction by Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2 Tax in Primary Human Glial Cells

ABSTRACT Infection with human T-cell leukemia virus type 1 (HTLV-1) can result in the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic inflammatory disease of the central nervous system (CNS). HTLV-2 is highly related to HTLV-1 at the genetic level and shares a high degree of sequence homology, but infection with HTLV-2 is relatively nonpathogenic compared to HTLV-1. Although the pathogenesis of HAM/TSP remains to be fully elucidated, previous evidence suggests that elevated levels of the proinflammatory cytokines in the CNS are associated with neuropathogenesis. We demonstrate that HTLV-1 infection in astrogliomas results in a robust induction of interleukin-1β (IL-1β), IL-1α, tumor necrosis factor alpha (TNF-α), TNF-β, and IL-6 expression. HTLV encodes for a viral transcriptional transactivator protein named Tax that also induces the transcription of cellular genes. To investigate and compare the effects of Tax1 and Tax2 expression on the dysregulation of proinflammatory cytokines, lentivirus vectors were used to transduce primary human astrocytomas and oligodendrogliomas. The expression of Tax1 in primary human astrocytomas and oligodendrogliomas resulted in significantly higher levels of proinflammatory cytokine gene expression compared to Tax2. Notably, Tax1 expression uniquely sensitized primary human astrocytomas to apoptosis. A Tax2/Tax1 chimera encoding the C-terminal 53 amino acids of the Tax1 fused to the Tax2 gene (Tax221) demonstrated a phenotype that resembled Tax1, with respect to proinflammatory cytokine gene expression and sensitization to apoptosis. The patterns of differential cytokine induction and sensitization to apoptosis displayed by Tax1 and Tax2 may reflect differences relating to the heightened neuropathogenicity associated with HTLV-1 infection and the development of HAM/TSP.

Human T-Cell Lymphotropic Virus Type 1 Infection of CD34+ Hematopoietic Progenitor Cells Induces Cell Cycle Arrest by Modulation of p21cip1/waf1 and Survivin

Human T‐cell lymphotropic virus type 1 (HTLV‐1) is an oncogenic retrovirus and the etiologic agent of adult T‐cell leukemia (ATL), an aggressive CD4+ malignancy. HTLV‐2 is highly homologous to HTLV‐1; however, infection with HTLV‐2 has not been associated with lymphoproliferative diseases. Although HTLV‐1 infection of CD4+ lymphocytes induces cellular replication and transformation, infection of CD34+ human hematopoietic progenitor cells (HPCs) strikingly results in G0/G1 cell cycle arrest and suppression of in vitro clonogenic colony formation by induction of expression of the cdk inhibitor p21cip1/waf1 (p21) and concurrent repression of survivin. Immature CD34+/CD38− hematopoietic stem cells (HSCs) were more susceptible to alterations of p21 and survivin expression as a result of HTLV‐1 infection, in contrast to more mature CD34+/CD38+ HPCs. Knockdown of p21 expression in HTLV‐1‐infected CD34+ HPCs partially abrogated cell cycle arrest. Notably, HTLV‐2, an HTLV strain that is not associated with leukemogenesis, does not significantly modulate p21 and survivin expression and does not suppress hematopoiesis from CD34+ HPCs in vitro. We speculate that the remarkable differences in the activities displayed by CD34+ HPCs following infection with HTLV‐1 or HTLV‐2 suggest that HTLV‐1 uniquely exploits cell cycle arrest mechanisms to establish a latent infection in hematopoietic progenitor/hematopoietic stem cells and initiates preleukemic events in these cells, which eventually results in the manifestation of ATL.

Humanized NOG mice as a model for tuberculosis vaccine‐induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4+ and CD8+ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG‐C, a liposome‐based formulation containing the M. tuberculosis antigen ESAT‐6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T‐cell response. Humanized mice provide a crucial pre‐clinical platform for evaluating human T‐cell immune responses in vaccine development against M. tuberculosis.

Abstract 405: HTLV-1 infection of humanized SCID mice recapitulates adult T-cell leukemia/lymphoma (ATLL) development

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Human T-lymphotropic virus type-1 (HTLV-1) is the etiologic agent of Adult T-cell leukemia/lymphoma (ATLL), an aggressive CD4+/CD25+ T cell malignancy. The early molecular events induced by HTLV-1 infection as well as the role of various viral genes in the induction of leukemia remain unclear, predominantly due to the lack of an animal model that recapitulates ATLL development. We have previously demonstrated that HTLV-1 is capable of infecting human hematopoietic progenitor and stem cells (CD34+ HP/HSCs) and that infection of CD34+ HPCs has dramatically different biological effects in comparison to infection of mature T lymphocytes. To determine if HTLV-1 infection of CD34+ HP/HSCs recapitulates leukemogenesis in vivo, human hematopoiesis was reconstituted in NOD/SCID mice by injection of human CD34+ HPCs infected ex vivo with HTLV-1. Humanized NOD/SCID (HU-SCID) mice infected with HTLV-1 (HTLV-1-HU-SCID) consistently developed CD4+CD25+ T cell lymphomas with clinical characteristics associated with ATLL at ∼10 weeks post-reconstitution and show significantly elevated levels of HTLV-1 infected human CD4+ T cells in the thymus, mesenteric lymph node, spleen and peripheral blood. Lymphoma cells successfully engrafted in naive NOD/SCID mice when injected into the peritoneal cavity and maintain the expression of viral proteins, gp46env and p19gag. Moreover these infected mice showed hyperproliferation of infected human stem cells (CD34+CD38−) in the bone marrow suggesting that HP/HSCs represents viral reservoir target cells which maintain HTLV-1 infection for extended periods of time in vivo. We speculate that HTLV-1 infection of hematopoietic stem cells establishes a virally-infected “cancer stem cell” which subsequently gives rise ATL in patients. Notably, CD34+ HPCs isolated from HTLV-1 infected patient PBL demonstrate proviral integrations, suggesting that these cells harbor infection in humans. CD34+ HPCs transduced with a lentivirus vector expressing the HTLV-1 Tax gene (Tax1) also results in CD4+/CD25+ T cell leukemia/lymphoma in HU-SCID mice, suggesting that Tax1 expression is sufficient for lymphomagenesis. HTLV-1 infection of humanized NOD/SCID mice represents a novel in vivo model which recapitulates viral lymphomagenesis and provides a compelling system to investigate and characterize molecular events in human stem cells in the initiation and progression to ATLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 405.

Abstract 403: Deletion of HBZ alters lymphomagenesis in HTLV-1 infected humanized SCID mice

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of Adult T-cell Leukemia (ATL), an aggressive, mature CD4+/CD25+ T-cell malignancy. The early molecular events induced by HTLV-1 infection as well as the role of various viral genes in the induction of leukemia remain unclear, predominantly due to the lack of an animal model that recapitulates ATL development. We have previously demonstrated that HTLV-1 is capable of infecting human hematopoietic progenitor and stem cells (CD34+ HPCs). Humanized NOD/SCID (HU-SCID) mice generated by injection with HTLV-1-infected CD34+ HPCs demonstrate persistent infection and reproducibly develop CD4+ T-cell lymphomas (∼80%) with clinical characteristics associated with ATL. The HTLV-1 bZIP protein (HBZ) is consistently expressed in all ATL cells and has been implicated in the maintenance of leukemogenesis. HBZ has previously been demonstrated to modulate Tax activity and to interact with the cellular factors CREB, Jun and p53. Transduction of HBZ into CD34+ HP/HSCs using a lentivirus vector suppresses clonogenic colony formation in vitro. Moreover, infection of CD34+ HP/HSCs with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ) results in elevated clonogenic colony formation in vitro, suggesting that HBZ suppresses hematopoiesis. To establish the role of HBZ in HTLV-1 replication and leukemogenesis in vivo, HU-SCID mice were infected with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ). HTLV-1ΔHBZ-infected HU-SCID mice developed lymphoproliferations that were phenotypically characterized as a mixture of immature lymphoid progenitors (CD90+) and mature B-cells (CD19+) starting at ∼10 weeks post-reconstitution, in contrast to wild-type HTLV-1 infection. Lymphoproliferations induced by HTLV-1ΔHBZ successfully engraft and expand in naive NOD/SCID mice when injected into the peritoneal cavity and leukemic cells and lymphoma cells express HTLV-1 gp46env and p19gag. FACS-purified CD90+CD19− HTLV-1ΔHBZ infected cells also successfully engraft in naive NOD/SCID mice and mature into CD19+ cells suggesting that these cells may be leukemic progenitors with the capability of maturing in vivo. In addition, a majority of HTLV-1ΔHBZ-infected HU-SCID mice also develop a progressive, flaccid hind limb paralysis with features similar to the neurological disorder, HAM/TSP. These results suggest that HBZ is dispensable for HTLV-1 replication and modulates lymphomagenesis in the HU-SCID model. The humanized SCID mouse provides a novel in vivo platform for analysis of the role of HTLV-1 genes in viral replication and pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 403.