Rates of CTL killing in persistent viral infection in vivo.

The CD8+ cytotoxic T lymphocyte (CTL) response is an important defence against viral invasion. Although CTL-mediated cytotoxicity has been widely studied for many years, the rate at which virus-infected cells are killed in vivo by the CTL response is poorly understood. To date the rate of CTL killing in vivo has been estimated for three virus infections but the estimates differ considerably, and killing of HIV-1-infected cells was unexpectedly low. This raises questions about the typical anti-viral capability of CTL and whether CTL killing is abnormally low in HIV-1. We estimated the rate of killing of infected cells by CD8+ T cells in two distinct persistent virus infections: sheep infected with Bovine Leukemia Virus (BLV) and humans infected with Human T Lymphotropic Virus type 1 (HTLV-1) which together with existing data allows us to study a total of five viruses in parallel. Although both BLV and HTLV-1 infection are characterised by large expansions of chronically activated CTL with immediate effector function ex vivo and no evidence of overt immune suppression, our estimates are at the lower end of the reported range. This enables us to put current estimates into perspective and shows that CTL killing of HIV-infected cells may not be atypically low. The estimates at the higher end of the range are obtained in more manipulated systems and may thus represent the potential rather than the realised CTL efficiency.

Viral expression directs the fate of B cells in bovine leukemia virus-infected sheep.

The host immune response is believed to tightly control viral replication of deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). However, this assumption has not been definitely proven in vivo. In order to further evaluate the importance of the immune response in the BLV model, we studied the fate of cells in which viral expression was transiently induced. Using a dual fluorochrome labeling approach, we showed that ex vivo induction of viral expression induces higher death rates of B cells in vivo. Furthermore, cyclosporine treatment of these animals indicated that an efficient immune response is required to control virus-expressing cells.

Reduced levels of reactive oxygen species correlate with inhibition of apoptosis, rise in thioredoxin expression and increased bovine leukemia virus proviral loads.

BACKGROUND: Bovine Leukemia virus (BLV) is a deltaretrovirus that induces lymphoproliferation and leukemia in ruminants. In ex vivo cultures of B lymphocytes isolated from BLV-infected sheep show that spontaneous apoptosis is reduced. Here, we investigated the involvement of reactive oxygen species (ROS) in this process. RESULTS: We demonstrate that (i) the levels of ROS and a major product of oxidative stress (8-OHdG) are reduced, while the thioredoxin antioxidant protein is highly expressed in BLV-infected B lymphocytes, (ii) induction of ROS by valproate (VPA) is pro-apoptotic, (iii) inversely, the scavenging of ROS with N-acetylcysteine inhibits apoptosis, and finally (iv) the levels of ROS inversely correlate with the proviral loads. CONCLUSION: Together, these observations underline the importance of ROS in the mechanisms of inhibition of apoptosis linked to BLV infection.

Emphasis on cell turnover in two hosts infected by bovine leukemia virus: a rationale for host susceptibility to disease.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects and induces accumulation of B-lymphocytes in the peripheral blood and lymphoid tissues of cattle, leading to leukemia/lymphoma. BLV can also be experimentally transmitted to sheep, in which disease appears earlier and at higher frequencies. Abnormal accumulation of leukemic B-lymphocytes results from an alteration of different parameters that include cell proliferation and death as well as migration to lymphoid tissues. Interestingly, B lymphocyte turnover is increased in BLV-infected sheep but reduced in cattle, revealing a potential relationship between cell kinetics and disease progression.

Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human.

In 1871, the observation of yellowish nodules in the enlarged spleen of a cow was considered to be the first reported case of bovine leukemia. The etiological agent of this lymphoproliferative disease, bovine leukemia virus (BLV), belongs to the deltaretrovirus genus which also includes the related human T-lymphotropic virus type 1 (HTLV-1). This review summarizes current knowledge of this viral system, which is important as a model for leukemogenesis. Recently, the BLV model has also cast light onto novel prospects for therapies of HTLV induced diseases, for which no satisfactory treatment exists so far.

Cell dynamics and immune response to BLV infection: a unifying model.

Bovine Leukemia virus (BLV) is the natural etiological agent of a lymphoproliferative disease in cattle. BLV can also be transmitted experimentally to a related ruminant species, sheep, in which the pathogenesis is more acute. Although both susceptible species develop a strong anti-viral immune response, the virus persists indefinitely throughout life, apparently at a transcriptionally silent stage, at least in a proportion of infected cells. Soon after infection, these humoral and cytotoxic activities very efficiently abolish the viral replicative cycle, permitting only mitotic expansion of provirus-carrying cells. Short term cultures of these infected cells initially indicated that viral expression protects against spontaneous apoptosis, suggesting that leukemia is a process of accumulation of long-lived cells. This conclusion was recently reconsidered following in vivo dynamic studies based on perfusions of nucleoside (bromodeoxyuridine) or fluorescent protein markers (CFSE). In sheep, the turnover rate of infected cells is increased, suggesting that a permanent clearance process is exerted by the immune system. Lymphocyte trafficking from and to the secondary lymphoid organs is a key component in the maintenance of cell homeostasis. The net outcome of the immune selective pressure is that only cells in which the virus is transcriptionally silenced survive and accumulate, ultimately leading to lymphocytosis. Activation of viral and/or cellular expression in this silent reservoir with deacetylase inhibitors causes the collapse of the proviral loads. In other words, modulation of viral expression appears to be curative in lymphocytic sheep, an approach that might also be efficient in patients infected with the related Human T-lymphotropic virus type 1. In summary, a dynamic interplay between BLV and the host immune response modulates a complex equilibrium between (i) viral expression driving (or) favoring proliferation and (ii) viral silencing preventing apoptosis. As conclusion, we propose a hypothetical model unifying all these mechanisms.

Quantifying lymphocyte kinetics in vivo using carboxyfluorescein diacetate succinimidyl ester (CFSE).

The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify cell kinetics. It is particularly important in studies of lymphocyte homeostasis where its labelling of cells irrespective of their stage in the cell cycle makes it preferable to deuterated glucose and BrdU, which only label dividing cells and thus produce unrepresentative results. In the past, experiments have been limited by the need to obtain a clear separation of CFSE peaks forcing scientists to adopt a strategy of in vitro labelling of cells followed by their injection into the host. Here we develop a framework for analysis of in vivo CFSE labelling data. This enables us to estimate the rate of proliferation and death of lymphocytes in situ, and thus represents a considerable advance over current procedures. We illustrate this approach using in vivo CFSE labelling of B lymphocytes in sheep.

Chemoresistance to Valproate Treatment of Bovine Leukemia Virus-Infected Sheep; Identification of Improved HDAC Inhibitors.

We previously proved that a histone deacetylase inhibitor (valproate, VPA) decreases the number of leukemic cells in bovine leukemia virus (BLV)-infected sheep. Here, we characterize the mechanisms initiated upon interruption of treatment. We observed that VPA treatment is followed by a decrease of the B cell counts and proviral loads (copies per blood volume). However, all sheep eventually relapsed after different periods of time and became refractory to further VPA treatment. Sheep remained persistently infected with BLV. B lymphocytes isolated throughout treatment and relapse were responsive to VPA-induced apoptosis in cell culture. B cell proliferation is only marginally affected by VPA ex vivo. Interestingly, in four out of five sheep, ex vivo viral expression was nearly undetectable at the time of relapse. In two sheep, a new tumoral clone arose, most likely revealing a selection process exerted by VPA in vivo. We conclude that the interruption of VPA treatment leads to the resurgence of the leukemia in BLV-infected sheep and hypothesize that resistance to further treatment might be due to the failure of viral expression induction. The development of more potent HDAC inhibitors and/or the combination with other compounds can overcome chemoresistance. These observations in the BLV model may be important for therapies against the related Human T-lymphotropic virus type 1.

Preventive and therapeutic strategies for bovine leukemia virus: lessons for HTLV.

Bovine leukemia virus (BLV) is a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1). BLV is a major animal health problem worldwide causing important economic losses. A series of attempts were developed to reduce prevalence, chiefly by eradication of infected cattle, segregation of BLV-free animals and vaccination. Although having been instrumental in regions such as the EU, these strategies were unsuccessful elsewhere mainly due to economic costs, management restrictions and lack of an efficient vaccine. This review, which summarizes the different attempts previously developed to decrease seroprevalence of BLV, may be informative for management of HTLV-1 infection. We also propose a new approach based on competitive infection with virus deletants aiming at reducing proviral loads.

Earlier onset of delta-retrovirus-induced leukemia after splenectomy.

Infection by delta-retroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) is mostly asymptomatic. Indeed, only a minority (<5%) of delta-retrovirus infected hosts will develop either lymphoproliferative or neurodegenerative diseases after long latency periods. In fact, the host immune response is believed to tightly control viral replication but this assumption has not been definitely proven in vivo. Here, we provide direct experimental evidence demonstrating that integrity of the spleen is required to control pathogenesis. In the BLV model, we show that asplenia decreases efficiency of the immune response and induces an imbalance in cell dynamics resulting in accelerated onset of leukemia. These observations enlighten a potential threat in splenectomized HTLV-1 carriers and justify a regular preventive evaluation.

Gene activation therapy: from the BLV model to HAM/TSP patients.

HTLV-1 (human T-lymphotropic virus type 1) and BLV (bovine leukemia virus) are two related retroviruses infecting CD4+ and B lymphocytes in humans and ruminants, respectively. During infection, the host-pathogen interplay is characterized by very dynamic kinetics resulting in equilibrium between the virus, which attempts to proliferate, and the immune response, which seeks to exert tight control of the virus. A major determinant of disease induction by both viruses is the accumulation of provirus in peripheral blood. In the absence of viral proteins, virus infected cells escape recognition and destruction by the host immune response. We propose a novel therapeutic strategy based on transient activation of viral expression using epigenetic modulators; this exposes infected cells to the immune response and results in significant reductions in proviral loads. In the absence of satisfactory therapies, this viral gene-activation strategy might delay progression, or even be curative, for HTLV-1 induced myelopathy / tropical spastic paraparesis (HAM/TSP).

Even attenuated bovine leukemia virus proviruses can be pathogenic in sheep.

Based on a reverse genetics approach, we previously reported that bovine leukemia virus (BLV) mutants harboring deletions in the accessory R3 and G4 genes persist at very low proviral loads and are unable to induce leukemia or lymphoma in sheep, indicating that these R3 and G4 gene sequences are required for pathogenesis. We now show that lymphoma can occur, albeit infrequently (1 case of 20) and after extended periods of latency (7 years). Direct sequencing and reinfection experiments demonstrated that lymphomagenesis was not due to the reversion of the mutant to the wild type. Similar observations with another type of attenuated mutant impaired in the transmembrane protein (TM) YXXL signaling motifs were made. We conclude that the R3 and G4 genes and the TM YXXL motifs are not strictly required for pathogenesis but that their integrity contributes to disease frequency and latency.

Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep.

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.

Peripheral blood B-cell death compensates for excessive proliferation in lymphoid tissues and maintains homeostasis in bovine leukemia virus-infected sheep.

The size of a lymphocyte population is primarily determined by a dynamic equilibrium between cell proliferation and death. Hence, lymphocyte recirculation between the peripheral blood and lymphoid tissues is a key determinant in the maintenance of cell homeostasis. Insights into these mechanisms can be gathered from large-animal models, where lymphatic cannulation from individual lymph nodes is possible. In this study, we assessed in vivo lymphocyte trafficking in bovine leukemia virus (BLV)-infected sheep. With a carboxyfluorescein diacetate succinimidyl ester labeling technique, we demonstrate that the dynamics of lymphocyte recirculation is unaltered but that accelerated proliferation in the lymphoid tissues is compensated for by increased death in the peripheral blood cell population. Lymphocyte homeostasis is thus maintained by biphasic kinetics in two distinct tissues, emphasizing a very dynamic process during BLV infection.

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.