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.     

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 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.     

CD5 is dissociated from the B-cell receptor in B cells from bovine leukemia virus-infected, persistently lymphocytotic cattle: consequences to B-cell receptor-mediated apoptosis

Bovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2, can induce persistent nonneoplastic expansion of the CD5(+) B-cell population, termed persistent lymphocytosis (PL). As in human CD5(+) B cells, we report here that CD5 was physically associated with the B-cell receptor (BCR) in normal bovine CD5(+) B cells. In contrast, in CD5(+) B cells from BLV-infected PL cattle, CD5 was dissociated from the BCR. In B cells from PL cattle, apoptosis decreased when cells were stimulated with antibody to surface immunoglobulin M (sIgM), while in B cells from uninfected cattle, apoptosis increased after sIgM stimulation. The functional significance of the CD5-BCR association was suggested by experimental dissociation of the CD5-BCR interaction by cross-linking of CD5. This caused CD5(+) B cells from uninfected animals to decrease apoptosis when stimulated with anti-sIgM. In contrast, in CD5(+) B cells from PL animals, in which CD5 was already dissociated from the BCR, there was no statistically significant change in apoptosis when CD5 was cross-linked and the cells were stimulated with anti-sIgM. Disruption of CD5-BCR interactions and subsequent decreased apoptosis and increased survival in antigenically stimulated B cells may be a mechanism of BLV-induced PL.     

Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.     

Involvement of glutathione as a mechanism of indirect protection against spontaneous ex vivo apoptosis associated with bovine leukemia virus

Viruses have developed strategies to counteract the apoptotic response of the infected host cells. Modulation of apoptosis is also thought to be a major component of viral persistence and progression to leukemia induced by retroviruses like human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). Here, we analyzed the mechanism of ex vivo apoptosis occurring after isolation of peripheral blood mononuclear cells from BLV-infected sheep. We show that spontaneous apoptosis of ovine B lymphocytes requires at least in part a caspase 8-dependent pathway regardless of viral infection. Cell death is independent of cytotoxic response and does not involve the tumor necrosis factor alpha/NF-kappaB/nitric oxide synthase/cyclooxygenase pathway. In contrast, pharmaceutical depletion of reduced glutathione (namely, gamma-glutamyl-l-cysteinyl-glycine [GSH]) by using ethacrynic acid or 1-pyrrolidinecarbodithioic acid specifically reverts inhibition of spontaneous apoptosis conferred indirectly by protective BLV-conditioned media; inversely, exogenously provided membrane-permeable GSH-monoethyl ester restores cell viability in B lymphocytes of BLV-infected sheep. Most importantly, intracellular GSH levels correlate with virus-associated protection against apoptosis but not with general inhibition of cell death induced by polyclonal activators, such as phorbol esters and ionomycin. Finally, inhibition of apoptosis does not correlate with the activities of GSH peroxidase and GSH reductase. In summary, our data fit into a model in which modulation of the glutathione system is a key event involved in indirect inhibition of apoptosis associated with BLV. These observations could have decisive effects during therapeutic treatment of delta-retroviral pathogenesis.     

A mutant form of the tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity,increases expression and propagation of BLV in vitro but not in vivo

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.     

Human immunodeficiency virus type 1 induces apoptosis in CD4(+) but not in CD8(+) T cells in ex vivo-infected human lymphoid tissue

Progression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4(+) T cells along with death and/or dysfunction of CD8(+) T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4(+) and CD8(+) T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4(+) T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8(+) T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4(+) T cells, neither viral isolate induced apoptosis in CD8(+) T cells. Moreover, in both infected and control tissues we found similar numbers of CD8(+) T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3-anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4(+) T cells, the death of CD8(+) T cells apparently requires additional factors.     

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.     

VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics

Tumor expression of the lymphangiogenic factor VEGF-C is correlated with metastasis and poor prognosis, and although VEGF-C enhances transport to the draining lymph node (dLN) and antigen exposure to the adaptive immune system, its role in tumor immunity remains unexplored. Here, we demonstrate that VEGF-C promotes immune tolerance in murine melanoma. In B16 F10 melanomas expressing a foreign antigen (OVA), VEGF-C protected tumors against preexisting antitumor immunity and promoted local deletion of OVA-specific CD8(+) T cells. Naive OVA-specific CD8(+) T cells, transferred into tumor-bearing mice, were dysfunctionally activated and apoptotic. Lymphatic endothelial cells (LECs) in dLNs cross-presented OVA, and naive LECs scavenge and cross-present OVA in vitro. Cross-presenting LECs drove the proliferation and apoptosis of OVA-specific CD8(+) T cells ex vivo. Our findings introduce a tumor-promoting role for lymphatics in the tumor and dLN and suggest that lymphatic endothelium in the local microenvironment may be a target for immunomodulation.     

Dissemination of bovine leukemia virus-infected cells from a newly infected sheep lymph node

To investigate the early establishment of bovine leukemia virus (BLV) infection, we injected BLV-infected or mock-infected allogeneic cells into the shoulder of sheep in which an efferent lymphatic duct of the draining prescapular lymph node had been cannulated. Rare mononuclear cells acting as centers of BLV infection in culture were present within 4 to 6 days in efferent lymph and within 6 to 10 days in blood. Soon after BLV injection, immunoglobulin M+ (IgM+) and CD8+ cells increased in efferent lymph and oscillated reciprocally in frequency. CD8+ blasts increased on days 4 to 6, when infectious centers increased 100-fold in lymph. On days 6 and 7, both lymph and blood were enriched with CD8+ cells that were labeled late on day 5 with an intravenous pulse of 5-bromo-2'-deoxyuridine (BrdU). Lymph, but not blood, was enriched with BrdU+ B cells on day 7. Capsid-specific antibodies became detectable in efferent lymph on days 6 to 8 and surface glycoprotein-specific antibodies on day 9, preceding their detection in serum by 9 to 14 days. Systemic dissemination of BLV-infected cells was thus accompanied by an increase in proliferating CD8+ cells and the onset of BLV-specific antibodies in lymph. Infectious centers reached maximum frequencies of 0.2% in lymph by days 11 to 13, and then their frequencies increased by 5- to 40-fold in blood cells, suggesting that many infected blood cells do not recirculate back into lymph. Beginning on days 10 to 13, a subpopulation of B cells having high levels of surface IgM increased sharply in peripheral blood. Such cells were not present in lymph. After a day 16 pulse of BrdU, recently proliferated cells that stained intensely for surface IgM appeared in blood within 15 h. Predominantly B lymphocytes contained the viral capsid protein when lymph and blood cells were cultured briefly to allow BLV expression. However, both early in lymph and later in blood, BrdU+ B cells greatly exceeded productively infected cells, indicating that new BLV infections stimulate proliferation of two different populations of B cells.     

The influence of ovine MHC class II DRB1 alleles on immune response in bovine leukemia virus infection

We have reported previously that the alleles of the ovine leukocyte antigen (OLA)-DRB1 gene that encode the Arg-Lys (RK) motif and the Ser-Arg (SR) motif at positions beta70/71 of the OLA-DRbeta1 domain are associated with resistance and susceptibility, respectively, to development of bovine leukemia virus (BLV)-induced ovine lymphoma. Here, to investigate the different immune response in sheep that carried alleles associated with resistance and susceptible for 30 weeks after infection with BLV, we selected sheep that had the RK/RK or SR/SR genotype among the 52 sheep analyzed by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing of PCR product for the OLA-DRB1 exon 2 and infected them with BLV. Although the number of BLV-infected cells and virus titer had been maintaining low levels throughout the experimental period, the sheep with the RK/RK genotype could induce expansion of CD5- B-cells and rapid production of neutralizing antibody in the early phase of infection. The level of incorporation of [3H]thymidine by peripheral blood mononuclear cells from the sheep with RK/RK genotype gave a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-helper epitope of the BLV envelope glycoprotein gp51. In contrast, the sheep with SR/SR genotype showed a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-cytotoxic and B-cell epitopes. In such cases, the animals with the RK/RK strongly expressed IFN-gamma, the animals with SR/SR genotype strongly expressed IL-2. To determine the proliferating cells, we tried a blocking assay with monoclonal antibodies such as anti-CD4, -CD8 and -DR molecule. We found that these proliferating cells were MHC-restricted CD4+ T-cells.     

Apoptosis by neglect of CD4+ Th cells in granulomas: a novel effector mechanism involved in the control of egg-induced immunopathology in murine schistosomiasis

In infection with Schistosoma mansoni, parasite eggs precipitate an intrahepatic granulomatous and fibrosing inflammation that is mediated by CD4(+) Th cells. Compared with CBA mice, C57BL/6 mice develop smaller granulomas composed of cells that exhibit reduced proliferative responses to schistosome egg Ags. In the present study, we investigated CD4(+) T cell apoptosis as a possible mechanism that could account for this subdued response. We found throughout the course of several infection weeks a markedly higher proportion of apoptotic CD4(+) T cells in granulomas from C57BL/6 mice than in those from CBA mice ex vivo; the apoptosis further increased upon cell cultivation in vitro. Activation-induced cell death or CD8(+) T cells failed to account for the enhanced apoptosis as infected Fas-, Fas ligand,- and CD8-deficient mice exhibited similar apoptosis to that seen in wild-type counterparts. However, a strikingly lower IL-2 production by schistosome egg Ag-stimulated C57BL/6 granuloma and mesenteric lymph node cells suggested the possibility of apoptosis due to growth factor deprivation. Indeed, the CD4(+) T cell apoptosis was significantly reversed by addition of rIL-2 in vitro, or by injection of rIL-2 in vivo, which also resulted in significant exacerbation of granulomatous inflammation. These findings indicate that apoptosis by neglect can represent a significant means of controlling CD4(+) T cells that mediate the immunopathology in schistosomiasis.     

Clonality and longevity of CD4+CD28null T cells are associated with defects in apoptotic pathways

CD4(+)CD28(null) T cells are oligoclonal lymphocytes rarely found in healthy individuals younger than 40 yr, but are found in high frequencies in elderly individuals and in patients with chronic inflammatory diseases. Contrary to paradigm, they are functionally active and persist over many years. Such clonogenic potential and longevity suggest altered responses to apoptosis-inducing signals. In this study, we show that CD4(+)CD28(null) T cells are protected from undergoing activation-induced cell death. Whereas CD28(+) T cells underwent Fas-mediated apoptosis upon cross-linking of CD3, CD28(null) T cells were highly resistant. CD28(null) T cells were found to progress through the cell cycle, and cells at all stages of the cell cycle were resistant to apoptosis, unlike their CD28(+) counterparts. Neither the activation-induced up-regulation of the IL-2R alpha-chain (CD25) nor the addition of exogenous IL-2 renders them susceptible to Fas-mediated apoptosis. These properties of CD28(null) T cells were related to high levels of Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein, an inhibitor of Fas signaling that is normally degraded in T cells following activation in the presence of IL-2. Consistent with previous data showing protection of CD28(null) cells from spontaneous cell death, the present studies unequivocally show dysregulation of apoptotic pathways in CD4(+)CD28(null) T cells that favor their clonal outgrowth and maintenance in vivo.     

"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.     

Involvement of bovine leukemia virus in induction and inhibition of apoptosis

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G, that has an enhanced capacity to transactivate the long terminal repeat (LTR) of BLV and the cellular proto-oncogene c-fos when compared with wild-type Tax (wt-Tax). We demonstrate here that an infectious strain of BLV containing the mutant D247G form of Tax also differs in its capacity to modulate cell survival both positively and negatively. When peripheral blood mononuclear cells (PBMCs) infected with wild-type or mutant BLV are cultured ex vivo with staurosporine, an agent known to induce a mitochondrial caspase cascade pathway regulating apoptosis, the rate of apoptosis is reduced to a greater extent in cells infected with mutant BLV than wild-type BLV, consistent with previous observations in cultures without staurosporine. The increase in survival was associated with an increase in expression of mRNA of bcl-xl but not bcl-2 and bax ex vivo. In contrast, when a tissue culture-adapted cell line, 293T, was transiently transfected with either wild-type or mutant BLV, apoptosis was induced. The increase in the rate of apoptosis was higher in cells transfected with mutant BLV. The same difference was noted in cells transiently transfected with wild-type and mutant D247G Tax, suggesting that the observed positive and negative modulation of cell survival is attributed to the functional characteristics of mutant D247G Tax.     

In vivo leukocyte tropism of bovine leukemia virus in sheep and cattle.

Bovine leukemia virus (BLV), an oncovirus related to human T-cell leukemia virus type I, causes a B-cell lymphoproliferative syndrome in cattle, leading to an inversion of the T-cell/B-cell ratio and, more rarely, to a B-cell lymphosarcoma. Sheep are highly sensitive to BLV experimental infection and develop B-cell pathologies similar to those in cattle in 90% of the cases. BLV tropism for B cells has been well documented, but the infection of other cell populations may also be involved in the BLV-induced lymphoproliferative syndrome. We thus looked for BLV provirus in other leukocyte populations in sheep and cattle by using PCR. We found that while B cells harbor the highest proviral load, CD8+ T cells, monocytes, and granulocytes, but not CD4+ T cells, also bear BLV provirus. As previously described, we found that persistent lymphocytosis in cows is characterized by an expansion of the CD5+ B-cell subpopulation but we did not confirm this observation in sheep in which the expanded B-cell population expressed the CD11b marker. Nevertheless, BLV could be detected both in bovine CD5+ and CD5- B cells and in sheep CD11b+ and CD11b- B cells, indicating that the restricted BLV tropism for a specific B-cell subpopulation cannot explain its expansion encountered in BLV infection. Altogether, this work shows that BLV tropism in leukocytes is wider than previously thought. These results lead the way to further studies of cellular interactions among B cells and other leukocytes that may intervene in the development of the lymphoproliferative syndrome induced by BLV infection.     

Complete Bovine Leukemia Virus (BLV) Provirus Is Conserved in BLV-Infected Cattle throughout the Course of B-Cell Lymphosarcoma Development

Bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) belong to the same subfamily of oncoviruses. Defective HTLV-1 proviral genomes have been found in more than half of all patients with adult T-cell leukemia examined. We have characterized the genomic structure of integrated BLV proviruses in peripheral blood lymphocytes and tumor tissue taken from animals with lymphomas at various stages. Genomic Southern hybridization with SacI, which generates two major fragments of BLV proviral DNA, yielded only bands that corresponded to a full-size provirus in all of 23 cattle at the lymphoma stage and in 7 BLV-infected but healthy cattle. Long PCR with primers located in long terminal repeats clearly demonstrated that almost the complete provirus was retained in all of 27 cattle with lymphomas and in 19 infected but healthy cattle. However, in addition to a PCR product that corresponded to a full-size provirus, a fragment shorter than that of the complete virus was produced in only one of the 27 animals with lymphomas. Moreover, when we performed conventional PCR with a variety of primers that spanned the entire BLV genome to detect even small defects, PCR products were produced that specifically covered the entire BLV genome in all of the 40 BLV-infected cattle tested. Therefore, it appears that at least one copy of the full-length BLV proviral genome was maintained in each animal throughout the course of the disease and, in addition, that either large or small deletions of proviral genomes may be very rare events in BLV-infected cattle.     

The development of colitogenic CD4(+) T cells is regulated by IL-7 in collaboration with NK cell function in a murine model of colitis

We previously reported that IL-7(-/-)RAG(-/-) mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4(+) T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4(+) T cells. To further investigate these roles of NK cells, RAG(-/-) and IL-7(-/-)RAG(-/-) mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (T(EM)) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44(+)CD62L(-) T(EM) and unique CD44(-)CD62L(-) T cell subsets were observed in the T cell-reconstituted RAG(-/-) recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44(+)CD62L(-) T(EM) subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG(-/-) and IL-7(-/-)RAG(-/-) recipient mice through targeting of colitogenic CD4(+)CD44(+)CD62L(-) T(EM) and, possibly, of the newly observed CD4(+)CD44(-)CD62L(-) subset present at the early stage of T cell development.