Humanized Mouse Models for Immuno-oncology Drug Discovery.

Breakthroughs in cancer treatment with immunotherapeutics have provided long-term patient benefits for many different types of cancer. However, complete response is not achieved in many patients and tumor types, and the mechanisms underlying this lack of response are poorly understood. Despite this, numerous new targets, therapeutics, and drug combinations are being developed and tested in clinical trials. Preclinical models that recapitulate the complex human tumor microenvironment and the interplay between tumor and immune cells within the cancer-immunity cycle are needed to improve our understanding and screen new therapeutics for efficacy and safety/toxicity. Humanized mice, encompassing human tumors and human immune cells engrafted on immunodeficient mice, have been widely used for many years in immuno-oncology, with developments to improve both the humanization and the translational value central to the next generation of models. In this overview, we discuss recent advances in humanized models relevant to immuno-oncology drug discovery, the advantages and limitations of such models, the application of humanized models for efficacy and safety assessments of immunotherapeutics, and the potential opportunities. © 2023 Crown Bioscience. Current Protocols published by Wiley Periodicals LLC.

Co-clinical Modeling of the Activity of the BET Inhibitor Mivebresib (ABBV-075) in AML.

BACKGROUND/AIM: The therapeutic potential of bromodomain and extra-terminal motif (BET) inhibitors in hematological cancers has been well established in preclinical and early-stage clinical trials, although as of yet, no BETtargeting agent has achieved approval. To add insight into potential response to mivebresib (ABBV-075), a broadspectrum BET inhibitor, co-clinical modeling of individual patient biopsies was conducted in the context of a Phase I trial in acute myeloid leukemia (AML). MATERIALS AND METHODS: Co-clinical modeling involves taking the patient's biopsy and implanting it in mice with limited passage so that it closely retains the original characteristics of the malignancy and allows comparisons of response between animal model and clinical data. Procedures were developed, initially with neonate NOD/Shi-scid-IL2rγnull (NOG) mice and then optimized with juvenile NOG-EXL as host mice, eventually resulting in a robust rate of engraftment (16 out of 26, 62%). RESULTS: Results from the co-clinical AML patient-derived xenograft (PDX) modeling (6 with >60% inhibition of bone marrow blasts) were consistent with the equivalent clinical data from patients receiving mivebresib in monotherapy, and in combination with venetoclax. The modeling system also demonstrated the activity of a novel BD2-selective BET inhibitor (ABBV-744) in the preclinical AML setting. Both agents were also highly effective in inhibiting blast counts in the spleen (10/10 and 5/6 models, respectively). CONCLUSION: These findings confirm the validity of the model system in the co-clinical setting, establish highly relevant in vivo models for the discovery of cancer therapy, and indicate the therapeutic value of BET inhibitors for AML and, potentially, myelofibrosis treatment.

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.

Experimental animal modeling for immuno-oncology.

Immuno-oncology (I/O) research has intensified significantly in recent years due to the breakthrough development and the regulatory approval of several immune checkpoint inhibitors, leading to the rapid expansion of the new discovery of novel I/O therapies, new checkpoint inhibitors and beyond. However, many I/O questions remain unanswered, including why only certain subsets of patients respond to these treatments, who the responders would be, and how to expand patient response (the conversion of non-responders or maximizing response in partial responders). All of these require relevant I/O experimental systems, particularly relevant preclinical animal models. Compared to other oncology drug discovery, e.g. cytotoxic and targeted drugs, a lack of relevant animal models is a major obstacle in I/O drug discovery, and an urgent and unmet need. Despite the obvious importance, and the fact that much I/O research has been performed using many different animal models, there are few comprehensive and introductory reviews on this topic. This article attempts to review the efforts in development of a variety of such models, as well as their applications and limitations for readers new to the field, particularly those in the pharmaceutical industry.

C7a, a biphosphinic cyclopalladated compound, efficiently controls the development of a patient-derived xenograft model of adult T cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a highly aggressive disease that occurs in individuals infected with the human T lymphotropic virus type 1 (HTLV-1). Patients with aggressive ATLL have a poor prognosis because the leukemic cells are resistant to conventional chemotherapy. We have investigated the therapeutic efficacy of a biphosphinic cyclopalladated complex {Pd(2) [S(-)C(2), N-dmpa](2) (µ-dppe)Cl(2)}, termed C7a, in a patient-derived xenograft model of ATLL, and investigated the mechanism of C7a action in HTLV-1-positive and negative transformed T cell lines in vitro. In vivo survival studies in immunocompromised mice inoculated with human RV-ATL cells and intraperitoneally treated with C7a led to significantly increased survival of the treated mice. We investigated the mechanism of C7a activity in vitro and found that it induced mitochondrial release of cytochrome c, caspase activation, nuclear condensation and DNA degradation. These results suggest that C7a triggers apoptotic cell death in both HTLV-1 infected and uninfected human transformed T-cell lines. Significantly, C7a was not cytotoxic to peripheral blood mononuclear cells (PBMC) from healthy donors and HTLV-1-infected individuals. C7a inhibited more than 60% of the ex vivo spontaneous proliferation of PBMC from HTLV-1-infected individuals. These results support a potential therapeutic role for C7a in both ATLL and HTLV-1-negative T-cell lymphomas.

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.

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.

Human T-cell lymphotropic virus type 1 infection of CD34+ hematopoietic progenitor cells induces cell cycle arrest by modulation of p21(cip1/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 G(0)/G(1) cell cycle arrest and suppression of in vitro clonogenic colony formation by induction of expression of the cdk inhibitor p21(cip1/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.

Human T-cell leukemia virus type 1 (HTLV-1) p12I down-modulates ICAM-1 and -2 and reduces adherence of natural killer cells, thereby protecting HTLV-1-infected primary CD4+ T cells from autologous natural killer cell-mediated cytotoxicity despite the reduction of major histocompatibility complex class I molecules on infected cells.

Although natural killer (NK) cell-mediated control of viral infections is well documented, very little is known about the ability of NK cells to restrain human T-cell leukemia virus type 1 (HTLV-1) infection. In the current study we show that NK cells are unable to kill HTLV-1-infected primary CD4+ T cells. Exposure of NK cells to interleukin-2 (IL-2) resulted in only a marginal increase in their ability to kill HTLV-1-infected primary CD4+ T cells. This inability of NK cells to kill HTLV-1-infected CD4+ T cells occurred despite the down-modulation of major histocompatibility complex (MHC) class I molecules, one of the ligands for the major NK cell inhibitory receptor, by HTLV-1 p12(I) on CD4+ T cells. One reason for this diminished ability of NK cells to kill HTLV-1-infected cells was the decreased ability of NK cells to adhere to HTLV-1-infected cells because of HTLV-1 p12(I)-mediated down-modulation of intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. We also found that HTLV-1-infected CD4+ T cells did not express ligands for NK cell activating receptors, NCR and NKG2D, although they did express ligands for NK cell coactivating receptors, NTB-A and 2B4. Thus, despite HTLV-1-mediated down-modulation of MHC-I molecules, HTLV-1-infected primary CD4+ T cells avoids NK cell destruction by modulating ICAM expression and shunning the expression of ligands for activating receptors.

Proinflammatory cytokine gene induction by human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 Tax in primary human glial cells.

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-1beta (IL-1beta), IL-1alpha, tumor necrosis factor alpha (TNF-alpha), TNF-beta, 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 (Tax(221)) 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.

KSHV/HHV-8 infection of human hematopoietic progenitor (CD34+) cells: persistence of infection during hematopoiesis in vitro and in vivo.

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV, purified HPCs were exposed to KSHV, and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells, and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection, persistence, and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo.

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.

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 G(0)/G(1) cell cycle arrest and resulted in the concomitant suppression of multilineage hematopoiesis in vitro. Tax1 induced transcriptional upregulation of the cdk inhibitors p21(cip1/waf1) (p21) and p27(kip1) (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 (Tax(221)) 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.

Comparative biology of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2.

HTLV-1 and HTLV-2 are highly related complex retroviruses that have been studied intensely for nearly three decades because of their association with neoplasia, neuropathology, and/or their capacity to transform primary human T lymphocytes. The study of HTLV also represents an attractive model that has allowed investigators to dissect the mechanism of various cellular processes, several of which may be critical steps in HTLV-mediated pathogenesis. Both HTLV-1 and HTLV-2 can efficiently immortalize and transform T lymphocytes in cell culture and persist in infected individuals or experimental animals. However, the clinical manifestations of these two viruses differ significantly. HTLV-1 is associated with adult T-cell leukemia (ATL) and a variety of immune-mediated disorders including the chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). In contrast, HTLV-2 is much less pathogenic with reports of only a few cases of variant hairy cell leukemia and neurological disease associated with infection. The limited number of individuals shown to harbor HTLV-2 in association with specific diseases has, to date, precluded convincing epidemiological demonstration of a definitive etiologic role of HTLV-2 in human disease. Therefore, it has become clear that comparative studies designed to elucidate the mechanisms by which HTLV-1 and HTLV-2 determine distinct outcomes are likely to provide fundamental insights into the initiation of multistep leukemogenesis.

Human T lymphotropic virus type 1 accessory protein p12I modulates calcium-mediated cellular gene expression and enhances p300 expression in T lymphocytes.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia/lymphoma (ATLL), an aggressive CD4+ T lymphocyte malignancy. Activation of T lymphocytes is required for effective retroviral integration into the host cell genome and subsequent viral replication, but the molecular mechanisms involved in HTLV-1-mediated T cell activation remain unclear. HTLV-1 encodes various accessory proteins such as p12I, which has been demonstrated to be critical for HTLV-1 infectivity in vivo in rabbits and in vitro in quiescent primary human T lymphocytes. This hydrophobic protein localizes in the endoplasmic reticulum, increases intracellular calcium, and activates nuclear factor of activated T cell-mediated transcription. To further elucidate the role of p12I in regulation of cellular gene expression, we performed gene array analysis on stable p12I-expressing Jurkat T cells, using Affymetrix U133A arrays. Our data indicate that p12I altered the expression of genes associated with a network of interrelated pathways including T cell signaling, cell proliferation, and apoptosis. Expression of several calcium-regulated genes was found to be altered by p12I, consistent with known properties of the viral protein. Gene array findings were confirmed by semiquantitative RT-PCR in Jurkat T cells and primary CD4+ T lymphocytes. Furthermore, dose-dependent expression of p12I in Jurkat T cells resulted in significant increases in p300 and p300-dependent transcription. This is the first report of a viral protein influencing the transcription of p300, a rate-limiting coadapter critical in HTLV-1-mediated T cell activation. Collectively, our data strongly indicate that HTLV-1 p12I modulates cellular gene expression patterns to hasten the activation of T lymphocytes and thereby promote efficient viral infection.

Inhibition of HHV-8/KSHV infected primary effusion lymphomas in NOD/SCID mice by azidothymidine and interferon-alpha.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus type-8 (KSHV/HHV-8) is associated with primary effusion lymphomas (PEL), a rare form of B-cell lymphoma. PEL cell lines infected with HHV-8, but negative for Epstein-Barr virus (EBV), were analyzed for their tumorigenic potential in a small animal model system. Inoculation of PEL cell lines into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice results in efficient engraftment and tumorigenesis in vivo. PEL-engrafted NOD/SCID (PEL/SCID) mice displayed malignant ascites development with notable abdominal distension, consistent with the clinical manifestations of PEL in humans. Azidothymidine (AZT, zidovudine) and interferon-alpha (IFN-alpha) induce apoptosis in HHV-8+/EBV- PEL cells in culture, by induction of a tumor necrosis factor-related apoptosis inducing ligand (TRAIL) mediated suicide program and has been proposed as a therapy for herpesvirus-associated lymphomas. Daily injection of AZT and IFN-alpha significantly increased mean survival time (MST) of PEL/SCID mice suggesting that induction of apoptosis in PEL cells in vivo may be exploited as an effective relatively non-toxic therapy targeting HHV-8 infected PEL. These data demonstrate that the PEL/SCID mouse is an important preclinical model to characterize efficacy and anti-tumor mechanisms of new therapeutic targets in vivo and will be useful in the design and testing of agents in viral lymphoproliferative diseases.

Human T lymphotropic virus type-1 p30II alters cellular gene expression to selectively enhance signaling pathways that activate T lymphocytes.

BACKGROUND: Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in the virus life cycle or HTLV-1 pathogenesis. Proviral clones of the virus with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. Exogenous expression of p30II differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and represses tax/rex RNA nuclear export. RESULTS: Herein, we further characterized the role of p30II in regulation of cellular gene expression, using stable p30II expression system employing lentiviral vectors to test cellular gene expression with Affymetrix U133A arrays, representing approximately 33,000 human genes. Reporter assays in Jurkat T cells and RT-PCR in Jurkat and primary CD4+ T-lymphocytes were used to confirm selected gene expression patterns. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling, apoptosis and cell cycle in p30II expressing Jurkat T cells. In all categories, p30II appeared to be an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes. CONCLUSIONS: We are the first to demonstrate that p30II, while repressing the expression of many genes, selectively activates key gene pathways involved in T-cell signaling/activation. Collectively, our data suggests that this complex retrovirus, associated with lymphoproliferative diseases, relies upon accessory gene products to modify cellular environment to promote clonal expansion of the virus genome and thus maintain proviral loads in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 tax oncoproteins modulate cell cycle progression and apoptosis.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. HTLV-2, although highly related to HTLV-1 at the molecular level, has not been conclusively linked to development of lymphoproliferative disorders. Differences between the biological activities of the respective tax gene products (Tax1 and Tax2) may be one factor which accounts for the differential pathogenicities associated with infection. To develop an in vitro model to investigate and compare the effects of constitutive expression of Tax1 and Tax2, Jurkat T-cell lines were infected with lentivirus vectors encoding Tax1 and Tax2 in conjunction with green fluorescent protein, and stably transduced clonal cell lines were generated by serial dilution in the absence of drug selection. Jurkat cells that constitutively express Tax1 and Tax2 (Tax1/Jurkat and Tax2/Jurkat, respectively) showed notably reduced kinetics of cellular replication, and Tax1 inhibited cellular replication to a higher degree in comparison to Tax2. Tax1 markedly activated transcription from the cdk inhibitor p21(cip1/waf1) promoter in comparison to Tax2, suggesting that upregulation of p21(cip1/waf1) may account for the differential inhibition of cellular replication kinetics displayed by Tax1/Jurkat and Tax2/Jurkat cells. The presence of binucleated and multinucleated cells, reminiscent of large lymphocytes with cleaved or cerebriform nuclei often seen in HTLV-1- and -2-seropositive patients, was noted in cultures expressing Tax1 and Tax2. Although Tax1 and Tax2 expression mediated elevated resistance to apoptosis in Jurkat cells after serum deprivation, Tax1 was unique in protection from apoptosis after exposure to camptothecin and etoposide, inhibitors of topoisomerase I and II, respectively. Characterization of the unique phenotypes displayed by Tax1 and Tax2 in vitro will provide information as to the relative roles of these oncoproteins and their contribution to HTLV-1 and -2 pathogenesis in vivo.

Human T-cell leukemia virus type 1 tax oncoprotein suppression of multilineage hematopoiesis of CD34+ cells in vitro.

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis, bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2, respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors, and transduced cells were cultured in a semisolid medium permissive for the development of erythroid, myeloid, and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro, in contrast to Tax2-transduced cells, which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected, suggesting that Tax1 inhibited the maturation of relatively early, uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis, lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells, an activity that is not displayed by Tax2, and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo, the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2.

Human T-cell lymphotropic virus type 1 p12I enhances interleukin-2 production during T-cell activation.

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL) and a variety of lymphoproliferative disorders. The early virus-cell interactions that determine a productive infection remain unclear. However, it is well recognized that T-cell activation is required for effective retroviral integration into the host cell genome and subsequent viral replication. The HTLV-1 pX open reading frame I encoding protein, p12(I), is critical for the virus to establish persistent infection in vivo and for infection in quiescent primary lymphocytes in vitro. p12(I) localizes in the endoplasmic reticulum (ER) and cis-Golgi apparatus, increases intracellular calcium and activates nuclear factor of activated T cells (NFAT)-mediated transcription. To clarify the function of p12(I), we tested the production of IL-2 from Jurkat T cells and peripheral blood mononuclear cells (PBMC) expressing p12(I). Lentiviral vector expressed p12(I) in Jurkat T cells enhanced interleukin-2 (IL-2) production in a calcium pathway-dependent manner during T-cell receptor (TCR) stimulation. Expression of p12(I) also induced higher NFAT-mediated reporter gene activities during TCR stimulation in Jurkat T cells. In contrast, p12 expression in PBMC elicited increased IL-2 production in the presence of phorbal ester stimulation, but not during TCR stimulation. Finally, the requirement of ER localization for p12(I)-mediated NFAT activation was demonstrated and two positive regions and two negative regions in p12(I) were identified for the activation of this transcription factor by using p12(I) truncation mutants. These results are the first to indicate that HTLV-1, an etiologic agent associated with lymphoproliferative diseases, uses a conserved accessory protein to induce T-cell activation, an antecedent to efficient viral infection.