Complete Rescue of HTLV-1p12KO Infectivity by Depletion of Monocytes Together with NK and CD8+ T Cells.

The transient depletion of monocytes alone prior to exposure of macaques to HTLV-1 enhances both HTLV-1WT (wild type) and HTLV-1p12KO (Orf-1 knockout) infectivity, but seroconversion to either virus is not sustained over time, suggesting a progressive decrease in virus expression. These results raise the hypotheses that either HTLV-1 persistence depends on a monocyte reservoir or monocyte depletion provides a transient immune evasion benefit. To test these hypotheses, we simultaneously depleted NK cells, CD8+ T cells, and monocytes (triple depletion) prior to exposure to HTLV-1WT or HTLV-1p12KO. Remarkably, triple depletion resulted in exacerbation of infection by both viruses and complete rescue of HTLV-1p12KO infectivity. Following triple depletion, we observed rapid and sustained seroconversion, high titers of antibodies against HTLV-1 p24Gag, and frequent detection of viral DNA in the blood and tissues of all animals when compared with depletion of only CD8+ and NK cells, or monocytes alone. The infection of macaques with HTLV-1WT or HTLV-1p12KO was associated with higher plasma levels of IL-10 after 21 weeks, while IL-6, IFN-γ, IL-18, and IL-1ß were only elevated in animals infected with HTLV-1WT. The repeat depletion of monocytes, NK, and CD8+ cells seven months following the first exposure to HTLV-1 did not further exacerbate viral replication. These results underscore the contribution of monocytes in orchestrating anti-viral immunity. Indeed, the absence of orf-1 expression was fully compensated by the simultaneous depletion of CD8+ T cells, NK cells, and monocytes, underlining the primary role of orf-1 in hijacking host immunity.

In Vivo Treatment with Insulin-like Growth Factor 1 Reduces CCR5 Expression on Vaccine-Induced Activated CD4+ T-Cells.

At the heart of the DNA/ALVAC/gp120/alum vaccine's efficacy in the absence of neutralizing antibodies is a delicate balance of pro- and anti-inflammatory immune responses that effectively decreases the risk of SIVmac251 acquisition in macaques. Vaccine efficacy is linked to antibodies recognizing the V2 helical conformation, DC-10 tolerogenic dendritic cells eliciting the clearance of apoptotic cells via efferocytosis, and CCR5 downregulation on vaccine-induced gut homing CD4+ cells. RAS activation is also linked to vaccine efficacy, which prompted the testing of IGF-1, a potent inducer of RAS activation with vaccination. We found that IGF-1 changed the hierarchy of V1/V2 epitope recognition and decreased both ADCC specific for helical V2 and efferocytosis. Remarkably, IGF-1 also reduced the expression of CCR5 on vaccine-induced CD4+ gut-homing T-cells, compensating for its negative effect on ADCC and efferocytosis and resulting in equivalent vaccine efficacy (71% with IGF-1 and 69% without).

Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques.

The human immunodeficiency virus epidemic continues in sub-Saharan Africa, and particularly affects adolescent girls and women who have limited access to antiretroviral therapy. Here we report that the risk of vaginal simian immunodeficiency virus (SIV)mac251 acquisition is reduced by more than 90% using a combination of a vaccine comprising V1-deleted (V2 enhanced) SIV envelope immunogens with topical treatment of the zinc-finger inhibitor SAMT-247. Following 14 weekly intravaginal exposures to the highly pathogenic SIVmac251, 80% of a cohort of 20 macaques vaccinated and treated with SAMT-247 remained uninfected. In an arm of 18 vaccinated-only animals without microbicide, 40% of macaques remained uninfected. The combined SAMT-247/vaccine regimen was significantly more effective than vaccination alone. By analysing immune correlates of protection, we show that, by increasing zinc availability, SAMT-247 increases natural killer cytotoxicity and monocyte efferocytosis, and decreases T-cell activation to augment vaccine-induced protection.

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC.

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

Transient Viral Activation in Human T Cell Leukemia Virus Type 1-Infected Macaques Treated With Pomalidomide.

Human T cell leukemia virus type 1 (HTLV-1) persists in the host despite a vigorous immune response that includes cytotoxic T cells (CTL) and natural killer (NK) cells, suggesting the virus has developed effective mechanisms to counteract host immune surveillance. We recently showed that in vitro treatment of HTLV-1-infected cells with the drug pomalidomide (Pom) increases surface expression of MHC-I, ICAM-1, and B7-2, and significantly increases the susceptibility of HTLV-1-infected cells to NK and CTL killing, which is dependent on viral orf-I expression. We reasoned that by restoring cell surface expression of these molecules, Pom treatment has the potential to reduce virus burden by rendering infected cells susceptible to NK and CTL killing. We used the rhesus macaque model to determine if Pom treatment of infected individuals activates the host immune system and allows recognition and clearance of HTLV-1-infected cells. We administered Pom (0.2 mg/kg) orally to four HTLV-1-infected macaques over a 24 day period and collected blood, urine, and bone marrow samples throughout the study. Pom treatment caused immune activation in all four animals and a marked increase in proliferating CD4+, CD8+, and NK cells as measured by Ki-67+ cells. Activation markers HLA-DR, CD11b, and CD69 also increased during treatment. While we detected an increased frequency of cells with a memory CD8+ phenotype, we also found an increased frequency of cells with a Treg-like phenotype. Concomitant with immune activation, the frequency of detection of viral DNA and the HTLV-1-specific humoral response increased as well. In 3 of 4 animals, Pom treatment resulted in increased antibodies to HTLV-1 antigens as measured by western blot and p24Gag ELISA. Consistent with Pom inducing immune and HTLV-1 activation, we measured elevated leukotrienes LTB4 and LTE4 in the urine of all animals. Despite an increase in plasma LTB4, no significant changes in plasma cytokine/chemokine levels were detected. In all cases, however, cellular populations, LTB4, and LTE4 decreased to baseline or lower levels 2 weeks after cessation of treatment. These results indicated that Pom treatment induces a transient HTLV-1-specific immune activation in infected individuals, but also suggest Pom may not be effective as a single-agent therapeutic.

NK cells and monocytes modulate primary HTLV-1 infection.

We investigated the impact of monocytes, NK cells, and CD8+ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1WT) or to the HTLV-1p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8+ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8+ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1WT. In contrast, HTLV-1p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1WT and HTLV-1p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 "don't-eat-me" signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.

Functional properties and sequence variation of HTLV-1 p13.

Human T cell leukemia virus type-1 (HTLV-1) was the first retrovirus found to cause cancer in humans, but the mechanisms that drive the development of leukemia and other diseases associated with HTLV-1 infection remain to be fully understood. This review describes the functional properties of p13, an 87-amino acid protein coded by HTLV-1 open reading frame II (orf-II). p13 is mainly localized in the inner membrane of the mitochondria, where it induces potassium (K+) influx and reactive oxygen species (ROS) production, which can trigger either proliferation or apoptosis, depending on the ROS setpoint of the cell. Recent evidence indicates that p13 may influence the cell's innate immune response to viral infection and the infected cell phenotype. Association of the HTLV-1 transcriptional activator, Tax, with p13 increases p13's stability, leads to its partial co-localization with Tax in nuclear speckles, and reduces the ability of Tax to interact with the transcription cofactor CBP/p300. Comparison of p13 sequences isolated from HTLV-1-infected individuals revealed a small number of amino acid variations in the domains controlling the subcellular localization of the protein. Disruptive mutations of p13 were found in samples obtained from asymptomatic patients with low proviral load. p13 sequences of HTLV-1 subtype C isolates from indigenous Australian patients showed a high degree of identity among each other, with all samples containing a pattern of 5 amino acids that distinguished them from other subtypes. Further characterization of p13's functional properties and sequence variants may lead to a deeper understanding of the impact of p13 as a contributor to the clinical manifestations of HTLV-1 infection.

p30 protein: a critical regulator of HTLV-1 viral latency and host immunity.

The extraordinarily high prevalence of HTLV-1 subtype C (HTLV-1C) in some isolated indigenous communities in Oceania and the severity of the health conditions associated with the virus impress the great need for basic and translational research to prevent and treat HTLV-1 infection. The genome of the virus's most common subtype, HTLV-1A, encodes structural, enzymatic, and regulatory proteins that contribute to viral persistence and pathogenesis. Among these is the p30 protein encoded by the doubly spliced Tax-orf II mRNA, a nuclear/nucleolar protein with both transcriptional and post-transcriptional activity. The p30 protein inhibits the productive replication cycle via nuclear retention of the mRNA that encodes for both the viral transcriptional trans-activator Tax, and the Rex proteins that regulate the transport of incompletely spliced viral mRNA to the cytoplasm. In myeloid cells, p30 inhibits the PU-1 transcription factor that regulates interferon expression and is a critical mediator of innate and adaptive immunity. Furthermore, p30 alters gene expression, cell cycle progression, and DNA damage responses in T-cells, raising the hypothesis that p30 may directly contribute to T cell transformation. By fine-tuning viral expression while also inhibiting host innate responses, p30 is likely essential for viral infection and persistence. This concept is supported by the finding that macaques, a natural host for the closely genetically related simian T-cell leukemia virus 1 (STLV-1), exposed to an HTLV-1 knockout for p30 expression by a single point mutation do not became infected unless reversion and selection of the wild type HTLV-1 genotype occurs. All together, these data suggest that inhibition of p30 may help to curb and eventually eradicate viral infection by exposing infected cells to an effective host immune response.

Role of HTLV-1 orf-I encoded proteins in viral transmission and persistence.

The human T cell leukemia virus type 1 (HTVL-1), first reported in 1980 by Robert Gallo's group, is the etiologic agent of both cancer and inflammatory diseases. Despite approximately 40 years of investigation, the prognosis for afflicted patients remains poor with no effective treatments. The virus persists in the infected host by evading the host immune response and inducing proliferation of infected CD4+ T-cells. Here, we will review the role that viral orf-I protein products play in altering intracellular signaling, protein expression and cell-cell communication in order to escape immune recognition and promote T-cell proliferation. We will also review studies of orf-I mutations found in infected patients and their potential impact on viral load, transmission and persistence. Finally, we will compare the orf-I gene in HTLV-1 subtypes as well as related STLV-1.

JAG1 overexpression contributes to Notch1 signaling and the migration of HTLV-1-transformed ATL cells.

BACKGROUND: HTLV-1 is a retrovirus that infects over 20 million people worldwide and is responsible for the hematopoietic malignancy adult T cell leukemia (ATL). We previously demonstrated that Notch is constitutively activated in ATL cells. Activating genetic mutations were found in Notch; however, Notch signaling was also activated in the absence of genetic mutations suggesting the existence of other mechanisms. METHODS: We analyzed the expression of Notch receptor ligands in HTLV-I-transformed cells, ATL patient-derived cell lines, and fresh uncultured ATL samples by RT-PCR, FACS, and immunohistochemistry. We then investigated viral and cellular molecular mechanisms regulating expression of JAG1. Finally, using shRNA knock-down and neutralizing antibodies, we investigated the function of JAG1 in ATL cells. RESULTS: Here, we report the overexpression of the Notch ligand, JAG1, in freshly uncultured ATL patient samples compared to normal PBMCs. We found that in ATL cells, JAG1 overexpression relies upon the viral protein Tax and cellular miR-124a, STAT3, and NFATc1. Interestingly, our data show that blockade of JAG1 signaling dampens Notch1 downstream signaling and limits cell migration of transformed ATL cells. CONCLUSIONS: Our results suggest that targeting JAG1 can block Notch1 activation in HTLV-I-transformed cells and represents a new target for immunotherapy in ATL patients.

MicroRNAs-based Therapy: A Novel and Promising Strategy for Cancer Treatment.

MicroRNAs are short non-coding RNAs involved in critical biological processes. In the past decade, the deregulation of miRNAs has been well-documented in a wide range of human diseases, including cancer. Overexpression and downregulation of miRNAs affect cellular pathways that contribute to carcinogenesis and tumor progression. This evidence makes miRNAs a suitable candidate for therapeutic applications and leads to developing strategies to manipulate their expression. Consistently, in vitro and in vivo studies show that Let-7, miR-10b, miR-21, miR-34, miR-155 and miR-221 are promising targets to develop miRNAs-based therapy for human malignancies. The purpose of this review is to discuss the different approaches that can be used to restore or reduce miRNAs expression in human cancer and the therapeutic implications.

Clinical significance of microRNAs in chronic and acute human leukemia.

Small non-coding microRNAs (miRNAs) are epigenetic regulators that target specific cellular mRNA to modulate gene expression patterns and cellular signaling pathways. miRNAs are involved in a wide range of biological processes and are frequently deregulated in human cancers. Numerous miRNAs promote tumorigenesis and cancer progression by enhancing tumor growth, angiogenesis, invasion and immune evasion, while others have tumor suppressive effects (Hayes, et al., Trends Mol Med 20(8): 460-9, 2014; Stahlhut and Slack, Genome Med 5 (12): 111, 2013). The expression profile of cancer miRNAs can be used to predict patient prognosis and clinical response to treatment (Bouchie, Nat Biotechnol 31(7): 577, 2013). The majority of miRNAs are intracellular localized, however circulating miRNAs have been detected in various body fluids and represent new biomarkers of solid and hematologic cancers (Fabris and Calin, Mol Oncol 10(3):503-8, 2016; Allegra, et al., Int J Oncol 41(6): 1897-912, 2012). This review describes the clinical relevance of miRNAs, lncRNAs and snoRNAs in the diagnosis, prognosis and treatment response in patients with chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML) and acute adult T-cell leukemia (ATL).

Mutation of epigenetic regulators TET2 and MLL3 in patients with HTLV-I-induced acute adult T-cell leukemia.

BACKGROUND: Epigenetic regulators play a critical role in the maintenance of specific chromatin domains in an active or repressed state. Disruption of epigenetic regulatory mechanisms is widespread in cancer cells and largely contributes to the transformation process through active repression of tumor suppressor genes. While mutations of epigenetic regulators have been reported in various lymphoid malignancies and solid cancers, mutation of these genes in HTLV-I-associated T-cell leukemia has not been investigated. METHOD: Here we used whole genome next generation sequencing (NGS) of uncultured freshly isolated ATL samples and identified the presence of mutations in SUZ12, DNMT1, DNMT3A, DNMT3B, TET1, TET2, IDH1, IDH2, MLL, MLL2, MLL3 and MLL4. RESULTS: TET2 was the most frequently mutated gene, occurring in 32 % (10/31) of ATL samples analyzed. Interestingly, NGS revealed nonsense mutations accompanied by loss of heterozygosity (LOH) in TET2 and MLL3, which was further confirmed by cloning and direct sequencing of DNA from uncultured cells. Finally, direct sequencing of matched control and tumor samples revealed that TET2 mutation was present only in ATL tumor cells. CONCLUSIONS: Our results suggest that inactivation of MLL3 and TET2 may play an important role in the tumorigenesis process of HTLV-I-induced ATL.

Small PARP inhibitor PJ-34 induces cell cycle arrest and apoptosis of adult T-cell leukemia cells.

BACKGROUND: HTLV-I is associated with the development of an aggressive form of lymphocytic leukemia known as adult T-cell leukemia/lymphoma (ATLL). A major obstacle for effective treatment of ATLL resides in the genetic diversity of tumor cells and their ability to acquire resistance to chemotherapy regimens. As a result, most patients relapse and current therapeutic approaches still have limited long-term survival benefits. Hence, the development of novel approaches is greatly needed. METHODS: In this study, we found that a small molecule inhibitor of poly (ADP-ribose) polymerase (PARP), PJ-34, is very effective in activating S/G2M cell cycle checkpoints, resulting in permanent cell cycle arrest and reactivation of p53 transcription functions and caspase-3-dependent apoptosis of HTLV-I-transformed and patient-derived ATLL tumor cells. We also found that HTLV-I-transformed MT-2 cells are resistant to PJ-34 therapy associated with reduced cleaved caspase-3 activation and increased expression of RelA/p65. CONCLUSION: Since PJ-34 has been tested in clinical trials for the treatment of solid tumors, our results suggest that some ATLL patients may be good candidates to benefit from PJ-34 therapy.

The Emerging Role of miRNAs in HTLV-1 Infection and ATLL Pathogenesis.

Human T-cell leukemia virus (HTLV)-1 is a human retrovirus and the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a fatal malignancy of CD4/CD25+ T lymphocytes. In recent years, cellular as well as virus-encoded microRNA (miRNA) have been shown to deregulate signaling pathways to favor virus life cycle. HTLV-1 does not encode miRNA, but several studies have demonstrated that cellular miRNA expression is affected in infected cells. Distinct mechanisms such as transcriptional, epigenetic or interference with miRNA processing machinery have been involved. This article reviews the current knowledge of the role of cellular microRNAs in virus infection, replication, immune escape and pathogenesis of HTLV-1.

STAT1: A Novel Target of miR-150 and miR-223 Is Involved in the Proliferation of HTLV-I-Transformed and ATL Cells.

We have previously reported on the deregulation of cellular microRNAs involved in hematopoiesis and inflammation in human T-cell lymphotropic virus type 1 (HTLV-I)-transformed cells. In this study, we demonstrate that miR-150 and miR-223 specifically target the signal transducer and activator of transcription 1 (STAT1) 3' untranslated region, reducing STAT1 expression and dampening STAT1-dependent signaling in human T cells. The effects of miR-150 and miR-223 on endogenous STAT1 were confirmed using inducible cell lines. Our studies also showed that miR-150 expression is upregulated by interleukin-2 signaling in adult T cell leukemia/lymphoma (ATL) cells. HTLV-I-transformed and ATL-derived cells have reduced levels of miR150 and miR223 expression, which coincide with increased STAT1 expression and STAT1-dependent signaling. Knockdown of STAT1 by short hairpin RNA demonstrated that the constitutive activation of STAT1 is required for the continuous proliferation of HTLV-I-transformed cells. Our studies further demonstrate that increased expression of STAT1 in ATL cells is associated with higher levels of major histocompatibility complex class I expression. Previous studies have demonstrated that the pressure exerted by natural killer (NK) cells in vivo can edit leukemic tumor cells by forcing an increased expression of major histocompatibility complex class I to escape immune clearance. STAT1-expressing tumor cells produce more aggressive tumors because they cannot be eliminated by NK cells. Our results suggest that therapeutic approaches using combined targeting of STAT1 and MHC class I may be an effective approach to activate NK cell-mediated clearance of ATL tumor cells.

On enzyme-based anticancer molecular dietary manipulations.

Evidence from both epidemiological and experimental observations has fuelled the belief that the high consumption of fruits and vegetables rich in nutrients and phytochemicals may help prevent cancer and heart disease in humans. This concept has been drastically simplified from the dietary approaches to the use of single bioactive components both as a single supplement or in functional foods to manipulate xenobiotic metabolism. These procedures, which aim to induce mutagen/carcinogen detoxification or inhibit their bioactivation, fail to take into account the multiple and paradoxical biological outcomes of enzyme modulators that make their effects unpredictable. Here, we show that the idea that the physiological roles of specific catalysts may be easily manipulated by regular long-term administration of isolated nutrients and other chemicals derived from food plants is not viable. In contrast, we claim that the consumption of healthy diets is most likely to reduce mutagenesis and cancer risk, and that both research endeavours and dietary recommendations should be redirected away from single molecules to dietary patterns as a main strategy for public health policy.