Nazli Azimi

Requirement for IRF-1 in the microenvironment supporting development of natural killer cells

Natural killer (NK) cells are critical for both innate and adaptive immunity. The development of NK cells requires interactions between their progenitors and the bone-marrow microenvironment; however, little is known about the molecular nature of such interactions. Mice that do not express the transcription factor interferon-regulatory factor-1 (IRF-1; such mice are IRF-1−/− mice) have been shown to exhibit a severe NK-cell deficiency,. Here we demonstrate that the lack of IRF-1 affects the radiation-resistant cells that constitute the microenvironment required for NK-cell development, but not the NK-cell progenitors themselves. We also show that IRF-1−/− bone-marrow cells can generate functional NK cells whencultured with the cytokine interleukin-15 (refs 9-12) and that the interleukin-15 gene is transcriptionally regulated by IRF-1. These results reveal, for the first time, a molecular mechanism by which the bone-marrow microenvironment supports NK-cell development.

Onset of natural killer cell lymphomas in transgenic mice carrying a truncated HMGI-C gene by the chronic stimulation of the IL-2 and IL-15 pathway

Rearrangements of the high mobility group protein I-C (HMGI-C) gene, consisting in the loss of the carboxyl-terminal tail, have been frequently detected in benign human tumors of mesenchymal origin. We have previously demonstrated that transgenic (TG) mice carrying a truncated HMGI-C construct (HMGI-C/T) exhibit a giant phenotype together with a predominantly abdominal/pelvic lipomatosis. Here, we report that HMGI-C/T TG mice develop natural killer (NK)-T/NK cell lymphomas starting from 12 months of age. We found an increased expression of IL-2 and IL-15 proteins and their receptors in these lymphomas, and we demonstrate that HMGI-C/T protein positively regulates their expression in vitro. Therefore, the HMGI-C/T-mediated chronic stimulation of the IL-2/IL-15 pathway could be responsible for the onset of NK-T/NK cell lymphomas in HMGI-C/T TG mice.

Human T Cell Lymphotropic Virus Type I Tax Activates IL-15Rα Gene Expression Through an NF-κB Site

IL-15 mRNA levels are increased in diseases caused by human T cell lymphotropic virus type I (HTLV-I). In this study, we demonstrated that IL-15Rα, the IL-15-specific binding receptor, mRNA and protein levels were also elevated in HTLV-I-infected cells. We showed that transient HTLV-I Tax expression lead to increased IL-15Rα mRNA levels. In addition, by using a reporter construct that bears the human IL-15Rα promoter, we demonstrated that Tax expression increased promoter activity by at least 4-fold. Furthermore, using promoter deletion constructs and gel shift analysis, we defined a functional NF-κB-binding motif in the human IL-15Rα promoter, suggesting that Tax activation of IL-15Rα is due, in part, to the induction of NF-κB. These data indicate that IL-15Rα is transcriptionally regulated by the HTLV-I Tax protein through the action of NF-κB. These findings suggest a role for IL-15Rα in aberrant T cell proliferation observed in HTLV-I-associated diseases.

Overexpression of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its association with cyclin A/cdk2.

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5′-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G1, S, or G2/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G1 phase prior to gamma-irradiation, arrested in G1 and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G1/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.

IL-15 plays a major role in the persistence of Tax-specific CD8 cells in HAM/TSP patients.

IL-15 is a critical cytokine for the maintenance of memory-phenotype CD8 cells in mice. Here, we investigated the role of IL-15 in the neurological disease termed human T cell lymphotropic virus I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The high number of viral-specific CD8 cells in these patients is associated with inflammatory responses in the central nervous system. Because IL-15 is overexpressed in these patients, we asked whether IL-15 contributes to the persistence of human T cell lymphotropic virus I viral-specific CD8 cells. Using ex vivo cultures of HAM/TSP peripheral blood mononuclear cells, we demonstrated that in the majority of patients examined here blocking IL-15 action resulted in a decrease in the number of viral-specific CD8 cells. This decrease was caused by both inhibition of proliferation and induction of apoptosis in these cells. The data indicate that IL-15 plays a major role in the maintenance of viral-specific CD8 cells in HAM/TSP.

Regulation of IFN Regulatory Factor 4 Expression in Human T Cell Leukemia Virus-I-Transformed T Cells

IFN regulatory factor (IRF)-4 is a lymphoid/myeloid-restricted member of the IRF transcription factor family that plays an essential role in the homeostasis and function of mature lymphocytes. IRF-4 expression is tightly regulated in resting primary T cells and is transiently induced at the mRNA and protein levels after activation by Ag-mimetic stimuli such as TCR cross-linking or treatment with phorbol ester and calcium ionophore (PMA/ionomycin). However, IRF-4 is constitutively upregulated in human T cell leukemia virus type I (HTLV-I) infected T cells as a direct gene target for the HTLV-I Tax oncoprotein. In this study we demonstrate that chronic IRF-4 expression in HTLV-I-infected T lymphocytes is associated with a leukemic phenotype, and we examine the mechanisms by which continuous production of IRF-4 is achieved in HTLV-I-transformed T cells. IRF-4 expression in HTLV-1-infected cells is driven through activation of the NF-κB and NF-AT pathways, resulting in the binding of p50, p65, and c-Rel to the κB1 element and p50, c-Rel, and NF-ATp to the CD28RE element within the −617 to −209 region of the IRF-4 promoter. Furthermore, mutation of either the κB1 or CD28RE sites blocks Tax-mediated transactivation of the human IRF-4 promoter in T cells. These experiments constitute the first detailed analysis of human IRF-4 transcriptional regulation within the context of HTLV-I infection and transformation of CD4+ T lymphocytes.

Repression of IRF-4 target genes in human T cell leukemia virus-1 infection.

The human T cell leukemia/lymphotropic virus-1 (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4+ T lymphocytes. Interferon regulatory factor-4 (IRF-4) was shown previously to be constitutively expressed in T cells infected with HTLV-1. In this study, we investigated the role of IRF-4 gene regulation in the context of HTLV-1 infection using gene array technology and IRF-4 expressing T cells. Many potential IRF-4 regulated genes were identified, the vast majority of which were repressed by IRF-4 expression. Cyclin B1, a G2-M checkpoint protein identified as an IRF-4 repressed gene in the array, was further characterized in the context of HTLV-1 infection. All HTLV-1 infected cell lines and ATL patient lymphocytes demonstrated a dramatic decrease in cyclin B1 levels; subsequent analysis of the cyclin B1 promoter identified two sites important in IRF-4 binding and repression of cyclin B1 expression. Furthermore, IRF-4-mediated repression of cyclin B1 led to a significant decrease in CDC2 kinase activity in HTLV-1 infected T cells. IRF-4 expression in HTLV-1 infected T cells also downregulated other genes implicated in the mitotic checkpoint as well as genes involved in actin cytoskeletal rearrangement, DNA repair, apoptosis, metastasis and immune recognition. Several of the identified genes are dysregulated in ATL and may provide important mechanistic information concerning pathways critical to the emergence of ATL.

IFN Regulatory factor 4 participates in the human T cell lymphotropic virus type I-mediated activation of the IL-15 receptor α promoter

IL-15Rα mRNA and protein levels are increased in human T cell lymphotropic virus type-I (HTLV-I)-associated adult T cell leukemia. Previously, we demonstrated that IL-15Rα expression was activated by HTLV-I Tax, in part, through the action of NF-κB. However, there appeared to be additional motifs within the IL-15Rα promoter that were responsive to HTLV-I Tax. In this study, we demonstrated that IL-15Rα mRNA expression was activated in human monocytes by IFN treatment, suggesting a role for IFN regulatory factors (IRFs) in IL-15Rα transcription. In addition, an IRF element within the Tax-responsive element of the IL-15Rα promoter was necessary for maximal Tax-induced activation of this promoter. Furthermore, we demonstrated that IRF-4, a transcription factor known to be elevated in HTLV-I-infected cells, activated the IL-15Rα promoter. Inhibition of IRF-4 action lead to reduced Tax-induced activation of the IL-15Rα promoter, while inhibition of both IRF-4 and NF-κB severely inhibited the Tax-induced activation of this promoter. These findings suggest a role for both NF-κB and IRF-4 in the transcriptional regulation of IL-15Rα by HTLV-I Tax. It is possible that the HTLV-I Tax-mediated induction of IL-15Rα and IL-15 may lead to an autocrine cytokine-mediated stimulatory loop leading to the proliferation of HTLV-I infected cells. This loop of proliferation may facilitate viral propagation and play a role in HTLV-I-mediated disease progression.

How Does Interleukin 15 Contribute to the Pathogenesis of HTLV Type 1-Associated Myelopathy/Tropical Spastic Paraparesis?

HTLV-1 is the etiological agent of a neurological disease named HAM/TSP that has clinical characteristics similar to those of multiple sclerosis (MS). The PBMC obtained from HAM/TSP patients undergo spontaneous proliferation in the absence of addition of any exogenous cytokines in an ex vivo culture. This spontaneous proliferation has been thought to be due to the proliferation of T cells. It was demonstrated that this proliferation is, in part, due to the production of IL-2 and its receptor (IL-2Ralpha) by HTLV-1-infected T cells. In this review, we demonstrate that IL-15 production also contributes to the spontaneous proliferation of T cells obtained from HAM/TSP PBMC. We provide data indicating that IL-15 expression is elevated in HAM/TSP PBMC when compared to that of the normal donors. Furthermore, we demonstrate that IL-15 overexpression by HTLV-1 is due to Tax trans-activation of its promoter and induction of NF-kappaB transcription factors. On the basis of these studies, we propose a model in which HTLV-1 infection of T cells results in the production of both IL-2 and IL-15 cytokines, growth factors that support the proliferation of T cells.

Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical spastic paraparesis

Significance IL-2 and IL-15, members of the gamma chain family of cytokines, are prominently deregulated in human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and underlie many of the characteristic immune abnormalities such as spontaneous lymphocyte proliferation, increased STAT5 phosphorylation in the lymphocytes, and increased frequency and cytotoxicity of virus-specific CD8+ T lymphocytes (CTLs). In this in vitro study, we demonstrate that selective and simultaneous blockade of IL-2 and IL-15, with a γ-chain antagonistic peptide, reduces spontaneous lymphocyte proliferation (SP), STAT5 phosphorylation, and more important, the degranulation of CD8+ T cells and the frequency of HTLV-1-specific CTLs. Thus, selective cytokine blockade with antagonistic peptides might be a therapeutic approach in HAM/TSP and is potentially applicable to multiple other conditions in which cytokines are pathogenic. Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive inflammatory myelopathy occurring in a subset of HTLV-1-infected individuals. Despite advances in understanding its immunopathogenesis, an effective treatment remains to be found. IL-2 and IL-15, members of the gamma chain (γc) family of cytokines, are prominently deregulated in HAM/TSP and underlie many of the characteristic immune abnormalities, such as spontaneous lymphocyte proliferation (SP), increased STAT5 phosphorylation in the lymphocytes, and increased frequency and cytotoxicity of virus-specific cytotoxic CD8+ T lymphocytes (CTLs). In this study, we describe a novel immunomodulatory strategy consisting of selective blockade of certain γc family cytokines, including IL-2 and IL-15, with a γc antagonistic peptide. In vitro, a PEGylated form of the peptide, named BNZ132-1-40, reduced multiple immune activation markers such as SP, STAT5 phosphorylation, spontaneous degranulation of CD8+ T cells, and the frequency of transactivator protein (Tax)-specific CD8+ CTLs, thought to be major players in the immunopathogenesis of the disease. This strategy is thus a promising therapeutic approach to HAM/TSP with the potential of being more effective than single monoclonal antibodies targeting either IL-2 or IL-15 receptors and safer than inhibitors of downstream signaling molecules such as JAK1 inhibitors. Finally, selective cytokine blockade with antagonistic peptides might be applicable to multiple other conditions in which cytokines are pathogenic.