Bovine Leukemia Virus-Induced Persistent Lymphocytosis in Cattle Does Not Correlate with Increased Ex Vivo Survival of B Lymphocytes

Bovine leukemia virus (BLV) is an oncogenic retrovirus associated with B-cell lymphocytosis, leukemia, and lymphosarcoma in the ovine and bovine species. We have recently reported that in sheep, BLV protects the total population of peripheral blood mononuclear cells (PBMCs) from ex vivo spontaneous apoptosis. This global decrease in the apoptosis rates resulted from both direct and indirect mechanisms which allow extension of cell survival. Although sheep are not natural hosts for BLV, these animals are prone to develop virus-induced leukemia at very high frequencies. Most infected cattle, however, remain clinically healthy. This difference in the susceptibilities to development of leukemia in these two species might be related to alterations of the apoptotic processes. Therefore, we designed this study to unravel the mechanisms of programmed cell death in cattle. We have observed that PBMCs from persistently lymphocytotic BLV-infected cows were more susceptible to spontaneous ex vivo apoptosis than cells from uninfected or aleukemic animals. These higher apoptosis rates were the consequence of an increased proportion of B cells exhibiting lower survival abilities. About one-third of the BLV-expressing cells did not survive the ex vivo culture conditions, demonstrating that viral expression is not strictly associated with cell survival in cattle. Surprisingly, culture supernatants from persistently lymphocytotic cows exhibited efficient antiapoptotic properties on both uninfected bovine and uninfected ovine cells. It thus appears that indirect inhibition of cell death can occur even in the presence of high apoptosis rates. Together, these results demonstrate that the protection against spontaneous apoptosis associated with BLV is different in cattle and in sheep. The higher levels of ex vivo apoptosis occurring in cattle might indicate a decreased susceptibility to development of leukemia in vivo.     

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis.

Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo. Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV.     

"Shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence

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.     

Ex vivo survival of peripheral blood mononuclear cells in sheep induced by bovine leukemia virus (BLV) mainly occurs in CD5- B cells that express BLV

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis (EBL). In a previous report, we found that in a sheep model, only CD5(-) B cells proliferated clonally, while CD5(+) B cells rapidly decreased when the disease progressed to the lymphoma stage. We demonstrate here that, although both CD5(+) and CD5(-) B cells, but not CD4(+) T, CD8(+) T and gammadeltaTCR(+)T cells, are protected from spontaneous ex vivo apoptosis in sheep infected with wild-type and a mutant BLV that encodes a mutant Tax D247G protein with elevated trans-activation activity, only CD5(-) B cells become the main target for ex vivo survival when the disease proceeds to the persistent lymphocytotic stage, which showed an increased expansion of the CD5(-) B cells. In addition, we identified, by four-color flow cytometric analysis, that in CD5(-) B cells, the apoptotic rates of cells that expressed wild-type and mutant BLV were greatly decreased compared with those of BLV-negative cells. There was only a slight reduction in the apoptotic rates in BLV-positive cells from CD5(+) B cells. In addition, supernatants from peripheral blood mononuclear cell (PBMC) cultures from wild-type- and mutant BLV-infected sheep mainly protected CD5(-) B cells from spontaneous apoptosis. Our results suggest that, although BLV can protect both CD5(+) and CD5(-) B cells from ex vivo apoptosis, the mechanisms accounting for the ex vivo survival between these two B-cell subsets differ. Therefore, it appears that the phenotypic changes in cells that express CD5 at the lymphoma stage could result from a difference in susceptibility to apoptosis in CD5(+) and CD5(-) B cells in BLV-infected sheep.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

Cellular pathways involved in the ex vivo expression of bovine leukemia virus.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression.     

Latency of viral expression in vivo is not related to CpG methylation in the U3 region and part of the R region of the long terminal repeat of bovine leukemia virus

Bovine leukemia virus (BLV) is silent in most cells detectable in vivo, and the repression of its expression allows BLV to evade the host's immune response. In this study, we examined whether CpG methylation of DNA might be involved in the regulation of the expression of BLV in vivo. To investigate the effects of CpG methylation on the activity of the long terminal repeat (LTR) of BLV, we measured the transactivation activity of this region after treatment with the CpG methyltransferase SssI by using a luciferase reporter system. The activity of methylated LTR was significantly lower than that of nonmethylated LTR. Therefore, we examined the extent of CpG methylation of the U3 region and part of the R region of the LTR in BLV-infected cattle and in experimentally BLV-infected sheep at various clinical stages by the bisulfite genomic sequencing method. We detected no or minimal CpG methylation at all stages examined in cattle and sheep, and our results indicate that CpG methylation probably does not participate in the silencing of BLV in vivo.     

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.     

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.     

Bovine leukemia virus transmembrane protein gp30 physically associates with the down-regulatory phosphatase SHP-1

In B lymphocytes, the down-regulatory phosphatase SHP-1 associates with CD22 and CD32b (also known as FcgammaRIIB) and acts as a critical negative regulator of B-cell receptor signaling. Bovine leukemia virus, a retrovirus of the HTLV/BLV group, causes persistently increased numbers of peripheral blood B lymphocytes, known as persistent lymphocytosis (PL) and, in some animals, progression to B-cell leukemia and/or lymphoma. Here, we show that SHP-1 associates with the bovine leukemia virus transmembrane protein, gp30. This interaction is either direct or indirect. The interaction is dependent on tyrosine phosphorylation, and the interaction increases after cell stimulation with sodium pervanadate. The gp30-SHP-1 interaction is seen in all of the BLV-infected, PL animals tested, but is not seen in uninfected animals or in most BLV-infected, non-PL animals, which do not express significant quantities of gp30. However, one BLV-infected, non-PL animal expressed large quantities of gp30, yet no gp30-SHP-1 interaction was detected, suggesting that there may be other factors in cells from the PL animals that facilitate the gp30-SHP-1 interaction. The association of gp30 and SHP-1 suggests the hypothesis that gp30 may act as a decoy to sequester SHP-1, resulting in up-regulation of B-cell receptor signaling. The implication of this could be a novel mechanism of viral activation of lymphocytes by removal of a down-regulatory phosphatase.     

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.     

Bovine Leukemia Virus-Induced Lymphocytosis and Increased Cell Survival Mainly Involve the CD11b+ B-Lymphocyte Subset in Sheep

In this study, we show that bovine leukemia virus (BLV)-induced persistent lymphocytosis (PL) results from the in vivo expansion of the CD11b⁺ B-lymphocyte population. This subset shares phenotypic characteristics with murine and human B-1 cells. BLV interactions with the sheep B-1-like subset were explored. We found that B-1- and B-2-like cells are initially infected to similar extents. However, in long-term-infected sheep, the viral load is higher in B-1-like cells and only B-1- and not B-2-like cells show increased ex vivo survival compared to that in uninfected sheep. Ex vivo viral expression was found in both B-1- and B-2-like cells, indicating that both cell types support viral replication. Finally, cycloheximide and a protein kinase C inhibitor (H7) that blocks the ex vivo activation of viral expression did not affect the increased survival in B-1-like cells, suggesting that resistance to apoptosis is acquired in vivo. Collectively, these results indicate a peculiar susceptibility of sheep B-1-like cells to BLV transforming effects and further support the involvement of increased survival in BLV pathogenesis.     

T-cell responses to highly conserved CD4 and CD8 epitopes on the outer membrane protein of bovine leukemia virus: relevance to vaccine development.

Bovine leukemia virus (BLV) is a retrovirus that infects cattle and sheep and may provide a model for studying human leukemia. Cell-mediated immune mechanisms may play a major role in protection against BLV infection. We describe here for the first time the identification of proliferative (CD4) and cytotoxic T-lymphocyte (CD8) epitopes of the gp51 envelope (env) protein of BLV. This protein and a recombinant form expressed by a vaccinia virus construct have been shown to be potential vaccine candidates. A complete series of overlapping peptides, 20 amino acids in length, was prepared to identify epitopes from gp51. These peptides were tested for the ability to elicit peripheral blood lymphocyte proliferation and cytotoxic T-lymphocyte responses in infected and uninfected cattle and sheep. Peptides 51-70 and 61-80 produced a proliferative response in lymphocytes from only uninfected animals (both sheep and cattle), and this was shown by T-cell subset deletion to be a CD4-mediated response. Seven BLV-infected sheep did not show a response to either peptide. Cytotoxic T-lymphocyte activity, however, was associated only with peptides 121-140 and 131-150. In this case, the response was demonstrated to be CD8 dependent and was found only in BLV-infected animals (sheep). Knowledge of the location of these T-cell recognition domains will complement data available on B-cell epitopes in gp51 and may be useful in the design of a subunit vaccine.